T cell subsets - University of Oxford

T cell subsets

A synopsis of the lecture by Dr. Steve Cobbold for the FHS Physiology Immunology Option

1/ Definitions and relationships of different T cell subsets

There are many different, and sometimes confusingly named, T cell and related cell subsets, although much of the confusions arises
where subsets have been separately defined on the basis of molecular markers (eg. CD4 T cell) and functional properties (eg.
cytotoxic T cell) and the two definitions "overlap" (eg. most cytotoxic T cells are CD8, but CD4 T cells can be cytotoxic in
appropriate assays too). The only absolute definition is that a T cell must express either the gd or ab T cell receptor (as this is a
genetically irreversible decision) - all other subset definitions are somewhat dependent on the assay used to measure them!

2/ Subsets of T helper cells (Th1 and Th2) defined by cytokines

The original observation that mouse CD4+ T cell clones could be divided into two different sets based on their pattern of cytokine
expression has become the paradigm for heterogenenity within the T cell response in vitro and in vivo. Th1 and Th2 cells are
thought to derive from a non-polarised, naive Th0 precursor that makes a wide range of cytokines, that can differentiate after
activation in the presence of IL-12 and IL-18 (from DCs) into Th1 cells that secrete IL-2, IFN-g and lymphotoxin (LT) or in the
presence of IL-4 (from B cells or lymphoid DCs?) into Th2 cells that secrete IL-4, IL-5 and IL-10. This initial polarisation of the
response towards Th1 or Th2 is then self perpetuating as Th1 cytokines enhance further Th1 responses and down regulate Th2
cytokines, and vise versa.

3/ Antigen presentation for Th1 versus Th2 responses

It is still not clear how the initial polarisation towards either Th1 or Th2 is controlled, but it is thought to involve a number of factors
including the route of immunisation and the type of APC (eg. skin -> LC -> Th1 or i.v. -> B cell -> Th2), the antigen density/affinity
(v. high or v. low antigen dose -> Th2), and the innate immune response (NK cells -> IFN-g -> Th1 or NKT cells -> IL-4 -> Th2).
With respect to T cells interacting with the APC it seems that CD40 -> CD40L promotes Th1 responses, while B7 (CD80, CD86) ->
CD28 is more important as costimulation for Th2 cells. Once a Th1 or Th2 response has been polarised and established the antigen
may be presented directly or indirectly for activation of different effector cells - macrophages, neutrophils and cytotoxic T cells for
Th1 responses and eosinophils, mast cells and B cells for the Th2 response. B cells are also dependent on cytokines to promote
maturation and isotype switching, with IL-2 and IFN-g promoting IgG2, IL-4 promoting IgG1 and IgE, and IL-5 promoting IgA.

4/ Phenotypic and functional markers for Th1 and Th2 cell subsets

Th1 and Th2 clones were defined by their cytokine production pattern in vitro, and there has been much effort to find good markers
to identify Th1 or Th2 cells in vivo, but with overall little success. The CD4-like molecule LAG-3 was thought to be a surface
marker for Th1 cells, while CD30 (TNF-R family) and ST2L (IL-1R family) were thought to be specific for Th2, but all these
markers probably more accurately reflect IFN-g or IL-4 responsive cells respectively, as does the expression of the cytokine receptor
signalling molecules Stat-4 and Stat-6. The loss of the IL-12Rb chain is thought to be a marker for Th2 commitment, while the
transcription factor GATA-3 is lost on Th1 cells. It was originally thought that chemokine receptor expression would reliably
distinguish the different subsets, and although there is some functional division (CCR1, CCR5 and CXCR3 on Th1 while Th1
express CCR3 and CCR4), it is becoming clear that this association is less well defined in vivo. The best way to identify Th1 and
Th2 cells is therefore immunofluorescent staining for the appropriate cytokines on fixed and permeabilised cells, after a brief
activation step in vitro in the presence of Brefeldin or Monensin that amplify staining by holding the cytokines in the golgi.

5/ Th1 and Th2 responses in disease

The Th1/Th2 paradigm has been used to explain a wide variety of disease and pathological conditions, both in expreimental rodent
models and in man. The classical example is that of Leishmania infection of C57Bl/6 mice that induces a protective Th1 response,
compared to BALB/c mice that insetad make an ineffective Th2 response and die from a progressive infection (the genetic basis is
still not clear). A similarly classical example in man is that of Tuberculoid Th1 lesions compared to lepromatous Th2 disease.
Generally speaking, Th1 responses protect from invasive bacterial, protozoal and viral infections, and cause autoimmune diseases,
while Th2 responses protect from extracellular parasites, helminths, and cause allergic responses in atopic individuals.

