1. Advanced Immunology Th Cell Subsets and Cytokines
Dale T. Umetsu, MD, PhD
Stanford University
February 26, 2003

2. Humoral immunity is essential for the elimination of extracellular bacterial infections
Bacteria are opsonized with antibody
and complement

3. Cellular immunity is required for infections with intracellular pathogens

4. Reciprocal relationship between humoral and cellular immunity
Humoral immunity
Cell mediated immunity
Dose of Flagellin
Parish and Liu, 1972

5. Adaptive Immunity Humoral Immunity (CD4) (extracellular organisms)
IgG, IgA, IgM, IgE
Cell-mediated Immunity (intracellular organisms)
Delayed type hypersensitivity (CD4)
Cytotoxic responses (CD8)

6. 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.

7. Expression of cytokines by CD4 T cell subsets
Cytokine Th1 Th2 Th0 Thp
IFN-g IL
Lymphotoxin IL IL
GM-CSF
TNF-a IL IL IL IL

8. Effector Functions of Th1 Cells
IFN-g
IFN-g
IFN-g 1
IFN-g
IgG2a, IgG3

9. Effector Functions of Th2 Cells

10. Generation of Th Cell lines
OVA + APC
rIL-12
Anti-IL-4 mAb
Th1
Isolate
clonotypic
CD4+ T cells
OVA + APC
rIL-4
Anti-IL-12 mAb
Th2
OVA + APC
rIL-4
rIFN-g
Th0
DO11.10
OVA-specific TCR Tg

11. Markers of Th1 and Th2 Cells
IFN-
IL-12R2
IL-18R
P- and E- selectin receptors
CXCR3, CCR5
Tim3
Stat4, T-bet
IL-4
IL-1R, IFN- receptor
T1/ST2 (homolog of IL-1R)
CCR4, CCR8
Tim1
ICOS
Stat6, GATA-3, c-maf

12. TIM family of genes
TIM-1 encodes a 305 aa protein, regulates the development of asthma and allergy (McIntire, et al Nature Immunol. 2:1109) (McIntire, et al Nature 425:576) .
In mice, TIM-3 encodes a 281 aa protein, regulates the development of autoimmune diseases (Monney et al Nature. 415:536).
TIM-1 is preferentially expressed in Th2 cells.
TIM-3 is preferentially expressed in Th1 cells (Sanchez-Fueyo, et al. Nature Immunol. Oct 2003).

13. TIM: T cell, Immunoglobulin domain, Mucin domain.

14. Infection with Leishmania
Strain result cytokines produced
C3H/HeN resistant IFN-g, IL-2
BALB/c susceptible IL-4, IL-5, IL-13

15. Disease States Attributed to an Imbalance in Th1/Th2 Cells
Over production Over production
of Th2 cytokines of Th1 cytokines
Infections
Leishmania
TB, leprosy
Fungal infection
HIV
Allergy and asthma
Ulcerative colitis
Autoimmune diseases
Multiple sclerosis
Rheumatoid arthritis
Diabetes mellitus
Crohn’s disease
Graft rejection
Recurrent abortions
(Helminth infection)

16. 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.

17. Th1 Cells Cross Regulate Th2 Cells
Th1 cytokines
IL-2
IFN-g
IL-12
Th2 cytokines
IL-4
IL-5
IL-13
IL-12
IFN-g
IL-4
IL-10

18. 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

19. Signals that influence Th1 differentiation
IL-18
CD8a+ DC
B7.1, DC1 (human)
IL-12
IL-12, TGF-b
IFN-g
IL-4, IL-10, TGF-b inhibits

20. 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-g production in naïve T cells.
Pflanz et al Immunity. 16:779.

21. IL-23 p19 subunit + IL-12 p40 subunit = IL-23
IL-23 binds to IL-12Rb1 + another novel subunit.
Produced by activated dendritic cells.
Induces proliferation and IFN-g production by memory (Th1) cells.
Some shared activities with IL-12 (p35/p40).
Oppmann et al Immunity. 13:715

22. Signals that influence Th2 differentiation
TSLP (humans)
Humoral responses
Allergic responses
IL-4, IL-25
B7.2, OX40L, ICOSL
CD8a- DC,
DC2 (human)
IL-12, IL-18, TGF-b inhibits

23. 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 IL4Ra signaling.
Fort et al Immunity. 15:985

24. Thymic Stromal Lymphopoietin (TSLP)
Short-chain four a-helical bundle cytokine.
Produced by human epithelial cells.
Binds to TSLPR + IL-7Ra.
Activates DCs, which then induce Th cell production of IL-4, IL-5, IL-13, reduces production of IL-10 and IFN-g, and induces production of Th2 attracting chemokines.
Highly expressed in skin of patients with atopic dermatitis.
Soumelis et al Nature Immunol. 3:673

25. Role of Immature Dendritic Cells
CD8a
CD40
CD86
Anergy

26. ICOS-ICOSL pathway 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.
APC
T Cell
Nature Rev. Immunol :116.

27. Inhibitory receptors may influence Th cell differentiation
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)
B7x
BTLA
B7-H3
APC
T Cell
Nature Rev. Immunol :116.

