Clinical Immunology_ Principles and Practice ( PDFDrive )

1212 Part ten Prevention and Therapy of Immunological Diseases


persons (Fig. 90.1). Other opponents of vaccination were those
with financial interests in lucrative variolation practices. When
vaccination in England was made compulsory by the Vaccination
Act of 1853, an organized antivaccination movement arose almost
19
immediately. Incredibly, even in the present day, despite all the
evidence supporting the safety and effectiveness of licensed vaccines,
organized antivaccination movements continue to challenge
contemporary clinicians and public health officials. The explosive
growth of the Internet in the last 2 decades has made it relatively
easy to self-publish anything and everything, and this has resulted
in rapid and wide dissemination of misinformation about vaccines,
antivaccination propaganda, and pseudoscience that obstructs
traditional scientific evaluation and further feeds the general
public’s fears and misunderstandings.
It is worth noting that Jenner’s work of 220 years ago encapsulates
many of the elements of today’s translational vaccinology.
FIG 90.1 Antivaccination Caricature by James Gillray From
1802 Entitled “The Cow-Pock—or—The Wonderful Effects KeY COnCePtS
of the New Inoculation!” Directed Against Jenner and His Edward Jenner’s Work Relevant to Translational
Work to Promote Smallpox Vaccination. (Originally by James Vaccinology Today
Gillray, The Cow-Pock—or—the Wonderful Effects of the New
Inoculation! (1802). vide. the Publications of the Anti-Vaccine • Disease burden, surveillance, epidemiology: A significant and
Society. Print (color engraving) published June 12, 1802 by H. unacceptable burden of smallpox disease drove development of a
safer intervention to improve health.
Humphrey, St. James’s Street. Library of Congress, Prints & • Innovation: Jenner’s innovation resulted from the need for an improved
Photographs Division.) biomedical intervention given the significant risk of harm associated
with the centuries old variolation practice.
• Clinical insight: An observation that dairymaids who had recovered
from an occupational illness (cowpox) were seldom affected by smallpox
stock. Young Phipps survived both the vaccination and the led to Jenner’s promotion of smallpox vaccination. The observation
challenge, and Jenner first reported his experiment in 1797 in a of the protected state (immunity) in dairymaids led to a concept that
short communication to the Royal Society. After Jenner’s initial was tested and promoted by Jenner.
report submitted for publication was rejected, he added a few • Postvaccination challenge: After the vaccination procedure, Jenner’s
more cases of his vaccine experiment (including one on his own subjects were subsequently intentionally exposed to (challenged with)
wild-type smallpox and observed for safety and disease outcomes.
son), and in 1798, he reported his findings in a self-published This would not be permissible today in smallpox research, although
booklet entitled “An inquiry into the causes and effects of the human challenge experiments are performed for certain self-limiting
variolae vaccinae, a disease discovered in some of the western or treatable infectious diseases.
counties of England, particularly Gloucestershire, and known • Presentation of experimental results: To disseminate the scientific
by the name of the cow pox.” 18 information and advocate for wider vaccination deployment, Jenner
presented his work to the Royal Society. He self-published his manu-
script after it was rejected by the Society.
CLInICaL reLeVanCe • Branding: The name “vaccination” was applied to the intervention.
Vacca is the Latin word for “cow.”
Why vaccines are critical interventions for every • antivaccination movement and conflicts of interest: Jenner
medical practice experienced significant opposition to his vaccine from groups opposed
to the new technique and from individuals who had vested interests
• Vaccines are highly effective interventions for preventing infectious in variolation practices and stood to lose their business if the concept
diseases and have public health importance. of vaccination caught on.
• Both individual protection and community (herd) immunity result from
vaccination programs.
• The reductions in disease burden (morbidity and mortality) achieved Although Jenner’s smallpox challenge experiment would not
through implementation of childhood vaccination programs are be approved by today’s institutional research boards (IRBs, or
extraordinary.
• In the last several years, new adolescent and adult vaccines have ethics committees), certain human challenge studies remain both
become available and are now recommended. Vaccination is not just acceptable and valuable today. The postvaccination challenge
for children anymore. experiment can be an important process to efficiently obtain
• Clinicians of all specialties should take vaccine histories and provide preliminary protective efficacy information for a vaccine and
access to vaccines relevant to their patients’ ages and medical condi- has been shown in early-phase clinical trials to be safe, well
tions. Access can be provided through referral or by stocking and tolerated, and immunogenic. Human challenge studies are
administering the indicated vaccines.
performed for self-limiting and/or treatable infections to study
vaccine protection or to characterize host response to infection
20
21
22
This self-published report and Jenner’s subsequent use of in detail (e.g., influenza, primary dengue, norovirus, and
23
cowpox vaccination were greeted with mixed reaction. Jenner’s malaria ). A human challenge experiment can rapidly provide
work was considered controversial by some because of the feedback to vaccine developers and public health officials to help
introduction of a cow virus into humans. A famous satirical cartoon prioritize resource-intensive field trial evaluations of promising
by James Gillray depicts cow parts emerging from vaccinated candidate vaccines. If an encouraging preliminary efficacy signal

CHaPter 90 Vaccines 1213


is observed in a postvaccination human challenge trial, it can (antibodies) taken from an immune donor patient or animal,
provide additional evidence and justification to funding agencies without administering a vaccine to the patient. Early work by
for proceeding to large and expensive field trials. This is particu- Emil von Bering (1854–1917) with small-animal serum therapy
larly advantageous in the context of an epidemic, where rapid experiments led to the development of human serum therapies
vaccine development and deployment are needed for optimal for passive immunization. In 1900, von Bering used horse sera
impact on morbidity and mortality. A contemporary example from immune horses to cure and prevent diphtheria caused by
is Zika virus vaccine development—for which postvaccination Corynebacterium diphtheria. The very first Nobel Prize in Physiol-
human challenge experiments are under consideration. ogy or Medicine in 1901 was awarded to von Behring “for his
The history of vaccination entered its second phase in the work on serum therapy, especially its application against
nineteenth century. The miasma theory (which postulated that diphtheria, by which he has opened a new road in the domain
infectious diseases were caused by a noxious form of bad air) of medical science and thereby placed in the hands of the physician
was gradually replaced by the development of the germ theory a victorious weapon against illness and deaths.” 25
(according to which infectious diseases were caused by micro- During the second half of the nineteenth century, scientists
organisms too small to be seen without magnification). Animal were discovering the immune system’s defense mechanisms. The
experiments and laboratory cultivation of microbes were key Nobel Prize in Physiology or Medicine for 1908 was awarded to
advances relating to the germ theory in the second half of the Ilya Ilyich Mechnikov (1845–1916) and Paul Ehrlich (1854–1915)
nineteenth century. Robert Koch (1843–1910) and the great “in recognition of their work on immunity” and for establishing
French chemist Louis Pasteur (1822–1895) made significant the concepts of cell-mediated and humoral immunities. Mech-
contributions through their many key observations and experi- nikov described the abilities of certain white blood cells (WBCs)
ments in both agricultural and human infectious diseases and of starfish larvae to perform phagocytosis or engulfment and
vaccines. Koch’s famous four postulates laid out the requirements destruction of harmful bacteria and other microbes. Ehrlich
for establishing causality of infectious diseases by microbes and worked on serum therapy against diphtheria with von Bering
proved that Bacillus anthracis was the cause of anthrax. This and speculated that certain WBCs have receptors that bind toxins
provided the first proof of a microbial etiology of a specific formed by bacteria, and when these receptors separate from the
24
disease. Pasteur’s work was credited with saving the silkworm cells, they become antibodies.
industry, reducing the spoilage of wine, stopping epidemics among In 1924, Gaston Ramon (1886–1963) developed the method
agricultural herds, and the development of vaccines against human of chemical inactivation of bacterial toxins with formaldehyde
infectious diseases, such as rabies and anthrax. Through attenu- and heat to produce toxoids of the pathogenic toxins of diphtheria
ation or inactivation of wild-type microbes, Pasteur produced and tetanus, producing safer vaccine antigens that retained their
vaccines that induced protection against a number of diseases. immunogenic potential.
He performed a number of classic vaccination and challenge The Nobel Prize in Physiology or Medicine 1951 was awarded
experiments in farm animals; these experiments were designed to Max Theiler (1899–1972) “for his discoveries concerning yellow
to show that in experimental challenges, his altered (attenuated fever and how to combat it.” Theiler, through passage of yellow
or inactivated) cultures of microbes could be delivered as vaccines fever virus in mice, developed an attenuated live yellow fever
that offered protection to susceptible animals and herds against virus variant designated 17D, which became a highly effective
organisms that could be devastating, including veterinary vaccine.
pathogens, such as chicken cholera and anthrax, and human Other innovators who exploited the potential of microbial
pathogens, such as rabies. 24 cultivation were Enders et al., who, in the 1940s, discovered
Twenty-first century vaccinology is a multitasker’s dream, methods for cultivation of poliovirus in cell culture. This removed
encompassing multiple fields, including microbiology, immunol- the obstacles in the field of poliovirus vaccine development,
ogy, medicine, epidemiology, statistics, policy, manufacturing, which then advanced relatively quickly after decades of very slow
molecular biology, public health, and ethics. But vaccinology progress. Laboratory growth of poliovirus permitted the develop-
was not always so. Certainly in the 1700s and earlier centuries, ment of both the inactivated polio vaccine (IPV; Salk, licensed
the variolators and the great Jenner knew they had created a in 1955) and the live attenuated oral polio vaccine (OPV; Sabin,
26
protected condition in their patients through their intervention monovalent licensed in 1961, trivalent in 1963). As a result of
only if their patient survived that intervention. But they had no these vaccines, still in use today, poliovirus type 2 was eradicated
specific knowledge of the killed or attenuated microbe they were in 1999, and no wild-type poliovirus type 3 has been detected
administering, of the virulent microbe they were protecting since 2012. Only poliovirus type 1 is still endemic in 2016 in
against, or of the immune system changes induced in the bodies just two countries, Pakistan and Afghanistan. The progress toward
of their patients (or even of the existence of the immune system). polio eradication is impressive, as the final steps are being taken,
In the nineteenth century, Pasteur and Koch certainly knew but final eradication will require great persistence in vaccinating
that microbes were the cause of infectious diseases and that the populations in these countries and sustained attention to
weakened forms of microbes (vaccines) could create a protected surveillance. 27
state after administration to livestock or humans. But they had In their considerations of the power of vaccines and other
no specific knowledge of the changes produced in the vaccine preventive interventions to protect humans and reduce infectious
recipient’s immune system. disease incidences, public health officials have described the stages
In the early twentieth century, passive immunization was leading to the ultimate goal of ending human suffering caused
developed as a therapy for infectious diseases. Active immuniza- by infectious diseases: control, elimination, eradication, and
28
tion involves administering a vaccine to trigger a subsequent extinction of infectious diseases (Table 90.1). The long road,
cascade of changes in the patient’s own immune system leading which will hopefully culminate soon in poliovirus eradication,
to a protected state (immunity), whereas passive immunization began with successful poliovirus cultivation in cells in the labora-
is direct transfer of the protective immune effector molecules tory. “For their discovery of the ability of poliomyelitis viruses

1214 Part ten Prevention and Therapy of Immunological Diseases



TABLE 90.1 Stages of reduction of Army’s A/H1N1 vaccine, which had been successfully used for
Infectious Disease Incidence by Vaccination several years, became significantly less effective in 1947 as a result
and Other Preventive Interventions of antigenic drift. In 1958, the type A virus, which was circulating
at that time, entirely changed its HA and NA proteins to new
33
Control. The reduction of disease incidence and prevalence to a subtypes, a process termed antigenic shift. With increased
locally acceptable level as a result of vaccination and/or other recognition that it was necessary to regularly update the vaccine
interventions; continued interventions are needed to maintain the composition to match the changing circulating strains, in 1973,
reduction. Example: diarrheal diseases.
elimination of disease. Reduction to zero of the incidence of a the World Health Organization (WHO) began providing annual
specified disease in a defined geographical area as a result recommendations for the composition of the seasonal influenza
vaccination and/or other interventions; continued measures are vaccine based on the current circulating subtypes and strains.
required. Example: neonatal tetanus. In 1978, the first trivalent vaccine was licensed (two influenza
elimination of infection. Reduction to zero of the incidence of A strains and one influenza B strain). Beginning in 2013, the
infectious disease in a defined geographical area as a result of WHO began including a second type B strain and recommended
vaccination and/or other interventions; continued measures to
prevent reestablishment of transmission are required. Example: four vaccine strains annually (A/H3N2, A/H1N1, B/Victoria,
poliomyelitis elimination from North America. and B/Yamagata), which enabled production and licensure of
eradication. Permanent reduction to zero of the worldwide incidence the new quadrivalent influenza vaccines.
of infection caused by a specific agent as a result of vaccination Several other live, attenuated viral vaccines, such as measles,
and/or other prevention efforts; interventions are no longer needed. mumps, and rubella vaccine (MMR), were developed in the
Example: smallpox. second half of the twentieth century and became staples of
extinction. An infectious agent no longer exists in nature or in the childhood vaccination programs both in the United States and
laboratory. Example: none.
globally. Serial passages of wild-type viruses led to viral adaptation
(Adapted from Dowdle WR. The principles of disease elimination and eradication. Bull for growth in cell cultures and a diminished ability to cause
W H O 1998;76 Suppl 2:22–5.) disease in humans. Importantly, the cell culture–passaged viruses
that were useful as vaccines were not only well tolerated and
to grow in cultures of various types of tissue,” John Enders, safe in humans, but they retained the ability to produce protective
Thomas Weller, and Frederick Robbins jointly received the Nobel immune responses. Later in the twentieth century, the develop-
Prize in Physiology or Medicine in 1954. 29 ment of the Oka strain of the varicella-zoster virus led to live
Recognition and subsequent exploitation in vaccines of key attenuated vaccines for both chicken pox in children and herpes
antigenic substructures rather than whole microbes was another zoster in seniors.
technical advance. For example, the studies of Oswald Avery
(1877–1955), Rebecca Lancefield (1895–1981), and others of the KeY COnCePtS
polysaccharide capsules of Streptococcus pneumoniae 30,31 and the
32
M proteins of the Streptococcus species, respectively, led to the Historical Highlights
characterization, isolation, and serotyping of these bacterial • Edward Jenner promotes use of the smallpox vaccine in 1798.
structures and their recognition as key antigens in immunity to • “Morbid matter of various kinds, when absorbed into the system,
streptococcal diseases. Such observations eventually led to safer may produce effects in some degree similar; but what renders the
vaccination with components (subunits) of pathogens, as opposed cow-pox virus so extremely singular is that the person who has
to entire microbes (e.g., the bacterial polysaccharide capsules been thus affected is forever after secure from the infection of the
from S. pneumoniae or Neisseria meningitidis, or the viral surface small-pox; neither exposure to the variolous effluvia, nor the insertion
18
of the matter into the skin, producing this distemper.”
antigens [hemagglutinins, or HA] in influenza split-virus vac- • World Health Organization certifies eradication of smallpox from the
cines). When delivered as vaccines, these isolated microbial world in 1980.
components produced protective antibodies and cellular immune • “The world and its peoples have won freedom from smallpox,
responses in vaccinated hosts but did not cause the disease induced which was a most devastating disease sweeping in epidemic form
by the complete wild-type organisms. through many countries since earliest time, leaving death, blindness
The great influenza A/H1N1 pandemic of 1918 (“Spanish and disfigurement in its wake and which only a decade ago was
rampant in Africa, Asia and South America.”
40
flu”) caused an estimated 50 million deaths globally. This led to
efforts to develop vaccines against influenza, and in 1933, the
33
virus that causes influenza was identified. That discovery quickly The decades following World War II have been described as
led to the first-generation live attenuated and killed monovalent a golden age for vaccinology. Maurice Hilleman (1919–1985)
influenza vaccines. A bivalent vaccine was produced in 1942 developed a number of vaccines against viruses while working
after the discovery of a second influenza type, type B. The ability for large pharmaceutical companies during the middle of the
to grow influenza viruses in embryonated hen’s eggs facilitated twentieth century. His highly productive career included inventions
vaccine production and provided the basis for killed and split- of vaccines against measles, mumps, and rubella; Haemophilus
33
virus influenza virus vaccines that came into use in the 1940s. influenzae type b; hepatitis A; hepatitis B; and chickenpox. 34
Over the following years that followed, there was increasing The polysaccharide vaccines developed for the prevention of
awareness of the multiple types of influenza (A, B, and C), bacterial diseases caused by H. influenzae, N. meningitidis, and
subtypes of influenza A (e.g., A/H1N1, A/H3N2), and lineages S. pneumoniae were welcome advances. Over time it was recog-
of influenza B (Victoria and Yamagata). In addition, recognition nized that these polysaccharide antigens were T-cell independent
that influenza virus antigen drift (caused by changes in the two because they contained no peptide/protein antigens. Furthermore,
surface proteins HA and neuraminidase [NA]) could lead to unlike T cell–dependent protein antigens, these carbohydrate
vaccine mismatch with an altered circulating strain and thus antigens failed to produce B-cell memory responses. The polysac-
decreased vaccine effectiveness was first described when the US charide vaccines could not be used in neonates because infants

CHaPter 90 Vaccines 1215


cannot mount an effective immune response against polysac- TABLE 90.2 Historical Comparisons of
charide antigens alone. The brilliant technical development that Morbidity and Mortality for Vaccine-
overcame this disadvantage for bacterial polysaccharide vaccines Preventable Diseases in the United States
was covalent coupling, or conjugation, of the glycan structure
to a protein carrier, such as tetanus or diphtheria toxoids. This Before
maneuver converted the T cell–independent polysaccharide Vaccination: after
vaccines into T cell–dependent protein–polysaccharide conjugate estimated Vaccination:
vaccines, and this resulted in B-cell memory, much improved annual annual Cases
immunity, usefulness in newborns, and even reduction in average (reported or
nasopharyngeal carriage (not seen with the pure polysaccharide number of estimated) in
35
vaccines), thus creating herd immunity. The observation that Disease Cases Year 2006 % reduction
protein conjugation to polysaccharide improved the immune Diphtheria 21053 0 100
36
response was made first by Avery and Goebel in 1929 but was Measles 530217 55 99.9
not utilized until Robbins et al. conjugated the H. influenzae Mumps 162344 6584 95.9
type B polysaccharide. 37 Pertussis 200752 15632 92.2
Increasingly in the late twentieth century and early twenty-first Paralytic 16316 0 100
century, the molecular biology revolution and the fundamental poliomyelitis
99.9
dissections of the innate and adaptive immune responses are Rubella 47745 11 100
29005
Smallpox
0
being exploited to produce new-generation vaccines. Some of Tetanus 580 41 92.9
these dimensions are discussed further in the sections below. Hepatitis A 117333 15296 87
Acute hepatitis B 66232 13169 80.1
<50
ACCOMPLISHMENTS OF VACCINATION Invasive Hib 20000 41550 99.8
34.1
63067
Invasive
pneumococcal
It is generally believed that elimination of an infectious disease disease
from circulation in human populations through vaccination can Varicella 4085120 48445 85
only be achieved in the case of pathogens that have no animal
reservoir and vaccination against which induces long-lasting Hib, Haemplhilus influenzae type b.
immunity (see Table 90.1). Smallpox eradication was, in fact, (Adapted from Roush SW, Murphy TV, Vaccine-Preventable Disease Table Working
Group. Historical comparisons of morbidity and mortality for vaccine-preventable
achieved after a successful worldwide vaccination campaign and diseases in the United States. JAMA 2007;298:2155–63.)
is the signature accomplishment of vaccination. The fields of
medicine and public health celebrate this remarkable vaccination
42
success as an example of the power of vaccination to improve 99.9%, 99.9%, and 95.9%, respectively. It is estimated that for
human health. Smallpox was an infection that was a scourge of each annual birth cohort of approximately four million children
humanity for millennia, disfiguring and blinding survivors and in the United States, vaccines in the childhood immunization
38
killing 30% of those infected. The last reported case of smallpox schedule prevent an estimated 20 million cases of disease and
43
in the United States was in 1949. The world’s last known patient 42 000 deaths. Furthermore, although it is true that a consider-
with naturally occurring smallpox was Ali Maow Maalin in able investment of resources is required to complete the annual
39
Somalia in 1977. After the disease was eliminated, routine programs of childhood vaccination, vaccines do result in very
vaccination of the general public against smallpox was discon- significant cost savings, and hence are highly cost-effective
tinued because it was no longer necessary for prevention of this interventions. For each yearly US birth cohort, vaccines result
disease. In 1972, the United States halted routine smallpox in nearly $14 billion in annual net direct cost savings and $69
immunization of the American public. In 1980, the WHO certified billion in annual net societal cost savings (which include savings
that smallpox had been eradicated: “The world and its peoples such as reductions in costs of missed work by parents caring for
have won freedom from smallpox, which was a most devastating an ill child). 43
disease sweeping in epidemic form through many countries since Saving lives and reducing morbidity and human suffering is
earliest time, leaving death, blindness and disfigurement in its the major accomplishment of vaccination. Although there are
wake and which only a decade ago was rampant in Africa, Asia many human infectious diseases yet to be controlled by vaccina-
40
and South America.” Small quantities of smallpox virus are tion (some are discussed below), the accomplishments to date
stored in secure research laboratories at the Centers for Disease are truly spectacular. For example, the CDC estimates that US
Control and Prevention (CDC, Atlanta, GA) and in Russia. childhood vaccination programs will prevent 21 million hospi-
It has been over 200 years since Jenner’s self-published report talizations and 732 000 deaths among children born in the last
17
on smallpox vaccination. As the dawn of the third millennium 20 years. 44
approached, the CDC designated vaccination as first on the list Vaccines are not given solely to protect individuals against
of the 10 greatest public health achievements of the twentieth diseases. Another purpose of vaccination is to protect communities
41
century. In addition to smallpox eradication, the control of by reducing transmission of disease-causing microbes from
many common childhood infections and attendant reductions vaccinated persons to unvaccinated persons. The term for this
45
in morbidity and mortality ranked as very high achievements. protection is herd immunity or community immunity. A disease
Implementation of routine US childhood immunization programs that has been studied closely with regard to community immunity
led to major reductions from mid-twentieth century disease is measles. Measles is very highly contagious and its epidemic
peaks to the record low levels of several infectious diseases today form is easily recognizable. Clustering of poor vaccination
(Table 90.2). For example, in the United States, the number of coverage often occurs in particular communities, and this was
46
polio, measles, rubella, and mumps cases declined by 100%, highlighted in recent measles outbreaks in the United States.