6/ Th1 and Th2 in transplantation and pregnancy

The Th1/Th2 paradigm has also been applied to transplantation, with Th1 responses being implicated in most forms of acute
rejection and graft versus host disease, while Th2 responses have been variably associated with either protection or chronic rejection.
However, cloned Th1 or Th2 cells have a similar capacity to reject skin grafts in experimental models, and Tr1/Treg cells are now
being implicated in protection and tolerance induction. The foetus is also analogous to an allograft, and Th2 or Treg responses are
thought to be protective, while Th1 may lead to resorption or spontaneous abortion.

T cell subsets - selected references

The primary literature on Th1, Th2 and other T cell subsets is massive, and I have therefore provided mainly
review articles for this lecture, and these will obviously contain original references to those particular
aspects of the field that may interest you further.

Mosmann, T.R., and Sad, S.
The expanding universe of T-cell subsets: Th1, Th2 and more.
Immunology Today 17: 138-146 (1996)

Fallon, P.
Immunopathologyof schistosomiasis: a cautionary tale of mice and men.
Immunology Today 21: 29-35 (2000)

Murphy, K.M.
T lymphocyte differentiation in the periphery.
Current Opinion in Immunology 10: 226-232 (1998)

Louis, J., Himmelreich, H., et al.
Regulation of protective immunity against Leishmania major in mice.
Current Opinion in Immunology 10: 459-464 (1998)

Abbas, A.K., Murphy, K.M., Sher, A.
Functional diversity of helper T lymphocytes.
Nature 383: 787-793 (1996)

Sallusto, F., Lanzavecchia, A., and Mackay, C.R.
Chemokines and chemokine receptors in T cell priming and Th1/Th2-mediated responses.
Immunology Today 19: 568-574 (1998)

Zelenika, D., Mellor, A., Simpson, E., Stockinger, B., Adams, E., Waldmann, H., & Cobbold, S.
Rejection of H-Y disparate skin grafts by monospecific CD4+ T helper 1 (Th1) and T helper 2 (Th2) cells:
no requirement for CD8+ T cells or B cells.
J. Immunol., 161: 1868-1874 (1998)

Hammond, KJ., Pelikan, SB., et al.
NKT cells are phenotypically and functionally diverse.
Eur. J. Immunol. 29: 3768-81 (1999)

Carter, LL., Swain, SL.
Single cell analyses of cytokine production.
Current Opinion in Immunology 9: 177-82 (1997)

Groux-H ; O'Garra-A; Bigler-M; Rouleau-M; Antonenko-S; de-Vries-JE; Roncarolo-MG
A CD4+ T-cell subset inhibits antigen-specific T-cell responses and prevents colitis.
Nature 389: 737-42 (1997)

Lymphocyte Subset: CD4 T-cell Lymphocyte Subset: CD8 T-cell
Defined by: CD8 (Lyt-2/3)
Defined by: CD4 (L3/T4)

Also expresses: CD3, CD2, TCR (usually ab ) Also expresses: CD3, CD2, TCR (usually ab)

Function: MHC-class II restricted Function: MHC-class I restricted
T-cell responses T-cell responses

Ag Ag

TCR MHC-II TCR MHC-I

T-cell APC T-cell APC

CD4 CD8

Lymphocyte Subset: Helper T- cell Lymphocyte Subset: Cytotoxic T-cell

Defined by: Functions Defined by: MHC restricted killing

Also expresses: CD3, CD2, TCR, CD4 (usually) Also expresses: CD3, CD2, TCR , CD4 or CD8

Function: Help for antibody (B- cells) Function: Killing of virus infected cells,
Help for T-cells (lymphokines) FasL -> Fas (and rejection of foreign tissue).

Lymphokines (IL-2, IL-4 etc) Perforin Killing
Ag Granzymes Ag
TCR MHC
TCR MHC-II APC T-cell
Target
Helper (Macrophage, cell
T-cell
dendritic cell,

or B-cell)

CD4 CD4 or CD8

(or CD8/MHC-I for Tc2?)

