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Cytokines and Lymphocyte Subsets
Micro 204
Rich Locksley
Nov 2016
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/
Differentiation
IL-3, GM-CSF, G-CSF, M-CSF, IL-34
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-g, IL-18
Immune Regulation
IL-4, IL-10, TGFb
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.
Cytokine headaches
Regulated upon activation, normal T cell expressed and presumably secreted
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? Do we
really know what some of these do?
Cytokine headaches
Science 2016
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
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: TGFb, IL-10, soluble receptors
Advantages:
Biologically grouped, easier to remember
4a Helix: IL-2 and gc 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: (IFNa/b, IFNg, IFNl, 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-1a), IL-1F2 (IL-1b)..IL-1F4 (IL-18)…IL-1F5 (IL36Ra)…IL-1F6/8/9 (IL-36a/b/g).. IL-1F7 (IL-37)…IL1F11 (IL-33)
IL-17 family: IL-17A…IL-17F, IL-25
TNF family: TNFSF1 (LTa)…..TNFSF18 (AITR-L)
Chemokine family: CXC…, CC…., C (lymphotactin 1/2), CX3C (fractalkine)
Short
B
D
Autocrine
Long
C A
C
B D
A
N
Paracrine
C
N
Autocrine
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
A
F
B
D
C
E
4a-Helix Face
E
B
C
F
D
A
IFN a/b
IFN-g/IFN-l 1-3
IL-10 superfamily
Conserved cysteines
WSXWS motif
Box 1 (JAKs)
Box 2 (STATs)
Type I Cytokine Receptors
Type II Cytokine Receptors
R2
R1
R1
R2
R2
R1
R1
R2
R1
R2
R2 R1
JAK
JAK
Usually constitutively associated with JAK-box motif
(JAK3 can be induced)
Four JAKs: JAK1, JAK2, JAK3, Tyk2
Receptor Oligomerization
JAK
P Y
P
JAK
Y
Tyrosine phosphorylation of cytokine signaling chain
Receptor Oligomerization
JAK
P
Y
P Y
STAT
P
STAT
JAK
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
Receptor Oligomerization
JAK
P Y
P
STAT nuclear translocation,
gene transcription
JAK
Y
P
STAT
Y
STAT
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
a chain
Adapter
gc
2, 4, 7, 9, 15, 21
JAK-3
bc
3, 5, GM-CSF
JAK-2
IL-6 family
JAK-1, JAK-2
gp 130
gp-130-like
IL-12 p40 family
JAK-2, Tyk-2
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
Combinatorial – but engagement sequence matters
LePorte, SL et al. Cell 132:259-272, 2008.
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/
IFNAR2
IFNGR1/
IFNGR2
IL-10R2
Jak1/Tyk2 Jak1/Jak2
STAT1-3 STAT1
IL-10R1/
IL-20R1/
IL-10R2
Jak1/Tyk2
STAT1/3
IL-20R2
Jak1?
STAT3/5
IL-22R/ IL-22BP
IL-10R2
IL22R/
IL-20R1/
IL-20R2
Jak1/Tyk2
Jak1?
STAT3/5
?
IL-10R2
Tyk2?
STAT3/5
Chromosome
9
21 21
12
6
21
19
6
21
1
11
1/1/1
21
6
3
12
1
1/1
21
6
1
3
12
21
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
Ig Domains
b-Trefoil (IL-1F)
TIR Domain
Adapters
MyD88 {MAL,
TRIF, TRAM,
SARAM}; IRAKs;
IRFs
CRDs
Jellyroll (TNF)
TRAFs
DD
Cysteine Knot
(TGF-b)
Ser-Thr
Kinase
DD, TRAFs 1-6
SMADs
JAK
JAK
Stat
Stat
Stat
JAK
Stat
SOCS
JAK
Stat
Linossi EM, SE Nicholson. 2015. Immunological Reviews 266:123-133.