1216 Part ten Prevention and Therapy of Immunological Diseases


In a measles outbreak in California in 2014–2015, of the 110 yellow fever, and influenza viruses). In a few cases, attenuated
patients, 49 were unvaccinated; in that subgroup, 24% were zoonotic organisms closely related to human pathogens were
children too young to be vaccinated, and 67% were intentionally employed to produce cross-reactive protective responses in
unvaccinated (mostly children) as a result of parental beliefs. humans (e.g., vaccinia, an animal poxvirus utilized as a vaccine
Such outbreaks point out the importance of community immunity against human smallpox, and Bacille Calmette-Guérin [BCG],
to protect the vulnerable (unvaccinated) members of our com- an agent of bovine tuberculosis [TB] developed as a human TB
munities. Given that many of the recent measles outbreaks in vaccine).
the United States have been linked to imported cases, another Later, split-virus vaccines utilized partially purified protein
important lesson is that as long as a vaccine-preventable, highly antigens derived from whole killed viruses (e.g., split-virus
transmissible infectious disease exists anywhere in the world, it influenza vaccines). The polysaccharide capsules of bacteria were
remains a potential threat everywhere—and thus vaccination purified from cultures of serologically distinguishable strains,
programs will continue to be important to ensure the health of or serotypes, within single bacterial species leading to polyvalent
all community members in any part of the world. polysaccharide vaccines (e.g., the 23-valent pneumococcal
Another powerful example of vaccine-induced community polysaccharide and the quadrivalent meningococcal polysac-
immunity comes from pneumococcal vaccines. There are many charide vaccine). Bacterial toxins were purified from cultures
specific challenges associated with pneumococcal vaccines: the and made harmless by application of heat or chemical treatment
large number of circulating serotypes, the suboptimal immu- to produce toxoid vaccines (e.g., tetanus and diphtheria
nogenicity of polysaccharide only vaccines, and noninvasive vaccines).
carriage of the organism—all of which lead to significant problems During the closing years of the twentieth century and in the
in establishing community immunity. However, in spite of those early twenty-first century, the advances in genetics, molecular
challenges, introduction of the pneumococcal conjugate vaccines biology, immunology, and microbiology have led to new theory-
in infants in 2000 not only led to reduction in invasive diseases based (so-called rational) approaches to vaccine development.
among the vaccinated population of children but also produced Perhaps “informed empiricism” is an apt description of much
a significant reduction in adults, particularly among seniors, in of early twenty-first century vaccine development: experimental
1
whom this bacterium frequently causes pneumonia. This result approaches that are heavily influenced by our now-imperfect,
highlights the effectiveness of community immunity produced always evolving knowledge of innate and adaptive immune
by vaccines. responses and of microbial antigens but are still dependent on
Other recently introduced vaccines have made a significant an iterative system of trial and error to discover safe, well-tolerated,
impact in relatively brief periods. The 2006 implementation of and efficacious vaccines. Vaccine developers today also benefit
routine rotavirus vaccination prevents an estimated 40 000 to from the cumulative body of knowledge and experience in
47
60 000 rotavirus hospitalizations in the United States annually. vaccinology that has accumulated over the past century.
The human papillomavirus (HPV) vaccine is a recombinant There have been numerous changes in the design of vaccine
virus-like particle (VLP) vaccine for primary prevention of cancer. immunogens. The formation of links between bacterial polysac-
The CDC Advisory Committee on Immunization Practices (ACIP) charides and protein carriers (conjugation) led to dramatic
recommended routine HPV vaccination for girls in 2006 and improvements in protection against certain bacterial diseases.
for boys in 2011. Within 6 years of the introduction of vaccine Conjugation of H. influenzae, N. meningitidis, and, most recently,
for girls, there was a 64% decrease in the prevalence of the four S. pneumoniae polysaccharides to proteins improved these vaccines
types of HPV contained in the vaccine among females 14–19 by converting them from T cell–independent antigens to T
years of age and a 34% decrease among those 20–24 years of cell–dependent antigens.
48
age. This example shows the power of an excellent new- Adjuvants are materials added to vaccine antigens to enhance
generation viral vaccine, even when uptake is incomplete. At the the recipient’s immune response. Recent advances in our under-
end of the first decade of the current century, cases of hepatitis standing of the innate immune system have led to an appreciation
A, hepatitis B, and varicella have been reported to be at record that adjuvants act through their effects on innate immunity.
low levels 49,50 (see Table 90.2). Adjuvant-triggered innate signals enhance the quantity and quality
of the downstream adaptive immune responses to the vaccine
RECENT CHANGES IN VACCINE antigen. Today’s vaccinologists are developing vaccines with
DEVELOPMENT STRATEGIES specific molecular adjuvants (e.g., Toll-like receptor-5 [TLR-5],
CD-40L, and interleukin-12 [IL-12]), intended to shape the
Vaccines were originally developed with no knowledge of the immune response to provide a presumed best fit for protective
immunological correlates of protection or, indeed, of the existence immunity against the targeted pathogen.
of the immune system. Similarly, the earliest vaccines were The advent of molecular biology in the mid-twentieth century
developed without an in-depth understanding of microbial resulted in many new avenues for vaccine development. Advances
antigens and epitopes, which are critical to the production of in molecular biology allowed for cloning of microbes’ genes and
protection against disease. Rather than having a theoretical or their expression in recombinant molecular systems, and vaccines
purely logical basis, for a century and a half after Jenner, vaccina- could consequently be designed on the basis of the in vitro
tion continued to be based on experience and clinical observation. expression of one or a few genes. For example, the hepatitis B
The observations and experiences led to a concept that was vaccine, originally developed by Hilleman, was purified hepatitis
evaluated by a trial-and-error approach. The desired outcomes B surface antigen (HBsAg) from the blood of humans with chronic
of the trial-and-error, or empirical, approach to vaccine develop- infection. Soon thereafter, a second licensed hepatitis B vaccine
ment were protection against disease and survival after exposure was produced in yeast cells through recombinant DNA methods
to the pathogen. Vaccines were initially attenuated or killed that inserted the HBsAg gene into yeast organisms for expression
versions of the whole wild-type human pathogens (e.g., rabies, and purification. The recombinant hepatitis B vaccine is in use

CHaPter 90 Vaccines 1217


in the United States and offers advantages, such as protein purity Year-round
(because the genes-of-interest are expressed in relative isolation) WHO surveillance and epidemiology of circulating influenza strains
and vaccine safety (because it is no longer necessary to derive Production timeline
vaccines by partially purifying the HBsAg from paid-donor plasma Feb Mar Apr May Jun Jul Aug
of humans chronically infected with hepatitis B virus and
potentially other viruses). Six months
Another example of a highly effective recombinant vaccine Feb
is the HPV vaccine. Recombinant HPV L proteins expressed in WHO announces influenza strain selections for trivalent and quadrivalent
recombinant systems form VLPs that are purified and formulated seasonal vaccines
with or without an adjuvant. The most recent polyvalent vaccine
expresses VLPs representing nine HPV serotypes. HPV vaccines Mar
are truly remarkable for their efficacy and safety and because Manufacturers obtain influenza vaccine viruses from WHO
they offer primary prevention against several types of cancer in
both females and males. Apr
It is notable that several countries have recently licensed the Cloning of recombinant viruses and adaptation for
51
first dengue vaccine. It is a recombinant live viral vector that efficient egg growth
is based on the yellow fever virus vaccine strain 17D but in which May
the E and preM genes of 17D have been replaced by the E and Testing egg-grown viruses for antigenic stability;
preM genes of dengue serotypes 1, 2, 3, and 4. Four separate Expansion and growth of stocks
recombinant vectors were developed, one for each of the four June
dengue serotypes. The fact that the dengue virus and yellow Production and purification of millions of
fever virus are both flaviviruses with similar genetic organization vaccine doses
facilitated development of this chimeric yellow fever–dengue
vaccine. The final formulation is a tetravalent mixture of recom- July
binant viruses representing all four dengue serotypes. Vialing, quality control, and
potency testing
Over the last decade, systems biology, or systems vaccinology,
approaches to vaccine development have captured considerable August
interest. 52-55 The use of new high-throughput assays to assess Shipment and delivery to
multiple dimensions of innate and adaptive immune responses clinical sites
has generated very large data sets. In addition to the traditional
cellular and humoral immunological assays (antibodies, T cells, Sep – Mar
B cells), multiple “-omics” assays, including transcriptomics, Annual vaccine campaign
proteomics, metabolomics, lipidomics, and glycomics, among FIG 90.2 An Approximation of the Current Annual Influenza
others, may be performed. These detailed assessments are being Vaccine Production Process for the Northern Hemisphere.
applied in a variety of infectious and noninfectious disease states. This is the process for egg-based vaccine production. A few
One advantage for vaccine systems biology studies that enroll manufacturers’ recombinant vaccine techniques have recently
generally healthy study participants is the ability to obtain one eased the tight annual timeline, but most vaccines remain egg
or more baseline, preperturbation (prevaccination) sets of samples based. Development of a universal vaccine would obviate this
for analysis. Postvaccination changes can therefore be compared challenging and time-constrained annual process.
with the undisturbed condition. Analyses and integration of the
huge amounts of collected data require multidisciplinary col-
laboration with computational biologists and informaticians.
One application of the systems vaccine approach is to dissect vaccine once, followed by tetanus boosters every 10 years; zoster
the “-omics” responses produced by protective vaccines to identify vaccination at age 60 years; and pneumococcal vaccination at
associated changes at the RNA, protein, metabolite, lipid, and/ age 65 years. It should be noted that although the US Food and
or glycans levels. The promise, and some might say “hype,” of Drug Administration (FDA) has licensed a zoster vaccine for
systems vaccinology is both exciting and unfulfilled—and will use at age 50 years, the ACIP recommendations do not always
be realized once actual improvements in human health have align completely with FDA indications (hence the ACIP recom-
been achieved as a result of the systems approach. mendation for the zoster vaccine at age 60 years).
The adult schedule also provides recommendations for vaccines
CURRENT RECOMMENDATIONS indicated for certain risk factors, including medical conditions
(e.g., immunocompromising conditions, kidney failure, diabetes)
Currently in the United States, clear guidelines recommend or life style, occupational, or other conditions (e.g., pregnancy,
vaccines for children, adolescents, and adults. Each February, men who have sex with men, health care personnel). Live vaccines
56
the CDC publishes two immunization schedules based on the (varicella, zoster, and MMR) are contraindicated in pregnant
recommendations of the CDC’s Advisory Committee on Immu- women, immunocompromised hosts, and those with human
nization Practices (ACIP). One ACIP schedule of immunizations immunodeficiency virus (HIV) infection when the CD4 T-cell
57
provides the adult immunization recommendations (Fig. 90.3). absolute count is below 200 cells/µL.
The adult schedule has recommendations for each vaccine based The second ACIP immunization schedule of immunizations
on the age of the patient. For example, the ACIP recommends covers the period from birth to 18 years and includes recom-
that all adults (persons ≥19 years of age) receive annual influenza mendations for children or adolescents who have not received
58
vaccination; a tetanus–diphtheria–acellular pertussis (Tdap) recommended vaccines (Fig. 90.4). The ministries of health in

1218 Part ten Prevention and Therapy of Immunological Diseases


many European countries publish their own country-specific computation, and science will result in improvements in human
immunization schedules, and vaccination guidelines that are health remains to be seen. At present there is some (healthy)
published by the WHO are utilized by many developing countries. skepticism about the big data systems biology approach. The
The schedules are generally similar but with some region-specific field must show that the massive data can be analyzed
differences. For example, the 2016 US ACIP immunization and integrated and that the approach is more than a fishing
schedule for children recommends vaccinations against 10 viral expedition but, rather, is an exploratory engine that is hypothesis
diseases: hepatitis B, rotavirus infection, polio, influenza, measles, generating and also leads to models that will be tested and will
58
mumps, rubella, varicella, hepatitis A, and HPV infection. produce new knowledge, resulting in improved vaccines that
Preventive viral vaccines in the WHO-recommended routine benefit human health.
immunization schedule for children include the same 10 viral For outstanding scientists to be attracted to and retained in
vaccines (although four of those, mumps, influenza, varicella, the field of vaccinology, it is essential that science funding agencies
and hepatitis A vaccines, are recommended only for country- provide and maintain robust support for critical discovery research
specific immunization programs with certain characteristics). projects (investigator-initiated research projects) that form the
The WHO schedule also recommends some additional vaccines engine of innovation that drives all of science. At the same time,
(e.g., rabies, yellow fever, Japanese encephalitis, and tick-borne targeted big science vaccine program projects and networks have
encephalitis vaccines are recommended for certain high-risk the potential to synergistically pool their approaches in an intense
populations). 59 focused effort to tackle major vaccine needs and challenges (e.g.,
development of HIV and TB vaccines). To translate basic advances
SOME PRESENT AND FUTURE CHALLENGES and laboratory science into improved health care for patients,
having well-trained translational physician-scientists lead
The science of vaccinology is exciting and strong, having entered clinical–translational human research programs is an additional
a new era with the high-throughput analytical and data approaches component of essential infrastructure. There is a need for
being applied today. Whether these advances in technology, postdoctoral training programs that focus on vaccinology, but


Vaccine 19–21 years 22–26 years 27–59 years 60–64 years ≥ 65 years

Influenza 1 1 dose annually
Td/Tdap 2 Substitute Tdap for Td once, then Td booster every 10 yrs
MMR 3 1 or 2 doses depending on indication

VAR 4 2 doses
HZV 5 1 dose

HPV–Female 6 3 doses
HPV–Male 6 3 doses
PCV13 7 1 dose

PPSV23 7 1 or 2 doses depending on indication 1 dose
HepA 8 2 or 3 doses depending on vaccine
HepB 9 3 doses
MenACWY or
MPSV4 10 1 or more doses depending on indication
MenB 10 2 or 3 doses depending on vaccine
Hib 11 1 or 3 doses depending on indication

Recommended for adults who meet the Recommended for adults with additional
age requirement, lack documentation of medical conditions or other indications No recommendation
vaccination, or lack evidence of past infection
A
FIG 90.3 (A) Recommended Immunization Schedule for Adults Aged 19 Years or Older by Age
Group. The US Centers for Disease Control and Prevention (CDC) Recommended Immunization
Schedule for Adults Aged 19 Years and Older, United States, 2017, Became Effective in February,
2017 as Recommended by the Advisory Committee on Immunization Practices (ACIP) of the
CDC. When Used in Immunization Practice, the Extensive Footnotes Provided With the Schedules
(and Referenced in the Figure) Should be Consulted at www.cdc.gov/ vaccines/schedules/hcp/
index.html.

CHaPter 90 Vaccines 1219



Immuno- HIV infection Asplenia, Kidney failure, Heart or
compromised CD4+count persistent end-stage renal lung disease, Men who
(excluding HIV (xells/µL) 3–7,9–11 complement disease,on chronic Chronic liver Healthcare have sex
Vaccine Pregnancy 1–6,9 infection) 3–7,11 < 200 ≥ 200 deficiencies 7,10,11 hemodialysis 7,9 alcoholism 7 disease 7–9 Diabetes 7,9 personnel 3,4,9 with men 6,8,9
Influenza 1 1 dose annually
1 dose
Td/Tdap 2 Tdap each Substitute Tdap for Td once,then Td booster every 10 yrs
pregnancy
MMR 3 contraindicated 1 or 2 doses depending on indication

VAR 4 contraindicated 2 doses
HZV 5 contraindicated 1 doses

HPV–Female 6 3 doses through age 26 yrs
3 doses
HPV–Male 6 3 doses through age 26 yrs 3 doses through age 21 yrs through age
26 yrs
PCV13 7 1 dose

PPSV23 7 1, 2, or 3 doses depending on indication

HepA 8 2 or 3 doses depending on vaccine
HepB 9 3 doses
MenACWY or
MPSV4 10 1 or more doses depending on indication

MenB 10 2 or 3 doses depending on vaccine
3 doses
Hib 11 post-HSCT 1 dose
recipients only
Recommended for adults who meet the Recommended for adults with additional
age requirement, lack documentation of medical conditions or other indications Contraindicated No recommendation
vaccination, or lack evidence of past infection
B
FIG 90.3, cont’d (B) Recommended immunization schedule for adults aged 19 or older by medical
condition and other indications. The following acronyms are used for vaccines recommended for
adults: HepA, hepatitis A vaccine; HepAHepB, hepatitis A and hepatitis B vaccines; HepB, hepatitis
B vaccine; Hib, Haemophilus influenzae type b conjugate vaccine; HPV vaccine, human papillomavirus
vaccine; HZV, herpes zoster vaccine; IIV, inactivated influenza vaccine; LAIV, live attenuated
influenza vaccine; MenACWY, serogroups A, C, W, and Y meningococcal conjugated vaccine;
MenB, serogroup B meningococcal vaccine; MMR, measles, mumps, and rubella vaccine; MPSV4,
serogroups A, C, W, and Y meningococcal polysaccharide vaccine; PCV13, 13-valent pneumococcal
conjugate vaccine; PPSV23, 23-valent pneumococcal polysaccharide vaccine; RIV, recombinant
influenza vaccine; Td, tetanus and diphtheria toxoids; Tdap, tetanus toxoid, reduced diphtheria
toxoid, and acellular pertussis vaccine; VAR, varicella vaccine. (Courtesy CDC; https://www.cdc
.gov.)


only a few are in existence. The future of the field depends on vaccination history, recommend and discuss needed vaccines,
attracting and training a next generation of highly qualified and bring our patients’ vaccinations up to date.
vaccinology research leaders. Training of health care professionals and education of the
Patients’ and parents’ confidence in and uptake of vaccines public are essential dimensions for vaccination to achieve its
derives most of all from the advice and education provided by potential as a driver of improved health. Clearly a vaccine that
their trusted physicians and other clinicians. But because of the stays in its vial has no use at all. Health care providers must be
high time pressures of office or hospital encounters, clinicians inspired and trained to serve as advocates and champions of
often find it challenging to prioritize patient education about vaccination; to remember to discuss vaccination at every patient
vaccination. Nonetheless the clinician–patient encounter is the encounter; to stock vaccines in their refrigerators and freezers;
most potent opportunity for education and influence, and this and to use electronic reminder systems to ensure compliance
opportunity must be seized for vaccine programs to be successful. with recommended vaccinations based on the patient’s age and
Every physician/clinician encounter is an opportunity to review medical or other indications.

1220 Part ten Prevention and Therapy of Immunological Diseases


Public awareness and education about vaccination must
be enhanced through professional marketing campaigns. The A Vaccine Against Human Immunodeficiency Virus
negative consequences of propaganda by antivaccination groups, The development of a vaccine against human immunodeficiency
alternative or delayed schedules of vaccination, and parental virus/acquired immunodeficiency syndrome (HIV/AIDS) has
hesitancy regarding vaccination can all result in resurgence of long been recognized as a top HIV research global priority at
infectious diseases, increased morbidity, and increased vaccine- the US National Institutes of Health (NIH). Strong and simple
preventable deaths. treatments for those who are living with HIV infection are now
With modern air travel, which makes it possible to go from in existence in the United States and have been made available
one continent to another in a matter of a few hours, it is no even in developing countries. It has been shown that the
stretch to say we dwell in a global village, where maintaining treatment-as-prevention approach, wherein the viral load is
good health and avoiding highly contagious infectious diseases lowered to an undetectable level by antiretroviral treatment of
depend on the health of communities, whether local or global. infected persons, results not only in benefit to the infected patient
Therefore it is in our self-interest to maintain high levels of but also in up to 96% reduction in HIV incidence among sexual
vaccination in our home communities and to advocate and partners. 60,61
support vaccination efforts around the globe. Clearly, vaccination More recently, antiretroviral drugs (ARDs) have been tested
is both a personal and a social, or community, endeavor. globally and licensed in the United States as a once-daily pill (a
A few specific challenges for vaccination are highlighted below combination of tenofovir and emtricitabine) for HIV/AIDS
62
to illustrate the general points made above. prevention in higher-risk individuals. Known as preexposure




13-15
11-12
Vaccine Birth 1 mo 2 mos 4 mos 6 mos 9 mos 12 mos 15 mos 18 mos 19-23 2-3 yrs 4-6 yrs 7-10 yrs 11-12 13-15 16 yrs 17-18
yrs
mos
yr
yrs
s
yrs
yr
s
nd
rd
st
1
Hepatitis B (HepB) 1 dose 2 dose 3 dose
2
Rotavirus ,(RV) RV1 (2-dose st nd See
series);RV5 (3-dose series) 1 dose 2 dose footnote 2
Diphtheria, tetanus, & acellular st nd rd th th
3
pertussis (DTaP: <7 yrs) 1 dose 2 dose 3 dose 4 dose 5 dose
th
rd
+DHPRSKLOXV LQIOXHQ]DH 1 dose 2 dose See 3 or 4 dose,
nd
st
4
type b (Hib) footnote 4 See footnote 4
Pneumococcal conjugate 5 st nd 3 dose 4 dose
rd
th
(PCV13) 1 dose 2 dose
Inactivated poliovirus 6 1 dose 2 dose 3 dose 4 dose
th
rd
nd
st
(IPV: < 18 yrs)
7 Annual vaccination (IIV)
Influenza (IIV) Annual vaccination (IIV) 1 or 2 doses
1 dose only
nd
8
st
Measles,mumps,rubella (MMR) See footnote 8 1 dose 2 dose
9
st
nd
Varicella (VAR) 1 dose 2 dose
10
HepatitisA (HepA) 2-dose series, See footnote 10
11
Meningococcal (Hib-MenCY
st
nd
≥6 weeks; MenACWY-D ≥9 mos; See footnote 11 1 dose 2 dose
MenACWY-CRM ≥2 mos)
Tetanus, diphtheria,& acellular Tdap
12
pertussis (Tdap: ≥7 yrs)
13
Human papillomavirus (HPV) See footnote
13
See footnote 11
Meningococcal B 14
Pneumococcal polysaccharide 15 See footnote 5
(PPSV23)
Range of recommended Range of recommended ages Range of recommended ages Range of recommended ages for non-high-risk No recommendation
ages for all children for catch-up immunization for certain high-risk groups groups that may receive vaccine subject to
A
FIG 90.4 CDC Recommended Immunization Schedule for Children and Adolescents Aged
18 Years or Younger, United States, 2017 Became Effective in January, 2017, as Recom-
mended by the CDC’s AICP. When used in immunization practice, the extensive footnotes
provided with the schedules (and referenced in the figure) should be consulted at www.cdc.gov/
vaccines/schedules/hcp/index.html, which also provides a schedule for persons aged 4 months
through 18 years who start late or who are more than 1 month behind. (A) Recommended
immunization schedule for children and adolescents aged 18 years or younger by age.

CHaPter 90 Vaccines 1221



HIV infection
CD4+ count
µ
(cells/ L) Asplenia and
Immunocompromised <15% of ≥15% of Kidney failure, end- CSF leaks/ persistent complement Chronic
status (excluding HIV total CD4 total CD4 stage renal disease, on Heart disease, cochlear component liver
VACCINE INDICATION Pregnancy infection) cell count cell count hemodialysis chronic lung disease implants deficiencies disease Diabetes
Hepatitis B 1
Rotavirus 2 SCID*
Diphtheria, tetanus, and
3
acellular pertussis (DTaP)
Haemophilus influenzae
type b 4
Pneumococcal conjugate 5
Inactivated poliovirus 6

Influenza 7
Measles,mumps,rubella 8
Varicella 9

Hepatitis A 10
Meningococcal ACWY 11
Tetanus, diphtheria, and
12
acellular pertussis (Tdap)
Human papillomavirus 13
Meningococcal B 11
Pneumococcal
polysaccharide 5
Vaccination is recommended,
Vaccination according to the Recommended for persons with and additional doses may be No recommendation Contraindicated Precaution for vaccination
routine schedule recommended an additional risk factor for which necessary based on medical
the vaccine would be indicated
condition. See footnotes.
B
FIG 90.4, cont’d (B) Vaccines that might be indicated for children and adolescents aged 18 years
or younger based on medical indications. (Courtesy CDC; https://www.cdc.gov.)




prophylaxis (PrEP), this approach, in an ideal setting with years of major efforts since the identification of the viral etiological
unlimited resources and complete patient compliance, could have agent of AIDS in 1984, a licensed HIV vaccine with proven
a truly dramatic impact on HIV incidence. However, to date, protective efficacy continues to be elusive. The field has completed
uptake has been low, and patient adherence to treatment continues (only) five efficacy trials, which did not achieve protection in
to be a concern. Many of those most at risk for HIV infection vaccinated higher-risk subjects relative to placebo recipients, 64-68
do not have medical insurance and cannot afford to purchase and in two of the trials (which tested replication-deficient adeno-
medications. virus serotype 5 recombinant vaccine vectors expressing HIV
Although the advances in HIV treatment, treatment-as- Gag, Pol, and Nef but not Env), there were reports of higher
prevention, and PrEP have been significant, the numbers of new rates of HIV infection in some subsets of participants in the
infections globally remain unacceptably high, with 2.1 million vaccine groups compared with the placebo groups. 69,70
new infections reported in 2015 and a total of 36.7 million people Importantly, modest vaccine efficacy was observed in a sixth
63
71
living with HIV infection. In the United States, progress on efficacy trial reported in 2009. This 16 000-person study, known
the prevention of HIV infections through the use of condoms, as RV144, which was conducted by the US Army in collaboration
education, and evidence-based interventions has plateaued at with the government of Thailand, evaluated a prime-boost
≈44 000 new infections annually. Another remarkable achievement regimen of a nonreplicating canarypox vector prime (expressing
of ARDs has been the dramatic reduction in AIDS-related deaths HIV Gag, protease and gp120) followed by boosting with the
in the United States, from a peak of ≈55 000 per year to ≈13 000 same vector plus a bivalent gp120 protein adjuvanted in alum.
per year. As a consequence of the unabated incidence of HIV To the surprise of some early critics of the trial and many in the
infection and the reduced mortality, the number of people living field, the RV144 regimen produced modest (31.2%) protection
with HIV in the United States is now increasing at a rate of in the general population of Thailand at 3.5 years after vaccination,
≈30 000 per year. and 61% protection in the first year after vaccination. 71
The global need for an HIV vaccine remains strong. However, This first evidence of human protection provided by an HIV
there are large scientific challenges, and despite more than 30 vaccine proved that development of an HIV vaccine would be

1222 Part ten Prevention and Therapy of Immunological Diseases


possible, and this has significantly reenergized the field. In
follow-up laboratory studies, an NIH-funded international Improved Influenza Vaccines
collaborative program that assessed the immunological correlates Seasonal influenza A imposes a significant disease burden, with
of RV144 infection risk revealed that the level of nonneutralizing the highest morbidity occurring in children and the highest
immunoglobulin G (IgG) antibody targeting a V1V2 loop antigen mortality occurring in persons over 65 years of age. The CDC
from the HIV Env gp120 protein correlated inversely with risk estimates that in the United States, annually influenza is respon-
of infection, whereas the level of IgA antibody against gp120 sible for 200 000 hospitalizations in infants and children and
72
protein directly correlated with infection. The IgG antibody 3000 to 49 000 deaths (depending on the year), with 90% occur-
was capable of mediating antibody-dependent cellular cytotoxicity ring in seniors. Influenza case numbers peak in February during
(ADCC), whereas the IgA antibody competed with IgG for binding most years, and 10% of the population can become infected in
72
to gp120 V2. The US National Institute of Allergy and Infectious a season. In the United States, starting from 2010, it is recom-
Diseases (NIAID)–-funded HIV Vaccine Trials Network (HVTN), mended that all persons ≥6 months of age receive an annual
the US Army, and country level and industry collaborators have influenza vaccine. If fully implemented, this would entail
formed the Pox-Protein Public-Private Partnership (also known administration of 350 million doses each year.
as “P5”) to plan an intensive series of follow-up human studies There are two types of influenza, A and B, responsible for
to confirm and fully investigate the important leads provided most human disease, and multiple subtypes of influenza A, which
73
to the field by RV144. Efficacy trials that will test next-generation are categorized on the basis of the amino acid (AA) sequence
HIV vaccine regimens of clade C–expressing poxvirus vectors homology within the HA and NA proteins. Currently 19 HA
with adjuvanted protein boosts and may include DNA vector and nine NA subtypes are recognized, and these can combine
priming are being planned for southern Africa. In that region, to form many viral subtypes (e.g., H1N1, H3N2, H5N1), which
the HIV clade C (the most prevalent global clade) predominates, are further defined as specific strains based on AA sequence.
and the population has a higher risk of HIV acquisition compared Influenza B is further categorized into two lineages, Yamagata
with Thailand, where the clades E and B predominate. The coming and Victoria.
decade in HIV vaccine research will be highly interesting. Will Influenza is a negative-stranded RNA virus that lacks a proof-
the “improved versions” of the RV144 regimen provide protection reading function in its viral polymerase; hence it is highly mutable.
in southern Africa among the higher-risk, genetically distinct One of the truisms about influenza is that the only thing predict-
population of Africans exposed to C clade HIV? able about influenza is that it is unpredictable. Because of the
Discovery of a vaccine immunogen that induces broadly RNA genome mutations that continually accumulate during
neutralizing antibodies remains the holy grail of HIV vaccination. influenza replication, the WHO issues a recommendation every
Such antibodies do occur naturally in up to 15% of chronically February for a new vaccine strain composition. Vaccine manu-
infected persons, but only after years of infection. Although seen facturers in the Northern Hemisphere then race to produce the
in natural infection, no vaccine has been able to induce these year’s trivalent or quadrivalent seasonal vaccine by late summer
broadly neutralizing antibodies in vaccinated humans. However, to be ready for the winter influenza season. There are multiple
several broadly neutralizing monoclonal antibodies (mAbs) have challenges and needs with regard to this repeated (endless) annual
been cloned, and a few have been tested for safety and pharma- process of influenza vaccine prediction, production, distribution,
cokinetics in early-phase human trials. The antibody genes are implementation, uptake, and protection (Fig. 90.2).
highly mutated from their germline with ≈20–25% mutations, For the segment of the population that has the highest mortal-
indicating that extensive somatic hypermutation (SHM) has ity, that is, older adults, currently available vaccines provide only
occurred in the lymph-node germinal centers (GCs). They also relatively weak protection against influenza diagnosed with
tend to have short complementarity-determining region 1 (CDR1) polymerase chain reaction (PCR). One approach to this challenge
arms and as a result of autoreactivity against self lipids, these has been vaccination of persons ≥65 years of age with a fourfold
clones are frequently deleted. 74 higher dose of antigen, 60 µg (rather than 15 µg) for each strain
The NIAID, through two of its HIV/AIDS clinical trials based on HA protein content. This high-dose vaccine was shown
networks (the HVTN and the HIV Prevention Trials Network to increase both immunogenicity (ref) and efficacy and was
[HPTN]), is conducting a phase IIB efficacy trial of VRC01, one licensed for use in seniors in the United States. 11,12
of the broadly neutralizing mAbs. In laboratory virus neutraliza- In European and many other countries, for a number of years,
tion assays, this antibody has activity against >90% of 343 strains an oil-in-water adjuvant, MF59, has been licensed for use in
of HIV-1 across all clades at an IC80 of <1 µg/mL. 75,76 The influenza vaccine formulations specifically for seniors. The toler-
ongoing VRC01 efficacy trial (or the antibody-mediated protec- ability and safety of this adjuvanted vaccine has been demonstrated
tion [AMP] study) is a randomized, double-blind, placebo- in tens of millions of recipients, and the increases in immune
13
controlled clinical trial, where the antibody will be infused response and efficacy are well documented. In 2015, on the
intravenously every other month for 18 months at doses of 0 mg/ basis of safety and antibody response data from clinical trials,
kg (placebo), 15 mg/kg, or 30 mg/kg. If the VRC01 antibody the US FDA approved this oil-in-water adjuvant, designated as
14
does protect at-risk humans against HIV acquisition, this will MF59, in a formulation of an influenza vaccine for use in seniors.
provide a clear impetus to the HIV vaccine field to continue and Therefore for seasonal influenza vaccination of seniors in the
intensify efforts to develop immunogens capable of inducing United States, clinicians will now, as in many other countries,
similar or further improved broadly neutralizing antibodies. have an adjuvanted influenza vaccine option that provides
Notably, the preparation used in the VRC01 trial is a single enhanced immunogenicity.
antibody, and many experts feel that a combination of mAbs Another approach to increased protection of seniors against
targeting different epitopes on the gp120 envelope protein mortality caused by is better community (herd) immunity. The
structure may provide an even stronger result. The AMP study CDC’s ACIP recommendation for universal influenza vaccination
result is eagerly awaited. of all persons >6 months of age has the potential to reduce

