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 SP (CD4+CD8-, single positive) thymocytes were inefficiently generated in PS-deficient thymocytes with a polyclonal TCR repertoire and severely reduced with MHC2-restricted AND or 5C.C7 TCR transgenes. Both pre-selected and selected PS-deficient DP (CD4+CD8+) thymocytes showed impaired responses to TCR stimulation in vitro, while Notch gain-of-function mutant thymocytes mediated enhanced responses. CD4 SP 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 the positive selection, promoting the generation of mature CD4 T cells. Similar to Notch, functions of the zinc finger transcription factor, Gata3, are linked to TCR signaling and thymocyte development. Expression of Notch1 and Gata3 are similarly developmentally regulated in thymocytes. Also, similar to a PS-deficiency, conditional deletion of the Gata3 gene inhibits CD4 SP thymocyte development;however, the block is much more severe. Up-regulation of TCR and CD69 expression is defective in PS- and Gata3-deficient DP thymocytes, and these functions are reciprocally increased in Notch1 and Gata3 gain-of-function mutant thymocytes. Collectively, these results indicate that both Notch and Gata3 promote TCR signaling and positive selection in vivo. We find, in addition, that CD69 up-regulation, calcium mobilization, and pErk activation in response to in vitro TCR stimulation is attenuated when DP thymocytes are deprived of Presenilin or Gata3. Responses are decreased also in pre-selected DP thymocytes, demonstrating that this impairment is not a consequence of defective thymic selection. While it was reported that TCR signaling can regulate Gata3 expression in DP thymocytes (Immunity 19:863), our results indicate that the reverse is also true;that is, Gata3 promotes TCR signaling in these cells. Given that impairment of TCR signaling is evident even in pre-selected thymocytes, these results support the existence of a reinforcing loop in which GATA3 promotes TCR signaling as well as to be regulated by TCR signaling in thymocyte selection. Thus, we propose that Gata3 and TCR up-regulate each other during positive selection, accounting for the high levels of Gata3 and TCR in CD4hiCD8lo and CD4 SP thymocyte subsets. In light of the similar phenotypes of Notch and Gata3 mutant thymocytes, we considered a report showing that Notch binds to the Gata3 locus and regulates Gata3 expression and function in peripheral CD4 T helper 2 (Th2) differentiation (Immunity 27:100, 2007). Collectively, these observations suggest coordinate or interactive roles for Notch and Gata3 in TCR signaling and CD4 T cell development, raising the question, does Notch facilitate TCR signaling in positive selection (and CD4 T cell development) via regulation of Gata3;or alternatively, do Notch and Gata3 independently promote these processes? To address whether Notch regulates Gata3 in DP thymocytes, we used both Notch and Gata3 loss- and gain-of-function mutant mice. Expression of these mutations with a single TCR allowed functional analyses of developing thymocytes in the presence or absence of selecting MHC. We characterized: 1) GATA3 expression;2) TCR activation in vitro by Ca flux, CD69 induction, and pErk;and 3) in vivo indicators of positive selection, TCR expression and CD69 up-regulation;as well as CD4/CD8 SP development. Supporting the hypothesis that Notch signaling promotes Gata3 expression in DP thymocytes, Gata3 protein is reciprocally under and over expressed in Notch loss- and gain-of-function DP thymocytes, respectively, undergoing positive selection, Moreover, enforced Gata3 expression rescues in vivo indicators of positive selection in Presenilin-deficient DP thymocytes;and significantly, CD4 SP development. These data support a model in which Notch influences thymocyte selection and maturation by promoting Gata3 expression/function. Previous reports suggested that Notch signaling controls Th1/Th2 differentiation of peripheral CD4 T cells, although the mechanism responsible for this regulation is controversial. We investigated this question in collaboration with W. Paul's lab, using a mouse model bearing a single 5C.C7 TCR with deletion of Presenilin genes targeted to peripheral T cells. We also generated mouse lines with the same TCR, targeting deletion of Pofut1 (an enzyme essential for surface expression of Notch receptors) or CSL (a DNA-binding protein regulating transcription of Notch target genes). Similarity in phenotypes of these three mutant mouse strains could corroborate that the observed defects were a consequence of impaired Notch signaling as well as to distinguish CSL-dependent vs. -independent mechanisms. In this study, naive T cells were stimulated with low doses of specific peptide presented by antigen presenting cells (APC) under non-polarizing conditions, inducing Th2 differentiation . All three Notch mutants proliferated poorly in this system and failed to secrete IL-4 in response to peptide. Defects in IL-4 production could be rescued by addition of exogenous IL-2, suggesting IL-2 to be the major defect early in the response. This decrease in IL-2 production was correlated with less nuclear translocation of NFATc2. Although IL2 secretion was impaired with low dose antigen, high dose antigen induced normal levels of IL2, demonstrating that the effect of Notch mutation was to attenuate rather than ablate IL2 secretion. Also, capture assay revealed that the fraction of CD4 T cells secreting IL2 in response to low dose peptide was diminished when T cells were deprived of Notch signals. Conversely, a gain-of-function experiment showed that the frequency of IL2-producing cells was increased in assays using wild type T cells and APC (P13.9) stably transfected with Notch ligand. Notably, this function was elicited from normal T cells by delivery of an extrinsic Notch signal, making it unlikely that the impaired IL2 responses in Notch loss-of-function mutants were due to pre-existing developmental defects. Collectively, these studies indicate that the primary influence of Notch on Th2 differentiation is to optimize IL-2 production. We addressed also the question of which Notch ligands are important for T cell activation. We observed that the frequency of IL2-secreting cells was higher when naive T cells were stimulated with splenic myeloid dendritic cells (DCs), as compared to lymphoid DCs. Analysis of a panel of surface molecules expressed by DCs revealed that expression of Notch ligand, DLL4 (Delta-like ligand 4) is higher on myeloid than lymphoid DCs, perhaps accounting for the more efficient T cell activation elicited by these DCs. To determine physiological relevance, we generated a mutant mouse strain, targeting deletion of DLL4 to CD11c-expressing dendritic cells. We generated lines expressing this mutation with different haplotypes for in vitro stimulation of two MHC-restricted TCRs and for in vivo experiments. Without DLL4 on DCs, T cell activation and IL2 secretion were markedly impaired. Taken together, our results indicate that IL2 secretion is similarly impaired whether T cells are deprived of Notch signals or APCs are deprived of Notch ligand.
