SOX13, A γδ T Cell-Specific Gene, Is a WNT-Signaling Antagonist Regulating T Cell Development: A Dissertation

Mature αβ and γδ T cells arise from a common precursor population in the thymus. Much debate has focused on the mechanism of T cell lineage choice made by these multi-potential precursor cells. It is widely believed that the decision of these precursor cells to commit to the γδ or αβ T cell lineages is regulated primarily by a specific instructive signal relayed through the appropriate T cell receptor. Contrary to this model, we present evidence for a TCR-independent lineage commitment process. Comparison of global gene expression profiles from immature αβ and γδ lineage thymocytes identified Sox13, an HMG-box transcription factor, as a γδ T cell-specific gene. Unlike other HMG-box transcription factors such as TCF1, LEF1 and SOX4, that are critical for proper αβ T cell development, Sox13 expression is restricted to early precursor subsets and γδ lineage cells. Importantly, SOX13 appears to influence the developmental fate of T cell precursors prior to T cell receptor expression on the cell surface. Transgenic over-expression of Sox13 in early T cell precursors strongly inhibits αβ lineage development, in part, by inhibiting precursor cell proliferation and concomitantly, leading to increased cell death among αβ lineage subsets. Steady-state γδ T cell numbers, however, appear unaffected. Strikingly, the DP αβ lineage cells that do develop in Sox13 transgenic mice are imprinted with a γδ- or precursor-like molecular profile, suggesting that SOX13 plays an active role in the lineage fate decision process or maintenance. Sox13-deficient mice, on the other hand, have selectively reduced numbers of γδ thymocytes, indicating that SOX13 is essential for proper development of γδ T cells. We present additional data demonstrating that SOX13 is a canonical WNT signaling antagonist modulating TCF1 activity, raising a strong possibility that WNT signals, and their modulators, are at the nexus of γδ versus αβ T cell lineage commitment

CD5 levels define functionally heterogeneous populations of naïve human CD4+ T cells

Studies in murine models show that subthreshold TCR interactions with self-peptide are required for thymic development and peripheral survival of naïve T cells. Recently, differences in the strength of tonic TCR interactions with self-peptide, as read-out by cell surface levels of CD5, were associated with distinct effector potentials among sorted populations of T cells in mice. However, whether CD5 can also be used to parse functional heterogeneity among human T cells is less clear. Our study demonstrates that CD5 levels correlate with TCR signal strength in human naïve CD4+ T cells. Further, we describe a relationship between CD5 levels on naïve human CD4+ T cells and binding affinity to foreign peptide, in addition to a predominance of CD5hi T cells in the memory compartment. Differences in gene expression and biases in cytokine production potential between CD5lo and CD5hi naïve human CD4+ T cells are consistent with observations in mice. Together, these data validate the use of CD5 surface levels as a marker of heterogeneity among human naïve CD4+ T cells with important implications for the identification of functionally biased T- cell populations that can be exploited to improve the efficacy of adoptive cell therapies

Pre-existing chromatin accessibility and gene expression differences among naive CD4+ T cells influence effector potential

CD4+ T cells have a remarkable potential to differentiate into diverse effector lineages following activation. Here, we probe the heterogeneity present among naive CD4+ T cells before encountering their cognate antigen to ask whether their effector potential is modulated by pre-existing transcriptional and chromatin landscape differences. Single-cell RNA sequencing shows that key drivers of variability are genes involved in T cell receptor (TCR) signaling. Using CD5 expression as a readout of the strength of tonic TCR interactions with self-peptide MHC, and sorting on the ends of this self-reactivity spectrum, we find that pre-existing transcriptional differences among naive CD4+ T cells impact follicular helper T (TFH) cell versus non-TFH effector lineage choice. Moreover, our data implicate TCR signal strength during thymic development in establishing differences in naive CD4+ T cell chromatin landscapes that ultimately shape their effector potential

Comparative Study of Hematopoietic Differentiation between Human Embryonic Stem Cell Lines

Directed differentiation of human embryonic stem cells (hESCs) into any desired cell type has been hailed as a therapeutic promise to cure many human diseases. However, substantial roadblocks still exist for in vitro differentiation of hESCs into distinct cell types, including T lymphocytes. Here we examined the hematopoietic differentiation potential of six different hESC lines. We compare their ability to develop into CD34+ or CD34+CD45+ hematopoietic precursor populations under several differentiation conditions. Comparison of lymphoid potential of hESC derived- and fetal tissue derived-hematopoietic precursors was also made. We found diverse hematopoietic potential between hESC lines depending on the culture or passage conditions. In contrast to fetal-derived hematopoietic precursors, none of the CD34+ precursors differentiated from hESCs were able to develop further into T cells. These data underscore the difficulties in the current strategy of hESC forward differentiation and highlight distinct differences between CD34+ hematopoietic precursors generated in vitro versus in vivo

