During FY15, the TCDS provided further evidence that the Notch signaling pathway is an important regulator of adaptive immune responses. Notch and Notch ligands are widely expressed and play an essential role in nearly every cell type and organ system. Gain and loss of function mutations have deleterious effects; therefore, there is tremendous therapeutic value in understanding this pathway whose dysregulation can lead to a broad spectrum of diseases. The development of safe and effective treatments targeting Notch signaling requires an understanding of Notch deprivation in specific cell types and the associated secondary effects. To this end, we genetically abrogated components of the Notch signaling pathway in T cells. We used the helminth parasite, Schistosoma mansoni, as a model antigen to assess the role of Notch signaling in T cells contributing to the host response. Chronic infection with the parasite can cause hepatosplenic disease. An appropriate granulomatous response protects the host from egg-induced damage, and generation of high affinity immunoglobulin is protective against re-infection. A sub-sufficient adaptive immune response can be fatal, but an overaggressive granulomatous response leads to fibrosis. Although specific cell targets have not been determined, some reports indicate that Notch inhibition has anti-fibrotic effects in preclinical models of asthma, hepatic fibrosis, and systemic sclerosis. In previous work, we inhibited Notch signaling in T cells genetically by inducing deletion of three genes, each encoding a protein involved in the Notch signaling pathway, Presenilin 1/2 (PS), Pofut, or RPBJ, as well as deleting Notch ligand DLL4 in a subset of dendritic cells. These mice were used to examine the cell-intrinsic effects of Notch signaling on CD4 T cell activation and differentiation and the granulomatous and inflammatory response to S. mansoni. Since it closely mimics the natural route and sequence of infection, mice were first exposed cutaneously to S. mansoni cercariae. Although there was considerable variation in the responses of individual mice, the tendency for fewer Notch-deficient T cells to make Th2 cytokines suggested that Notch signaling in T cells could impact the response. To circumvent the variability in antigen burden that occurs with cutaneous exposure, we directly injected S. mansoni eggs to mimic the deposition of eggs that occurs during natural infection when eggs from the liver are shunted to the lung and lodge in pre-capillary arteries. After infection, the mean number of granulomas in lung per animal was the same, but the mean volume of the granulomas was decreased in mice with Notch-deficient T cells. Also the number of total CD4 T cells in the lung was reduced, as was the frequency of IL-13+ T cells. IL-13 is believed to be a mediator of fibrosis because of its ability to stimulate collagen deposition by fibroblasts. Assay of collagen fibers in lung tissue confirmed that mice with Notch signaling-deficient T cells had fewer collagen fibers. The balance of Th1:Th2 cytokines is also critical to the extent of fibrosis in part because Type 1 cytokines antagonize IL-13 effector functions. Notably, the proportion of IFNg+ T cells was unchanged, resulting in a lower IL-13:IFNg ratio; however, in all cases the response of the mutant mice was skewed towards a Th2 response. These data suggested that Notch influenced the magnitude, rather than the nature of the T cell response. Neither the frequency of IL-17+ or of IL-10+ CD4 T cells in the lung accounted for the decreased fibrosis/pathology. Although the proportion IL-5+ cells was significantly reduced, there was no diminution in the frequency of eosinophils in granulomas. Notch signaling is reported to directly regulate transcription of the Il-4 gene. The frequency of IL-4+ T cells in lungs of mice exposed to S. mansoni eggs was diminished, but the amount of IL-4 expressed on a per cell basis was not reduced. T cell-derived IL-4 is required for B cell class-switching to produce IgE, and elevated serum IgE is a hallmark of helminth infection. Mice with Notch-deficient T cells exposed to S. mansoni eggs had significantly less serum IgE. Although serum IgE was decreased, total cellularity, number of CD4 T cells, and frequency of germinal center B cells (GCB) in the lung-draining LN were equivalent, suggesting that the ability of B cells to undergo class-switching to IgE was impaired. CD4 T follicular helper cells (Tfh) provide necessary contact dependent signals to GCB to facilitate isotype switching. The frequency of Tfh in lung-associated LN of egg immunized mice was significantly reduced with Notch signaling-deficient T cells. Although there were less Tfh cells, they were not totally absent. These results suggested that a subset of Tfh develops independently of Notch or that some T cells manage to escape Cre-mediated deletion of the targeted gene. To address this issue, we introduced a YFP reporter to reveal Cre-mediated function. The majority of conventional CD4 T cells in the LN of Cre+ animals were YFP+, indicating that on a population level most CD4 T expressed Cre at sufficient levels to mediate gene deletion. In contrast, Tfh cells were highly enriched for YFPneg cells. This was the case for Tfh in the draining LN immunized with soluble egg antigens and in lung-associated LN of mice receiving whole live eggs. There was no evidence for a similar selective pressure in the residual CD4 T cells producing Th2 cytokines isolated from lung tissue of egg immunized mice. These data provided strong evidence that Notch signaling is a critical regulator of Tfh development, whereas the requirement for Notch signaling in Th2 differentiation is less rigorous. By limiting Notch signaling deficiency to T cells, we demonstrated CD4 T cell-intrinsic effects in vivo on T cell activation, Th2 cytokine expression, generation of Tfh cells, and magnitude of responses to egg antigens. We also observed indirect effects on granuloma volume, collagen deposition, and class switching to IgE. In most cases, the phenotype of mice with PS-, Pofut-, or RBPJ-deficient T cells was similar, indicating that the canonical Notch signaling pathway in T cells contributed to the response. In a second project, we extended our studies on the role of the transcription factor, GATA3, in T cell development. We observed that MHC2 generates stronger TCR signals and more GATA3 than MHC1 in thymocytes undergoing selection. Our work also indicated that GATA3 functions in CD4 versus CD8 T lineage commitment, as manipulation of GATA3 expression could redirect the CD4/CD8 lineage choice. These data suggested that levels of GATA3 may regulate the key lineage specifying factors, ThPOK (CD4 lineage) and Runx3 (CD8 lineage). Although ThPOK antagonizes Runx3 in thymocytes committed to the CD4 lineage, what induces Runx3 was unknown. We found that TCR stimulation induced and up-regulated Runx3 expression during and prior to thymocyte selection. These data begged the question of how Runx3 was negatively regulated in MHC2-selected thymocytes prior to ThPOK expression. We hypothesized that the high levels of GATA3 attained in MHC2-selected thymocytes committing to the CD4 lineage suppresses Runx3 and induces ThPOK. In support of our hypothesis, GATA3 binds the ThPOK locus and ThPOK induction correlates with high levels of GATA3. Importantly, we observed that GATA3 overexpression suppressed Runx3; whereas Runx3 expression was enhanced when GATA3 was deficient, demonstrating that GATA3, as well as TCR, regulate Runx3 expression in thymocytes undergoing selection. Collectively, these data establish GATA3 is a pivotal regulator of lineage commitment, providing a link between TCR signal strength and the CD4/CD8 T cell fate in T cell development.
