Advanced ImmunologyTh Cell Subsets and Cytokines

Dale T. Umetsu, MD, PhD

Stanford University

February 26, 2003

Humoral immunity is essential for the elimination of extracellular bacterial infections

www.cellsalive.com

Bacteria are opsonized with antibody and complement

Cellular immunity is required for infections with intracellular pathogens

Reciprocal relationship between humoral and cellular immunity

Dose of Flagellin

Parish and Liu, 1972

Humoral immunity

Cell mediated immunity

Adaptive Immunity

Humoral Immunity (CD4) (extracellular organisms)

– IgG, IgA, IgM, IgE Cell-mediated Immunity

(intracellular organisms)

– Delayed type hypersensitivity (CD4)

– Cytotoxic responses (CD8)

Th Cell Subsets and Cytokines

Characteristics of Th1/Th2 subsets. Situations in which Th subsets are

important. How these subsets arise and differentiate. Modifications of the Th1/Th2 paradigm.

– Related T cell subsets– Regulatory T cell subsets.

IFN- + - + -

IL-4 - + +-

Lymphotoxin + --

IL-2 + - ++

Expression of cytokines by CD4 T cell subsets

IL-3 + + + -

GM-CSF + + +

TNF- + +

Cytokine Th1 Th2 Th0 Thp

IL-5 - + +-

IL-9 - +IL-10 - +

-IL-13 - +

1

IFN-

IFN- IFN-

IgG2a, IgG3

IFN-

Effector Functions of Th1 Cells

Effector Functions of Th2 Cells

Generation of Th Cell lines

DO11.10OVA-specific TCR Tg

Isolate clonotypicCD4+ T cells

OVA + APCrIL-12Anti-IL-4 mAb

OVA + APCrIL-4Anti-IL-12 mAb

OVA + APCrIL-4rIFN-

Th1

Th2

Th0

Markers of Th1 and Th2 Cells

Th1 Th2

IFN-IL-12R2IL-18RP- and E- selectin receptorsCXCR3, CCR5

Tim3

Stat4, T-bet

IL-4IL-1R, IFN- receptorT1/ST2 (homolog of IL-1R)

CCR4, CCR8

Tim1ICOSStat6, GATA-3, c-maf

TIM family of genesTIM family of genes

TIM-1TIM-1 encodes a 305 aa protein, regulates the encodes a 305 aa protein, regulates the development of asthma and allergy development of asthma and allergy (McIntire, et al. 2001. (McIntire, et al. 2001.

Nature Immunol. 2:1109) (McIntire, et al. 2003. Nature 425:576) Nature Immunol. 2:1109) (McIntire, et al. 2003. Nature 425:576) . . In mice, TIM-3 encodes a 281 aa protein, In mice, TIM-3 encodes a 281 aa protein,

regulates the development of autoimmune regulates the development of autoimmune diseases diseases (Monney et al. 2002. Nature. 415:536)(Monney et al. 2002. Nature. 415:536)..

TIM-1 is preferentially expressed in Th2 cells. TIM-1 is preferentially expressed in Th2 cells. TIM-3 is preferentially expressed in Th1 cells TIM-3 is preferentially expressed in Th1 cells

(Sanchez-Fueyo, et al. Nature Immunol. Oct 2003)(Sanchez-Fueyo, et al. Nature Immunol. Oct 2003). .

TIM: T cell, Immunoglobulin domain, Mucin domain.

Infection with Leishmania

C3H/HeN resistant IFN-, IL-2

BALB/c susceptible IL-4, IL-5, IL-13

Strain result cytokines produced

Disease States Attributed to an Imbalance in Th1/Th2 Cells

Autoimmune diseasesMultiple sclerosisRheumatoid arthritisDiabetes mellitusCrohn’s disease

Graft rejectionRecurrent abortions(Helminth infection)

InfectionsLeishmaniaTB, leprosyFungal infectionHIV

Allergy and asthmaUlcerative colitis

Over production Over production of Th2 cytokines of Th1 cytokines

T Cell Subsets: when are they important?

During chronic antigen stimulation– Chronic infection

– Chronic response to auto antigens.

– Chronic exposure to allergens. Not following a single antigenic

stimulation, not with fulminant infections.

