VBI Immunology Research Thrust

Ken J. Oestreich, Ph.D.Assistant Professor, VTCRIAssistant Professor, Dept. of Biomedical Sciences and Pathobiology, Virginia Tech

CD4+ T cell differentiation:An experimental viewExpanding view of specialized CD4+ T cell subtypes Basics and historical perspectiveStable versus flexible T helper cell typesMechanisms that regulate differentiation decisionsComputational modeling as a valuable tool

Development in the Immune SystemCD4+ T helper cellsorCD8+ Cytotoxic cellsOriginal Helper T cell modelIdentification of two T helper cell subtypes

Unique immune functions

Antagonistic to each others function

Extracellular pathogensIntracellular pathogensMosmann and Coffman model 1986Historical perspective: RL Coffman. 2006. Nat. Immunol. 7: 539-541.

T helper cell differentiationAdapted from Liu et al. Immunol Rev 20131986-2003

T helper cell differentiationAdapted from Liu et al. Immunol Rev 2013Present day

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

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

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

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

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

T helper cell differentiationAdapted from Liu et al. Immunol Rev 2013Reasons for expecting there may be flexibility (co-expression of lineage-defining factors)T-bet and Gata3 co-expression T-bet and Bcl-6 co-expressionFoxP3 and T-bet/Gata3/Bcl-6/Rorgt co-expression-Chaudhry, A. et al. Science 326, 986991 (2009).-Chung, Y. et al. Nature Med. 17, 983988 (2011).-Hwang, E. S., Szabo, S. J., Schwartzberg, P. L. & Glimcher, L. H. Science 307, 430433 (2005).-Hegazy, A. N. et al. Immunity 32, 116128 (2010).-Linterman, M. A. et al. Nature Med. 17, 975982 (2011).-Oestreich, K. J., Mohn, S. E. & Weinmann, A. S. Nature Immunol. 13, 405411 (2012).-Oldenhove, G. et al. Immunity 31, 772786 (2009).-Osorio, F. et al. Eur. J. Immunol. 38, 32743281 (2008).-Wang, Y., Su, M. A. & Wan, Y. Y. Immunity 35, 337348 (2011).-Zhang, F., Meng, G. & Strober, W. Nature Immunol. 9, 12971306 (2008).-Oestreich, K. J., Huang, A. C. & Weinmann, A. S. J. Exp. Med. 208, 10011013 (2011).-Zhou, L. et al. Nature 453, 236240 (2008).-Pepper, M., Pagan, A. J., Igyarto, B. Z., Taylor, J. J. & Jenkins, M. K. Immunity 35, 583595 (2011).-Nakayamada, S. et al. Immunity 35, 919931 (2011).-Lu, K. T. et al. Immunity 35, 622632 (2011).

Specialized CD4+ T cell subtype flexibilityCurrent Opinion in Immunology Volume 24, Issue 3 2012 297 - 302How can co-expression of factors be explained?

What are the functional outcomes of co-expression?

How can co-expression of factors be explained?

What are the functional outcomes of co-expression?

Muranski et al. Blood , 2013.Defining the stability versus flexibility ofspecialized CD4+ T cell subtypesEpigenetic insights

Defining the stability versus flexibility ofspecialized CD4+ T cell subtypesEpigenetic insightsWhat is the epigenetic status of lineage-defining factor loci?

Wei et al. Immunity, 2009.H3K4me3 (Active) H3K27me3 (Repressed) T-bet (Th1)Gata3 (Th2)Defining the stability versus flexibility ofspecialized CD4+ T cell subtypesEpigenetic insights-Bivalent Chromatin StateCell Type

Wei et al. Immunity, 2009.H3K4me3 (Active) H3K27me3 (Repressed) T-bet (Th1)Gata3 (Th2)Defining the stability versus flexibility ofspecialized CD4+ T cell subtypesEpigenetic insights-Bivalent Chromatin StateCell Type

Wei et al. Immunity, 2009.H3K4me3 (Active) H3K27me3 (Repressed) T-bet (Th1)Gata3 (Th2)Defining the stability versus flexibility ofspecialized CD4+ T cell subtypesEpigenetic insights-Bivalent Chromatin StateCell Type

