During FY14, we demonstrated that the Notch signaling pathway is an important regulator of adaptive immune responses. Immune responses are initiated when naive CD4 T cells engage specific peptide antigens on MHC2 together with co-stimulatory molecules on antigen presenting cells (APC). Although it was known that Notch signaling influences effector functions in differentiated CD4 T helper and regulatory cells, whether and how ligand-induced Notch signaling influences the initial priming of CD4 T cells had not been addressed. Studies of T cell priming necessitates the use of T cells that have not been previously activated, and un-manipulated T cells have the added benefit that Notch expression and signaling remain subject to endogenous regulatory mechanisms. To ascertain if Notch ligand DLL4 expressed on APC was functionally relevant in vivo, we generated mice in which DLL4 was conditionally deleted in a subset of dendritic cells and tested their ability to elicit a CD4 T cell-dependent immune response using a classic adoptive transfer protocol. These experiments revealed that DLL4-bearing APC enhanced both the frequency of CD4 T cells that responded and the quality of the response on a per cell basis. To dissect the mechanisms underlying in vivo phenotypes, we devised an in vitro system with an APC line such that Notch ligand, MHC2, and co-stimulatory molecules were clearly defined and amenable to manipulation. This system allowed us to characterize priming of a synchronized population of CD4 T cells with or without DLL4 on APC. DLL4-induced Notch signaling allowed more T cells to respond at lower doses of antigen, increasing the magnitude of the primary response. The higher levels of nutrient receptors and increased biomass indicated that Notch signaling also improved responses on a per cell basis. DLL4-deficient APC were less potent stimulators of CD4 T cell metabolism, proliferation, and cytokine secretion, particularly when the concentration of antigen was low. With IL-2 capture assays or an Il2-GFP reporter, we observed that Notch signaling accelerated the kinetics and increased the magnitude of the response by increasing the frequency of IL-2 expressing cells, resulting in greater IL-2 secretion. Unexpectedly, all of the co-stimulatory effects of DLL4 that we observed were predicated on PI3K signaling downstream of TCR plus CD28. In the absence of antigen, CD28 engagement, or PI3K activity;DLL4 had minimal effect on any of the parameters measured. Thus, DLL4-Notch acted as co-stimulators of CD28-mediated co-stimulation. One of the major signaling cascades downstream of CD28 involves PI3K and mTOR. To formally establish that PI3K activity was linked to Notch signaling, we confirmed that phosphorylation of several downstream targets of mTOR were decreased when T cells were stimulated in the absence of DLL4. We also investigated NFAT expression because sustained signaling via TCR plus CD28 leads to the synthesis, activation, and nuclear translocation of NFATc1. Using imaging flow cytometry, we established that NFATc1 was localized to the nucleus of activated T cells, irrespective of DLL4 expression. However, the frequency of T cells induced to express NFATc1 was significantly higher in the presence of DLL4. We considered the possibility that increased PI3K signaling was secondary to the increased IL-2 because IL-2R signaling also activates the PI3K signaling cascade. Our experiments using IL-2-deficient T cells indicated that DLL4-mediated increases in PI3K signaling were a consequence of Notch reinforcing signals downstream of CD28 and occurred independently of DLL4's ability to increase the availability of IL-2. Because our data suggested that Notch signals enhance CD4 T cell priming which could critically alter the course of a physiological immune response, we employed a tumor rejection model in which eradication of the tumor is dependent on cross-presentation of tumor antigens to CD4 T cells by host APC. In the absence of T cells, tumor cells injected in to control and DLL4-deficient recipients grew at a similar rate and resulted in 100% mortality in less than 5 weeks. Adoptively transferred T cells responded to the tumor-derived antigen and constrained tumor growth;however, the anti-tumor effect of T cells was severely impaired in DLL4-deficient mice where tumors grew markedly larger, leading to a significant increase in mortality. The inability of T cells to control tumor growth in the absence of DLL4-bearing APCs could not be attributed to a failure of T cells to survive in these mice, as the frequency of CD4 T cells in the tumor draining lymph node or infiltrating the tumor itself were equivalent or higher than controls. Thus, DLL4 expressed on APC was essential for efficient activation of CD4 T cells and an effective anti-tumor response. In a second project, we extended our studies on the role of the transcription factor, GATA3, in T cell development. We had found that GATA3 promotes TCR signal transduction and positive selection in developing thymocytes. Taken with results from others showing that TCR signaling up-regulates GATA3 expression, we propose that GATA3 and TCR operate in a positive reinforcing loop with each amplifying the activity of the other. Since MHC2 generates stronger TCR signals (and thus more GATA3) than MHC1 in thymocytes undergoing selection, this model explains the preferential loss of CD4 T cells in GATA3-deficient mice. Although GATA3-deficiency mainly impacts CD4 T cell development, we found that TCR signal transduction is attenuated also in developing CD8 T cells. Moreover, when gata3 gene deletion was targeted to peripheral T cells, both CD4 and CD8 T cells exhibited defective TCR signal transduction upon activation. Our previous work indicated that GATA3 functions also in CD4 versus CD8 T lineage commitment as GATA3 deficiency resulted in the re-direction of MHC2-selected thymocytes from the CD4 to the CD8 T cell lineage. These results led us to question whether GATA3 regulates the key lineage specifying factors, ThPOK for the CD4 lineage and Runx3 for the CD8 lineage. Although both ThPOK and Runx3 were thought to be induced late in thymocytes undergoing selection, we found that Runx3 was induced earlier in TCR transgenic mice and prior to ThPOK expression. While it was established that ThPOK could suppress Runx3 in thymocytes committed to the CD4 lineage, what induced Runx3 was unknown. We discovered that TCR stimulation could induce and up-regulate Runx3 expression prior to selection. These data raised the issue of how Runx3 is negatively regulated in MHC2-selected thymocytes before ThPOK expression. We surmised that GATA3 was responsible since Runx3 expression was higher in GATA3-deficeint thymocytes undergoing MHC2 selection, and lower when GATA3 was overexpressed during MHC1 selection. Also, the maximum levels of GATA3 were higher in thymocytes undergoing MHC2- than MHC1-selection, correlating with the levels of TCR. These findings suggest a model in which GATA3 regulates the critical specifying factors in the CD4/CD8 T cell fate decision. TCR signaling up-regulates both GATA3 and Runx3. The higher levels of GATA3 attained in MHC2-selected thymocytes committing to the CD4 lineage suppress Runx3 and induce ThPOK. In spite of the Runx3 repression observed when GATA3 was over expressed with a MHC1-restricted TCR, there was no effect on lineage commitment. Since previous results indicated that MHC1 recognition is significantly CD8-dependent, we forced expression of both GATA3 and CD8 transgenes. This manipulation resulted in the re-direction of MHC1-selected thymocytes from the CD8 to the CD4 lineage. Collectively, our data support a model in which the levels of GATA3 in developing T cells serve as a pivotal switch factor controlling CD4/CD8 T lineage commitment.