Th1 Cells Cross Regulate Th2 Cells

Th1 cytokines IL-2 IFN- IL-12 Th2 cytokines

IL-4 IL-5 IL-13

IL-4IL-10

IL-12IFN-

What are the instructive signals for the development of Th1/Th2 cells

Cytokine microenvironment Antigen dose/TCR signaling

– Altered peptide ligands APC type

– Route of antigen administration Costimulatory signals Host genetic factors

Signals that influence Th1 differentiation

IL-12, TGF-

IFN-

IL-4, IL-10, TGF- inhibits

CD8+ DCB7.1, DC1 (human)

IL-18

IL-12

IL-27

Heterodimeric cytokine, related to IL-12. p28 (IL-12p35-related) + EBI3 (IL-12p40-

related) = IL-27. Long chain four-helix bundle cytokine. Early product of activated APC. Drives expansion of naïve but not memory

CD4 T cells. Synergistic with IL-12 in triggering IFN-

production in naïve T cells.

Pflanz et al. 2002. Immunity. 16:779.

IL-23

p19 subunit + IL-12 p40 subunit = IL-23 IL-23 binds to IL-12R1 + another novel

subunit. Produced by activated dendritic cells. Induces proliferation and IFN- production

by memory (Th1) cells. Some shared activities with IL-12 (p35/p40).

Oppmann et al. 2000. Immunity. 13:715

Signals that influence Th2 differentiation

IL-12, IL-18, TGF- inhibits

Humoral responsesAllergic responses

B7.2, OX40L, ICOSL

CD8- DC, DC2 (human)

TSLP (humans)

IL-4, IL-25

IL-25 Related to IL-17. Produced by Th2 cells. Induces production of IL-4, IL-5, IL-13,

eotaxin, and increases IgE, IgG1, eosinophilia.

Induces IL-13 and IL-5 from accessory cell population.

Affects lungs, GI tract; effects dependent on IL4R signaling.

Fort et al. 2001. Immunity. 15:985

Thymic Stromal Lymphopoietin (TSLP)

Short-chain four -helical bundle cytokine. Produced by human epithelial cells. Binds to TSLPR + IL-7R. Activates DCs, which then induce Th cell

production of IL-4, IL-5, IL-13, reduces production of IL-10 and IFN-, and induces production of Th2 attracting chemokines.

Highly expressed in skin of patients with atopic dermatitis.

Soumelis et al. 2002. Nature Immunol. 3:673

Anergy

CD8CD40CD86

Role of Immature Dendritic Cells

ICOS-ICOSL pathway

Nature Rev. Immunol. 2002. 2:116.

ICOS is the third member of the CD28 family.– Expressed on activated T cells

(particularly on Th2 cells).

ICOS binds to ICOS-ligand (B7h,

B7RP-1, B7-H2). ICOS upregulates CD40L.

– Important for isotype switch.

ICOS engagement promotes Th cell differentiation and effector function– Induces IL-4, IL-5, esp. IL-10

production.

– Also important for tolerance induction.

APCT Cell

Inhibitory receptors may influence Th cell differentiation

Nature Rev. Immunol. 2002. 2:116.

B7 binds CTLA-4. PD-L1 (B7-H1) and PD-L2

(B7-DC) bind to PD-1. PD-1-/- mice develop SLE

glomerulonephritis. B7x (B7-H4) binds to

BTLA, the 5th member of the CD28 family.

BTLA-/- mice develop enhanced sensitivity to EAE.

B7-H3 (inhibitory pathway)

APC T Cell

B7x BTLA

B7-H3

Tc1 and Tc2 Polarized CD8 Cells

CD8 cells cultured with IL-12 produce IFN-. CD8 cells cultured with IL-4 produce IL-4 and

IL-5, IL-10 and some IFN-. – CD8 cells make 100 fold less IL-4 than CD4 cells.– CD8 cells make 3-5 fold less IL-5 than CD4 cells.

Tc1 CD8 but not Tc2 effector cells protect against viral infection.

Croft et al. 1994.. J Exp Med. 180:1715. Cerwenka et al. 1999. J Exp Med. 189:423

Be1 and Be2 Polarized Cell Subsets

Harris et al. 2000. Nature Immunol. 1:465.

Signals that influence Th differentiation

NK T cell

eosinophil

NK T cell

iNKT CellsiNKT Cells Express cell surface markers characteristic of both NK

cells and conventional T cells. In mice, most NKT cells express an invariant V14-

J18 TCR (V14 iNKT cells) (V24J15 in humans). Recognize glycolipid antigens presented by the MHC

class I protein, CD1d. When activated, NK T cells rapidly produce large

amounts of IL-4 and IFN-. NKT cells regulate the development of autoimmune

diseases, such as diabetes, EAE, cancer and asthma.

NKT Cells in Autoimmunity (tolerance?) and Cancer

Type 1 diabetes– Reduced number of NKT cells in NOD mice and patients

with diabetes. – Treatment of NOD mice with GalCer protects.