Integrated morphogen signal inputs in gammadelta versus alphabeta T-cell differentiation

Morphogens, a class of secreted proteins that regulate gene expression in a concentration-dependent manner, are responsible for directing nearly all lineage fate choices during embryogenesis. In the thymus, morphogen signal pathways consisting of WNT, Hedgehog, and the transforming growth factor-beta superfamily are active and have been implicated in various developmental processes including proliferation, survival, and differentiation of maturing thymocytes. Intriguingly, it has been inferred that some of these morphogen signal pathways differentially affect gammadelta and alphabeta T-cell development or maintenance, but their role in T-cell lineage commitment has not been directly probed. We have recently identified a modulator of morphogen signaling that significantly influences binary gammadelta versus alphabeta T-cell lineage diversification. In this review, we summarize functions of morphogens in the thymus and provide a highly speculative model of integrated morphogen signals, potentially directing the gammadelta versus alphabeta T-cell fate determination process

Strength and Numbers: The Role of Affinity and Avidity in the ‘Quality’ of T Cell Tolerance

The ability of T cells to identify foreign antigens and mount an efficient immune response while limiting activation upon recognition of self and self-associated peptides is critical. Multiple tolerance mechanisms work in concert to prevent the generation and activation of self-reactive T cells. T cell tolerance is tightly regulated, as defects in these processes can lead to devastating disease; a wide variety of autoimmune diseases and, more recently, adverse immune-related events associated with checkpoint blockade immunotherapy have been linked to a breakdown in T cell tolerance. The quantity and quality of antigen receptor signaling depend on a variety of parameters that include T cell receptor affinity and avidity for peptide. Autoreactive T cell fate choices (e.g., deletion, anergy, regulatory T cell development) are highly dependent on the strength of T cell receptor interactions with self-peptide. However, less is known about how differences in the strength of T cell receptor signaling during differentiation influences the ‘function’ and persistence of anergic and regulatory T cell populations. Here, we review the literature on this subject and discuss the clinical implications of how T cell receptor signal strength influences the ‘quality’ of anergic and regulatory T cell populations

Stable Interactions and Sustained TCR Signaling Characterize Thymocyte–Thymocyte Interactions that Support Negative Selection

Negative selection is one of the primary mechanisms that render T cells tolerant to self. Thymic dendritic cells play an important role in negative selection, in line with their ability to induce migratory arrest and sustained TCR signals. Thymocytes themselves display self-peptide/MHC class I complexes, and although there is evidence that they can support clonal deletion, it is not clear whether they do so directly via stable cell-cell contacts and sustained TCR signals. In this study, we show that murine thymocytes can support surprisingly efficient negative selection of Ag-specific thymocytes. Furthermore, we observe that agonist-dependent thymocyte-thymocyte interactions occurred as stable, motile conjugates led by the peptide-presenting thymocyte and in which the trailing peptide-specific thymocyte exhibited persistent elevations in intracellular calcium concentration. These data confirm that self-Ag presentation by thymocytes is an additional mechanism to ensure T cell tolerance and further strengthen the correlation between stable cellular contacts, sustained TCR signals, and efficient negative selection

Differential expression of tissue-restricted antigens among mTEC is associated with distinct autoreactive T cell fates

T cell tolerance is established in the thymus via interactions with medullary thymic epithelial cells (mTEC) expressing tissue-restricted self antigens. Here, the authors suggest, using new transgenic mouse lines and single cell transcriptome analyses, that specific mTEC subsets are associated with distinct T cell fates

Stable Interactions and Sustained TCR Signaling Characterize Thymocyte–Thymocyte Interactions that Support Negative Selection

Negative selection is one of the primary mechanisms that render T cells tolerant to self. Thymic dendritic cells play an important role in negative selection, in line with their ability to induce migratory arrest and sustained TCR signals. Thymocytes themselves display self-peptide/MHC class I complexes, and although there is evidence that they can support clonal deletion, it is not clear whether they do so directly via stable cell-cell contacts and sustained TCR signals. In this study, we show that murine thymocytes can support surprisingly efficient negative selection of Ag-specific thymocytes. Furthermore, we observe that agonist-dependent thymocyte-thymocyte interactions occurred as stable, motile conjugates led by the peptide-presenting thymocyte and in which the trailing peptide-specific thymocyte exhibited persistent elevations in intracellular calcium concentration. These data confirm that self-Ag presentation by thymocytes is an additional mechanism to ensure T cell tolerance and further strengthen the correlation between stable cellular contacts, sustained TCR signals, and efficient negative selection