Signals through the TCR and other surface receptors promote cell fate decisions in thymocytes and mature T cells. To investigate Notch receptor functions in T cell development, we generated Presenilin (PS) conditionally mutant mice in which all Notch activity could be prohibited in specifically targeted tissues. Our previous studies showed that mature CD4 T cells were inefficiently generated from PS deficient precursor thymocytes with a polyclonal TCR repertoire and severely reduced with MHC2-restricted TCR transgenes. Both pre-selected and MHC selected PS-deficient thymocyte precursors showed impaired responses to TCR stimulation in vitro, while Notch gain-of-function mutant thymocytes mediated enhanced responses. CD4 T cell development in PS deficient mice was improved with higher affinity TCR or increased density of selecting MHC ligand, indicating that impaired differentiation was due to defective positive selection. From these data, we proposed that Notch reinforces or potentiates TCR signaling in positive selection, promoting the generation of mature CD4 T cells. One of our major aims has been to determine how Notch and TCR signaling pathways are linked in developing thymocytes. Like Notch, the GATA3 transcriptional factor has been associated with TCR signaling in thymocyte development. Conditional GATA3 deficiency, like the PS deficiency, interferes with CD4 T cell development, although the block is much more severe. Regulation of Notch1 and GATA3 expression are also similar at successive stages of thymocyte development. From our analyses of loss- and gain-of-function mutant mice, both Notch and GATA3 promote TCR activation and thymocyte development in vivo, as well as TCR signal transduction in vitro. Also notable, Notch had been reported to bind the locus and regulate GATA3 expression in peripheral T cell responses. These collective observations suggested coordinate or interactive roles for Notch and GATA3 in the selection and maturation of T cells, causing us to investigate whether Notch facilitates TCR signaling and thymocyte selection via direct regulation of GATA3. By our most quantitative and sensitive assays, the levels of GATA3 expression were normal in pre-selected PS deficient thymocytes that already manifested abnormalities in TCR signaling. Although enforced GATA3 expression in PS deficient mice appeared to restore thymocyte development/maturation in vivo with polyclonal TCR, GATA3 failed to rescue the in vitro defects in TCR signal transduction. Since calcium mobilization and Erk activation occur within seconds to minutes of TCR stimulation, our results were inconsistent with a model in which Notch potentiates TCR signaling through direct regulation of the GATA3 gene. Although these studies are still ongoing, these data suggested that Notch and GATA3 function cooperatively in thymocytes but by distinct mechanisms. Another goal of the lab is to determine how TCR and Notch signaling pathways cooperate to guide cell fate decisions in peripheral T cell responses. We had observed that the frequency of activated T cells was higher when stimulated with myeloid dendritic cells (DCs) as compared to lymphoid DCs. Analysis of a panel of surface molecules expressed by these splenic antigen presenting cells (APC) revealed that expression of Notch ligand, DLL4 (Delta-like ligand 4) was higher on myeloid than lymphoid DCs, which correlated with their superior ability to stimulate naive T cells. To further analyze the physiological importance of this result, we generated a mutant mouse line, targeting deletion of DLL4 to CD11c-expressing dendritic cells. DCs bearing this mutation and the appropriate MHC haplotype were used to stimulate normal naive MHC1- or MHC2-restricted T cells. Without DLL4 on DCs, T cell activation was impaired as shown by decreased proliferation, metabolism, expression of early activation markers, and IL2 secretion. Similar results were obtained when normal DCs presented antigen to T cells with targeted deletion of genes encoding various Notch signaling components (Presenilin, Pofut1, or CSL). Taken together, the results indicate that T cell activation was similarly impaired when signaling through Notch receptors was disrupted in T cells or DCs lacked Notch ligand. These loss-of-function experiments utilized T cells or APCs with targeted disruption of genes essential to the Notch signaling pathway. We developed a complementary gain-of-function system by transfecting an artificial APC cell line generated by R. Germain (NIAID/LI), with the Notch ligand, DLL4. These experiments showed that the frequency of activated T cells was enhanced by Notch ligand as revealed by increases in proliferation, metabolism, expression of early activation markers, and IL2 production. Thus, Notch signaling and Notch ligand mutants all reveal that Notch facilitates TCR activation and IL2 production in naive CD4 T cells. Previous reports suggested that Notch signaling controls T helper (Th1/Th2) differentiation of CD4 T cells, although the mechanism responsible for this regulation was controversial. We hypothesized that Notch signaling augments T helper responses by facilitating the primary activation of naive CD4 T cells. In collaboration with W. Paul's lab (NIAID/LI), we tested this hypothesis using an in vitro assay for Th2 differentiation. When stimulated with APC and low doses of specific peptide, Notch mutant T cells failed to secrete IL4. Defects in IL4 production could be rescued by addition of exogenous IL2, suggesting IL2 to be the major defect in the generation of a Th2 response. We extended those findings in vivo in collaboration with T. Wynn's (NIAID/LPD) lab, using a mouse model of Schistosoma mansoni infection. When mice with T cell specific deletion of Presenilin, Pofut1, or CSL genes were exposed to S. mansoni eggs;granuloma formation, eosinophil recruitment, and serum IgE were all reduced. Moreover, fewer lung resident CD4 T cells produced Th2 cytokines;IL4, IL5, and IL13. Given our in vitro findings that link disrupted Notch function to defects in IL2 production, experiments are in progress to identify and quantify the IL2 secreting cells in this disease model. We wanted to determine the mechanism(s) by which Notch signaling augments naive T cell responses. Imaging experiments, carried out in collaboration with R. Varma's lab (NIAID/LCMI), demonstrated that Notch/DLL4 interactions do not significantly enhance adhesion between T cells and peptide bearing APCs. Moreover, antigen responses were attenuated in the absence of Notch signaling even though surface levels of Notch receptor on T cells and Notch ligand on APC were maintained. Although Notch functions as a transcriptional regulator, our findings suggested that Notch does not regulate IL2 transcription directly but does so via TCR activation since defective responses from mutant T cells with normal APC or mutant APC with normal T cells were antigen specific. Notably, analysis of expression and cellular localization of several transcription factors by Imagestream technology revealed that the decreased proportion of T cells responding to peptide antigen in the absence of Notch signaling correlated with the reduced fraction of cells activating transcription factor families of NFAT, AP1, and NFkB.