Lymphocyte Subset: Activated T-cell Lymphocyte Subset: Memory T-cells
(Th1, Th2 and cytotoxic T-cells)

Defined by: Activation markers Defined by: "Secondary responses in vitro"
Also expresses: eg. CD25 (IL-2 receptor) but note Also expresses:
may also be expressed on Treg CD3, CD2, CD4 or CD8, TCR
Function: CD45R0 (man), Pgp-1 (mouse CD44)
CD2R, CD26, CD71 (Tf-rec) CD29 (VLA antigens), increased integrins
CD3, CD4 or CD8, TCR CD45RB low and L-Selectin low
also MHC-II in man, rat, NOT mouse
Immunity to previous Ag encounter
Function: Proliferation in response to antigen

IL-2 receptor High affinity IL-2 receptor + _ + CDw49 + CD45RA
beta chain (p75) IL-2 receptor beta chain (p75) CD45RA CD45RA CD_45R0_ CD29 CD_45R0_ CD45RB
CD45RB+ (RA ,RB )
B CD25 B B + (RA ,RB ) Long-Term
CD45RB Memory
T-cell
'Th1' or AB

L-selectin 'Th2'

Naive Antigen Activated IL-2 etc Memory Naive Antigen Activated IL-2 etc Memory ?Time?
T-cell T-cell T-cell T-cell T-cell T-cell

CD44 CD44

Lymphocyte Subset: T- regulatory cell

Lymphocyte Subset: Suppressor T-cell

Poorly defined as yet! Defined by: "Suppression" of responses
Defined by: Th3 = TGFb, Tr1 = IL-10, Treg = CD25/anergy in vitro or transfer in vivo

Also expresses: CD3, CD2, TCR, CD4 (usually)
CD45RB low, CD38, CD25, CTLA4?
Also expresses: CD8 (or CD4), CD3, CD45RA?

Functions: Down regulation of Th1 (and Th2?) Ag presentation? Function: Immuno-regulation?
Existence controversial
Control of autoreactivity

Oral tolerance to food and gut flora

Transplantation tolerance a) Anti-idiotype?

Anti-inflammatory TCR TCR

cytokines (IL-10, TGF-beta) Antigen-
specific
Ag "Suppressor" or T-cell Ag-MHC
T-cell
TCR MHC-II APC

Type 3 (B cells, MHC-I
or T reg 1 dendritic cell?) CD8

T-cell b) Anti-TCR CDR peptides + MHC

CD4

Lymphocyte Subset: NK- cell Lymphocyte Subset: K- cell

Defined by: Killing of K562 (human) YAC-1 (mouse) Defined by:
Also expresses:
Also expresses: Killer inhibitory receptors (KIRs) CD16 (FcR-III)
HNK-1 (CD57), NKH-1 (CD56 / NCAM) Function:
CD2, CD3+/-, CD8+/-
CD2, CD3+/-, CD8+/-, CD16+/-
Antibody dependent
Function: Innate response/Th1 amplification cell mediated cytotoxicity
Killing of syngeneic tumours?
(ADCC)
FasL -> Fas
Perforin Killing

Granzymes or MHC-I K-cell CD16 Ag
KIR HLA-E
CD16 Target Target
Cross- cell Lymphocyte cell
linking inhibits or
activates
NK- cell Macrophage

K562 Ag
Antibody
CD2 Adhesion molecules
(LFA-1->ICAM-1 etc)

Interferong

Lymphocyte Subset: NKT - cell Lymphocyte Subset: Gamma/delta T-cell

Defined by: NK1.1, CD4 low (mouse) Defined by: TCR gamma/delta expression
Also expresses:
Also expresses: CD3, CD2, limited TCR CD3, CD2, CD7
Va14 (Ja281)/Vb7 (mouse) Function: usually CD4 negative, CD8+/-

Va 24 (Ja Q)/Vb11 (man) Non-classical MHC antigen presentation
Responses to Mycobacterial Glycolipids
Function: Amplification of Th2
T-cell responses?