CHaPter 90 Vaccines 1223


mortality in seniors, even when a senior citizen’s antibody level week in most cases, but longer durations of viral RNA detection
remains suboptimal after vaccination. If nearly everyone around are reported in semen and urine. 79,80
a senior citizen is vaccinated and protected, a susceptible senior In summer 2016, most Zika virus infections in the continental
would be much less likely to come in contact with the influenza United States were diagnosed in travelers who returned from
virus. Enhancing uptake of the annual vaccine by all children Zika virus epidemic areas in Latin America or in their sexual
and adults is thus an effective approach to improving protection contacts. However, a few cases of local transmission had occurred
of the vulnerable population of seniors. by midsummer 2016 in southern United States. These southern-
Seasonal influenza vaccine effectiveness varies year to year most parts of the country will likely experience ongoing limited
for each influenza A subtype or influenza B lineage, depending local transmissions as a result of the broad susceptibility of the
on the degree of match between the vaccine strains and the population and the presence of the primary mosquito vector
circulating strains. To allow vaccine manufacturers the 6 months Aedes aegypti. The projection for only limited local transmission
currently required for egg-based vaccine production methods, in the United States is based on recent prior experiences with
the vaccine strains for each subtype and lineage must currently dengue and chikungunya, which are also spread by the same
be selected in February for the following season’s vaccine cam- mosquito vector. However, the risk and magnitude of harm that
paign. The burden of annual revaccination of the entire popula- will occur in the United States because of this emerging virus
tion against a variable viral target is high, both logistically and is truly unknown.
financially. Furthermore, uptake of the annual seasonal influenza Although the virus was first identified nearly 70 years earlier
vaccine in the general population remains suboptimal. Over the in 1947 in the Zika forest in Uganda, only rare human infections
past decade, considerable effort has been put into the develop- had been documented in Africa and Asia for 60 years. Relatively
ment of the so-called universal influenza vaccine. The universal little scientific work was conducted. Then in 2007, a Zika virus
81
vaccine would target conserved regions on the HA protein (e.g., outbreak occurred in the Yap Islands in Micronesia, and in
82
the highly conserved stalk rather than the hypervariable head) 2013, there was an outbreak in French Polynesia. By 2015, large
or other conserved viral proteins (e.g., the M protein). Some of numbers of persons in Brazil were becoming infected, and in
the universal influenza vaccine candidates are moving into 2016, the virus continued to spread across the Americas and the
early-phase human safety and immunogenicity clinical trials. If Caribbean islands. Associations of the Zika virus with increased
a safe and effective universal influenza vaccine can be developed, incidences of microcephaly in newborns and GBS in adults led
the goal would be to replace annual revaccination against the the WHO to declare a “Global Public Health Emergency of
current season’s strains with initial vaccination and periodic International Concern” on February 1, 2016. Several governments
boosting with a universal vaccine that should protect against in the affected regions have advised women to delay pregnancy.
any influenza variant that arises each year (e.g., a booster every No vaccine or treatment is currently available.
decade as for tetanus). A further advantage would be pandemic As a response to the Zika virus epidemic, the development
preparedness and prevention because the universal influenza of a vaccine has emerged as a top priority of the US government.
vaccine, by targeting conserved viral domains present on all Preclinical work has demonstrated efficacy and human vaccine
viruses, should also protect against avian strains, such as A/ trials are on-going. A WHO website indicates that over 15
H5N1 or A/H7N9. 77,78 Finally, recent studies comparing a pharmaceutical and governmental entities are working on finding
standard-dose conventional (egg-produced) quadrivalent vaccine a Zika virus vaccine.
to a recombinant quadrivalent HA protein vaccine produced The vaccine field has a sense of optimism that a Zika virus
by a cell-line in serum-free medium demonstrated better protec- vaccine can be developed relatively quickly, given that there are
tion in older adults with the recombinant than the conventional several existing flavivirus vaccines in use around the world. The
vaccine, thus suggesting the possibility of a newer chapter in live attenuated yellow fever virus vaccine 17D, developed by
vaccine production. 78a Theiler, has been extensively used since 1927. Chimeric yellow
fever virus 17D–dengue virus vaccines have been licensed in
Zika Virus several countries, and a live attenuated dengue vaccine was recently
A current challenge that also encapsulates the recent history of shown to be protective in a human challenge study. 21,51 Further-
pathogens reemerging and spreading globally from continent more, tickborne encephalitis virus vaccines have been in use for
to continent is the ongoing Zika virus epidemic. Zika virus is a decades in Russia and Europe, and a West Nile virus vaccine
reemerging flavivirus closely related to dengue virus. As of 2015, that expresses viral antigens from a DNA vector was developed
this single-stranded positive-sense RNA virus has been causing by the NIAID and was shown to have acceptable safety and
an epidemic of mosquito-borne dengue-like illness across South promising immunogenicity in humans.
and Central Americas and the Caribbean. Infections in pregnant
women, with resulting fetal microcephaly or other birth anomalies, Other Needed Vaccines
are the most feared and serious consequences of Zika virus It is beyond the scope of this chapter to describe in detail all the
7
infection. A mosquito-borne viral cause of birth defects has new or improved vaccines that are needed to address significant
never existed before. In general, healthy adults with symptomatic infectious disease burdens. Brief mention is given to a few more
infections experience a mild to moderate self-limiting viral illness needed vaccines that have been reviewed elsewhere recently.
that has been described as “mild dengue” and is mostly character- The Ebola epidemic of 2014–2015 highlighted the importance
ized by fever, rash, conjunctivitis, and arthritis. An increased of having a vaccine ready for the next inevitable outbreak of
association of Guillain-Barré syndrome (GBS) with Zika virus that recurrent periodic killer disease. Major field trials of candidate
infection has been reported in multiple countries, and research Ebola vaccines were launched in 2015, but before the major
is ongoing to determine if a causal relationship exists. Interestingly, efficacy endpoints were achieved, the epidemic waned. However,
it is believed that 80% of Zika virus infections are asymptomatic. a preliminary result of strong efficacy for the recombinant
In symptomatic infected adults, viremia persists for less than a vesicular stomatitis virus (rVSV)–Ebola glycoprotein vaccine was

1224 Part ten Prevention and Therapy of Immunological Diseases



On tHe HOrIZOn REFERENCES
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CHaPter 90 Vaccines 1226.e1


MUL t IPL e -CHOIC e QU e S t IO n S

1. The physician often recognized as the “father of vaccinology” recommendations for each vaccine based on the age of the
is: patient, and other indications. In the 2017 schedule, these
A. James Phipps included:
B. Robert Koch A. All adults (persons ≥19 years of age) should receive annual
C. Edward Jenner influenza vaccination; a tetanus–diphtheria–acellular
D. Louis Pasteur pertussis (Tdap) vaccine once, followed by tetanus boosters
every 10 years; zoster vaccination at 60 years of age; and
2. The polysaccharide vaccines developed for the prevention of
bacterial diseases caused by pathogenic organisms, such as pneumococcal vaccination at 65 years of age.
Haemophilus influenzae, Neisseria meningitidis, and Streptococ- B. Other vaccines indicated for certain risk factors, including
cus pneumoniae, became much more useful upon conjugation medical conditions (e.g., immunocompromising conditions,
with: kidney failure, diabetes) or life style, occupational, or other
A. Lipids conditions (e.g., pregnancy, men who have sex with men,
B. Small molecule metabolites health care personnel).
C. Proteins C. Live vaccines (varicella, zoster, and measles–mumps–rubella
D. Polymers [MMR]) are contraindicated for pregnant women,
immunocompromised hosts, and in those with human
3. The Advisory Committee on Immunization Practices immunodeficiency virus [HIV] infection when the CD4+
(ACIP) of the US Centers for Disease Control and Prevention T-cell absolute count is below 200 cells/µL.
(CDC) in its adult immunization schedule makes annual D. All of the above are correct.

91









Immunotherapy of Allergic Disease



Anthony J. Frew







Specific-allergen immunotherapy (SIT), or allergen desensiti- that SIT for one allergy can alleviate symptoms caused by different
zation, is used to treat various forms of allergic disease that allergens. Before deciding to use SIT, it is, therefore, essential to
involve type I hypersensitivity. SIT involves the administration of assess the patient’s condition carefully, especially the role of
allergen extracts to modify or abolish symptoms associated with allergic triggers.
exposure to relevant allergens. SIT is used for three distinct types SIT was pioneered over 100 years ago and developed as an
of allergic condition: anaphylaxis, rhinoconjunctivitis, and asthma. empirical science of pollen vaccination, based on the mistaken
1
In anaphylaxis, the patient is completely well between episodes; concept that hay fever was caused by infection. Early studies
exposure is infrequent and unexpected but provokes an immediate found that giving large doses of pollen would trigger allergic
and potentially catastrophic response. However, since exposure reactions, so regimes that start with extremely low doses and
only occurs occasionally, it can be difficult to know whether the build up to a maintenance dose that is given every few weeks
treatment has been effective until an accidental exposure event were developed. A great deal has been learned about the immu-
occurs. Allergic rhinoconjunctivitis is clearly driven by allergen nological events associated with clinically successful SIT, but
exposure, which is low level and may be either continuous or uncertainties remain about which of these events is truly
intermittent. Exposure and clinical relevance are most obvious important in delivering the clinical benefit. New and modified
for seasonal rhinitis (caused by pollens or molds), occupational forms of SIT have been developed to deliver these immunological
rhinitis, and for rhinitis caused by animal danders. Because there effects more efficiently, but none of these, as yet, has the efficacy
is no seasonal variation in perennial rhinitis, it is more difficult to of the standard approach.
be sure that allergen exposure drives symptoms—in some cases it Following initial reports from the United Kingdom, SIT was
clearly does, but in others there may be nonallergic or structural developed in the 1920s–30s in North America, where some
causes for the symptoms. The value of SIT for rhinoconjunctivitis differences in practice emerged compared with European practice.
is generally clearer when exposure is predictable, but assessing In particular, American allergists tend to treat patients for all
efficacy is complicated by year-to-year variations in pollen sensitivities identified on skin testing, using personalized mixtures
exposure because of weather patterns and individual activity. of extracts prepared from bulk vials, whereas in Europe, patients
The third indication for SIT is asthma, where the role of allergen are normally treated with single allergens, which are supplied
exposure is different from that in rhinoconjunctivitis. As discussed directly by the manufacturer. However, pollens from different
elsewhere, allergen sensitization is a risk factor for developing grasses or trees are often combined. Mixtures of allergens from
asthma in childhood, but it is less clear whether allergen exposure different sources (e.g., mites and pollens) are used in some parts
has a role in ongoing asthma. At the very least, the fact that of Europe as custom mixes from manufacturers. Different
allergen avoidance measures are not usually effective in asthma approaches are also taken for standardization of extracts. Allergen
begs the question whether modifying the response to allergen extracts used in Europe are standardized by their ability to elicit
will influence established disease. It is inherently more risky to a weal, whereas US standardization has been based on erythema
use SIT to treat patients with asthma compared with treating rather than weal.
those who do not have asthma, so the benefits and risks must Whichever form of extract is chosen, patients are started on
be weighed carefully when considering the use of SIT to treat a very low dose of allergen, and the dose is then increased, usually
patients with asthma. at weekly intervals, until reaching the maintenance dose, which
The general principles of managing any allergic condition is then given at 4- to 6-weekly intervals for 3–5 years. Alternative
are to make an accurate diagnosis, identify relevant trigger factors, induction protocols may involve several doses on each day
institute appropriate interventions to reduce the impact of those (semirush), or the whole series of incremental injections may
triggers, and control symptoms and disease progression. Allergen be given in a single day (rush). The main drawback to rush and
avoidance measures may help but are rarely sufficient to avoid semirush protocols is the frequency of adverse reactions, which
the need for other therapies. Drug treatments can be very effective, occur much more often than with conventional protocols.
but they only work as long as the drugs are taken. SIT is the However, full protection can be attained in a few days compared
only current therapy that modifies the immune response to with the 3 months required in the conventional regime. Normally,
allergens. It is specific in that treatment is targeted at those the doses are given by subcutaneous injection, but since 1990,
allergens recognized by the patient and physician as responsible there has been increasing interest in SIT administered by the
for symptoms. Although claims have been made for bystander sublingual route, which is more convenient for patients and has
benefits on unrelated allergens, there is little convincing evidence some advantages in terms of safety.

1227

1228 Part tEN Prevention and Therapy of Immunological Diseases


MECHANISMS OF SIT specific immunoglobulin G (IgG) antibodies, which increase
progressively during treatment. This led to suggestions that SIT
might work by inducing antibodies that intercept the allergen
KEY CONCEPtS and “block” the allergic response. In patients treated for venom
anaphylaxis, the development of allergen-specific IgG antibody
Possible Mechanisms of Immunotherapy correlates with clinical efficacy, but for other allergens, the
magnitude of the IgG response is not closely related to the degree
• Induction of immunoglobulin G (IgG) (blocking) antibodies of efficacy. Moreover, the rise in IgG follows, rather than precedes,
• Reduction in specific IgE (long-term)
• Reduced recruitment of effector cells onset of clinical benefit. Allergen-specific IgE antibodies increase
3
• Altered T-cell cytokine balance (shift to T-helper cell-1 [Th1] from Th2) initially, but the usual seasonal rise in IgE is blunted. Over several
• T-cell anergy years, the amount of allergen-specific IgE declines but does not
• B-cell suppression disappear. In keeping with this, there is little effect on immediate
skin test responses to allergen. In contrast, the late-phase skin
test response is virtually abolished after successful SIT. Similar
patterns are observed for late-phase nasal and airway responses. 4
The primary reason for studying the mechanisms of SIT is to SIT also affects allergen-specific T cells. In both skin and the
identify features that are biologically important and thus to devise nose, successful SIT is accompanied by a reduction in T-cell
new forms of immunotherapy that may improve efficacy, increase and eosinophil recruitment in response to allergen challenge. In
safety margins, shorten treatment courses, or achieve more durable parallel, the balance of T-helper cell-1 (Th1) and Th2 cytokine
results from SIT. At least three distinct phases can be identified expression (Chapter 16) alters at allergen-challenged sites. Th2
2
in SIT, each with distinct immunological mechanisms. Initially, cytokine expression is not affected, but an increased proportion
there is pharmacological desensitization, in which basophils and of the recruited T cells express the Th1 cytokines interleukin-2
mast cells are rendered tolerant of the allergen. This is achieved (IL-2), interferon-γ (IFN-γ), and IL-12. In addition, there is induc-
quickly, within a few days if the rush protocol is followed. Clini- tion of allergen-specific CD4 Tregs that express CD25, FOXP3,
2
cally, this not only allows the patient to tolerate the maintenance and IL-10. IL-10 is induced within a few days of starting SIT
dose of allergen but also protects against acute exposure to allergen and has a number of biological effects that could explain the
(e.g., a wasp sting). This state of pharmacological tolerance is beneficial effects of immunotherapy. These include modulation of
not permanent and rapidly disappears if SIT is stopped. During IL-4–induced B-cell IgE production in favor of IgG4, inhibition of
the first year of maintenance SIT, T-cell tolerance is achieved, IgE-dependent mast cell activation, inhibition of human eosino-
with abrogation of late-phase responses to allergen exposure phil cytokine production and survival, suppression of IL-5, and
and induction of regulatory T cells (Tregs). Finally, with prolonged induction of antigen-specific anergy (Fig. 91.1). Taken together,
treatment, B-cell tolerance develops, and the level of sensitizing these findings suggest that SIT modulates allergen-specific T
antibodies decreases. cells, which explains why clinical and late-phase responses are
Following subcutaneous injection of allergen extracts, a small attenuated without much effect on allergen-specific antibody
proportion of the allergenic material is taken up by phagocytic levels. Further work in this area is now concentrating on finding
cells and carried to regional lymph nodes. The process is fairly more efficient ways of inducing allergen-specific Tregs. Some
inefficient: <1% of a radiolabeled dose actually reaches the lymph caution is needed: Most of the evidence of Treg induction is from
nodes. Almost all studies have shown that SIT induces allergen- peripheral blood rather than from target organs, and although




Allergen immunotherapy Degranulation of mast cells


- IL-4
Th2 IL-13 Th2 B cell IgE
+
IL-5 Eosinophil
-
+
Th0 Allergic mucosal
inflammation -
- and symptoms
Th1 IL-12, αIFN
-
+ IgG, IgA, blocking Ab’s
T cell ‘anergy’


IL-10
Tr cells TGF-β Th2 B cell

-
FIG 91.1 Immunological mechanisms of specific immunotherapy.

CHaPtEr 91 Immunotherapy of Allergic Disease 1229


there are group effects, there is no direct relationship to clinical factors to consider are the potential risks of emergency treat-
efficacy in individuals. ment with epinephrine and medical contraindications to VIT
(see below). Large local reactions to stings are not regarded as
SIT for Venom Anaphylaxis indications for VIT, as they are not life-threatening and do not
Anaphylaxis to Hymenoptera venom is relatively rare but can predict future problems, but there is some evidence they can be
be fatal. Venom-specific IgE antibodies can be found in 30–40% attenuated by VIT. 6
of adults for a few months following a sting, but usually these VIT accelerates the process of risk reduction and confers rapid
disappear subsequently. Some individuals develop high concentra- protection against field and laboratory stings. There is a low risk
tions of venom-specific antibodies, which may persist for many of systemic reaction (≈10%) for many years after discontinuing
years without further stings. This group of patients is at risk of VIT. Although most reactions to stings after completing VIT are
anaphylaxis to subsequent stings: a small number die of ana- mild, fatalities have been reported in patients with other risk
phylaxis each year. Fatality rates are hard to estimate, perhaps factors (mastocytosis, severe historical reactions, systemic reaction
10–20 deaths/year in the United States. during VIT). This raises the question whether VIT should be
Before embarking on venom immunotherapy (VIT), the life long, although lifelong treatment would alter the cost–benefit
patient needs to be carefully assessed, and some account should analysis. In children, the chance of systemic sting reactions remains
5
be taken of the natural history of the venom allergy. Patients <5% for up to 20 years after discontinuing VIT.
who have experienced systemic symptoms after stings are at In summary, desensitization with venom preparations provides
much greater risk of anaphylaxis on subsequent stings, compared protection against field and laboratory stings, but some patients
with patients who have only had large local reactions. The fre- will still react despite completing VIT. The residual risk of systemic
quency of systemic sting reactions in children and adults with reactions to stings after VIT is about 10%, but these are usually
a history of large local reactions is about 5–10%, whereas the mild. Patients undergoing VIT for venom anaphylaxis are usually
risk in patients with previous systemic reactions is about 30–70%. offered injectable epinephrine and other antiallergic medication
In general, there is a lower risk of repeated systemic reactions to use if stung during VIT, but this is unnecessary once they are
in children and in those with a history of milder reactions. In on maintenance doses.
adults, the risk of systemic reaction to field stings diminishes
over 10–20 years toward 15–30% (Fig. 91.2), but does not return Assessing Effectiveness in Clinical Trials of SIT for
to the background general population prevalence (3%). Children Asthma and Rhinitis
with a history of cutaneous systemic reactions had <5% risk of The two main keys to success in designing any clinical trial are
anaphylaxis during observation for 10–20 years. No test can application of appropriate criteria for subject recruitment and
accurately predict the outcome of the next sting. Live sting selection of appropriate clinical endpoints (Table 91.1). In allergic
challenges have been used in research but are not practical or rhinitis, efficacy is generally assessed in terms of symptom scores,
7
acceptable in clinical practice. Unfortunately, sting challenges recorded daily on diary cards or personal organizers. Several
are not absolutely predictive, since patients who do not react to symptom scales are in use, which all rely on a categorical grading
laboratory challenge stings may react to subsequent field stings. system (e.g., 1 = mild, 2 = moderate, 3 = severe, etc.). These
In deciding to recommend VIT, account should be taken of measures carry the implicit assumption that the step from 1 to
occupational and geographical factors that affect the likelihood of 2 is clinically equivalent to the step from 2 to 3. By assigning
future stings. Bee stings are much more common in beekeepers, numerical values to nonparametric variables, one may either
their families, and neighbors, whereas wasp stings tend to be overestimate or underestimate the true effects of therapy. Reduc-
sporadic but are an occupational hazard for bakers, gardeners, tion in rescue medication use is also measured, as, in theory,
outdoor caterers, greengrocers, and other similar trades. Other symptoms can improve because patients use more medication.
In practice, most studies that showed improved symptom scores
showed parallel reductions in medication use, but some studies
70 Hx + Untreated showed reductions in medication use without a statistically
VIT 1-2 years
Risk of systemic reaction (%) 40 TABLE 91.1 Endpoints for trials of
60
VIT 5 years
50
Specific Immunotherapy in asthma
30
Spirometry
Air flow obstruction (forced expiratory volume per
second [FEV 1 ], peak expiratory flow rate [PEFR])
20
+
SPT
Bronchial irritability (histamine/methacholine PC 20 )
10
Wheeze, nocturnal waking, cough,
Symptoms
breathlessness, time off work/school (diary
0 cards or visual analogue scales)
-2 0 2 4 6 8 10 12 14 Drug use Bronchodilator use as surrogate for symptoms
Cost–benefit ratio Reduction in other drugs, lost work, etc.
Years compared with costs of specific-allergen
FIG 91. 2 Risk of systemic reaction (SR) from wasp stings over immunotherapy (SIT) and time lost due to SIT
time after an anaphylactic event, and the effect of specific-allergen Safety Frequency of local and systemic adverse events
immunotherapy for 1–2 years or 5 years on the natural history. Immunology Allergen-specific immunoglobulin G4 (IgG4)
VIT, venom immunotherapy. (Adapted from Golden DB, et al. Skin test responses
Survey of patients after discontinuing venom immunotherapy. Nasal allergen challenge responses
J Allergy Clin Immunol 2000; 105: 385–90.) Inhalation allergen challenge responses