28. Tc1 and Tc2 Polarized CD8 Cells
CD8 cells cultured with IL-12 produce IFN-g.
CD8 cells cultured with IL-4 produce IL-4 and IL-5, IL-10 and some IFN-g.
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 J Exp Med. 180: Cerwenka et al J Exp Med. 189:423

29. Be1 and Be2 Polarized Cell Subsets
Harris et al Nature Immunol. 1:465.

30. Signals that influence Th differentiation
NK T cell
NK T cell
eosinophil

31. iNKT Cells
Express cell surface markers characteristic of both NK cells and conventional T cells.
In mice, most NKT cells express an invariant Va14-Ja18 TCR (Va14 iNKT cells) (Va24Ja15 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-g.
NKT cells regulate the development of autoimmune diseases, such as diabetes, EAE, cancer and asthma.

32. 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 aGalCer protects.
EAE
Treatment with aGalCer 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.

33. NK T Cell Subsets CD4+ NK T cells produce IL-4, IL-5, IL-13, IFN-g
CD4- CD8- NK T cells produce IFN-g
Gumperz et al J Exp Med. 195:625.

34. NKT Cells are Required for Oxazolone Colitis, a Th2 Colitis Model
Immunity :629.

35. NKT Cells are Required for the Development of AHR
Akbari. Nature Medicine :582.

36. NKT Cells Bridge Innate and Acquired Immunity

37. Transcription factors that influence Th differentiation
Pearce, EL et al Control of effector
CD8 T cell function by the transcription factor
eomesodermin. Science 302:1041.

38. Agarwal, Rao Immunity. 9:765

39. Stat6, GATA3 Acylated histones Normal inactive chromatin
(ground state)
nucleosome
DNA

40. Monoallelic Gene Expression
activation
One allele expressed
(monoallelic expression)
sustained
activation
Both alleles expressed
IL-4 Gene
Both alleles inactive
Allele 1
Allele 2

41. 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).

42. Some Gene Polymorphisms Affecting Cytokine Production
IL-4 receptor a (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 :603).
CTLA-4 alternative splice form (chromosome 2q33) (Graves disease, hypothyroidism, type 1 diabetes). (Nature 423:506)
TIMs (Nature :576)

43. Lafaille, et al. J. Exp. Med 186:307, 1997.
Th1(5x106)
Th2 (5x106)
Th1(0.2x106)
Th2(0.2x106)
Lafaille, et al. J. Exp. Med 186:307, 1997.

44. Attempts to tolerize with MOG results in exacerbation.
Induce Th2 response
Control
Attempts to tolerize with MOG results in exacerbation.
Genain, et al Science. 274:2054

45. Transfer of diabetes with both Th1 and Th2 cells
Pakala, et al JEM. 186:299.

46. Th1 cells do not inhibit Th2 cell-induced airway hyperreactivity
Hansen, et al J.C.I. 1997

47. 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.

48. Other CD4 Subsets that “Regulate”
CD4 + CD25+ cells
TR1 TR
NKT cells

49. Th3 Cells Generated by oral tolerance induction with low dose antigen.
Produce TGF-b, IL-4
Express regulatory/suppressive activity.
TGF-b production may be enhanced by cross-linking of CTLA-4.
Chen, Y, et al Regulatory T cell clones induced by oral tolerance: suppression of
autoimmune encephalomyelitis. Science 265:1237.

50. MBP specific Th3 Cells Inhibit EAE
Th3 cells + anti-TGF
Th3 cells
Science :1237.

51. CD45RBlow Cells, CD25+ Cells
CD25+ (IL-2Ra-chain), CTLA-4, GITR, foxp3.
When transferred, have strong regulatory/suppressive activity for colitis, diabetes, thyroiditis.
Appear to require TGF-b, IL-10 and CTLA-4 to function.
In vitro activity depends on cell contact.
Antigen specificity?

53. Tr1 Cells Develop after stimulation with IL-10.
Produce IL-10, IL-5, (TGF-b).
Low proliferative capacity.
Inhibitory for experimental colitis.
Anti-IL-10 mAb reverses inhibitory effect.
Groux et al Nature. 389:737.

54. DCs from mice exposed to i.n. OVA produce IL-10
Akbari, et al Nature Immunol.

55. IL-10 producing DCs induce development of T cell lines producing IL-10
IFN-g
Akbari, et al. 2002, Nature Medicine

58. 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.

59. 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?

60. References
Hawiger 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:
Steinman R, Hawiger D, Nussenzweig M. Tolerogenic dendritic cells. Annu Rev Immunol 2003; 21:
Sharpe AH, Freeman GJ. The B7-CD28 superfamily. Nat Rev Immunol 2002; 2:
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:
Pasare C, Medzhitov R. Toll pathway-dependent blockade of CD4+CD25+ T cell-mediated suppression by dendritic cells. Science 2003; 299:
Wilson SB, Kent SC, Patton KT, Orban T, Jackson RA, Exley M, et al. Extreme Th1 bias of invariant V24JQ T cells in type 1 diabetes. Nature 1998; 391:
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:

61. 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:
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:
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:
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: