Clinical Immunology_ Principles and Practice ( PDFDrive )

430 ParT THrEE Host Defenses to Infectious Agents


in part through TLR2 and possibly TLR4. CpG motifs in the T. pathway, T-cell or macrophage suppressor activity, and PGE 2
cruzi genome have also been shown to activate TLR9. Several production. Within the foci of myocarditis, apoptosis of both
other TLR-independent mechanisms of innate immunity, includ- parasites and host cells occurs. The phagocytosis of these apoptotic
ing the activation of nucleotide-binding oligomerization domain cells by macrophages leads to their acquisition of an M2 phe-
(NOD)–like receptors, have been identified. 20 notype, which enables parasite replication and persistence. The
rapid escape of the parasite from the phagosome into the
Adaptive Immunity cytoplasm through the action of acid-activated porins enables
The innate immune response, through production of IL-12, type the organism to avoid phagolysosomal enzymatic destruction.
I IFNS, and other proinflammatory mediators, is critically linked
to the generation of an effective adaptive immune response. Toxoplasma gondii
Antibodies contribute to immunity within the bloodstream
through opsonization, complement activation, and antibody- Pathogenesis
dependent cellular cytotoxicity. Transmission occurs via the ingestion of oocysts, which are shed
Several lines of evidence establish the importance of T cells in the feces of felines or via tissue cysts present in undercooked
in adaptive immunity to T. cruzi infection. Parasite-specific CD4 meat. Following the oral ingestion of cysts, phagocytes recruited to
and CD8 T cells are activated during infection, and mice lacking the gut lumen facilitate transepithelial migration into the lamina
CD4 or CD8 T cells have impaired parasite control. CD8 T cells propria. These phagocytes have been termed “Trojan Horses,”
with cytotoxic activity against T. cruzi–infected cells have been as they carry the parasite to new, unsuspecting host cells. Host-
identified in infected mice, and these cells confer protection cell invasion starts with loose attachment facilitated by laminin.
21
when passively transferred to naïve mice. In the early stage of Intracellular tachyzoites replicate within a parasitophorous
infection, CD4 T cells are the predominant subset recruited to vacuole (PV) and ultimately leave the cell by using an active
the myocardium, but activated CD8 T cells soon dominate the egress pathway. The released tachyzoites can disseminate to invade
inflammatory process in cardiac tissue. IFN-γ and TNF production virtually any nucleated cell type, but mononuclear phagocytes are
by parasite-specific CD8 T cells is more important than cytolytic the preferred host cells. The toxoplasma SAG proteins mediate
activity in the control of infection. 21 parasite attachment, and the transmembrane adhesins, MIC and
T. cruzi infection leads to a mixed Th1/Th2 cytokine response, AMA1, and RON family proteases play major roles in the myosin
and in general, the Th1/Th2 balance determines resistance or motor-driven process of host-cell invasion. Under pressure from
susceptibility. As noted earlier, IL-12/STAT4-dependent IFN-γ the host immune response, tachyzoite replication is controlled
production by NK cells in early infection, and later by T cells, and tissue cysts, containing slowly replicating bradyzoites, are
is critically important to protection. IL-4 does not appear to formed. The tissue cysts persist as a chronic latent infection
play a major role in susceptibility to T. cruzi infection, but IL-10 as long as the host immune function is intact. If the latently
promotes parasite replication by inhibiting macrophage trypano- infected person is immunosuppressed, reactivation occurs, and
cidal activity. IL-10 also plays a critical role in minimizing tachyzoites are released to infect more cells. Because tissue cysts
inflammation-mediated tissue pathology by regulating the Th1 (bradyzoites) are found in proportionately larger numbers in the
and TNF responses. Polymorphisms in the IL-10 gene that lead brain, reactivation of latent infection in the immunocompromised
to reduced IL-10 production are associated with increased severity host most commonly manifests as encephalitis.
of cardiomyopathy in patients with Chagas disease. Similarly,
TGF-β has been shown to inhibit macrophage trypanocidal Innate Immunity
activity and increase parasitemia and mortality. In addition to Similar to immunity to Plasmodium and Leishmania, IL-12,
these regulatory cytokines, the secretion of prostaglandins and IFN-γ, TNF-α, and NK cells contribute to the control of the
+
23
NO, the induction of apoptosis of T and B cells, and the expansion early stages of T. gondii infection. CD8α DCs are the primary
of a myeloid suppressor cell population serve to control the producers of IL-12, which is essential to host resistance. IL-12 is
intensity of the immune response. produced through a MyD88-dependent mechanism, at least in
part, through the interaction of parasite-derived proteins and
Evasion of Host Immunity TLR2 and TLR11, and activation of CCR5. IL-12-dependent
24
A significant part of the pathogenesis following T. cruzi infection activation of NK cells leads to IFN-γ production, which, in turn,
is its dissemination through the bloodstream to many tissues. activates macrophages to limit parasite replication. T cells express-
T. cruzi bloodstream trypomastigotes resist complement lysis ing the γδ receptor and neutrophils are also an early source of
via a complement regulatory protein (GP-160). This parasite IFN-γ and TNF-α. Recruitment of inflammatory macrophages
protein is functionally similar to mammalian decay-accelerating to the site of infection is crucial to control of parasite replica-
factor in that it inhibits C3 convertase formation and activation tion and dissemination. IFN-γ-induced macrophage activation is
of the alternate complement pathway. T. cruzi invades host cells, the key effector mechanism leading to parasite killing. In mice,
in particular cardiomyocytes, by subverting a host plasma the induction of immunity-related GTPases (IRG), which
membrane repair pathway that promotes parasite persistence damage the parasitophorous vacuole membrane and kill T.
and tissue tropism. 22 gondii within the cytosol, is the primary macrophage effector
The establishment of chronic infection by T. cruzi is favored mechanism. The generation of reactive nitrogen and oxygen
by a generalized depression of T-cell responses. A number of intermediates, degradation of tryptophan, and the production
different mechanisms may contribute to this, including low IL-2 of LTs have also been implicated in the control of T. gondii in
production or IL-2 receptor expression; downregulation of human macrophages. However, humans lack the entire IRG family,
components of the T cell–receptor complex; T-cell receptor as well as TLR11, and the mechanism(s) of early IFN-γ–medi-
dysfunction; apoptosis of T cells; defects in the processing and ated macrophage activation and parasite control in humans is
presenting of antigens in the MHC class II (but not the class I) unclear.

CHaPTEr 30 Host Defenses to Protozoa 431


microvilli shortening and apical separation that allows the parasite
Adaptive Immunity to penetrate between the epithelial cells and degrade connective
Although serum antibodies can be used in the diagnosis of T. tissue, resulting in ulceration of the mucosa and submucosa (Fig.
gondii infection, the systemic antibody response does not play 30.3). The trophozoites can lyse multiple cell types, including
a role in adaptive immunity. Mucosal IgA, however, does provide neutrophils, which release enzymes that further damage the tissue.
resistance to oral infection with T. gondii cysts. CD4 and CD8 The cytotoxic effects of amebae are mediated by a secreted cysteine
T cells are highly activated during infection and are essential for protease, the Gal/GalNAc lectin, phospholipase A, and contact-
adaptive immunity. As such, patients with defects in T cell– dependent cytolysis, in which an ion channel (amebapore) is
mediated immune responses (e.g., patients with AIDS) are at inserted into the membrane of target cells. E. histolytica can induce
risk for reactivation of latent infection. apoptosis in mammalian cells by a caspase-dependent, TNF-α/
IL-12 from DCs drives the differentiation of T cells into type-1 Fas–independent process. Amebic liver abscesses develop when
CD4 and CD8 T cells that are essential to adaptive immunity. trophozoites erode through the intestinal submucosa, enter the
28
CD4 and CD8 T cells act synergistically to prevent cyst reactivation portal circulation, and disseminate to the liver. Comparison of
during chronic latent infection. Parasite-specific cytolytic T cells the transcriptional profiles of virulent and nonvirulent Entamoeba
have been demonstrated; however, CD8 T cells mediate protection species has identified novel virulence factors in E. histolytica. 29
primarily through the generation of IFN-γ. IL-12 drives the
generation of terminally differentiated CD8 effector T cells, which Innate Immunity
express the killer cell lectin-like receptor G1 (KLRG1) and high Adherence of trophozoites to intestinal epithelial cells stimulates
levels of granzyme B and IFN-γ. The combination of IL-7 and the release of a variety of proinflammatory mediators, including
IL-15 is required for T cm -cell differentiation. IL-1α, IL-1β, IL 6, IL-8, and TNF-α, which triggers the recruit-
Antiinflammatory molecules, particularly IL-10 and IL-27 ment of neutrophils and macrophages to the site of invasion.
made by Th1 cells, play an important role in modulating the Following activation by IFN-γ and TNF-α, neutrophils and
adaptive immune response and restricting host tissue damage. 25 macrophages become amebicidal through the release of reactive
28
oxygen species and production of nitric oxide, respectively.
Evasion of Host Immunity Invariant natural killer T (iNKT) cells, which comprise about
25
T. gondii escapes early macrophage killing in a number of ways. 30% of hepatic lymphocytes, are stimulated by amebic lipopep-
Virulent parasites are protected by localization to the parasi- tidephosphoglycan (LPPG) to release IFN-γ. Production of IFN-γ
tophorous vacuole, which does not fuse with host cell lysosomes reduces parasite burden and controls abscess formation. 28
(probably because the PV membrane proteins are of parasite
rather than host origin), and so the vacuole is not acidified to Adaptive Immunity
kill the parasite. The infected macrophage is also a suboptimal Secretory IgA responses against E. histolytica have been well
target for T cell–induced immunity because of reduced expression characterized and shown to correlate with protection against
of MHC class II and costimulatory molecules. Infection also infection and disease. For this reason, vaccines designed to
induces the production of counterregulatory molecules, such as generate IgA antibodies against the Gal/Gal/NAc lectin have been
IL-10, TGF-β, lipoxin A4. These not only downregulate a poten- highly efficacious in preventing experimental E. histolytica infec-
28
tially pathological host inflammatory response but also inhibit tion in mice and baboons. However, the role of antilectin IgG
Th1 induction and macrophage antimicrobial activity. In addition, is unclear, and protection may be subclass-dependent. Cell-
T. gondii interferes with normal macrophage signaling. For mediated immunity is also critical for host defense against E.
example, infection inhibits DNA binding of signal transducer histolytica. IFN-γ–producing CD4 T cells provide protection
and activator of transcription 1 (STAT1) and NF-κB by interfering through their ability to activate macrophages and neutrophils
with chromatin structure, and promotes antiinflammatory to the parasite. CD8 T cells mediate protection through the
pathways downstream of the suppressor of cytokine synthesis secretion of IL-17, a key player in the secretion of mucin and
proteins. The parasite also has several virulence proteins, including antimicrobial peptides, recruitment of neutrophils, and IgA
ROP5 and ROP16, which bind to the parasitophorous vacuole transport across the epithelial barrier. Impairment of cell-mediated
and reduce accumulation of the IRGs. ROP proteins also activate immunity results in parasite dissemination. Indeed, patients with
host STAT3 and STAT6 driving an M2 macrophage phenotype human immunodeficiency virus infection and coinfected with
that is permissive to infection. 26 E. histolytica have high rates of invasive amebiasis, liver abscesses,
and seroconversion. 29
Entamoeba histolytica Evasion of Host Immunity

Pathogenesis E. histolytica utilizes a number of strategies to circumvent the
E. histolytica causes asymptomatic intestinal colonization, acute immune defenses of the host. It resists complement-mediated
diarrhea, dysentery, colitis, liver abscess, and, rarely, disseminated lysis during hematogenous spread by proteolytic degradation of
disease. Susceptibility to amebiasis is determined by the host’s C3a and C3b. In addition, the Gal/GalNAc lectin binds to C8
27
nutritional status, intestinal microflora, genetics, and gender. and C9, preventing assembly of the C5b-9 membrane attack
E. histolytica cysts are ingested through consumption of food or complex. The cytolytic capability of E. histolytica affords protection
water contaminated by feces. After excystation, the trophozoites from neutrophils, macrophages, and eosinophils, unless these
degrade the colonic mucus barrier through secretion of proteases cells are activated. Cytolysis by E. histolytica can occur via necrosis
and glycosidases. The trophozoites adhere to the colonic epithelial and apoptosis. Trophozoites also inhibit the macrophage respira-
cells by a galactose/N-acetylgalactosamine-inhibitable lectin (Gal/ tory burst and the production of IL-1 and TNF-α. A protective
GalNAc), which leads to NF-κB activation and proinflammatory antibody response is subverted by the degradation of IgA and
cytokine release. Epithelial cells with adherent amebae undergo IgG by amebic cysteine proteases and by capping, ingesting, or

432 ParT THrEE Host Defenses to Infectious Agents


Pathogen Innate immune response Intestinal response

Trophozoite
Complement Villous atrophy and
Giardia Inflammatory crypt hyperplasia
Macrophage mediators
IL-6, IL-8, IL-1
Neutrophil Epithelial
Sporozoite GM-CSF, GROα, damage
Eosinophil prostaglandins
Cryptosporidium
NK cell ROI, RNI
Trophozoite
Proteases
Erosions and
Enterocyte ulcerations
Entamoeba (Cryptosporidium)

Cytokines

B- and T-lymphocyte activation Secretion
Malabsorption
Acquired immune response
Exudation
Host protection Diarrhea
FIG 30.3 Immunopathogenesis of Intestinal Protozoal Pathogens. After adherence (Giardia
and Entamoeba) or epithelial invasion (Entamoeba and Cryptosporidium), there is release of
various inflammatory mediators from macrophages and neutrophils. This causes the activation
of resident phagocytes and recruitment of phagocytes into the lamina propria. Enterocyte death
can be due to direct action of the parasites or to immune-mediated damage from complement,
cytotoxic lymphocytes, proteases, and reactive oxygen and nitrogen intermediates (ROI and RNI,
respectively). The inflammatory mediators also act on enterocytes and the enteric nervous system,
inducing the secretion of water and chloride. In response to enterocyte damage, under the
influence of activated T lymphocytes, the crypts undergo hyperplasia, and the villi become shorter
(villous atrophy). The immature hyperplastic cells have poor absorptive ability but retain secretory
ability. Damage to the epithelium can cause leakage (exudation) from lymphatics and capillaries.
Similar mechanisms are probably responsible for the diarrhea that occurs in infection with Cyclospora
and Isospora. Isospora is unique in causing an eosinophilic infiltrate.






27
shedding ameba-specific antibodies. Amebic proteases can also chronic watery diarrhea, epigastric pain, nausea, vomiting, and
cleave the Fc region so that interaction with host cell surface weight loss, depending on host factors and the virulence of the
receptors is avoided. Another secreted product of E. histolytica, Giardia strain. 30,31 Recent studies suggest even with treatment
monocyte locomotion inhibition factor (MLIF), inhibits monocyte the parasite can elicit intestinal complications that persist for
locomotion and the monocyte and neutrophil respiratory burst years. Younger age, malnutrition, and immunodeficiency increase
31
and NO production; enhances antiinflammatory cytokine and the risk of severe disease. Infection is initiated following the
chemokine release from host cells; and alters adhesion molecule ingestion of food or water contaminated with G. lamblia cysts.
expression on macrophages. The suppression of host macrophage Exposure to stomach acids induces the excystation process that
NO production by an array of trophozoite secretory products, releases two trophozoites into the lumen of the proximal small
including parasite-derived PGE 2 , is a major factor in the persis- intestine. Colonization occurs when the parasite attaches to the
tence of amebic liver abscesses. In chronic infection, E. histolytica intestinal epithelium and begins to reproduce by binary fission.
promotes the development of Tregs that suppress the proliferation Trophozoites remain within the lumen and do not invade the
of responder T cells by releasing IL-10, TGF-β, and IL-35. 28 epithelial barrier. Parasite migration into the lower intestine
triggers encystation, allowing the organism to survive when
Giardia lamblia excreted into the environment.
The G. lamblia trophozoite initiates adherence to the intestinal
Pathogenesis epithelium via a surface mannose-binding lectin. Histopathologi-
Recent studies of Giardia lamblia have identified eight geno- cal changes in symptomatic giardiasis range from a normal
30
types, two of which infect humans (assemblages A and B). appearance to increased crypt-villous ratios, epithelial damage,
The severity of giardiasis ranges from asymptomatic carriage to and chronic inflammatory infiltrate in the lamina propria (see

CHaPTEr 30 Host Defenses to Protozoa 433


Fig. 30.3). The factors responsible for the structural changes in Cryptosporidium parvum AND
the small bowel are not well defined but may include injury Cryptosporidium hominis
from adherence, parasite-induced apoptosis of epithelial cells,
and the release of cytotoxins, including proteases. Additional In humans, there are four intestinal coccidians that are intracel-
epithelial damage may be mediated by the host cellular immune lular parasites of enterocytes: Isospora belli, Cyclospora cayetanensis,
response. Diarrhea arises from epithelial barrier dysfunction, and two species of Cryptosporidium, C. parvum, and C. hominis.
reduction in microvillous surface area, chloride hypersecretion, Of the four coccidians, Cryptosporidium has the greatest epide-
and glucose and sodium malabsorption. 31 miological significance: in 1993, a huge outbreak involving 403
000 persons occurred in Milwaukee, Wisconsin, in the United
Innate Immunity States. Because of their similarity, only the immunology of
As G. lamblia does not invade the intestinal epithelium, host cryptosporidiosis will be discussed.
defense and immune factors present within the lumen are essential
for preventing and controlling infection. Increased intestinal Pathogenesis
motility contributes to G. lamblia clearance, by impairing the Cryptosporidium typically causes self-limited (but often prolonged)
ability of the parasite to attach to the epithelium and resist the diarrhea in the immunocompetent host. However, in the
luminal bulk flow. Antimicrobial peptides derived from Paneth immunocompromised host Cryptosporidium can cause severe
cells, including cryptdins, neutrophil defensin, and cathelicidin, diarrhea, with malabsorption and wasting, and cholangiopathy.
effectively kill G. lamblia trophozoites in vitro. Infection begins with the ingestion of food or water contaminated
NO, produced by both epithelial cells and macrophages, with oocysts. The acidic environment of the stomach induces
inhibits excystation trophozoite division. Despite the protective excystation and the release of four sporozoites into the small
effect of NO, the parasite can circumvent this defense by compet- intestines. Glycoproteins on the surface of sporozoites facilitate
30
ing with host cells for arginine uptake. Mast cells have a sig- attachment and invasion of epithelial cells. After entry into
nificant role in protecting against the parasite. Mice deficient in epithelial cells, the parasite resides within a unique intracellular
mast cells fail to clear G. lamblia infection, in part because they but extracytoplasmic vacuole, that protects the pathogen from
are unable to mount parasite-specific IgA. Mast cells also con- environmental and host insults. Inside a vacuole the sporozoites
tribute to B-cell survival, activation and differentiation into develop into trophozoites and undergo schizogony, with a resultant
plasma cells, and together with NO induce peristalsis. merozoite-containing schizont. The merozoites are extruded and
invade neighboring epithelial cells. The merozoites may continue
Adaptive Immunity an asexual cycle or develop into macro- or microgametes that
Several lines of evidence suggest the importance of the humoral fuse to form oocysts. Before being excreted in feces, oocysts
immune response in the control of giardiasis. Infection with undergo sporulation to become infectious. Histologically, infection
Giardia results in the production of anti-Giardia antibodies causes villous atrophy and blunting, and crypt hyperplasia with
in the serum and mucosal secretions. Patients with severe increased infiltration of lymphocytes, macrophages, and plasma
33
B-cell defects or selective IgA deficiency have an increased risk cells. Intraepithelial lymphocytes are uncommon; neutrophils
30
of developing chronic infections. Studies in mice have dem- and occasional eosinophils are present between the epithelium
onstrated key functions of secretory IgA and the polymeric and the lamina propria. Disorganized cells undergoing necrosis
immunoglobulin receptor, which is responsible for IgA transport replace normal enterocyte architecture (see Fig. 30.3). There is
into the intestinal lumen in controlling parasite burden and an association between the degree of intestinal injury and
eliminating infection. 30 malabsorption and the intensity of infection, as measured by
There is also evidence for a role of T cell–dependent immunity oocyst excretion.
in the control of giardiasis. A reduction or absence of CD4 T The neuropeptide substance P, which is produced by endo-
cells can lead to chronic infection. IFN-γ- and IL-17A-producing thelial cells, lymphocytes, and monocytes in the lamina propria,
32
CD4 T cells develop following infection and are important for contributes to diarrhea by increasing intestinal chloride secretion
mediating parasite clearance. Although the mechanisms are still and glucose malabsorption. Increased expression of substance
being defined, IL-17A is likely involved in modulating transport P is observed in patients with AIDS as well as cryptosporidiosis
of IgA into the intestinal lumen. Epidemiological studies indicate and severe diarrhea. 34
that partial immunity is acquired from Giardia infection, which
leads to reduced risk and severity of subsequent infections. 30 Innate Immunity
The type I and type II IFNs play a key role in the innate
Evasion of Host Immunity protective response against cryptosporidium. Because of
35
Giardia evades the host humoral immune response by undergoing the parasite’s intracellular location near the luminal surface
surface antigenic variation by altering a group of variant-specific of the enterocyte, the macrophages of the lamina propria are
surface proteins (VSPs). Selection occurs by an immune-mediated spatially isolated from the parasite. Thus the intestinal epithe-
process because switching occurs when intestinal anti-VSP IgA lium mounts its own assault on the invading microbe through
30
responses are first detected. G. lamblia also produces a protease TLR2/TLR4–dependent activation of NF-κB and release of the
that cleaves IgA. Although Giardia activates dendritic cells for microbicidal peptide β-defensin-2, TNF-α, and the chemokines
antigen presentation, it also inhibits IL-12 production, in part IL-8, RANTES, and GRO-α, which act as chemoattractants and
by enhancing IL-10 release; the net result is the dampening of activators of neutrophils. In patients with AIDS and crypto-
30
a local antiparasitical inflammatory response. The trophozoite sporidiosis, the HIV Tat protein may sabotage host defense
also releases arginine deiminase, which degrades arginine, making against Cryptosporidium by inhibiting cholangiocyte TLR4
it less available for host NO production. 31 expression. 33

434 ParT THrEE Host Defenses to Infectious Agents


IL-15, produced by activated monocytes, stimulates NK-cell Trichomonas vaginalis
proliferation, cytotoxicity, and cytokine production, including
IFN-γ. There is significant IL-15 expression in the jejunal mucosa Pathogenesis
in immunocompetent patients with cryptosporidiosis, and IL-15 Trichomonas vaginalis is a flagellated protozoan parasite of the
levels inversely correlate with parasite burden. However, in patients human urogenital tract that exists only as a trophozoite. It causes
with AIDS who have chronic uncontrolled cryptosporidiosis, vaginitis, cervicitis, and urethritis. Its adherence to the vaginal
IL-15 is undetectable. 33 squamous epithelium is facilitated by a number of adhesins.
Mannose-binding lectin (MBL) is a serum protein that binds Trichomonas causes tissue damage by contact-dependent cytolysis
to various pathogens, including Cryptosporidium. Upon binding, caused by pore-forming proteins and proteases, and secretion
MBL activates complement, thereby promoting opsonization of a glycoprotein cell-detaching factor that causes sloughing of
and phagocytosis. Low serum levels of MBL, which may result the vaginal epithelium. Levels of the cell-detaching factor correlate
from malnutrition or polymorphisms in the MBL2 gene, increase with the severity of the disease, and vaginal antibodies directed
33
susceptibility to cryptosporidiosis. Infected intestinal cells also against this factor modulate its effects. Inflammation in the genital
release TGF-β, which decreases necrosis and stimulates the mucosa and submucosa leads to copious secretions, and the
synthesis of extracellular matrix proteins, thereby limiting surface epithelium may slough, causing focal erosions and
epithelial damage. hemorrhage.
Prostaglandins E 2 and F 2 α, released by infected enterocytes, The increased risk of HIV transmission in women with
not only promote secretory diarrhea but also upregulate mucin trichomoniasis may result from increased recruitment of inflam-
production, which may hinder parasite attachment. In addition, matory cells, mucosal erosion, or degradation of secretory
these prostaglandins stimulate the release of β-defensin-2, which leukocyte protease inhibitor (SLPI) by trichomonal proteases.
has direct anticryptosporidial activity and is chemotactic for T Lower levels of SLPI are found in the vaginal fluid of women
cells and DCs. 33 with trichomoniasis, which can lead to increased tissue damage
36
and HIV transmission. The lipophosphoglycan of Trichomonas
Adaptive Immunity induces production of the chemokines IL-8 and CCL20, which,
Cell-mediated immunity plays an important role the resolution which can also facilitate HIV infection by promoting DC
of cryptosporidiosis and protection from reinfection. In immu- recruitment.
nocompetent, adult mice CD4 intraepithelial lymphocytes (IELs)
initiate early control of infection, whereas cytotoxic CD8 IELs Innate Immunity
appear later and function in parasite elimination. Resolution of Although trichomoniasis has recently received increased attention
infection depends on a balance of Th1 cytokines (IFN-γ, IL-18) as a risk factor for HIV transmission and obstetrical complications,
needed to control the infection and Th2 cytokines (IL-4, IL-10, there is little known about the protective immune response against
and IL-13) that limit immunopathological damage. In mice, γδ this organism. Trichomonas secretes a factor that promotes
T cells are rapidly recruited to control cryptosporidial infection, neutrophil chemotaxis, causing profuse leukorrhea, but the
but their role in human infection is unknown. Severe intestinal oxidative microbicidal mechanisms of the neutrophils have
disease or biliary involvement is usually seen in patients with decreased efficacy in the anaerobic vaginal environment. Activated
33
AIDS whose CD4 count is <50/µL. Patients with HIV infection macrophages can destroy trichomonads in a T and B cell–
who have CD4 counts >200/µL usually experience self-limited independent manner and release IL-lβ and TNF-α, which are
37
disease. chemotactic for neutrophils. Trichomonas induces neutrophil
The role of humoral immunity is less clear. Secretory antibodies apoptosis, and macrophage clearance of these apoptotic cells
produced in the intestines are thought to impair parasite attach- causes release of IL-10, which may contribute to resolution of
ment to epithelial cells. Indeed, immunoglobulin deficiencies the inflammatory response. 38
are often associated with persistent or recurrent infections.
However, specific anti-Cryptosporidium IgA levels have been Adaptive Immunity
reported in patients with AIDS who are infected with the parasite, Repeated infections with T. vaginalis do not induce immunity;
suggesting polyreactive T cell–independent antibodies are not however, the infection is self-limited in most cases, so there are
33
sufficient for parasite eradication. In experimentally infected effective mechanisms of host defense. T. vaginalis induces the
human volunteers, serum IgM and IgG directed toward sporozoite production of antibodies in both the serum and vaginal secretions.
proteins protect against the development of symptoms, but not The serum antibody response correlates with active infection,
infection. and serum, but not vaginal, IgG from infected patients displays
complement-mediated lytic activity against trichomonads in
Evasion of Host Immunity culture. 38
Cryptosporidium evades host defenses primarily by exerting control
over infected enterocyte apoptosis. One of the upregulated genes Evasion of Host Immunity
is osteoprotegerin, which inhibits apoptosis by acting as a decoy Although T. vaginalis activates the alternative pathway of comple-
34
receptor for TNF-related apoptosis inducing ligand (TRAIL). ment, the cervical mucus and menstrual blood are low in comple-
Control of host apoptosis is complex; early inhibition of apoptosis ment. Menstrual blood also supplies iron, which upregulates
by NF-κB activation allows the parasite to complete its life cycle, trichomonal adhesins and cysteine proteases, causing the degrada-
whereas the late promotion of apoptosis facilitates merozoite tion of complement component C3 bound to the surface of the
release. Nevertheless, the infected cells secrete FasL, which parasite. Parasite virulence is thus enhanced, and the symptoms
promotes apoptosis in uninfected bystander cells. In this way, are exacerbated during menses. Cysteine proteases secreted by
the host counters the antiapoptotic activity of the parasite by T. vaginalis also degrade immunoglobulins, sabotaging the
surrounding the parasitized cells by a zone of apoptotic cells. antibody response. The parasite also secretes soluble antigens

CHaPTEr 30 Host Defenses to Protozoa 435


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2. Bethony JM, Cole RN, Guo X, et al. Vaccines to combat the neglected amebiasis. PLoS Pathog 2013;9:e1003489.
tropical diseases. Immunol Rev 2011;239:237–70. 28. Nakada-Tsukui K, Nozaki T. Immune response of amebiasis and immune
3. Crompton PD, Pierce SK, Miller LH. Advances and challenges in malaria evasion by Entamoeba histolytica. Frontiers Immunol 2016;7:175.
vaccine development. J Clin Investig 2010;120:4168–78. 29. Baxt LA, Singh U. New insights into Entamoeba histolytica pathogenesis.
4. Vazquez-Chagoyan JC, Gupta S, Garg NJ. Vaccine development against Curr Opin Infect Dis 2008;21:489–94.
Trypanosoma cruzi and Chagas disease. Adv Parasitol 2011;75:121–46. 30. Lopez-Romero G, Quintero J, Astiazaran-Garcia H, et al. Host defences
5. Gazzinelli RT, Kalantari P, Fitzgerald KA, et al. Innate sensing of malaria against Giardia lamblia. Parasite Immunol 2015;37:394–406.
parasites. Nat Rev Immunol 2014;14:744–57. 31. Cotton JA, Beatty JK, Buret AG. Host parasite interactions and
6. Crompton PD, Moebius J, Portugal S, et al. Malaria immunity in man pathophysiology in Giardia infections. Int J Parasitol 2011;41:925–33.
and mosquito: insights into unsolved mysteries of a deadly infectious 32. Saghaug CS, Sornes S, Peirasmaki D, et al. Human memory CD4+ T cell
disease. Annu Rev Immunol 2014;32:157–87. immune responses against Giardia lamblia. Clin Vaccine Immunol
7. Schwenk RJ, Richie TL. Protective immunity to pre-erythrocytic stage 2016;23:11–18.
malaria. Trend Parasitol 2011;27:306–14. 33. Kothavade RJ. Challenges in understanding the immunopathogenesis of
8. Langhorne J, Ndungu FM, Sponaas AM, et al. Immunity to malaria: more Cryptosporidium infections in humans. Eur J Clin Microbiol Infect Dis
questions than answers. Nat Immunol 2008;9:725–32. 2011;30:1461–72.
9. Stanisic DI, Barry AE, Good MF. Escaping the immune system: how the 34. Pantenburg B, Dann SM, Wang HC, et al. Intestinal immune response to
malaria parasite makes vaccine development a challenge. Trend Parasitol human Cryptosporidium sp. infection. Infect Immun 2008;76:23–9.
2013;29:612–22. PubMed PMID: 24176554. 35. Barakat FM, McDonald V, Foster GR, et al. Cryptosporidium parvum
10. Guizetti J, Scherf A. Silence, activate, poise and switch! Mechanisms of infection rapidly induces a protective innate immune response involving
antigenic variation in Plasmodium falciparum. Cell Microbiol 2013;15: type I interferon. J Infect Dis 2009;200:1548–55.
718–26. 36. Thurman AR, Doncel GF. Innate immunity and inflammatory response
11. Gurung P, Kanneganti TD. Innate immunity against Leishmania to Trichomonas vaginalis and bacterial vaginosis: relationship to HIV
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Science 2008 15;321:970–4.