1230 Part tEN Prevention and Therapy of Immunological Diseases


significant reduction in symptoms. Combining symptom and not used in routine clinical practice but may be useful for assessing
medication scores is fraught with difficulty. It is almost impossible effectiveness in clinical trials.
to agree on equivalences between different drugs, let alone the The effectiveness of SIT in seasonal allergic rhinitis has been
relative value of one antihistamine tablet versus a day when confirmed in many trials, using grass, ragweed, and birch pollen.
symptoms are moderate rather than mild. Moreover, SIT has been shown to be clinically effective even in
When assessing improvement in asthma, it is important to patients with severe seasonal rhinitis that is resistant to conven-
10
remember that the majority of patients with atopic asthma have tional drug therapy. Data regarding the long-term efficacy of
relatively mild disease that is usually easy to control with standard SIT for allergic rhinitis are limited, but formal studies have shown
11
doses of conventional antiasthma drugs. Cases of severe asthma that the effects last for at least 3 years after discontinuing therapy.
are relatively few in number, and the role of allergy in driving Longer-term studies are difficult to conduct, but efficacy has
their disease is often less obvious than in mild cases. In mild been shown for up to 10 years in open studies. The benefits of
cases, resting spirometry will usually be close to predicted values, SIT for perennial rhinitis are less well established than for seasonal
so there is little scope for improvement in simple spirometric rhinitis. In part, this reflects the difficulty in determining the
measures, such as forced expiratory volume per second (FEV 1 ). extent to which allergy is responsible for perennial symptoms.
Peak expiratory flow rates (PEFRs) may show improvement after Allergy to house dust mite (HDM) is common, but only about
SIT, especially the early morning values, which are more likely 50% of those with positive skin tests have symptoms. Conversely,
to be reduced below the patient’s best possible value compared there are other causes of perennial rhinitis, including vasomotor
with evening peak flow rates. Objective measures of bronchial instability, infection, aspirin sensitivity, and so on. Nevertheless,
irritability are more useful markers of improvement and a shift clinical trials have shown a definite benefit provided the subjects
12
in the concentration of histamine or methacholine needed to are appropriately selected. Clearer evidence of efficacy has been
induce a 20% fall in FEV 1 (PC 20 ) is commonly used. This measure- obtained in rhinitis caused by pet allergy. Several studies have
ment can be quite variable within individuals, so the minimum shown a marked improvement in tolerance of cat exposure after
change that would be clinically significant is a change of at least SIT, confirmed both on challenge tests and simulated natural
one doubling dilution in the group geometrical mean PC 20 . Other exposure. 13
bronchial challenge tests, such as exercise challenge or adenosine As with any therapy, the risks and cost-effectiveness of SIT
inhalation, may provide additional insights into the ability of need to be assessed on a case-by-case basis. Current drug therapy
an antiasthma treatment to prevent acute attacks. Both these for rhinitis can be very effective, but about 60% of patients with
parameters depend indirectly on the PC 20 , so again, it is important seasonal allergic rhinitis report inadequate symptom control,
to consider statistical and biological significance separately. even when taking maximal doses of antihistamines and intranasal
Although these challenge models are useful surrogates for glucocorticoids. Others experience nose bleeds caused by intra-
evaluating antiasthma treatment, more weight should be placed nasal steroids and drowsiness caused by antihistamines. Moreover,
on improvements in the symptoms or progression of the disease we are now more aware of the adverse effects of rhinitis on
than on protection against laboratory challenges. Lately, there quality of life. SIT offers a useful option for these patients as
has been a move to assess efficacy in terms of improvements in well as a logical approach to dealing with the underlying problem.
quality of life or health gain per unit cost. These analyses are
becoming increasingly sophisticated but require careful validation SIT for Asthma
to ensure that the quality-of-life instrument is sensitive to the Immunotherapy has been widely used to treat allergic asthma,
illness being studied and that the economic model is realistic. but evidence of severe adverse reactions, including a small number
Finally, many trials of SIT for asthma and rhinitis have studied of fatalities, has led to SIT being abandoned for asthma treatment
laboratory surrogate markers, such as changes in IgG subclasses, in the United Kingdom since 1986, although asthma remains a
T-cell function, or skin test responses. Although such paraclinical common indication for SIT in North America and continental
measures do change during SIT and can shed light on the Europe. 12,14 There is no doubt that SIT is effective on asthma
mechanisms of successful SIT, they cannot be used to provide symptoms and protects against specific and nonspecific triggers.
direct evidence of clinical efficacy. Studies that show antibody However, the role of allergic sensitization in ongoing asthma is
or T-cell changes without direct clinical evidence of efficacy less clear than for rhinitis, and the risks of therapy are undoubtedly
neither support nor undermine the established evidence that greater in asthma. Current drug therapies for asthma aim to
SIT works, albeit in mysterious ways, to improve the symptoms suppress the airways inflammation and smooth muscle contrac-
of appropriately selected patients. Recent research on paraclinical tion, which are characteristic features of asthma. None of these
measures has identified four areas that may prove useful in future: treatments is curative, and asthma recurs rapidly upon cessation
8
dendritic cell (DC) markers in sublingual immunotherapy (SLIT) ; of treatment. Moreover, current drug therapies are directed against
innate lymphoid cells (ILCs) favoring Th2 development (ILC-2), downstream mechanisms rather than against the agents that
which have been show to decrease in frequency after successful might cause asthma. Allergen avoidance has been proposed as
SIT; decreased expression of diamine oxidase—the enzyme a potentially useful maneuver in those with allergic asthma;
responsible for making histamine; and IgG antibodies that block however, although asthma control can be improved by extreme
IgE-mediated functional antigen presentation. 9 forms of allergen avoidance (e.g., admission to hospital, sending
children to holiday homes at altitude), there is little evidence
SIT for Allergic Rhinitis that similar benefits can be achieved by using the type of allergen
Allergic rhinitis is the main indication for SIT worldwide. As avoidance that can be achieved in suburban homes. Thus there
with all uses of SIT, it is important to select patients appropriately. is scope for improving asthma care and for identifying allergen-
The allergic basis of the rhinitis should be carefully assessed specific therapies. SIT offers the possibility of diverting the
during history taking and skin or blood tests for IgE, and other immune response away from the allergic pattern and toward a
relevant causes should be excluded. Tests of nasal sensitivity are more protective or less damaging response.

CHaPtEr 91 Immunotherapy of Allergic Disease 1231


The efficacy of SIT in adult asthma has been assessed in effect on surrogate markers suggests that: (i) current forms
many trials over the last 60 years. The results of these studies of oral immunotherapy (OIT) for food allergy are achieving a
are difficult to interpret, either because poor quality allergen temporary remission; (ii) strategies that are more efficacious
extracts were used or because of poor study design. Many trials against basophil reactivity may offer a more sustained state of
15
were not placebo controlled; they were either open or single- tolerance. In parallel, work is ongoing to develop injection SIT
blind studies; and in most cases, only small numbers of patients for fish allergies. 16
14
were treated. A 2010 meta-analysis reviewed all of the relevant
papers published between 1954 and 2008. Many of the earlier Comparison of SIT With Other Therapies
trials had methodological flaws, but there are sufficient data The majority of clinical trials of SIT for asthma have compared
for general conclusions to be drawn. Symptom scores improved SIT either with untreated historical controls or with a matched
in the treated groups—it was necessary to treat four patients placebo-treated group. To date, the effectiveness of SIT in asthma
to prevent one from experiencing exacerbation of symptoms, has rarely been compared with conventional management (avoid-
and to treat five to prevent one from needing an increase in ance measures and inhaled or oral antiasthma drugs).
medication. SIT reduced the airways response to inhalation
of specific allergen and also improved nonspecific bronchial Effects of SIT on the Natural History of Allergic Disease
reactivity. In recent years, there has been increasing interest in the possibility
In double-blind placebo-controlled (DBPC) trials in patients that SIT may have disease-modifying or preventive effects. These
with grass pollen asthma, active treatment led to a 60–75% are important not only in their own right as desirable outcomes
reduction in symptom scores compared with placebo-treated but also as factors in the economic evaluation of SIT.
patients. Similar results were found in ragweed allergy with Children often start with a limited range of allergic sensitivities
improved peak flow rates during the pollen season and reduced and progress over time to develop IgE against a wider range of
hay fever symptoms and sensitivity to laboratory challenge with inhaled allergens. Treatment with SIT may limit this tendency
ragweed pollen extracts. However, the associated economic analysis to acquire new sensitizations, although the clinical benefit of
indicated that the cost saving in asthma drugs was less than that this preventive effect is not clear. In the most widely cited study,
in SIT. The value of SIT is thus dependent on its ability to improve children with asthma who were sensitized only to HDM were
symptoms and quality of life rather than to reduce the costs of treated with HDM-SIT. Of the 19 treated children, 10 did not
other medications. develop new sensitizations, whereas all 22 untreated children
In patients with asthma who are sensitive to cats, SIT with acquired new sensitivities, in many cases to more than one new
17
standardized aqueous extracts reduces the early asthmatic response allergen. A long-term study of treatment with a grass pollen
to inhaled allergen and attenuates responses to room exposure allergoid has confirmed this observation, with 50% fewer new
18
to cats. Interestingly, these studies showed a clear delay in onset sensitizations 12 years after completion of the course of SIT.
of symptoms and an overall reduction in symptoms and peak Similar results have been found in a study of children treated
flow recordings after simulated exposure to cats, but there was with SLIT, in which 3% of SLIT-treated children and 34% of
19
no protection against allergen-induced increases in nonspecific control subjects developed new sensitizations within 3 years.
bronchial hyperresponsiveness. Another study of cat SIT for It seems probable that this effect is indirect. In other words,
asthma using a depot preparation found a reduction in both it is unlikely that HDM-SIT directly affects the B cells that
specific and nonspecific bronchial reactivity (to cat extract and recognize cat or grass pollen, but by treating the HDM allergy,
histamine, respectively). There is, however, no hard evidence SIT may reduce inflammation in the nose and hence modify
that SIT is of benefit to treat asthma caused by dog allergy. the likelihood that exposure to other allergens will lead on to
Several DBPC studies have examined the effects of SIT in sensitization.
patients with asthma who are sensitive to HDM. Early studies Each year, a proportion of patients with allergic rhinitis go on
used tyrosine-adsorbed or depot preparations, which yielded to develop asthma. This rate of progression has been estimated
20
conflicting results, with reductions in drug requirements in at 5% per annum in college students, but this remains, perhaps
children but no improvement in asthma control in adults. surprisingly, an area of considerable uncertainty. Several long-term
Subsequent studies using more modern extracts have found epidemiological studies are now in progress, under the auspices
beneficial effects on symptoms, antiasthma medication use, and of the International Study of Asthma and Allergies in Childhood
bronchial hyperresponsiveness. (ISAAC), and these should eventually shed light on the rate
of progression at different ages and the extent of regional and
SIT for Food Allergy international variation. It has been suggested that SIT may modify
After several false starts, research has now shown that it is possible the natural history of asthma in children who have allergic
to tolerize patients to food allergens, such as peanut. Arguments rhinitis but have not yet developed asthma. Data to support
continue over what to call this, as the tolerance achieved seems this proposition are limited. In the key study, a group of 205
to be temporary, and patients become fully sensitive again if children aged 6–14 years, without previously diagnosed asthma,
they stop treatment. However, the possibility to render patients were treated with SIT for birch or grass pollen allergy in an
with life-threatening food allergy tolerant of the relevant food open randomized design. Three years after completing treatment,
is a useful step forward. In line with the clinical time course, it 45% of the untreated group had developed asthma, whereas
has been shown that basophil sensitivity is suppressed early during only 26% of the treated group had asthma. These results have
peanut tolerization but returns to baseline when treatment is been sustained out to 7 years after completing therapy. Thus in
stopped. In contrast, in patients undergoing SLIT for aeroallergen terms of number needed to treat, four children had to be treated
sensitivity their basophil reactivity takes longer to drop away to prevent one case of asthma, which makes this an extremely
21
after starting treatment, but once achieved, the effect on basophils effective therapy. Similar results have been reported with SLIT:
is sustained for months or years after ceasing SLIT. This differential After 3 years of SLIT, 8 of 45 SLIT-treated children and 18 of 44

1232 Part tEN Prevention and Therapy of Immunological Diseases


control subjects had developed asthma, representing a relative risk intercurrent viral illness, so doses should be delayed if the patient
of 3.8 for untreated subjects compared with the actively treated is unwell or has any signs of active asthma.
group. 22 On a separate note, there is general agreement that SIT should
SIT may also modify the progression of established asthma. not be used in patients with autoimmune disorders or malignant
An early open study using uncharacterized mixed allergen extracts disease. Although there is no hard evidence that SIT is actually
supported this view, with about 70% of treated children losing harmful in these groups, it seems unwise to attempt manipulation
their asthma after 4 years therapy, compared with about 19% of the immune system in such patients, not least because of the
of untreated controls, a result that was sustained up to the age risk that spontaneous and unrelated variations in the autoimmune
of 16 years. The proportion of children whose asthma was severe disorder or cancer may be blamed on SIT. Other medical con-
23
at age 16 years was also much lower in the treated group. In traindications to SIT include significant coexistent cardiac disease,
this period, when clinical trials were conducted differently, well which may be exacerbated by any adverse reactions to SIT. Patients
before the days of ethics committees, Johnstone and Dutton on beta-blockers should also not receive SIT. Although they are
randomized all children attending their clinic between August not at increased risk of adverse reactions, their physiological
1953 and January 1955 to one of four treatments. Neither the response to the cardiovascular component of anaphylaxis will
subjects nor their parents were aware of the treatment being be impaired, and they will not respond to the epinephrine that
given. Compared with those who received placebo or very dilute is used to treat adverse reactions to SIT.
allergens, those treated with the two higher doses of SIT were
much more likely to lose their asthma after 4 years, and among SUBLINGUAL IMMUNOTHERAPY
those whose asthma persisted, the likelihood of having severe
disease was much lower in those who had received active treat- High-dose topical immunotherapy regimes were used in the
ment. Although it would be very difficult to conduct such a first half of the twentieth century but then lost ground to injection
study today, the data are useful in confirming the dose–response immunotherapy in conventional medical practice. Over the past
relationship in SIT and its potential for reducing the duration 20 years, there has been a resurgence of interest in SLIT, which
and severity of asthma. A prospective, nonrandomized, open-label is based on the concept that allergens given via the mucosal
study of SLIT has reported that children with asthma who received surface can induce immunological tolerance, without carrying
SLIT had less asthma compared with those on standard medica- the same risk of systemic reactions that is found with injection
tion after 4–5 years, and this difference persisted for 5 years after SIT. Moreover, SLIT is more appealing to patients, especially to
cessation of treatment. 24 those who cannot easily find time to attend clinic for injection
27
In contrast, there is no current evidence that SIT influences SIT. In animal models, IgE responses to allergens can be reduced
the evolution of established asthma in adults. In part, this reflects or prevented by oral administration of allergen. Most of these
the reluctance of physicians to use SIT to treat patients with models involve feeding in early life to prevent subsequent acquisi-
severe asthma. Studies that have investigated withdrawal of therapy tion of sensitivity, so this is a different scenario from the usual
have reported rapid recurrence of asthma symptoms, although clinical context of treating established allergic disease. The precise
rhinitis symptoms seem to show much more sustained relief mechanisms by which “oral tolerance” is induced remain unclear,
after SIT. 25 but it seems likely that the route of allergen processing and
In summary, there is persuasive evidence that both subcutane- presentation is a critical determinant of the subsequent T-cell
ous and sublingual immunotherapy can modify the course of response (Chapter 6). In mice, locally administered allergen is
allergic disease, by reducing the incidence of new sensitizations, taken up by mucosal DCs and then presented to T cells together
and by preventing or slowing the development of clinical asthma. with IL-12, biasing the response toward a Th1-like profile and
The mechanisms that underlie these observations are not fully away from the pro-IgE Th2 profile. In contrast to the animal
understood. It is likely that a combination of immunological models, the immunological response to SLIT in human studies
and structural alterations is involved. Economic evaluations of has been relatively modest. Some changes have been found in
the benefits of SIT and SLIT need to take on board these preven- skin sensitivity, but most studies have not found any change in
tive effects to arrive at a full cost–benefit analysis. systemic parameters, such as specific IgE, specific IgG, or T-cell
cytokine balance. 28
SAFETY Nevertheless, a body of evidence has accumulated from well-
conducted clinical trials, indicating that SLIT can be effective,
The main factor preventing the wider adoption of SIT is the with up to 30–40% reductions in symptom scores and rescue
29
risk of serious adverse reactions. In general, SIT is quite safe in medication use in seasonal allergic rhinitis. SLIT treatment
patients who do not have asthma, but a significant number of regimes typically involve a rapid buildup phase followed by
deaths have been reported in the United Kingdom and the United treatment about three times per week with rapidly dissolving
States when SIT was used to treat patients whose asthma was tablets containing allergen extracts. Some preparations are sup-
26
unstable. The incidence of systemic reactions in patients receiv- plied in liquid form, with a calibrated dropper. Several large
ing SIT for asthma varies among study series and has been clinical trials of SLIT have shown clinically and statistically
reported to range from 5% to 35%. In contrast, serious systemic significant reductions in symptom and medication scores, sup-
reactions occur after about 1 in 500 injections in patients with porting the conclusions of previous meta-analyses of SLIT, which
rhinitis. Some adverse events are caused by administration of assessed the earlier, less well-designed studies that were individu-
the wrong dose, which underscores the importance of checking ally small and inconclusive. The meta-analysis estimated the
doses carefully before each injection. Others occur during efficacy of SLIT as being about two-thirds of that seen in
changeover from one vial to another. Most allergists, therefore, comparable studies of injection SIT, but the most recent studies
cut back on the dose when changing vials to minimize this risk. have shown levels of efficacy that are equal to the best recent
Systemic reactions are much more likely if the patient has an multicenter trials of injection SIT. However, the efficacy may be

CHaPtEr 91 Immunotherapy of Allergic Disease 1233


30
greater for pollens than for HDM allergies. Local side effects be possible to manipulate allergens and reduce their ability to
are common with SLIT but are generally well tolerated. Systemic bind to IgE or to create peptide fragments, which will modulate
side effects of SLIT are very rare but have been reported, especially T cells without the risk of anaphylaxis. Cross-linking allergen
in patients who have previously had systemic reactions to injection proteins with aldehydes reduces their ability to bind antibodies.
SIT. SLIT is now being used routinely in some parts of Europe Such allergoids do not degranulate the basophils of sensitized
(especially Italy and France), but the doses and regimes being individuals in vitro but are recognized by allergen-specific T
prescribed are often different from those that were used in the cells. Clinically, allergoids have shown efficacy in rhinitis caused
clinical trials. Overall SLIT should widen the scope of SIT and by grass pollen or HDM. 36
allow more patients to be treated. As with all forms of immu- Short peptide sequences that should not be recognized by
35
notherapy, patient selection remains the key to ensuring that IgE antibodies can be used as vaccines as well. Peptide vaccines
therapy is targeted to those who are likely to benefit from it. can be either natural sequences or altered peptide ligands. If
high doses of natural peptides are given, these deceive the T cell
FUTURE DIRECTIONS into high dose tolerance. Altered peptide ligands induce anergy
by providing an incomplete activation signal. Both approaches
There is clearly scope to improve conventional SIT. Possible are affected by the MHC type of the individual undergoing
avenues include the use of recombinant allergens, which should treatment. By sequential alteration of HDM allergen Der p
make it easier to standardize allergen vaccines and may allow peptides, it is possible to suppress proliferation of T-cell clones
fine-tuning of vaccines for patients with unusual patterns of recognizing native Der p peptides, as well as suppressing their
reactivity. Most patients with allergies react to the same com- expression of CD40 ligand and their production of IL-4, IL-5,
ponents of an allergen extract, the so-called major allergens, and IFN-γ. These anergic T cells do not provide help to B cells
which are defined as those allergens recognized by over 50% of to switch class to make IgE; importantly, this anergy cannot be
sera from a pool of patients with clinically significant allergy to reversed by providing exogenous IL-4. In humans, the main
the material in question. However, not all patients recognize all focus has been on cat allergen (Fel d1) peptides, which can reduce
37
major allergens, and some patients only recognize allergens that the level of symptoms on exposure to cat dander. Similar studies
are not recognized by the majority of sera of patients with allergies. with the peptides of phospholipase A2 (PLA2, a major allergen
This latter group may not respond to standard extracts but might in bee venom) have also been reported. However, to date, peptide
be better treated by a combination of allergens to which they vaccines have not shown any greater efficacy compared with
are sensitive. Until the advent of molecular cloning, this has conventional vaccines.
been impossible to achieve. Now that recombinant allergens for In an animal model, intranasal application of genetically
SIT are available, the range of sensitivities can be better character- produced hypoallergenic fragments of Bet v1 produced mucosal
ized, and this may lead to patient-tailored vaccine products. tolerance with significant reduction of IgE and IgG1 antibody
Thus far, clinical trials have confirmed that recombinant allergen responses, as well as reduced cytokine production in vitro (IL-5,
cocktails are effective, but these have not yet shown superiority IFN-γ, IL-10). These reduced immunological responses were
31
over conventional vaccines. Regulations are the major barrier accompanied by inhibition of the cutaneous and airway responses
to this development: Under current regulations, each combination that were seen with the complete Bet v 1 allergen. The mechanisms
of allergen components would have to be developed as a freestand- of immunosuppression seemed to be different for the allergen
ing pharmaceutical. Until a different regulatory framework is fragments and the whole molecule, in that tolerance induced
agreed upon, recombinant allergens will only be tested as fixed with the whole Bet v 1 molecule was transferable with spleen
combination cocktails. cells, whereas that induced by the fragments was not. 38
Another alternative approach is to inject allergens directly From epidemiological and experimental studies, we know
into lymph nodes, under ultrasound guidance. Proponents that vaccines with mycobacteria have antiallergic properties. In
of this approach argue that after subcutaneous injection, less Japan, early vaccination with the Bacille Calmette-Guérin (BCG)
than 1% of the injected material reaches the lymphatic system; vaccine was associated with a substantial reduction in the risk
39
therefore if the benefit of SIT is driven by the dose, then direct of developing allergy, although similar associations were not
40
intranodal administration should be more efficient and also safer evident in studies in Sweden. In an animal model, administration
than conventional SIT. Early studies with doses of about 1% of of the BCG vaccine before or during sensitization to ovalbumin
standard SIT showed immunological efficacy, with some signs reduced the degree of airway eosinophilia that followed subse-
32
of clinical efficacy and reduced risks of side effects. However, quent challenge with ovalbumin. This effect was not mediated
subsequent clinical trials by other groups have not demonstrated through any direct effect on IgE production or blood eosinophil
efficacy. 33,34 numbers but was mediated through IFN-γ and could be reversed
Novel forms of allergenic molecules may be created: for by exogenous IL-5. 41
example, a recombinant trimer consisting of three covalently Two new approaches using DNA vaccines are also undergoing
linked copies of the major birch pollen allergen, Bet v 1 has been serious consideration. The first of these is a general approach,
made. This trimer is much less allergenic, even though it contains using CpG oligodeoxynucleotides (ODN), which mimic bacterial
the same B-cell and T-cell epitopes as the native molecules and DNA, binding to T-cell receptor-9 (TCR-9) and stimulating a
induces Th1 cytokine release and IgG antibodies, analogous to Th1-type cytokine response. This technology is essentially a
35
the antibody response to standard SIT. Folding variants and refinement of vaccination adjuvant technology, since CpG ODN
other modifications of the physical structure may also improve have been shown to be the principal activity from complete
the safety of SIT. Freund adjuvant. In a mouse model of asthma, preadministration
Since the epitopes recognized by IgE molecules are usually of CpG ODN prevented both airway eosinophilia and bronchial
three-dimensional, while epitopes recognized by T cells are short hyperresponsiveness. Moreover, these effects were sustained for
42
linear peptide fragments of the antigen (Chapter 6), it should at least 6 weeks after CpG ODN administration. Alternatively,

1234 Part tEN Prevention and Therapy of Immunological Diseases


CpG ODN can be coupled to the allergenic protein, which Please check your eBook at https://expertconsult.inkling.com/
enhances immunogenicity in terms of eliciting a Th1-type for self-assessment questions. See inside cover for registration
response to the allergen but reduces its allergenicity and stimulates details.
Th1 cytokine expression in cultured human peripheral blood
43
mononuclear cells (PBMCs). Initial clinical trials confirmed
44
that the hybrid vaccine elicits a Th1-pattern response, but the REFERENCES
results of subsequent trials have been inconclusive. A contrasting
approach is immunization with allergen-specific naked DNA 1. Frew AJ. 100 years of immunotherapy. Clin Exp Allergy 2011;41:1221–5.
2. Jutel M, Akdis CA. Immunological mechanisms of allergen-specific
sequences. This technology is still in its infancy, but preliminary immunotherapy. Allergy 2011;66:725–32.
data suggest that administration of naked DNA leads to produc- 3. Creticos P, Van Metre TE, Mardiney MR, et al. Dose-response of IgE and
45
tion of allergens from within the airway epithelial cells. It seems IgG antibodies during ragweed immunotherapy. J Allergy Clin Immunol
that the endogenously produced allergen elicits a Th1-type 1984;73:94–104.
response, due to the different handling pathways for endogenous 4. Iliopoulos O, Proud D, Adkinson NF, et al. Effects of immunotherapy on
and exogenous allergens. If this can be reproduced in humans the early, late and rechallenge nasal reaction to provocation with allergen:
with allergies, this may overcome the existing Th2-pattern changes in inflammatory mediators and cells. J Allergy Clin Immunol
response and eliminate the allergy. However, the potential for 1991;87:855–66.
generating a powerful Th1-type response to ubiquitous agents 5. Golden DB, Moffitt J, Nicklas RA, et al. Stinging insect hypersensitivity:
means that this approach needs careful evaluation in animal a practice parameter update 2011. J Allergy Clin Immunol 2011;127:
852–4.
models before it can be pursued in humans. 6. Golden DB, Kelly D, Hamilton RG, et al. Venom immunotherapy reduces
The recent introduction of monoclonal antibodies (mAbs) large local reactions to insect stings. J Allergy Clin Immunol
directed against IgE offers another option. Treatment with anti-IgE 2009;123:1371–5.
reduces immediate and late-phase responses to inhaled allergens 7. Bousquet J, Schünemann HJ, Bousquet PJ, et al. How to design and
and should also reduce the risk of adverse effects from SIT evaluate randomized controlled trials in immunotherapy for allergic
injections. Moreover, when anti-IgE is combined with conven- rhinitis: an ARIA-GA(2)LEN statement. Allergy 2011;66:765–74.
46
tional SIT, the effects on seasonal allergic rhinitis are additive. 8. Gueguen C, Bouley J, Moussu H, et al. Changes in markers associated
However, the high cost of anti-IgE and the need for regular with dendritic cells driving the differentiation of either TH2 cells or
injections are likely to limit its use to patients who have severe regulatory T cells correlate with clinical benefit during allergen
immunotherapy. J Allergy Clin Immunol 2016;137:545–58.
allergic disease that cannot be managed by other means, and 9. Shamji MH, Ljorring C, Francis JN, et al. Functional rather than
these patients are not generally suitable candidates for SIT. immunoreactive levels of IgG4 correlate closely with clinical response to
grass pollen immunotherapy. Allergy 2012;67:217–26.
CONCLUSIONS 10. Frew AJ, Powell RM, Corrigan CJ, et al. Efficacy and safety of specific
immunotherapy with SQ allergen extract in treatment-resistant seasonal
allergic rhinoconjunctivitis. J Allergy Clin Immunol 2006;117:319–25.
ON tHE HOrIZON 11. Durham SR, Walker SM, Varga EM, et al. Long-term clinical efficacy of
grass pollen immunotherapy. N Engl J Med 1999;341:468–75.
New Technologies for Immunotherapy 12. Cox L, Esch RE, Corbett M, et al. Allergen immunotherapy practice in the
• Recombinant allergens United States: guidelines, measures, and outcomes. Ann Allergy Asthma
• T-cell peptide vaccines Immunol 2011;107:289–99.
• T-helper cell-1 (Th1) immunostimulants (e.g., mycobacteria, CpG) 13. Varney VA, Edwards J, Tabbah K, et al. Clinical efficacy of specific
• Allergen-immunostimulant complexes immunotherapy to cat dander: a double blind placebo controlled trial.
• Anti–immunoglobulin E (IgE) Clin Exp Allergy 1997;27:860–7.
• New routes of administration: 14. Abramson MJ, Puy RM, Weiner JM. Injection allergen immunotherapy
• Sublingual for asthma. Cochrane Database Syst Rev 2010;(8):CD001186.
• Intralymphatic 15. Gorelik M, Narisety SD, Guerrerio AL, et al. Suppression of the
• Epicutaneous immunologic response to peanut during immunotherapy is often
• Liposome encapsulation transient. J Allergy Clin Immunol 2015;135:1283–92.
16. Zuidmeer-Jongejan L, Huber H, Swoboda I, et al. Development of a
hypoallergenic recombinant parvalbumin for first-in-man subcutaneous
SIT has been established as a treatment for allergic rhinitis and
for venom hypersensitivity but is more controversial for treating immunotherapy of fish allergy. Int Arch Allergy Immunol
2015;166:41–51.
allergic asthma. When used in appropriately selected patients, 17. Des Roches A, Paradis L, Menardo JL, et al. Immunotherapy with a
SIT is effective and acceptably safe, but care must be exercised standardized Dermatophagoides pteronyssinus extract. VI. Specific
in recognizing and treating adverse reactions. Appropriate training immunotherapy prevents the onset of new sensitizations in children. J
of allergists and SIT clinic support staff is essential. Despite a Allergy Clin Immunol 1997;99:450–3.
century of use, the precise mechanisms of action of SIT remain 18. Eng PA, Borer-Reinhold M, Heijnen IA, et al. Twelve-year follow-up after
uncertain. Current emphasis on the role of Tregs is leading to discontinuation of pre-seasonal grass pollen immunotherapy in
renewed attempts to simplify SIT regimes and reduce its risks. childhood. Allergy 2006;69:198–201.
Future directions in SIT include the development of vaccines 19. Marogna M, Tomassetti D, Bernasconi A, et al. Preventive effects of
that are better standardized and the use of recombinant allergens, sublingual immunotherapy in childhood: an open randomized controlled
study. Ann Allergy Asthma Immunol 2008;101:206–11.
both of which should improve the safety profile of SIT. In parallel, 20. Horak F. Manifestation of allergic rhinitis in latent sensitised patients. A
and perhaps for the longer term, we should explore the develop- prospective study. Arch Otorhinolaryngol 1985;242:242–9.
ment of general immunomodulatory therapies, which would be 21. Niggemann B, Jacobsen L, Dreborg S, et al. Five-year follow-up on the
particularly advantageous for those patients sensitized to multiple PAT study: specific immunotherapy and long-term prevention of asthma
allergens. in children. Allergy 2006;61:855–9.

