ken j. oestreich, ph.d. assistant professor, vtcri
DESCRIPTION
CD4+ T cell differentiation: An experimental view. Ken J. Oestreich, Ph.D. Assistant Professor, VTCRI Assistant Professor, Dept. of Biomedical Sciences and Pathobiology, Virginia Tech. Expanding view of specialized CD4 + T cell subtypes. Basics and historical perspective. - PowerPoint PPT PresentationTRANSCRIPT
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Ken J. Oestreich, Ph.D.Assistant Professor, VTCRI
Assistant Professor, Dept. of Biomedical Sciences and Pathobiology, Virginia Tech
CD4+ T cell differentiation:An experimental view
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Expanding view of specialized CD4+ T cell subtypes
Basics and historical perspective
Stable versus flexible T helper cell types
Mechanisms that regulate differentiation decisions
Computational modeling as a valuable tool
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Development in the Immune System
CD4+ T helper cellsor
CD8+ Cytotoxic cells
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Original Helper T cell model
1. Identification of two T helper cell subtypes
2. Unique immune functions
3. Antagonistic to each other’s function
Extracellular pathogens
Intracellular pathogens
Mosmann and Coffman model 1986
Historical perspective: RL Coffman. 2006. Nat. Immunol. 7: 539-541.
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T helper cell differentiation
Adapted from Liu et al. Immunol Rev 2013
1986-2003
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T helper cell differentiation
Adapted from Liu et al. Immunol Rev 2013
Present day
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T helper 1 (Th1) cells
Role in immune response
Effector function
Immune response against intracellular pathogens(viruses, bacteria-i.e. mycobacterium, salmonella)
Secretion of Interferon GammaRecruitment of CD8+ T cells, activate macrophages,promote B cell class switching
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Role in immune response
Effector function
Control of extracellular parasites (helminths)
Secretion of IL-4, IL-5, IL-13 (mucosal barrier)Recruitment of eosinophils
T helper 2 (Th2) cells
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T helper 17 (Th17) cells
Role in immune response
Effector function
Response against extracellular bacteria and fungi-Staphylococcus aureus, Klebsiella pneumonia(can promote inflammation/autoimmune disease)
Secretion of IL-17Recruitment of neutrophils
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T follicular helper (Tfh) cells
Role in immune response
Effector function
Promote antibody-mediated immune responsethrough interactions with B cells
Secretion of IL-21, B cell crosstalk in germinal centers
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T regulatory (Treg) cells
Role in immune response
Effector function
Suppress effector T cell responses(limit potential autoimmune disease)
Consume IL-2, Secrete anti-inflammatory IL-10, TGF-b
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T helper cell differentiation
Adapted from Liu et al. Immunol Rev 2013
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Reasons for expecting there may be flexibility (co-expression of lineage-defining factors)
T-bet and Gata3 co-expression
T-bet and Bcl-6 co-expression
FoxP3 and T-bet/Gata3/Bcl-6/Rorgt co-expression
-Chaudhry, A. et al. Science 326, 986–991 (2009).-Chung, Y. et al. Nature Med. 17, 983–988 (2011).
-Hwang, E. S., Szabo, S. J., Schwartzberg, P. L. & Glimcher, L. H.
Science 307, 430–433 (2005).-Hegazy, A. N. et al. Immunity 32, 116–128 (2010).
-Linterman, M. A. et al. Nature Med. 17, 975–982 (2011).
-Oestreich, K. J., Mohn, S. E. & Weinmann, A. S. Nature Immunol. 13, 405–411 (2012).
-Oldenhove, G. et al. Immunity 31, 772–786 (2009).
-Osorio, F. et al. Eur. J. Immunol. 38, 3274–3281 (2008).-Wang, Y., Su, M. A. & Wan, Y. Y. Immunity 35, 337–348 (2011).-Zhang, F., Meng, G. & Strober, W. Nature Immunol. 9, 1297–1306 (2008).
-Oestreich, K. J., Huang, A. C. & Weinmann, A. S. J. Exp. Med. 208, 1001–1013 (2011).
-Zhou, L. et al. Nature 453, 236–240 (2008).
-Pepper, M., Pagan, A. J., Igyarto, B. Z., Taylor, J. J. & Jenkins, M. K.
Immunity 35, 583–595 (2011).
-Nakayamada, S. et al. Immunity 35, 919–931 (2011).
