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