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22. Novembre E, Galli E, Landi F, et al. Coseasonal sublingual 36. Corrigan CJ, Kettner J, Doemer C, et al. Efficacy and safety of
immunotherapy reduces the development of asthma in children with preseasonal-specific immunotherapy with an aluminium-adsorbed
allergic rhinoconjunctivitis. J Allergy Clin Immunol 2004;114:851–7. six-grass pollen allergoid. Allergy 2005;60:801–7.
23. Johnstone DE, Dutton A. The value of hyposensitization therapy for 37. Norman PS, Ohman JL, Long AA, et al. Treatment of cat allergy with
bronchial asthma in children. A 14 year study. Pediatrics 1968;42:793–802. T-cell reactive peptides. Am J Respir Crit Care Med 1996;154:1623–8.
24. Di Rienzo V, Marcucci F, Puccinelli P, et al. Long-lasting effect of 38. Wiedermann U, Herz U, Baier K, et al. Intranasal treatment with a
sublingual immunotherapy in children with asthma due to house dust recombinant hypoallergenic derivative of the major birch pollen allergen
mite: a 10-year prospective study. Clin Exp Allergy 2003;33:206–10. Bet v 1 prevents allergic sensitization and airway inflammation in mice.
25. Shaikh WA. Immunotherapy vs inhaled budesonide in bronchial asthma: Int Arch Allergy Immunol 2001;126:68–77.
an open, parallel, comparative trial. Clin Exp Allergy 1997;27:1279–84. 39. Shirakawa T, Enomoto T, Shimazu SI, et al. The inverse association
26. Bernstein DI, Wanner M, Borish L, et al. Twelve-year survey of fatal between tuberculin responses and atopic disorder. Science 1997;275:77–9.
reactions to allergen injections and skin testing: 1990-2001. J Allergy Clin 40. Strannegaard IL, Larsson LO, Wennergren G, et al. Prevalence of allergy
Immunol 2004;113:1129–36. in children in relation to prior BCG vaccination and infection with
27. Frew AJ. Sublingual immunotherapy. N Engl J Med 2008;358:2259–64. atypical mycobacteria. Allergy 1998;53:249–54.
28. Scadding G, Durham SR. Mechanisms of sublingual immunotherapy. 41. Erb KJ, Holloway JW, Sobeck A, et al. Infection of mice with
Immunol Allergy Clin North Am 2011;31:191–209. Mycobacterium bovis-BCG suppresses allergen-induced airways
29. Calderon MA, Penagos M, Sheikh A, et al. Sublingual immunotherapy for eosinophilia. J Exp Med 1998;187:561–9.
allergic conjunctivitis: Cochrane systematic review and meta-analysis. 42. Sur S, Wild JS, Choudury BK, et al. Long-term prevention of allergic lung
Clin Exp Allergy 2011;41:1263–72. inflammation in a mouse model of asthma by CpG
30. de Bot CM, Moed H, Berger MY, et al. Sublingual immunotherapy is not oligodeoxynucleotides. J Immunol 1999;162:6284–93.
effective in house dust mite-allergic children in primary care. Pediatr 43. Marshall JD, Abtahi S, Eiden JJ, et al. Immunostimulatory sequence DNA
Allergy Immunol 2012;23:150–8. linked to the Amb a 1 allergen promotes Th1 cytokine expression while
31. Pauli G, Larsen TH, Rak S, et al. Efficacy of recombinant birch pollen downregulating Th2 cytokine expression in PBMCs from human patients
vaccine for the treatment of birch-allergic rhinoconjunctivitis. J Allergy with ragweed allergy. J Allergy Clin Immunol 2001;108:191–7.
Clin Immunol 2008;122:951–60. 44. Creticos PS, Eiden JJ, Broide D, et al. Immunotherapy with
32. Senti G, Prinz Vavricka BM, Erdmann I, et al. Intralymphatic allergen immunostimulatory oligonucleotides linked to purified ragweed Amb a 1
administration renders specific immunotherapy faster and safer: a allergen: effects on antibody production, nasal allergen provocation and
randomized controlled trial. Proc Natl Acad Sci USA 2008;105:17908–12. ragweed seasonal rhinitis. J Allergy Clin Immunol 2002;109:743–4.
33. Witten M, Malling HJ, Blom L, et al. Is intralymphatic immunotherapy 45. Hartl A, Hochreiter R, Stepanoska T, et al. Characterisation of the
ready for clinical use in patients with grass pollen allergy? J Allergy Clin protective and therapeutic efficiency of a DNA vaccine encoding the
Immunol 2013;132:1248–52. major birch pollen allergen Bet v1a. Allergy 2004;59:65–73.
34. Patterson AM, Bonny AE, Shiels WE, et al. Three-injection intralymphatic 46. Rolinck-Werninghaus C, Hamelmann E, Keil T, et al. The co-seasonal
immunotherapy in adolescents and young adults with grass pollen application of anti-IgE after preseasonal specific immunotherapy
rhinoconjunctivitis. Ann Allergy Asthma Immunol 2016;116:168–70. decreases ocular and nasal symptom scores and rescue medication use in
35. Valenta R, Campana R, Focke-Tejkl M, et al. Vaccine development for grass-pollen allergic children. Allergy 2004;59:973–9.
allergen-specific immunotherapy based on recombinant allergens and
synthetic allergen peptides: Lessons from the past and novel mechanisms
of action for the future. J Allergy Clin Immunol 2016;137:351–7.

CHaPtEr 91 Immunotherapy of Allergic Disease 1235.e1


MUL t IPLE-CHOICE QUES t IONS

1. Successful immunotherapy may work through induction of: 3. Changes in allergen-specific immunoglobulin G4 (IgG4):
A. B-cell tolerance A. Reflect the dose of allergen injected
B. Allergen-specific immunoglobulin A (IgA) antibodies B. Predict response at the individual level
+
C. FOXP3 regulatory T cells C. Are caused by induction of interleukin-17 (IL-17)
+
D. Tryptase mast cells D. Can be used to predict clinical efficacy
2. Venom immunotherapy: 4. Immunotherapy is a proven treatment for allergy to:
A. Prevents large local reactions A. Dog dander
B. Accelerates the rate of tolerance B. Walnuts
C. Confers long-lasting efficacy after 1 year of treatment C. Hen’s egg
D. Permanently prevents systemic reactions D. Tree pollen

92









Flow Cytometry



Sergio D. Rosenzweig







Flow cytometry has become a standard laboratory tool in the activation and cell-mediated cytotoxicity studies, cell cycle analysis,
evaluation of hematopoietic cells, including the identification apoptosis detection, and multimer technology, focusing on the
of leukocyte populations and subpopulations, a method referred appropriate application of these approaches as well as their
to as immunophenotyping. The clinical application of this technol- limitations.
ogy has been facilitated by the development of instruments and
data analysis systems suitable for routine use in diagnostic labo- INSTRUMENTATION
ratories. In addition, the expanded range of monoclonal antibodies
(mAbs) specific for lymphocyte (and other hematopoietic cell) The basic components of a flow cytometer, as shown in Fig.
surface antigens directly conjugated to a number of different 92.1, include an illumination source, an optical bench, a fluidic
1
fluorescent indicators (fluorochromes) provide an extensive panel system, electronics, and a computer. Briefly, stained cells flow
of reagents that facilitate multicolor (polychromatic) studies. into single file by the fluidic system and are interrogated by one
The clinical needs that pushed this technology date back to or more light sources; these sources generate light signals, which
the emergence of absolute CD4 T-cell counts as a critical measure are directed by the optical system to the photodetectors, which,
for disease assessment and follow-up in managing patients infected in turn, convert light into electronic signals for storage and
with human immunodeficiency virus (HIV). Flow cytometry subsequent analysis. This process is discussed further in the
applied in the monitoring of HIV infection was followed by the section below.
routine application of cell characterization by flow cytometry The fluidic system lies at the heart of the flow cytometer and
in the evaluation of hematologic malignancies and, more recently, consists of isotonic sheath fluid that moves the sample stream
in the study of immunodeficiency disorders and other immune- containing the cells. This is accomplished by injecting the cell
mediated diseases. sample into flowing sheath fluid, establishing a hydrodynamically
Recent advances in instrumentation and fluorochrome focused single-file flow of cells (particles) that move through
chemistry now allow for routine polychromatic flow cytometry the analysis point while maintaining the cell stream in a constant,
2
studies, with concomitant assessment of cell surface markers central location. The centrally focused cell stream ensures that
and intracellular parameters, including intracellular proteins, the illumination of all cells is virtually equivalent. Thus the
phosphoproteins, and cytokines, as well as identification of difference in magnitude of the emission signal(s) generated from
changes linked to cellular activation and apoptosis. Intracellular each cell reflects biological differences between the cells (rather
flow cytometry also can be applied to evaluate cell cycle status than reflecting the variation in the illumination energy if the
(i.e., G 0-G 1, S, G 2 -M) based on DNA staining, which is useful in cells were not tightly focused). The use of hydrodynamic focusing
evaluating tumor cells and assessing the in vitro lymphocyte has the additional advantage of producing little or no change
response to various stimuli. Additionally, evaluation of lymphocyte in cell shape, although it may have an effect on cell orientation.
proliferation can be performed with cell tracking dyes that allow The consistency in maintaining cell shape facilitates distinguishing
quantitation of the rounds of cell division associated with cell “architectural” differences between specific leukocyte types (see
3
activation, and techniques to assess immune cell mediated Gating section). However, this method can generate single-file
cytotoxicity have also been developed. Finally, characterization cell rows with precision only up to a flow rate of 60 to 100 µL/
of antigen-specific T cells following immunization or associated min, which can lead to long acquisition times for the detection
with normal and/or abnormal immune responses in association of very rare events. To overcome this problem, recently introduced
with disease states can be accomplished by using multimer flow cytometry instruments utilize acoustic focusing, which align
technology as well as intracellular cytokine detection following cells through the use of sound waves, allowing sample flow rates
antigen exposure. of up to 1000 µL/min, without loss of signal quality. 4,5
This chapter focuses on the basic concepts of flow cytometry, Illumination in standard clinical instruments is gener-
including instrument characteristics, data management, lym- ated by two or more lasers, each of which provides a specific
phocyte gating, and directed use of test reagents. In addition, it monochromatic light source (e.g., a sapphire laser generates a
provides a brief overview of intracellular protein detection, cell 488-nm-wavelength [blue] beam). Modern lasers are small and
available in multiple wavelengths, including ultraviolet (350 nm),
violet (405 nm), blue (488 nm), green (532 nm), yellow (560 nm),
This work was supported in part by the Intramural Research Program orange (610 nm), and red (633 nm), permitting the simultane-
of the National Institutes of Health Clinical Center. ous use of multiple fluorochromes having different excitation

1239

1240 Part ElEvEn Diagnostic Immunology



Photodetectors: 1000
side scatter signal
Filters Fluorescent signals
PerCP 800 Granulocytes
PE
Laser FITC 600
light source Flow cell
Photodetector:
forward scatter signal SSC-H: SSC-Height 400 Monocytes





Focused cell stream 200
FIG 92.1 Simplified Design of A Flow Cytometer with One
Illumination Source (Laser) Set Up to Collect Five Parameters. Lymphocytes
These include the two nonfluorescent parameters (blue light)
forward and side scatter, as well as three fluorescent parameters, 0 0 200 400 600 800 1000
green (FITC), orange (PE), and red (PerCP) light.
A FSC-H: FSC-Height


6
requirements. The point where the light illuminates the cell in 10 5
analytical instruments occurs within a flow cell, while in cell
sorters, the beam intersects cells flowing as a stream in air. The
optical bench contains lenses that shape and focus the illumination
beam to ensure consistent excitation energy at the analysis point. 10 4 Monocytes
The illumination of a cell generates both nonfluorescent and
fluorescent signals, which are collected and measured by optically
coupling the signal to a detection system consisting of filters,
each of which is linked to a photodetector. The filters are chosen <FITC-A>:CD14 10 3
to allow the nonfluorescent signals to be measured at the same Granulocytes
wavelength as the excitation signal (e.g., 488 nm from a blue
light source) for the forward- and side-scatter channels (see
Gating section), whereas those for the fluorescence channels 10 2
utilize specific filters that allow passage of light with wavelengths Lymphocytes
specific to each fluorochrome (e.g., green, orange, or red; see
Fluorochrome section). The number and arrangement of the
photodetectors allows for the simultaneous evaluation of multiple 10 0
colors (parameters) for each cell, with a report describing a 0 10 2 10 3 10 4 10 5
modified clinical instrument capable of evaluating 17 or more B
colors simultaneously from each cell evaluated. 7 <APC-efluor 780-A>:CD45
The internal electronics in the flow cytometer provides the FIG 92.2 (A) Forward- and side-scatter dot plots on a lysed
system for converting analog light signals (photoelectrons) whole blood sample, demonstrating the basic three-part leukocyte
received at the photodetectors into digital signals for acquisition differential with lymphocytes, monocytes, and granulocytes.
and storage in a computer. The intensity of these converted (B) Dot plot with DC45/CD14 gating reagents showing the fluo-
signals is measured on a relative scale that is generally set in rescence distribution of all the three leukocyte types identified
either 256 or 1024 equal increments (referred to as channels) to include lymphocytes, monocytes, and granulocytes, as well
for display and analysis. A number of specialized analysis programs as a small number of nonlysed red blood cells and/or debris.
are available, and results are depicted graphically as single-
parameter histograms displaying specific light (fluorescence)
intensity (x-axis) versus cell number (y-axis) (Fig. 92.2), or individual cell contained within a large number of cells present
two-color displays where the x-axis and the y-axis reflect the in the test sample, and these are typically accrued at rates of
light intensity of the two colors, and the cell numbers are rep- 1000–2000 (or more) cells per second.
resented via dot, pseudocolor, contour, or density plots (Fig.
92.3). Most analysis programs enable the operator to evaluate FLUORESCENCE REAGENTS
the number and percentage of events, mean and/or median
channel fluorescence, and selected statistical measures for each Standard mAb reagents for clinical use are typically directly
identified cell, and these can be aggregated into specific popula- conjugated to a fluorochrome, a dye that absorbs and emits light
tions and/or subpopulations of cells. Thus a flow cytometer of different wavelengths based on the energy lost during the
provides a platform with the capacity to assess multiple pieces return of excited electrons to their ground state associated with
of discrete information (parameters) generated from each illumination by a specific wavelength of light. Thus the emitted

CHaPtEr 92 Flow Cytometry 1241


light has a longer wavelength (lower energy) than the wavelength subpopulation is an accurate measurement. Without gating, data
of the excitation beam. The number of commercially available can also be negatively impacted by coexpression of surface antigens
fluorochromes has increased dramatically with the routine use on different cell lineages (e.g., CD4 is found on lymphocytes
8
of dye conjugates and instruments with three or more lasers. and monocytes at various densities). In addition, nonspecific
Commonly used fluorochromes in clinical immunophenotyping binding of monoclonal reagents through Fcγ receptors, and the
include the organic dyes fluorescein isothiocyanate (FITC), level of cytophilic human immunoglobulin (Ig), varies between
phycoerythrin (PE), peridin chlorophyll protein (PerCP), and cell types, making appropriate gating crucial to generate valid
allophycocyanin (APC). Conjugations of PE and APC to cyanines data. These techniques are also used to focus the evaluation on
(Cy5, Cy5.5, and Cy7) and Alexa Fluor dyes produce tandem other hematopoietic cells, including monocytes, granulocytes,
dyes with additional emission spectra, based on energy transfer eosinophils, erythrocytes, and platelets.
from one fluorochrome to the second fluorochrome serving as Initial gating to focus on a specific leukocyte population
the source of emitted light. This allows for the simultaneous typically involves the use of two nonfluorescent parameters,
3
evaluation of 6–8 colors in most current clinical instruments forward scatter (FSC) and side scatter (SSC) (see Fig. 92.2A).
with only two or three lasers. FSC is a reflection of cellular cross-sectional area (direct relation-
One recent advance in the field was the development of a ship to cell size), whereas SSC is an indication of the cellular
new class of inorganic fluorescent semiconductor nanocrystals, granularity. The combination of these two nonfluorescent
9
named quantum dots (QDs). These particles are perfectly suited parameters provides a three-part differential in red blood cell
for polychromatic flow cytometry, as they have broad excitation (RBC)–lysed whole blood that distinguishes between normal
spectra (525–800 nm) and sharp, discrete emission spectra that lymphocytes, monocytes, and granulocytes. As can be seen in
9
varies depending on their core size. This means that QDs of Fig. 92.2A, among leukocytes, lymphocytes have the lowest FSC
different sizes (and consequently of different colors) can be excited and SSC, monocytes have higher FSC and SSC, and granulocytes
9
by the same laser source, allowing simpler multiplexing. In have the greatest SSC. This method is effective in distinguishing
addition, QDs have high quantum yield, high molar extinction a relatively pure population of lymphocytes under most circum-
coefficients, and extraordinary resistance to photodegradation stances. However, the presence of nucleated RBCs, large platelets,
and chemical degradation. These qualities make them perfectly basophils, or other particulate debris can produce contaminating
suitable for use in biological studies, including intracellular in events (cells) within this “lymphocyte gate.” Furthermore,
vivo imaging, fluorescence resonance energy transfer (FRET) malignant or activated lymphoid cells may not fit into the previ-
analysis, and dynamic imaging of single proteins for longer ously outlined standard light scatter patterns.
periods. 9 A method for confirming the integrity of the light scatter–based
Additional dyes are available for functional studies and include lymphocyte gate uses the directly conjugated monoclonal “gating”
13
calcium-sensitive dyes (e.g., fluo-3), glutathione-sensitive dyes reagents anti-CD45 and anti-CD14. These two mAbs more
(e.g., monochlorobimane), and hydrogen peroxide (H 2 O 2 )–respon- accurately identify the three-part differential. Lymphocytes have
sive dyes (e.g., dihydrorhodamine 123 [DHR123]). 10,11 Assessment the highest level of CD45 binding but are negative for CD14;
of DNA content can be performed with dyes that intercalate granulocytes have a lower level of CD45 binding and an intermedi-
double-stranded DNA and RNA, including propidium iodide ate level of CD14 expression; and monocytes have high levels
12
(PI) and ethidium bromide. In addition, there are ultraviolet- of both CD45 and CD14 expression (see Fig. 92.2B). Importantly,
excited dyes that are highly specific for DNA, including Hoechst nonleukocytes, including erythrocytes and platelets, are negative
33258 and 4,6-diamidino-2-phenylindole (DAPI); acridine orange for these markers. However, malignant leukocytes that have
is used for simultaneous staining of DNA/RNA. 12 characteristics of early precursor cells often have altered CD45
and/or CD14 expression, which must be recognized when studying
DATA ANALYSIS hematological malignancies. Gating reagents provide a reliable
means of checking the light scatter–based lymphocyte gate for
Gating the frequency of nonlymphocytes within the gate as well as the
extent of lymphocyte exclusion from the gate. Guidelines for an
KEY COnCEPtS acceptable degree of contamination within the lymphocyte gate,
Gating as well as the level of lymphocyte exclusion, are contained in
the US Clinical and Laboratory Standards Institute guideline
14
• Method for defining cell population of interest for lymphocyte immunophenotyping. With the expanded use
• Typically performed using forward scatter and side scatter and lineage- of polychromatic flow cytometry, some centers now include
specific antibodies anti-CD45 in every tube to refine the gate and prevent cell
contamination as described above.
Data Display
The proper assessment of specific cell types within a mixture
requires initial identification of lineage-specific cells, an approach KEY COnCEPtS
referred to as gating. In practical terms, immunophenotyping Data Presentation
focused on lymphocytes requires minimizing the nonlymphocytes
included in the evaluation, and this is accomplished by lymphocyte • Fluorescence intensity is plotted versus cell number.
gating. The standard sample for clinical studies is anticoagulated • Flow cytometry can present cumulative data on more than one
whole blood; directing the study to lymphocytes requires eliminat- parameter for each cell.
ing the great majority of nonlymphocytes from the collected • Multicolor data presentation can increase cell subpopulation
resolution.
data such that the expression of a percentage for a specific cell

1242 Part ElEvEn Diagnostic Immunology


The simplest method for demonstrating flow cytometry data is cellular autofluorescence together with any nonspecific binding
the single-parameter histogram (see Fig. 92.2), a graphic presenta- of the monoclonal reagent(s), and the magnitude of both
tion of cell number on the y-axis versus fluorescence (light) phenomena varies among different cell types. Interpretation of
intensity from a single fluorochrome on the x-axis. Integration data is simplified when there are two distinct cell populations
of curve areas provides the number of cells, and often there are (i.e., negative and positive), but the evaluation of two overlapping
two distinct distributions, one referred to as negative identifies distributions is more difficult.
cells that are not bound specifically by the monoclonal reagent, Multiparameter data can be evaluated by using a series of
and the second represents cells bound by the antibody. Negative single-parameter histograms that consider each fluorochrome
distribution actually reflects low-level fluorescence resulting from independently. However, it is more informative to present two
parameters simultaneously by using a correlated display (Fig.
92.4), and two-color displays are recommended for clinical flow
15
cytometry. This approach enables the simultaneous visualization
+
−
+
−
−
+
+
-
of four different populations: A /B , A /B , A /B , and A /B . More
recently, these displays were developed further to include a mixture
of logarithmic (for higher intensity expression) and linear (for
lower intensity expression) intensity for each axis to allow for
300 better interpretation of events with very low, zero, or negative
fluorescence. This combined-display approach resolves the
previous problem of a large number of events being displayed
compacted against the axes, even with properly compensated
samples, and will be used in the illustrations throughout this
# Cells 200 CD3+ chapter. 16
The simultaneous use of n monoclonal reagents can identify
n
a total of 2 subpopulations. These different subsets can be
CD3- identified sequentially by first dividing the cells into those that
are positive versus those that are negative with one reagent and
100
then evaluating the defined subpopulations for the remaining
two reagents using a two-color approach. Alternatively, more
modern software can represent multiple populations as poly-
17
chromatic plots, which can simplify data analysis. The poly-
chromatic approach can provide a means to further resolve
0 subpopulations and has been particularly useful in the evaluation
0 10 2 10 3 10 4 10 5
of cellular differentiation, activation, and functional correlates,
<FITC-A>:CD3 as well as clarifying overlapping cell subpopulations.
FIG 92.3 Single-parameter histogram for CD3 expression on
lymphocytes demonstrating the negative, non–T-cell population Positive–Negative Discrimination
(B cells, natural killer [NK] cells), and a positive T-cell population. The evaluation of clinical immunophenotyping data requires
Integrating the area under each curve would provide the numbers establishing criteria for the boundaries between negative or
and percentage of cells present in each respective subpopulation. nonstained (autofluorescence plus nonspecifically stained)


10 5 10 5 10 5
A-B+
10 4 4 4
<APC-A>: CD8 10 <APC-A>: CD8 10
A+B+
10 3 10 3 10 3


10 2 A-B- 10 2 10 2
0 A+B- 0 0
B
0 10 2 10 3 10 4 10 5 0 10 2 10 3 10 4 10 5 0 10 2 10 3 10 4 10 5
A
FIG 92.4 Examples of Dot Pseudocolor (Left), Density (Middle), and Contour (Right) Displays
Based on the Same Two-Color Parameter Data. All three techniques enable simultaneous
evaluation of both parameters, in this case evaluating the expression of markers A and B. These
plots identify four populations of cells, those expressing only A or B, those expressing both A
and B (very few), and those expressing neither A nor B.