-Lu, K. T. et al. Immunity 35, 622–632 (2011).
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Specialized CD4+ T cell subtype flexibility
Current Opinion in Immunology Volume 24, Issue 3 2012 297 - 302
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How can co-expression of factors be explained?
What are the functional outcomes of co-expression?
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How can co-expression of factors be explained?
What are the functional outcomes of co-expression?
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Muranski et al. Blood , 2013.
Defining the stability versus flexibility ofspecialized CD4+ T cell subtypes
Epigenetic insights
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Defining the stability versus flexibility ofspecialized CD4+ T cell subtypes
Epigenetic insights
What is the epigenetic status of lineage-defining factor loci?
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Wei et al. Immunity, 2009.
H3K4me3 (Active)
H3K27me3 (Repressed)
T-bet (Th1) Gata3 (Th2)
Defining the stability versus flexibility ofspecialized CD4+ T cell subtypes
Epigenetic insights-Bivalent Chromatin State
Cell Type
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Wei et al. Immunity, 2009.
H3K4me3 (Active)
H3K27me3 (Repressed)
T-bet (Th1) Gata3 (Th2)
Defining the stability versus flexibility ofspecialized CD4+ T cell subtypes
Epigenetic insights-Bivalent Chromatin State
Cell Type
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Wei et al. Immunity, 2009.
H3K4me3 (Active)
H3K27me3 (Repressed)
T-bet (Th1) Gata3 (Th2)
Defining the stability versus flexibility ofspecialized CD4+ T cell subtypes
Epigenetic insights-Bivalent Chromatin State
Cell Type
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Wei et al. Immunity, 2009.
H3K4me3 (Active)
H3K27me3 (Repressed)
T-bet (Th1) Gata3 (Th2)
Defining the stability versus flexibility ofspecialized CD4+ T cell subtypes
Epigenetic insights-Bivalent Chromatin State
Cell Type
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Oestreich et al. Curr Top Microbiol Immunol , 2013.
Defining the stability versus flexibility ofspecialized CD4+ T cell subtypes
Uni-directional Plasticity
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Wei et al. Immunity, 2009.
Rorgt (Th17) Foxp3 (Treg)
Defining the stability versus flexibility ofspecialized CD4+ T cell subtypes
Epigenetic insights-Bivalent Chromatin State
Cell Type
H3K4me3 (Active)
H3K27me3 (Repressed)
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H3K4me3
H3K27me3
Lu et al. Immunity 35, 2011.
Epigenetic patterns of transcription factor genes in CD4+ T cell subtypes
Bcl6 (Tfh)
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H3K4me3
H3K27me3
Lu et al. Immunity 35, 2011.
Epigenetic patterns of transcription factor genes in CD4+ T cell subtypes
Bcl6 (Tfh)
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Cannons et al. Trends in Immunology , 2013.
Defining the stability versus flexibility ofspecialized CD4+ T cell subtypes
Multi-directional Plasticity
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How can co-expression of factors be explained?
What are the functional outcomes of co-expression?
At the epigenetic level, lineage-defining factors maintain the capacity to be expressed in multiple T helper cell subsets.
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How can co-expression of factors be explained?
What are the functional outcomes of co-expression?
At the epigenetic level, lineage-defining factors maintain the capacity to be expressed in multiple T helper cell subsets.