CHaPTEr 30 Host Defenses to Protozoa 435.e1


MULTIPLE-CHOICE QUESTIONS


1. Which one of the following is characteristic of the immuno- 3. Which one of the following is a component of the innate
pathogenesis of cerebral malaria? immune response to Cryptosporidium infection?
A. Absence of Plasmodium-specific antibodies A. Decreased intestinal mucin production
B. Impaired interferon (IFN)-γ mediated production of nitric B. Increased type I and type II interferon production
oxide C. Decreased nuclear factor-kappa B (NF-κB) activation
C. Cytoadhesion of parasites and leukocytes to vascular D. Antigen-specific CD8 intraepithelial T-cell responses
endothelium E. Antigen-specific immunoglobulin A (IgA) responses
D. Immune-mediated destruction of infected erythrocytes
E. Failure of leukocyte production of leukotriene/tumor
necrosis factor (LT/TNF)
2. Which one of the following is NOT a mechanism through
which Leishmania can evade the host immune response?
A. Complement resistance mediated by the parasite’s surface
lipophosphoglycan
B. Blunted interferon (IFN)-γ-mediated macrophage activation
through the disruption of signal transduction pathways
C. Increased synthesis of the immunosuppressive molecules
interleukin (IL)-10, transforming growth factor (TGF)-β,
and prostaglandin (PG)E 2
D. Depletion of L-arginine through the activity of host
arginase
E. Suppression of an early neutrophil response at the site of
infection

31









Immune Responses to Helminth Infection



Subash Babu, Thomas B. Nutman







Parasitical helminths are complex eukaryotic organisms, character- nematode infection, or skin/subcutaneous tissue and draining
ized by their ability to maintain long-standing, chronic infections lymph nodes in onchocerciasis) or systemic (lymphatic filariasis
in human hosts, sometimes lasting decades. Hence, parasitical or schistosomiasis). Moreover, the migration patterns of the
helminths are a major health care problem worldwide, infect- parasite might elicit varied cutaneous, pulmonary, and intestinal
ing more than 2 billion people, mostly in resource-constrained inflammatory pathologies, as seen, for example, in Ascaris or
countries (Fig. 31.1). Common helminth infections include those Strongyloides infection during their migratory phase. This is
with intestinal helminths, and filarial and schistosome infec- further complicated by the fact that human hosts are often exposed
tions are a major medical, social, and economic burden to the to multiple lifecycle stages of the parasite at the same time. Thus
countries in which these infections are endemic. Chemotherapy, a patient with chronic infection with lymphatic filariasis harboring
although highly successful in some areas, still suffers from the adult worms and microfilariae might be exposed to insect bites,
disadvantages of the length of treatment, the logistics involved thereby transmitting the infective-stage parasite. The immune
in the distribution of drugs, and, in some cases, the emergence response that ensues will not only be a reaction to the invading
of drug resistance. Vector control measures are at best an adjunct organism but will also bear an imprint of the previous exposures
measure in the control of helminth infections but also suffer and the concurrent infection.
from the same social, logistic, and economic obstacles as those
for mass chemotherapy. Therefore the study of the immune KEY CONCEPTS
responses to helminth infections attains great importance both
in terms of understanding the parasite strategies involved in Helminth Infection
establishing chronic infection and in the delineation of a successful Divided into nematodes, trematodes, and cestodes
host immune response to develop protective vaccines against Produce chronic infections that can persist for decades
infection. Characteristically cause morbidity rather than mortality
Multicellular parasites that do not multiply in the definitive host but can
reproduce sexually to produce larval stages that ensure continued
SPECTRUM OF HOST–PARASITE INTERACTIONS transmission
Helminths have characteristically complex lifecycles with many
1
developmental stages. Thus the host is exposed during the course Helminth infections can elicit a spectrum of clinical manifesta-
of a single infection to multiple lifecycle stages of the parasites, tions mirroring diversity in host immune responses. For example,
each stage with a shared as well as a unique antigenic repertoire. in lymphatic filariasis, most infected individuals remain clinically
Thus Schistosoma mansoni infection begins with penetration of asymptomatic despite harboring significant worm burdens; this
the skin of humans exposed to infested waters by the free- is thought to reflect the induction of parasite-specific tolerance
swimming cercariae, which then develop into tissue-dwelling in the immune system. Others exhibit acute manifestations,
schistosomula. In the liver and mesenteric veins, schistosomula including fever and lymphadenopathy, and this is thought to
differentiate into sexually dimorphic adult worms, which then reflect inflammatory processes induced by incoming larvae, dying
mate, and the resultant eggs produced migrate through tissues worms, or superadded infections. Individuals who mount a strong
into the lumen of the intestine or bladder for environmental but inappropriate immune response end up with lymphatic
release. Similarly, in lymphatic filarial infection, the host is exposed damage and subsequent immune-mediated pathology—hydrocele
to infective-stage larvae in skin, lymph nodes, and lymphatics; and elephantiasis. Finally, a group of infected individuals mount
to adult worms in lymph nodes and lymphatics; and finally to exuberant immune responses that often result in unusual pathol-
microfilariae in the peripheral circulation. Hence, the host– ogy, such as tropical pulmonary eosinophilia. Thus the clinical
helminth interaction is complex not only because of the multiple manifestations of lymphatic filariasis exemplify the spectrum of
lifecycle stages of the parasite but also because of the tissue host–parasite interactions that occur during helminth infections
tropism of the different stages. (Fig. 31.2).
Antigenic differences among the lifecycle stages can lead to Another hallmark of all helminth infections is their chronic
distinct immune responses that evolve differentially over the nature, with many helminths surviving in the host for decades.
course of a helminth infection. In addition, depending on the For example, adult schistosomes and filariae may survive in host
location of the parasite, the responses are compartmentalized tissues for as long as 30 years, producing eggs and larval stages
(intestinal mucosa and draining lymph nodes in intestinal throughout this time. Similarly, Strongyloides stercoralis, with its

437

438 ParT ThrEE Host Defenses to Infectious Agents


ability to “autoinfect,” can maintain its lifecycle for decades. the infection, helminths appear to reflect a harmonious host–
Chronic infections certainly reflect an adaptation that leads to parasite interface such that relatively asymptomatic carriers are
“parasitism,” as mortality induced in the host would prevent available as reservoirs for ongoing transmission. Of course, failure
parasite transmission if the host died before larval release or egg to establish this harmonious coexistence does occur, leading to
production could occur. In addition to the long-lived nature of pathological conditions exemplified by cirrhosis and portal
hypertension in schistosomiasis and elephantiasis associated with
lymphatic filariasis.
Schistosoma mansoni
> 200 million infected
Echinococcus, Taenia spp PROTOTYPICAL HOST RESPONSES TO HELMINTHS
>100 million infected
The canonical host immune response to all helminths is of the
T-helper 2 (Th2) type and involves the production of cytokines
interleukin (IL)-4, IL-5, IL-9, IL-10, and IL-13; the antibody
Brugia malayi Trematodes Cestodes isotypes immunoglobulin G1 (IgG1), IgG4, and IgE; and expanded
populations of eosinophils, basophils, mast cells, type 2 innate
Onchocerca volvulus lymphoid cells, and alternatively activated macrophages (Chapter
2
Wuchereria bancrofti Ancylostoma duodenale
157 million infected Nematodes Nector americanus 16). However, it is also being increasingly recognized that while
576 million infected the predominant response is Th2 in nature, a large regulatory
component involving both regulatory cytokines and cells are
3
also part of this repertoire. The Th2 response induced by
helminth parasites is quite stereotypical, but its initiation, progres-
sion, and culmination of this response requires interaction with
many different cell types, most notably (i) epithelial/stromal
cells, (ii) innate lymphoid cells (ILCs), (iii) dendritic cells (DCs)
and macrophages; (iv) T cells; (v) B cells; (vi) eosinophils; (vii)
Ascaris lumbricoides
>1 billion infected Trichinella spiralis mast cells/basophils; and (viii) neutrophils (Chapter 2). In addi-
Trichuris trichiura tion, the host–helminth interactions can lead to a variety of
>600 million infected modulated immune responses that are mediated largely by the
FIG 31.1 Common and medically relevant helminth infections induction of regulatory T cells (Tregs) and alternatively activated
and their global prevalence. macrophages (AAMs) (Fig. 31.3).


















A B



















C D
FIG 31.2 The clinical manifestations of lymphatic filariasis, including (A) mild lymphedema,
(B) severe lymphedema, (C) elephantiasis, and (D) hydrocele.

ChaPTEr 31 Immune Responses to Helminth Infection 439







Epithelial cells Langerhans cells


TSLP/other cytokines?
TSLP TSLP IL-33/ IL-25
Basophil TSLP Relmα/FIZZ1
Natural
helper cell
Dendritic IL-4 IL-13
cells Alternatively activated Eosinophil
YM-1 macrophages
Th0 IL-4 IL-13 Arginase-1
IL-5 Eosinophil differentiation
IL-4 Promotes Th2 differentiation,
IgE production
Th2 IL-10 Role in immunomodulation
Treg IL-9 Role in mastocytosis

IL-10

Bystander suppression Tfh B cell IgG1
(allergy, autoimmunity, etc.)
IL-4 IgE Basophil
FIG 31.3 Regulation of the T-cell response in helminth infection. IL, interleukin; Th0, precursor
T-helper cell; Th2, T-helper 2 cell; Treg, Regulatory T cell; TfH, T-follicular helper cell, RELM-α,
Resistin-like molecule-α; Chi3l, Chitinase 3 like protein; IDO, indoleamine 2,3 dioxygenase; TSLP,
thymic stromal lymphopoietin.






Helminths and Epithelial Cells Helminths and Innate Lymphoid Cells
Epithelial cells are the first barrier layer exposed to or breached The ILC family includes ILC1, which predominantly express
by most helminths, and the capacity of these cells to respond IFN-γ; ILC2, which predominantly express IL-5 and IL-13; and
4
5
by initiating an “alarm” response has recently been recognized. ILC3; which predominantly express IL-22 and/or IL-17. ILC2
These epithelial cells mount a prototypical response comprising are defined by their expression of the IL-33 receptor (IL-33R)
chemokines and cytokines, such as IL-1, IL-25, IL-33, and thymic and the transcriptional regulators, Id2, RORα, GATA-3, and
stromal lymphopoietin (TSLP), as well as alarmins, such as uric Bcl11b. Unlike T cells, ILC2 rely on cytokines to drive activation
acid, ATB, HMGB1, and S100 proteins. These signals program rather than on cognate interactions mediated by antigen-specific
DCs to mount Th2 cell–mediated immunity and in doing so receptors. ILC2 are a critical innate source of type 2 cytokines,
boost type 2 innate lymphoid cell (ILC2), basophil, and mast including moderately large quantities of IL-5 and IL-13, but also
cell function. Epithelial cells produce chemokines, including of IL-4, IL-9, granulocyte macrophage–colony-stimulating factor
CCL17 and CCL22 (acting on ILC2, basophils, Th2 cells, and (GM-CSF), and amphiregulin. These cytokines potently induce
Tregs), and eotaxins, such as CCL11, CCL24, and CCL26 (acting eosinophilia, mucus production from goblet cells, activation of
5
on eosinophils and Th2 cells). They also produce prostaglandin AAM, muscle contractility, mastocytosis, and tissue repair. They
D2 (PGD2), which acts on the CRTH2 receptor to recruit ILC2, are dependent on IL-2 and IL-7 for their development and
basophils, mast cells, and Th2 cells. More recently, tuft cells, a activation. In addition, the transcription factors GATA-3 and
specialized secretory cell type of the intestinal epithelium, has RORα have been found to be essential for the development of
been identified as a major player in anthelmintic immunity. In ILC2. Although the function of ILC2 and Th2 cells appear to be
addition, epithelial cells in the intestine, for instance, are in largely overlapping, the kinetic differences in the ability to secrete
constant contact with both beneficial and pathogenic bacteria cytokines rapidly and in profuse amounts allows for a coordinated
and hence ideally located for immunological surveillance of the interaction between the two cell types. Moreover, ILC2 can directly
intestinal lumen. This recognition of signals by intestinal epithelial regulate the activation of T cells through their expression of
cells is essential to mucosal homeostasis, implicating these cells major histocompatibility complex (MHC) Class II molecules
as central modulators of inflammatory responses. Finally, the and the accessory molecules, CD80 and CD86, albeit less efficiently
production of mucus and mucus-associated bioactive molecules compared with DCs. Finally, recent reports have linked ILC2
(Mucin5AC, trefoil factor-2, and resistin-like molecule-β [RELM- with metabolic homeostasis, obesity, and dietary stress, providing
β]) are important in promoting protection against intestinal an indirect link by which helminths might modulate host meta-
helminth infection. bolic function.

440 ParT ThrEE Host Defenses to Infectious Agents


helminth infection and promotes inflammatory responses and
Helminths and Dendritic Cells increased susceptibility.
DCs are professional antigen-presenting cells (APCs) that play an
essential role in presenting antigen to T cells to initiate immune Helminths and T Cells
2
responses. Although the role of DCs in inducing Th1, Th17, Typically, infections with helminths induce a robust Th2 response
and Treg responses is well established, their role in inducing manifested by enhanced expression of IL-4, IL-5, IL-9, IL-10,
Th2 responses has remained relatively unclear. Nevertheless, a and IL-13 in response to live parasites, parasite antigens, or
7
series of studies have shown that DCs are required for optimal mitogens. The central player in Th2 immunity is certainly the
+
Th2 responses in vivo. Thus in vivo depletion of DCs has been CD4 Th2 cell. It is clear that IL-4Rα, a component of both the
shown to inhibit the induction of Th2 responses to S. mansoni IL-4 and IL-13 receptors, is at the epicenter of Th2 immunity,
or Heligmosomoides polygyrus. Helminth products can prime since IL-4 and IL-13, together or individually, are absolutely
DCs for the induction of Th2 responses by interaction with critical for resistance to most helminth parasites. Recent work
pattern recognition receptors (PRRs), such as Toll-like receptors has reported that the Th2 cell population is heterogeneous,
−
+
(TLRs) and C-type lectin receptors (CLRs). This interaction, containing both IL-5 and IL-5 Th2 cells that express IL-4 and
which depends on TLR and CLR signaling, can promote Th2 IL-13. In addition, IL-4 and IL-13 production is spatially separated,
responses by suppressing antigen presentation, costimulation, with IL-13 expression being marked in tissues and IL-4 expression
and/or expression of Th1-promoting cytokines by directly being pronounced in the lymph nodes within the Th2 cell
interfering with these pathways. DCs that drive Th2 responses compartment. Finally, induction of GATA-3 and downregulation
typically exhibit specialized markers, such as CD301b, PDL2, of T-box expressed in T cells (T-bet) has been shown to be an
and CD11b, and several receptors for the Th2-related cytokines important step in T resistin-like molecule cell differentiation to
IL-4R, IL-13R, IL-25R, TSLP-R, and IL-33R. Additionally, the the Th2 phenotype in helminth infections. Interestingly, chronic
extracellular signal-regulated kinase (ERK) and signal transducer helminth infections are associated with downmodulation of
and activator of transcription 4 (STAT4) pathway upregulates the parasite antigen-specific proliferative responses as well as IFN-γ
costimulatory molecules, CD40, OX40L, and Jagged. Activation and IL-2 production but with intact IL-4 responses to parasite
of the major transcription factors interferon regulatory factor antigens and global downregulation of both Th1 and Th2
4 (IRF4) and KLF4 inhibits IL-12 production and increased responses to live parasites. Finally, the receptor NLRP3 has been
IL-10 secretion. In addition, DCs expressing FcεRIII can induce shown to be a key transcription factor in Th2 differentiation.
murine IgG1-related Th2 responses. These factors typically Although the role of tissue resident memory T (T RM ) cells is
act individually or in concert to orchestrate Th2 responses in well established in viral and bacterial infections, very little is
helminth infections. Although Th2 cell–mediated immunity known about the role of these cells in helminth infections.
+
+
requires IRF4-dependent CD301b CD11b DCs in the mouse, However, it has been shown that tissue-resident Th2 cells can
Langerhans cells are the predominant inducers of Th2 cells ex exert innate (TCR-independent and IL-33–dependent) functions
vivo in humans. The modulation of DC function by helminth upon appropriate stimuli and confer protection against helminth
antigens appears to be generalizable and has been shown to infection. In addition, although multifunctionality (ability to
impair their ability to respond to other infectious stimuli (e.g., produce two or more cytokines) has not been well described in
Mycobacterium tuberculosis). the Th2 cell compartment, helminth infections are known to be
associated with an antigen-dependent enhancement of mono- and
Helminths and Macrophages dual-functional Th2 cells and its reversal after treatment. Of
+
+
Macrophages are the other important class of APCs that can interest, a stable subset of parasite induced T-bet , GATA-3 ,
serve as protective effector cells in bacterial and protozoan Th1/Th2 hybrid T cells has been described to develop directly
infections by their production of nitric oxide and other mediators. from naïve precursors and to play a role in limiting pathological
Helminth interaction with macrophages induces a population inflammation in animal models of helminth infection.
of cells preferentially expressing arginase instead of nitric oxide Recently, a new subset of T cells expressing IL-9 and IL-10,
as a result of increased activation of arginase-1 by IL-4 and but not IL-4 (and therefore different from Th2 cells), has been
6
IL-13. These AAMs are characterized by their ability to upregulate described in allergic inflammation and in response to intestinal
8
arginase-1, chitinase 3-like proteins 3 and 4 (also known as Ym1 parasites. These cells appear to be under the control of TGF-β
and Ym2, respectively), and RELM-α. These AAMs are known and IL-4 and are dependent on STAT6, GATA-3, IRF4, and PU.1.
to be important in wound healing and have been postulated to Th9 cells have been recently shown to be associated with host
play a potential role in repairing wound damage that occurs protection in Nippostrongylus brasiliensis and Trichuris muris
during tissue migration of helminth parasites. In fact, there infection. Finally, Th9 cells have also been shown to be predomi-
appears to be two distinct populations of AAMs, one derived nantly associated with lymphatic pathology in filariasis. T-follicular
from blood and functioning in an immune regulatory role and helper (Tfh) cells are a subset of CD4 T cells that migrate to
the other derived from tissue-resident macrophages apparently B-cell follicles after activation and promote germinal center
9
responsible for much of the fibrosis seen in chronic helminth formation and B-cell isotype switching. These cells, which form
infections. By virtue of the expression of regulatory molecules, an independent lineage of CD4 T cells, have been recently identi-
such as IL-10, TGF-β, and programmed cell death 1 ligand 2 fied to be the predominant IL-4 producing T cells early in
(PDL2), these AAMs may have a predominantly regulatory role helminth infection. In addition, Tfh are major producers of IL-21,
in helminth infections. These antiinflammatory macrophages a cytokine that plays a crucial role in supporting polarized Th2
function through arginase-1, PDL2, triggering receptor expressed responses in vivo.
on myeloid cells 2 (TREM2) and RELM-α to inhibit classic Th17 cells, another subset of CD4 T cells, express the prototypi-
macrophage inflammation and recruitment and T-cell responses. cal cytokine—IL-17. In terms of helminth infections, the role
Similarly, macrophage-derived human resistin is induced by of Th17 cells has been primarily studied in animal models of S.

ChaPTEr 31 Immune Responses to Helminth Infection 441


mansoni, where it has been strongly associated with infection- influence of ILC2. Apart from the rapid kinetics of recruitment,
10
induced, immune-mediated pathology. More recently, it has eosinophils in blood and tissue also exhibit morphological and
also been demonstrated in human infections, in which children functional changes attributable to eosinophil activation. Eosino-
with S. hematobium–associated pathology have higher Th17 phils possess a range of immunomodulatory factors that are
responses compared with those who are pathology-free. Similarly, released upon cell activation, including cytokines, growth factors,
a strong association of Th17 responses with pathological responses and chemokines. Unlike T and B cells, eosinophils can rapidly
has also been demonstrated in lymphatic filariasis. Finally, Th22 release cytokines within minutes in response to stimulation, since
cells are yet another subset of CD4 T cells that typically secrete most of the cytokines are stored in a preformed fashion in
IL-22. To date, only a few studies have examined the role of secretory vesicles. Moreover, eosinophils can participate in the
11
Th22 cells in helminth infections. IL-22 was shown to be induced regulation of IgE and goblet cell mucus production; they also
in the intestinal mucosa after infection with T. trichiura or Necator serve as effector cells in protective immune responses and as
americanus in humans, whereas the frequency of Th22 cells was regulatory cells influencing both innate and adaptive immunity
shown to be higher in individuals with filarial infection compared in helminth infections.
with endemic healthy controls.
Helminths and Basophils/Mast Cells
Helminths and B Cells Basophils are an important component of the immune response
15
Helminth interactions with B cells occur both at the B-cell to helminth infections. Basophils are capable of secreting a
cytokine level and at the level of antibody production. Interactions variety of mediators, including histamines, cytokines, chemokines,
at the cellular level primarily result in B-cell activation and and lipid mediators that promote Th2 responses. Basophils in
cytokine production, most notably by the induction of IL-10. B humans and mice also readily generate large quantities of IL-4
cells have been shown to be important for the Th2 responses to in IgE-dependent and IgE-independent manners. Basophils appear
certain helminths, with IL-2 producing B cells supporting optimal to play an important role in protective immunity to secondary
development of effector and memory Th2 cells and LTα1β2- infection (similar to eosinophils) with N. brasiliensis, H. polygyrus
expressing B cells supporting the recruitment of a Th2 promoting bakeri, and L. sigmodontis; they also play an active role in resistance
12
DCs. Immune regulation by B cells has also been recognized to primary infection (through secretion of IL-4 and IL-13) with
in schistosome infection, where B-cell deficiency leads to enhanced T. muris and T. spiralis. In addition, basophils have been shown
Th2 cell–dependent immunopathology. However, it is at the level to be critical APCs for driving Th2 cell differentiation in different
of antibody production that B cells play a profound role in models of helminth infection.
helminth infections. Susceptibility to secondary infection is Mast cells may contribute to inflammatory reactions directed
increased in the absence of B cells in infection with Litomosoides against invasive helminth parasites. These cells express high
sigmodontis, S. mansoni, T. muris, and Heligmosomoides polygyrus affinity Fcε receptors that are sensitized with parasite antigen–
13
bakeri. IgG is reported as an antibody isotype that is important specific IgE and can be triggered by parasite antigens. It has
for protection against intestinal helminths, and IgM (typically been postulated that cytokines and other mediators released by
produced in a T cell–independent manner) has been linked to sensitized mast cells contribute to (i) the recruitment and activa-
timely elimination of filarial parasites. One of the most consistent tion of effector eosinophils; (ii) increased local concentrations
findings in helminth infections, both in mice and humans, is of antibody and complement; and (iii) enhanced mucus hyper-
the elevated level of IgE that is observed after exposure to hel- secretion and increased peristalsis of the gastrointestinal (GI)
minths. Most of the IgE produced is not antigen specific, perhaps tract that plays an important role in resistance to certain GI
16
representing nonspecific potentiation of IgE-producing B cells nematode infections. More recently, a role for mast cells (in
or deregulation of a normally well-controlled immune response. an IgE-independent manner) in mediating the secretion of
Interestingly, these IgE antibodies persist many years after the epithelial-derived cytokines (IL-25, IL-33, and TSLP) and optimal
infection has been treated, indicating the presence of long-lived migration of DCs was shown in H. polygyrus bakeri infection.
memory B cells or plasma cells in helminth infections. IgE
production both in mice and humans is absolutely dependent Helminths and Neutrophils
on IL-4 or IL-13. Other isotypes that are commonly elevated in Although neutrophils are typically considered more important
humans with chronic helminth infection are IgG4 and IgG1, the in bacterial and fungal infections, a number of studies have
former being most dependent on both IL-4 and IL-10. Recent revealed that neutrophils can act in conjunction with macrophages
17
studies have highlighted the role of regulatory B cells in sup- to contain or kill helminth parasites. Thus neutrophils are major
pression of immune responses to helminth parasites. This B-cell components of the granulomas forming around filarial parasites
function involves the secretion of IL-10 and IL-35 and is similar and the cysts containing larvae of intestinal helminths. Neutrophils
to the regulatory activity of B cells in autoimmune diseases. have been demonstrated to collaborate with macrophages in the
immobilization and killing of S. stercoralis larvae in a process
Helminths and Eosinophils that is complement dependent and involving neutrophil extracel-
Blood and tissue eosinophilia is characteristic of helminth infec- lular traps (NETs). Similarly, neutrophils contribute in the early
tion and is mediated by IL-5 (probably in concert with IL-3 and antifilarial response through oxidative burst, degranulation, and
GM-CSF). Recruitment of eosinophils to the site of infection NETosis and protect against infective larvae in skin. A seminal
occurs very early in experimental helminth infection—as early study reported that neutrophils adopt an “N2” phenotype during
as 24 hours after exposure. Kinetics of blood eosinophilia in experimental infection with N. brasiliensis in the lung and express
humans is harder to determine but is postulated to occur as the genes for IL-13, IL-33, RELM-α, and Ym1. These “N2”
early as 2–3 weeks after infection, as demonstrated in experimental neutrophils can train macrophages to acquire a memory phe-
14
infections of volunteers. Both basal eosinophil levels and tissue notype that protects against secondary infection. Finally, it was
accumulation during helminth infection appear to be under the also shown that even during primary infection, the absence of

