Lineage Commitment During T Cell Development Art Weiss & Mark Anderson 10/19/16

Summary of Thymic Development Zuniga-Pflucker, NRI, 2004

What do we mean by the term “Lineage Commitment”?

What do we mean by the term “Lineage Commitment” What do we mean by the term “Lineage Commitment”? Commitment represents the loss in the ability of a cell to make alternative lineage choices under permissive conditions.

Name the Lineage Commitment Choices that are made during T cell Development

Lineage Commitment Decisions During T Cell Development To become a T cell To become an TCRab cell vs a TCRgd T cell To become a CD4 or CD8 T cell To become one of the minor T cells subset NK T cell CDaa T cell nTreg

First Lineage Commitment Decision in T Cell Development To be, or not to be, a T cell

Seeding the Thymus from Precursors in the Blood Bhandoola, et al., Immunity, 26:678-689, 2007 HSC Hematopoietic Stem Cell MPP Multipotent Progenitor ELP Early Lymphoid Progenitor CLP Common Lymphoid Progenitor CMP Common Myeloid Progenitor CTP Circulating T cell Progenitor TSP Thymus Settling Progenitors ETP Early Thymic Progenitor

Is the ETP, recently arrived in the thymus, already committed to the T cell lineage?

Alternative Fates of the Early Thymic Progenitor Yui and Rothernberg, NRI, 2014 A critical role for Notch in T cell lineage commitment

Zuniga-Pflucker, NRI, 2004

How would you demonstrate the importance of Notch in T cell lineage commitment of ETPs?

OP9 stromal cells transfected with Deltex-1 can support T Cell Lineage Commitment Zuniga-Pflucker, NRI, 2004 Schmitt, et al, JEM, 2004

ab T Cell Development Yui and Rothernberg, NRI, 2014

Notch signaling is critical for the induction of multiple transcription factors Yui and Rothernberg, NRI, 2014

Early Notch signaling induces TCF-1 (Tcf7) and Gata3 at The DN1 -> DN2 transition Yui and Rothernberg, NRI, 2014

How would you test the importance of one of the transcription factors (for instance, TCF-1) that is upregulated during early Notch signaling for T cell lineage development?

Impaired development of TCF-1-deficient BM HSC into CD25+ (i. e Impaired development of TCF-1-deficient BM HSC into CD25+ (i.e.,DN2) thymocytes in OP9 cultures Weber, et al., Nature, 2011 Defects also observed in vivo using competitive repopulation studies of WT vs TCF-1 KO BM

Evolving Transcriptional Networks as Notch Influences Early T Cell Development Yui and Rothernberg, NRI, 2014

DN Cells (CD4-/CD8-) DN1 DN2 DN3 DN4

Sequential Rearrangement of TCR ab Genes DN DP Abbas & Lichtman. Cellular and Molecular Immunology, 5th ed. W. B. Saunders 2003

The pre-TCR is Expressed in DN cells g Pre-Ta functions as a surrogate for the a chain during thymic development Expressed in DN cell and heterodimerizes with a functional b chain - assists in quality control for b chain rearrangement The pre-Ta/b chain dimer promotes increased CD3 expression and induces a ligand- independent signal, perhaps because of constitutive localization to lipid rafts or constitutive dimerization (unusual preTalpha structure), that is responsible for maturation and probably shut off RAG expression and further rearrangement, resulting in  chain allelic exclusion

How would you test the ligand-independency of signaling by the pre-TCR?

Reconstitution of rag1-/- mice with various forms of the pre-TCR Irving, et al., Science, 1998

Ligand-Independent Dimerization of the pre-TCR Pang, et al, Nature 2010 Extended structure of Pre-Ta compared to TCR Ca Dimer of Heterodimers of pre-Ta and TCR b

Two Lineages of Cells Expressing Distinct TCRs Develop in the Thymus: Stages of gd and ab T Cell Development Modified from Ciofani and Zuniga-Pflucker, Nature Rev. Immunol., 2010 (C-Kit)

Two Lineages of T cells (cont.) Stochastic Instructive model Recent data suggest that receptor expression results in stronger signal that can provides instructional cue for cell to become  lineage ( Reviewed in Ciofani, et al., Nat. Rev. Immunol. 2010)

Why might the pre-TCR and the TCRgd signal strengths be different differently?