Initiators
IL-1, TNF
Adhesins, Activation
Chemokines
Migration
IL-6, IL-17
Neutrophils then Monocytes,
Acute Phase Response, Fever
IFN-b
DC Activation, Antiviral state
Regulators
IL-10
TGFb
Effectors
IFN-g, IL-4
Take Home Message:
LN, Spleen
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 (IFNgR, IL-12, etc), autoantibodies (GMCSF, IFNg, IL-17, etc), interferonopathies (deficiencies
of Trex1, MDA5, STING, ISG15, etc), NLRP3
(hereditary periodic fevers, etc)
Th1
Th2
IFN-g
IL-4
IL-2
IL-5
Lymphotoxin
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-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.
DC
Type 1
NK
IL-27
IL-18
IL-12
IFNg
Systemic Immunity
Th1
IFNg
LT
Phagocyte activation
Opsonizing Ab
CTL, NK cells
Antigen Elimination
Naïve
CD4+
T cell
Type 2
DC
Barrier Immunity
IL-4
Th2
IL-13
IL-25
IL-33
TSLP
Baso
Mast
cell
IL-4
IL-5
IL-9
IL-13
Mast cells, basophils
Eosinophils
IgE, IgA
Allergy & asthma
Eo
Antigen Sequestration
Th subsets: an epigenetic differentiation model
Cytokine loci are methylated and associated chromatin is hypoacetylated
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
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
IL-4
8 kb
4 kb
IL-13
CNS-1 (401 bp)
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
Regulatory elements in the IL-4/13 locus
Mini-locus of all elements enhanced by co-expression of GATA-3
IL-4
IL-13
RAD50
CNS-1
3’ Enhancer Silencer Intronic Enhancer Promoter Intergenic Enhancer CD8 ROG site LCR
TCR specificity
Th2
Weak enhancer
specificity
Th2 specificity, weak enhancer
TCR
Strong enhancer
CD8 Silencing
Position
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.
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 4:616-623, 2003.
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
GATA-family zinc finger transcription factor
Discovered in Flavell lab using differential display
Necessary and sufficient for Th2 differentiation
T-bet
T-box family transcription factor, Tbx21
Discovered in Glimcher lab using yeast 1-hybrid
Necessary and sufficient for Th1 differentiation
Initiation of Transcription
Stabilization
From
Hardwired
Murphy and Reiner, The lineage decisions of helper T cells, Nature Rev Immunol 2002, 2:933.
Terminal differentiation in Th subsets is associated with
repositioning of cytokine genes
naïve CD4+
Th2
Th1
IL-4
g-satellite
Positioning near areas of
condensed, nontranslated,
chromatin
CD4 T cell-dependent
collagen
arthritis
myelin
EAE
IL-12Rb1 ko
IL-12/23 p40 ko
IL-12: p35/p40 :: IL-12Rb1/IL-12Rb2
IL-23: p19/p40 :: IL-12Rb1/IL-23R
DC
Type 1
IL-12
Systemic Immunity
Th1
IFNg
Phagocyte activation
Opsonizing Ab
CTL, NK cells
IFNg
LT
-
Naïve TGFb
IL-21 IL-6
CD4
T cell
Antigen Elimination
Type 17
Inflammatory Immunity
IL-23
Th17
IL-17A, -17F
IL-22, -26
IL-21
Neutrophils
Monocytes
Acute Inflammation
T cells differentiate using positive and negative regulatory modules (i.e.; there is
no master regulator)
Ciofani M…DR Litmman. 2012. A validated regulatory network for Th17 cell specification. Cell 151:289-303.