442 ParT ThrEE Host Defenses to Infectious Agents


neutrophils resulted in greater worm burdens because of lack by vaccination with irradiated cercariae. This resistance is
of immunity in the lungs. Thus neutrophils appear to play an dependent on a Th1-mediated immune response consisting of
unexpected role in immunity to helminths that certainly merits macrophages and endothelial cells activated by IFN-γ and TNF-α,
further investigation. producing nitric oxide and Th1-associated antibodies—IgG2a
and IgG2b. In contrast, studies in rats and epidemiological studies
PROTECTIVE IMMUNITY AGAINST HELMINTHS in humans suggest Th2-mediated effector mechanisms involving
IgA and IgE antibodies as well as eosinophils are thought to be
The mechanism of protective immunity to helminths is dependent central to protective immunity. Protective immunity to filarial
18
on the location of the helminth infection. Clearly, T cells are infections in mice is dependent primarily on Th2 responses in
central to resistance against helminths. For example, T cells are mice. Thus mice lacking IL-4, IL-4R, or Stat6 are all susceptible
essential in mediating the expulsion of GI nematodes. Mice to infection with Brugian parasites.
lacking T cells are defective in their ability to expel T. muris, but In tissue-invasive helminth infections, effector mechanisms
resistance can be reconstituted by transfer of T cells from normal involve multiple innate immune cells, with antibodies acting as
mice. In addition, CD4 T cells from infected mice can transfer initiators of immunity by activating Fc receptor expressing cells.
protective immunity to severe combined immunodeficiency Basophils, by their ability to produce high levels of IL-4, act as
(SCID) mice (lacking both B and T cells), indicating that CD4 effectors to promote helminth killing in secondary or challenge
T cells, not CD8 T cells, were important for protective immunity. infections. For example, basophils are important in immunity
Similarly, T cells were shown to be required for expulsion in N. to the skin invasive stages of intestinal helminths and, through
brasiliensis infection. Both nude mice (lacking T cells alone) and IL-4 release, promote the activation of macrophages that trap
SCID mice are susceptible to infection with Brugian parasites, larvae in an arginase-dependent manner. Although eosinophils
whereas mice that lack either CD4 T cells or CD8 T cells are are crucial players in producing IL-4 early in infection, they are
not. In schistosome-infected mice, T cells are essential in forming also amplifiers of immune responses, rather than being critical
host-protective granulomas around the eggs deposited in the mediators of primary immunity, since depletion of eosinophils
liver. does not alter the course of many helminth infections in murine
The role of cytokines in protective immunity has been models. The mechanism of protection mediated by eosinophils
extensively studied in murine models of both GI helminths and is thought to be by antibody-dependent, cell-mediated cytotoxicity,
19
tissue-invasive helminths. In general, type 2 (Th2) cytokines as observed in S. mansoni studies in vitro or through release of
target epithelial cells, goblet cells, smooth muscle cells, and eosinophil granule contents. In addition, eosinophils play an
macrophages, which together coordinate parasite expulsion by important role in the protective immunity against primary
increasing fluid and mucus production, encapsulation and barrier infection with B. malayi and/or secondary infection with either
formation, epithelial cell turnover, smooth muscle cell contraction, T. spiralis or N. brasiliensis. The two most abundant granular
and production of anthelmintic effector molecules, such as proteins, major basic protein (MBP) and eosinophil peroxidase
RELM-β. The cytokines involved in both responses are IL-4, (EPO), are required for protective immunity against S. stercoralis
IL-5, IL-9, and IL-13. Most of the studies examining resistance and L. sigmodontis. Similarly, neutrophils can attack helminth
to intestinal helminths involve four parasitical GI nematode larvae in response to IL-4 and IL-5, but their importance in
infections of rodent models—T. spiralis, H. polygyrus bakeri, N. resistance to primary helminth infections is not known.
brasiliensis, and T. muris. These studies show that (i) CD4 T cells Antibodies play a major role in mediating protection to some
18
are crucial for host protection; (ii) IL-4 is required for host but not all helminth infections. Antibody-mediated passive
protection and limiting host pathology; (iii) IL-13 can substitute immunity has been demonstrated in animal models for A.
for IL-4 in some but not all infections; (iv) IL-2 and IFN-γ inhibit caninum, Schistosoma species, Taenia species, Ascaris suum, S.
protective immunity; and (v) IL-4 and IL-13 have multiple effects ratti, T. muris, N. brasiliensis, and H. polygyrus bakeri. Passive
on the immune system and gut physiology leading generally to immunity has also been shown by using IgG monoclonal antibod-
protection. Type 2 cytokines mobilize a broad range of down- ies (mAbs) specific for Fasciola hepatica and S. mansoni; IgM
stream effector mechanisms. Epithelial cells in the gut, specifically (mAbs) specific for B. malayi; and IgG or IgA mAbs specific for
tuft cells, promote goblet cell differentiation, enhancement of T. spiralis. Again using genetically manipulated mouse models,
mucus secretion, and the production of RELM-β, an innate IgM has been shown to be crucial for host protection against B.
effector molecule with direct anthelmintic activity. Goblet cells malayi and to S. stercoralis. B1B cells, a subset of B cells that
can also secrete gel-forming mucins, which are major macro- secrete IgM, appear to be an important component of this
molecular components of the mucus barrier. Two of these mucins protective axis. Finally, antibodies have the capacity to trap tissue
have been shown to be critical in resistance to intestinal nematode migrating helminth larvae and prevent tissue damage by driving
infection—Muc2 and Muc5AC. IL-4Rα activation also leads an IL-4Rα–independent alternative differentiation of macro-
increased intestinal smooth muscle hypercontractility and phages, in a process dependent on CD11b and FcγR1.
accelerated epithelial turnover to promote an effector response In terms of protection, a major mechanism appears to be the
akin to an “epithelial escalator,” which, together with epithelial formation of multicellular, immune cell aggregates, called granu-
18
secretions, helps expel intestinal helminths. Mucosal mast cells lomas, around incoming infectious larvae or eggs. In murine
release proteases that can degrade epithelial tight junctions, models of schistosomiasis and filariasis, granulomas are primarily
thereby increasing fluid flow as part of the “weep and sweep” composed of T cells (which help in the recruitment of other cell
response. AAMs in the gut can also entrap intestinal worms and types and mediate alternative activation of macrophages), B cells
cause death by compromising worm vitality. (particularly the B1 subset), and macrophages and eosinophils.
Although the role of Th2 cytokines in immunity to GI helminth Although the exact mechanism by which granulomas mediate
infection is well defined, their role in protective immunity to killing of the parasite remains unknown, it is clear that formation
18
tissue-invasive helminths is not as clear. In murine models of of these structures is an important host defense mechanism. One
schistosomiasis, protective immune responses can be generated cell type that can mediate effector functions within granulomas

ChaPTEr 31 Immune Responses to Helminth Infection 443



KEY CONCEPTS Granulomatous Reactions
Helminth-Induced Immune Responses Granuloma formation is the mainstay of the protective immune

Characterized by immunoglobulin E (IgE) antibody production, tissue and response to certain helminths, but it can also lead to deleteri-
peripheral blood eosinophilia, mast cell involvement, innate lymphoid ous effects in the form of pathology. Although granulomatous
cell type 2 and Th2 cell expansion, and production of type 2 reactions occur in many helminth infections (e.g., toxocariasis,
cytokines. Angiostrongylus infections and lymphatic filariasis), parasitical
Implicated both in pathogenesis of helminth infections and in mediating granulomata have been best studied in S. mansoni infections,
immunological protection.
In mucosal immunity to helminths, T-helper 2 (Th2) cell responses are where granulomatous and fibrosing reactions against tissue-
initiated and sustained by innate populations (including tuft cells and trapped eggs is orchestrated by CD4 T cells and the fibrosis
innate lymphoid cells) through interleukin (IL)-25, IL-33, and thymic that results from the cellular response is the principal cause
stromal lymphopoietin (TSLP). of morbidity in infected individuals. The severity of the
In tissues, helminths are acted upon by the host innate effectors, including inflammatory process markedly varies both in humans and in
macrophages, neutrophils, eosinophils, and basophils. experimental animal models, with severe pathology associated
Regulated by T cells and other cells producing IL-4, IL-5, IL-9, IL-10, and/ with Th1 and Th17 responses and milder pathology with Th2-
or IL-13. 21
Characterized by the induction of regulatory T cells (Tregs) that mediate dominant responses. Studies in murine models of granuloma
downmodulation of immune responses to helminth infections and formation have demonstrated the important roles of IL-13 and
impact bystander phenomena, such as allergy and autoimmunity. TNF-α.
Fibrosis
Fibrosis is commonly associated with chronic helminth infections
is the AAM, which is exemplified by the targeting of the glycan that result in chronic inflammation and dysregulated wound
22
chitin that is frequently expressed by helminths but not by the healing. These infections activate macrophages and fibroblasts,
host. The chitinase and fizz family proteins (ChaFFs), which resulting in the production of TGF-β, platelet-derived growth
include chitinase and chitinase-like secreted proteins, are prime factor (PDGF), IL-1β, and other factors. Macrophages also
candidates for mediating host resistance. These proteins include promote inflammation by recruiting and activating monocytes
acidic mammalian chitinase (AMCase) and the RELM family and neutrophils, as well as activating CD4 T cells. In addition,
proteins and are capable of enzymatic activities that potentially fibroblasts are stimulated to synthesize matrix metalloproteinases
damage certain helminths. (MMPs) and tissue inhibitors of metalloproteinases (TIMPs),
leading to extracellular matrix remodeling and fibrosis. Another
PATHOLOGY ASSOCIATED WITH IMMUNE consequence of chronic schistosomiasis is pulmonary arterial
RESPONSES IN PARASITIC HELMINTH INFECTION hypertension, which has been shown to be associated with
IL-4– and IL-13–mediated type 2 inflammation resulting in
Typically, pathological findings associated with each parasitic TGF-β−induced pulmonary vascular disease. In the same manner,
infection are different and relate to the presence of the parasites IL-10 and IL-12 are known to modulate IL-13–mediated fibrosis;
in host tissues, but there are pathological reactions that stem in the combined absence of IL-10, IL-12, and IL-13Rα, IL-13–
directly from the host response. dependent fibrosis in chronic schistosomiasis proceeds rapidly
to lethal cirrhosis. Infection with Wuchereria bancrofti (one of
Immune Complexes the causative agents of lymphatic filariasis) is associated with
Immune complexes are potent mediators of localized inflam- similar fibrotic reactions.
matory processes that form in many parasitic infections presum-
ably as a result of the chronic low-dose antigen release seen in Toll-Like Receptors
these infections. Circulating immune complexes have been Immunopathology in lymphatic filariasis is associated with the
identified in both experimental and human filarial and schisto- presence of an endosymbiotic, Rickettsia-like bacteria called
somal infections. These have been shown to induce lymphatic Wolbachia. Wolbachia are known to stimulate immune cells
inflammation and vasculitis in filarial infections as a result of through TLR2 and TLR4 and release proinflammatory cytokines,
their deposition. In addition, a common manifestation of immune as well as vascular endothelial growth factors (VEGFs), which
23
complex–mediated pathology, immune complex glomerulone- might contribute to lymphatic pathology. Wolbachia-TLR4
phritis (ICGN), has been documented by renal biopsy in patients interaction has also been shown to be the major mechanism of
with schistosomiasis and filarial infections. Other manifestations corneal inflammation in onchocerciasis, and a TLR signaling
of immune complex–mediated damage, such as reactive arthritis molecule, IL-1 receptor associated kinase-2 (IRAK-2), regulates
and dermatitis, have also been described in patients with helminth pathogenic Th17-cell development in S. mansoni infection.
infections.
Immediate Hypersensitivity Responses
Autoantibodies and Molecular Mimicry Immediate hypersensitivity responses are associated with the
Autoantibodies have been implicated as causing disease in a early and/or acute phase of infections with invasive helminth
variety of helminth infections, including filarial infections, parasites, such as Ascaris, hookworm, schistosomes, or filariae.
schistosomiasis, and hookworm infection, and are thought to Patients do manifest symptoms suggestive of allergic reactivity,
reflect a polyclonal B cell expansion that often accompanies these such as wheezing or urticaria. Furthermore, in clinical syndromes
20
infections. Autoantibodies against nuclear material have been associated with Loa loa infection (with its angioedematous Calabar
found in a vast majority of patients with chronic schistosomiasis, swellings), with tropical pulmonary eosinophilia, and with larva
and antibodies against human calreticulin and defensin have currens in strongyloidiasis, IgE-mediated reactions are thought
24
been found in onchocerciasis. to underlie these signs and symptoms. Anaphylaxis is a severe,

444 ParT ThrEE Host Defenses to Infectious Agents


life-threatening, generalized, or systemic hypersensitivity reaction, MECHANISMS OF EVASION AND IMMUNE
and is associated with IgE interaction with high-affinity IgE REGULATION BY HELMINTH PARASITES
receptors on basophils and mast cells (Chapter 23). The risk of
anaphylaxis in individuals with helminth infections can vary, Helminths exert profound immunoregulatory effects on the host
depending on the parasite, tending to occur more frequently immune system with parasite antigen–specific immune suppres-
with echinococcosis or after Anisakis infection, while being sion as well as more generalized levels of immune suppression.
extremely rare in most other helminth infections. It has been shown that patients with schistosomiasis or filariasis
have markedly diminished responses to parasite antigens and
Wound Healing to some measurable attenuation in responses to bystander antigens
Recent studies have shown a close association of type 2 cytokine and routine vaccinations. Thus host immunosuppression is usually
responses with many aspects of wound healing and repair. 25,26 antigen specific, whereas chronic infection can be associated
It has been proposed that the type 2 cytokine response has evolved with some spillover effects. Among the mechanisms utilized by
to not only mediate resistance to helminth infection but also parasites to avoid immune-mediated elimination are those of
activate the wound healing apparatus to repair and reconstruct evasion—the use of sequestration, camouflage, and antigenic
tissue, since tissue damage is intricately associated with helminth variation—and suppression, regulation, or blockade of immune
infections. Thus AAMs are intimately involved in this process effector pathways.
as they produce MMPs, Arginase-1, insulin-like growth factor
1 (IGF1), VEGF, and TGF-β, which together promote myofibro- Parasite-Derived Factors
blast activation, angiogenesis, epithelial cell turnover, and Parasite-derived products play a very important role in host
extracellular matrix deposition. immune evasion. 29,30 Parasite products, such as the schistosome-
secreted proteins, alpha-1 and omega-1, promote Th2 differ-
Lymphangiogenesis entiation. Alpha-1 (also known as IL-4–inducing principle of
The anatomical changes in the architecture of lymphatics, which schistosome eggs [IPSE]), released by schistosome eggs, induces
range from lymphangiectasia and granulomatous responses to IL-4 release and degranulation by human and mouse basophils by
the development of collaterals, suggest that active lymphatic cross-linking surface IgE. Omega-1 is a ribonuclease abundantly
remodeling involving endothelial cell growth, migration, and secreted by eggs, shown to condition DCs to drive Th2 polariza-
proliferation is an important feature of early lymphatic filarial tion. Omega-1 binds to and is internalized by DCs in a mannose
disease. Live filarial parasites (and their excretory/secretory receptor–dependent process and then suppresses protein synthesis
products) have been shown to induce activation, proliferation, through degradation of messenger RNA (mRNA).
and tube formation in lymphatic endothelial cells (LECs) and Phosphorylcholine (PC) is a small hapten-like moiety present
27
their differentiation into tubelike networks. This was found to in the excretory/secretory products of many helminths, and one
be associated with significantly increased levels of MMPs and particular PC-containing molecule, called ES-62, from filarial
TIMPs. Recent studies have also implicated the VEGF family in worms has been shown to have a wide variety of immunomodula-
lymphangiogenesis, with VEGF-C being associated with lymph- tory properties. Thus ES-62 can inhibit the proliferation of CD4
23
edema and VEGF-A with hydrocele. Finally, TLR-mediated T cells and conventional B cells, decrease IL-4 and IFN-γ produc-
events are considered to be the main drivers of this angiogenic/ tion, promote proliferation and IL-10 production by B1B cells,
lymphangiogenic process in filarial disease. 27 modulate complement activation, and condition APCs to drive
Th2 differentiation with concomitant inhibition of Th1 responses.
Carcinogenesis ES-62 has also been shown to exhibit bystander antiinflammatory
Infection with Opisthorchis viverrini, Clonorchis sinensis, and activity in collagen-induced arthritis, rheumatoid arthritis,
Schistosoma hematobium are classified as group 1 biological chemical contact sensitivity, lupus-associated atherosclerosis, ear
carcinogens (i.e., definitive causes of cancer). The former (liver inflammation, chronic asthma, and airway hyperreactivity.
fluke) is associated with cancer of the bile duct (cholangio- Helminths utilize glycans within glycoproteins and glycolipids,
carcinoma) and cancer of the liver (hepatocarcinoma), and which mimic host glycans, to regulate host immune responses.
the latter is associated with carcinoma of the urinary bladder. In addition, these host-like helminth glycans can directly interact
The mechanisms of helminth-induced cancer include chronic with host glycan–binding proteins, such as C-type lectin receptors
inflammation, sustained cellular proliferation, modulation of and galectins, to shape innate and adaptive immune responses.
the host immune system, reprogramming of glucose metabo- Similarly, helminth lipids have also been implicated in immune
lism and redox signaling, induction of genomic instability and modulation; schistosome phosphatidylserine induces DCs to
destabilization of tumor proteins, stimulation of angiogen- polarize IL-4–producing T cells, whereas schistosome lysophos-
esis, resistance to apoptosis, and activation of invasion and phatidyl serine induces DCs to induce IL-10–secreting Tregs.
metastasis. 28 Helminth parasites utilize mechanisms involving cytokine
mimicry and interference to establish chronic infection. Thus
Epileptogenesis parasites produce cytokine- and chemokine-like molecules to
Neurocysticercosis, caused by the larval form of Taenia solium, is interfere with the function of host innate immune products.
the most common preventable risk factor for epilepsy worldwide The first helminth cytokines were found to be homologues of
and accounts for nearly 30% of all epilepsies in some endemic TGF-β expressed by B. malayi, and both schistosomes and filarial
areas. The manifestations are variable, depending on the parasites express members of the TGF-β receptor family. Similarly,
location, number, and size of the cysts in the central nervous E. granulosus expresses a TGF-β ligand, and thus all helminth
system as well as the degree of accompanying inflammation, groups might have the potential to exploit TGF-β–mediated
provoked by cyst degeneration, calcification, and/or perilesional immune suppression. Various helminths, including B. malayi,
edema. produce homologues of macrophage migration inhibitory factors

ChaPTEr 31 Immune Responses to Helminth Infection 445


(MIFs), which are known to activate an antiinflammatory pathway can then act as messengers of communication between the parasite
through SOCS-1, a molecule involved in cytokine signaling. T. and the host cell. These exosomes (containing microRNA) can
muris is known to express a homologue of IFN-γ, which binds then enter the host cell and modulate host gene expression. 32
to the IFN-γ receptor in vitro and induces signaling. As T. muris
is expelled by IL-4, secretion of an IFN-γ–like protein can prolong Host-Related Factors
its survival. Regulatory T and B Cells
Similarly, helminth parasites utilize chemokine- or chemokine- Evidence for the involvement of Tregs in helminth-mediated
receptor like proteins to evade protective immunity. Ascaris suum downmodulation of the immune response has been accumulating
33
is known to express a neutrophil chemoattractant with chemokine in recent years (Chapter 18). IL-10 and TGF-β, both factors
binding properties. S. mansoni eggs secrete a protein (S. mansoni associated with Tregs, are elicited in response to helminth infec-
chemokine-binding protein [smCKBP]) that binds the chemo- tions, and in vitro neutralization of IL-10 and TGF-β at least
kines CXCL8 and CCL3 and inhibits their interaction with host partially restores T-cell proliferation and cytokine production
chemokine receptors and their biological activity, resulting in in lymphatic filariasis. Similar reversals of immunosuppression
31
suppression of inflammation. Similarly, B. malayi (and all of are observed in onchocerciasis and schistosomiasis, with IL-10
the other filariae sequenced to date) has been shown to express producing natural Tregs (nTregs) from egg-induced granulomas
galectins that can bind host immune cells in a carbohydrate- (in S. mansoni infection) being important for host survival. More
dependent manner. recently, Tregs from patients with filarial infections have also
Helminths secrete two major classes of protease inhibitors been shown to express high levels of other suppressive molecules,
called cystatins and serpins, each with proposed immunomodula- such as CCL4, IL-29, LAG-3, and Foxo3. Moreover, filarial infec-
tory roles. Cystatins inhibit cysteine proteases (cathepsins and tion is associated with an expansion of T cells expressing the
aspartyl endopeptidases) required for antigen processing and IL-10 superfamily cytokine members (IL-19 and IL-24), and
presentation and therefore inhibit T-cell activation. They also inhibition of these cytokines results in increased Th1 and Th2
elicit the regulatory cytokine IL-10, leading to direct impairment responses. Tregs play a vital role in limiting host pathology by
of T-cell proliferation. The serpins are serine protease inhibitors, downregulating harmful Th1/Th17 responses in filarial infection
which can cause specific inhibition of the neutrophil proteinases and schistosomiasis. In addition, low level Treg activity is essential
cathepsin G and neutrophil elastase. Aspartic proteases from for type 2 effector immunity to expel certain intestinal helminths,
Ascaris lumbricoides have been shown to block efficient antigen and transdifferentiation of Th17 cells into Tregs during helminth
processing that is dependent on proteolytic lysosomal enzymes. infection is important for resolution of inflammation.
Other parasite products mediate their effect by blocking A number of studies have recently reported that B cells might
effector functions, including recruitment and activation of have an active regulatory role in helminth infections. For example,
inflammatory cells and limiting the destructive potential of purified B cells from mice infected with B. malayi produce IL-10
hi
activated granulocytes or macrophages in the local extracellular in response to filarial antigens. Similarly, IL-10 producing CD1d
milieu. For example, the host chemoattractant platelet-activating B cells are induced in both mice and humans and are suppressed
factor (PAF) is inactivated by a complementary enzyme PAF after anthelmintic treatment. This induction occurs, in part,
hydrolase secreted by N. brasiliensis. Eotaxin-1, a potent eosinophil through a mechanism involving the ICOS-B7RP-1 pathway.
chemoattractant, is degraded by metalloproteases from hook-
worms. A. caninum secretes a protein called neutrophil inhibitory Hyporesponsive T Cells
factor, which binds the integrins CD11b/CD18 and blocks Effector T cell responses can be turned off or modulated through
adhesion of activated neutrophils to vascular endothelial cells a variety of mechanisms, including through cytotoxic T lym-
and also the release of hydrogen peroxide (H 2 O 2 ) from activated phocyte antigen-4 (CTLA-4) and programmed death-1 (PD-1).
neutrophils. N. americanus ES products also bind to host NK Interestingly, increased expression of CTLA-4 and PD-1 has been
cells and augment the secretion of IFN-γ, which might cross- demonstrated in filarial infections, and blocking of CTLA-4 can
regulate deleterious Th2 responses. Other modulators, such as partially restore a degree of immunological responsiveness in
prostaglandins, and other arachidonic acid family members, such cells from infected individuals. Moreover, T cells have decreased
as PGE2 and PGD2, are known to inhibit IL-12 production by induction of T-bet, the Th1 master regulatory gene indicating a
DCs. Finally, helminths susceptible to oxidant-mediated killing failure at the transcriptional level to differentiate into Th1 cells.
express both secreted and membrane-associated enzymes, such Finally, T cells from individuals with filarial infection exhibit
as superoxide dismutase, glutathione-S-transferase, and gluta- classic signs of anergy, including diminished T-cell prolifera-
thione peroxidase, molecules that are thought to play a significant tion to parasite antigens, lack of IL-2 production, and increased
role in assisting parasite survival in inflamed tissues. Recently, expression of E3 ubiquitin ligases. Similarly, anergic T cells are
a family of helminth defense molecules secreted by parasitic found in both humans and mice with F. hepatica infection and
helminths has been shown to exhibit biochemical and functional schistosomiasis; in the latter case, these T cells express high
characteristics similar to human antimicrobial peptides. These levels of the anergy molecule GRAIL (gene related to anergy in
molecules can modulate innate cell activation by classic TLR lymphocytes).
ligands, such as lipopolysaccharide.
It has been also been reported that parasitical helminths can Modulation of Apc Function
produce exosomes and other secretory vesicles that facilitate the DCs are the first APCs usually to encounter parasites, and
transfer of intracellular cargo. Exosomes derived from helminths helminth modulation of DC function has been well character-
34
have been shown to possess immunomodulatory capacity with ized. Filarial parasites induce downregulation of MHC class I
the ability to modulate immune responses by altering the function and class II molecules, as well as cytokines and other genes
of ILC2. In addition, secreted vesicles from B. malayi, O. volvulus, involved in antigen presentation, thereby rendering DCs sub-
and S. mansoni have been shown to contain microRNAs that optimal in their ability to activate CD4 T cells. Schistosomes

446 ParT ThrEE Host Defenses to Infectious Agents


have similar effects on DCs, with subsequent Th2 polarization animal models have revealed the protective effect of helminth
and inhibited responses to Th1-inducing TLR ligands. In addition, infections against atopy and asthma. Several mechanisms have
schistosomes modulate the activation of Nlrp3 (NLR family, been proposed for the helminth-induced protection, the chief
pyrin domain containing 3) inflammasome and thus IL-β produc- of which are the induction of Treg activity, regulatory B-cell
tion. Excretory/secretory antigens produced by helminths can activity, and immunosuppressive cytokines including IL-10 and
inhibit DC synthesis of proinflammatory cytokines, chemokines, TGF-β. Similarly, exposure to helminth parasites has been shown
and costimulatory molecules and promote DC production of to prevent the onset of Th1-mediated diseases, such as multiple
the regulatory cytokines IL-10 and TGF-β. Helminth infection sclerosis (MS), diabetes mellitus, and Crohn disease in experi-
37
has also been shown to induce in vivo differentiation of a CD103 - mental animal models. Finally, recent studies in mice have
lo
CD11c population of regulatory DCs, which are inefficient in shown that type 2 immunity, induced by helminth infection,
priming effector T cells and instead favor the generation of Tregs. can maintain adipose tissue homeostasis and promote adipose
AAMs are able to markedly suppress target cell proliferation, as tissue beiging, protecting against obesity and metabolic dysfunc-
well as mediate repair of tissue that has been damaged by parasites. tion; that the immunomodulatory glycan LNFPIII, which is
In addition, human filarial infection is associated with the secreted by helminths, can alleviate hepatosteatosis and insulin
expansion of the nonclassic monocyte subset, as well as an resistance; and that there exists an inverse association between
immunoregulatory monocyte subset. Helminth antigens can the presence of helminth infections and the prevalence of type
modulate MHC class II and CD80/86 expression on “antigen- 2 diabetes.
presenting” basophils to induce the development of Th2 cells.
Finally, a heterogeneous population of immature myeloid cells HELMINTH THERAPY FOR
that share the common property of suppressing immune responses INFLAMMATORY DISEASES
are termed myeloid-derived suppressor cells (MDSCs). Although
these MDSCs have been well characterized in cancer immunology, To date, two species of helminths have been tested as clinical
their role in helminth infections is still being explored. treatment for therapy of inflammatory diseases: T. suis ova and
infection with N. americanus. Currently, 28 clinical trials of
Apoptosis helminth therapy in 10 autoimmune diseases and allergic or
38
Another mechanism of immune evasion is the ability of some related conditions have been planned, started, or completed.
helminths to induce host cell apoptosis (Chapter 13). Apoptosis These include Crohn disease, ulcerative colitis, MS, celiac disease,
has been described as a host regulatory mechanism in various autism, plaque psoriasis, peanut and tree nut allergy, asthma,
helminth infections, including schistosomiasis, lymphatic filariasis, rhinoconjunctivitis, and rheumatoid arthritis. Although well
and onchocerciasis. tolerated, recent results suggested that this therapy did not achieve
improvement in disease activity or remission rates in Crohn
HELMINTHS AND THE MICROBIOTA disease. Studies using N. americanus include fairly small trials
with patients with Crohn disease or individuals with celiac disease
Recent work has highlighted the importance of the microbiota (gluten allergy). Additionally, a few studies have been performed
in influencing host immunological and metabolic functions examining the effect of helminth therapy on asthma/allergy. The
(Chapter 14). Helminths secrete a variety of products that can one minor success of helminth therapy in humans is in the
directly influence the composition and function of the microbiota, treatment of MS, wherein small trials have successfully demon-
whereas changes in microbiota can have an impact on susceptibil- strated lower relapses and lower magnetic resonance imaging
ity to helminth infection, indicating that helminth–microbiota (MRI) activity, prompting larger phase I or II trial involving
35
cross-talk can regulate a variety of host processes. Recently, it either T. suis or N. americanus.
was reported that infection with helminths (H. polygyrus or T.
muris) results in alteration of the composition of the microbiota,
with expansion of Lactobacillacea and Enterobacteriacea in the ON ThE hOrIZON
gut, and that the removal of helminths resulted in restoration Identification and synthesis of helminth products that can be useful as
of the original composition of the microbiota. The consensus immune therapy in a variety of inflammatory disorders
in humans is yet to emerge, with some studies showing an effect Deciphering the three way cross-talk among host immunity, helminths,
of helminth infection on microbial diversity and others failing and the microbiota
to reveal any differences. Conversely, it has also been demonstrated Elucidation of the detailed mechanisms by which helminths manipulate
that introducing higher level of certain commensal microbes immune responses to bystander antigens
enhances susceptibility, whereas removal of microbiota by Development of clues to production of novel vaccine candidates to protect
against not only helminth infection but also helminth-induced
antibiotics decreases susceptibility to helminth infections. morbidity
Combined approaches involving genomics, transcriptomics, proteomics,
REGULATION OF ALLERGY, AUTOIMMUNITY, AND and metabolomics for assessment of host–helminth interactions
METABOLIC DISEASES IN HELMINTH INFECTION
VACCINES AGAINST HELMINTH PARASITES
The hygiene hypothesis postulates that the stimulation of the
immune system by microbes or microbial products protects from Vaccines against helminth infections are a necessary tool for
39
36
the development of inflammatory and atopic disorders (Chapter their elimination and eradication for several different reasons.
1). Human studies have demonstrated that people living in areas Currently, a total of five human anthelmintic vaccines have
endemic for helminth infections have a decreased reactivity to advanced from discovery through manufacture and are now in
40
37
skin tests for allergens and milder forms of asthma. Experimental phase I or II clinical testing. These include three Schistosome