Strength of signal: a fundamental mechanism for cell fate specification Comparison of pre‐T‐cell receptor (preTCR) and γδTCR surface expression on immature thymocytes. Histograms show CD3 levels on gated TCRβ+ (purple histogram) or TCRγδ+ (blue histogram) CD25+CD4–CD8– thymocytes from TCRα–/– mice. Shaded histogram depicts CD3 staining of CD25+CD4–CD8– thymocytes from CD3ɛ–/– mice. © IF THIS IMAGE HAS BEEN PROVIDED BY OR IS OWNED BY A THIRD PARTY, AS INDICATED IN THE CAPTION LINE, THEN FURTHER PERMISSION MAY BE NEEDED BEFORE ANY FURTHER USE. PLEASE CONTACT WILEY'S PERMISSIONS DEPARTMENT ON PERMISSIONS@WILEY.COM OR USE THE RIGHTSLINK SERVICE BY CLICKING ON THE 'REQUEST PERMISSION' LINK ACCOMPANYING THIS ARTICLE. WILEY OR AUTHOR OWNED IMAGES MAY BE USED FOR NON-COMMERCIAL PURPOSES, SUBJECT TO PROPER CITATION OF THE ARTICLE, AUTHOR, AND PUBLISHER. Immunological Reviews Volume 209, Issue 1, pages 170-175, 31 JAN 2006 DOI: 10.1111/j.0105-2896.2006.00356.x http://onlinelibrary.wiley.com/doi/10.1111/j.0105-2896.2006.00356.x/full#f3

Reciprocal regulation of Syk and ZAP-70 expression during thymocyte development Palacios & Weiss, JEM, 2007 WT DN3 -selection Development TCR selection CD44- CD25+ DN3

Strength of signal: a fundamental mechanism for cell fate specification Signal strength model for αβ/γδ lineage choice. Quantitative differences in T‐cell receptor (TCR) signal strength specify cell fate, such that an immature double negative (DN) thymocyte expressing the preTCR receives a ‘weak’ signal and chooses the αβ lineage, whereas one expressing the γδTCR receives a ‘strong’ signal and chooses the γδ lineage. © IF THIS IMAGE HAS BEEN PROVIDED BY OR IS OWNED BY A THIRD PARTY, AS INDICATED IN THE CAPTION LINE, THEN FURTHER PERMISSION MAY BE NEEDED BEFORE ANY FURTHER USE. PLEASE CONTACT WILEY'S PERMISSIONS DEPARTMENT ON PERMISSIONS@WILEY.COM OR USE THE RIGHTSLINK SERVICE BY CLICKING ON THE 'REQUEST PERMISSION' LINK ACCOMPANYING THIS ARTICLE. WILEY OR AUTHOR OWNED IMAGES MAY BE USED FOR NON-COMMERCIAL PURPOSES, SUBJECT TO PROPER CITATION OF THE ARTICLE, AUTHOR, AND PUBLISHER. Immunological Reviews Volume 209, Issue 1, pages 170-175, 31 JAN 2006 DOI: 10.1111/j.0105-2896.2006.00356.x http://onlinelibrary.wiley.com/doi/10.1111/j.0105-2896.2006.00356.x/full#f1

How might you test the signaling hypothesis?

Starting with a TCRgd Transgene: Increasing signaling strength by elimination of a negative regulator, CD5, favors gd lineage commitment Hayes, et al., Immunity, 2005

Instructing ab vs gd Lineage Commitment via Strength of Signal Ciofani and Zuniga-Pflucker, Nat. Rev. Immunol. 2010

Some caveats to the strength of signaling Stochastic Instructive model SOX13, an SRY-related HMG-box transcription factor, is preferentially expressed in gd T cells. Its KO, decreases gd development. Its over-expression in DN thymocytes impairs DN -> DP transition and ab T cell development. How SOX13 is regulated is not clear. Bcl11b, a zinc finger transcription factor, is preferentially expressed in ab lineage T cells and is induced in DN2a-DN2b, before the is expressed. It is low in the gd lineage. The KO of Bcl11b has little effect on gd T cell development but completely impairs ab lineage T cell development.

Checkpoints in Thymocyte Development Modified from Carpenter and Bosselut, Nature Immunology 2010 Notch Pre-TCR TCRab

Linking CD4 (helper) to CD8 (killer) T cell lineage commitment to the recognition of class I versus class II MHC ensures that T cell effector functions are directed appropriately. MHC class I pathway samples intracellular antigens (e.g. viruses, intracellular bacteria). MHC class II pathway samples extracellular antigens Cytotoxic CD8 T cells can kill invaded host cells before pathogens can replicate and spread. Helper T cells regulate the activity of other cells of the immune system that have endocytosed pathogens.

Recognition of MHC-1 or MHC-2 during positive selection in the thymus determines the CD4 versus CD8 T cell lineage choice.

TCR transgenic mice and MHC gene ko mice. Recognition of MHC-1 or MHC-2 during positive selection in the thymus determines the CD4 versus CD8 T cell lineage choice. CD4 CD8 MHC-2 MHC-1 Early evidence of the link between TCR specificity for MHC-1 vs. MHC-2 and the CD4 vs. CD8 lineage choice: TCR transgenic mice and MHC gene ko mice.

Generation of transgenic mice bearing rearranged TCR genes with defined specificity provided important tools for study of positive selection.