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
Josefowicz S, L-F Lu, AY Rudensky. 2012. Regulatory T cells: mechanisms of differentiation and function. Annu Rev
Immunol 30:531-64
CD4 T cell ‘help’ orchestrates immunity
Lymphoid tissues
Peripheral tissues
Naïve CD4 T cell
TFH
Lots and lots
of papers
Critical for
every
successful
human
vaccine
IL-4 Priming
IL-4 Secretion
IL-13 Secretion
Lymph node IL-4-secreting T cells are TFH cells
Reinhardt et al., Nat Immunol 10:385,
2009:
EDTA
TFH
B
Cytokine production by TFH cells and Th2 cells
Mediastinal LN
Lung
IL13
IL-4
GATA-3lo
Bcl6
Intracellular GATA-3
LN
GATA-3hi
Stat6
IL-4/13
IL-13
IL-5
Blimp
Periphery
Lee Reinhardt, Hong-Erh Liang
Tissue IL-13-secreting cells are Th2 effector cells
Nippo
ROSA26 locus
X
Day 8
Tissue Eosinophils
Day 12
Lung CD4 cells
yetCre13
DTa
yetCre13xDTa
IL-5
YFP+
IL13
yfp-
Humoral
response
sustained
IgE mg/ml
Hong-Erh Liang, Lee Reinhardt
Th2
Th1
Th17
IL-4
IFNg
TGFb
Nimmerjahn and Ravetch, Immunity 24:19-24, 2006
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 Lymphoid Cells, 2016
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).
IL-12, IL-15, IL-18, etc.
IL-33, TSLP, IL-25, etc.
IL-1, IL-6, IL-23, Ahr ligands, etc.,
ILC1
ILC2
ILC3
Group 1: T-bet+
IEL ILC1
ILC1
Group 2: GATA3hi
Th1
NK-22
Natural helper cells
Nuocytes
Ih2
cNK
IFNγ
Group 3: RORγt+
Ex-RORγt
LTi
IL-5
IL-13
Th2
IL-17A IL-22
Th17
Th22
IFNγ
ExTh17
‘Innate’ TCR+ lymphoid cells
Berg LJ, Signalling through TEC kinases regulates conventional versus innate CD8+ T cell development. Nat Rev Immunol
7:479, 2007.
McDonald BD et al., Nature Immunol 14:1110, 2013
Cell 164:1198, 2016
Cell 150:1235-48, 2012
Localization of invariant effector lymphocytes in draining LNson
Kastenmuller W…RN Germain, Cell 150:1235-48, 2012.
Localization of invariant effector lymphocytes at sites of tissue stresson
IL-17-producing gd T cells in tendon entheses
Sherlock JP…DJ Cua, Nature Med 18:1069-76, 2012.
days post-birth
wean
7
0
14
21
neonatal flora/metabolites
adult flora/metabolites
?
CP
Induction
of CPs
RORg/IL7R/gc
LTa/LTbR
RANKL (SI)
AHR
iILF
Activation
Of ILC cytokines
mILF
Induction
ILFs
Microbiota/MyD88/NOD1
IL-22
CCR6/CCR7/CXCL13
a4b7/MAdCAM1
ILC3
Modified from Eberl & Sawa,
Trends Immunol 31:50-5, 2010
days post-birth
wean
7
0
14
21
neonatal flora/metabolites
adult flora/metabolites
?
CP
Induction
of CPs
RORg/IL7R/gc
LTa/LTbR
RANKL (SI)
AHR
iILF
Activation
Of ILC cytokines
mILF
Induction
ILFs
Microbiota/MyD88/NOD1
IL-22
CCR6/CCR7/CXCL13
a4b7/MAdCAM1
ILC3
Catch22
colon
Modified
from
Eberl & Sawa,
Trends Immunol 31:50-5, 2010
Treg
Homeostasis
TGF-b, retinoic acid, etc
IL-10, IL-35
ILC
Treg
Perturbation
IL-6/IL-1 plus TGF-b, retinoic acid, etc
(+/- adaptive immune response)
ILC
IL-17, etc
Treg
Repair
IL-6 (TGF-b exhausted from environment)
ILC
Treg
Homeostasis
TGF-b, retinoic acid, etc
ILC
IL-22, IL-10,
etc
IL-10, IL-35
Resident memory adaptive effectors displace innate
effectors at peripheral sites of injury
Zaid et al, PNAS
111:5307, 2014
Commensals and Pathogens
Tissue Homeostasis
Latent organisms
Immune System
Stem Cells
Clarity from darkness…
Members of the Locksley Lab at UCSF