ChaPTEr 31 Immune Responses to Helminth Infection 447


vaccine candidates: (i) S. hematobium Sh28GST (phase II); (ii) 18. Anthony RM, Rutitzky LI, Urban JF Jr, et al. Protective immune
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11. Bouchery T, Kyle R, Ronchese F, et al. The differentiation of CD4(+) and partners of the microbiota in host intestinal homeostasis. Curr Opin
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ChaPTEr 31 Immune Responses to Helminth Infection 447.e1


MULTIPLE-C h OICE QUESTIONS

1. The immunity to helminths typically involves a T-helper 2 3. These cells are a novel cell type recently shown to play a
(Th2) cell response with what other component of the T-cell pivotal role in the initiation of immunity to helminth
response? parasites:
A. Th1 response A. Natural killer (NK) cells
B. Th17 response B. Type 1 innate lymphoid cells
C. Regulatory T-cell response C. Type 2 innate lymphoid cells
D. Th22 response D. Type 3 innate lymphoid cells
2. The classic antibody isotypes induced by helminths typically
include:
A. Immunoglobulin E (IgE) and IgG4
B. IgE and IgG3
C. IgE and IgD
D. IgG1 and IgG2

32






Approach to the Evaluation of the Patient

With Suspected Immunodeficiency



Javier Chinen, Mary E. Paul, William T. Shearer







Clinical immunologists are often consulted to evaluate patients KEY CONCEPTS
for suspected immune defects, usually because such patients
have an unusual frequency or severity of infectious illnesses. Secondary Immunodeficiencies
Indeed, immunodeficiency presents with increased susceptibility Immunodeficiency is often secondary or transient, caused by nonimmune
to infection but may also manifest with conditions that reflect factors, including:
dysregulation of the immune response, such as allergies, autoim- • Previous use of high-dose steroids, or other immunosuppressive
munity, or lymphoproliferation. Prompt diagnosis is essential to medications
reduce the risk of organ damage caused by preventable severe • Previous use of monoclonal antibodies (mAbs), such as rituximab
infections. Primary immunodeficiencies (PIDs) are congenital (anti-CD20)
diseases that might affect any aspect of the immune response and • Immunoglobulin losses via the gastrointestinal or urinary tract
• Severe illness requiring critical care
are often diagnosed in childhood. Examples of PIDs include severe • Malnutrition
combined immune deficiencies (SCIDs), complete DiGeorge • Human immunodeficiency virus (HIV) infection
syndrome, and chronic granulomatous disease (CGD). In
contrast to PIDs, secondary immunodeficiencies (Chapters 38,
39) present at any age, as a result of a wide variety of factors
that affect the immune function, such as environmental factors, increase the frequency of infections include allergic inflammation,
metabolic disease, anatomical abnormalities, or infectious agents. HIV infection, and the use of immunosuppressive drugs. Examples
The most known and significant secondary immunodeficiency of nonimmunological conditions include those that disrupt the
is caused by human immunodeficiency virus (HIV). The assess- usual mucosal clearance mechanisms, such as posterior urethral
ment of a patient for PID should include history and physical valves or urethral stenosis in a patient with recurrent urinary
examination to direct immunology laboratory testing to confirm a tract infection, or cystic fibrosis in individuals who have recurrent
diagnosis. sinusitis or pneumonia and/or diarrhea. Disruption of natural
barriers can lead to increased risk of infection, for example, in
EPIDEMIOLOGY—PRIMARY patients with skin lesions caused by eczema or burns or in
IMMUNODEFICIENCIES ARE NOT UNCOMMON individuals with cerebrospinal fluid (CSF) leaks following bony
injury to the cranium. Patients presenting with low Ig levels
Estimates of the incidence of PIDs or congenital immuno- might have loss of antibodies as a result of a protein-losing
deficiencies varies from selective immunoglobulin A (IgA) enteropathy, nephropathy, or massive protein loss through skin,
1
5
deficiency, a relatively common condition, (1/223–1/1000 people) such as in severe eczema or burns. Secondary immunodeficiency
to the less common SCID. Recent analysis from 11 state programs can also result from other conditions affecting cell metabolism
established for universal newborn screening for T-cell deficiencies (e.g., malnutrition, diabetes mellitus, and sickle cell anemia) or
in the United States reported an incidence of SCID of 1/58 000 could be secondary to predictable or idiopathic adverse effects
2
live births, comparable with childhood leukemia. A household- of drugs. Optimal management of these conditions often results
based telephone survey suggested that 1 in 1200 persons in the in improved immunity.
3
United States has been diagnosed with a PID. Although significant
progress has been made to stop the acquired immunodeficiency EVALUATING PATIENTS FOR IMMUNODEFICIENCY
syndrome (AIDS) epidemic, HIV infection continues to be the
most prevalent cause of immunodeficiency worldwide, with an The evaluation of patients for immunodeficiency is based on a
estimated 36.9 million people living with HIV. 4 careful assessment of patient history and physical examination,
with very limited initial laboratory testing. With this information,
PRIMARY VERSUS SECONDARY the clinician can often tell patients (or parents) whether their
IMMUNODEFICIENCY (or their child’s) immune system is significantly compromised.
The immunologist must take on a role of counselor and advisor
Because of their common occurrence, acquired and nonim- of patients and explain the many factors that may result in
munological causes for recurrent infections should be first increased frequency of infections (Fig. 32.1). The limitations of
considered in the differential diagnosis of the patient with a available clinically validated testing need to be considered, as
suspected immune disorder. Acquired conditions that might such tests may not be sensitive or specific to identify uncommon

451

452 ParT FOur Immunological Deficiencies



General screening of immunity
History 1. CBC, differential, platelets Physical examination
1. Number, type, course of infection 2. IgG, IgA, IgM, IgE levels 1. Growth and development
2. Family history 3. Baseline antibody titers: 2. Associated abnormalities
3. Age of onset, sex To immunizations (i.e., DiGeorge facies, rash, telangiectasia)
4. Environmental exposure to infection (e.g., tetanus, pneumococcus) 3. Presence or absence of lymphoid tissue
Isohemagglutinins

Suspicious finding Suspicious finding Suspicious finding
or red flag or red flag or red flag


Disease-specific testing of the immune system
FIG 32.1 Evaluation of Immunity in a Patient for Immunodeficiency Starts With a Careful
History and Physical Examination. Clues in the history for further evaluation include an excess
of respiratory tract infections, of unusual severity; life-threatening infections; infections with
unusual organisms; a family history of immunodeficiency. The outlined laboratory tests provide
an adequate screen of immunity in a patient with no specific findings.




immune defects, such as impairments in phagocyte function exposure to indoor allergens, such as dust mites and molds, often
other than oxidative burst deficiency. The medical history and worsens mucosal congestion and increases the risk of sinusitis
initial laboratory testing often provide clues that suggest a specific and otitis media.
immune disorder, and the examination of specific component
of the immune response or a diagnostic test for specific Immunization and Previous Infections
immunodeficiencies may be indicated. For example, an increased The immunization history provides significant clues, since the
frequency of infections affecting only the respiratory tract and efficacy of vaccines depends on intact immunity. An incom-
caused by encapsulated bacteria direct the exploration to defects plete schedule of immunizations easily explains the incidence
in humoral immunity and complement; in contrast, a history of preventable diseases. A history of an adverse reaction to a
of Aspergillus pneumonia would suggest neutropenia and CGD. live viral vaccine is suspicious for immunodeficiency, as infants
According to the severity of the illness, clinical immunologists with T-cell defects, B-cell defects, and combined T- and B-cell
may recommend an initial exploration of the major components defects are susceptible to potentially fatal or severe infections
of the immune system: lymphocyte subset distribution, antibody from live attenuated vaccines. These infections include measles
responses, T-cell function, phagocyte oxidative burst, and the and chicken pox pneumonitis, rotavirus vaccine-induced diar-
8
complement system. rhea, and lymphadenitis caused by the Bacille Calmette-Guérin
9
(BCG) vaccine. Historical information regarding the frequency,
EXPLORING THE MEDICAL HISTORY type, and severity of illnesses and infections should be sought.
Individuals with immunodeficiency may have infections with
Age and Environment unusually prolonged courses or unusual severity or that may
The differential diagnosis of immunodeficiency varies with the age present as unexpected complications (Fig. 32.2). Recurrent infec-
of onset of symptoms. Pediatric patients are more likely to present tions that involve multiple sites are more suspicious for immune
a PID than a secondary immunodeficiency. Infants from birth to deficiency than those involving a single site. Also suggestive of
3 months of age have maternal Igs acquired through the placenta, immune compromise are severe and invasive infections, such
unless they were born prematurely. Therefore deficiencies in the as recurrent pneumonia, meningitis, sepsis, septic arthritis,
immune system at this age presenting with frequent infections osteomyelitis, or abscess and infections with organisms of low
most probably result from severe deficiencies in other immune pathogenicity in normal individuals, such as Candida albicans
components, such as neutrophils, complement components, or T or Pneumocystis jiroveci (Table 32.1) Patients with antibody
cells. Older patients might present with increased risk of infections deficiency disorders tend to present with infections caused by
secondary to comorbid conditions, such as allergic inflammation extracellular pyogenic organisms, such as Haemophilus spp.,
or diabetes mellitus, or to a normal decline of immune responses, a Pneumococcus spp., and Streptococcus spp. In contrast, patients
6
process known as immunosenescence (Chapter 38). Environmental with defects in cell-mediated immunity are more likely to also
conditions can influence the risk of infection. Infants frequently present with recurrent infections with viruses, fungi, protozoa,
exposed to other infants with infections, such as in the setting of and mycobacteria. Furthermore, infections by catalase-positive
a daycare facility, have more infectious illnesses compared with bacteria, such as Serratia marcescens, may indicate a possible
those who are not exposed. By inducing an inflammatory response neutrophil oxidative burst defect. Recurrent neisserial infections
of the respiratory mucosa, passive cigarette smoke inhalation also may be found in individuals deficient in the terminal complement
predisposes to infections, including otitis media, pneumonia, and components. A relatively normal incidence of infections followed
7
bronchitis. The hygiene practices of the patient, caregivers, and by a sudden occurrence of repeated infections in an adult or
family members have an impact on the frequency of infections, adolescent suggests a secondary immunodeficiency, including
such as impetigo and furunculosis. For patients with allergies, HIV infection.

CHaPTEr 32 Approach to the Evaluation of the Patient With Suspected Immunodeficiency 453



TABLE 32.1 Clinical Clues of Significance for the Diagnosis of Immunodeficiencies
T-Cell Function antibody Granulocyte
Defect Defect Defects Complement Defect IFN-γ/IL-12 Defect
Recurrent or severe bacterial infections X X X (catalase-positive) X (encapsulated bacteria)
Systemic mycobacterial infections X X
Recurrent or severe viral infections X X X X
Invasive fungal infections X X
Opportunistic infections X X X
Failure to thrive X X X
Autoimmunity X X X X
Lymphoma X X (CVID)

CVID, common variable immunodeficiency; IFN, interferon; IL, interleukin.


TABLE 32.2 Nonimmunological Clinical
Findings Present in Immunodeficiency
Syndromes

Nonimmunological
Clinical Finding Immunodeficiency
Small platelets, thrombocytopenia, Wiskott-Aldrich syndrome (WAS)
eczema
Conical teeth, ectodermal Nuclear factor κB (NF-κB)
dysplasia essential modulator (NEMO)
defect
Delayed shedding of primary Autosomal dominant hyper-IgE
teeth, frequent fractures, syndrome (AD-HIES)
hyperextensibility
Cerebellar ataxia, telangiectasia Ataxia–telangiectasia (AT)
Hypoparathyroidism, conotruncal DiGeorge syndrome
heart defect, velopalatal
insufficiency
Short limbs Cartilage-hair hypoplasia
FIG 32.2 Computed Tomography (CT) Scan in an Infant With Microcephaly DNAse IV–deficient severe
Chronic Granulomatous Disease (CGD). Multiple nodular combined immunodeficiency
opacities are seen throughout both lung fields as a result of (SCID), Nijmegen syndrome
fungal pneumonia in an infant with CGD. (From Seeborg FO, Silvery hair, albinism Pigment dilution disorders
et al. A 5-week-old HIV-1-exposed girl with failure to thrive and Pectum carinatum, skeletal Shwachman-Bodian-Diamond
diffuse nodular pulmonary infiltrates. J Allergy Clin Immunol dysostosis, pancreatic syndrome (SBDS)
2004; 113: 629, with permission from Elsevier.) insufficiency


Comorbid Conditions or enteropathies that result in protein losses and secondary
Apart from frequent infections, other important aspects of the hypogammaglobulinemia.
history and physical examination may suggest congenital syn-
dromes associated with immune defects (Table 32.2). Neutrophil Use of Medications
adhesion defects lead to delayed (beyond 2 weeks of age) umbilical Use of particular drugs might also cause immunodeficiency,
cord separation because of omphalitis and poor wound healing. which could be predictable, such as the use of rituximab
Patients with DiGeorge syndrome are usually diagnosed in the (anti-CD20 antibody) resulting in B-cell depletion and potential
neonatal period with hypocalcemic seizures associated with antibody deficiency, or idiopathic, such as hypogammaglobu-
hypoparathyroidism, velopalatal insufficiency, or cardiovascular linemia that might develop with the use of anticonvulsants. 11
malformations, rather than with recurrent infections. Infants
with Wiskott-Aldrich syndrome (WAS)—immunodeficiency, Family and Social Histories
thrombocytopenia, and eczema—present with petechiae and Family history is essential in the evaluation of suspected immu-
bruises similarly in the neonatal period. Obtaining a history of nodeficiency. A history of early infant deaths and possible
atopic disease is helpful. Atopic dermatitis has been associated consanguinity should be sought. A clear pattern of inheritance
with high risk of infection, including nondermatological infec- may be found to define an X-linked, autosomal dominant, or
10
tions. For some patients with allergic rhinitis, management of autosomal recessive genetic syndrome. Many of the most common
allergies reduces the frequency of upper respiratory tract infection. PIDs have X-linked inheritance patterns. Family members of
In addition, it is important to inquire for a history of recurrent patients with immunodeficiencies might also have a history of
wheezing. At times, an initial history of recurrent pneumonia autoimmune disease or of connective tissue disease. Familial
is, in fact, secondary to reactive airways disease or asthma. Other cases of selective IgA deficiency and common variable immu-
significant conditions include renal disease causing proteinuria nodeficiency (CVID) have been reported, and a susceptibility

454 ParT FOur Immunological Deficiencies


trait can sometimes be traced back to many generations. A social
history should be obtained for risk factors associated with
increased risk of acquiring HIV infection. Socioeconomic factors
often determine malnutrition, known to be of a significant impact
on immune function. 12

KEY CONCEPTS
Genetic Immunodeficiencies
• Diagnosis of genetic immunodeficiencies require high clinical suspicion
and confirmation with molecular studies.
• In a minority of cases, genetic immunodeficiencies might not present
with all clinical elements of classic descriptions. For example, patients
with Bruton agammaglobulinemia might have residual B-cell and
immunoglobulin production.


PHYSICAL EXAMINATION FINDINGS
The physical examination might provide findings that indirectly
address the immune system; for example, scarred bilateral tympani
suggest recurrent ear infections. More commonly, patients with
immunodeficiency might otherwise look like normal individuals, FIG 32.3 Severe gingivostomatitis and dental erosion in a 2-year-
unless severe infections had produced an organ damage or had old child with leukocyte adhesion defect (LAD). (Courtesy of Dr.
delayed growth and development. However, attention to details D.C. Anderson.)
in the physical examination may supply important clues that
suggest immune dysfunction. In a normal child, a paucity of
lymphoid tissue, such as tonsils and lymph nodes, might reflect age-specific ranges. Leukocytosis, neutropenia, lymphopenia, and
impaired development resulting from immune deficiency. This abnormalities in WBC morphology can be detected from this
is especially seen in patients with X-linked agammaglobulinemia. test. Persistent neutrophilia might suggest LAD. Anemia may be
Certain physical findings are suggestive of syndromic immuno- present in children with chronic disease. Platelet counts may be
deficiencies, such as with telangiectasia over the bulbar conjunc- abnormally low in children with poor bone marrow function or
tivae and face with or without ataxia in ataxia–telangiectasia autoimmune disease, and platelets will be reduced in number
(AT); chronic eczema and delayed shedding of primary teeth in and morphologically small in children with WAS. Chemistry
hyper-IgE syndrome (HIES); severe eczema in immunodeficiency, panels, including serum liver enzymes levels, might suggest organ
polyendocrinopathy and enteropathy, X-linked (IPEX) syndrome, compromise as a result of infections or autoimmunity associated
and WAS; chronic periodontitis in defects of the neutrophils; with immunodeficiency. Low protein levels suggest malnutrition
or silvery hair, pale skin, and photophobia in Chediak-Higashi and conditions associated with protein losses, which may cause
syndrome. Investigation for Shwachman-Bodian-Diamond hypogammaglobulinemia. Examination of the posteroanterior
syndrome (SBDS) should be considered in patients with neu- and lateral chest radiographs to look for a thymic shadow can be
tropenia, especially if they also present with skeletal dysplasia. helpful because its absence suggests impaired T-cell development.
Patients with DiGeorge syndrome and nuclear factor-κB (NF-κB) This is especially useful in infants because the thymus mass
essential modulator (NEMO) deficiency present with characteristic normally involutes with age. In addition, the thymus may shrink
facies. in response to such stresses as surgery, infection, or high-dose
Children with severe immune defects are small for their age, steroid treatment.
with growth delay secondary to recurrent infections. Hepato- HIV infection can be ruled out by screening with measurement
splenomegaly and diffuse lymphadenopathy might suggest HIV of anti-HIV antibodies, by the enzyme-linked immunosorbent
infection or a disorder of immune dysregulation. Children with assay (ELISA) or the rapid HIV test. In those individuals with
leukocyte adhesion defect (LAD) can present with severe gingi- suspected humoral immunity defect and in children younger
vostomatitis and dental erosion as a consequence of abnormal than 18 months of age, a polymerase chain reaction (PCR)–based
leukocyte function (Fig. 32.3). Multiple scars from skin abscesses test to detect HIV viremia should be performed to avoid false-
might suggest neutrophil defects, and scarred tympani with negative results and confounding maternal anti-HIV antibodies,
reduced hearing might indicate a history of recurrent otitis media, respectively.
which can be associated with antibody deficiency.
Immunology Testing
LABORATORY TESTING FOR IMMUNE FUNCTION Specific immunological testing is guided by clues obtained from
the history and physical examination and common screening
Results from commonly ordered tests might provide with a great laboratory tests.
deal of information about the immune system. The complete
blood count (CBC) with differential and platelet determination Serum Immunoglobulin Levels
is ordered to quantitate the total white blood cell (WBC) count The levels of IgG, IgA, IgE, and IgM can be measured in serum.
and total numbers of neutrophils, lymphocytes, eosinophils, The IgA level is especially helpful in that it is low in all permanent
and platelets. Abnormal counts should be determined by using types of agammaglobulinemia and in selective IgA deficiency.

CHaPTEr 32 Approach to the Evaluation of the Patient With Suspected Immunodeficiency 455



KEY CONCEPTS normally present in titers greater than 1 : 10; individuals with
Diagnosis of Immune Deficiencies poor antibody production may have low or absent titers. Specific
IgG antibody production can be measured following immuniza-
Features of Congenital antibody Deficiencies tion with protein antigens, such as toxoids derived from Tetanus
• Free of infections until 6–9 months of age, when maternal antibodies and Diphtheria organisms, and polysaccharide antigens, such as
that passed through the placenta to infant are below protective levels those produced by pneumococci and Haemophilus influenzae.
• Severe infections with bacterial organisms, especially sinusitis, otitis For pneumococcal immunization, there are two vaccines that
media, and pneumonias caused by encapsulated bacteria, such as need to be differentiated. The conjugated vaccine containing 13
Streptococcus pneumoniae pneumococcal serotypes (PREVNAR13, Wyeth) is currently
included in the universal schedule of immunizations for infants
Features of Congenital T-Cell Immunodeficiency and toddlers and induces a robust, T cell–dependent immune
• Onset of thrush, diarrhea, and failure to thrive in the first months of response. The unconjugated 23-valent pneumococcal polysac-
life
• Severe infections with opportunistic microorganisms, such as Pneu- charide vaccine (Pneumovax, Merck) is available for immunization
mocystis jiroveci, Candida albicans, adenovirus, cytomegalovirus (CMV), to adults and children aged 2 years and older. The immune
Epstein-Barr virus (EBV). “BCG-itis” in areas where Bacille Calmette- response for this vaccine is considered less dependent on T cells
Guérin (BCG) immunization is mandatory and also less lasting than the conjugated vaccine. The pneumococ-
• Absolute and relative lymphopenia cal antigen challenge using the unconjugated vaccine is not
recommended for children under 2 years of age because healthy
Screening Tests for Suspected Immunodeficiency children are not thought to reliably respond to the unconjugated
• Evaluation for neutropenia, lymphopenia, thrombocytopenia, and/or pneumococcal antigen at this age. However, this view has been
small platelets
• Immunoglobulin levels and specific antibodies to childhood challenged by data showing that 1-year-old children produce
13,14
immunizations normal antibody responses to this unconjugated vaccine.
• Lateral chest radiography in infants for thymus shadow Normal antibody responses are usually demonstrated with an
• Consider flow cytometry to quantify T cells, T-cell subsets, B cells, over twofold rise in specific antibody levels within 2–3 weeks
and natural killer (NK) cells (especially in infants) for protein antigens and within 4–6 weeks for polysaccharide
• Measurement of CH50 activity antigens. Patients with agammaglobulinemia are expected not
15
• Test for oxidative burst in phagocytes
to produce antibody responses, whereas others, such as those
with IgG2 subclass deficiency and normal levels of total IgG,
may only have difficulty with antibody production following
IgE level measurement is of significance for the diagnosis of immunization with polysaccharide antigens. Patients with selective
HIES. Serum IgG subclasses levels can be determined. However, IgA deficiency, alone or with transient hypogammaglobulinemia
rather than using IgG subclass levels to screen for immunode- of infancy, have normal specific IgG antibody production, by
ficiency, they are best utilized when patients have clinical condi- definition. The pneumococcal serotypes included in the current
tions associated with specific antibody deficiencies but normal conjugated antipneumococcal vaccine, serotypes 1, 3, 4, 5, 6A,
total IgG levels. In some of these patients, IgG subclass deficiency, 6B, 7F, 9V, 14, 18C, 19A, 19F, and 23F, were estimated to be
particularly IgG2 and IgG3 deficiencies, might be present. IgG2 responsible for approximately 90% of invasive pneumococcal
16
subclass deficiency has been linked with selective IgA deficiency disease in children less than 5 years of age worldwide. Previous
and deficiency of antipolysaccharide antibodies. IgA subclass immunization with the conjugate vaccine does not preclude use
low levels, IgA1, IgA2, have not been associated with a specific of the unconjugated pneumococcal polysaccharide vaccine. The
immune defect, and there is no validity for measuring these. 23-valent polysaccharide vaccine provides the potential for
The variation of normal ranges of human serum Igs with age stimulation and measurement of a protective immune response
is an important consideration in children, since IgA and IgG to additional 11 serotypes (2, 8, 9N, 10A, 11A, 12F, 15B, 17F, 20,
subclass levels may not reach normal adult reference ranges until 22F, 33F) not included in the conjugated vaccine. Testing for
6 years of age. 1 antibodies against serotypes not included in the two vaccines
and comparing the antibody titers in the pre- and postimmuniza-
B-Cell Function: Specific Antibody Production tion blood samples helps in the assessment of specific increase
To properly assess B-cell function, specific antibody production of particular antiserotype antibody titers as a response to the
must be measured. Patients with normal Ig and Ig subclass levels vaccine administration.
might exhibit deficient antigen-dependent antibody responses.
An initial screen of antibody production may involve the quan- Evaluation of Cellular Immunity
tification of isohemagglutinins. Isohemagglutinins occur in all Cellular immune function had been historically screened with
individuals except those with blood type AB; isohemagglutinins the use of the delayed-type hypersensitivity (DTH) skin tests.
are natural IgM antibodies to polysaccharide blood group antigens DTH reactions occur 48–72 hours after antigen exposure, although
A and/or B, which are not expressed in the red blood cells (RBCs) antigens used to test DTH can occasionally produce immediate
of the patient tested. Individuals form isohemagglutinins as a hypersensitivity reactions. The clinical response and time of
result of environmental exposure to ubiquitous antigens that observation were to discriminate between immediate and delayed
share epitopes with blood antigens. Children less than 1 year of reactions. DTH reactions involve the production of local edema
age do not reliably have measurable serum isohemagglutinins and vasodilation as a result of inflammatory cytokines secreted
because of the limited exposure to the environment. A patient by antigen-specific T cells, followed by lymphocyte infiltration
with blood type A should have anti-B IgM; patients with blood and maximal induration 48 hours after antigen intradermal
type B should have anti-A IgM; and patients with blood type O injection of an antigen. Generally, DTH skin tests are performed
should have both anti-A and anti-B IgM. These antibodies are using vaccine antigens or microbial agents to which the patient

456 ParT FOur Immunological Deficiencies



10 3 10 3
A1 A2 A1 A2


10 2 10 2



CD4 PE 10 1 CD4 PE 10 1




10 0 A3 A4 10 0 A3 A4





10 0 10 1 10 2 10 3 10 0 10 1 10 2 10 3
A CD3 FITC B CD3 FITC
FIG 32.4 Histograms of Fluorescently Stained Lymphocytes. The quadrant of interest, A2,
shows lymphocytes that are positive for labeling with both fluorescein isothiocyanate (FITC)–tagged
monoclonal antibodies specific for CD3 and phycoerythrin (PE)–tagged monoclonal antibodies
(mAbs) specific for CD4. The histogram on the left shows normal fluorescence as a result of
CD4 T lymphocytes present in quadrant A2. The histogram on the right shows absence of CD4
T lymphocytes in an infant with SCID.



