Cytokines and Th Subsets Micro 204 Rich Locksley 10/28/15.
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Transcript of Cytokines and Th Subsets Micro 204 Rich Locksley 10/28/15.
Cytokines and Th Subsets
Micro 204
Rich Locksley
10/28/15
No Antigen DNP-GPA DNP-BCG DNP-BA
DNP-GPA T cell media
DNP-BCG T cell media
System for cell communication in the absence of cell:cell contact
J David/B Bloom, 1966
Soluble
Cell Contact
Cytokine functions
Cell-cell communication
Response amplification
Response regulation
Cytokine Function - Examples
Cell Growth/ IL-3, GM-CSF, G-CSF, M-CSF, IL-34
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 IL-17, IFN-, IL-18
Immune Regulation IL-4, IL-10, TGF
Cytokine biosynthesis - general features
1. Absent from unstimulated cells.
2. Rapid transcriptional activation with stimulation.
3. Can have post-translational control; TNF,TGF-.
4. Rapid secretion.
5. Short-lived transcripts and proteins.
6. Transcription ceases when stimulus abates.
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?
Regulated upon activation, normal T cell expressed and presumably secreted
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/genefamily/il.php
http://www.gene.ucl.ac.uk/nomenclature/genefamily/interleukins.html
http://www.genenames.org/genefamily/il.php
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
Cytokine solutions II.
Order in functionally-related Families:
Inflammatory/Early: IL-1, IL-6, TNF, IL-12, IL-17
Growth/Differentiation: CSF1-3, Epo, IL-7, kit-L
Migratory: Chemokines
Immuno-activating: IFNs, IL-4
Immuno-de-activating: TGF, IL-10, soluble receptors
Advantages:
Biologically grouped, easier to remember
4 Helix: IL-2 and c superfamily (IL-2,4,7,9,13,15,21,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, IL-35)
IFN/IL-10 superfamily: (IFN/, IFN, IFN, IL-10,19,20, 22,24,26)
Not called interleukins: CSFs, LIF, Flt-3L, EPO…..
Non-interleukins: GH1, PRL, LPT,….
IL-1 family: IL-1F1 (IL-1), IL-1F2 (IL-1)..IL-1F4 (IL-18)…IL-1F5 (IL-36Ra)…IL-1F6/8/9 (IL-36//).. IL-1F7 (IL-37)…IL1F11 (IL-33)
IL-17 family: IL-17A…IL-17F, IL-25
TNF family: TNFSF1 (LT)…..TNFSF18 (AITR-L)
Chemokine family: CXC…, CC…., C (lymphotactin 1/2), CX3C (fractalkine)
Short
Long
A AD DB
C
CN
NC
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
B
F B
D C E
E C D
B F A
A
4-Helix Face
IFN /
IFN-/IFN- 1-3
IL-10 superfamily
Box 1 (JAKs)
Box 2 (STATs)
Conserved cysteines
WSXWS motif
Type I Cytokine Receptors Type II Cytokine Receptors
R1 R1R2 R2
R1 R1R2 R2
R1 R1R2 R2
JAK JAK
Usually constitutively associated with JAK-box motif (JAK3 can be induced)
Four JAKs: JAK1, JAK2, JAK3, Tyk2
JAKJAK
Receptor Oligomerization
P
P
Y
Y
Tyrosine phosphorylation of cytokine signaling chain
STAT
JAKJAK
Receptor Oligomerization
P Y
P YSTAT
P Y
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
JAKJAK
Receptor Oligomerization
P Y
P Y
STAT
P Y
PY
STAT
STAT nuclear translocation, gene transcription
Common paradigms underpinning redundancy
Unique recognition receptor chain
Common signaling receptor chain
Combinatorial use of each
Combinatorial use underpins cytokine redundancy
Type 1 cytokine SF
Common chain chain Adapter
c 2, 4, 7, 9, 15, 21 JAK-3
c 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
Combinatorial use underpins cytokine redundancy
Type 2 cytokine SF
chains Adapters
IFNAR1, 2 Tyk-2, JAK-1
IFNGR 1,2 JAK-1, JAK-2
IL-10R JAK-1
IL-10R Tyk-2
Hormones (dimeric chains) JAK-2
Combinatorial – but engagement sequence matters
LePorte, SL et al. Cell 132:259-272, 2008.
IFN-/ IFN- IL-28/29 IL-10 IL-19/IL-20/IL-24 IL-22 IL-20/IL-24 IL-26
(IFN-1-3)
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 21
IFN/IL-10 superfamily of cytokines and cytokine receptors
Combinatorial – but receptors matter
Ouyang W et al. Regulation and functions of the IL-10 family of cytokines in inflammation and disease. Annu Rev Immunol 29:71-109, 2011.