TCR specificity for MHC I or II determines CD4 versus CD8 lineage commitment OT1, HY, F5, etc OT2, DO11.10, AND, etc

MHC deficient mice provide evidence for positive selection. WT CD4 CD8 . . . . . . . . .. . … . . MHC Class II o Lack of MHC class II expression prevents development of CD4 cells And lack of MHC class I expression (b2-microglobulin deficient mice) prevents development of CD8 T cells. MHC class I and II double deficient mice lack both CD4 and CD8 mature T cells, but have normal numbers of DP thymocytes.

How does a DP decide it is a CD4 or CD8? MHC-1 MHC-2 CD8 CD4 What are the gene expression programs that determine the CD4 or CD8 T cell fate? How are distinct gene expression programs linked to TCR recognition of MHC class I or class II?

CD4 CD8 MHC-1 MHC-2 CD8 CD4 What are the gene expression programs that determine the CD4 or CD8 T cell fate? How are distinct gene expression programs linked to TCR recognition of MHC class I or class II?

How would you identify transcription factors involved in CD4 versus CD8 lineage commitment?

How would you identify transcription factors involved in CD4 versus CD8 lineage commitment? Gene profiling mature CD4 vs CD8 T cells to identify differentially expressed TF. Identify TF that regulate key CD4 vs CD8 specific target genes (CD4, CD8, perforin, CD40L, etc) Gene KO of candidate TF and assess impact on CD4, CD8 T cell development. (embryonic lethality, blocks at earlier stages of development (T commitment, TCRb checkpoint, etc) Serendipity

Th-POK (c-KROX)--a “master regulator” of CD4 T cell lineage commitment 1997: “HD” mouse strain: lacks CD4 T cells spontaneous mutation in D. Kappes’ animal colony Autosomal recessive Not due to defect in MHC Class II Genetic mapping and BAC complementation used to discover the mutant gene: Th-POK The “HD” allele has a single point mutation in a zinc-finger domain Block in CD4 T cell development, or “lineage swap”? How could you tell? 1997: “HD” mouse strain: lacks CD4 T cells spontaneous mutation in D. Kappes’ animal colony Autosomal recessive Not due to defect in MHC Class II Genetic mapping and BAC complementation used to discover the mutant gene: Th-POK The “HD” allele has a single point mutation in a zinc-finger domain Transgene caused “redirection of Class I restricted T cells” to the CD4 lineage. This is the first transcription factor shown to directly regulate lineage commitment, I.e., act as a master regulator Gata-3 is necessary but not sufficient for complete development of positively selected thymocytes to the CD4 lineage. Runx factors regulate coreceptor expression; an additional proposed role in lineage determination remains to be proven. Now we just need to figure out how TCR/co-receptor signaling talks to these Txn factors! CD4 CD8 He et al. Nature 2005

Uncoupling between positive selection and lineage commitment. Mutation in ThPOK leads to the development of “mismatched” class II specific, CD8 T cells CD4 CD8 OT1 TCR tg OT1 x ThPOK tg MHC-2 ko MHC-2 ko x ThPOK tg Over-expression of ThPOK in thymocytes lead to reciprocal phenotype: CD4 T cells with TCR specific for MHC-1.

TRANSCRIPTION FACTOR NETWORK CONTROLING CD4 VS CD8 FATE: THE SIMPLE VERSION MHC-2 positive selection MHC-1 positive selection ThPOK RUNX3 Mutual antagonism Auto- regulation CD4 CD8

CD4 CD8 MHC-1 MHC-2 CD8 CD4 What are the gene expression programs that determine the CD4 or CD8 T cell fate? How are distinct gene expression programs linked to TCR recognition of MHC class I or class II?

Relating the transcription factor networks that control CD4 versus CD8 to recognition of MHC-1 or MHC-2 during positive selection.

An “instructive” model? More prolonged signal More transient signal A kinetic model for CD4/CD8 development: duration of TCR signals influence lineage choice.

Medulla SP (CD4) DP Cortex DN SP (CD8) Medulla SP (CD4) Medulla Exit to the body DN DP Cortex SP (CD8)

Class II KO mice inject in vivo with anti-CD3: Look in the thymus Kappes et al. Immunity 2008

Instructive model: A key transition step CD4+CD8lo Al Singer Curr Opinion Immunology 2002

CD4 loop CD8 loop MHC-1 recognition: weaker, intermittent signal MHC-2 recognition: stronger, continuous signal Maintain CD4 expression allowing for late MHC-2 recognition Maintain CD8 expression allowing for late MHC-1 recognition GATA3 RUNX3 RUNX3 CD8 ThPOK ThPOK CD8 CD4 Loops can be initiated by biasing signals, and/ or stochastic fluctuations. Positive feedback makes lineage choices more robust and allows for re-verification.