has had previous exposure. Commonly used antigens include provide support for the diagnosis of an specific immunodeficiency.
tetanus toxoid, mumps, and extracts from Candida albicans and For example, the proportion of class-switched B cells has a
Trichophyton spp. The purified protein derivative (PPD), or predictive value for autoimmune and granulomatous complica-
tuberculin, can serve as a negative control in most patients in tions in CVID.
developed countries with a low incidence of tuberculosis. Likewise, In flow cytometry, the fluorescence intensity corresponding
a positive PPD result indicates sensitivity to mycobacteria as to cells labeled with each specific antibody is obtained (Fig. 32.4)
well as robust cellular immunity. Virtually all children and adults and the percentage of the specific lymphocyte subset can be
with previous exposure should respond to at least one antigen estimated. A reference range is available for each subset defining
in a panel of tetanus toxoid, mumps, and C. albicans. Anergy, or normal values as those whose values fall between the fifth and 95th
nonresponse to the antigen following previous exposure, may percentages for this population. Separate ranges should be used
indicate a cellular defect. A nonresponder should be retested for children because infants and children generally have higher
with an in vitro lymphocyte evaluation. absolute numbers of T-cell subsets and higher percentages of CD4
Lymphocyte subset enumeration. Quantitation of B- and T cells (Fig. 32.5 and Appendix 2). Nonimmune factors, such as
T-cell subsets narrows the differential diagnosis and provides age, gender, and adrenocorticoid levels, influence the expression
evidence for the diagnosis of combined, cellular, or antibody of blood lymphocyte subset populations. Therefore interpretation
immunodeficiency (Chapters 34, 35, 93, 94, 95,-96). Both T and of lymphocyte phenotyping should take into consideration the
B cells can be identified and labeled by using flow cytometry and clinical status of the patient. For example, transient moderate
fluorescent monoclonal antibodies (mAbs) (Chapter 92). T-cell lymphopenia with predominance of T cells and NK cells might
enumeration involves the use of a pan–T cell mAb specific for be seen in patients admitted to intensive care units. HIV infection
CD3. The CD4 marker serves as identification for T-helper (Th) causes progressive depletion of CD4 T cells. These abnormalities
cells. CD8 marker characterizes cytotoxic T cells. B cells can be resolve when the patient’s condition improves.
identified by using mAbs against the cell surface markers CD19 Lymphocyte functional analysis. To test lymphocyte function
or CD20. Natural killer (NK) cells can be identified by using in the laboratory, mitogen- and antigen- induced lymphocyte
mAbs against CD16 and CD56. Specialized clinical laboratories proliferation or transformation studies are performed (Chapter
are available to measure lymphocyte markers of importance to 93). For these studies, lymphocytes are stimulated to proliferate
particular diseases; for instance, the proportion of αβ T-cell involving new DNA synthesis and cell division. Lymphocytes
+
receptor (αβTCR) and γδTCR double-negative CD3 T cells are from immunized or previously exposed individuals will normally
of relevance in the diagnosis of autoimmune lymphoproliferative proliferate in response to antigens to which they are sensitized.
syndrome (ALPS). T cell subsets can be characterized as naïve This response in vitro correlates with the in vivo DTH response.
or activated based on the expression of CD45RA and CD45RO Mitogens, such as concanavalin A (ConA), phytohemagglutinin
antigens. (PHA), and pokeweed mitogen (PWM), stimulate proliferation of
B-cell panels and NK-cell panels. These panels have been normal T cells, as can allogeneic histocompatibility antigens when
designed to characterize the maturation stage of these cells and leukocytes from two donors are mixed in culture. Proliferation

CHaPTEr 32 Approach to the Evaluation of the Patient With Suspected Immunodeficiency 457


P1009: CD4 Oxidase activity can be detected by a flow cytometry assay
8000 measuring the oxidation of dihydrorhodamine (DHR) 123 in
19
phagocytes, resulting in fluorescent rhodamine-123. The
nitroblue tetrazolium (NBT) test measures oxidative burst activity
6000
as well, but it is a more subjective test and can miss the diagnosis
of CGD. For patients with suspected LAD-1 deficiency, neutrophils
Counts 4000 are labeled with mAb directed against the adhesion molecule
CD11/CD18 heterodimer. Absence of fluorescence intensity
indicates lack of expression of the adhesion molecule. In addition,
2000 an increase of fluorescence intensity after stimulation can be
documented in normal individuals, indicating the normal
20
0 upregulation of this molecule after cell activation. Other labora-
0 5 10 15 tory techniques used to identify phagocytic defects include assays
for chemotaxis and bactericidal activity. A major pitfall for
Age (years) neutrophil studies is the spontaneous cell activation that might
FIG 32.5 Change in Distribution of Peripheral Blood CD4 occur in vitro when cells are not tested within a few hours of
T-Cell Subsets With Age in Healthy Children. Scatter plot when the sample was drawn, resulting in artifactual values that
indicates peripheral blood CD4 T-cell counts (cells/µL) by age, might falsely suggest poor function.
with lowest curves in healthy children from birth to 18 years of
age. (From Shearer WT, et al. Lymphocyte subsets in healthy Complement
children from birth through 18 years of age: The Pediatric AIDS Laboratory tests for complement components include tests for
Clinical Trials Group P1009 study. J Allergy Clin Immunol 2003; functional activity of the classical pathway with a CH50 assay
112: 973, with permission from Elsevier.) and the alternative pathway with an AH50 assay, as well as
immunochemical methods to measure complement component
21
levels. The CH50 evaluation tests the ability of fresh serum
from the patient to lyse antibody-coated sheep erythrocytes.
This reflects the activity of all numbered components of the
of lymphocytes can be evaluated by the demonstration of cell classical complement pathway, C1–C9, and terminal components
division or by increased DNA synthesis reflecting this cell process. of the alternative complement pathway. A total deficiency of one
Increased DNA synthesis is monitored by the incorporation of of the classical complement pathway components will result in
radiolabeled nucleotides, usually tritiated thymidine, in culture a CH50 assay approaching zero (Chapter 21). Patients with
media. A measure of the amount of radioactivity in the cells corre- complement deficiency are rare, and complement test abnormali-
lates with DNA synthesis. Other assays to assess mitogen-induced ties are often transient because of increased consumption or
cell proliferation measure deoxybromouridine incorporation, activation. It is usually recommended that that in case of an
change in pH, or adenosine triphosphate (ATP) concentration abnormal result, the complement test be repeated if the sample
of the culture media. These assays are being increasingly used as was taken when the patient had an acute illness. Quantitative
surrogate markers of cellular immunity; however, a comparison tests for components C3 and C4 are utilized in testing for comple-
with the traditional assay based on radiolabeled nucleotide is ment deficiencies and for evaluation of complement activation
not available. Of note, a flow cytometry assay that measures cell (Chapter 21).
division with the use of carboxyl fluorescein succinyl ester (CFSE),
a fluorescent compound that distributes evenly in cells and specific Innate Immunity: Interferon-γ Levels, Toll-Like
17
antibodies, is also increasingly used in clinical immunology. Receptor Assay
CFSE is distributed equally in dividing cells, and each progeny The importance of the many components of innate immunity
cell has half the fluorescence intensity of CFSE compared with are increasingly recognized, as single gene defects in this immune
the parent cell, providing the basis to identify these dividing compartment have been found to cause susceptibility to specific
22
cells. After mitogen or antigen stimulation, mononuclear cells infections. For example, patients with defects in the proteins
can be stained with specifically labeled antibodies, allowing the that are part of the interferon-γ (IFN-γ) receptor may have
identification of cell subsets that proliferate. elevated serum IFN-γ levels, even when there is no infection to
explain these levels. The IFN-induced response associated kinase
Phagocytes 4 (IRAK4) defect, observed with susceptibility to pneumococcal
The laboratory evaluation of a patient with a suspected phagocyte infection, might be accompanied with abnormal Toll-like receptor
deficiency (Chapters 22, 94) should always begin with a CBC. (TLR) assay responses. It should be noted that the clinical value
Neutropenia is the most frequently encountered disorder of the of most of these innate immunity tests as screening or diagnostic
18
phagocyte system. Neutrophilia, at values exceeding those tools for immune defects has not been clearly established.
associated with acute infection, is a common finding in LAD The testing of lymphocyte apoptosis in a patient who may
type 1 (LAD-1). Abnormalities of WBC function involve difficulty have ALPS and the evaluation of NK-cell function for suspected
with adherence, locomotion, deformability, recognition, attach- familial hemophagocytic lymphohistiocytosis are examples of
ment, engulfment, phagosome formation, phagocytosis, degranu- specific functional assays that suggest immunodeficiency syn-
lation, microbial killing, and elimination of engulfed material. dromes. Many patients with increased frequency of infections
Clinical assays to evaluate neutrophil function are limited in may not have abnormal results in clinically available immunologi-
number. CGD is diagnosed by demonstrating absent or markedly cal testing, which may not give clear evidence of a secondary
reduced oxidase activity in neutrophils in response to stimulation. etiology in the medical evaluation. In these difficult cases, referral

458 ParT FOur Immunological Deficiencies


to tertiary care and research centers for investigation of rare the clinical presentation of these disorders. The medical history,
diseases is recommended. particularly the frequency, severity, and etiology of infections,
is most helpful to orient the diagnostic workup. Commonly
MOLECULAR TESTING FOR PRIMARY ordered tests in primary care, such as a CBC and serum Ig levels,
IMMUNE DEFECTS are helpful to support possible diagnosis and referral to the clinical
immunologist. Immunological testing according to clues obtained
Molecular testing for specific PIDs is available through commercial from the medical history helps narrow the differential diagnoses
23
and research laboratories. Biochemical and genetic testing should to specific immunodeficiencies, which are confirmed by molecular
be considered. If autosomal recessive SCID is suspected, the methods. Description of new T-cell subsets (e.g., Th17 and
adenosine deaminase (ADA) and purine nucleoside phosphorylase regulatory T cells [Tregs]) has helped explain the immunopatho-
(PNP) enzyme activities in the RBCs should be determined. genesis of certain clinical manifestations, such as the occurrence
White blood cells must be used to measure the activity of these of autoimmunity in patients with combined immunodeficiency,
enzymes in recently transfused individuals, since donor RBCs and “cold abscesses” in the autosomal dominant HIES. Testing
will elevate the enzyme activity in deficient patients. AT has the for these lymphocyte phenotypes is being integrated in the clinical
consistent laboratory finding of elevated alpha-fetoprotein (AFP) evaluation. Identification of genetic defects can now be accom-
levels along with variable abnormalities in B- and T-cell function. plished by increased availability of whole exome sequencing, as
Nearly 300 defective genes and gene products have been identified an alternative to genetic analysis of candidate genes. Technological
24
to result in congenital immunodeficiency syndromes. In these advances are making molecular diagnosis available for most
cases, the diagnosis can be confirmed with molecular genetic patients with immunodeficiency conditions.
analysis (Chapter 33). For example, gene mutations in BTK leading
to the absence of Bruton tyrosine kinase (BTK) results in arrest
of B-cell development at the pre–B cell stage in congenital X-linked ON THE HOrIZON
agammaglobulinemia. Similarly, abnormal T-cell development • Early diagnosis of severe combined immunodeficiency (SCID) and
leading to SCID results from mutations in at least 15 genes, non-SCID is the key to successful hematopoietic stem cell transplanta-
including IL2RG and JAK3. Patients with gene mutations that tion (HSCT).
may result in disease but have not been investigated need to be • Development of DNA sequence analysis for >300 types of primary
immunodeficiency (PID).
carefully evaluated to demonstrate the pathogenic nature of the • Wide availability of whole-genome sequencing methods to determine
genetic change. Some genetic changes do not have clinical sig- molecular defects for difficult to diagnose immunodeficiency
nificance and are known as single nucleotide polymorphisms syndromes
(SNPs). Standard protocols using Southern, Northern, and
Western blot analyses, PCR analysis, and DNA sequence analysis
are helpful to identify affected patients, affected fetuses prenatally, ILLUSTRATIVE CASES
and carriers of genetic mutations (Chapter 96). Most recently,
the use of whole exome sequencing for immunodeficiency Case 1
syndromes has facilitated the identification of new genes causing A 6-month-old Caucasian female infant was brought with a
immunodeficiencies by examining all known gene exons without history of rash and otitis media that had been recurrent since
bias, and simultaneously. This methodology for diagnosis is 2 weeks of age. The infant had poor weight gain, frequent spitting
particularly helpful when the clinical presentation does not match up, coughing spells, and persistent diarrhea. Maternal HIV testing
any of the already described immunodeficiency syndromes. 25 showed negative results. Physical examination revealed an emaci-
ated infant without palpable lymphoid tissue and severe oral
CONCLUSIONS thrush. Cystic fibrosis was ruled out by using genetic studies.
Stool viral cultures were persistently positive for rotavirus.
Immunodeficiency was suspected because of the infant’s failure
CLINICaL PEarLS to thrive, persistent chronic diarrhea, and recurrent infection.
Management of Immunodeficiency Patients An evaluation of the immune system was performed (Table
32.3). Humoral immunity testing showed Ig levels below or
• Lymphopenia is a hallmark of T-cell immunodeficiency in infancy. just above the lower limit of normal. Isohemagglutinins were
Unexplained lymphopenia should be recognized and evaluated.
• Normal range for immunoglobulin levels and lymphocyte counts varies not tested as the patient was less than 1 year old. As she had not
with age; age-matched controls should be used for interpretation. been immunized, specific antibody titers to vaccines were not
• Survival and morbidity outcomes of hematopoietic stem cell transplanta- tested. Stool α 1 -antitrypsin levels were normal, suggesting that
tion (HSCT) for severe combined immunodeficiency (SCID) are best protein was not being lost in stool, and urinalysis did not show
when performed in the first 4 months of age. protein loss. CBC revealed profound lymphopenia and absolute
• Delays in treatment of immunodeficiency leads to infections causing neutropenia. The neutropenia had resolved upon subsequent
permanent damage to organ systems, such as the lungs, where evaluations, but the lymphopenia persisted. A thymic shadow was
bronchiectasis or bronchiolitis obliterans may develop secondary to
recurrent pneumonias. not present on the chest radiograph. Evaluation of lymphocyte
• Implementation of universal screening of newborn infants by DNA subsets by flow cytometry revealed that the CD4 and CD8
analysis of dried blood spots on Guthrie cards (T-cell receptor excision T-cell and B-cell numbers were all markedly low. The patient’s
circle [TREC]) detect all T-cell deficiencies. lymphocytes had no proliferative response when stimulated with
phytohemagglutinin. The history and the physical examination,
The approach to the patient with suspected immune deficiency along with laboratory values, including low Ig levels and markedly
requires knowledge of developmental pathways and function of low lymphocyte numbers with poor proliferative response to
the different compartments of the immune system, as well as mitogen stimulation, strongly suggested the diagnosis of SCID.

CHaPTEr 32 Approach to the Evaluation of the Patient With Suspected Immunodeficiency 459



TABLE 32.3 results of Screening Tests and Special Evaluations of Illustrative Cases*
CaSE NuMBEr
Test 1 2 3 4
antibody Function
Serum immunoglobulin (mg/dL)
IgG 235 (208–686) Normal 41 (256–1067) 748 (520–1340)
IgA <8 (10–62) Normal 7 (12–103) 40 (25–81)
IgM <7 (43–183) Normal 14 (47–173) 78 (64–275)
Isohemagglutinins ND ND Negative Normal
Functional antibody tests
Antitetanus ND Normal Undetectable 0.28
Antidiphtheria ND Normal Undetectable 0.12
Anti-Pneumococcus ND 2 of 14 protective ND Normal

Lymphocyte Numbers
Lymphocyte count (×10 cell/µL) 0.217 (2.5–16.5) 2.8 (2.3–5.4) 2.56 (4.0–13.5) 10.60 (3.0–9.5)
3
CD4 number (×10 cell/µL) 0.009 (1.6–4.0) 1.27 (0.9–2.4) 1.72 (1.4–4.3) ND
3
3
CD8 number (×10 cell/µL) 0.008 (0.5–1.7) 1.5 (0.5–1.5) 0.73 (0.5–1.7) ND
3
CD19 number (×10 cell/µL) 0.010 (0.3–2.0) 0.023 (0.4–1.4) 0.107 (0.6–2.6) ND
Lymphocyte Proliferation (counts/min)
PHA, Phytohemagglutin 546 (55494) Normal 210973 (177195) ND
ConA, Concanavalin A ND Normal 124680 (143591) ND
PWM, Pokeweed mitogen ND Normal 162646 (138684) ND
Complement Function
Total hemolytic complement (U/mL) ND ND 618 (300–500) <100 (300–500)
Phagocyte Function
Neutrophil count (×10e3 cell/mL) 0.109 (1.0–9.0) 0.570 (1.5–8.5) 3.3 (1.0–8.5) 4.2 (1.5–8.5)
Monocyte count (×10e3 cell/mL) 0.546 Normal 0.11 0.65
Oxidative activity (DHR) ND Normal Normal ND

*Normal values are reported in parentheses. Normal values may vary from laboratory to laboratory as a result of different techniques used and may vary according to patient age.
ConA, concanavalin A; DHR, dihydrorhodamine; ND, not done; PHA, phytohemagglutinin; PVVM, .


The patient had low levels of ADA and increased levels of the being fully immunized. Lymphocyte phenotyping revealed low
toxic nucleotide metabolites. SCID as a result of ADA deficiency B cells and NK cells. A diagnosis of Schwachman-Bodian-Diamond
was diagnosed. syndrome (SBDS) was made on the basis of the patient’s unique
Following ADA enzyme replacement therapy and Ig infusions, clinical manifestations. Genetic sequencing analysis of the SBDS
the patient thrived and had fewer infections. Her lymphocyte gene identified one mutated allele that had not been previously
numbers and function improved. reported, and molecular investigations for chromosomal breakage
defects, other skeletal genetic syndromes, and mitochondrial
Case 2 defects showed negative results. The patient was treated with
A 4-year-old female child was presented with a history of short pancreatic enzyme replacement, antibiotic prophylaxis, and close
stature and frequent upper and lower respiratory infections. hematological follow-up.
Family history was not contributory. Physical examination was
remarkable for height and weight less than the fifth percentile Case 3
for age and a significant chest deformity (Fig. 32.6). Radiological A 12-year-old Caucasian male child had a history of diarrhea
examination demonstrated metaphyseal dysplasia and pectum that lasted for 1 month, nuchal rigidity, left facial weakness,
carinatum, with significant bilateral lung atelectasis. Hematological and a left sixth cranial nerve palsy when he was 8 months of
evaluation showed severe neutropenia, a normal lymphocyte age, although he had been well for the first 6 months of life. The
count, and mild thrombocytopenia (see Table 32.3). Examination family history was negative for immunodeficiency. The patient
of bone marrow revealed mild dysplasia with the presence of had received his childhood immunizations, including the live
three lineages and no evidence of chronic viral infections. Evalu- polio virus vaccine. Physical examination revealed absence of
ation for failure to thrive was significant for low levels of pancreatic tonsillar tissue, and the patient had no palpable lymph nodes.
enzymes. A sweat test was done to screen for cystic fibrosis, and The presenting neurological symptoms resolved, but the patient
the result was normal. subsequently developed a lower-extremity flaccid paralysis that
Immunological evaluation showed normal serum levels of has persisted.
IgG, IgA, and IgM, with mildly increased IgE levels. Specific Immunodeficiency was suspected because of the chronicity
antibody responses to childhood vaccines were protective against of the illness, the lack of lymphoid tissue on physical examination,
tetanus, diphtheria, and H. influenzae antigens, and to only two and the finding of an unusual infectious agent causing severe
of 14 pneumococcal serotypes were measured despite the child sequelae to infection. The age at presentation was typical for

460 ParT FOur Immunological Deficiencies





















A B





















C
FIG 32.6 A 4-Year-Old Girl Presenting With Congenital Neutropenia, Small Stature, and
Pancreatic Insufficiency. Genetic sequencing analysis confirmed diagnosis of Schwachman-
Bodian-Diamond syndrome. (A) Pectus carinatum. (B) Chest computed tomography (CT) scan.
(C) Bone marrow biopsy (hematoxylin and eosin stain; original magnification, ×100). (From Shah
SS, et al. Diagnosis of primary immunodeficiency: let your eyes do the talking. J Allergy Clin
Immunol 2009;124:1363–4, with the permission from Elsevier.)


patients with a defect in humoral immunity. Immune system diagnosed immunodeficiency, although two siblings had a history
evaluation (see Table 32.3) showed a normal absolute neutrophil of recurrent otitis media and bronchitis. The physical examination
count and a low absolute lymphocyte count. The CH50 was revealed normal tonsillar tissue and the presence of small lymph
mildly elevated; this result ruled out a complement component nodes in the cervical region.
deficiency from the differential diagnosis. Ig levels were low. Laboratory values (see Table 32.3) included normal numbers
Antibody levels to diphtheria and tetanus were very low. A thymus of granulocytes and lymphocytes. Serum Ig levels were normal.
shadow was present on the chest radiograph. Lymphocyte evalu- The patient had been immunized and had adequate antibody
ation showed normal numbers and percentages of T cells and titers to tetanus, diphtheria, and Pneumococcus antigens. The
absent numbers and percentages of B cells. Proliferative responses CH50 level was below the lowest measurable level. C3 and C4
of lymphocytes to mitogens were normal. Molecular testing levels were normal, but the level of C2 was 0. The patient’s father,
showed a mutation in the BTK gene. The CSF was remarkable mother, and brother had levels of C2 that were half the normal
for lymphocytic pleocytosis, but the protein and glucose levels value, consistent with being heterozygous for an abnormal C2
were normal. Stool and throat cultures revealed vaccine-strain allele. C2 deficiency was diagnosed. The patient has had chronic
poliovirus. The patient was diagnosed with vaccine-acquired sinusitis and bronchitis despite antibiotic prophylaxis. C2
polio virus infection and X-linked agammaglobulinemia. deficiency is most frequently associated with autoimmune disease;
The patient receives monthly Ig replacement therapy and is this patient has not had autoimmune disease and undergoes
otherwise well. yearly surveillance examinations.
Case 4
The patient was a 3-year-old girl who was hospitalized for Please check your eBook at https://expertconsult.inkling.com/
pneumonia. Her past medical history was significant for recurrent for self-assessment questions. See inside cover for registration
otitis media and bronchitis. The family history was negative for details.

CHaPTEr 32 Approach to the Evaluation of the Patient With Suspected Immunodeficiency 461


REFERENCES 14. Balloch A, Licciardi PV, Russell FM, et al. Infants aged 12 months can
mount adequate serotype-specific IgG responses to pneumococcal
1. Yel L. Selective IgA deficiency. J Clin Immunol 2010;30:10–16. polysaccharide vaccine. J Allergy Clin Immunol 2010;126:395–7.
2. Kwan A, Abraham RS, Currier R, et al. Newborn screening for severe 15. Orange JS, Ballow M, Stiehm ER, et al. Use and interpretation of
combined immunodeficiency in 11 screening programs in the United diagnostic vaccination in primary immunodeficiency: a working group
States. JAMA 2014;312:729–38. report of the Basic and Clinical Immunology Interest Section of the
3. Boyle JM, Buckley RH. Population prevalence of diagnosed primary American Academy of Allergy, Asthma & Immunology. J Allergy Clin
immunodeficiency diseases in the United States. J Clin Immunol Immunol 2012;130:S1–24.
2007;27:497–502. 16. Johnson HL, Deloria-Knoll M, Levine OS, et al. Systematic evaluation of
4. UNAIDS. AIDS by the numbers 2015. Joint United Nations Programme serotypes causing invasive pneumococcal disease among children under
on HIV/AIDS (UNAIDS) and World Health Organization (WHO); 2015. five: the pneumococcal global serotype project. PLoS Med 2010;7:pii:
5. Agarwal S, Mayer L. Diagnosis and treatment of gastrointestinal disorders e1000348.
in patients with primary immunodeficiency. Clin Gastroenterol Hepatol 17. Rakha A, Todeschini M, Casiraghi F. Assessment of anti-donor T cell
2013;11:1050–63. proliferation and cytotoxic T lymphocyte-mediated lympholysis in living
6. Goronzy JJ, Weyand CM. Understanding immunosenescence to improve donor kidney transplant patients. Methods Mol Biol 2014;1213:355–64.
responses to vaccines. Nat Immunol 2013;14:428–36. 18. Henry M, Sung L. Supportive care in pediatric oncology: oncologic
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and the increased risk of bronchitis, bronchiolitis and other lower North Am 2015;62:27–46.
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Respir Res 2011;12:5. Am 2013;27:89–99.
8. Shearer WT, Fleisher TA, Buckley RH, et al. Medical Advisory Committee 20. Etzioni A. Defects in the leukocyte adhesion cascade. Clin Rev Allergy
of the Immune Deficiency Foundation, Recommendations for live viral Immunol 2010;38:54–60.
and bacterial vaccines in immunodeficient patients and their close 21. Grumach AS, Kirschfink M. Are complement deficiencies really rare?
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9. Marciano BE, Huang CY, Joshi G, et al. BCG vaccination in patients with approach. Mol Immunol 2014;61:110–17.
severe combined immunodeficiency: complications, risks, and vaccination 22. Rosenzweig SD, Holland SM. Recent insights into the pathobiology of
policies. J Allergy Clin Immunol 2014;133:1134–41. innate immune deficiencies. Curr Allergy Asthma Rep 2011;11:369–77.
10. Silverberg JI, Silverberg NB. Childhood atopic dermatitis and warts are 23. https://www.genetests.org/tests/.
associated with increased risk of infection: a US population-based study. J 24. Picard C, Al-Herz W, Bousfiha A, et al. Primary Immunodeficiency
Allergy Clin Immunol 2014;133:1041–7. Diseases: an Update on the Classification from the International Union of
11. Guerra IC, Fawcett WA, Redmon AH, et al. Permanent intrinsic B cell Immunological Societies Expert Committee for Primary
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predictors. Clin Immunol 2010;137:374–83.

CHaPTEr 32 Approach to the Evaluation of the Patient With Suspected Immunodeficiency 461.e1


M u LTIPLE-CHOICE Q u ESTIONS

1. Which of the following diagnoses is most likely to present C. Neisseria spp.
with Pneumocystis jiroveci pneumonia during the first year D. Mycobacterium tuberculosis
of life? E. Cryptosporidium parvum
A. Partial DiGeorge syndrome 3. Which of the following conditions is MOST likely to contribute
B. Human immunodeficiency virus (HIV) infection to frequent respiratory infections?
C. Common variable immunodeficiency (CVID) A. Adolescence
D. Chronic granulomatous disease (CGD) B. Peanut allergy
E. Complement deficiency
C. Dust mite allergy
2. Deficiencies of terminal component of the complement system D. Homeschooling
is associated with frequent or severe infections caused by: E. Protein-rich diet
A. Candida spp.
B. Epstein-Barr virus

33









Human Genomics in Immunology



Robert L. Nussbaum, Jennifer M. Puck






The completion of the Human Genome Project in 2003, 50 years challenges to completing the human genome sequence are posed
after the landmark 1953 publication of the double-helical structure by regions that contain segmental duplications of nearly the same
of DNA by James Watson and Francis Crick, is correctly deemed sequence. 1
a major milestone in modern biology. The sequence provided
a first comprehensive and accurate view of the genetic makeup HUMAN VARIATION
of humans, with a low error rate and only a few hundred gaps
of indeterminate sequence. The first detailed picture of the The first publicly available human genome sequence was con-
structure of a composite human genome is for human genetics structed from a small number of individuals. Nonetheless, the
what Vesalius’s publication of a consensus structure of the human “reference” human genome sequence available at public websites
body, De humani corporis fabrica, was for anatomy. And, like is a consensus, composite haploid sequence and not the sequence
Vesalius’s work, it continues to serve as a foundation for further of any one individual. It is neither a “normal” genome nor a
scientific discovery in such areas as genetic variation, gene func- “control” genome; instead, it serves as a reference, providing
tion, human physiology, and the genetic basis for disease. The a universally available sequence against which the genomes of
years following the completion of the first human genome other individuals, as well as other species, can be compared and
sequencing have seen progress in all of these areas. any differences (“variants”) determined. Even before the original
human genome sequence was completed, the need to discover
KEY CONCEPTS as broad a range of human variations as possible in populations
from around the world was clearly recognized to be essential if
The sequence of the human genome and catalogue of extensive genome
variation among humans has revolutionized our approach to heritable we were to begin to understand how variations in the genome
immune disorders. lead to differences in phenotypic variations in traits and disease
Genomic variation between humans and other species illuminates susceptibilities. The first concerted efforts to catalogue human
conserved areas that are critical for normal function of gene genetic diversity after completion of the Human Genome Project
products. was the dbSNP (database of single nucleotide polymorphisms)
A variety of types of DNA variation are recognized, including single project, followed by the 1000 Genomes project. These initial
nucleotide changes, insertions or deletions of a few to many nucleo-
tides, copy number variants, and structural variants. catalogues that surveyed the extent of variation in both the
Interpretation of the significance of an observed variant in DNA sequence coding and the noncoding portions of the genome have been
may require consideration of its location, frequency in the population, supplemented enormously by more comprehensive sequencing
inheritance in a family, and specific effect on the resulting gene product. efforts, which produced the NHLBI GO Exome Sequencing
Project (ESP) and Exome Aggregation Consortium (EXaC).
These projects focus on variation within exomes of hundreds
GENOME ANNOTATION of thousands of individuals and have made a vast number of
variants and their frequency publicly available to researchers
A consensus sequence of the human genome is only the first and clinicians worldwide.
step in furthering our understanding of normal biological Variants can be classified as rare or common (polymorphic)
functions and how mutations lead to abnormal functions that according to their frequency in a population under study. Any
cause disease. The Human Genome Project has now matured variant present in >2% of the population (i.e., constituting >1%
into a number of important basic and applied research areas: (i) of the alleles at any locus in that population) is arbitrarily
acquiring a comprehensive catalogue of human variation and designated as polymorphic. With the advent of comprehensive
the impact of such variation on phenotype, including disorders DNA sequencing of larger and larger numbers of people from
of human development; (ii) comparing the genomes of humans across the globe, it has become clear that most variants (85%)
with those of other organisms, including model organisms and at any one base pair (bp) or segment of the genome have allele
human ancestors; and (iii) learning how to interpret all the frequencies substantially below the 1% cutoff for being a poly-
sequence elements within the genome, not just the codons. morphism and are, instead, considered rare, sometimes restricted
Even now, over a dozen years after “completion” of the human to a single ethnic group or even a single kindred. 2
genome sequence, a complete, accurate, and single contiguous DNA variation can also be classified according to the type of
stretch representing a reference human haploid genome is DNA change seen in the variant (Table 33.1). Single nucleotide
still being constructed, and updated versions of the genome variants (SNVs), insertion/deletion variants (indels), copy number
sequence continue to be released. As described below, the greatest variants (CNVs), and structural variants (SVs) can have different

