Advanced Immunology
Th 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
Humoral immunity
Cell mediated immunity
Dose of Flagellin
Parish and Liu, 1972
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.
Expression of cytokines by CD4 T cell subsets
Cytokine
IFN-g
Lymphotoxin
IL-2
IL-3
GM-CSF
TNF-a
IL-4
IL-5
IL-9
IL-10
IL-13
Th1
Th2
Th0
Thp
+
+
+
+
+
+
-
+
+
+
+
+
+
+
+
+
+
-
+
+
+
+
+
-
Effector Functions of Th1 Cells
IFN-g
IFN-g
IFN-g
1
IFN-g
IgG2a, IgG3
Effector Functions
of Th2 Cells
Generation of Th Cell lines
Isolate
clonotypic
CD4+ T cells
DO11.10
OVA-specific TCR Tg
OVA + APC
rIL-12
Anti-IL-4 mAb
Th1
OVA + APC
rIL-4
Anti-IL-12 mAb
Th2
OVA + APC
rIL-4
rIFN-g
Th0
Markers of Th1 and Th2 Cells
Th1
Th2
IFN-g
IL-12R2
IL-18R
P- and E- selectin receptors
CXCR3, CCR5
IL-4
IL-1R, IFN-g receptor
T1/ST2 (homolog of IL-1R)
Tim3
Tim1
ICOS
Stat6, GATA-3, c-maf
Stat4, T-bet
CCR4, CCR8
TIM family of genes




TIM-1 encodes a 305 aa protein, regulates the
development of asthma and allergy (McIntire, et al. 2001.
Nature Immunol. 2:1109) (McIntire, et al. 2003. Nature 425:576) .
In mice, TIM-3 encodes a 281 aa protein,
regulates the development of autoimmune
diseases (Monney et al. 2002. 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).
TIM:
T cell, Immunoglobulin domain, Mucin domain.
Infection with Leishmania
Strain
result
cytokines produced
C3H/HeN resistant
IFN-g, IL-2
BALB/c
IL-4, IL-5, IL-13
susceptible
Disease States Attributed to an
Imbalance in Th1/Th2 Cells
Over production
of Th2 cytokines
Infections
Leishmania
TB, leprosy
Fungal infection
HIV
Allergy and asthma
Ulcerative colitis
Over production
of Th1 cytokines
Autoimmune diseases
Multiple sclerosis
Rheumatoid arthritis
Diabetes mellitus
Crohn’s disease
Graft rejection
Recurrent abortions
(Helminth infection)
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-g
IL-12
IL-4
IL-10
Th2 cytokines
IL-4
IL-5
IL-13
IL-12
IFN-g
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-18
CD8a+ DC
B7.1, DC1 (human)
IL-12
IL-12, TGF-
IFN-g
IL-4, IL-10, TGF- inhibits
IL-27
Heterodimeric cytokine, related to IL-12.
 p28 (IL-12p35-related) + EBI3 (IL-12p40related) = 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. 2002. Immunity. 16:779.
IL-23
p19 subunit + IL-12 p40 subunit = IL-23
 IL-23 binds to IL-12R1 + 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. 2000. Immunity. 13:715
Signals that influence Th2 differentiation
TSLP (humans)
Humoral responses
Allergic responses
B7.2, OX40L, ICOSL
CD8a- DC,
DC2 (human)
IL-12, IL-18, TGF- inhibits
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. 2001. Immunity. 15:985
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. 2002. Nature Immunol. 3:673
Role of Immature Dendritic Cells
CD8a
CD40
CD86
Anergy
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
APC
production.
– Also important for tolerance induction.
T Cell
Nature Rev. Immunol. 2002. 2:116.
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. 2002. 2:116.
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. 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
NK T cell
eosinophil
iNKT Cells





Express cell surface markers characteristic of both
NK cells and conventional T cells.
In mice, most NKT cells express an invariant Va14Ja18 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.
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.
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. 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
10
BALB/c PBS
BALB/c OVA
7.5
CD1 KO PBS
PenH
CD1 KO OVA
5
2.5
0
0
10
20
30
40
50
Methacholine (mg/ml)
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
Acylated histones
Normal inactive chromatin
(ground state)
nucleosome
DNA
Stat6, GATA3
Monoallelic Gene Expression
IL-4 Gene
Allele 1
activation
sustained
activation
Allele 2
Both alleles inactive
One allele expressed
(monoallelic expression)
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 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 2001. 411:603).

CTLA-4 alternative splice form (chromosome 2q33)
(Graves disease, hypothyroidism, type 1 diabetes).
(Nature 423:506)

TIMs (Nature.
2003. 425:576)
Th1(5x106)
Th2 (5x106)
Th1(0.2x106)
Th2(0.2x106)
Lafaille, et al. J. Exp.
Med 186:307, 1997.
Induce Th2 response
Control
Attempts to tolerize with MOG results in exacerbation.
Genain, et al. 1996. Science. 274:2054
Transfer of diabetes with both Th1 and Th2 cells
Pakala, et al. 1997. JEM. 186:299.
Th1 cells do not inhibit Th2 cell-induced
airway hyperreactivity
800
transferred cells:
600
% above baseline
Th2
Th1+Th2 (1:1)
400
Th1+Th2 (2:1)
Th1
200
none
0
0
10
20
30
Methacholine (mg/ml)
40
50
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 cells + anti-TGF
Th3 cells
Science. 1994. 265:1237.
low
CD45RB





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-, 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.
Intracellular staining
tin
-a
c
10
IL-
A
OVA( i.p.+i.n.)
RN
tin
-a
c
10
IL-
RN
A
tin
-a
c
10
IL-
IL-
OVA-Alum i.d.
PBS i.n.
A
10
-a
cti
n
RN
A
OVA i.n.
RN
a
DCs from mice exposed to i.n.
OVA produce IL-10
Intracellular staining
33%
2 .3 %
IL-1 0
Akbari, et al. 2001 Nature Immunol.
IL-10 producing DCs induce development of T
cell lines producing IL-10
IL-10
IL-4
IFN-g
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?
References
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cells that induce tolerance and T regulatory 1 cell differentiation in vivo. Immunity 2003, 18:605617.
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.
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pathway. Nat Immunol 2000; 1:515-20.
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CD25(+)CD4(+) regulatory T cells through GITR breaks immunological self-tolerance.
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