Structure Receptor Family Adapters
TIR Domain
Ig Domains
CRDs
TRAFs
DD
Ser-Thr Kinase
-Trefoil (IL-1F)
Jellyroll (TNF)
Cysteine Knot (TGF-)
MyD88 {MAL, TRIF, TRAM, SARAM}; IRAKs; IRFs
DD, TRAFs 1-6
SMADs
JAK
JAKJAK JAK
StatStat
Stat
Stat
StatSOCS
Adhesins, Activation
Migration
Neutrophils then Monocytes, Acute Phase Response, Fever
DC Activation, Antiviral state
LN, Spleen
IL-1, TNF
Chemokines
IL-6, IL-17
IFN-
Initiators
IFN-, IL-4
Effectors
IL-10
TGF
Regulators
Take Home Message:
Cytokines Mediate Immunity
Therapeutic Potential of Cytokines
Harnessing cytokines as therapeutics
Anti-TNF, IL-1, IL-6, IL-12/23, IL-5, IL-13, IL-17…
Hematopoietic growth factors for stem cell mobilization
JAK inhibitors (JAKinibs)
Insights from cytokinopathies
Genetic loss (IFNR, IL-12, etc), autoantibodies (GM- CSF, IFN, IL-17, etc), interferonopathies (deficiencies of Trex1, MDA5, STING, ISG15, etc), NLRP3
(hereditary periodic fevers, etc)
Th1
Th2
IFN-
IL-2
Lymphotoxin
IL-4
IL-5
IL-13
IL-10
IL-9
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
Th1/Th2 - definitions
Original Stable CD4+ clones
General Usage Any response polarized towards IFN- 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.
TSLP
IL-33
IL-25
IL-13
Th1
Th2
Systemic ImmunityPhagocyte activationOpsonizing AbCTL, NK cells
Barrier ImmunityMast cells, basophilsEosinophilsIgE, IgA
Allergy & asthma
IFNLT
IL-4IL-5IL-9IL-13
NaïveCD4+T cell
Type 2
Type 1IL-27
IL-18
IL-12
IL-4
DC
Antigen Elimination
Antigen Sequestration
DC
Eo
Baso
Mast cell
NK
IFN
Th subsets: an epigenetic 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 9:229-37, 1998; Agarwal and Rao, Immunity 9:765-75, 1998
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
IL-4 IL-13 IL-5 IL-3
100 kb
~ 460 kb
8 kb 4 kb
CNS-1 (401 bp)
GM-CSF
IL-4 IL-13
Conserved non-coding sequence CNS-1: regulatory element for IL-4 and IL-13
conservation: IL-4/13 50 %CNS-1 89 %
Loots et al, Science 2000
CNS-1-/- mice have impaired Th2 responses
Mohrs et al, Nat Immunol, 2001
IL-13 RAD50IL-4
3’ Enhancer Silencer Intronic Enhancer Promoter Intergenic Enhancer CD8 ROG site LCRTCR specificity Th2 Th2 specificity, weak enhancer TCR Strong enhancer CD8 Silencing Position
Weak enhancer specificity Effect
Notch target
Lee et al., Immunity 14:447-59,2001; Lee et al., Immunity 19:145-53,2003; Omori et al., Immunity 19:281-94,2003; Ansel et al., Nat Immunol 5:1251,2004.
Mini-locus of all elements enhanced by co-expression of GATA-3
CNS-1
Regulatory elements in the IL-4/13 locus
KM Ansel et al, An epigenetic view of helper T cell differentiation. Nature Immunol 4:616-623, 2003.
Looping-out model for coordinate gene expression at the type 2 cytokine locus
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 24:369-79, 2006.
GATA-3
T-bet
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
Initiation of Transcription Stabilization Hardwired
From Murphy and Reiner, The lineage decisions of helper T cells, Nature Rev Immunol 2002, 2:933.
Th2naïve CD4+
Th1
IL-4
-satellite
Positioning near areas of condensed, nontranslated, chromatin
Terminal differentiation in Th subsets is associated with repositioning of cytokine genes
collagen
myelin
arthritis
EAE
IL-12R1 ko
IL-12/23 p40 ko
IL-12: p35/p40 :: IL-12R1/IL-12R2
IL-23: p19/p40 :: IL-12R1/IL-23R
CD4 T cell-dependent
IL-21
IL-23
Th1
Th17
Systemic ImmunityPhagocyte activationOpsonizing AbCTL, NK cells
Inflammatory ImmunityNeutrophilsMonocytes
IFNLT
IL-17A, -17FIL-22, -26IL-21
NaïveCD4T cell
Type 17
Type 1IL-12
DC
Antigen Elimination
Acute Inflammation
DC
IL-6TGF
IFN
-
Ciofani M…DR Litmman. 2012. A validated regulatory network for Th17 cell specification. Cell 151:289-303.
T cells differentiate using positive and negative regulatory modules (i.e.; there is no master regulator)
Transcriptional networks direct epigenetic fate
Ciofani M…DR Litmman. 2012. A validated regulatory network for Th17 cell specification. Cell 151:289-303.
Epigenetic fate directs phenotype – position, function, lifespan
Ciofani M…DR Litmman. 2012. A validated regulatory network for Th17 cell specification. Cell 151:289-303.