EAE– Treatment with GalCer protects, but not in IL-4-/- or IL-10-/-

mice. – Reduced number of NKT cells in MS patients.

Tumor immunity– Lack of iNKT cells increased tumor metastasis (melanomas

and hepatomas). – In one system, IL-13 production by NKT cells inhibited

tumor immunity.

NK T Cell Subsets

CD4+ NK T cells produce IL-4, IL-5, IL-13, IFN-

CD4- CD8- NK T cells produce IFN-

Gumperz et al. 2002. J Exp Med. 195:625.

NKT Cells are Required for Oxazolone Colitis, a Th2 Colitis Model

Immunity. 2002. 17:629.

NKT Cells are Required for the Development of AHR

0

2.5

5

7.5

10

Pe

nH

0 10 20 30 40 50

Methacholine (mg/ml)

CD1 KO OVA

CD1 KO PBS

BALB/c OVA

BALB/c PBS

Akbari. Nature Medicine. 2003. 9:582.

NKT Cells Bridge Innate and Acquired Immunity

Transcription factors that influence Th differentiation

Pearce, EL et al. 2003. Control of effector CD8 T cell function by the transcription factor eomesodermin. Science 302:1041.

Agarwal, Rao. 1998. Immunity. 9:765

nucleosome

DNA

Normal inactive chromatin(ground state)

Acylated histonesStat6, GATA3

Monoallelic Gene Expression

IL-4 Gene

Both alleles inactive

Allele 1

Allele 2

activation

One allele expressed(monoallelic expression)

sustainedactivation

Both alleles expressed

Genetic Effects

Individuals differ in their susceptibility to autoimmune disease, infection, and allergic disease.

Susceptibility is in part regulated by differences in the capacity to generate Th1 or Th2 cytokines.

Susceptibility genes are identified by DNA sequence differences between individuals (humans) or strains (mice).

Some Gene Polymorphisms Affecting Cytokine Production

IL-4 receptor (SNPs present in coding region). IL-13 (SNPs present in coding region) MHC linked to some autoimmune diseases. NOD2 linked to Crohn’s disease (LPS receptor)

(Nature 2001. 411:603). CTLA-4 alternative splice form (chromosome 2q33)

(Graves disease, hypothyroidism, type 1 diabetes). (Nature 423:506)

TIMs (Nature. 2003. 425:576)

Lafaille, et al. J. Exp. Med 186:307, 1997.

Th1(5x106)Th2 (5x106)

Th1(0.2x106)

Th2(0.2x106)

Attempts to tolerize with MOG results in exacerbation. Genain, et al. 1996. Science. 274:2054

Induce Th2 response Control

Transfer of diabetes with both Th1 and Th2 cellsPakala, et al. 1997. JEM. 186:299.

Th1 cells do not inhibit Th2 cell-induced Th1 cells do not inhibit Th2 cell-induced airway hyperreactivityairway hyperreactivity

0

200

400

600

800

% a

bove b

aselin

e

0 10 20 30 40 50

Methacholine (mg/ml)

none

Th1

Th1+Th2 (2:1)

Th1+Th2 (1:1)

Th2

transferred cells:

Hansen, et al J.C.I. 1997

The Th1/Th2 Paradigm and Disease Regulation

Both Th1 and Th2 cells can cause disease. The “opposite” of a Th1 cell is not always a

Th2 cell. The Th1/Th2 paradigm cannot fully explain

immune regulation in all situations. Additional regulatory cells must exist to

regulate immune responses.

Other CD4 Subsets that “Regulate”

Th3 CD4 + CD25+ cells TR1

TR

NKT cells

Th3 Cells

Generated by oral tolerance induction with low dose antigen.

Produce TGF-, IL-4 Express regulatory/suppressive activity. TGF- production may be enhanced by

cross-linking of CTLA-4.

Chen, Y, et al 1994. Regulatory T cell clones induced by oral tolerance: suppression of autoimmune encephalomyelitis. Science 265:1237.

MBP specific Th3 Cells Inhibit EAE

Th3 cellsTh3 cells + anti-TGF

Science. 1994. 265:1237.

CD45RBlow Cells, CD25+ Cells

CD25+ (IL-2R-chain), CTLA-4, GITR, foxp3. When transferred, have strong

regulatory/suppressive activity for colitis, diabetes, thyroiditis.

Appear to require TGF- IL-10 and CTLA-4 to function.

In vitro activity depends on cell contact. Antigen specificity?