463

464 ParT FOUr Immunological Deficiencies



TABLE 33.1 Types of DNa Variation
Type Description ability to Detect Frequency in an Individual
Single nucleotide variants A sequence change where, compared with a reference High 3–4 000 000
(SNVs) sequence, one nucleotide is substituted for another
nucleotide
Deletion or duplication A sequence change involving between 2 and ~1000 base Moderate 1 000 000?
variants (Del/Dups) pairs (bp) in which reference nucleotides are either not
present (deleted) or are duplicated and inserted directly 3’
of the original copy of the sequence
Insertion/deletion variants A sequence change involving between 2 and ~1000 bp in Moderate
(Indels) which one or more reference nucleotides are replaced by
one or more other nucleotides and is not an SNV or SV
Copy number variants (CNVs) Del/Dup or Indel arbitrarily set as larger than ~1000 bp Difficult
Structural Variants (SVs) Very difficult
From Human Variome Society, Sequence Variant Nomenclature. 2016. Available at: http://varnomen.hgvs.org.


consequences, depending on their location and the number and AAATAAAT … AAAT. The units are repeated between two and
identity of nucleotides affected. a few dozen times at a particular site in the genome. Some
The simplest and most common of all variants are SNVs, in variation in microsatellite length is common enough to constitute
which one nucleotide in the reference sequence is substituted a polymorphism, defined as an allele or alleles other than the
by another. A locus characterized by an SNV usually has only reference sequence, found in ≥2% of the population. Many tens
two alleles, corresponding to the more common (major allele) of thousands of polymorphic microsatellites are known to exist
and less common (minor allele) bases found at that particular throughout the human genome. There are, however, STR DNA
location in the genome, although, theoretically, four alleles at segments present within exons or splice junctions of >90% of
any one base position are possible. SNVs are observed on average genes associated with human disease. They rarely, but famously,
once every 1000 bp in the genome but are not distributed evenly can expand to become hundreds or thousands of nucleotides
throughout and are most often not equally frequent throughout long, thereby causing such human disorders as fragile X syndrome
all populations. Most SNVs are found in the 98% of genomic or Huntington disease. Even without expansion, STRs within
sequence that is not within exons, including the ~20% of the exons, some of which may be as small as 9–25 bp, have an outsized
genome inside genes, in introns, with the remaining ~80% impact on the frequency of human disease, since they confer a
between genes. Nonetheless, many SNVs are in coding portions five- to sixfold increase in the frequency of rare disease-causing
of genes and other known functional elements in the genome. indel mutations compared with neighboring exon sequences that
Just under half of these do not alter the predicted amino acid do not contain an STR. 5
sequence of the encoded protein and are thus termed synonymous, One subclass of indel variants arises from mobile elements.
whereas the remainder do alter the amino acid sequence and Nearly half of the human genome sequence consists of families
are said to be nonsynonymous. Other SNVs introduce or change of repetitive elements dispersed throughout the genome, of
a stop codon, and yet others alter a known splice site; such SNVs which the two most common are the Alu (a short interspersed
are expected to have significant functional consequences. nuclear element [or SINE]-usually about 300 bp) and LINE (long
A second general class of variation is the result of insertion interspersed nuclear element) families of repeats. Although most of
and/or deletion of reference sequence ranging from 1 up to an the copies of these repeats are stationary, some of them contribute
arbitrary cutoff of ~300–1000 bp, that is, a variant size that can to human genetic diversity through retrotransposition, a process
be detected by the most commonly used next-generation sequenc- that involves insertion of a DNA segment generated through
ing (NGS) technology. When reference nucleotides are simply transcription of an Alu or LINE element into an RNA that is then
deleted or duplicated, the variant is referred to as a “del/dup.” reverse-transcribed into a DNA sequence that is inserted into the
When reference sequence has some nucleotides deleted and genomic DNA. Each mobile element indel consists of two alleles,
replaced by other inserted sequence, the variant is referred to as one with and one without the inserted mobile element. Mobile
an “indel.” Approximately half of all del/dups are referred to as element polymorphisms are found on all human chromosomes;
“simple” because they have only two alleles—that is, the presence although most are found in nongenic regions of the genome,
or absence of the inserted or deleted segment. Each individual a small proportion exist within genes. At least 5000 of them
is known to carry hundreds of thousands of indels, but this are known to be frequent enough to be polymorphisms, and
estimate is suspected to be too low, and a corrected estimate for insertion frequencies of >10% occur in various populations; other
3
indels per individual may be upward of a million. Up to 30–40% mobile elements are rare and have been implicated in causing
of actual indels are likely to be missing from catalogues because insertional mutations in human disease genes. As with other
of the technical difficulties of distinguishing many indels from indels, difficulties with sequencing, in particular the challenges
sequencing errors using the current sequencing technologies. 4 posed to NGS by repetitive DNA, result in underestimation of
Some del/dup variants are multiallelic because of variable Alu and LINE indels throughout the genome. 3
numbers of the identical segment of DNA inserted in tandem Another important type of human variation includes CNVs.
at a particular location, thereby constituting what is referred to These consist of variations in the number of copies of segments
as a microsatellite (also referred to as a short tandem repeat [STR]). of the genome that are larger than those involved in indels and
Microsatellites are segments of DNA composed of units of 2, 3, range in size from 1000 bp to many hundreds of kilobase (kb)
or 4 nucleotides, such as TGTG …TG, CAACAA …CAA, or pairs. Variants larger than 500 kb are found in 5–10% of

CHaPTEr 33 Human Genomics in Immunology 465


individuals in the general population, whereas variants encom- of DNA in the offspring of inversion carriers because of aberrant
6
passing more than a million bases (1 Mb) are found in 1–2%. recombination during meiosis, leading to serious syndromes
Smaller CNVs, in particular, may have only two alleles (i.e., the brought about by chromosomal imbalance. Furthermore, if
presence or absence of a segment), similar to indels. Larger CNVs inversions interfere with normal gene expression by disrupting
tend to have multiple alleles because of the presence of different a gene or altering the physical relationship between a gene and
numbers of copies of a segment of DNA in tandem. The content its regulatory elements, it may be detrimental to health. 9,10
of any two human genomes can differ by as much as 50–100 Mb
as a result of copy number differences at CNV loci, compared CLINICAL IMPACT OF HUMAN VARIATION
with some 3–4 Mb of differences because of SNVs and indels.
Thus simply on the basis of the number of nucleotides involved One of the greatest challenges facing human geneticists is linking
11
in the variation, CNVs represent vastly more human variation variation to phenotype. The significance for the health of the
than do SNVs or indels. vast majority of variants of any type is unknown, and yet this
Not only are CNVs a very significant contributor to human knowledge is essential if we are to apply genomics to clinical
7
variation and disease, the areas of the genome where they are care. The impact of variants ranges from completely benign to
8
found are often the sites of segmental duplications, some of the highly pathogenic, the latter causing devastating disorders of
most difficult regions in which to develop an accurate reference the immune system that may occur as new mutation dominants
sequence. Segmental duplications are ubiquitous throughout the or as autosomal recessively inherited syndromes. Even common
genome. It is difficult to construct an accurate contiguous genome polymorphic variants may affect health or longevity, although
sequence in regions of segmental duplications because the most their being common means that they are likely to produce a
widely used massively parallel sequencing technologies generate relatively subtle alteration of disease susceptibility rather than
12
only short reads of a few hundred base pairs and therefore may not directly cause a serious illness. Working out the functional
be able to differentiate between the sequences of nearly identical impact of human variation will occupy genomics researchers
DNA segments using unique DNA that flanks them. The assembly for many years to come. An essential component of this work
algorithms, which always try to create the simplest assembly is to make databases of genetic variants and their impact on
possible, may consider two nearly identical segments of DNA that human health available to the research and clinical communities,
have been duplicated in the genome, but differ at a few locations as is being done with the ClinVar database hosted by the National
in their sequence, to be the same sequence and collapse them Library of Medicine in the United States and the Leiden Open-
into a single segment of DNA. The fixed sequence differences source Variation Database (LOVD). 13-15
between the duplicated segments are incorrectly interpreted as
polymorphic differences between two alleles of a nonduplicated COMPARATIVE GENOMICS
locus instead of being real sequence differences between different
copies of duplicated DNA. The fact that there is a segmental Evolution at work is nowhere better illustrated than in the field
duplication is therefore overlooked. Conversely, the sequence of comparative genomics, which deals with similarities in the
of a unique DNA segment with many SNVs compared with the sequence, structure, and chromosomal location of genes between
reference may be incorrectly interpreted as having come from different species whose evolutionary paths diverged from a few
distinct but highly similar duplicated segments, leading to the years ago to many hundreds of millions of years ago. Direct
supposition that there are paralogues or duplicated segments. As sequence comparison has revealed that an enormous number of
a result, segmental duplications are liable to both false-negative human proteins have orthologues (genes derived from a common
and false-positive representations in the human genome assembly. ancestor) in other organisms, ranging from 87% in chimps to
CNVs are particularly difficult to detect and interpret if the 79% in mice, 63% in zebrafish, 39% in the fruit fly (Drosophila
number of segments in the reference is incorrect or ambiguous. melanogaster), and 31% in the nematode (Caenorhabditis elegans)
Many CNVs include genes, from one up to several dozens; (Fig. 33.1). The study of the human genome and the genomic
thus CNVs are frequently implicated in diseases that result from basis for human disease has benefited from studies done in other
7
altered gene dosage. One well-known human immunological organisms, particularly mice, in which many decades of inbreeding
multisystem disorder, DiGeorge syndrome, is caused by a deletion and gene manipulation have permitted study of the genes and
CNV that occurs de novo in about 1 in 5000 individuals. When underlying mechanisms by which mutations responsible for
a CNV is more frequent, it represents a background of common immune defects lead to phenotypes. Mouse models of immunity
polymorphism that must be understood if to properly interpret have been highly relevant and informative for humans.
alterations in copy number observed in patients. As with all DNA However, orthologous genes may serve different functions in
variations, the significance of different CNV alleles in health and different species; therefore one cannot assume that a disease-
in disease susceptibility is the subject of intensive investigation. causing mutation in humans will cause a similar defect when
The most common SVs in the human genome sequence are its orthologue is similarly mutated in mice, and vice versa. For
inversions, which differ in size from a few base pairs to large example, the V(D)J recombination activating genes RAG1 and
regions of the genome (up to several Mb) that can be present RAG2 in humans and Rag1 and Rag2 in mice appear to have
in either of two orientations in the genomes of different individu- identical functions, with knockout mice showing the same inability
8
als. As with CNVs, inversions are usually flanked by regions of to recombine T- and B-lymphocyte antigen receptor genes as
sequence homology, which indicates that recombination of these humans with RAG1 or RAG2 deficient severe combined immu-
16
homologous regions is likely involved in the origin of the inver- nodeficiency (SCID; Ch. 35). As a result, the lymphocyte profile
7,8
sions. Most inversions do not involve gain or loss of DNA; in in both species is absent T and B cells with normal natural killer
− −
+
this case, the two alleles corresponding to the two orientations (NK) cells, referred to as T B NK SCID. A contrasting situation
can achieve substantial frequencies in the general population. occurs in other SCID genotypes. For example, humans lacking
Such inversions can, however, cause significant gains or losses the common γ chain (γc) of receptors for interleukin-2 (IL-2)

466 ParT FOUr Immunological Deficiencies


and other cytokines, caused by mutations in the X-linked gene are highly prone to developing autoimmunity, while others such
IL2RG have SCID in which T and NK cells are absent but as C57BL/6 (B6) are resistant. 18
nonfunctional B cells are present in normal to high numbers,
− +
−
T B NK SCID. Mice with the orthologous gene Il2rg mutated FUNCTIONAL GENOMICS
or removed can make T cells but have no B cells, which gives
−
+ −
them a T B NK phenotype. 17 Immediately after the Human Genome Project was declared to
Genes other than IL2RG alone must be responsible for this have been completed in 2003, an important follow-on project
difference between species. Such genes, known as modifiers, have was launched to identify the functional segments of DNA,
not yet been identified. Notably, different strains of mice can particularly portions in the 98–99% lying outside of the coding
also have important phenotypic differences in the presence of exons of genes, for which there was no simple sequence code
a single gene mutation under study; some strains, for example, that could be understood the way the triplet codon code can be
nonobese diabetic (NOD) and Murphy Roths Large (MRL) mice read. This project, termed ENCODE (for “encyclopedia of DNA
elements”), began as a pilot project to identify functional elements
in 1% of the genome, but it was quickly expanded to include
100% the entire human genome as well as the genomes of model
90% 19
80% organisms. Before ENCODE, estimates were that 3–8% of the
70% human genome had some role in function, given that this fraction
60% of the genome appeared to be highly conserved among species
50% with only very limited variation. This estimate was clearly too
40% low, as it did not take into account rapidly evolving functional
30% elements or those restricted to particular evolutionary lineages.
20% This estimate also did not include segments of DNA that were
10% too small to show conservation with statistical significance, nor
0% did it include the functional elements in repetitive DNA that
Chimp Dog Mouse Rat Chicken Pufferfish Zebrafish Fruitfly Mosquito C. elegans Rice Amoebae E. coli are not reliably scored as being evolutionarily conserved.
Bee
C. cerevisiae
Mustard weed
Since the same genome is present but functions differently
FIG 33.1 Homology Between Human Genes and Genes in in different cells of an individual, ENCODE used a number of
different tissues for its studies (Fig. 33.2). A comprehensive
Multiple Other Organisms. Comparisons were made by calculat- catalogue of every segment of DNA that is transcribed into RNA
ing gene homology or similarity between reference protein in any tissue, including all splice isoforms, was required, including
sequences from human and another species, with homology RNAs that code for a protein or are noncoding and function in
considered present when the probability of an interspecies match gene regulation. ENCODE is analyzing not only total whole-cell
-30
by chance alone was <10 . (Reproduced with permission from RNAs but also those located in the nucleus or cytosol because
Nussbaum R. Human genomics and development. In: Epstein subcellular localization of RNAs is important in how RNA is
CJ, Erickson RP, Wynshaw-Boris A, editors. Inborn errors of processed and functions. Other assays for functionality of seg-
development. New York: Oxford University Press; 2008). ments of DNA include biochemical evidence, such as identifying




Hypersensitive CH
sites 3
RNA
polymerase

CH CO
3
CH 3

WGBS Computational
5C DNase-seq ChIP-seq RRBS predictions and RNA-seq CLIP-seq
ChIA-PET FAIRE-seq methyl450k RT-PCR RIP-seq



Genes


Long-range regulatory elements Promoters Transcripts
(enhancers, repressors/
silencers, insulators)
FIG 33.2 Diagram of Various Transcriptional and Chromatin Assays Being Used in the
ENCODE Project. The assays can be used to identify regions of the genome involved in the
regulation of gene expression. (With permission from https://www.encodeproject.org.)

CHaPTEr 33 Human Genomics in Immunology 467


(i) segments of DNA located in chromatin loops that allow
chromatin–chromatin interaction; (ii) regions of open chromatin,
which are accessible to transcription; (iii) motifs that bind
transcription factors; (iv) regions that are associated with histones A A a
that have been modified to either promote or suppress transcrip- b Y b B
tion; and (v) regions with differential methylation of cytosine
residues in different tissues, with methylation being associated
with inactivity. *
Now, approximately 15 years into the project, ENCODE has * *
created a large database of more than 4 million functional regions
of the genome. Mostly on the basis of biochemical evidence, it
has been estimated that 80% of the genome may be of functional a A A A a A A A a a A A
20
significance, but this estimate is probably too high and has b Y b b b B B Y b Y b b B Y b b
been strongly criticized. For example, evidence of transcription FIG 33.3 An Example of Family-Based Linkage Analysis as
of a segment does not necessarily mean that the transcript plays a Means to Identify a Disease Gene. The pedigree of a family
any functional role, nor is it likely to be true that the millions with X-linked severe combined immunodeficiency (SCID) caused
of vestigial repetitive DNA sequences that are undergoing muta- by an IL2RG mutation in the proximal long arm of the X chromo-
tion without any apparent selection could have important some (marked with black bar) is shown. Affected males have
21
functional roles. A more stringent threshold for ascribing a filled in square; female carriers are designated by dot in the
functional role to DNA segments (i.e., direct effect on gene center of their symbols. Two loci, one with alleles A and a, and
expression and phenotype of at least one human cell type) reduces the other with alleles B and b, are shown in the proximal and
that fraction to the range of 10–20%. Much more research will distal long arm of the X (Xq). There are no recombinations during
be needed before the ENCODE project delivers its final assessment female meiosis between allele a and the IL2RG mutation in any
of the fraction of the human genome that plays a role in gene of the eight children, while three children (marked with asterisk)
regulation—that is, in how different cells use their genomes. 22
show a recombination with the locus on the distal Xq. The
father’s X chromosome is passed on without any recombinations
APPLYING HUMAN GENOMICS TO to each of his daughters and is shown on the left of each pair
of X chromosomes in the daughters.
UNDERSTANDING DISORDERS OF
THE HUMAN IMMUNE SYSTEM

Genome research has provided geneticists with a catalogue responsible for association to have been carried along, on the
of all known human genes, knowledge of their location and same chromosome in a haplotype block, through many genera-
structure, and an ever-growing list of variants in DNA sequences tions (Fig. 33.4). Association analysis does not require pedigrees
found among individuals in different populations. In the past, and is particularly useful for complex diseases that do not show
geneticists followed two approaches to identifying the genetic strict mendelian inheritance.
basis for human disorders. The first approach, linkage analysis, Linkage analysis and association studies have limitations in
is family based (Fig. 33.3). Linkage analysis takes advantage of investigating the genetic basis for human immunological disorders.
family pedigrees to follow the inheritance of a disease among Linkage analysis is problematic if the disorder is a rare autosomal
family members and test a few hundred DNA variants distributed recessive condition such that there are not enough families with
throughout the genome for consistent, repeated coinheritance, two carrier parents to enable such a study; nonetheless, the
or segregation, with the disease. A demonstration of significant increased frequency of consanguineous matings in some popula-
coinheritance with a variant or variants located in a particular tions has been utilized to overcome this limitation. 23,24 Another
region of the genome indicates that the disease-causing mutation challenge with linkage analysis is if the disorder is genetically
is also located in a gene within or near this region. The variants lethal so that it is never inherited and always occurs sporadically
showing coinheritance with the disease are usually not the variants as a result of a new mutation. Detecting an association in a
responsible for the disorder. Marker variants need, however, to case-control study is also a problem when the frequency of any
be located close enough that recombination between the marker particular allele associated with the disease is too low among
and the gene mutation responsible for the disease is sufficiently the cases to give a detectable association. For example, if the
rare that cosegregation is observed over a few generations. disorder arises from different, independent mutations and if
The second approach, genome-wide association analysis these mutations are found on many different haplotypes in
(GWAS), is population based. A sample of affected individuals, affected individuals, it may be very difficult to establish a sig-
or “cases,” taken from the population, is chosen along with a nificant association with any one variant.
matched set of unaffected “control” individuals from the same When linkage and association studies are not possible, as in
population. Then, a large number, in the order of a million or very rare mendelian disorders, a third approach is now available—
more, variants are examined individually for an increased or genome sequencing. Vastly improved methods of DNA sequencing
decreased frequency of cases compared with controls. The alleles have cut the cost of sequencing six orders of magnitude over
used to test for association need not be the actual variants what was spent generating the Human Genome Project’s reference
functionally responsible for the disease association—in fact, it is sequence, opening up new possibilities to discover the genes and
highly unlikely this would be the case. Instead, GWAS, like linkage, mutations responsible for rare mendelian disorders. One can
depends on the vast number of marker alleles that can be tested generate a whole-genome sequence (WGS) or, in what has often
for association to be located close enough to the alleles functionally proven a cost-effective compromise, a sequence of only about

468 ParT FOUr Immunological Deficiencies



Mutation on founder chromosome X

X X X X

Fragmentation of original
chromosome by recombination X X X
as population expands through
multiple generations
X X X
X
X X X X
X
X X X X
X
X X X X
X
X X X

X X X X

Mutation located within region of
linkage disequilibrium

A B
FIG 33.4 An Example of a Population-Based Genome-Wide Association Analysis–—As a
Means to Identify a Disease Gene. (A) A mutation (X) that predisposes to a disease first occurs
on a chromosome with a certain set of alleles at polymorphic loci along that chromosome (symbol-
ized by the blue color). With each generation, meiotic recombination exchanges the alleles that
were initially present at polymorphic loci on the “blue chromosome” for other alleles present
on homologous chromosomes (symbolized by other colors). Over many generations, the only
alleles that remain associated with the mutation are those at loci so close to the mutant locus
that recombination between them is very rare. These alleles constitute a disease-associated
haplotype. (B) Affected individuals in the current generation (arrows) carry the mutation (X) are
enriched for the disease-associated haplotype (individuals in blue) compared with unaffected
individuals. Depending on the age of the mutation and other population genetic factors, a disease-
associated haplotype ordinarily spans a region of DNA of a few kilobases to a few hundred
kilobases. (From Nussbaum R, McInnes RR, Willard HF. Thompson and Thompson Genetics in
medicine. 8th ed. Toronto, Canada: Elsevier Canada; 2016: 177, Fig. 10.8, with permission.)



2% of the genome, the part containing the exons of genes, referred in the immunodeficiency and dwarfing syndrome cartilage
to as a whole exome sequence (WES). hair hypoplasia). However, these are currently more difficult
As an example of what is now possible, suppose that there is to assess, and thus, as a simplifying assumption, it is reasonable
a family “trio” consisting of a child affected with a rare immu- to focus initially on protein-coding genes.
nodeficiency and his or her parents. WES is carried out on the 2. Population frequency. Keep rare variants from Step 1 and
child and the parents, yielding typically over 4 million SNV, discard common variants with allele frequencies <0.05 (or
indel, and CNV differences in the child compared with the human some other arbitrary number between 0.01 and 0.1) because
genome reference sequence. Which of these variants would be common variants are highly unlikely to be responsible for a
responsible for the disease? The extraction of useful information disease whose population prevalence is much less than the
2
from such a massive amount of data relies on creating a variant q predicted by the Hardy- Weinberg equilibrium.
filtering scheme based on a variety of reasonable assumptions 3. Deleterious nature of the mutation. Keep variants from Step
about likely responsible explanations for the disease. 2 that cause nonsense or nonsynonymous changes in codons
One example of a filtering scheme that can be used to sort within exons, cause frame-shift mutations, or alter highly
through these variants is shown in Fig. 33.5, in which exome conserved splice sites. Discard synonymous changes that have
sequence was performed for two parents and their offspring, no predicted effect on protein function (unless there is reason
two affected and one unaffected. to suspect that they influence splicing or expression, such as
1. Location with respect to protein-coding genes. Keep variants the last nucleotide of an exon, which is typically “G”).
that are within or near exons of protein-coding genes and 4. Consistency with likely inheritance pattern. If the disorder is
discard variants deep within introns or intergenic regions. It considered most likely to be autosomal recessive, keep any
is possible, of course, that the responsible mutation might variants from Step 3 for which an affected child has 2 variants
lie in a noncoding RNA gene (e.g., RMRP, the gene mutated in the same gene and each parent has one of the variants.

CHaPTEr 33 Human Genomics in Immunology 469


FILTERS or in an animal model with the disease? Finding mutations in
one of the candidate genes also in other patients would confirm
that this gene was the responsible gene trio under study. In some
Known 1° cases, one gene remaining on the list in Step 4 may become a
Recessive or
Sequence immuno- leading candidate because its involvement makes biological or
quality MAF≤0.05 de novo deficiency genetic sense or it is known to be mutated in other affected
pedigree
genes individuals. In other cases, however, the gene responsible may
turn out to be entirely unanticipated on biological grounds or
17613 14353 88 2
protein high 3901 recessive variants in may not be mutated in other affected individuals because of
altering quality rare or de novo one gene-> locus heterogeneity (i.e., mutations in other as yet undiscovered
variants variants variants variants ZAP-70 genes can cause a similar disease).
3260 10452 3813 86 Such variant assessments require extensive use of public
genomic databases and software tools. These include the human
genome reference sequence, databases of variants with their allelic
frequencies, software that assesses how deleterious an amino
acid substitution might be to gene function, collections of known
disease-causing mutations, and databases of functional networks
Set aside and biological pathways. The enormous expansion of this
FIG 33.5 Variant Filtering Can Be Used to Sort Through Whole- information over the past few years has played a crucial role in
Exome Sequence Analysis of a Familial Disease. Protein-altering facilitating gene discovery of rare mendelian disorders.
variants detected in the affected children, their unaffected sibling, In a recent report of the clinical use of WES in rare disorders,
25
and parents were first filtered by sequencing quality and allele 2000 individuals with a variety of disorders that had escaped
frequency to yield a set of 88 rare, damaging or nonsynonymous diagnosis despite thorough conventional clinical evaluations
variants that fit an autosomal recessive inheritance pattern and underwent WES. A likely causative mutation or pair of mutations
were then candidates for causing the autoimmune disorder. This was found in 504 (25.2%). Of these mutations (of which many
group was then further analyzed for genes involved in immune were severe developmental defects), half were de novo, not present
function, which brought two variants in ZAP-70, for which the in either parent. Also of interest was that 23 (4.6%) of these 504
affected children were both compound heterozygotes, to the patients in whom a diagnosis was made through WES had two
top of the list as possible causative mutations. (Adapted from different genetic disorders, resulting in a combined phenotype,
Chan et al. A novel human autoimmune syndrome caused by most likely obscuring the diagnosis of either of the individual
combined hypomorphic and activating mutations in ZAP-70. J disorders had they been present alone.
Exp Med 2016;213:155–65, with permission.) In some cases, the difficulty in making a diagnosis was the
result of there being a mutation in a gene, previously unknown
to cause human disease, but implicated by finding mutations
The child need not be homozygous for the same deleterious that fit the inheritance pattern (de novo dominant or autosomal
variant but could be a compound heterozygote for two different recessive), and then later confirmed by finding other mutations
deleterious mutations in the same gene. in phenotypically similar patients. In others, the gene involved
In the example in Fig. 33.5, the two affected children were had been previously described, but not in association with the
compound heterozygous for variants, with each parent and the phenotype for which the patient was undergoing diagnostic
unaffected sister carrying one of the variants. If there were exome analysis. For example, two children affected with an
consanguinity in the parents, the candidate genes and variants undiagnosed autoimmune syndrome consisting of early-onset
might be further filtered by requiring that the child be a true bullous pemphigoid, nephritis, autoimmune antifactor VIII
homozygote for the same mutation derived from a single common hemophilia, and inflammatory bowel disease. WES was performed
ancestor. If the autosomal recessive mode of inheritance is correct, for both children, their unaffected sister, and both unaffected
26
then the parents should both be heterozygous for the variants. parents. Eighty-eight genes were found in which the two affected
The disease-causing variant in a male could also be X-linked, children were compound heterozygotes for two different rare
in which case any variant found in an X-linked gene for which mutations, whereas the unaffected sister and both parents were
the mother is a heterozygote would be a candidate. carriers for only one of the two mutations found in the children.
For either the autosomal or X-linked case, the disorder could Of these, only one gene was known to have any immunological
be the result of a new dominant mutation; in this case, keep function: ZAP70, previously known to be mutated in combined
variants from Step 3 that are de novo changes in the child and immunodeficiency but not with strictly autoimmune disease of
are not present in either parent. this type (see Fig. 33.5). Both affected children were compound
In the end, millions of variants can often be filtered down to heterozygotes for two missense mutations, p.R192W and p.R360P,
a handful of SNVs, indels, or CNVs affecting a reasonable number in ZAP70. The mother carried the R192W allele, whereas the
of genes. Once the filtering reduces the number of genes and father and unaffected sister carried the R360P allele. Neither
alleles to a manageable number, they can be assessed individually mutation had been reported previously, nor were they near
for other characteristics. Do any of the genes have a known mutations in the same gene that had been previously associated
function or tissue expression pattern that would be expected if with human disease. Functional studies revealed the p.R192W
it were the potential disease gene? Is the gene already implicated had reduced activity, whereas the R360P encoded a modestly
in other disease phenotypes, or does it have a role in pathways hyperactive protein because of disruption of an autoinhibitory
in which mutations are known to cause similar or different mechanism. The combination of these alleles, as proven in labora-
phenotypes? Finally, is the same gene mutated in other patients tory studies, fully explained the autoimmune phenotype.

