A multiple redundant genetic switch locks in the transcriptional signature of T regulatory cells

The transcription factor FoxP3 partakes dominantly in the specification and function of FoxP3+CD4+ T regulatory cells (Tregs), but is neither strictly necessary nor sufficient to determine the characteristic Treg signature. Computational network inference and experimental testing assessed the contribution of other transcription factors (TF). Enforced expression of Helios or Xbp1 elicited specific signatures, but Eos, Irf4, Satb1, Lef1 and Gata1 elicited exactly the same outcome, synergizing with FoxP3 to activate most of the Treg signature, including key TFs, and enhancing FoxP3 occupancy at its genomic targets. Conversely, the Treg signature was robust to inactivation of any single cofactor. A redundant genetic switch thus locks-in the Treg phenotype, a model which accounts for several aspects of Treg physiology, differentiation and stability

Coreceptor gene imprinting governs thymocyte lineage fate

Double-positive (CD4+CD8+) thymocytes differentiate into CD4+ helper T cells and CD8+ cytotoxic T cells. A knock-in approach replacing CD8-coding sequences with CD4 cDNA shows that it is the expression kinetics of CD8, and not the identity of the coreceptor, that governs thymocyte-lineage fate

Regulation of coreceptor gene transcription and CD4/CD8 lineage choice in the thymus

CD4 and CD8 T cells express αβ T cell receptors (TCR) and respectively function as helpers and cytotoxic effector cells during an immune response. Importantly, CD4 T cells recognize peptides presented by major histocompatibility complex class II (MHCII) molecules and CD8 T cells recognize peptides presented by MHCI molecules. Intriguingly, both CD4 and CD8 T cells arise from immature CD4+CD8+ (double positive, DP) thymocytes in the thymus. However, how DP thymocytes successfully differentiate into CD4 lineage T cells expressing MHCII-specific TCR and CD8 lineage T cells expressing MHCI-specific TCR remains an unresolved and intensely debated aspect of T cell biology. Previous investigations on the mechanism of the CD4/CD8 lineage decision have either focused on signals contributed by individual coreceptors or by the TCR itself. Given their distinct regulatory elements, I hypothesized that the kinetics of Cd4 and Cd8 gene transcription during T cell differentiation in the thymus determines specification of the CD4 and CD8 T cell fates. In this dissertation, I found that the process of CD4/CD8 lineage choice in the thymus is not stochastic. Significantly, I revealed a novel integration of TCR and cytokine signals by cis-regulatory Cd8 gene enhancers during positive selection and CD8 T cell lineage commitment. Specifically, during positive selection, TCR signals terminated the activity of cis enhancers that promote CD8 expression in pre-selection thymocytes while Stat5-dependent cytokines (notably IL-7) promote the activity of cis-regulatory Cd8 enhancers in post-selection T cells. Indeed, cis-regulatory enhancers that promote CD8 expression in post-selection thymocytes are critical in reactivating Cd8 expression in CD4+CD8 - intermediate thymocytes that give rise to mature T cells. Consistent with the hypothesis that CD4/CD8 lineage choice is determined by the Cd4 and Cd8 transcriptional machinery, MHCII-selected T cells in mice in which the Cd8a gene was engineered to encode CD4 protein differentiated into cytotoxic CD8 lineage T cells. Based on the data presented in this dissertation, I conclude that CD4/CD8 lineage specification in the thymus is dictated by the kinetics of coreceptor gene transcription and not by the identity of the coreceptor

Regulation of coreceptor gene transcription and CD4/CD8 lineage choice in the thymus

CD4 and CD8 T cells express αβ T cell receptors (TCR) and respectively function as helpers and cytotoxic effector cells during an immune response. Importantly, CD4 T cells recognize peptides presented by major histocompatibility complex class II (MHCII) molecules and CD8 T cells recognize peptides presented by MHCI molecules. Intriguingly, both CD4 and CD8 T cells arise from immature CD4+CD8+ (double positive, DP) thymocytes in the thymus. However, how DP thymocytes successfully differentiate into CD4 lineage T cells expressing MHCII-specific TCR and CD8 lineage T cells expressing MHCI-specific TCR remains an unresolved and intensely debated aspect of T cell biology. Previous investigations on the mechanism of the CD4/CD8 lineage decision have either focused on signals contributed by individual coreceptors or by the TCR itself. Given their distinct regulatory elements, I hypothesized that the kinetics of Cd4 and Cd8 gene transcription during T cell differentiation in the thymus determines specification of the CD4 and CD8 T cell fates. In this dissertation, I found that the process of CD4/CD8 lineage choice in the thymus is not stochastic. Significantly, I revealed a novel integration of TCR and cytokine signals by cis-regulatory Cd8 gene enhancers during positive selection and CD8 T cell lineage commitment. Specifically, during positive selection, TCR signals terminated the activity of cis enhancers that promote CD8 expression in pre-selection thymocytes while Stat5-dependent cytokines (notably IL-7) promote the activity of cis-regulatory Cd8 enhancers in post-selection T cells. Indeed, cis-regulatory enhancers that promote CD8 expression in post-selection thymocytes are critical in reactivating Cd8 expression in CD4+CD8 - intermediate thymocytes that give rise to mature T cells. Consistent with the hypothesis that CD4/CD8 lineage choice is determined by the Cd4 and Cd8 transcriptional machinery, MHCII-selected T cells in mice in which the Cd8a gene was engineered to encode CD4 protein differentiated into cytotoxic CD8 lineage T cells. Based on the data presented in this dissertation, I conclude that CD4/CD8 lineage specification in the thymus is dictated by the kinetics of coreceptor gene transcription and not by the identity of the coreceptor