Tr1 Cells

Develop after stimulation with IL-10. Produce IL-10, IL-5, (TGF-. Low proliferative capacity. Inhibitory for experimental colitis. Anti-IL-10 mAb reverses inhibitory

effect.

Groux et al. 1997. Nature. 389:737.

DCs from mice exposed to i.n. OVA produce IL-10

. . .

H P R TIL - 1 0RN A H P R TIL - 1 0RN AH P R TIL - 1 0RN AH P R TIL - 1 0RN A H P R TIL - 1 0RN A

Day 1 Day 3 Day 7 Day 10 Day 14

K J I . 2 6

CFSE CFSE

Day 3 Day 7

K J I . 2 6

b

RN

AIL

-10

RN

AIL

-10

RN

A

PBS i.n. OVA-Alum i.d.OVA i.n. OVA(i.p.+i.n.)

33%

2.3%

Intracellular staining Intracellular staining

a

IL-1

0

IL-10

RN

AIL

-10

-ac

tin

-ac

tin

-ac

tin

-ac

tin

Akbari, et al. 2001 Nature Immunol.

IL-10 producing DCs induce development of T cell lines producing IL-10

KJ1-26

IFN

IL-4

IL-10

0 1 2 3 4

IL-4

IL-10

IFN-

Akbari, et al. 2002, Nature Medicine

Summary-T cell subsets

A major theme in immunology, and documented feature of the immune system.

Subsets of T cells express restricted cytokine profiles

Cells with restricted cytokine profiles (CD4, CD8, NK, NK T, B) have distinct effector functions and regulate immune responses.

Questions that remain

What regulatory cells “balance” Th1 and Th2 cells? What downregulates polarized responses?

How Th subsets are involved in tolerance? What additional molecular mechanisms regulate

cytokine synthesis? Are there Th2 PAMPs? What activates NKT

cells? What are the host/genetic factors that regulate

cytokine production?

ReferencesHawiger D, Inaba K, Dorsett Y, Guo M, Mahnke K, Rivera M, et al. Dendritic cells induce peripheral T cell unresponsiveness under steady state conditions in vivo. J Exp Med 2001; 194:769-79.

Steinman R, Hawiger D, Nussenzweig M. Tolerogenic dendritic cells. Annu Rev Immunol 2003; 21:685-711

Sharpe AH, Freeman GJ. The B7-CD28 superfamily. Nat Rev Immunol 2002; 2:116-26.

Weiner HL. The mucosal milieu creates tolerogenic dendritic cells and Tr1 and Th3 regulatory cells. Nature Immunol. 2001; 2:671-2.

Hori S, Nomura T, Sakaguchi S. Control of Regulatory T Cell Development by the Transcription Factor Foxp3. Science 2003; 299:1057-61.

Pasare C, Medzhitov R. Toll pathway-dependent blockade of CD4+CD25+ T cell-mediated suppression by dendritic cells. Science 2003; 299:1033-6.

Wilson SB, Kent SC, Patton KT, Orban T, Jackson RA, Exley M, et al. Extreme Th1 bias of invariant V24JQ T cells in type 1 diabetes. Nature 1998; 391:177-81.

Wakkach A, Fournier N, Brun V, Breittmayer JP, Cottrez F, Groux H: Characterization of dendritic cells that induce tolerance and T regulatory 1 cell differentiation in vivo. Immunity 2003, 18:605-617.

References

Heller F, Fuss I, Nieuwenhuis E, Blumberg R, Strober W. Oxazolone colitis, a Th2 colitis model resembling ulcerative colitis, is mediated by IL-13-producing NK-T cells. Immunity 2002; 17:629-38.

Terabe M, Matsui S, Noben-Trauth N, Chen H, Watson C, Donaldson D, et al. NKT cell-mediated repression of tumor immunosurveillance by IL-13 and the IL-4R-STAT6 pathway. Nat Immunol 2000; 1:515-20.

Ansel KM, Lee, DU, Rao A. An epigenetic view of helper T cell differentiation. Nature Immunol. 4:616.

Shimizu J, Yamazaki S, Takahashi T, Ishida Y, Sakaguchi S: Stimulation of CD25(+)CD4(+) regulatory T cells through GITR breaks immunological self-tolerance. Nat Immunol 2002, 3:135-142.

Levings MK, Sangregorio R, Sartirana C, Moschin AL, Battaglia M, Orban PC, Roncarolo MG: Human CD25+CD4+ T suppressor cell clones produce transforming growth factor beta, but not interleukin 10, and are distinct from type 1 T regulatory cells. J Exp Med 2002, 196:1335-1346.