Wei et al. Immunity, 2009.H3K4me3 (Active) H3K27me3 (Repressed) T-bet (Th1)Gata3 (Th2)Defining the stability versus flexibility ofspecialized CD4+ T cell subtypesEpigenetic insights-Bivalent Chromatin StateCell Type

Oestreich et al. Curr Top Microbiol Immunol , 2013.Defining the stability versus flexibility ofspecialized CD4+ T cell subtypesUni-directional Plasticity

Wei et al. Immunity, 2009.Rorgt (Th17)Foxp3 (Treg)Defining the stability versus flexibility ofspecialized CD4+ T cell subtypesEpigenetic insights-Bivalent Chromatin StateCell TypeH3K4me3 (Active) H3K27me3 (Repressed)

H3K4me3H3K27me3Lu et al. Immunity 35, 2011.Epigenetic patterns of transcription factor genes in CD4+ T cell subtypesBcl6 (Tfh)

H3K4me3H3K27me3Lu et al. Immunity 35, 2011.Epigenetic patterns of transcription factor genes in CD4+ T cell subtypesBcl6 (Tfh)

Cannons et al. Trends in Immunology , 2013.Defining the stability versus flexibility ofspecialized CD4+ T cell subtypesMulti-directional PlasticityHow 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.

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.

Bi-directional competition Interplay between lineage-specifying transcription factorsGata3 T-betIL-4,IL-5, IL-13 Early fate decision-one factor wins over another (Th1 cell) Hwang et al. Science. 2005 Bi-directional competition Interplay between lineage-specifying transcription factorsGata3 T-betIL-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. 2010Hwang et al. Science. 2005 Bi-directional competition Interplay between lineage-specifying transcription factorsGata3 T-betIL-4,IL-5, IL-13 Early fate decision-one factor wins over another (Th1 cell) Bcl6 T-betTcf7 ,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. 2012Oestreich et al. J Exp Med. 2011Hegazy et al. Immunity. 2010Hwang et al. Science. 2005 Bi-directional competition Interplay between lineage-specifying transcription factorsGata3 T-betIL-4,IL-5, IL-13 Early fate decision-one factor wins over another (Th1 cell) Bcl6 T-betTcf7 ,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. 2012Oestreich et al. J Exp Med. 2011Hegazy et al. Immunity. 2010Hwang et al. Science. 2005T helper cell differentiation

How does T-bet repress the gene expressionprograms of alternative T helper cell types?

T-bet and Bcl-6 physically interact to form a complex to appropriately regulate Th1 gene expression patterns

Primary Th1 Co-IPaT-betIB:InputIP: Bcl-6IP: V5WTWTWTWTT-bet-/-T-bet-/-

123456Bcl6 T-betAnti TH1 Gene K4Me2K4Me2STAT T-betPro TH1 Gene K4Me2K4Me2Developing TH1 CD4+ T cell Oestreich et al. identified two classes of T-bet target genes (J Exp Med, 2011).T helper cell differentiation

Bcl-6If two lineage-defining transcription factors aresimultaneously expressed in the same cell, how is cellfate determined?

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 domainT-bet is a T-box transcription factor encodedby the Tbx21 geneT-bet mediates both chromatin remodelingactivities and direct gene transactivation T-bet promotes Th1 development by activating canonical Th1 genes Ifng, Cxcr3 Bcl-6 (Lineage-defining transcription factor for the Tfh cell)

Bcl6BTB/POZZF1706130520681PEST 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

The C-terminal zinc finger domain is required for the interaction with T-bet

Co-IPBcl61234InputIP: T-betaV5IB:5678Bcl6DBTBBcl6DZFBcl6DB/ZFBcl6Bcl6DBTBBcl6DZFBcl6DB/ZF

Bcl6BTB/POZZF1706130520681Bcl6DBTBBcl6DZFBcl6DB/ZFPESTOestreich et al. Nat Immunol 2012IP: Bcl6aV5IB:InputCo-IPT-betT-betDNT-betDCT-betT-betDNT-betDC123456

C-terminal domain of T-bet required for its interaction with Bcl-6T-betT-betDNT-betDC T-box(DNA-binding)Oestreich et al. Nat Immunol 2012T-bet and Bcl-6 physically interact through theC-terminal ZF domain of Bcl-6