Transcriptional modules as paradigms for parsing positioning and effector function
Gao Y et al, Molecular mechanisms underlying the regulation and functional plasticity of FoxP3+ regulatory T cells. Genes & Immunity 13:1-13, 2012
Peripheral tissues Lymphoid tissues
Naïve CD4 T cell
TFH
Lots and lots of papers
Critical for every
successful human vaccine
CD4 T cell ‘help’ orchestrates immunity
IL-4 Priming
IL-4 Secretion IL-13 Secretion
TFH B
EDTA
Reinhardt et al., Nat Immunol 10:385, 2009:
Lymph node IL-4-secreting T cells are TFH cells
IL-4
Mediastinal LN Lung
IL-13
Stat6
IL-4/13
Lee Reinhardt, Hong-Erh Liang
Intracellular GATA-3
Bcl6
GATA-3lo
LN
Cytokine production by TFH cells and Th2 cells
IL-13
IL-5
GATA-3hi
Periphery
Blimp
Tissue IL-13-secreting cells are Th2 effector cells
Lung CD4 cells Lung CD4 cells
YFP+YFP+ yfp-yfp-
IL-5
IL-13
X
ROSA26 locus
Hong-Erh Liang, Lee Reinhardt
Nimmerjahn and Ravetch, Immunity 24:19-24, 2006
IL-4 IFN TGF
Th2 Th1 Th17
Th subsets coordinate distinct populations of innate cells that share effector functions
Th2 cells: Basophils, eosinophils, mast cells, alternatively activated macrophages, (IgE, IgG1)
Th1 cells: NK cells, CD8 T cells, activated macrophages, (IgG2a)
Th17 cells: PMN, monocytes, (IgG2b)
Implies role for Th cells in attraction, organization, sustaining survival of myeloid cells.
‘Innate’ TCR+ lymphoid cells
Berg LJ, Signalling through TEC kinases regulates conventional versus innate CD8+ T cell development. Nat Rev Immunol 7:479, 2007.
Berg LJ, Signalling through TEC kinases regulates conventional versus innate CD8+ T cell development. Nat Rev Immunol 7:479, 2007.
ALSO: B1 cells and Marginal zone B cells (germline antibodies/immediate effector function, etc.
McDonald BD et al., Nature Immunol 14:1110, 2013
Netea MG et al., Nature Immunol 16:675, 2015
Cell 150:1235-48, 2012
Kastenmuller W…RN Germain, Cell 150:1235-48, 2012.
Localization of invariant effector lymphocytes in draining LNson
Sherlock JP…DJ Cua, Nature Med 18:1069-76, 2012.
Localization of invariant effector lymphocytes at sites of tissue stresson
IL-17-producing T cells in tendon entheses
Innate Lymphoid Cells, 2014
Lineage-negative, Id2-dependent cells that arise from a common lymphoid precursor.
Comprise cells that mediate lymph node organogenesis during fetal development and become tissue-resident effector cells expressing cytokine programs driven by transcriptional modules associated with typical CD4 helper subsets (Th1, Th2, Th17, etc).
Roles in homeostasis (establishing commensals, responding to dietary signals, responding to circadian cues, etc).
Roles in inflammation and disease (facilitate programs mediated by established Th subsets).
Group 1: T-bet+
IFNγ
Group 2: GATA3hi Group 3: RORγt+
IL-5 IL-13 IL-17A IL-22
Ex-RORγt
IEL ILC1
LTi
NK-22
ILC1 cNK
IFNγ
Natural helper cellsNuocytes
Ih2
ILC1 ILC2 ILC3
IL-12, IL-15, IL-18, etc. IL-33, TSLP, IL-25, etc. IL-1, IL-6, IL-23, Ahr ligands, etc.,
Modified from Eberl & Sawa, Trends Immunol 31:50-5, 2010
7 14 210
neonatal flora/metabolites adult flora/metabolites
days post-birth
ROR/IL7R/cLT/LTR
RANKL (SI)AHR
Microbiota/MyD88/NOD1IL-22
CCR6/CCR7/CXCL1347/MAdCAM1
Inductionof CPs
ActivationOf ILC cytokines
InductionILFs
?
wean
CPiILF mILF
ILC3
Modified from Eberl & Sawa, Trends Immunol 31:50-5, 2010
7 14 210
neonatal flora/metabolites adult flora/metabolites
days post-birth
ROR/IL7R/cLT/LTR
RANKL (SI)AHR
Microbiota/MyD88/NOD1IL-22
CCR6/CCR7/CXCL1347/MAdCAM1
Inductionof CPs
ActivationOf ILC cytokines
InductionILFs
?
wean
CPiILF mILF
ILC3Catch22 colon
Homeostasis TGF-, retinoic acid, etc
Treg
ILC
IL-10, IL-35
Perturbation IL-6/IL-1 plus TGF-, retinoic acid, etc (+/- adaptive immune response)
Treg
ILC IL-17, etc
Repair IL-6 (TGF- exhausted from environment)
Treg
ILC IL-22, IL-10, etc
Homeostasis TGF-, retinoic acid, etc
ILC
Treg IL-10, IL-35
Resident memory adaptive effectors displace innate effectors at peripheral sites of injury
Zaid et al, PNAS 111:5307, 2014
Immune System
Tissue Homeostasis
Commensals and Pathogens
Latent organisms
Stem Cells
Clarity from darkness…
Members of the Locksley Lab at UCSF