470 ParT FOUr Immunological Deficiencies



ON THE HOrIZON 6. McCarroll SA, et al. Common deletion polymorphisms in the human
genome. Nat Genet 2006;38:86–92.
As the costs of deep sequencing fall, examination of whole-genome 7. Stankiewicz P, Lupski JR. Structural variation in the human genome and
sequence should allow for examination of noncoding regulatory regions. its role in disease. Annu Rev Med 2010;61:437–55.
Data storage and computer speed will need to be improved to handle 8. Carvalho CM, Lupski JR. Mechanisms underlying structural variant
50-fold more data from each individual. formation in genomic disorders. Nat Rev Genet 2016;17:224–38.
New analytical paradigms for the analysis of variation in noncoding DNA 9. Lakich D, et al. Inversions disrupting the factor VIII gene are a common
and the interpretation of DNA variants that are not clearly associated cause of severe haemophilia A. Nat Genet 1993;5:236–41.
with deleterious effects will be required. 10. Puig M, et al. Human inversions and their functional consequences. Brief
Proving causality of new gene variants in primary immunodeficiencies Funct Genomics 2015;14:369–79.
will depend on future cataloguing of deleterious mutations in each 11. Amendola LM, et al. Performance of ACMG-AMP Variant-interpretation
gene and on molecular studies of the direct effects of the variants. guidelines among nine laboratories in the Clinical Sequencing
Exploratory Research Consortium. Am J Hum Genet 2016;99:247.
Since the application of WGS or WES to rare mendelian 12. Tryka KA, et al. NCBI’s Database of Genotypes and Phenotypes: dbGaP.
disorders was first described in 2009, hundreds of such disorders Nucleic Acids Res 2014;42:D975–9.
have been studied and the causative mutations found in nearly 13. Landrum MJ, et al. ClinVar: public archive of interpretations of clinically
relevant variants. Nucleic Acids Res 2016;44:D862–8.
27
500 previously unrecognized disease genes. The pace of discovery 14. Harrison SM, et al. Using ClinVar as a resource to support variant
will only increase, and we can anticipate that the number of interpretation. Curr Protoc Hum Genet 2016;89:8.16.1–23.
known mendelian disorders, including those characterized by 15. Fokkema IF, et al. LOVD v.2.0: the next generation in gene variant
primary immunodeficiency and/or autoimmunity, will grow over databases. Hum Mutat 2011;32:557–63.
coming years. It has been predicted that the genetic basis for 16. Revy P, et al. The repair of DNA damages/modifications during the
every human single gene disorder will be elucidated in the near maturation of the immune system: lessons from human primary
future. However, the genetic basis for complex disorders that immunodeficiency disorders and animal models. Adv Immunol
show increased familial occurrence, and yet are not single gene 2005;87:237–95.
disorders and do not follow mendelian inheritance, remains 17. Kovanen PE, Leonard WJ. Cytokines and immunodeficiency diseases:
elusive and will require another leap in technology and genetic critical roles of the gamma(c)-dependent cytokines interleukins 2, 4,
7, 9, 15, and 21, and their signaling pathways. Immunol Rev 2004;202:
analytical tools to be fully unraveled. The data being generated 67–83.
through human and model organism genomics will certainly 18. Choi Y, Simon-Stoos K, Puck JM. Hypo-active variant of IL-2 and
aid in the elucidation of these more challenging disorders of the associated decreased T cell activation contribute to impaired apoptosis in
human immune system. New tools available to immunologists autoimmune prone MRL mice. Eur J Immunol 2002;32:677–85.
and geneticists will open great opportunities for discovery and 19. Diehl AG, Boyle AP. Deciphering ENCODE. Trends Genet 2016;32:
understanding of genetic disorders of immunity which, in turn, 238–49.
will deepen our knowledge of immune system networks. 20. ENCODE Project Consortium. An integrated encyclopedia of DNA
elements in the human genome. Nature 2012;489:57–74.
Please check your eBook at https://expertconsult.inkling.com/ 21. Doolittle WF. Is junk DNA bunk? A critique of ENCODE. Proc Natl Acad
Sci USA 2013;110:5294–300.
for self-assessment questions. See inside cover for registration 22. Eddy SR. The ENCODE project: missteps overshadowing a success. Curr
details. Biol 2013;23:R259–61.
23. Al-Herz W. Primary immunodeficiency disorders in Kuwait: first report
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1. Steinberg KM, et al. Single haplotype assembly of the human genome 24. Al-Herz W, et al. Parental consanguinity and the risk of primary
from a hydatidiform mole. Genome Res 2014;24:2066–76. immunodeficiency disorders: report from the Kuwait National Primary
2. Gonzaga-Jauregui C, Lupski JR, Gibbs RA. Human genome sequencing in Immunodeficiency Disorders Registry. Int Arch Allergy Immunol
health and disease. Annu Rev Med 2012;63:35–61. 2011;154:76–80.
3. Jiang Y, Turinsky AL, Brudno M. The missing indels: an estimate of indel 25. Yang Y, et al. Molecular findings among patients referred for clinical
variation in a human genome and analysis of factors that impede whole-exome sequencing. JAMA 2014;312:1870–9.
detection. Nucleic Acids Res 2015;43:7217–28. 26. Chan AY, et al. A novel human autoimmune syndrome caused by
4. Huddleston J, Eichler EE. An incomplete understanding of human genetic combined hypomorphic and activating mutations in ZAP-70. J Exp Med
variation. Genetics 2016;202:1251–4. 2016;213:155–65.
5. Madsen BE, Villesen P, Wiuf C. Short tandem repeats in human exons: a 27. Chong JX, et al. The genetic basis of mendelian phenotypes: discoveries,
target for disease mutations. BMC Genomics 2008;9:410. challenges, and opportunities. Am J Hum Genet 2015;97:199–215.

CHaPTEr 33 Human Genomics in Immunology 470.e1


MULTIPLE-CHOICE QUESTIONS

1. Which of the following variations in a gene expressed in T 2. True or False: A human inherited immunodeficiency caused
lymphocytes is MOST likely to be deleterious, capable of by mutation of single gene can reliably be reproduced in the
causing a human immune disorder if found in homozygosity: mouse by knockout of the orthologous murine gene.
A. A single nucleotide substitution AAA (lysine) ≥ TAA (stop) 3. DiGeorge syndrome is most commonly caused by:
in an early exon of a gene required for T-cell receptor A. A single nucleotide variation
rearrangement B. A copy number variation
B. A single nucleotide substitution CTC (leucine) ≥ ATC C. A retrotransposition
(isoleucine) in a gene encoding a cytokine receptor D. An indel mutation
C. Deletion of 6 amino acids (CAACAA) in a short tandem
repeat (STR) consisting of (CAA) 10 in the 3’ untranslated
portion of a gene expressed in all lymphocytes

34









Primary Antibody Deficiencies



Tracy Hwangpo, Harry W. Schroeder, Jr.







Primary antibody deficiency diseases are characterized by an CLINICAL PEARLS
inability of the humoral immune system to produce sufficient
quantities of protective antibodies to properly protect the host Clinical Manifestations of Antibody Deficiency
1
from hazardous antigens. This inability may be evident from • Recurrent bacterial infections
birth, or it may manifest at a later age. In some cases, the deficiency • Early in untreated disease, infections are primarily caused by
2
may either resolve or worsen with time. Many of these diseases encapsulated pyogenic bacteria (e.g., Streptococcus pneumoniae
are caused by mutations that alter the function of genes that and Haemophilus influenzae type b).
regulate B-cell development or homeostasis. Others reflect • Later in untreated disease, damage to mucosal surfaces engenders
mutations in the immunoglobulin (Ig) genes themselves. In some susceptibility to staphylococci, nontypable H. influenzae, and gram-
negative rods, as well.
of the most common conditions, a genetic predisposition has • Recurrent viral infections
been well documented, but the underlying defect remains unclear. • In most cases, viral infections are cleared normally, but protective
The typical patient will present with a history of recurrent upper immunity does not develop. For example, recurrent shingles can
or lower respiratory infections and reduced serum concentrations be a common symptom in untreated patients.
of one or more classes of immunoglobulin (IgM, IgG, or IgA). • In some cases, patients may continue to excrete virus after resolution
However, patients with normal serum Ig levels may exhibit specific of their clinical symptoms.
deficits in their ability to mount a protective response against • Increased prevalence of other immunological disorders
• Paradoxical increased risk of antibody-mediated autoimmune dis-
certain antigens, and some patients with agammaglobulinemia orders, such as idiopathic thrombocytopenia, autoimmune thyroiditis,
can be remarkably asymptomatic. A classification of primary systemic lupus erythematosus, and celiac disease.
antibody deficiency diseases can be found in Table 34.1. • Lymphoid hypertrophy
Primary immune deficiency disorders are the consequence • Increased risk of allergic disorders, especially among patients with
of specific defects in B-cell development (Chapter 7). B-cell IgA deficiency.
production begins in the fetal liver and shifts to bone marrow
in the latter stages of fetal life (Fig. 34.1). As B cells mature, they
leave the liver and bone marrow and migrate via blood into patients that are deficient in neutrophil function or in the pivotal
secondary lymphoid organs, including spleen, lymph nodes, and third component of complement (C3), all three of these arms
other peripheral and mucosal tissues. Contact with a polymeric of the host defense system should be evaluated in patients who
cognate antigen, such as a polysaccharide, can activate the B cell suffer with recurrent bacterial infections.
and allow it to differentiate into an antibody-producing plasma The clinical course of uncomplicated primary infections with
cell. In contrast, the response to protein antigens, including toxins viruses, such as varicella zoster or mumps virus, does not differ
and viral proteins, requires T-cell help. In the germinal centers, significantly from that of the normal host. However, patients
B cells can replace an upstream heavy (H) chain constant domain with antibody deficiency have difficulty generating long-lasting
with a downstream one (e.g., µ to γ1), altering effector function immunity; thus chickenpox can repeatedly recur as shingles.
(Chapter 15). They can also introduce mutations at a high level The general rule is that T cells typically control established viral
into the variable (V) domains, tailoring the antibody to the infections, whereas antibodies limit initial viral dissemination
antigen, a process termed affinity maturation. and cell entry, thereby preventing reinfection. As with all rules,
there are exceptions. Patients with hypogammaglobulinemia can
CLINICAL MANIFESTATIONS have difficulty clearing hepatitis B virus from the circulation,
poliovirus from the gut, and enterovirus from the brain, leading
Patients with antibody deficiencies most commonly present with a to progressive and sometimes fatal outcomes.
history of recurrent sinusitis, bronchitis, and pneumonia. Patients Because sinopulmonary infections are also commonly seen
can also present with recurrent cellulitis, boils, gastrointestinal in normal infants and children, in individuals with allergies, in
(GI) discomfort, myalgias, arthralgias, fatigue, and depression. smokers, and in patients with other diseases, such as cystic fibrosis,
The respiratory infections typically involve encapsulated bacterial the threshold for an extensive evaluation for immunodeficiency
pathogens, such as Streptococcus pneumoniae and Haemophilus can be a matter of clinical judgment. However, two or more
influenzae. Protection against these bacteria requires production episodes of bacterial pneumonia within a 5-year period, unex-
of antipolysaccharide antibodies, which does not require T-cell plained bronchiectasis, H. influenzae meningitis in an older child
help. Because a similar susceptibility for infection is seen among or adult, chronic otitis media in an adult, recurrent intestinal

471

472 PARt fouR Immunological Deficiencies



TABLE 34.1 Primary Antibody Deficiencies
Disorder Gene or Locus Chromosome
IgA deficiency / Common variable immunodeficiency (CVID) MHC 6p21.3
CVID1: ICOS deficiency (AR) ICOS 2q33
CVID2/IgAD2: TACI deficiency (AD/AR) TNFRSF13B 17p11.2
CVID3: CD19 deficiency (AR) CD19 16p11.2
CVID4: BAFF-R (AR) TNFRSF13C 22q13.1-q13.31
CVID5: CD20 deficiency (AR) CD20 11q13
CVID6: CD81 deficiency (AR) CD81 11p
CVID7: CD21 (AR) CD21 1q32
CVID8: LRBA (AR) LRBA 4q31.3
CVID9: PKCδ (AR) PRKCD 3p21.31
CVID10: NF-κB2 (AD) NFkB2 10q24
CVID11: IL-21 (AR) IL21 4q27
CVID12: NF-κB1 (AD) NFkB1 4q24
CTLA-4 (AD) CTLA-4 2q33
TWEAK (AD) TNFSF12 17p13
P13K (AD) PIK3CD 1p36.2
PI3K regulatory subunit (AD) PIK3R1 5q13.1
BLK BLK 8p23-p22
IRF2BP2 IRF2BP2 1q42.3
IKAROS (AD) IKZF1 7p12.2
Kabuki syndrome (AD) KMT2D, KDM6A 12q13.12, Xp11.2
Transient hypogammaglobulinemia of infancy (THI) Unknown Unknown
X-linked agammaglobulinemia (XL) BTK Xq21.3-q22
X-linked agammaglobulinemia with growth hormone deficiency (XL) BTK Xq21.3-q22
Hyper-IgM syndrome
HIGM1: X-linked hyper-IgM syndrome (XHM) (XL) CD154 or CD40L Xq26
HIGM2: Activation-induced cytidine deaminase deficiency (AR/AD) AID 12p13
HIGM3: CD40 deficiency (AR) CD40 20q12-q13.2
HIGM5: Uracil-DNA glycosylase (UNG) deficiency (AR) UNG 12q23-q24.1
HIGM6: XHM with ectodermal dysplasia (XHM-ED) (XL) NEMO Xq28
Autosomal Agammaglobulinemia (AGM)
AGM1: Immunoglobulin µ H-chain deficiency (AR) IGHG1 14q32.33
AGM2: Surrogate light-chain deficiency (AR) IGLL1/CD179B 22q11.21
AGM3: Ig-associated α(Igα) deficiency (AR) CD79A 19q13.2
AGM4: BLNK deficiency (AR) BLNK 10q23.2
AGM5: LRRC8 truncation (AD) LRRC8
AGM6: Ig-associated β (Igβ) deficiency (AR) CD79B 17q23
AGM7: PI3K regulatory subunit (AR) PIK3R1 5q13.1
AGM8: E47 transcription factor deficiency (AD) TCF3
Myelodysplasia with hypogammaglobulinemia Monosomy 7
Thymoma with immunodeficiency (Good syndrome) Trisomy 8
Selective IgG Subclass Deficiencies
Immunoglobulin γ H-chain deficiencies (AR) IGHG1 14q32.33
Selective κ light-chain deficiency (AR) IGKC 2p12
Selective κ light-chain deficiency (AR) IGLC 22q11.2
Antibody deficiency with normal serum immunoglobulin levels
Vκ A2 deficiency (AR) IGKV2D-29 2p12
Autosomal Dominant (AD), Autosomal Recessive (AR), X-linked (XL)

infections and diarrhea caused by Giardia lamblia, or a family autoimmunity, which is increased among patients with IgA
history of immunodeficiency all warrant evaluation by an deficiency (IgAD), common variable immunodeficiency (CVID),
immunologist. and HIGM1.
The purest forms of antibody deficiency result from mutations Clinical manifestations of the primary immunodeficiency can
that prevent the expression or function of the pre–B-cell receptor also be heavily influenced by the patient’s past medical history.
(Chapter 4). For example, a function-loss mutation of µ heavy A delayed diagnosis and failure to treat infections aggressively
chain or components of the surrogate light chain [VpreB, λ14.1 can lead to permanent damage to the respiratory or GI mucosa,
(λ5)] have been found to affect only the B-cell lineage. However, thus creating susceptibility to nontypable H. influenzae, staphy-
most of the diseases associated with primary antibody deficiency lococci, Pseudomonas spp., and enteric bacteria.
involve more than one cell lineage. For example, X-linked agam-
maglobulinemia (XLA) is the product of loss-of-function muta- PRINCIPLES OF DIAGNOSIS AND TREATMENT
tions in Bruton tyrosine kinase (BTK). Patients with X-linked
hyper-IgM syndrome (HIGM1) may also exhibit T-cell dysfunction, Diagnostic Tests and Their Interpretation
placing them at risk for infection with Pneumocystis jiroveci. Testing for immune deficiency should be performed when the
Immune deficiency also appears to place patients at risk for patient has a history of repeated infections that exceeds

CHAPtER 34 Primary Antibody Deficiencies 473


Antigen dependent

M M
IgM
CVID CVID

XLA M G
Antigen independent
Mψ L M M G IgG
µ D HIGM CVID CVID
TdT E
XLA XLA M
µ 0
IGLL1 0 HIGM E IgE
Pro-B Pre-B Immature Mature CVID CVID
cell cell B cell B cell
HIGM M A
A IgA
IgAD IgAD
CVID CVID
Class Mature Plasma
switching B cell cell
fIG 34.1 Defects in B-Cell Development Can Lead to Humoral Immune Deficiency. X-linked
agammaglobulinemia (XLA) and autosomal agammaglobulinemias (AGMs) result from either a
failure to generate a functional antibody receptor signaling complex or a failure to transmit signals
from this complex. These failures obstruct production of immature B cells. Hyper-IgM syndrome
(HIGM) results from either a failure to engage in proper cognate interactions with T cells or disrup-
tions in the genes that permit class-switch recombination or somatic hypermutation. These
failures inhibit class switching to IgG, IgA, and IgE, as well as limiting affinity maturation. Common
variable immune deficiency (CVID), IgG subclass deficiencies (IgSD), selective IgA deficiency
(IgAD), and transient hypogammaglobulinemia of infancy (THI) reflect a selective or generalized
failure to progress from the immature B-cell stage to the plasma cell stage.



KEY CoNCEPtS C3, C4, and mannose-binding lectin protein [MBL]), a complete
Hypogammaglobulinemia and blood count with differential (CBC/diff), and an erythrocyte
sedimentation rate (ESR). Lymphopenia is found most often in
Antibody Deficiency disorders that affect the production or function of T cells (Chapter
35) but can also occur in patients with CVID. Congenital absence
• The genetic mutations that underlie most primary antibody deficiencies
tend to affect genes that play key roles in the regulation of lymphocyte of an individual complement component will result in total
development and homeostasis. Clinically significant antibody deficiency absence of measurable complement-mediated hemolysis (Chapter
is not synonymous with hypogammaglobulinemia. 21). MBL deficiency increases susceptibility to respiratory infec-
3
• Serum immunoglobulin (Ig) concentrations vary widely with age. tions. The ESR is often, although not always, elevated in individu-
• Serum immunoglobulin levels vary with exposure to drugs (e.g., als with inflammatory disorders and can be useful in the
steroids), infectious agents, and other environmental stressors. evaluation of patients with a questionable or unclear history of
• A complete absence of IgG subclasses resulting from homozygous
deletions of heavy-chain genes has been observed in healthy recurrent or chronic infection.
individuals.
• Absence of a specific Vκ gene segment has been associated with
an increased risk of H. influenzae infection in spite of normal serum KEY CoNCEPtS
Ig levels. Testing for Immune Function

Testing for immune function should be performed:
• When the patient’s history suggests a rate or severity of infection
that exceeds normal expectations
expectations for normal individuals, when an opportunistic • When the organism responsible for infection is of low virulence or is
pathogen or one of a low virulence is responsible for an infection, considered to be an opportunistic pathogen
when a diagnosis of a disorder frequently associated with • When there is a diagnosis of a genetic syndrome or disorder associated
immunodeficiency has been made, or when there is a family with immune deficiency either in the patient or in the patient’s family
history of primary immunodeficiency. Table 34.2 illustrates four
levels of testing complexity and when such testing might com-
monly be undertaken. Interpretation of the significance of the quantitative Ig
Level I testing is both revealing and cost effective. It includes determinations requires appreciation of age-related changes in
4,5
measuring serum Ig (IgM, IgG, and IgA), complement (50% Ig concentrations (Fig. 34.2). At the end of the second trimester
hemolytic power of serum [CH 50 ] and complement components of pregnancy, active transport of IgG across the placental barrier

474 PARt fouR Immunological Deficiencies



TABLE 34.2 Laboratory Diagnosis of Primary Antibody Deficiency
Level test Application(s)
I CBC with differential Primary screening tests
Complement (CH 50 , C3, C4, mannose-binding lectin [MBL] protein)
Erythrocyte sedimentation rate (ESR)
Quantitative serum immunoglobulin (Ig)M, IgG, and IgA levels
Ia Urinalysis, 24-hour urine for protein Symptoms suggest protein loss
Stool for α-1-antitrypsin through kidneys or
gastrointestinal (GI) tract
II B-cell functional evaluation Level I normal but history suggests
Quantitative IgG subclasses, IgE Antibody deficiency
Natural or commonly acquired antibodies (isohemagglutinins, rubella, rubeola, tetanus) Better definition of a level I defect
Response to immunization
T cell–dependent antigens (tetanus)
T cell–independent antigens (unconjugated pneumococcal vaccine, unconjugated H. influenzae
B vaccine)
III Quantification of blood T-and B-cell subpopulations by immunofluorescence assays using Panhypogammaglobulinemia or
monoclonal antibody markers severely low IgM and IgA
T cells: CD3, CD4, CD8
B cells: CD19, CD20, CD21, Ig (µ, δ, κ, λ),
IV Disease-specific analysis Gene-specific diagnosis
Gene expression Genetic counseling
Gene sequencing



1300
IgG
1100 Maternal IgG
Average serum immunoglobulin concentration (mg/dL) 500 Child’s IgG IgA
900
700



300

250
200

150
100

50 IgM
25
0
0 2 4 6 8 2 4 6 810122 4 6 810 1525351255657585
Months Birth Months Years
fIG 34.2 Age-Related Changes in the Serum Concentration of Immunoglobulins (Ig). Shown
4,5
are average serum immunoglobulin concentrations of the major isotypes as a function of age.


begins and, at birth, the infant’s serum IgG concentration is IgG antibodies specific for diphtheria or tetanus become useful
typically 20–25% higher than that of the mother. Catabolism functional measures.
of maternal IgG coupled with the slow development of endog- IgM is the first isotype to reach young adult levels, followed
enous antibody function leads to a physiological nadir of serum by total IgG and then IgA. This physiological delay in the produc-
IgG in infants of 4–6 months of age. In normal infants, this loss tion of serum IgA can complicate the diagnosis of IgA deficiency
of maternal protection is often associated with the first appearance in infants and young children. Serum Ig concentrations in healthy
of otitis media or bronchitis. Thus the onset of sinopulmonary adults tend to remain remarkably constant but can increase
infections within the first 3 months of age should also raise the dramatically in response to infection and suffer a decline in
index of suspicion for immunodeficiency in the mother. After response to immunosuppressive agents, such as corticosteroid
age 6 months, maternally-derived IgG has largely been lost, and administration. With increasing age, serum immunoglobulin

CHAPtER 34 Primary Antibody Deficiencies 475



CLINICAL PEARLS should be assayed simultaneously to avoid confusion that may
Interpreting Quantitative Immunoglobulins (Igs) result from single-tube dilution differences at the time the assay
is performed. As a general rule, a high baseline titer or ≥4-fold
• Normal ranges of serum immunoglobulin levels vary with age (Appendix rise in a specific titer in individuals with a low baseline titer
2); hence evaluation should take the age of the patient into account. confirms that a specific humoral response is intact. The develop-
• As the mother’s transplacental contribution of IgG is catabolized, ment of conjugated polysaccharide vaccines complicates analysis
total serum IgG concentrations reach a nadir at 4–6 months of age for those who have received such a vaccine, although information
• IgG2 and IgG4 subclass levels rise more slowly than IgG1 and can still be gleaned from study of responses to polysaccharide
IgG3; hence reference to adult controls can lead to the false diagnosis antigens present only in multivalent unconjugated vaccines.
of IgG subclass deficiency in young children.
• Serum IgA concentrations typically do not achieve adult values until Because of its relatively low molecular weight and slow
puberty. They are often the first to decline in many primary turnover compared with other isotypes, protein loss through
immunodeficiencies the kidneys or GI tract can result in the selective loss of IgG.
Should symptoms warrant, 24-hour urine for protein or a stool
sample for alpha-1-antitrypsin level can document protein loss.
An elevated IgE level may support a suspicion of allergy as
4
may continue to rise. The physiological significance of this an underlying explanation of sinopulmonary symptoms. Serum
increase in older adults is unclear, although in some cases it IgE concentrations are often elevated in patients with IgA
reflects an accumulation of expanded B-cell clones or monoclonal deficiency. Extreme elevations of IgE suggest the hyper-IgE
gammopathies (Chapter 80). syndrome.
Enumeration of B cells and of T cells should be performed
KEY CoNCEPtS for any individual who has severe panhypogammaglobulinemia.
The most widely used method of demonstrating B cells relies
Functional Tests of Specific Antibody Production on immunofluorescent labeling of surface CD19, which is
restricted in expression to mature B cells. Because an infant may
• Immunoglobulin M (IgM) T-independent responses can be assessed
by measurement of serum isohemagglutinins (anti-A and anti-B titers) have serum IgG of maternal origin for the first several months
in patients who are not blood type AB. of life, the determination of the number of circulating B cells is
• IgG T-independent responses can be assessed by measurement of the single most useful test in making the presumptive diagnosis
antibodies produced in response to immunization with unconjugated of XLA, a disorder in which pre-B cells in bone marrow fail to
purified pneumococcal polysaccharide vaccine. develop to cells of mature phenotype. Absence of circulating B
• IgG T-dependent recall responses can be assessed by measurement cells also characterizes the immunodeficiency associated with
of ≥4-fold rise in titer of antibodies to diphtheria or tetanus toxoid
alter booster immunization. thymoma in adults, whereas adults with chronic lymphocytic
leukemia may have hypogammaglobulinemia with an overabun-
dance of circulating B cells that typically express CD5.
The common laboratory practice of defining the lower range HIGM1 represents the product of a loss-of-function mutation
of normal for serum Ig levels as two standard deviations below of the CD154 (CD40L) gene. CD154, a surface antigen found
the age-adjusted mean carries with it the risk of falsely labeling on activated T cells, binds CD40 on B cells to facilitate class
otherwise normal patients as having immunodeficiency. Ig levels switching, survival, and proliferation (Chapter 8). A fluorescent-
vary widely with environmental exposure, and normal biological labeled CD40 fusion protein can be used to evaluate the expression
variation is much broader than that defined by the mean of the of functional CD154 on T cells by flow cytometry. Confirmation
population. Patients with a combined deficiency of IgA and an of the diagnosis, carrier detection, and prenatal diagnosis often
IgG subclass may benefit from more aggressive therapy; thus depend on molecular or sequence analysis of the gene.
quantitative measurements of all four IgG subclasses—IgG1,
IgG2, IgG3, and IgG4—can be useful in fully defining the extent Replacement Therapy With Human Immunoglobulin
of humoral immune deficiency. There are a number of commercial preparations of human Ig
Among patients with borderline serum IgG levels, tests to that are approved by the US Food and Drug Administration
evaluate the host’s ability to produce functional specific antibody (FDA) and available in the United States (Chapter 84). No com-
should be performed before making a decision to institute a mercial preparations in this country are available to supplement
more aggressive therapy, especially among patients receiving IgM or IgA exclusively, but some of the preparations contain
corticosteroids, which can lower total serum IgG while preserving minute amounts. All commercial human Ig preparations are
function. The most commonly employed tests include measure- effective in treating patients with immunodeficiency disorders.
ment of isohemagglutinins (naturally occurring IgM antibodies to Clinically relevant differences relate to the route of administration,
the polysaccharide antigens that define the ABO blood type system which can be by intravenous or subcutaneous means, to the
on red blood cells [RBCs]) and postimmunization responses to method of stabilization and storage, and to quantities of con-
polysaccharide antigens (e.g., Pneumovax23 or unconjugated taminating serum IgA in the preparations. Low IgA content is
H. influenzae B vaccine) and protein antigens (e.g., tetanus or a concern for those rare individuals with immunodeficiency and
diphtheria toxoids). IgM is made by the newborn, and most absent IgA, who manufacture IgG or IgE antibodies directed
infants can generate isohemagglutinins, making determination of against IgA and have a history of anaphylactic reactions upon
anti-A and anti-B titers a useful measurement of B-cell function infusion of IgA-containing blood products. 7
even in infants. In older children and adults, isohemagglutinin Ig replacement therapy is not indicated for patients whose
titers <1 : 8 are considered significantly decreased. 6 immune deficiency is limited to the selective absence of IgA
Serum for specific antibody titers should be obtained before because of the risk of anaphylaxis upon receipt of IgA-containing
7
and 4–6 weeks after immunization. Optimally, the paired sera products, even though such reactions are extremely rare. However,

