Cytokines and Th Subsets

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Cytokines and Th Subsets Micro 204
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Cytokines discovery of MIF
No Antigen DNP-GPA DNP-BCG
DNP-BTA
DNP-GPA T cell media DNP-BCG T cell media
System for cell communication in the
absence of cellcell contact
J David/B Bloom, 1966
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Cytokines inform cells
Soluble Cell Contact
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Cytokine functions
Cell-cell communication Response
amplification Response regulation
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Cytokine Function - Examples
Cell Growth/ IL-3, GM-CSF, G-CSF,
M-CSF Differentiation IL-2, IL-5, IL-7,
IL-15, IL-21 Cell Death TNF,
FasL Inflammation IL-1, IL-6, TNF, IL-8, IL-12
IFN-g, IL-18 Immune Regulation IL-4,
IL-10, TGFb
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Cytokine biosynthesis - general features
1. Absent from unstimulated cells. 2. Rapid
transcriptional activation with stimulation. 3.
Can have post-translational control
TNF,TGF-b. 4. Rapid secretion. 5. Short-lived
transcripts and proteins. 6. Transcription
ceases when stimulus abates.
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Cytokine headaches
1. How many? 2. Who named these (TNF, RANTES,
etc.)? 3. How do you get to be an IL? 4.
Redundancy and Pleiotropy - do we care
about any individual cytokine?
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Official Interleukin Names HUGO (Human Genome
Organization) and IUIS (International Union of
Immunological Sciences)
HUGO Genomics basis IUIS Functional
basis
http//www.gene.ucl.ac.uk/nomenclature/genefamily/
interleukins.html http//www.genenames.org/genefam
ily/il.php
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Cytokine solutions I.
Order in structurally-related Families - reflects
origin through gene duplication and
inter-chromosomal spread Advantages Common
Receptor Families Common Signaling
Adapters Functional Linkage Genomic Linkage
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Cytokine solutions II.
Order in functionally-related Families Inflammat
ory/Early IL-1, IL-6, TNF, IL-12 Growth/Differe
ntiation CSF1-3, Epo, IL-7, kit-L Migratory
Chemokines Immuno-activating IFNs, IL-4
Immuno-de-activating TGFb, IL-10, soluble
receptors Advantages Biologically grouped,
easier to remember
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Major cytokine families - structures
4a Helix IL-2 and gc superfamily
(IL-2,4,7,9,15,21,30,TSLP) IL-6/gp130
superfamily (IL-6, IL-11, G-CSF, leptin, LIF,
OSM, CNTF, CT-1, IL-31) IL-12/gp130-related
superfamily (IL-12, IL-23, IL-27) IFN/IL-10
superfamily (IFNa/b, IFNg, IFNl, IL-
10,19,20,22,24,26) Not called
interleukins CSFs, LIF, Flt-3L.. Non-interleuk
ins GH1, PRL, LPT,. IL-1 family IL-1F1
(IL-1a), IL-1F2 (IL-1b)..IL-1F4 (IL-18).IL1F11
(IL-33) IL-17 family IL-17AIL-17F,
IL-25 TNF family TNFSF1 (LTa)..TNFSF18
(AITR-L) Chemokine family CXC, CC., C
(lymphotactin), CX3C (fractalkine)

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Type 1 cytokines 4?-helix bundle
Short
Long

B
A
A
C
D
D
B
C
N
N
C
Autocrine Autocrine Paracrine Paracrine IL
-2, 4, 7, 9, 13, 15, 21 Endocrine IL-3, 5,
GM-CSF IL-6 family Immune System IL-12
p40 family Hormones, EPO, TPO
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Type 2 cytokine family - 6 helix structure

F
B
A
D
C
E
4a-Helix Face
E
C
D
IFN a/b IFN-g/IFN-l 1-3 IL-10 superfamily
A
B
F
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4? helix cytokines bind to cytokine receptor
superfamily members

Conserved cysteines
WSXWS motif
Box 1 (JAKs) Box 2 (STATs)
Type I Cytokine Receptors
Type II Cytokine Receptors
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Paradigm for cytokine/cytokine receptor
interactions