Upregulation of CD4 Expression during MHC Class II-Specific Positive Selection Is Essential for Error-free Lineage Choice

SummaryThe lineage fate of developing thymocytes is determined by the persistence or cessation of T cell receptor (TCR) signaling during positive selection, with persistent TCR signaling required for CD4 lineage choice. We show here that transcriptional upregulation of CD4 expression is essential for error-free lineage choice during major histocompatibility complex class II (MHC II)-specific positive selection and is critical for error-free lineage choice in TCR-transgenic mice whose thymocytes compete for the identical selecting ligand. CD4 upregulation occurred for endogenously encoded CD4 coreceptors, but CD4 transgenes were downregulated during positive selection, disrupting MHC II-specific TCR signaling and causing lineage errors regardless of the absolute number or signaling strength of transgenic CD4 proteins. Thus, the kinetics of CD4 coreceptor expression during MHC II-specific positive selection determines the integrity of CD4 lineage choice, revealing an elegant symmetry between coreceptor kinetics and lineage choice

The transcription factor XBP1 is selectively required for eosinophil differentiation

The transcription factor XBP1 has been linked to the development of highly secretory tissues such as plasma cells and Paneth cells, yet its function in granulocyte maturation has remained unknown. Here we discovered an unexpectedly selective and absolute requirement for XBP1 in eosinophil differentiation without an effect on the survival of basophils or neutrophils. Progenitors of myeloid cells and eosinophils selectively activated the endoribonuclease IRE1α and spliced Xbp1 mRNA without inducing parallel endoplasmic reticulum (ER) stress signaling pathways. Without XBP1, nascent eosinophils exhibited massive defects in the post-translational maturation of key granule proteins required for survival, and these unresolvable structural defects fed back to suppress critical aspects of the transcriptional developmental program. Hence, we present evidence that granulocyte subsets can be distinguished by their differential reliance on secretory-pathway homeostasis

Targeting CD4 coreceptor expression to postselection thymocytes reveals that CD4/CD8 lineage choice is neither error-prone nor stochastic

The mechanism by which CD4/CD8 lineage choice is coordinated with TCR specificity during positive selection remains an unresolved problem in immunology. The stochastic/selection model proposes that CD4/CD8 lineage choice in TCR-signaled CD4+CD8+ thymocytes occurs randomly and therefore is highly error-prone. This perspective is strongly supported by "coreceptor rescue" experiments in which transgenic CD4 coreceptors were ectopically expressed on thymocytes throughout their development and caused significant numbers of cells bearing MHC-II-specific TCR to differentiate into mature, CD8 lineage T cells. However, it is not known if forced coreceptor expression actually rescued positively selected thymocytes making an incorrect lineage choice or if it influenced developing thymocytes into making an incorrect lineage choice. We have now reassessed coreceptor rescue and the concept that lineage choice is highly error-prone with a novel CD4 transgene (referred to as E8I-CD4) that targets expression of transgenic CD4 coreceptors specifically to thymocytes that have already undergone positive selection and adopted a CD8 lineage fate. Unlike previous CD4 transgenes, the E8I-CD4 transgene has no effect on early thymocyte development and cannot itself influence CD4/CD8 lineage choice. We report that the E8I-CD4 transgene did in fact induce expression of functional CD4 coreceptor proteins on newly arising CD8 lineage thymocytes precisely at the point in thymic development that transgenic CD4 coreceptors would putatively rescue MHC-II-specific thymocytes that incorrectly adopted the CD8 lineage. However, the E8I-CD4 transgene did not reveal any MHC-II-selected thymocytes that adopted the CD8 lineage fate. These results demonstrate that CD4/CD8 lineage choice is neither error-prone nor stochastic