Bcl6BTB/POZZF1706130520681PESTBcl-6Complex formation with T-bet T-betLocus X+1Bcl-6 Locus Y+1T-bet Bcl-6 T-bet Bcl-6 Model for differential targeting between lociBcl-6 T-bet Effector TH1 high IL-2

Effector TH1 high IL-2

Effector TH1 high IL-2

Effector TH1 high IL-2

Locus X+1Bcl-6 Locus Y+1T-bet Bcl-6 T-bet Bcl-6 Model for differential targeting between lociBcl-6 T-bet Locus X+1Bcl-6 Locus Y+1T-bet Bcl-6 T-bet Bcl-6 Model for differential targeting between lociBcl-6 Bcl-6 Bcl-6 Bcl-6 Bcl-6 T-bet Bcl6 T-betBcl6 T-betT-betT-betBcl6 Cxcr5 Btla Il6ra Cxcr5 Btla Il6ra Bcl6 Bcl6 Bcl6 Bcl6 T-betBcl6 T-betBcl6 T-betBcl6 T-betBcl6 Bcl6 Bcl6 TH1 polarizing conditions Bcl6 Bcl6 T-betT-betT-betBcl6 T-betT-betBcl6 Bcl6 Bcl6 Bcl6 Bcl6 Ifng T-betIfng Prdm1 Bcl6 Bcl-6 Bcl-6 Are there conditions that naturally induce Bcl-6 expression in TH1 cells?Prdm1 TH1 TFH-like? Bcl6 T-betBcl6 T-betT-betT-betBcl6 Cxcr5 Btla Il6ra Cxcr5 Btla Il6ra Bcl6 Bcl6 Bcl6 Bcl6 T-betBcl6 T-betBcl6 T-betBcl6 T-betBcl6 Bcl6 Bcl6 TH1 polarizing conditions Bcl6 Bcl6 T-betT-betT-betBcl6 T-betT-betBcl6 Bcl6 Bcl6 Bcl6 Bcl6 Ifng T-betIL-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?GAPDHBcl-6IL-2T-bet

Bcl6HI IL-2LO IL-2Relative ExpressionBcl-6 expression in TH1 cells is inhibited IL-2 signaling51TH1-TFH-like low IL-2

?Expression of Blimp1 and Bcl-6 inversely correlate in TH1 cells

HI IL-2LO IL-2Percent InputPrimary Th1 ChIP (a-Bcl-6) Prdm1 (Blimp1)53GAPDHBcl-6IL-2T-bet

Blimp-1

Primary WT TH1 qRT-PCR

Prdm1 (Blimp1)HI IL-2LO IL-2Relative Expression

HI IL-2LO IL-2Percent InputPrimary Th1 ChIP (a-Bcl-6) Prdm1 (Blimp1)Expression of Blimp1 and Bcl-6 inversely correlate in TH1 cells54TH1-TFH-like low IL-2

???Primary WT TH1 qRT-PCRThe expression of a subset of TFH-signature genes are enhanced in TH1 cells maintained in low IL-2

Cxcr5 BtlaHI IL-2LO IL-2Il6raRelative ExpressionRelative ExpressionRelative ExpressionT-bet and Bcl-6 complex formation generates a regulatory switch enabling Th1/Tfh flexibility

Therapeutic Strategies-Cytokine based immunotherapy

External cytokine environment

2. Internal transcriptional networks

3. Interactions between these factorsFactors influencing T helper cell differentiationExternal cytokine environment

2. Internal transcriptional networks

3. Interactions between these factorsThis complexity makes it an ideal problem to be solved by computational modeling Factors influencing T helper cell differentiation

How can computational modeling aid in our understanding of T helper cell differentiation?

Specialized CD4+ T cell subtype flexibilityCurrent Opinion in Immunology Volume 24, Issue 3 2012 297 - 302Oestreich LabKaitlin Read, M.S. Chandra BakerPaul McDonald, Ph.D. Ian CooleyAshlyn Anderson Emily Meeks

AcknowledgementsUABAmy Weinmann, Ph.D.University of Cal-BerkeleySarah Gilbertson

Josep Bassaganya-Riera, Ph.D.Bassaganya-Riera Lab