CHaPtEr 92 Flow Cytometry 1243


cells and positive (specifically stained) cells. A commonly used laboratories by the Clinical Laboratory Improvement Amendment
approach involves using directly conjugated control mAbs of of 1988 (CLIA 88).
the appropriate class or subclass (e.g., IgG1, IgG2a, IgG2b, or
IgM) that do not specifically react with human lymphocyte Methods
surface antigens (commonly referred to as “isotype controls”). Whole-blood lysis represents the most common technique used
The marker (discriminator) is set at the fluorescence histogram for sample preparation and consists of mixing a fixed volume
channel number such that it includes 98–99% of the negative of anticoagulated whole blood (or bone marrow) with one or
cells (Fig. 92.5A). more directly conjugated mAbs, followed by incubation at a
20
As previously noted, negative refers to the aggregate of baseline designated temperature and time. Next, RBCs are lysed, and
cellular autofluorescence plus nonspecific reagent binding, and the sample is washed and then run into the flow cytometer,
this can vary according to the cell type. For this reason, the use of usually following fixation in paraformaldehyde to reduce the
isotype controls may not correctly identify the positive–negative risk of infection. Nonlysed cells that remain include all peripheral
threshold for specific cell types, particularly when staining dimly blood leukocytes as well as any nonlysed RBCs, platelets, and
expressed proteins. Additionally, to perfectly mimic the specific debris. The heterogeneity of the sample necessitates careful
antibody used, the isotype control would need to have the same lymphocyte gating (see Gating section above) to generate accurate
antibody to fluorochrome ratio and brightness, something immunophenotyping data. The advantages of the whole-blood-
that is not easily accomplished. To overcome these difficulties, lysis method include fewer preparation steps, less sample handling,
an alternative method for positive–negative discrimination, and a lower likelihood of differential lymphocyte loss that can
fluorescence-minus-one (FMO), has been developed. 18 occur when density gradient techniques are used to prepare
FMO involves the staining of the sample with all the antibodies mononuclear cells for analysis. Alternative sources of cells (e.g.,
of interest, except the one targeted for positive–negative threshold. bone marrow, bronchial alveolar lavage fluid, fine-needle aspirates)
21
As an example, to define the negative threshold for the protein can also be evaluated with flow cytometry. Patient studies must
+
−
FAS (CD95) in CD8 and CD8 T cells, the FMO control tube be determined with the same methods and reagents as used in
would include the cell subset-specific markers (CD3, TCRαβ, the determination of the control ranges to ensure comparability.
and CD8) and omit anti-FAS. After appropriate gating on that The number of events (cells) analyzed typically ranges from
population, the threshold can be adequately defined, and it is 10 000–20 000 in routine clinical studies but must be increased
different for the two exemplified populations (see Fig. 92.5B, when evaluating very small subpopulations of cells to produce
right panel). One obvious limitation of this method is the higher statistically relevant data necessary in rare-event analysis.
cost, given that multiple control tubes need to be set up for each The application of control ranges must take into account the
sample. fact that significant changes take place in lymphocyte distribu-
tion and development during childhood, as well as changes in
Compensation lymphocytes that occur among the very old. 22,23 In addition,
The fluorescence signals emitted by different fluorochromes since immunophenotypic differences can be induced by drugs,
are not completely separated by the filters. This can lead to including tobacco products, information on current medications
signal overlap, which is corrected by electronic subtraction of should be obtained, whenever possible. Other factors can also have
the overlapping signal, a process referred to as compensation. an impact on lymphocyte distribution, including race, gender,
The overlap is particularly significant when using multiple diurnal variation, and recent or intercurrent infections. 24
18
fluorochromes, each with different spectral properties. The The choice of immunophenotyping reagents depends on the
compensation process involves subtraction of the “spillover” signal cells being targeted for study and the question being asked.
detected by the photodetector generated by samples stained with However, regardless of the specific setup, the inclusion of a tube
only one fluorochrome. Currently, most flow cytometry analysis with gating reagents (anti-CD45 and anti-CD14) to confirm the
13
software products allow for off-line compensation, where the integrity of the standard lymphocyte gate is recommended. In
single reagent stained tubes are used to create a compensation addition, control reagents should be included to establish the
matrix, which is then applied to all the tubes in the experiment. fluorescence intensity of negative cells. Important internal controls
This allows for much simplified compensation procedures, include a pan-T-, B-, and natural killer (NK)-cell marker for
without the need for any hardware compensation during data every sample (Table 92.1), based on the principle that the whole
collection. is the sum of its parts. Thus the total percentage of lymphocytes
in the gate determined by the gating reagents should approxi-
Quality Control mately equal the sum of the percentages for T, B, and NK cells.
Quality control is a critical component of clinical flow cytometry A technical or biological explanation must be identified when
19
to ensure optimal results. This includes monitoring instrument this relationship does not hold. Biological explanations for a
setup and performance, optimizing sample preparation and significant difference would include the presence of immature
reagents, and standardizing controls and data interpretation. or malignant cells that were not identified by standard pan-T-,
Quantitative flow cytometry based on a fluorescence standard B-, and NK-cell reagents. Also, if the gating reagents (CD45/
curve provides quantitative data in units referred to as molecular CD14) had not been included, contaminating cells (e.g., myeloid
equivalents of soluble fluorochrome (MESF). When properly precursors, nucleated RBCs, large platelets) with FSC and SSC
constructed standard curves are used, quantitative data for characteristics similar to those of lymphocytes could not be ruled
different reagents can be generated and compared. Finally, out. Potential technical problems include reagent or fluorochrome
participation in interlaboratory proficiency testing surveys, such degradation, failure to add a reagent, and a host of others. Evidence
as the triannual samples provided by the College of American for any major technical error should result in repeating the study.
Pathologists (CAP), is an important additional measure to monitor Additional data that can be used for controls depend on the
laboratory performance, and this is mandated in US clinical setup. For example, the availability of multiple antibodies that

1244 Part ElEvEn Diagnostic Immunology


Isotype control

10 4 10 4
0.26 0.09 12.1 5.76


10 3 10 3
FITC-A: IgG FITC-A 10 2 FITC-A: HLA-DR FITC-A 10 2







10 1 10 1



99 0.62 26 56.2
10 0 10 0
10 0 10 1 10 2 10 3 10 4 10 0 10 1 10 2 10 3 10 4
PE-Cy7-A: IgG PE-Cy7-A PE-Cy7-A: CD3 PE-Cy7-A


FMO control

20.0 10 4 20.0 10 4
20.0 20.0
Lymh gate 10 3 CD3+ Lymh gate 10 3 CD3+
20.0 20.0
Title Title 10 2 Title Title 10 2
20.0 20.0
10 1 10 1
20.0 20.0
0 0 0 0
0 30.0 30.0 30.0 30.0 30.0 0 10 1 10 2 10 3 10 4 0 30.0 30.0 30.0 30.0 30.0 0 10 1 10 2 10 3 10 4
Title Title Title Title
10 4 10 4




10 3 0.43 10 3 78.9 10.1
PE-A: subclass PE-A 10 2 0.37 PE-A: FAS PE-A 10 2







10 1 10 1




10 0 10 0
10 0 10 1 10 2 10 3 10 4 10 0 10 1 10 2 10 3 10 4
APC-A: CD8 APC-A APC-A: CD8 APC-A
FIG 92.5 Positive–Negative Discrimination Strategies. (A) Nonspecific antibodies (isotypes)
were used to stain the sample shown on left and the positivity threshold determined and applied
to the sample on the right. (B) Fluorescence-minus-one (FMO). The positivity threshold was
determined by staining the sample on the left with the population-specific markers (CD3, TCRαβ,
and CD8) and omitting FAS. The panel on the right contains FAS. Note that the positivity threshold
is slightly different for CD8 and CD8 cells.
+
−

CHaPtEr 92 Flow Cytometry 1245



TABLE 92.1 Selected lymphocyte Surface reporting of both percentages and absolute numbers is necessary
antigens for Immunophenotyping when immunophenotyping peripheral lymphocytes.
The objective of evaluating malignant cells is often to character-
t Cells ize the lineage and differentiation level of the abnormal cells,
Pan-T cell: CD3, CD2, CD7, CD5 rather than quantifying subpopulations. The pattern of reactivity
Major T-cell subset: CD4, CD8 combined with fluorescence intensity is often useful in identifying
Surface antigens associated with function: CD28, CD31, CD38,
CD45RA, CD45RO, CD62L, CXCR7 leukemic patterns, whereas the absolute number of cells usually
Activation antigens: CD25, CD40L, CD69, CD71, HLA-DR is not required. However, flow-cytometric detection and quantita-
tion of rare abnormal cells can be useful in evaluating for minimal
B Cells residual disease after therapy in lymphoproliferative disorders.
Pan-B cell: CD19, CD20, surface immunoglobulin
Major B-cell subset: CD5, CD21 PRACTICAL APPLICATIONS OF FLOW CYTOMETRY
Surface antigens associated with function: CD27, CD40, CD80, CD86
Activation antigens: CD23, CD25 Immunophenotyping Studies
nK Cells
Pan-natural killer (NK) cell: CD16, CD56 ClInICal rElEvanCE
NK subset: CD2, CD8, CD57 Immunophenotyping Studies
• Immunotyping studies can be used to identify cell subsets, lineage,
stage of cell differentiation, state of cell activation, and clonality.
• Lymphocyte results should be checked with T cells + B cells + natural
identify a similar cell subpopulation can serve as a useful check killer (NK) cells = 100%.
(e.g., total T cells by comparing CD3 and CD5 or CD2; total B • Immunophenotyping studies are not the equivalent of lymphocyte
function studies.
cells by comparing CD19 and CD20). In addition, the use of
specific reagents in >1 tube enables comparison between the
repeat values as a measure of consistency. The application of Most immunophenotyping studies aim to enumerate specific
internal checks should be performed by the flow operator as a cell subpopulations, evaluate for the presence or absence of
simple means of confirming the validity of the data. Insights particular surface antigens, identify the differentiation level of
regarding unusual biological findings may also be uncovered specific cells, determine cell lineage, evaluate for functional
through this type of evaluation (e.g., the presence of an increased correlates based on specific antigen expression, examine evidence
−
−
population of CD4 /CD8 double-negative T cells). 25 of cell activation, and/or establish evidence of monoclonality.
The challenge in performing immunophenotyping is to Quantification of a particular cell subpopulation can be readily
accurately identify cells with specific surface characteristics accomplished by flow cytometry together with a WBC count
(antigens). As previously noted, the capacity to discriminate cell and cell differential. The absolute lymphocyte count is used to
subpopulations is often enhanced through the directed use of generate population or subpopulation cell numbers based on
antibody combinations. The typical data generated consist of the percentages of the respective cell types generated with the
the percentage of negative versus positive cells when using one flow cytometer. The evaluation of absolute CD4 T-cell counts has
26
reagent and multiple subpopulations when using more than one formed the basis for monitoring patients with HIV infection.
+
reagent. Regardless of the experimental design, it is important The quantitation of CD34 hematopoietic stem cells (HSCs) in
to consider not only the percentage of cells within each subpopula- donor peripheral blood or bone marrow is used in many cellular
27
tion but also the absolute number of cells. This is most commonly reconstitution protocols. Subpopulation characterization can
obtained by multiplying the relevant percentage from the flow also be useful in the evaluation of patients with clinical history
28
cytometer by the absolute lymphocyte count obtained by using and laboratory findings suggestive of immune deficiency.
a white blood cell (WBC) count and differential. For example, This has taken on a higher level of significance in the setting
+
when assaying for CD4 T-cell counts, the percentage of CD4 of newborn screening for severe T-cell immune deficiency via
cells is multiplied by the peripheral absolute lymphocyte count the T-cell receptor excision circle (TREC) assay, which, when
to yield the absolute CD4 count. A potential problem with this abnormal, is typically followed by immunophenotyping to
approach is the requirement for two separate procedures (i.e., evaluate for naïve T cells based on CD45RA, CD127, or CD62L
dual platforms) to generate the final result. This introduces the expression.
possibility of additive error, based on the inherent errors of the When evaluating for the presence or absence of surface proteins
two different methods. It also has fueled a search for approaches associated with specific functional attributes, it is important to
that facilitate performing both tasks by flow cytometry (i.e., a realize that this approach does not assess the actual functional
single platform). One alternative involves the inclusion of a fixed status of cells. This point is clearly illustrated by the finding of
number of fluorescent beads (in a defined volume) in each tube normal B-cell numbers in most patients with common variable
as a reference standard to generate absolute numbers without immune deficiency despite the fact that these patients fail to
29
requiring the use of the complete blood count (CBC) and dif- produce Igs normally. However, changes in the characteristics
ferential to generate a lymphocyte count. An alternative approach of the B cells, particularly relative to memory B cells, provides
involves the use of impedance-based cell counting in the flow potential insight into different phenotypes of this disorder and
cytometer to generate an absolute lymphocyte count (dependent provides additional support for heterogeneity of patients with
on a fixed volume of sample being run) and then generating the disease. 29-31 Because of the limitations of immunophenotyping,
both percentage and absolute number of cells in each specific when evaluating the status of the immune system, it is common
population or subpopulation. Regardless of the approach, the practice to perform cell function testing in parallel.

1246 Part ElEvEn Diagnostic Immunology


Flow cytometry can be used to test for the presence or absence CD62L, and CXCR7 and memory T cells that express the alterna-
of a specific cell surface antigen. An example of this type of tive CD45 isoform, CD45RO (and varied CD62L, or CXCR7,
application is in the evaluation of a patient with a history of depending on whether the cells are central or effector memory
35
recurrent skin infections, delayed wound healing, and persistent cells). In addition, memory B cells can be detected by the
granulocytosis, which suggests a diagnosis of leukocyte adhesion expression of CD27 and can be further divided into isotype-
deficiency type 1 (LAD-1). 28,32 This disorder results from a defect switched and isotype-nonswitched memory cells on the basis
in the gene encoding CD18, preventing the expression of three of their pattern of surface immunoglobulin expression. 29,36
different heterodimeric adhesion molecules (β 2 integrins) each Defects associated with familial lymphohistiocytosis (FLH)
containing CD18 (Chapter 22). This disorder can usually be are generally associated with abnormal NK cell function. Many
diagnosed by studying granulocytes (and lymphocytes) for the of the FHL-causing defects can be determined by flow cytometry.
expression of CD18 (as well as the three isoforms of CD11). For example, signaling lymphocyte activation molecule (SLAM)
Patients often have decreased rather than absent CD18 expression associated protein (SAP) and X-linked inhibitor of apoptosis
and confirmation of the diagnosis can be accomplished by (XIAP) intracellular staining can be used to evaluate for X-linked
demonstrating failure of CD18 (and CD11a, 11b, 11c) upregula- lymphoproliferative (XLP) disorders types 1 and 2, respectively.
tion following granulocyte activation. 28 Likewise, lack of intracellular perforin expression in NK cells
Immunophenotyping can also help address questions regarding would be indicative of hemophagocytic lymphohistiocytosis
the level of cell differentiation. Antibodies specific to proteins (HLH) type 2. Additionally, the evaluation of CD107a surface
expressed by early (precursor) cells represent one approach and expression, which is normally expressed on cytoplasmic granules
would include evaluating for the thymocyte marker CD1 or the and upon incubation with specific target cells (e.g., K562 cells)
pre-B-cell marker CD10 (CALLA), to cite a few. However, defining gets expressed on the surface of NK cells, is useful in determining
the developmental level of a particular cell population or sub- the underlying genetic defect causing FHL. 37,38 Specifically, lack
population is best accomplished by using a panel of reagents of CD107a upregulation is suggestive of syntaxin-11 or
that span the natural history of the cell lineage. This approach MUNC-13.4 defects.
represents the standard for testing leukemias and lymphomas.
In addition to defining the presence or absence of specific antigens, INTRACELLULAR EVALUATION
evaluating their level of expression is also valuable, which may
be altered in abnormal cells. Malignant cells may also express Cellular Activation
antigens associated with different lineages and have altered FSC
and SSC characteristics, as well as diminished or absent CD45 ClInICal rElEvanCE
expression, requiring modified gating approaches.
Issues of monoclonality can be dealt with by using flow Intracellular Flow Cytometry
cytometry when analyzing B cells and, in some circumstances, • Activation-directed studies:
when studying T cells. Normally, B cells are a heterogeneous • Calcium flux
mixture of mutually exclusive κ or λ light-chain-positive cells. • Intracellular protein phosphorylation
Measuring the distribution of κ or λ light-chain-expressing B • Oxidative burst: neutrophils
cells or plasmocytes can be informative with respect to the • Intracellular cytokine studies:
33
presence or absence of monoclonality. The capacity to evaluate • Clarify the T-helper-1 (Th1)/Th2/Th17 status of an immune response
T-cell monoclonality by flow cytometry is less definitive and • Can be assessed in an in vitro antigen-specific response
consists of using T-cell antigen receptor β-variable (Vβ) chain- • Can be combined with evaluation of cell surface studies
specific reagents looking for evidence of significant overrepre-
sentation of one Vβ chain family. This approach currently consists
of setting up a series of tubes, each with three different Vβ Ligand binding and transmembrane signal transduction resulting
family–specific mAbs, one conjugated with FITC, one with PE, in cellular activation can be evaluated by using flow cytometry.
2 +
and the third with FITC plus PE. This combination enables Changes in intracellular ionic calcium concentration (Ca ) can
distinguishing the frequency of each of the three different Vβ be used to monitor cell activation after ligand binding. These
+
+
+
+
families per tube (green , orange , green /orange ) and represents changes are associated with the activation of phospholipase C
a flow-cytometric method to complement polymerase chain and protein kinase C. In general, three reagents have been used
2 +
reaction (PCR)–based spectratyping. 34 to measure Ca : quin 2, indo-1, and fluo-3. Quin 2 has a low
The state of lymphocyte activation can be addressed by excitation coefficient and is not useful for flow cytometry; indo-1
evaluating for the presence of surface antigens that are found requires ultraviolet excitation; fluo-3 can be excited by 488 nm
only on activated cells or are upregulated following activation. but does not permit ratiometric analysis. Nevertheless, because
These include receptors for specific growth factors (e.g., of its ease of use, fluo-3 is currently the most widely used probe
2 +
interleukin-2 [IL-2] receptor α chain, CD25), receptors for critical for intracytoplasmic Ca evaluation by flow cytometry. Strict
elements required for cell growth (e.g., transferrin receptor, CD71), attention must be paid to loading conditions, the presence or
2 +
ligands for cell–cell communication following activation (CD40 absence of free Ca in the medium, experimental temperature,
ligand [CD152] on activated CD4 T cells), and surface antigens baseline measurements, and calibration. This approach can be
that are upregulated as a result of activation (e.g., adhesion combined with cell surface marker or cell cycle evaluation. 10
molecules, HLA-DR, CD69). In addition, the memory status of Intracellular pH changes related to cellular activation also can
10
both T cells and B cells can be assessed on the basis of differential be evaluated. The most useful probe for pH is SNARF-1. This
surface molecule expression associated with prior antigen probe can be excited at 488 nm and allows for ratiometric analysis
encounter. This enables a distinction to be made between naïve with detection wavelengths set for 575 and 640 nm. Glutathione
T cells that express CD45RA, CD31 (recent thymic emigrants), (glutamylcysteinylglycine [GSH]), an important antioxidant

CHaPtEr 92 Flow Cytometry 1247


generated during cell activation, that can be measured by using approach allows for simultaneous detection of two or more
10
flow cytometry. The fluorescent probe monochlorobimane is intracellular cytokines in combination with cell surface markers
commonly used for this measurement, but it is complicated by or other intracellular markers. Important aspects of intracellular
the need to determine GSH by an independent method, such as cytokine detection include use of a protein transport inhibitor
high-performance liquid chromatography (HPLC). during activation, use of proper controls, and choice of antibodies.
Additional approaches for evaluation of cellular activation As there is little or no spontaneous cytokine production in
include assessment of intranuclear markers (Ki-67, PCNA) as circulating human lymphocytes, intracellular cytokine detection
well as surface proteins that are upregulated following cellular requires in vitro activation. Initial experience was based on
39
activation (e.g., CD69, CD25, CD71). Actual cell division can supraphysiological stimulation with use of PMA and ionomycin,
be evaluated by using lipophilic membrane dyes (e.g., PKH26, but antigen-specific activation systems have also proven to be
CFSE, Cell Trace Violet), also referred to as cell tracking dyes, feasible. It should be emphasized that regardless of the activation
which lose 50% of their fluorescence with each round of cell method, the duration of activation is an important variable, as
40
division. This approach has become more common in the individual cells reach maximum cytokine production at different
clinical assessment of lymphocyte function because of the capacity times. In addition, different cytokines have different optimal
to evaluate specific lymphocyte subpopulations responding to periods of activation. It is recommended that a proper kinetic
mitogenic and antigenic stimuli. Lipophilic membrane dyes also profile be established for the biological system or clinical condition
41
can be used to label target cells in cell-based cytotoxicity assays. being studied. 47
Recently, an approach to evaluate lymphocyte proliferation To increase the amount of intracellular cytokines, inhibitors
following cell stimulation has been described by using the modi- of intracellular protein transport (e.g., monensin or brefeldin)
fied nucleoside EdU. Detection of DNA synthesis induced by are commonly used, which leads to accumulation of proteins
the different activating agents is measured using a copper-catalyzed within the cell. Nonspecific binding of antibody reagents is an
click chemistry, which results in EdU being covalently bonded issue, as permeabilization allows access not only to the cytokine
42
to a fluorescent azide. This approach allows for assessment of of interest but also to other proteins present in much greater
cell proliferation at the cell population or subpopulation (e.g., quantities within the cell than on the cell surface. In addition,
CD3, CD4, CD8) level and can be used in association with mitogen fixation further increases nonspecific binding, and use of a
and recall-antigen stimulation. negative-control sample, which contains an excess of unlabeled
Functional evaluation of cell activation can be accomplished or “cold” anticytokine antibody, as well as a subclass-matched
with flow cytometry–directed detection of the generation of or FMO-control sample provides optimal control. When the
phosphorylated intracellular proteins associated with specific conjugated anticytokine is added to the negative-control sample,
activation signals. An example of this is the detection of phos- it can only bind to other proteins in a nonspecific manner, thereby
phorylated signal transducer and activator of transcription 1 (STAT1) providing a measure to discriminate between specific and
following interferon-γ (IFN-γ) stimulation of monocytes, which nonspecific binding. The use of directly conjugated anticytokine
has been found to be equal to or more sensitive than immunoblot- antibodies not only simplifies the staining procedure but also
43
ting. This type of assay requires fixation and permeabilization provides the best distinction between specific and nonspecific
to allow entry of the specific reagent and now has been extended binding. Because the fixation agent may change the native state
to a number of additional intracellular proteins that are phos- of certain epitopes, it is also important to use antibodies that
phorylated following exposure of selected cells to specific stimuli. recognize antigens after fixation when combining cell surface
Currently, a number of intracellular signaling proteins that characterization with intracellular cytokine evaluation.
undergo phosphorylation following a specific activation signal One of the main applications of intracellular cytokine detection
can be assessed with flow cytometry by using commercially by flow cytometry has been the study and refinement of the
available reagents, some of which are offered in kit form. T-helper (Th)1/Th2/Th17 paradigms. It has recently become
The assessment of oxidative burst following cell stimulation clear that the regulated secretion of cytokines can be used to
plays a central role in neutrophil function testing where the study the response of individual T cells to both polyclonal stimuli
hydrogen peroxide-sensitive dye DHR123 is used. This procedure and specific antigens. Measuring antigen-specific T-cell cytokine
involves loading granulocytes with the dye, stimulating with expression in response to specific antigen offers a useful alternative
phorbol myristate acetate (PMA), and evaluating for fluorescence to the multimer-based approach (discussed below) to quantify
with flow cytometry. 11,44 This test has proven to be extremely the frequency of antigen-specific T cells (Fig. 92.6). 48
accurate in diagnosing patients with chronic granulomatous
44
disease (CGD) and carriers of X-linked CGD. A major advantage Cell Cycle Analysis
is its sensitivity, which allows for detection of one normal cell
in a population of 1000 abnormal cells. This makes assessment ClInICal rElEvanCE
of oxidative burst a useful tool in monitoring allogeneic granu-
locyte survival after transfusion into patients with CGD, as well Cell Cycle Analysis
as a means of following donor chimerism in the setting of • Useful for screening percentage of S phase and aneuploidy
allogeneic stem cell transplantation. It also provides a method • Can be combined with cell surface studies
to identify corrected cells following gene therapy in CGD and • Can be combined with markers of apoptosis
has utility in predicting outcome. 45,46
Intracellular Cytokine Detection In addition to surface immunophenotyping and cytoplasmic
Flow cytometry affords a platform to evaluate cytokine production characterization, flow cytometry is also used in cell cycle analysis.
at the single-cell level by using cytokine-specific, directly con- PI is the most commonly used fluorochrome because of its
47
jugated mAbs after fixation and permeabilization of cells. This optimal linear DNA-binding capacity in a variety of different