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Paradigm for cytokine/cytokine receptor
interactions


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Paradigm for cytokine/cytokine receptor
interactions


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Type I and type II cytokine receptors signal via
JAKs and STATs


JAK
JAK
Usually constitutively associated with JAK-box
motif (JAK3 can be induced) Four JAKs JAK1,
JAK2, JAK3, Tyk2
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Type I and type II cytokine receptors signal via
JAKs and STATs

Receptor Oligomerization

JAK
JAK
P
Y
Y
P
Tyrosine phosphorylation of cytokine signaling
chain
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Type I and type II cytokine receptors signal via
JAKs and STATs

Receptor Oligomerization

JAK
JAK
STAT
P
Y
STAT
Recruitment and phosphorylation of STAT (dimers)
on tyrosine STAT signal transducers and
activators of transcription Latent cytosolic
transcription factors Seven STATs STAT1, 2, 3,
4, 5a, 5b, 6
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Type I and type II cytokine receptors signal via
JAKs and STATs

Receptor Oligomerization

JAK
JAK
P
Y
STAT nuclear translocation, gene transcription
STAT
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Common paradigms underpinning redundancy
Unique recognition receptor chain Common
signaling receptor chain Combinatorial use of
each
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Combinatorial use underpins cytokine redundancy
Type 1 cytokine SF Common chain a
chain Adapter gc
2, 4, 7, 9, 15, 21
JAK-3 bc 3, 5, GM-CSF
JAK-2 gp 130 IL-6 family JAK-1,
JAK-2 gp-130-like IL-12 p40 family JAK-2,
Tyk-2
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Combinatorial use underpins cytokine redundancy
Type 2 cytokine SF a chains
Adapters IFNAR1, 2
Tyk-2, JAK-1 IFNGR 1,2 JAK-1,
JAK-2 IL-10Ra JAK-1 IL-10Rb Tyk-2
Hormones (dimeric a chains) JAK-2
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IFN/IL-10 superfamily of cytokines and cytokine
receptors
IFN-a/b IFN-g IL-28/29 IL-10
IL-19/IL-20/IL-24 IL-22 IL-20/IL-24
IL-26 (IFN-l1-3)
IFNAR1/ IFNGR1/ IFNGR1/ IL-10R1/ IL-20R1/
IL-22R/ IL-22 BP IL22R/ IL-20R1/

IFNAR2
IFNGR2 IL-10R2 IL-10R2 IL-20R2
IL-10R2 IL-20R2 IL-10R2
Jak1/Tyk2 Jak1/Jak2 Jak1/Tyk2
Jak1? Jak1/Tyk2 Jak1?
Tyk2?
STAT1-3 STAT1 STAT1/3 STAT3/5
STAT3/5 ? STAT3/5
Chromosome 9 12 19
1 1/1/1 12
1/1 12 21 21
6 21 6 21 11 21 6 3
1 21 6 1 3
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Structural family paradigm
Structure Receptor Family Adapters

Ig Domains
MyD88 MAL, TRIF, TRAM, SARAM IRAKs
IRFs DD, TRAFs 1-6 SMADs
b-Trefoil (IL-1F) Jellyroll (TNF) Cysteine
Knot (TGF-b)
TIR Domain
CRDs
TRAFs DD
Ser-Thr Kinase
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SOCS proteins negative regulators of cytokine
SFRs
JAK
JAK
JAK
JAK
Stat
Stat
Stat
Stat
SOCS

Stat
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A working model
Adhesins, Activation Migration Neutrophils then
Monocytes, Acute Phase Response, Fever DC
Activation, Antiviral state
Take Home Message Cytokines Mediate Immunity