Glycolipid Ag? Ag (glycolipid)
TCR CD1
TCR CD1
gd
ab
APC
NK APC
T-cell

T-cell

CD4

High levels of Cytokines
(esp. IL-4)

Lymphocyte Subset: Helper T- cell (Th1) Lymphocyte Subset: Helper T- cell (Th2)

Defined by: Cytokine secretion Defined by: Cytokine secretion
Also expresses: IFN-g , LT (TNF-b ), IL-2 Also expresses: IL-4, IL-5, IL-10

CD3, CD2, TCR, CD4 (usually), LAG-3 CD3, CD2, TCR, CD4 (usually), CD30, ST2L

Functions: Activation of Type I effectors Functions: Activation of Type 2 effectors
(cytotoxic T cells, macrophages (B cells [esp. IgG1, IgE, IgA]

NK and K cells, neutrophils) eosinophils, mast cells)

Down regulation of Th1 response

Cytokines (IFN-g etc) Cytokines (IL-4, IL-5, IL-13 etc)

Ag IFN-g receptor Ag IL-4 receptor

Type 1 TCR MHC-II Stat-4 Type 2 TCR MHC-II Stat-6

Helper APC Helper APC
T-cell (Macrophage, T-cell (B cells,
dendritic cell) dendritic cell)

CD4 CD4

CD40L CD40 CD28 CD80/86

"Positive Feedback" Amplification Lymphocyte Subset: Type 2 Cytotoxic T-cell (Tc2)
of Cytokine Driven T-cell Responses

Defined by: CD8 plus Type 2 cytokines

Also expresses: CD3, CD2, TCR
Function:
Killing of infected cells
in ongoing Th2 response?

FasL -> Fas Killing
Perforin Ag
TCR MHC-I
Granzymes
Target
Tc2 cell
T-cell

CD8

IL-4, IL-5, IL-10 Help for B cells?

Regulation of Th1 versus Th2 responses

Microbial factors NK cells IFN-g
eg. LPS IFN-g IL-18
Actiavnadteddemndarcitricopcheallgs es LAG-3
IL-12R ab
IL-12 Th1 CCR1 RANTES
Stat-4 CCR5
CXCR3 Macrophage Inflammatory
Protein-1 (MIP-1 )a,b

IP-10

Secondary Lymphoid IL-2 Loss of
Chemokine (SLC) GATA-3
(irreversible?)
CCR7

Th0 CD62L AcTtihva0ted TGF- b
GATA-3
CXCR4 To Node IL-4

Stromal cell DC-CK1R IL-4
Derived Factor
Dendritic cell CD30
(SDF-1) chemokine 1 GATA-3 ST2L

IL-4R Th2 CCR3 Eotaxin
CCR4
Stat-6 Macrophage derived
Chemokine (MDC)
Oethge. rNsKoTucrceellss
Loss of
IL-12R b

(irreversible?)

Th1 versus Th2 responses in Disease

Beagc.teLeriiashl imnfaencitaion Macrophage

eVgir.aIlnifnlufeecntziaon IL-12 IFN-g IgG2 iNOS Protection from:
HIV Leishmania (C57Bl/6 mice)
Th1 IFN-g
IL-2 Macrophage Listeria
IL-4 HIV (early), Influenza
CTLIFNiN-Og S Tuberculosis, Candida
Th2 IgG2
Delayed
Neutrophil Hypersensitivity

Pathology generated:
Psoriasis, IDDM, RA
Tuberculoid Leprosy Lesions
Hepatosplenic Schistosomiasis
Graft rejection, foetal resorption?

Th0 AcTtihva0ted Pathology generated:
Leishmania (BALB/c mice)
egA.spDfe-A1rpll(e-a1rsgp(edenursgstiimlluist)e) IL-4 IgE
HIV (late)
Helminths Mucosal epithelium B cell Eosinophil Allergy: Asthma, Airway Disease
Lepromatous Leprosy Lesions
egN. Sipcphoissttoromnsgoymluess IgA
Chronic graft rejection
Mast cells
Immediate
Hypersensitivity

Protection from:
Schistosomiasis, Brugia etc

Malaria (late)
Autoimmunity: EAE, EAU ?

Monospecific female (A1xRAG-/-) anti-HY+H-2Ek
TCR Tg mice generate both Th1 and Th2 responses

as a consequence of rejecting male skin grafts

% male skin graft survival CD8-QR anti-mIg-FITC
CD4-PE IFN-g

CD44-QR IFNg-FITC
IL-2
IL-4

IL2-FITC IL4-FITC

Rejection of male (but not female) skin by A1(M)xRAG-1-/-
Th1 and Th2 lines

(by transfer into "empty" ATX-depleted CBA/Ca female mice)

1 R2.2 Th1 clone l MST = 11 days
2 R2.4 Th2 clone m MST = 13 days
3 D1 Treg clone MST > 25 days

All female grafts
lm

Treg

m

Th1 l m Th2

m