476 PARt fouR Immunological Deficiencies


Ig replacement therapy has been found to be beneficial in patients
with a combined deficit of IgA and IgG subclasses who exhibit
impaired antibody responses to carbohydrate antigens.
The goal of Ig replacement is to provide sufficient concentra-
tions of functional antibodies to prevent disease, not to achieve
a target IgG level in the serum. Specific approaches to and
protocols for Ig replacement therapy are described in detail in
Chapter 84. Note: The only indication for immunoglobulin replace-
ment in a patient with immunodeficiency is severe impairment of
the ability to produce functional antibody. Such impairment exists
in primary immunodeficiency diseases associated with low levels
of all five isotypes of immunoglobulin, such as XLA, CVID,
HIGM, and severe combined immunodeficiency (SCID) (Chapter
35). Patients with a documented inability to produce specific
antibodies after immunization with a history of significant
morbidity from infections are also candidates for intravenous
immunoglobulin (IVIG) therapy even though they may present
8
with normal or near-normal levels of IgG. This includes certain
cases of IgG subclass deficiency, such as those associated with
compound IgA, IgG2, and IgG4 deficiency, boys with Wiskott-
Aldrich syndrome, and patients with ataxia–telangiectasia. Since fIG 34.3 A Computed Tomography (CT) Scan, With Contrast,
most patients with transient hypogammaglobulinemia of infancy Demonstrates Bronchiectasis, Bronchitis, and Emphysema
can produce normal amounts of specific antibodies after immu- in the Lungs of a 36-Year-Old Man With X-Linked Agam-
nization despite having a low total serum IgG, they usually are maglobulinemia (XLA). Because of a left lower-lobe lobectomy,
not candidates for Ig replacement although those patients with the mediastinum has shifted to the left. As a result of bronchi-
a history of significant infections may benefit. ectatic scarring, the diameter of the bronchi in the right lung is
greater than the diameter of the corresponding blood vessels,
X-LINKED AGAMMAGLOBULINEMIA and the bronchi remain dilated in the periphery. Bronchial plugs
can be seen filling some of the bronchi on the right. Finally, as
Diagnosis a result of emphysema, the right upper lobe demonstrates greater
XLA, also known as Bruton agammaglobulinemia, is the proto- radiolucency. In addition to suffering from XLA, this patient has
9
typic humoral immunodeficiency. Function-loss mutations in a 30 pack-year history of smoking, which has exacerbated his
BTK lead to a block in B-cell maturation, a near total absence clinical condition.
of B cells in the periphery, and panhypogammaglobulinemia.
As a result of the transplacental transfer of maternal Ig, affected
boys typically do not begin to suffer recurrent pyogenic infections pyogenic encapsulated bacteria. Diarrhea caused by G. lamblia
until after age 6 months. The normal delay in endogenous Ig is also common, although less so than in CVID. Systemic infections
production and the presence of maternal IgG requires that testing include bacterial sepsis, meningitis, osteomyelitis, and septic
of infants known or suspected to have XLA should begin with arthritis. Mycoplasma and Chlamydia infections of the urogenital
examination of the number of B cells in blood. Deficient expres- tract may lead to epididymitis, prostatitis, and urethral strictures.
sion of BTK protein can be detected by flow cytometry, a technique Skin infections include cellulitis, boils, and impetigo.
that can also be used for carrier detection. For those cases where Although in patients with XLA most viral infections can be
protein is present but the phenotype suggests XLA, analysis of resolved, these patients are unusually sensitive to infections with
the BTK gene at the nucleotide level remains the definitive enteroviruses. Patients with XLA can develop paralytic polio-
diagnostic procedure. As with most X-linked lethal diseases, myelitis after vaccination with live virus. Echovirus and Coxsackie
approximately one-third of sporadic cases are caused by de novo virus infections may involve multiple organs with the patients
mutations, and diagnosis may require individual mutation going on to develop chronic meningoencephalitis, dermatomyo-
analysis. There can be significant variation in the manifestations sitis, and/or hepatitis.
of the disease in any given family member; thus a paucity of Untreated patients often complain of arthritis affecting the
symptoms should not prevent diagnostic evaluation even in large joints. This appears to have an infectious etiology because
10
adults. Some cases of XLA have been reported with mild muta- the arthritis typically resolves with Ig replacement therapy.
tions that cause infections late in life. 11 Enterovirus and mycoplasma have been identified in the affected
joints of these patients.
Clinical Manifestations Infections with opportunistic organisms, such as Mycobac-
Although patients begin to suffer recurrent infections by age 1 terium tuberculosis, Histoplasma, and P. jiroveci, and malignancies
year, with antibiotics and good hygiene, it is not uncommon to are rare, likely reflecting intact cell-mediated immunity.
delay suspicion of the diagnosis well into mid-childhood. Indeed,
diagnoses have been made in older adults, including aged male Origin and Pathogenesis
relatives of affected probands. 10,11 Recurrent upper and lower BTK belongs to a subfamily of the Src cytoplasmic protein-
respiratory tract infections are common. Untreated, these infec- tyrosine kinases. BTK is phosphorylated following activation of
tions may lead to bronchiectasis (Fig. 34.3), pulmonary failure, the B-cell receptor (BCR). It plays a critical role in the prolifera-
and death at an early age. The infections are typically caused by tion, development, differentiation, survival, and apoptosis of

CHAPtER 34 Primary Antibody Deficiencies 477


B-lineage cells. Individuals with XLA begin with normal numbers splicing. E12 and E47 are involved in regulation of immuno-
of early B-lineage progenitors in their bone marrow. These B-cell globulin gene expression. A mutation in the DNA-binding region
progenitors express the expected markers of B-cell differentiation, of E47 has been shown to result in agammaglobulinemia as a
including terminal deoxynucleotidyl transferase (TdT), CD19, and result of a dominant negative early block in B-cell development. 15
CD10. There is, however, a relative deficiency of cells containing
cytoplasmic µ heavy chains in bone marrow. Development of cells LRRC8
beyond the pre–B stage is even more severely impaired. Those cells An absence of B cells has also been reported in a young female
that make it through the gauntlet can produce antigen-specific patient with a truncation of leucine-rich repeat containing 8
16
antibodies. Although present in low numbers, these B cells in (LRRC8, AGM5), a gene of unknown function that is expressed
lymphoid tissues enable XLA patients to express endogenous Ig, in patients with progenitor B cells.
class switch, and even suffer allergic or autoantibody-mediated
reactions. PIK3R1
Patients have been described with an X-linked recessive form A homozygous nonsense mutation in the P1K3R1 gene, encoding
of agammaglobulinemia that is associated with growth hormone the p85a subunit of phosphoinositide 3-kinase (PI3K), was found
deficiency. Genetic analysis of the BTK gene in one such patient in a 19-year-old female with agammaglobulinemia, absent B
identified a frameshift mutation leading to a premature stop cells, and inflammatory bowel disease by exome sequencing. The
codon and the loss of carboxy-terminal amino acids. 12 loss of p85a resulted in decreased pro–B cells, a contrast to the
other agammaglobulinemia defect, which is localized to the
Treatment and Prognosis pre–B-cell stage. 17
The primary goal of therapy is to prevent damage to the lungs.
Human Ig replacement therapy should be started as soon as the Diagnosis and Treatment
diagnosis is made (Chapter 84). Patients treated with sufficient Diagnosis in each of these cases requires gene mutation analysis.
quantities (0.4–0.6 g/kg every 3–4 weeks for IVIG or 100–150 mg/ Treatment follows the same guidelines given for XLA.
kg every week for subcutaneous immunoglobulin [SQIG]) suffer
few lower respiratory tract infections. However, these patients HYPER-IGM SYNDROME
remain at risk for viral infections, including enteroviral menin-
goencephalitis. Since mucosal Ig cannot be replaced, the patients Diagnosis
also remain at risk for recurrent upper respiratory infections, Patients with the HIGM syndrome exhibit markedly reduced
which may require prophylactic antibiotic therapy. Ig-treated serum concentrations of IgG, IgA, and IgE with normal to elevated
18
patients may lead normal lives without concern about exposure levels of IgM and normal numbers of circulating B cells. The
13
to infectious agents in childcare settings or classrooms. Immu- altered distribution of Ig isotypes reflects a block in the ability
nizations of any type are unnecessary because the monthly of B lymphocytes to switch from IgM to the other isotypes.
replacement therapy will provide passive protection. Since patients Increased IgM reflects polyclonal expansion of IgM synthesis in
are unable to mount antibody responses, vaccines, especially live response to infection. Patients with HIGM suffer from the same
vaccines, carry some risk of untoward side effects, so these vaccines infections with encapsulated bacteria common to all patients
are relatively contraindicated. with antibody deficiency. The HIGM phenotype can be inherited
A patient with XLA who develops symptoms of enteroviral as an X-linked, autosomal recessive, or autosomal dominant
central nervous system (CNS) or neuromuscular infection should trait. The phenotype can also be acquired in association with
have appropriate culture of the involved organ system. For patients neoplasia or congenital rubella.
with agammaglobulinemia who have chronic enteroviral infec-
tions, Ig therapy should be given at higher doses and maintained HIGM Syndrome Type 1: CD40L (CD154) Deficiency
until symptoms cease and the virus can no longer be detected. Class switch recombination is a multistep process that requires
exquisite coordination between the B cell and its cognate T-helper
AUTOSOMAL AGAMMAGLOBULINEMIA (Th) cell. A key step in the initiation of the process is the binding
of constitutively expressed CD40 on the B cell to its ligand,
Origin and Pathogenesis CD40L (CD154), which is expressed on activated T cells. The
The Pre–B-Cell Receptor and Signal Transduction Axis most common form of the disease, X-HIGM1, results from
Expression of the pre-BCR is a key step in the maturation of loss-of-function mutations in CD154 (Xq26).
14
the pre–B cell (Chapter 7). Function-loss mutations in any one
of the genes that code for components of the pre-BCR and its HIGM Syndrome Type 2: AID Dysfunction
associated signaling complex can inhibit pre–B-cell development, Activation-induced cytidine deaminase (AID; 12p13), a member
leading to an absence of mature B cells. This phenotype is seen of the cytidine deaminase family, is required for class switch
in patients with biallelic function-loss mutations of the µheavy recombination between Ig H chain constant domains and for
chain region (µ 0 , AGM1), the λ-like surrogate light chain (IGLL1, somatic hypermutation of the Ig V domains (Chapter 4). The
AGM2), the Ig-associated α (Igα, CD79A, AGM3) and -β(Igβ, hyper-IgM phenotype (HIGM2) can result from either biallelic
CD79B, AGM6) chains, and the adaptor B-cell linker protein AID function-loss mutations or from a dominant negative
(BLNK, AGM4), which is a key cytoplasmic component of the mutation on only one of the AID alleles.
pre-BCR signaling pathway.
HIGM Syndrome Type 3: CD40 Deficiency
TCF3 The cognate partner for CD40L (CD154) is CD40, the gene for
The TCF3 gene, also called E2A, encodes 2 basic helix-loop-helix which (CD40) is located on an autosome (20q12-q13.2). Patients
(bHLH) transcription factors, E12 and E47, through alternative with HIGM3 with function-loss mutations on both alleles of

478 PARt fouR Immunological Deficiencies


the CD40 gene present with a phenotype indistinguishable from nutrition because of the presence of diarrhea or perirectal
HIGM1. abscesses. Oral ulcers, gingivitis, and perirectal ulcers are associated
with neutropenia, which may occur chronically or intermittently
HIGM Syndrome Type 4: As yet Unknown Causes in up to two-thirds of the patients. One-fifth of the patients
Patients presenting with a HIGM-like phenotype, but lacking develop sclerosing cholangitis that can lead to hepatic failure.
demonstrable mutations in genes previously associated with Cryptosporidiosis is present in half of these patients.
HIGM, such as CD40, CD154, NEMO, AID, and UNG, have Approximately one-quarter of patients with NEMO have
been grouped by some immunologists into a category termed autoimmunity or autoinflammation. An intestinal inflammatory
HIGM4. B cells from patients with HIGM4 demonstrate defective disorder may be the presenting problem with chronic diarrhea
class switch recombination, but normal somatic hypermutation. and abdominal pain, with a few having steroid dependence.
A genetic cause has yet to be identified, and spontaneous recovery Although originally distinguished by the high level of serum
has been reported. 19 IgM, IgM levels are often normal in affected individuals. IgG is
low in all patients. IgA and IgE are usually low but can be normal
HIGM Syndrome Type 5: UNG Deficiency or even elevated in one-tenth of the population. B- and T-cell
Activation-induced cytidine deaminase (AID) acts by deaminating counts are within the normal range in more than 90% of the
cytidine nucleotides in DNA, leaving a uracil nucleotide in its patients and depressed in the rest.
place. Uracil-DNA glycosylase (UNG; 12q23-24.1) can remove Lymphoid hyperplasia is a common finding in CD40–CD154
the uracil, permitting normal or error-prone repair. Patients axis patients with active infections. Individual nodes may become
with function-loss mutations on both alleles of the UNG gene extremely large, and some patients develop splenomegaly. Hilar
(HIGM5) have presented with a history of bacterial infections, adenopathy causes a diagnostic dilemma, as the risk of lymphoma
hyperplasia, increased serum IgM levels, and low IgG and IgA. is increased in HIGM. Although the lymphoid tissue is usually
histologically abnormal, reactive processes are far more common
NEMO than malignancy. Plasma cells can be abundant or sparse. Primary
The nuclear factor κB (NF-κB) essential modulator (NEMO) follicles are poorly developed. The most characteristic abnormality
plays a key role in the NF-κB pathway and consequently in the is the absence of germinal centers.
CD40 signal transduction pathway. NEMO acts as a scaffold for
two kinases important for the activation of NF-κB. The IKBKG AID–UNG Axis (HIGM2 and HIGM5)
gene encodes for NEMO and is located on the X-chromosome Infected patients with AID–UNG deficiency may present with
(Xq28). Hypomorphic mutations in the gene causes a syndrome giant germinal centers filled with highly proliferating B cells,
with affected individuals having ectodermal dysplasia. This results presumably as a result of intense antigen stimulation. Approxi-
in conical teeth, an absence of eccrine sweat glands, and a paucity mately one-fourth of patients with HIGM2, but not HIGM5,
of hair follicles. Patients with NEMO are considered to have a present with evidence of autoimmunity. Manifestations include
defect in innate immunity and cell-mediated immunity as a hemolytic anemia, thrombocytopenia, and autoimmune hepatitis.
result of defective NF-κB activation, which is important in Autoantibodies in these patients are of the IgM isotype.
Toll-like receptor (TLR) signaling. Laboratory abnormalities
include impaired natural killer (NK) cell function, impaired Origin and Pathogenesis
pneumococcal responses, hypogammaglobulinemia, antigen- CD40–CD154 Axis (HIGM1, HIGM3, and NEMO)
specific T-cell proliferative abnormalities, and increased serum CD154, a member of the tumor necrosis factor (TNF) family,
IgA levels. Less than 20% of patients with NEMO have HIGM is a type II transmembrane protein that is predominantly
and, thus in the past, have been grouped with the HIGM group. 20 expressed on mature, activated CD4 T cells. Its expression peaks
at 6–8 hours after activation and then falls to resting levels by
Clinical Manifestations 24–48 hours. CD154 is also expressed on CD4 thymocytes,
CD40-CD154 Axis (HIGM1, HIGM3, NEMO) activated CD8 T cells, NK cells, monocytes, basophils, mast cells,
The majority of patients with HIGM have inherited disruptions activated eosinophils, and activated platelets. Newborn T cells
in the CD40-CD154 axis. Lymph nodes and spleen are deprived are deficient in CD154 expression, although they can be induced
of germinal centers. Recurrent upper and lower respiratory tract to express the antigen if strongly stimulated.
infections are the most common clinical complaint. Patients CD40 is a member of the TNF receptor superfamily. It is
may also exhibit recurrent neutropenia with oral ulcers, perirectal constitutively expressed on pro-B, pre-B, and mature B cells, as
abscesses, and opportunistic infections with P. jiroveci, Toxoplasma well as on interdigitating cells, follicular dendritic cells (DCs),
gondii, or Cryptosporidium cholangitis. Autoimmunity is observed thymic epithelial cells, monocytes, platelets, and some carcinomas.
in approximately 20% of patients. Engagement of B-cell CD40 with CD154 on an activated T
Without prophylaxis, one third of patients develop P. jiroveci cell that also expresses Fas ligand (FasL or CD95L) leads to the
pneumonia, which can be the presenting problem in affected upregulation of Fas (CD95) on the B cell. NEMO is a part of
infants. These patients are also at risk for serious infections with the signaling pathway. If the B cell has concomitantly bound its
cytomegalovirus (CMV), adenovirus, Cryptococcus neoformans, cognate antigen and engaged the BCR-signaling pathway, it
or mycobacteria. These features signal a compromise in cell- becomes resistant to Fas-mediated apoptosis and expresses CD80/
mediated immunity as well as the characteristic flaw in antibody CD86 on the cell surface. The activated B cell can then engage
production, putting them in the spectrum of a combined CD28 on the T-cell surface and trigger the T cell to secrete its
immunodeficiency. cytokines. If the B cell fails to engage its BCR, the Fas pathway
Chronic diarrhea occurs in more than half of the patients. predominates, and the B cell is eliminated. With proper activation
Organisms include Cryptosporidium, G. lamblia, Salmonella, and of the CD40–CD154 pathway, exposure to interleukin-2 (IL-2)
Entamoeba histolytica. One quarter may require total parenteral and IL-10 induces production of IgM, IgG1, and IgA; and exposure

CHAPtER 34 Primary Antibody Deficiencies 479


to IL-4 induces production of IgG4 and IgE. This change in Ig frank IgG subclass deficiencies, or evidence of impairment of
isotype reflects both induction of switching and the enhanced T-cell function. Patients with IgA serum levels that fall >2 standard
survival and proliferation of the B cell. In the absence of CD154, deviations (SDs) below the mean serum level for their age are
B cells can express IgM but have difficulty switching and are considered to have partial IgA deficiency. These patients can also
likely to undergo apoptosis, rather than proliferate in response suffer from recurrent infections.
to antigen.
+
+
CD40–CD154 interactions between CD154 T cells and CD40 Clinical Manifestations
macrophages lead to enhanced production of IL-12, which then The likelihood that an individual with IgA deficiency that was
stimulates T cells to release interferon-γ (IFN-γ). Activation of identified serendipitously will require medical attention is difficult
this pathway appears necessary for the defense against P. jiroveci to assess because most studies in the literature reflect patients
and other opportunistic organisms. who were ascertained to have the condition as a result of clinical
symptoms. Among patients with IgAD referred to immunology
Treatment and Prognosis clinics, more than 85% present with recurrent infections, typically
The availability of human Ig replacement therapy has greatly with encapsulated bacteria. Among affected children, symptoms
improved the quality of life in patients with HIGM. Adequate may begin in the first year of life, although the physiological lag
replacement can result in the reduction of serum IgM concen- in serum IgA may delay the diagnosis until after the age of 2
trations, prevention of infections with encapsulated bacteria, years. In some patients, respiratory infections disappear with
resumption of growth, and the gradual resolution of splenomegaly maturity. In others, infections may persist throughout adult life.
and lymphoid hyperplasia. Autoimmune and lymphoproliferative Rarely, patients with IgAD may experience recurrent bronchitis,
complications may respond to anti-CD20 therapy (rituximab). 21 pneumonia, and even bronchiectasis. These more severely afflicted
Unfortunately, in spite of the improvement gained by Ig patients often exhibit concurrent IgG2 and IgG4 subclass deficien-
replacement, the prognosis of patients with defects in the cies. Some symptomatic patients have elevated IgE levels and
CD40–CD154 axis remains guarded. Death at a young age manifest allergic or asthmatic components to respiratory dysfunc-
continues to be common. It is primarily the result of opportunistic tion. The rise in IgE has been explained as a compensatory
infections, including Pneumocystis pneumonia, cholangitis, CMV response to the absence of IgA. This appears to be a double-edged
infection, mycobacterial infections, and cirrhosis secondary to sword because up to 20% of these patients complain of allergic
hepatitis. Prophylaxis with trimethoprim–sulfamethoxazole can rhinitis, conjunctivitis, urticaria, and atopic eczema. Allergic
significantly reduce the risk of Pneumocystis pneumonia and is reactions may be enhanced because of the lack of IgA-blocking
indicated in those with CD40L and CD40 deficiency. Regular antibodies in serum, and unusually severe asthma has also been
monitoring of GI manifestations and management of neutropenia associated with IgAD.
is mandatory. Neutropenia should be treated with a trial of Among those less common IgA-deficient patients that are
granulocyte macrophage–colony-stimulating factor (GM-CSF), truly devoid of IgA, as many as three-fifths produce IgG or IgE
24
since some have responded to this therapy. Bone marrow anti-IgA antibodies. These uncommon patients are at an
transplantation is a viable option for patients that fail to respond uncertain risk for adverse reactions following transfusion with
to supportive therapy. blood products (as mentioned previously), plasma from normal
donors, or from some preparations of Ig replacement therapy,
SELECTIVE IGA DEFICIENCY which, of course, contain IgA. Patients with high anti-IgA levels
(>1 : 1 000) typically have potent antibodies directed against all
Selective IgA deficiency (IgAD), selective IgG subclass deficiencies, IgA. These patients are at risk for severe anaphylaxis. Patients
CVID, and a syndrome of recurrent sinopulmonary infections with low anti-IgA antibody titers (<1 : 256) are often multiparous
(RESPIs) with normal serum Ig levels appear to share an overlap- women or those who had received multiple transfusions. These
22
ping set of gene defects. Clinically, these disorders are marked patients rarely demonstrate severe anaphylaxis after infusion
by an increased susceptibility to upper and lower respiratory with plasma or blood products but do present with hives and
infections with encapsulated bacteria. IgAD and CVID feature rashes.
similar B-cell differentiation arrests but differ in the extent of Patients with IgAD often develop autoimmune diseases. GI
Ig deficits. The correlation between serum Ig levels and severity disorders include pernicious anemia, inflammatory bowel disease,
of infection is not absolute. 23 intestinal disaccharidase deficiency, lactase deficiency, pancreatic
insufficiency, and celiac disease. The last, in particular, can be
Diagnosis difficult to diagnose without biopsy, since serological diagnosis
Approximately 1 in 600 individuals of European ancestry are often relies on detection of antitissue transglutaminase and
25
unable to produce detectable quantities of IgA1 and IgA2, making antiendomysial or antigliaden IgA antibodies. Hepatobiliary
selective IgAD the most frequently recognized primary immu- disorders include chronic active hepatitis, cholelithiasis, lupoid
nodeficiency in the Americas, Australia, and Europe. The diagnosis hepatitis, and primary biliary cirrhosis. Skin disorders include
is dependent on the sensitivity of the laboratory measurement. pyoderma gangrenosum, paronychia, and vitiligo. It is unclear
The clinical laboratory typically reports serum IgA levels of less whether this autoimmune diathesis is the end result of recurrent
than 7 mg/dL, the concentration below which nephelometry infections or is the product of recurrent insult by antigens that
becomes unreliable. would otherwise be cleared by IgA, or whether the underlying
Patients with uncomplicated IgAD have normal serum levels deficit that leads to IgAD also increases the risk of developing
of IgM, have normal or elevated levels of IgG, and demonstrate an autoimmune disorder. For example, autoimmune disorders,
normal cell-mediated immunity. A minority of patients may such as insulin-dependent diabetes mellitus and celiac disease,
demonstrate additional evidence of immune dysfunction, with are associated with the same major histocompatibility complex
inability to generate appropriate IgG2 anticarbohydrate antibodies, (MHC) haplotypes (Chapter 5) as IgAD and CVID.

480 PARt fouR Immunological Deficiencies


IgAD is associated with an increased risk for the development TABLE 34.3 other Conditions Associated
of malignant epithelial tumors, such as gastric and colonic With Humoral Immunodeficiency
adenocarcinoma, Hodgkin disease, and acute lymphoblastic
leukemia. Patients with chronic GI infections may demonstrate Genetic Disorders
a nodular lymphoid hyperplasia of the small intestine that can Monogenic diseases Ataxia–telangiectasia
lead to intestinal obstruction. Histological evaluation reveals Autosomal forms of severe combined
immunodeficiency (SCID)
active B-lymphocyte proliferation in the germinal centers of the Transcobalamin II deficiency and
Peyer patches. These “constipated” lymph nodes have been hypogammaglobulinemia
mistaken for lymphoma. In others, the simultaneous presence Wiskott-Aldrich syndrome
of IgAD and malignancy may simply reflect the high prevalence X-linked lymphoproliferative disorder (Epstein-
of IgAD in the Caucasian population. Barr virus [EBV] associated)
X-linked SCID
Origin and Pathogenesis Chromosomal Chromosome 18q- syndrome
anomalies Monosomy 22
IgAD, selective IgG subclass deficiencies, and CVID are diseases Trisomy 8
that are defined by a quantitative phenotype, a paucity of serum Trisomy 21
Igs of a given isotype in spite of the presence in the blood of B
lymphocytes bearing the missing isotypes. By definition, the Systemic Disorders
fundamental defect involves the failure of B lymphocytes bearing Malignancy Chronic lymphocytic leukemia
a given isotype to differentiate into plasma cells. These diseases Immunodeficiency with thymoma
appear to represent a common endpoint for multiple pathogenic T-cell lymphoma
processes. All three phenotypes may be acquired and many of Metabolic or physical Immunodeficiency caused by hypercatabolism of
immunoglobulin
loss
the recognized precipitating causes, such as phenytoin, are the Immunodeficiency caused by excessive loss of
same (Table 34.3). immunoglobulins and lymphocytes
IgA deficiency is associated with MHC haplotypes (6p21.3)
that are more common in European populations than in the Environmental Exposures
peoples of Sub-Saharan African and East Asia. In the United Drug-induced Antimalarial agents
States, the prevalence of IgAD among African Americans is Captopril
one-twentieth of that observed among Americans of European Carbamazepine
descent, and in Japan, the incidence is approximately 1 in 18 500. Glucocorticoids
Fenclofenac
IgA deficiency has also been observed in family members of Gold salts
patients with CVID with altered function of the transmembrane Imatinib
activator and CAML interactor (TACI, 17p11.2), which is a Levetiracetam
receptor for B cell–activating factor (BAFF). Penicillamine
Phenytoin
Treatment and Prognosis Sulfasalazine
Zonisamide
Most individuals with IgAD suffer respiratory infections no more Infectious diseases Congenital rubella
frequently than the average individual and thus require no special Congenital infection with cytomegalovirus (CMV)
treatment. All individuals with IgA deficiency should be warned Congenital infection with Toxoplasma gondii
of the risk of serious transfusion reactions caused by antibodies EBV
to IgA. Wearing a medical alert bracelet is recommended. Should Human immunodeficiency virus (HIV)
transfusion be necessary, the ideal donors are other individuals
with IgAD. Washed erythrocytes are safer than whole blood.
Patients with selective IgA deficiency who suffer from clinically
significant, recurrent upper respiratory infections often respond impaired. In the presence of infection, abortive differentiation
to prophylactic antibiotics with potency against encapsulated can lead to massive B-lymphocyte hyperplasia, splenomegaly,
bacteria. Treatment of allergy in those patients with a compensa- and intestinal lymphoid hyperplasia.
tory increase in IgE is helpful. Patients who present with combined With an estimated prevalence of 1 in 25 000, CVID is the
IgA and IgG subclass deficiencies and have a poor pneumococcal most prevalent human primary immunodeficiency requiring
26
antibody response may require IG replacement therapy. medical attention. Both sexes are equally affected. As with IgAD,
the prevalence among African Americans is one-twentieth that
COMMON VARIABLE IMMUNODEFICIENCY AND of Americans of European descent. Some patients present during
CVID-LIKE DISORDERS childhood, but most are diagnosed after the third decade of life.
The typical patient reports a normal pattern of recurrent otitis
Diagnosis media as an infant and toddler that resolved in childhood. During
The diagnostic category of CVID includes a heterogeneous group adolescence, respiratory infections appear and steadily increase
of patients older than age 4 years and exhibit deficient production in frequency and duration. Recurrent pneumonia as a young or
of more than one major antibody class and whose antibody middle-aged adult is often the precipitating complaint that brings
response to vaccination is significantly depressed or absent. These the patient to the attention of the clinical immunologist. Although
patients tend to have normal numbers of clonally diverse B CVID appears to be an acquired disorder, family studies have
lymphocytes in their blood. These B cells can recognize antigens clearly documented that susceptibility for the disease can be
and respond with proliferation, but their ability to develop into inherited and the manifestations of the disorder may change
memory B cells or mature plasma cells appears quantitatively with time. Transitions within the spectrum of normal serum Ig