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Th subsets - stable murine clones(Mosmann,
Coffman)
Th2
Th1
IL-4 IL-5 IL-13 IL-10 IL-9
IFN-g IL-2 Lymphotoxin
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Critical early concepts driven by Th1/Th2
paradigm
Cytokines mediate immune effector functions of
helper T cells Helper T cells produce patterns of
cytokines that mediate coordinate responses by
effector cells Distinct pathogens elicit distinct
Th cytokine patterns Effector patterns become
stabilized in recall responses
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Th1/Th2 - definitions
Original Stable CD4 clones General Usage Any
response polarized towards IFN-g or
IL-4 Operational Extension Type 1 immunity Host
response coordinated by Th1 cells and its
downstream targets, mainly activated phagocytes,
armed with complement-fixing Ig (IgG2a in
mouse), and CTL Type 2 immunity Host response
coordinated by Th2 cells and its downstream
targets, mainly activated eosinophils and mast
cells, armed with non-complement-fixing Ig (IgE,
IgG1) Paradigms Extended CD8 (Tc1/Tc2), NK, B
cells, DC, etc.
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The extended Th1/Th2 paradigm
Type 1
IL-27
Systemic Immunity Phagocyte activation Opsonizin
g Ab CTL, NK cells
IFN?
IL-18
Th1
IL-12
IFNg LT
Antigen Elimination
Naïve CD4 T cell
Type 2
Barrier Immunity Mast cells, basophils Eosinophi
ls IgE, IgA Allergy asthma
IL-4
Th2
IL-4 IL-5 IL-9 IL-13
IL-13
IL-25
IL-33
TSLP
Antigen Sequestration
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IFN-g and IL-4/13 are conserved regulators of
immunity
Mice Humans
Viruses Murine hepatitis virus, HSV
Stat1-/- lethal CMV, mycobacteria Bacteria M.
tb, salmonella, Listeria Partial
IFN-?R mycobacteria,

salmonella, Listeria,
legionella Systemic fungi Candida
Systemic
parasites Toxoplasma, Leishmania Worms
Trichuris, Nippostrongylus,
Polymorphisms allergy, asthma
Schistosoma Biting insects mites, ticks
IFN-g
IL-4
IL-13
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Th subsets a lineage differentiation model
Cytokine loci are methylated and associated
chromatin is hypo-acetylated Polarizing
conditions associated with chromatin acetylation,
new DNase I hypersensitivity sites (1-2 days) and
demethylation of target genes (4-7
days) Chromatin and DNA changes are
epigenetically stabilized in polarized clones
(irreversible) Current Paradigm Master
Regulators Modulate Chromatin and Mediate Changes
in DNA Methylation at Cytokine Genes by Targeting
Transcriptional Machinery Bird et al.,
Immunity 9229-37, 1998 Agarwal and Rao,
Immunity 9765-75, 1998
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Conserved Noncoding Sequences in the type 2
cytokine cluster on chromosome 11 (5q) Bold
Single-copy conserved in human, mouse, rat,
rabbit, dog, cow, pig, chicken, fugu
Loots et al, Science 2000
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Conserved non-coding sequence CNS-1 regulatory
element for IL-4 and IL-13
100 kb
IL-4
IL-13
IL-5
IL-3
GM-CSF
460 kb
8 kb
4 kb
IL-4
IL-13
CNS-1 (401 bp)
conservation IL-4/13 50 CNS-1 89
CNS-1-/- mice have impaired Th2 responses
Mohrs et al, Nat Immunol, 2001
Loots et al, Science 2000
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Regulatory elements in the IL-4 locus
Mini-locus of all elements enhanced by
co-expression of GATA-3
IL-13 RAD50
IL-4
CNS-1
3 Enhancer Silencer Intronic Enhancer Promoter
Intergenic Enhancer CD8 ROG site LCR
TCR specificity Th2 Th2
specificity, weak enhancer TCR
Strong enhancer CD8 Silencing
Position Weak enhancer
specificity
Effect Notch target
Lee et al., Immunity 14447-59,2001
Lee et al., Immunity 19145-53,2003 Omori et
al., Immunity 19281-94,2003 Ansel et al., Nat
Immunol 51251,2004.
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Looping-out model for coordinate gene expression
at the type 2 cytokine locus
KM Ansel et al, An epigenetic view of helper T
cell differentiation. Nature Immunol 4616-623,
2003.
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Tissue-specific expression regulated by
incorporation of LCR and induction of
lineage-specific factors
Lee GR et al T helper cell differentiation
regulation by cis elements and epigenetics.
Immunity 24369-79, 2006.
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Master regulators of Th differentiation
GATA-family zinc finger transcription
factor Discovered in Flavell lab using
differential display Necessary and sufficient for
Th2 differentiation T-box family transcription
factor, Tbx21 Discovered in Glimcher lab using
yeast 1-hybrid Necessary and sufficient for Th1
differentiation
GATA-3 T-bet
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Development of IFN? competence
1
2
Hlx

