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”? 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 TCR  cell vs a TCR  T cell To become a CD4 or CD8 T cell To become one of the minor T cells subset NK T cell CD  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: , 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 A critical role for Notch in T cell lineage commitment Yui and Rothernberg, NRI, 2014

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

 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.,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

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

The pre-TCR is Expressed in DN cells Pre-T  functions as a surrogate for the  chain during thymic development Expressed in DN cell and heterodimerizes with a functional  chain - assists in quality control for  chain rearrangement The pre-T  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-T  compared to TCR C  Dimer of Heterodimers of pre-T  and TCR 

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

Two Lineages of T cells (cont.) 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) Stochastic Instructive model

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

Strength of signal: a fundamental mechanism for cell fate specification Immunological Reviews Volume 209, Issue 1, pages , 31 JAN 2006 DOI: /j x Volume 209, Issue 1,

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

Strength of signal: a fundamental mechanism for cell fate specification Immunological Reviews Volume 209, Issue 1, pages , 31 JAN 2006 DOI: /j x Volume 209, Issue 1,

How might you test the signaling hypothesis?

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

Instructing  vs  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  T cells. Its KO, decreases  development. Its over-expression in DN thymocytes impairs DN -> DP transition and  T cell development. Bcl11b, a zinc finger transcription factor, is preferentially expressed in  lineage T cells and is induced in DN2a-DN2b. It is low in the  lineage. The KO of Bcl11b has little effect on  T cell development but completely impairs  lineage T cell development. There’s more to come.

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

MHC class II pathway samples extracellular antigens MHC class I pathway samples intracellular antigens (e.g. viruses, intracellular bacteria). Cytotoxic CD8 T cells can kill invaded host cells before pathogens can replicate and spread. 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. 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.

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

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

plate at 1 cell/well grow and expand Test for antigen specificity clone the TCR alpha and beta chain genes from the T cell clone Antigen (e.g., ovalbumin) T cells + DCs + ovalbumin Rest and restimulate with Ag and cytokines, rest, repeat 9 days T cell receptor transgenic mice:

T cell receptor alpha- & beta-chain genes specific for MHC class I or MHC class II OT-1 - CD8 T cells AND - CD4 T cells All T cells will express the same TCR  receptor

Increased development of CD4 SP thymocytes and T cells in mice expressing a rearranged class II MHC specific transgene CD8 CD4

plate at 1 cell/well grow and expand Test for antigen specificity clone the TCR alpha and beta chain genes from the T cell clone Expand anti-male CD8 T cells Rest and restimulate with Ag and cytokines, rest, repeat 9 days Generating H-Y TCR transgenic mice (anti-male peptide TCR) Male H-2 b cells Female H-2 b mouse

5% 37% 46% 10% Increased development of CD8 SP thymocytes and T cells in mice expressing a rearranged class I MHC specific transgene

TCR specificity for MHC I or II determines CD4 versus CD8 lineage commitment OT1, HY, F5, etcOT2, DO11.10, AND, etc

MHC deficient mice provide evidence for positive selection. And lack of MHC class I expression (  2-microglobulin deficient mice) prevents development of CD8 T cells. WT CD4 CD ….. MHC Class II o CD4 CD8 Lack of MHC class II expression prevents development of CD4 cells MHC class I and II double deficient mice lack both CD4 and CD8 mature T cells, but have normal numbers of DP thymocytes.

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

What are the gene expression programs that determine the CD4 or CD8 T cell fate? CD4 CD8 CD4 CD8 MHC-2 MHC-1 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?

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, TCR  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? He et al. Nature 2005 CD4 CD8

Uncoupling between positive selection and lineage commitment. 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. Mutation in ThPOK leads to the development of “mismatched” class II specific, CD8 T cells CD4 CD8

ThPOK acts together with other transcription factors in a network that specifies the CD4 fate. GATA-3 is required for ThPOK to induce the CD4 fate (but not to repress CD8 fate). GATA-3 promotes ThPOK expression. Loss of GATA-3 can (sometimes) divert thymocytes with TCR specific for MHC-2 into the CD8 lineage. Wang et al Nat Immunol Oct;9(10):1122