1248 Part ElEvEn Diagnostic Immunology


No Ag chicken erythrocytes) should be used as an internal reference
10 4 for evaluating DNA content and evaluating the cell cycle
distribution.
It should be noted that several different computer algorithms
10 3 have been developed to determine the relative proportion of
each cell cycle compartment, and the selection of a software
program is not a trivial process. The major instrument manu-
facturers supply cell cycle analysis programs, and third-party
10 2 programs are also available. Generally, the optimal program
should be capable of modeling two or more aneuploid popula-
tions, subtracting debris (particularly if formalin-fixed, paraffin-
embedded [FFPE] archival material is used) and accurately
10 1 estimating S-phase cells. 49,50 The combination of a surface marker
and the cell cycle has been very useful in differentiating normal
IFN-γ cell populations from tumor cell populations. One example is
the use of anti-κ-, anti-λ-, or B-cell reagents to separate the
10 0 aneuploid B-cell clone from the remaining normal, reactive B
10 0 10 1 10 2 10 3 10 4 cells in a lymphoid cell mixture. Another uses cytokeratin as a
marker to distinguish between tumor cells and inflammatory
IL-4 cells.
The other major event that has occurred in cell cycle analysis
M.Tb has been the development of technology using the incorporation
10 4 of bromodeoxyuridine. This thymidine analogue is used to
51
directly determine the percentage of S-phase cells. Additionally,
when used in kinetic studies, it permits determination of indi-
10 3 vidual times for the components of the cell cycle and determina-
tion of the growth fraction. Finally, recent developments have
resulted in the availability of two anticyclin reagents to evaluate
cell cycle transition points in malignant cells. 52
10 2
APOPTOSIS DETECTION
Flow cytometry has become the method of choice for the detection
10 1 and quantification of cellular apoptosis, in part because of its
53
capacity for rapid assessment of a large number of cells and
IFN-γ samples. Many distinct features of an apoptotic cell can be
evaluated by using flow cytometry based on light scatter, plasma
10 0 membrane changes, mitochondrial transmembrane potential,
10 0 10 1 10 2 10 3 10 4
DNA content, and DNA integrity.
The light scattering properties of a cell undergoing pro-
IL-4
+
FIG 92.6 Two-color dot plots of CD3 T cells evaluated for grammed cell death are the simplest attributes that can be assessed
intracytoplasmic interferon (IFN)-γ and interleukin (IL)-4 expression. by flow cytometry. Dying cells typically shrink, producing a loss
The donor had a positive skin test to purified protein derivative in FSC and, despite an initial transient increase in SSC, ultimately
(PPD) and demonstrated a T-helper-1 (Th1) pattern of cytokine demonstrate a decrease in SSC (see Fig. 92.7B). The use of light
expression (IFN-γ) in response to Mycobacterium tuberculosis scatter can be combined with cell surface staining to help
antigen, with an absence of a Th2 cytokine pattern (IL-4). characterize the dying cells. However, scatter changes alone are
(Courtesy of Calman Prussin, MD.) not specific to apoptosis and should be accompanied by an
additional characteristic associated with cell death. Live cells
have phospholipids asymmetrically distributed in inner and outer
plasma membranes, with phosphatidylcholine and sphingomyelin
cell types. Thus a single-parameter histogram of DNA content on the outer surface and phosphatidylserine (PS) on the inner
using PI readily permits the determination of cell cycle compart- side. Early during apoptosis cells lose asymmetry, exposing PS
ments, expressed as the percentage of cells in G 0 –G 1 , S, and G 2 –M on the outside. Annexin V is a protein that binds preferentially
(Fig. 92.7A). In addition to these conventional parameters, the to negatively charged phospholipids, such as PS, and directly
presence or absence of aneuploidy can be determined by inspec- conjugated annexin V is a useful reagent for the specific detection
tion of the G 0 –G 1 peak and/or use of a DNA index (ratio of of apoptotic cells. 53
abnormal DNA content to a diploid DNA standard). Also, eleva- Another characteristic of plasma membranes associated with
tion in the S and/or G 2 –M phase can be detected. The optimal live cells is that they exclude charged cationic dyes, such as PI
display of these data uses a combination of SSC versus DNA and 7-amino-actinomycin-D (7-AAD). Consequently, only cells
content. Cells observed on the histogram in the area below the in a late stage of apoptosis, with ruptured cell membranes, will
49
level of G 0–G 1 may be undergoing apoptosis. When dealing take up these dyes. Thus the combined use of cationic dyes (e.g.,
with DNA staining, a consistent cellular source of DNA (e.g., PI) with annexin V allows for discrimination between live cells

CHaPtEr 92 Flow Cytometry 1249



600 1000 10 4
G1
800 10 3
400
600 Late
# Cells SSC Live PI 10 2 Early

200 400
S G2-M 1
200 10
Live
Dead
0 0 10 0
0 200 400 600 800 1000 0 200 400 600 800 1000 10 0 10 1 10 2 10 3 10 4
A DNA content B FSC C Dioc6 (3)
FIG 92.7 (A) Assessment of DNA content as a reflection of cell cycle demonstrating cells in G 1 ,
S, and G 2 –M phases. (B) Assessment of live versus dead cells based on forward-(FSC) and
side-scatter characteristics (SSC). (C) Assessment of cell apoptosis using propidium iodide (PI)
and Dioc6 (3) identifying cells that recently initiated apoptosis (early), cells that are dead (late)
and cells that are alive (live).

(annexin V negative/ PI negative), early apoptotic cells (annexin of one chain of the MHC molecule, which, after combining with
V positive/ PI negative), and late apoptotic cells (annexin V a specific antigenic peptide, is bound by avidin or streptavidin.
positive/ PI positive). As both avidin and streptavidin have four biotin-binding sites,
Assessment of mitochondrial transmembrane potential (Δψm) the result is a tetrameric peptide–MHC complex that serves as
is yet another technique used to identify apoptotic cells. Cells a ligand for T cells specific for both the peptide and the MHC.
decrease Δψm very early in the apoptotic process, before rupture Flow-cytometric detection is achieved by labeling streptavidin
of the plasma membrane, losing the ability to accumulate with a fluorochrome. The major pitfall of this approach is the
potential-dependent dyes, such as rhodamine 123, JC-1, or need to know the antigen-derived peptide and its HLA restrictions,
3
3,3’ ’-dihexyloxacarbocyanine iodide (Dioc6 ). These dyes can as well as the HLA type of each subject studied. Since the initial
also be used with PI to detect cells in the different stages of report, an increasing number of tetramer-based studies have
apoptosis (see Fig. 92.7C). been performed. Most have focused on the MHC class I–mediated
Measurement of DNA content can also be employed to immune response, in both mice and humans, to a variety of
distinguish live cells from dead cells, as described above (see infectious agents, including cytomegalovirus (CMV), HIV,
Cell cycle analysis). This kind of analysis has to be done by using Epstein-Barr virus (EBV), and others. Since the initial description
a linear scale, not a logarithmic scale, to discriminate dying cells with class I–restricted recognition, detection of antigen-specific
from debris. DNA cleavage also exposes −OH termini associated CD4 T cells with tetramers of soluble MHC class II molecules
with the DNA breaks, and these can be detected via the attachment and covalently linked peptide has also been reported. 55
of fluorochrome-conjugated deoxynucleotides, in a reaction In addition to demonstrating the feasibility of this approach,
catalyzed by exogenous TdT, a technique called TUNEL. published studies have provided several new insights into the
MHC class I–mediated immune response (Chapter 5). For
PEPTIDE–MHC MULTIMERS example, it has become clear that the extent of the MHC class
I–mediated cellular response is much greater than previously
estimated. Furthermore, the extensive proliferation of CD8 T
KEY COnCEPtS cells during an acute infection is not the result of bystander
Peptide–MHC Tetramers activation but represents an expansion of antigen-specific CD8
T cells. Peptide–MHC tetramer assays have shown promise in
• Useful for assessing the number of antigen-specific T cells the study of the kinetics of primary and secondary immune
+
+
• Can be directed at both CD4 and CD8 T cells responses and have resulted in better understanding of such
• Requires information about the antigenic peptide and human leukocyte concepts as immunodominance and clonal exhaustion.
antigen (HLA) (major histocompatibility complex [MHC]) restriction
An obviously attractive aspect of this technology is that
tetramer staining can be combined with a variety of cell surface
In contrast to B cells, direct visualization of antigen-specific T and intracellular phenotypic and functional markers. Indications
cells ex vivo has, until recently, been unsuccessful. In 1996, Altman that the phenotype of antigen-specific T cells varies among
et al. introduced a novel flow cytometry–based methodology individuals and among different phases of the immune response
that enables the direct visualization and quantification of antigen- are already present. In addition, tetramer-positive T cells can be
54
specific T cells. Soluble peptide–MHC multimers are generated sorted for further analysis, such as cytotoxicity assays or in vitro
such that multiple T-cell receptors (TCRs) are engaged at the expansion. Tetramer-based technology has not only proven useful
same time, which means that the avidity of these multimeric for the study of the immune response to infectious agents, it
ligands for the peptide-specific TCR is greatly increased. The has also been applied to the study of oral tolerance, autoimmune
methodology involves engineering a biotinylation recognition conditions, and tumor immunology. It is likely that this highly
sequence on the −COOH terminus of the extracellular domain sensitive and specific technology and other approaches that define

1250 Part ElEvEn Diagnostic Immunology


the antigen-specific response will find many more applications 5. Ward M, Turner P, DeJohn M, et al. Fundamentals of acoustic cytometry.
and will lead to new discoveries and a reassessment of certain Curr Protoc Cytom 2009;49:1.22.21–21.22.12.
existing concepts. 56 6. Chattopadhyay PK, Hogerkorp CM, Roederer M. A chromatic explosion:
the development and future of multiparameter flow cytometry.
Immunology 2008;125:441–9.
CONCLUSIONS 7. Perfetto SP, Chattopadhyay PK, Roederer M. Seventeen-colour flow
cytometry: unravelling the immune system. Nat Rev Immunol
2004;4:648–55.
On tHE HOrIZOn 8. Giepmans BN, Adams SR, Ellisman MH, et al. The fluorescent toolbox
for assessing protein location and function. Science 2006;312:217–24.
• The combination of flow cytometry and mass spectrometry expands 9. Chattopadhyay PK, Yu J, Roederer M. Application of quantum dots to
the number of cell markers analyzed simultaneously (≥35 currently multicolor flow cytometry. Methods Mol Biol 2007;374:175–84.
performed), including extracellular and intracellular targets to provide 10. Rabinovitch PS, June CH, Kavanagh TJ. Introduction to functional cell
an extensive evaluation of functional markers.
• Flow cytometry utilizing spectral rather than conventional light analysis assays. Ann N Y Acad Sci 1993;677:252–64.
has the potential to allow more “colors” to be accurately evaluated 11. Vowells SJ, Sekhsaria S, Malech HL, et al. Flow cytometric analysis of the
without the need for complex compensation schemes to handle the granulocyte respiratory burst: a comparison study of fluorescent probes. J
overlapping light emissions from conventional fluorochromes. This Immunol Methods 1995;178:89–97.
could facilitate more extensive polychromatic studies to be performed 12. Darzynkiewicz Z, Halicka HD, Zhao H. Analysis of cellular DNA content
easily in the clinical laboratory. by flow and laser scanning cytometry. Adv Exp Med Biol
2010;676:137–47.
13. Loken MR, Brosnan JM, Bach BA, et al. Establishing optimal lymphocyte
Flow cytometry has become readily available in clinical labora- gates for immunophenotyping by flow cytometry. Cytometry
tories, and the application of this technology has moved forward 1990;11:453–9.
along with significant improvements in instrumentation and 14. US Clinical and Laboratory Standards Institute. Clinical applications
the availability of an array of monoclonal reagents. Properly of flow cytometry: quality assurance and immunophenotyping of
performed, flow cytometry can provide rapid and accurate peripheral blood lymphocytes. Villanova, PA: NCCLS Publication H24-T;
1992.
lymphocyte subpopulation identification. The primary clinical 15. Stewart CC, Stewart SJ. Multiparameter data acquisition and analysis of
indications of immunophenotyping remain quantifying CD4 leukocytes. Methods Mol Biol 2004;263:45–66.
T-cell counts in HIV infection, lineage assignment in leukemias 16. Parks DR, Roederer M, Moore WA. A new “Logicle” display method
and lymphomas, evaluation of other hematological cell types, avoids deceptive effects of logarithmic scaling for low signals and
and assessment of CD34 expression to identify stem cells for compensated data. Cytometry A 2006;69:541–51.
transplantation. Additional uses include characterization of 17. Roederer M, Moody MA. Polychromatic plots: graphical display of
immune deficiency disorders, evaluation of immune-mediated multidimensional data. Cytometry A 2008;73:868–74.
inflammatory diseases, and assessment of patients after organ 18. Roederer M. Spectral compensation for flow cytometry: visualization
transplantation. Generally, immunophenotyping does not artifacts, limitations, and caveats. Cytometry 2001;45:194–205.
represent a diagnostic procedure but, rather, plays a part in the 19. Owens MA, Vall HG, Hurley AA, et al. Validation and quality control of
evaluation and understanding of complex disorders and the immunophenotyping in clinical flow cytometry. J Immunol Methods
2000;243:33–50.
longitudinal evaluation of immunomodulatory therapy. 20. Schwartz A, Marti GE, Poon R, et al. Standardizing flow cytometry: a
It is critical to recognize that immunophenotyping is a means classification system of fluorescence standards used for flow cytometry.
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CHaPtEr 92 Flow Cytometry 1251.e1


MU lt IP l E-CHOICE QUES t IO n S

1. In flow cytometry compensation refers to a process that: A. Dedicator of cytokinesis 8 (DOCK8) deficiency
A. Distinguishes between leukocyte populations B. CD40 ligand deficiency
B. Eliminates overlapping fluorescent light signals C. Severe combined immunodeficiency
C. Provides the level of the cellular autofluorescence D. Wiskott-Aldrich syndrome
D. Converts analog light signals into digital signals
4. Which test would be indicated in a child with persistent
2. A flow cytometry setup with a combination of five different systemic nontuberculous infection not responsive to multiagent
monoclonal reagents, each with a different fluorochrome, antimicrobial drugs?
generates how many potential cell subsets? A. Dihydrorhodamine (DHR) assay
A. 8 B. Expression of CD18 on granulocytes
B. 16 C. Level of T-helper-1 (Th1) cell–based on intracellular
C. 32 interferon-γ (IFN-γ) expression
D. 64 D. Expression of the IFN-γ receptor type 1 on monocytes
3
3. The finding of a CD3 cell count of 285/mm and no
CD4/CD45RA cells in an infant is most consistent with which
diagnosis?

93






Assessment of Functional Immune

Responses in Lymphocytes



Roshini Sarah Abraham







The immune response is mediated by a complex network of induction of FOXP3 in conventional naïve T cells imparts
cells, soluble and membrane-bound biological mediators and suppressive function in vitro and in vivo, resulting in the genera-
4
receptors interacting within the context of specific tissues and tion of induced-regulatory (iTreg) T cells. Besides producing
organs to protect against pathogens. Although the immune system antibodies to neutralize pathogens as well as presenting antigen
has been broadly divided into innate and adaptive components, to T cells, B cells also exert immunomodulatory control of the
there is considerable overlap and interaction between the two, immune response, primarily via interleukin (IL)-10 production.
despite the highly specialized functions and kinetics of each in IL-10–producing B cells, classified as regulatory B (Breg) cells,
the immune response. T and B cells form the pillar of the adaptive play an important role in immune homeostasis and in protection
immune response, whereas natural killer (NK) cells are the effector against autoimmune responses and inflammatory damage. Breg
lymphocytes of the innate immune response. There are approxi- cells, in addition to producing IL-10, secrete other cytokines
12
mately 2 × 10 lymphocytes in the body, and typically the response that act on other effector T-cell subsets, Tregs, APCs such as
to a foreign antigen is in the context of initial activation of innate dendritic cells (DCs), and macrophages.
immune components. T cells recognize antigen primarily in the NK cells are considered to be innate immune effector cells,
context of antigen-specific T-cell receptors (TCRs) and specific but they straddle the threshold of innate and adaptive immunity.
peptides presented by molecules of the major histocompatibility NK cells are directly involved in cytotoxicity and cytokine secretion
complex (MHC), either class I or class II (Chapters 5 and 6). T upon activation, but they also function indirectly by regulating
cells are also capable of responding nonspecifically to polyclonal APC and the effector T-cells’ response. NK cell activation is
stimulators, such as mitogens (in vitro) or superantigens (in controlled by synergistic signals from activating and inhibitory
5
vivo) that do not initiate an antigen-specific proliferative response. receptors. The characteristics, responses, and assays to measure
T cells upon activation also produce cytokines that are crucial the activity and function of each of these subsets are described
to their effector functions including activating B cells and inducing in detail in the sections below.
antibody production, promoting differentiation of cytotoxic T
cells, activating macrophages, and promoting activation and T-CELL RESPONSE
migration of inflammatory cells. Cytokines play a role in the
induction of the T-cell response when produced by antigen- T cells form the cellular arm of the adaptive immune response,
presenting cells (APCs) at the time of antigen recognition. B and each T cell has unique specificity derived from the presence
cells recognize antigen directly via the B-cell receptor (involving of a functional, antigen-recognizing receptor on the cell surface.
surface immunoglobulin) and produce antibodies with help from Naïve T cells derived from the thymus can migrate through the
T cells (T-dependent antigens) or from the innate immune system peripheral circulation and secondary lymphoid tissues (spleen
(T-independent antigens). More recently, the granularity of the and lymph nodes), but they cannot effectively participate in the
antibody response has been further delineated, and T-dependent immune response to pathogens. To do so, naïve T cells must
antibody responses are divided into type 1, with help provided undergo a defined process of cellular activation, which involves
by T-follicular helper cells (Tfhs), and type 2, with help provided recognition of antigen by the TCR in the context of MHC
by NKTfh cells. Similarly, T-independent responses are classified molecules. The majority of circulating T cells express the α/β
into three groups: type 1, induced by recognition of microbial TCR, whereas a smaller proportion of cells express the γ/δ TCR.
antigens by the Toll-like receptors (TLRs) in the absence of On the surface of the T cell, the TCR associates with the CD3
Bruton’s tyrosine kinase (BTK); type 2, which requires the presence complex, comprised of four distinct subunits (γ, δ, ε, and ζ);
of BTK; and type 3, which has neutrophil B-helper cells. 1,2 the cytosolic components of the CD3 complex are involved in
Regulatory (Treg) cells have been shown to play a critical role the intracellular propagation of signals after ligation of the TCR.
in the control of both physiological and pathological immune Besides the CD3 complex, the TCR also clusters with a CD4 or
responses in a variety of contexts (Chapter 18). Treg cells exert CD8 coreceptor, depending on the type of T cell; CD4 T cells
a direct inhibitory effect on the development of autoimmune recognize antigen (Ag) in the context of MHC class II, whereas
disease because their absence leads to the development of a severe CD8 T cells recognize antigen presented on MHC class I. The
phenotype, as manifested by the FOXP3 deficiency, IPEX first signal or “cognate” signal, which is recognition of peptide-
3
(immune dysfunction/polyendocrinopathy/enteropathy/X-linked) MHC complex by the TCR, results in actin-mediated reorganiza-
(Chapter 35). Treg cells can suppress the proliferation of antigen- tion of the cytoskeleton in both the T cell and APC to form the
stimulated naïve T cells, as demonstrated by in vitro studies, and immunological synapse (Fig. 93.1). The synapse consists of the

1253

1254 Part eleven Diagnostic Immunology




















Antigen-presenting cell
(APC)



MHC CD80/86

CD4/CD8
TCR CD28
cSMAC
T cell APC
γ ε ε δ Fyn
TCR/CD3 MHC P P P
CD28 CD80/86 Lck P P
CD27 CD70 P P ζζ P LAT IP 3 PI3K
OX40 OX40L ZAP-70 P
4-1BB 4-1BBL PLC-γ P P
P P
P P
P Itk Ca2 + flux
pSMAC Gene transcription
T-cell activation
T cell APC
LFA-1 ICAM-1





FIG 93.1 Schematic of T-Cell Activation. T cells recognize antigen in the context of major
histocompatibility complex (MHC) molecules on antigen-presenting cells (APCs). Sustained interaction
between a T cell and APC results in the formation of the immunological synapse with membrane
reorganization and ordering of key molecules on both cells into a supramolecular activation
complex (SMAC) comprised of c (central) SMAC and p (peripheral) SMAC. Cognate interaction
with appropriate costimulatory signals results in intracellular signaling events that ultimately result
in T-cell activation. LAT, linker for activation of T cells; PLC-γ, phospholipase C-gamma; TCR, T-cell
receptor; IP3, inositol 1, 4, 5 triphosphate; P, phosphorylation; PI3K, phosphatidylinositol 3′-hydroxyl
kinase; Lck and Fyn are Src-family nonreceptor protein tyrosine kinases; Itk, IL-2–inducible T-cell
kinase; ZAP-70, ζ-chain–associated protein kinase 70. (Adapted from Figure 1, Pennock ND, et al.
Adv Physiol Educ 2013;37:273–83.)




TCR along with other costimulatory molecules and adhesion which drives T-cell proliferation in an autocrine and paracrine
receptors, which results in the formation of a large macromo- manner.
lecular structure called the supramolecular activation complex It is important to recognize that various T-cell processes and
(SMAC). The unique organization of the SMAC results in stages of differentiation are specifically regulated by metabolic
6
prolonged and stronger intracellular interactions and appropriate pathways. For example, naïve T cells rely on oxidative phos-
downstream signaling activity, including phosphorylation of the phorylation for their metabolism, with additional glycolytic
CD3 receptor components. The complete culmination of the metabolism during times of proliferation. On the other hand,
TCR-induced signals results in IL-2 production and secretion, activated effector T cells demonstrate aerobic glycolysis and

CHaPter 93 Assessment of Functional Immune Responses in Lymphocytes 1255


increased oxidative phosphorylation. T-helper cell-1 (Th1), Th2, +HDOWK\ FRQWURO
and Th17 CD4 effector T cells use glycolytic metabolism, whereas
regulatory T-cell and memory T-cell development is enhanced &' /
by fatty acid oxidation and catabolic metabolism. In particular,
memory T cells are quiescent and mainly use oxidative phos- &RXQW &RXQW &' XOJ
phorylation; however, on antigenic rechallenge, the use of oxidative
6
phosphorylation and glycolysis is rapidly facilitated. While naïve
T-cell activation and differentiation into effector cells is essential
for a normal immune response during exposure to acute antigenic
challenge, chronic antigenic stimulation via persistent antigen
exposure and/or inflammation causes an alteration of memory 3DWLHQW
T-cell differentiation, resulting in a state of T-cell exhaustion.
During T-cell exhaustion there is a progressive diminution of
effector functions, along with upregulation of several inhibitory &' /
receptors, metabolic changes, and a failure to transition to a &RXQW &RXQW &' XOJ
quiescent state and to obtain antigen-independent memory T-cell
homeostatic response. Exhausted T cells have only a limited
ability to clear pathogens or tumors; however, there is some
evidence that this is a reversible phenomenon, at least at a global
level. 7
FIG 93.2 Expression of CD40L on Activated CD4 T Cells.
CD40L is an early activation marker expressed on CD4 T cells,
ClInICal relevanCe and it is important for T-cell costimulation and isotype class-
Lymphocyte Function Evaluation switching. In healthy individuals, on T-cell activation CD40L
(CD154) is expressed on the cell surface of CD4 T cells and is
• Assessment of lymphocyte function typically refers to measurement capable of binding a soluble form of the receptor CD40µIg,
of T-cell function via cellular proliferation to nonspecific and specific which enables assessment of its function in vitro (top panel).
stimuli; however, it is much broader and more comprehensive and In patients with X-linked hyper-IgM syndrome due to CD40L
refers to any method that assesses any of the various effector or
regulatory aspects of any lymphocyte subset. deficiency, either there is no protein expression of CD40L on
• The robustness of clinical interpretation of lymphocyte function assays the cell surface and therefore no binding to the soluble receptor
is dependent on the analytical procedure, sample type, timing of (lower panel) or, as is the case with 20% of patients, the CD40L
collection, and clinical context, among other factors. Assays performed expressed on the cell surface is nonfunctional and cannot bind
in clinical diagnostic immunology laboratories are standardized to to the receptor (CD40µIg).
minimize inconsistencies in these variables.