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Bi-directional competition
Interplay between lineage-specifying transcription factors
Gata3 T-bet
IL-4,IL-5, IL-13
Early fate decision-one factor “wins” over another (Th1 cell)
Hwang et al. Science. 2005
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Bi-directional competition
Interplay between lineage-specifying transcription factors
Gata3 T-bet
IL-4,IL-5, IL-13
Early fate decision-one factor “wins” over another (Th1 cell)
Bcl6
Co-dominant competition
Factors cooperate to establisha hybrid T helper lineage(Th2+1 cell) Bcl6
T-bet
Ifng
IL-4,IL-5, IL-13
Gata3
Hegazy et al. Immunity. 2010
Hwang et al. Science. 2005
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Bi-directional competition
Interplay between lineage-specifying transcription factors
Gata3 T-bet
IL-4,IL-5, IL-13
Early fate decision-one factor “wins” over another (Th1 cell)
Bcl6 T-bet
Tcf7 ,Socs
Dominant competition
Factors cooperate to establishone T helper lineage(Th1 cell)
Bcl6
Co-dominant competition
Factors cooperate to establisha hybrid T helper lineage(Th2+1 cell) Bcl6
T-bet
Ifng
IL-4,IL-5, IL-13
Gata3
Oestreich & Weinmann Trends Immunol. 2012
Oestreich et al. J Exp Med. 2011
Hegazy et al. Immunity. 2010
Hwang et al. Science. 2005
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Bi-directional competition
Interplay between lineage-specifying transcription factors
Gata3 T-bet
IL-4,IL-5, IL-13
Early fate decision-one factor “wins” over another (Th1 cell)
Bcl6 T-bet
Tcf7 ,Socs
Dominant competition
Factors cooperate to establishone T helper lineage(Th1 cell)
Bcl6
Co-dominant competition
Factors cooperate to establisha hybrid T helper lineage(Th2+1 cell) Bcl6
T-bet
Ifng
IL-4,IL-5, IL-13
Gata3
Oestreich & Weinmann Trends Immunol. 2012
Oestreich et al. J Exp Med. 2011
Hegazy et al. Immunity. 2010
Hwang et al. Science. 2005
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T helper cell differentiation
How does T-bet repress the gene expressionprograms of alternative T helper cell types?
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T-bet and Bcl-6 physically interact to form a complex to appropriately regulate Th1 gene expression patterns
Primary Th1 Co-IP
aT-betIB:Input IP: Bcl-6IP: V5
WT WT WT WT T-bet-/-T-bet-/-
1 2 3 4 5 6
Bcl6 T-bet
Anti TH1 Gene
K4
Me2
K4
Me2
STAT
T-bet
Pro TH1 Gene
K4
Me2
K4
Me2
Developing TH1 CD4+ T cell
Oestreich et al. identified two classes of T-bet target genes (J Exp Med, 2011).
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T helper cell differentiation
Bcl-6
If two lineage-defining transcription factors aresimultaneously expressed in the same cell, how is cell
fate determined?
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T-bet (Lineage-defining transcription factor for Th1 cells)
T-bet
T-box(DNA-binding)
T-bet binds to DNA through the T-box DNA-binding domain
T-bet is a T-box transcription factor encodedby the Tbx21 gene
T-bet mediates both chromatin remodelingactivities and direct gene transactivation
T-bet promotes Th1 development by activating canonical Th1 genes Ifng, Cxcr3
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Bcl-6 (Lineage-defining transcription factor for the Tfh cell)
Bcl6BTB/POZ ZF1 706
130 520 681
PEST
Bcl-6 is a potent transcriptional repressor necessary for establishing the Tfh cell phenotype
N-terminal BTB/POZ required for dimerization and interactions with transcriptional co-repressors (N-CoR, BCoR, SMRT etc.)
C-terminal zinc finger domain necessary for some protein-protein interactions as well as for the DNA binding capabilities of Bcl-6
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The C-terminal zinc finger domain is required for the interaction with T-bet
Co-IP
Bcl
6
1 2 3 4
Input IP: T-bet
aV5IB:
5 6 7 8
Bcl
6D
BT
B
Bcl
6D
ZF
Bcl
6D
B/Z
F
Bcl
6
Bcl
6D
BT
B
Bcl
6D
ZF
Bcl
6D
B/Z
F
Bcl6BTB/POZ ZF1 706
130 520 681
Bcl6DBTB
Bcl6DZF
Bcl6DB/ZF
PEST
Oestreich et al. Nat Immunol 2012
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IP: Bcl6
aV5IB:
Input
Co-IP
T-b
et
T-b
etD
N
T-b
etD
C
T-b
et
T-b
etD
N
T-b
etD
C
1 2 3 4 5 6
C-terminal domain of T-bet required for its interaction with Bcl-6
T-bet
T-betDN
T-betDC
T-box(DNA-binding)
Oestreich et al. Nat Immunol 2012
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T-bet and Bcl-6 physically interact through theC-terminal ZF domain of Bcl-6
Bcl6
BTB/POZ ZF1 706
130 520 681
PEST
Bcl-6
Complex formation with T-bet
T-bet
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Locus X
+1
Bcl-6
Locus Y
+1
T-bet
Bcl-6 T-bet
Bcl-6
Model for differential targeting between loci
Bcl-6 T-bet
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Effector TH1 high IL-2
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Effector TH1 high IL-2
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Effector TH1 high IL-2
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Effector TH1 high IL-2
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Locus X
+1
Bcl-6
Locus Y
+1
T-bet
Bcl-6 T-bet
Bcl-6
Model for differential targeting between loci
Bcl-6 T-bet
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Locus X
+1
Bcl-6
Locus Y
+1
T-bet
Bcl-6 T-bet
Bcl-6
Model for differential targeting between loci
Bcl-6
Bcl-6
Bcl-6
Bcl-6
Bcl-6 T-bet
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Bcl6 T-bet
Bcl6 T-bet
T-bet T-bet
Bcl6
Cxcr5
Btla
Il6ra
Cxcr5
Btla
Il6ra
Bcl6
Bcl6 Bcl6
Bcl6 T-bet
Bcl6 T-bet
Bcl6 T-bet
Bcl6 T-bet
Bcl6
Bcl6
Bcl6
TH1 polarizing conditions
Bcl6 Bcl6
T-bet
T-bet
T-bet
Bcl6 T-bet
T-betBcl6
Bcl6
Bcl6
Bcl6 Bcl6
Ifng
T-bet
Ifng
Prdm1
Bcl6
Bcl-6 Bcl-6
Are there conditions that naturally induce Bcl-6 expression in TH1 cells?