ThPOK reinforces its own expression and acts together with other transcription factors in a network that specifies the CD4 fate. ThPOK opposes Runx repression of CD4 and ThPOK expression. Mutation of both Runx and ThPOK rescues CD4 development. Mutant ThPOK allele than cannot undergo positive autoregulation leads to “trans-differentiation” of CD4 cells to the CD8 lineage. Egawa and Littman and Miroi et al Nat Immunol. 2008

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

ThPOK CD4 RUNX3 CD8 TRANSCRIPTION FACTOR NETWORK CONTROLING CD4 VS CD8 FATE: MORE PLAYERS CD4 RUNX3 CD8 ThPOK GATA3 Tox MAZR cMyb Would you consider ThPOK to be a “master regulator” of the CD4 fate. Why or why not? MHC-2 positive selection MHC-1 positive selection

What are the gene expression programs that determine the CD4 or CD8 T cell fate? CD4 CD8 CD4 CD8 MHC-2 MHC-1 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.

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

But clearly not the whole story. Quantitative model cannot adequately account for the stringent relationship between MHC-1 vs MHC-2 specificity and positive selection.

Another mechanism to ensure that thymocytes adopt the CD4/CD8 fate appropriate for their TCR specificity: Some thymocytes make the “wrong” lineage choice, but a late check for co- receptor expression eliminate cells that turned down the wrong co-receptor. Thymocytes expressing TCR specific for MHC-1 A stochastic/selection model?

Davis et al ‘93, Itano et al ‘94, Robey et al ‘94, Baron et al ‘94, Corbella et al ‘94 Paterson et al ‘94, Chan et al ‘94, Rathemtulla et al, ‘02 Constitutive expression of CD8 (or CD4) leads to the (inefficient) development of “mismatched” T cells

How long does a thymocyte need to engage MHC and receive TCR signals in order to complete positive selection? How long does it take for a thymocyte to commit to the CD4 or CD8 lineage? Typical time required from initial encounter with extracellular ligand to changes in gene expression?

Multiple changes accompany positive selection Pre selection thymocytes are: In cortex CD4+CD8+ TCR neg-low rag+ low expression of activation markers (CD69-) Short-lived, dep on TCR signal post selection thymocytes are: In medulla CD4+CD8- or CD4-CD8+ TCR high rag- CD69+ Long-lived, indep of TCR signal These changes occur asynchronously over a period of 1-3 days. Would you say that a cortical CD4+CD8+ CD69+ rag- thymocyte has been positively selected? Can identify thymocytes exhibiting some, but not all signs of positive selection.

“Antigen-experienced” CD4+CD8+ thymocytes isolated from TCR trangenic positive selecting mice (HY TCR, H2b) and intrathymically injected into mice with or without the positive selecting ligand. How long does a thymocyte need to engage MHC and receive TCR signals in order to complete positive selection? Recent data from Art and Ellen’s lab showing that 2-3 days of continuous TCR signaling needed for efficient positive selection of CD8 T cells (Au-Yeong et al, Ross et al 2014) A single hit is not sufficient for positive selection of CD8 T cells:

Lingeage commitment represents the loss in the ability of a cell to make alternative lineage choices under permissive conditions. How long does it take for a thymocyte to commit to the CD4 or CD8 lineage?

Impact of removing ThPOK after CD4 lineage commitment:

TCR affinity (signal strength) Death by neglect Clonal deletion Positive selection CD8 lineage commitment CD4 lineage commitment T reg development (agonist selection) What does TCR “signal strength” really mean? TCR affinity for peptide-MHC complex? Duration, frequency, or intensity of TCR signaling? Presence/absence of co-stimulatory signals?

How do distinct temporal signaling patterns encode distinct T cell fates? days hours Temporal requirement for TCR signaling during CD8 T cell positive versus negative selection? Temporal pattern of TCR signaling during positive versus negative selection? intermittent continuous Why does positive selection take 2-3 days?

Positive selection and CD4 versus CD8 lineage development may be lengthy, reversible processes. Interpretation of TCR signaling difference and establishment of stable gene regulatory network for CD4 or CD8 fate likely occur at the same time and may be mechanistically linked.

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