MEASUREMENT OF T-CELL FUNCTION VIA but with aberrant function. These patients can be identified by
ACTIVATION MARKERS incorporating an additional component in the assay using a
soluble form of the receptor CD40-muIg to assess binding of
Activation of T cells results in the expression of several induced the ligand (Fig. 93.2). The flow cytometric assay for CD40L
markers, which can be used to ascertain the competence of T expression and function offers a rapid diagnostic test for XL-Hyper
9
cells participating in the immune response. These include CD69, IgM syndrome .
CD154 (CD40L), MHC class II, and CD25, which are expressed
8
in a sequential manner and can be assessed by flow cytometry.
In contrast to in vivo activation of T cells and subsequent expres- ASSESSMENT OF CELLULAR VIABILITY
sion of these markers, in vitro assessment of T-cell function IN LYMPHOCYTES
involves activation of T cells with nonspecific and polyclonal
stimulants such as phorbol myristate acetate (PMA) or phyto- The flow cytometry–based assays also allow determination of
hemagglutinin (PHA), which results in expression of these markers viable cells in the sample after peripheral blood mononuclear
8
in a kinetically regulated fashion. The expression of CD40L on cell (PBMC) isolation from whole heparinized blood, which is
activated T cells, besides being useful as a global marker for particularly important in the interpretation of results (Fig. 93.3).
T-cell activation, is used more specifically for the diagnosis of The use of annexin V in a flow cytometry assay enables visualiza-
a primary immunodeficiency, X-linked hyper-IgM syndrome or tion of apoptotic cells. An early event in cell apoptosis is the
CD40L deficiency (Chapter 35). CD40L on activated CD4 T translocation of membrane phosphatidylserine (PS) to the cell
2+
cells interacts with CD40, expressed constitutively on B cells, surface. Annexin V is a calcium (Ca )-dependent phospholipid-
and participates in isotype (class)-switching as well as providing binding protein, which has a strong affinity for PS, and a
costimulatory help to T cells. The expression of CD40L on fluorochrome-conjugated reagent directed at annexin V is
activated CD4 T cells can easily be ascertained in the laboratory used in a flow assay. In addition to visualizing apoptotic cells,
using an in vitro T-cell stimulation protocol (Fig. 93.2). In this dead cells can be assessed by simultaneously using 7-amino-
assay, CD69 is used as a control for early T-cell activation. The actinomycin-D (7-AAD), which is a membrane-impermeable
majority of patients (80%) with mutations in CD40LG have dye that is generally excluded from viable cells. When internalized
absent protein expression on activated CD4 T cells; however, into dying or dead cells, it binds to double-stranded DNA by
20% of mutations may remain permissive for protein expression, intercalating between base pairs in guanine (G)-cytosine (C)-rich

1256 Part eleven Diagnostic Immunology



10 3 KeY COnCePtS
Annexin V+7-AAD+
T-Cell Activation and Function
• Cognate recognition of peptide major histocompatibility complex (MHC)
10 2 complex on the APC by the T-cell receptor results in formation of the
immunological synapse.
• Activated T cells express early activation markers, such as CD69 and
CD40L. Other T-cell activation markers include CD25 and MHC class
10 1 II (human leukocyte antigen–D related [HLA-DR]).
• T cells can be stimulated to proliferate using nonspecific stimulants
such as plant lectins (mitogens) and cross-linking of the CD3 coreceptor,
along with other costimulatory molecules (e.g., anti-CD28) or in the
2 presence of exogenous interleukin-2 (IL-2).
• The magnitude of antigen-specific T-cell proliferation is dependent on
the starting frequency of antigen-specific T cells.
0 • Flow cytometry–based assays of T-cell proliferation offer the advantages
of high resolution, accounting for cellular dilution due to T-cell lym-
–2 phopenia and single-cell analysis.
Viable cells Annexin V+

0 10 0 10 1 10 2 10 3
FIG 93.3 Assessment of Cellular Viability for Lymphocyte
Function Studies. For all functional assessments of immune
response, it is essential and useful to measure the proportion with suspected IL-2 receptor (IL-2R)–associated signaling defects,
of viable cells at the initiation of the analysis and even at the it may be more helpful from a diagnostic perspective than the
end of the analysis, if required. Measurement of cell apoptosis use of anti-CD28 (Fig. 93.5). IL-2, an autocrine cytokine, has
and death can be achieved by flow cytometry analysis of annexin been demonstrated to be critical in T-cell proliferation and in
V positivity (apoptotic) and annexin V 7-amino-actinomycin-D regulation of T-cell growth through binding to a heterotrimeric
(7-AAD) dual positive (dead) cells. Cells that are negative for receptor complex consisting of 3 chains—α, β, and γ (IL-2Rα,
both these markers are viable cells, and their frequency can be IL-2Rβ, and IL-2Rγ)—on the surface of T cells. Triggering of
assessed in the flow assay. the TCR leads to synthesis of IL-2 in certain T-cell subsets with
induction of high-affinity IL-2Rs on antigen- or mitogen-activated
T cells; the binding of IL-2 to the IL-2R ultimately leads to T-cell
proliferation. The use of exogenous IL-2 in association with
regions. These two dyes can be combined to provide information anti-CD3 allows discrimination of T cells that cannot proliferate
on the proportion of viable cells in the starting cell mixture for to other mitogenic signals but can respond to a potent growth
proliferation assays (Fig. 93.3). factor such as IL-2.
Antigens including candida antigen (CA) and tetanus toxoid
MEASUREMENT OF T-CELL COMPETENCE (TT) have been widely used to measure antigen-specific recall
VIA PROLIFERATION (anamnestic) T-cell responses when assessing cellular immunity.
In fact, this may be more revealing about cellular immune
Mitogens are very potent stimulators of T-cell activation and compromise than assessing the response of lymphocytes to
induce polyclonal T-cell proliferation. It has been suggested that mitogens because the latter can induce T-cell proliferative
mitogens can induce T-cell proliferative responses even if the responses even if those T cells are incapable of responding
lymphocytes are incapable of responding adequately to antigenic adequately to antigenic (physiological) stimuli. Therefore
(physiological) stimuli. Therefore abnormal T-cell responses to abnormal T-cell responses to antigens are considered a diagnosti-
mitogens are considered a diagnostically less sensitive but more cally more sensitive, but less specific, test of aberrant T-cell
specific test of aberrant T-cell function. Lectin mitogens have function. Antigens used in recall assays measure the ability of T
been shown to bind to the TCR, thereby activating quiescent T cells bearing specific TCRs to respond to antigenic peptides
cells. Mitogenic stimulation induces increased intracellular calcium presented by APCs. The antigens used for assessment of the
2+
(Ca ) in T cells, which is essential for T-cell proliferation. Whereas cellular immune response are selected to represent antigens, seen
PHA is a strong T-cell mitogen (Fig. 93.4), PWM is a weak T-cell by a majority of the population either through natural exposure
mitogen that also induces B-cell activation and proliferation (CA) or as a result of vaccination (TT) (Fig. 93.6).
with a different time line for maximal stimulation. Mitogens In addition to measuring cellular proliferation as a readout
such as PHA activate T cells by binding to cell membrane gly- for T-cell function, the production of cytokines by activated T
coproteins, including the TCR-CD3 complex. In addition, there cells is another important component for evaluating T-cell
are a number of mitogenic or comitogenic antibodies, including functional activity. T cells typically are not monofunctional,
those directed against the CD3 coreceptor that can stimulate producing a single cytokine on activation; rather, they are multi-
T-cell proliferation. Typically, anti-CD3 antibodies provide an or polyfunctional, and the range of cytokines are produced in
initial activation signal and provide a variable proliferative a sequential manner rather than simultaneously, although a
response (Fig. 93.5). Addition of a costimulatory antibody population of stimulated T cells will have individual T cells
(anti-CD28) to anti-CD3 results in enhanced proliferation (Fig. producing different cytokines in a temporally regulated manner.
93.5). An exogenous T-cell growth factor, such as IL-2, can also These cytokines can be measured by intracellular flow cytometry
be used as an alternate to anti-CD28 costimulation, and in patients after T-cell activation with mitogens, in both CD4 and CD8

CHaPter 93 Assessment of Functional Immune Responses in Lymphocytes 1257


T-cell subsets or alternatively in the culture supernatant of (CTV) and Cell Proliferation Dye eFluor 670 (CPD), which have
activated cells. Typically, the cytokines measured in in vitro different excitation and emission spectra compared with CFSE.
stimulation assays include IL-2, interferon (IFN)-γ, IL-4, IL-5, These dyes also can be used for tracking lymphocyte proliferation
IL-6, IL-17, and tumor necrosis factor (TNF)-α. status in vivo, in animal models, permitting measurement of up
The process of T-cell activation as described above results in to 11 cell divisions.*
IL-2 production that drives T-cell proliferation. Measuring T-cell Another alternative to tritiated thymidine is the use of a
®
proliferation after stimulation in vitro with various specific and thymidine analog, 5-ethynyl-2′-deoxyuridine (Edu ), which can
nonspecific stimuli has been a staple of the diagnostic immunol- combine with a fluorescent azide in a copper-catalyzed cycload-
ogy repertoire for several decades. T-cell proliferation assays can dition reaction (referred to as “Click” chemistry) and permits
be divided into three categories: use of nonspecific mitogenic flow cytometric evaluation of lymphocyte proliferative responses
®
stimuli such as PHA, pokeweed mitogen (PWM), and concana- by assessing its incorporation into cellular DNA. Edu - labeling
valin A (Con A); physiological global stimuli based on CD3 has been shown to be a fast and sensitive method for measuring
coreceptor cross-linking (using anti-CD3, either soluble or cell proliferation and also facilitates identification of dividing
bead-bound) with or without CD28 cross-linking (using cells. It is relatively more photo-stable than CFSE and is added
anti-CD28, either soluble or bead-bound); and with or without to cells after completion of the stimulation period with the
exogenous IL-2. Antigen-specific stimulation depends on the measurement being comparable to the thymidine method in
®
use of common recall antigens. regards to a gain of signal is the endpoint. The Edu assay has
The commonly used method for assessing T-cell proliferation not shown the limitations of the CFSE method in the clinical
3
3
for decades involved measurement of H-thymidine ( H-T) laboratory and has been used to evaluate lymphocyte proliferation
incorporated into the DNA of proliferating cells. The results are in a spectrum of patients, including those with severe combined
expressed as either counts per minute (cpm) or disintegrations immunodeficiencies (SCIDs). In fact, this assay has been par-
per minute (dpm) of both activated and nonactivated cells ticularly useful in discriminating between functional T cells and
(background) cultured for a fixed period of time, usually 72 nonfunctional T cells in the context of severe T cell lymphopenia,
hours for mitogens. A reference range based on proliferation which cannot be achieved with the standard thymidine assay
results from a group of control subjects should be provided (Figs. 93.4-93.6). All the proliferation data shown in this chapter
®
along with the patients’ results (both background and poststimula- utilize this Edu -based measurement of T cell proliferation, and
tion results). Although this method remains widely employed, results are typically provided for both CD45 bright positive (total
several disadvantages exist, including but not limited to the use lymphocytes) and CD3 T cells, with the former being more
3
of radioactive material ( H-T); its inability to discriminate representative of the data generated with the standard thymidine
responder cell subsets or to account for cellular dilution, which assay.*
is particularly relevant in the context of T-cell lymphopenia;
and the lack of information on the contribution of cell death MEASUREMENT OF CELL-MEDIATED
after stimulation and its impact on the final result. To overcome CYTOTOXICITY
3
the intrinsic shortcomings of the H-T method, newer flow
cytometry assays are currently being used in the clinical diagnostic CD8 T cells are considered the representative cytotoxic T cell in
setting and are gaining popularity because of the additional the immune system, and cellular cytotoxicity is a mechanism to
information they provide. eliminate cells infected with intracellular pathogens, allogeneic
The flow cytometric methods for measuring cell prolifera- cells, or tumor cells. CD8 T cells, like CD4 T cells, recognize
tion include the use of fluorescent dyes to identify proliferating antigen via the TCR and kill target cells via cytotoxic protein
cells. One of the more commonly used dyes, carboxyfluorescein granule exocytosis and/or cytokine production. Over the past few
diacetate succinimidyl ester (CFSE), must be carefully used when decades, the cytotoxic potential of CD4 T cells has been described,
measuring lymphocyte proliferation in vitro since it can be toxic particularly in viral infections. Although cytotoxic CD4 T cells
to cells and nonoptimal labeling conditions can impact measure- are rare in the circulation of healthy individuals (<2%), they
ment of cell proliferation and interpretation of results. CFSE is can account for substantial proportions of total CD4 T cells in
10
a fluorescent cell-membrane permeable dye similar in physical certain viral infections, including but not limited to HIV. The
properties to the commonly used fluorochrome, fluorescein CD4 cytotoxic T cells appear to be a lineage of memory CD4 T
isothiocyanate (FITC). During cell proliferation, the intensity cells that contain cytotoxic granules with perforin and granzymes,
of staining in daughter cells is half that of parent cells, allowing and they are thought to arise in the context of chronic or potent
visualization of the number of rounds of cell divisions associated activation with viral infections such as cytomegalovirus (CMV),
with the successive decrease in fluorescence. The disadvantages Epstein-Barr virus (EBV), human immunodeficiency virus (HIV),
to using CFSE in the clinical laboratory are its photo-instability, or certain autoimmune diseases. 11
limitation in the number of cell divisions being identified Cellular cytotoxic activity has been conventionally measured
51
(≤7 cell divisions), and interpretation requires measuring loss of by release of chromium ( Cr) from labeled target (T) cells
signal rather than gain of signal. In addition, as noted above, CFSE cultured with various ratios of effector (E) cells (varied E:T
at concentrations of 37 nM to 10 µM can be toxic to cells resulting ratio). The percentage lysis is developed using the level of
in increased cell death. Furthermore, it can modulate expression radioactivity in the supernatant of target cells in comparison to
of activation markers, resulting in a decrease in CD69, HLA-DR,
and CD25 expression. Finally, it has been reported that there is
an increase in the number of false-positive results with CFSE
making it suboptimal for measuring lymphocyte proliferation *This section has been reproduced in part with permission from ASM
in patients with severe cellular immunodeficiencies. Alternatives Press (Abraham RS, Lymphocyte Activation) (copyright permission
to CFSE include related compounds, such as Cell Trace Violet obtained)

1258 Part eleven Diagnostic Immunology




Healthy donor PHA/CD45 PHA/CD3

80
60
60 Proliferating CD45+ lymphs Proliferating CD3+ T cells
Count 40 Count 40



20
20

0 0
0 10 0 10 1 10 2 10 3 0 10 0 10 1 10 2 10 3
Fluorescence Fluorescence
Patient with T cell lymphopenia
PHA/CD45 PHA/CD3
150

30
100 Proliferating CD45+ lymphs Proliferating CD3+ T cells
Count Count 20

50
10


0 0
0 10 0 10 1 10 2 10 3 0 10 0 10 1 10 2 10 3
Fluorescence Fluorescence
FIG 93.4 T-Cell Proliferation to Mitogenic Stimulants. T cells can respond polyclonally to in
vitro stimulation with various mitogens. T-cell proliferation assessment by flow cytometry allows
+
+
analysis in total lymphocytes (CD45 ) and T cells (CD3 ). The top panel demonstrates T-cell
proliferation to phytohemagglutinin (PHA) in both cell subsets. In patients with T-cell lymphopenia,
where cellular dilution may be a concern, single-cell analysis by flow cytometry allows discrimination
of functional versus nonfunctional T cells. In the lower panel, a patient with T-cell lymphopenia
has abnormal proliferation when total lymphocytes are assessed; however, the response is
normal when the T-cell compartment is specifically evaluated. Therefore this patient, who would
+
have been classified as abnormal based only on the CD45 lymphocyte response, can be reclassified
+
as having normal T-cell proliferation based on the CD3 T-cell response, but with significant T-cell
lymphopenia.

KeY COnCePtS
Cellular Cytotoxicity
• CD8 T cells and natural killer (NK) cells are involved in killing of cellular • NK cells recognize target cells that lack major histocompatibility complex
+
targets and contain intracellular granules with cytotoxic proteins, such (MHC) class I, e.g., viral cells or tumor cells that have downregulated
as perforin and granzymes. MHC class I.
+/−
+
++
• Cytotoxic CD4 T cells are a subset of memory T cells with cytolytic • The majority of circulating NK cells are cytotoxic (CD3 CD16 CD56 ),
−
++
−
potential and are usually observed in circulation in the context of chronic while a minority are cytokine producing (CD3 CD56 ).
viral infections, e.g., cytomegalovirus (CMV), human immunodeficiency • Interleukin (IL)-2 augments NK cell–mediated cytotoxicity (lymphokine-
virus (HIV). activated killing) and also induces interferon (IFN)-γ secretion by NK
• Tetramer-based assays have been used to quantify and delineate function cells.
of antigen-specific CD8 T cells (also CD4 T cells). • Regulatory T cells control NK cell activation and cytotoxic function by
+
+
• Cellular degranulation results in expression of CD107a on the cell surface limiting availability of IL-2.
+
of CD8 T cells and NK cells and is often used as a surrogate of cytotoxic
activity.

CHaPter 93 Assessment of Functional Immune Responses in Lymphocytes 1259




Anti-CD3/CD45 Anti-CD3+anti-CD28/CD45 Anti-CD3+IL-2/CD45
100
60
80
60 Proliferating CD45+ lymphs
Proliferating CD45+ lymphs Proliferating CD45+ lymphs
60 40
Count Count 40 Count

40
20
20
20

0 0 0
0 10 0 10 1 10 2 10 3 0 10 0 10 1 10 2 10 3 0 10 0 10 1 10 2 10 3
Fluorescence Fluorescence Fluorescence

Anti-CD3/CD3 Anti-CD3+anti-CD28/CD3 Anti-CD3+IL-2/CD3
100
60 50
80
Proliferating CD3+ T cells
40
Proliferating CD3+ T cells Proliferating CD3+ T cells
60 40
Count Count Count 30
40
20
20
20
10
0 0 0
0 10 0 10 1 10 2 10 3 0 10 0 10 1 10 2 10 3 0 10 0 10 1 10 2 10 3
Fluorescence Fluorescence Fluorescence
FIG 93.5 T-Cell Proliferation to Anti-CD3 Stimulants. Assessment of T-cell proliferation to CD3
coreceptor cross-linking provides a more physiological, yet global, evaluation of T-cell function.
T cells are stimulated with either soluble anti-CD3 alone or soluble anti-CD3 plus anti-CD28 or
+
with soluble anti-CD3 plus interleukin (IL)-2. In each case, proliferation is measured in total CD45
lymphocytes and CD3 T cells. An example of anti-CD3–stimulated T-cell response is shown in
+
a healthy donor.
controls consisting of target cells cultured alone (no lysis) and A tetramer-based approach to quantifying and measuring
target cells exposed to hypotonic conditions (e.g., distilled water, function of CMV-specific CD8 T cells has been available in the
saponin) representing 100% lysis. Similar to the radioactive assay clinical diagnostic immunology laboratory for over a decade
for measuring T-cell proliferation described previously, there (Fig. 93.7). However, the limitation of the tetramer approach in
are several limitations to the radioactive cytotoxicity assay, the clinical diagnostic setting is that it is constrained by the
including the hazard of radioactive material, the difficulty in number of HLA-peptide tetramer (multimer) combinations that
51
labeling target cells with Cr, and bulk quantitation of cytotoxic are available for use with a particular antigen (e.g., CMV, EBV).
activity, which does not allow single cell–specific analysis. Alterna- It also requires a priori knowledge of the patient’s HLA genotype,
tive assays have been developed, including flow cytometry and and very often it does not incorporate a comprehensive assessment
12
ELISPOT-based methods. More recently, a method of assessing of the CD8 and CD4 cytotoxic T-cell response. Although some
human CD8 T-cell cytotoxicity has been described in which laboratories use only a quantitative approach to determine
both the effector and target cells are primary cells (in contrast immune competence to pathogens, such as CMV with the tetramer
13
to using cell lines as target cells). In this particular assay, assay, more comprehensive assays are available that also provide
autologous B cells isolated from PBMC are used as target cells a functional assessment of these antigen-specific CD8 T cells
and cocultured with antigen-specific CD8 effector cells. The (Fig. 93.7).
killing of the fluorescently labeled target B cells (see NK cell An alternative approach is to evaluate for cytotoxic function
section) is used to estimate cytotoxic activity. Antigen-specific by assessing degranulation by evaluating for CD107a expression
effector cells are quantified in the total CD8 T-cell pool using by the effector T cell (described in further detail in the NK cell
MHC-peptide tetramers. 13 function section) and/or evaluating IFNγ production by activated

1260 Part eleven Diagnostic Immunology


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)OXRUHVFHQFH )OXRUHVFHQFH
FIG 93.6 Assessment of Antigen-Specific T-Cell Proliferation. Antigens such as candida antigen
(CA) and tetanus toxoid (TT) are frequently used to assess antigen-specific T-cell function. As
+
with mitogens and anti-CD3 stimulation, proliferation is measured in total CD45 lymphocytes
+
and CD3 T cells. An example in a healthy donor is shown.



cytotoxic CD8 T cells. As a positive control, CD8 T cells are CMV in organ transplant and allogeneic hematopoietic cell
polyclonally stimulated with PMA and ionomycin, and the same transplant (HCT) patients. However, this assay has theoretical
markers (CD107a expression and IFN-γ production) are measured. limitations in that assessing the production of a single cytokine
Approximately 10–60% of CD8 T cells in healthy adults are is unlikely to represent the breadth of the immune response to
activated to express CD107a and to secrete IFN-γ under these a complex specific antigen such as CMV.
conditions. An approach that avoids the use of tetramers involves
the stimulation of patient-specific PBMC with overlapping peptide NK CELL ACTIVATION AND FUNCTION
pools of the specific antigen and, subsequently, the evaluation
of the CD8 and CD4 T cells that proliferate in response to NK cells are innate immune lymphocytes that provide the cellular
antigenic stimulation. These T cells can also be assessed for basis for innate responses to specific viruses as well as to tumor
cytotoxic potential by measuring intracellular perforin and cells 15,16 (Chapter 3). Unlike T and B cells of the adaptive immune
granzyme expression as well as degranulation (CD107a expres- system, NK cells do not have antigen-specific recognition and
sion) after stimulation. 8 killing of target cells. NK cells participate directly in effector
A CMV-specific assay has been developed that measures IFN-γ functions via cytotoxicity and production of cytokines (e.g.,
production in response to CD8 T-cell stimulation with a pool IFN-γ) upon activation. Proinflammatory cytokines such as IL-12,
of MHC class I–restricted CMV peptides, the QuantiFERON- IL-15, and IL-18 trigger NK cell proliferation as well as cytotoxicity
14
17
CMV (Cellestis Ltd., Melbourne, Australia). This commercial and production of IFN-γ. The negative regulation of NK cells
assay is simpler to perform than the tetramer-based approach is controlled by receptors that recognize MHC class I molecules
for quantification and functional analysis of antigen-specific CD8 preventing NK cell–mediated cytotoxicity. In contrast, virally
T cells. It has been applied in the context of risk prediction for infected or tumor cells typically downregulate MHC class I,

CHaPter 93 Assessment of Functional Immune Responses in Lymphocytes 1261


making them appropriate targets of NK cell–mediated cytotoxicity [neural cell adhesion molecule]) and CD16 (FcγRIII). NK cells
in the presence of relevant ligands expressed by the target cell. can be subdivided into two major subsets based on their relative
18
NK cells, like cytotoxic T cells, contain granules with cytotoxic expression of CD56 and CD16: CD16 +++(bright) CD56 +/−(dim) NK
proteins including perforin and granzymes, which are serine cells, referred to as cytotoxic NK cells, and CD56 +++(bright) CD16
+/−
proteases that recognize different substrates. or CD16 NK cells, known as regulatory or cytokine-producing
NK cells (Fig. 93.8). The majority (~90%) of circulating human
NK CELL CYTOTOXICITY NK cells belong to the cytotoxic category, while a minority (10%)
represent cytokine-producing NK cells. Cytotoxicity can be
The majority of NK cells can be identified by a lack of CD3 on the subdivided into natural or spontaneous cytotoxicity directed
cell surface in conjunction with the expression of CD56 (NCAM largely toward virally infected cells or tumor cells, in the absence









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+/$ $ WHWUDPHU +/$ % WHWUDPHU % ,(
% SS


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&' &'
$
FIG 93.7a, B Quantitative of Antigen-Specific CD8 T Cells and Functional Evaluation Using
Major Histocompatibility Complex (MHC) Class I Tetramers. The tetramer (multimer) technology
has been useful for accurate quantitation of antigen-specific CD8 (or CD4 T cells, if MHC class
II tetramers are used). The top panels show identification of CD3 T cells from total lymphocytes
+
and subsequent segregation into CD3 CD8 T cells. In this assay, cytomegalovirus (CMV)-specific
+
+
+
CD8 T cells are quantitated using five MHC class I tetramers (human leukocyte antigen [HLA]
A1, A2, B7, B8, and B35), each recognizing a unique CMV peptide from three major CMV antigenic
proteins (pp50, pp65, and IE-1) as listed in the box. Based on the HLA class I haplotype of the
individual, one or more tetramers are used for stimulation. The bottom panels show an example
+
of a patient with HLA A2-specific CMV-CD8 T cells and another patient with HLA B8-specific
+
CMV-CD8 T cells. These CMV-specific CD8 T cells can be assessed for functional capacity by
+
gating on the tetramer-positive CD8 T cells and then measuring degranulation (CD107a expression)
+
and inteferon (IFN)-γ production after in vitro stimulation with the specific CMV peptide (lower
+
panel). The data are shown for CD107a expression and IFN-γ production in unstimulated CMV-CD8
+
T cells and peptide-stimulated CMV-CD8 T cells. Continued

1262 Part eleven Diagnostic Immunology


Unstimulated Stimulated
10 3 10 3
HLA B8 tetramer with
10 2 10 2 CD107 a expression after
CMV IE-1 peptide stimulation
Relevant tetramer 10 1 Relevant tetramer 10 1




10 0 10 0

0 0

0 10 0 10 1 10 2 10 3 0 10 0 10 1 10 2 10 3
CD107a/b CD107a/b

10 3 10 3

10 2 10 2
Relevant tetramer 10 1 Relevant tetramer 10 1 HLA B8 tetramer with
CD107 a expression after
CMV peptide stimulation

10 0 10 0


0 0

0 10 0 10 1 10 2 10 3 0 10 0 10 1 10 2 10 3
IFN-g IFN-g
B
FIG 93.7B, cont’d





of prior stimulation or immunization, and antibody-dependent (7-amino-actinomycin-D) (Fig. 93.10). IL-2 enhances cytotoxic
cellular cytotoxicity directed against antibody-coated target function with increased lytic potential against a broad range of
cells. NK cells go through a process of education or “licensing” target cells. IL-2 has also been shown to induce IFN-γ secretion
whereby NK cells that express inhibitory receptors to self-MHC by NK cells with upregulation of activation markers, such as
19
class I molecules are called licensed, which means they are more CD25 and CD69. Treg cells control NK cell activation and
20
functionally responsive to stimulation, whereas unlicensed NK cytotoxic function by limiting access to IL-2. Other methods
cells lack receptors for self-MHC class I and are hyporesponsive for measuring NK cell cytotoxic function that are primarily used
21
(Fig. 93.9). in the research setting include the use of image cytometry,
22
NK cell function is measured in the clinical laboratory by microchip screening, and flow-based assays using other dyes,
assessment of spontaneous (natural) NK cell cytotoxicity using such as calcein AM. 23
an MHC class I–deficient myelogenous leukemia cell line, K562. The direct measurement of NK cell cytotoxic function is useful
Traditional methods for measuring NK cell cytotoxicity are similar in a variety of clinical contexts, especially in patients with inherited
to those used for assessing cytotoxic T cell (CTL) function based immune defects affecting NK cells and function, including but
51
on varying effector:target ratios in a 4- to 16-hr Cr-release not limited to recurrent/persistent herpesvirus infections and
assay compared with the no-lysis and 100% lysis conditions as familial/primary hemophagocytic lymphohistiocytosis (FLH/
described for the T-cell cytotoxicity assay. However, there is HLH). There are other parameters that are widely used as sur-
interest in the use of flow cytometry to measure spontaneous rogates of cytotoxicity, including flow cytometric measurement
or IL-2–activated (lymphokine-activated killer [LAK]) NK cell of granule exocytosis/degranulation. The membrane of cytotoxic
cytotoxicity in the clinical laboratory to avoid radioactivity. One granules in both NK cells and CD8 T cells is composed of several
flow cytometric assay employed in the clinical laboratory involves proteins, including CD107a (lysosomal-associated membrane
®
the use of fluorescently labeled (Cell Tracker dyes) target cells protein 1 [LAMP-1]). On stimulation of NK cells and CD8
(K562) incubated with effector cells (donor or patient PBMC) cytotoxic T cells, CD107a is upregulated and expressed on the
in the absence (spontaneous) or presence of IL-2 (LAK). Following cell surface (Fig. 93.11) concomitantly with cytokine secretion
coincubation, the lysis of target cells is measured by using 7-AAD and target cell lysis, and therefore has been frequently used to