Prdm1
TH1 TFH-like?
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Bcl6 T-bet
Bcl6 T-bet
T-bet T-bet
Bcl6
Cxcr5
Btla
Il6ra
Cxcr5
Btla
Il6ra
Bcl6
Bcl6 Bcl6
Bcl6 T-bet
Bcl6 T-bet
Bcl6 T-bet
Bcl6 T-bet
Bcl6
Bcl6
Bcl6
TH1 polarizing conditions
Bcl6 Bcl6
T-bet
T-bet
T-bet
Bcl6 T-bet
T-betBcl6
Bcl6
Bcl6
Bcl6 Bcl6
Ifng
T-bet
IL-2R IL-2R
Ifng
Blimp1
Bcl6
Bcl-6 Bcl-6
IL-2 signaling?
Blimp1
TH1 TFH-like?
Are there conditions that naturally induce Bcl-6 expression in TH1 cells?
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GAPDH
Bcl-6
IL-2
T-bet
Bcl6
HI IL-2
LO IL-2
Re
lati
ve
Ex
pre
ss
ion
Bcl-6 expression in TH1 cells is inhibited IL-2 signaling
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TH1-TFH-like low IL-2
?
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Expression of Blimp1 and Bcl-6 inversely correlate in TH1 cells
HI IL-2
LO IL-2P
erc
en
t In
pu
t
Primary Th1 ChIP (a-Bcl-6)
Prdm1 (Blimp1)
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GAPDH
Bcl-6
IL-2
T-bet
Blimp-1
Primary WT TH1 qRT-PCR
Prdm1 (Blimp1)
HI IL-2
LO IL-2
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Primary Th1 ChIP (a-Bcl-6)
Prdm1 (Blimp1)
Expression of Blimp1 and Bcl-6 inversely correlate in TH1 cells
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TH1-TFH-like low IL-2
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Primary WT TH1 qRT-PCR
The expression of a subset of TFH-signature genes are enhanced in TH1 cells maintained in low IL-2
Cxcr5 Btla
HI IL-2
LO IL-2
Il6ra
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T-bet and Bcl-6 complex formation generates a regulatory switch enabling Th1/Tfh flexibility
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Therapeutic Strategies-Cytokine based immunotherapy
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1. External cytokine environment
2. Internal transcriptional networks
3. Interactions between these factors
Factors influencing T helper cell differentiation
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1. External cytokine environment
2. Internal transcriptional networks
3. Interactions between these factors
This complexity makes it an ideal problem to be solved by computational modeling
Factors influencing T helper cell differentiation
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How can computational modeling aid in our understanding of T helper cell differentiation?
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Specialized CD4+ T cell subtype flexibility
Current Opinion in Immunology Volume 24, Issue 3 2012 297 - 302
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Oestreich LabKaitlin Read, M.S. Chandra BakerPaul McDonald, Ph.D. Ian CooleyAshlyn Anderson Emily Meeks
Acknowledgements
UABAmy Weinmann, Ph.D.
University of Cal-BerkeleySarah Gilbertson
Josep Bassaganya-Riera, Ph.D.Bassaganya-Riera Lab