Initiation of Transcription
Stabilization TCR and
Cytokine-dependent

Activation Pathways
From Murphy and Reiner, The lineage decisions
of helper T cells, Nature Rev Immunol 2002, 2933.
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Gaining IL-4 Competence
Initiated by GATA3 from summated surface signals
(impede GATA3 protein degradation) Auto-amplifica
tion of GATA3 ICOS induction of c-maf directly
activates IL-4 gene
C-maf
From Murphy and Reiner, The lineage decisions
of helper T cells, Nature Rev Immunol 2002, 2933.
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Initiation of Transcription
Stabilization Hardwired
From Murphy and Reiner, The lineage decisions
of helper T cells, Nature Rev Immunol 2002, 2933.
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Terminal differentiation in T helper subsets is
associated with repositioning of cytokine genes
Th2
Th1
IL-4
Positioning near areas of condensed,
nontranslated, chromatin
?-satellite
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The Th1 paradigm
Naïve CD4 T cell
TLRs, CLRs
NODs
Maturation, Transit to LNs
IFNg LT
T-bet, etc.
Th1 cells
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Dendritic cells elaborate IL-12/23 p40 in vivo
Bi-cistronic GFP expression
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DC IL-12 accumulates on tissue side of HEV
MECA-79 HEV
YFP DC
IL-12 on DC and HEV
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Newer Th subsets
CD4 T cell-dependent
arthritis
collagen
myelin
EAE
IL-12R?1 ko IL-12/23 p40 ko
IL-12 p35/p40 IL-12R?1/IL-12R?2 IL-23
p19/p40 IL-12R?1/IL-23R
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Th17 cells mediate neutrophil inflammation
Type 1
IL-12
Systemic Immunity Phagocyte activation Opsonizin
g Ab CTL, NK cells
Th1
IFN?
IFNg LT
Antigen Elimination
-
TGF?
Naïve CD4 T cell
Type 17
IL-6
IL-21
Inflammatory Immunity Neutrophils Monocytes
Th17
IL-23
IL-17A, -17F IL-22, -26 IL-21
Acute Inflammation
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Where does polarization occur?
IFNg
LPS
Model 1 DC instruction
IL-4
Allergen
Lymph Node Tissue
IFNg
Model 2 Tissue instruction
IL-4
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Evidence for DC instruction
IFNg
Delta4
LPS
Cholera Toxin
Jagged1
IL-4

Amsen D,, RA Flavell. Instruction of distinct
CD4 T helper cell fates by different notch
ligands on antigen presenting cells. Cell
117515-526, 2004.
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Or, molecular PAMPs
IFNg
TLR ligands
LPS
Curdlan
IL-17
Cholera Toxin
Chitin

IL-4
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What about all the other subsets, plasticity,
reversibility, etc.?
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Locksley RM. Nine lives plasticity among T
helper cell subsets. J Exp Med 2061643, 2009.
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The need to appreciate anatomy
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Cytokine production in 4get x KN2 CD4 T cells
Th2
Th1
a-CD3
a-CD3
Primed
huCD2
GFP
huCD2
AAA
C
IRES
IL-4
AAA
C
Il4
Il4
GFP
Il4-IRES-GFP
cd2
IL-4
huCD2
AAA
C
IRES
AAA
C
Il4
cd2
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Cytokine production in 4get x KN2 mice in vivo
Uninfected
Lung PP
mesLN Spleen
K Mohrs et al, Immunity 23419-29, 2005
GFP
Heligomosomoides infection, 2 wks
Systemic dissemination of GFP-positive
(cytokine-competent) cells
Local induction of GFP/huCD2-positive
(cytokine-secreting) cells
huCD2
GFP
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Memory reflects rapid activation of systemically
dispersed IL-4-competent cells
Drug cure
2 wks
5 wks
H polygyrus
Lamina propria cells
Infection Immunity
K Mohrs et al, Immunity 23419-29, 2005
59
IL-4 priming in LNs occurs in the absence of
innate IL-4/13
but tissue Th2 cells are absent
Nippo
DO11 4get CD4 T cells

IL-4/IL-13-/-
WT 4get
OVA aerosol
9 days

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DO11.10 4get x KN2 transfer immunized with
OVA/Alum
Day 0
Day 3
Day 5
Day 7
Day 10
Green huCD2 Red KJ1-26 Blue B220
IL-4 secretion
Day 0
Day 3
Day 5
Day 7
Day 10
Green huCD2 Red B220
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IL-4-producing T cells reside in Germinal Centers
Day 5
Day 10
Green huCD2 Red PNA Blue B220
Lee Reinhardt
62
IL-4-positive CD4 T cells in BT cell conjugates
in LN
Draining LN
Sort GFP doublets
CD3 B220
CD4 singlets
Doublets
B220 singlets
EDTA-treated doublets
Lee Reinhardt
63
Express CXCR5
Express BCL6, SAP, IL-21
IL-21 ELISPOT
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Hong-Erh Liang
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IL-4 Priming
IL-4 Secretion
IL-13 Secretion
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Tissue IL-13-secreting cells are Th2 effector
cells
X
IL-5
IL-13
Hong-Erh Liang
67
Affinity maturation of IgG1 unaffected by
deletion of IL-13-expressing cells
NP-alum
14 days
IL-4 Deleted IL-13 Intact
IL-13-secreting cells deleted
IL-4, IL-13 Intact
IL-4, IL-13 Deleted
Lee Reinhardt
68
Cytokine-expressing CD4 effector cells after N.
brasiliensis infection (d9)
Mediastinal LN CD4 T cells
IL-13
IL-4
Lung tissue CD4 T cells
IL-13
IL-4
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LN (TFH)
Lung (Th2)
Lee Reinhardt
70
Paradigm for coordination of T helper function in
the periphery and in the germinal center -
microenvironments can dictate Th cell fates
B cell zone Tx Factor X Bcl6
CXCR5 IL-21 ICOS-L
Cytokines X
DC
GATA3 or T-bet or ROR?t
Peripheral tissues Cytokines X
PSGL-1 S1P-1 CRs
Tx Factor X
Matches isotype switching in LNs with FcR usage
by distinct myeloid cells in peripheral tissues
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FcRs bind IgG heavy chains with different
affinities

Nimmerjahn and Ravetch, Immunity 2419-24, 2006
72
Emerging Paradigm Th subsets coordinate
different populations of innate cells that share
effector functions
Th2 cells Basophils, eosinophils, mast
cells, alternatively activated
macrophages Th1 cells NK cells, CD8 T cells,
activated macrophages Th17 cells PMN, monocytes
Implies role for Th cells in attraction,
organization, sustaining survival of myeloid
cells.
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Th subsets and functional modules
NK, CTL, TiP DCs, Activated macrophages
B cells
IFNg LT
IL-21
T-bet
Bcl6

???
Macrophages Dendritic cells
PMN, Mono
IFN???
CXCL13
ROR?t
IFN?
IL-17A, IL-17F IL-22, IL-26 IL-21
???
TGF?
???
CD4
IL-6
IL-21
TGF?
Vit D3
???
IL-27
IL-2
Vit A
TGF?
GATA3
???
IL-4 IL-5 IL-9 IL-13
FoxP3
IL-10
Eo, Baso, Mast cells Alternatively activated
macrophages
Macrophages Dendritic cells
TGF? IL-10 IL-35
Dendritic cells
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Thank you
Members of the Locksley Lab at UCSF