During FY 2013, the TD section has focused on two major projects with the goal of elucidating mechanisms mediating activation, differentiation, and lineage commitment for specialized immune functions in thymocytes and peripheral T cells. In one project, we discovered that the transcription factor GATA3 is a key regulator of TCR signaling;facilitating thymocyte selection, CD4/CD8 lineage commitment, and peripheral T cell activation. Although previous studies demonstrated that GATA3 is required for the generation of CD4 T cells, the authors concluded that this factor functions in CD4 T cell maturation after selection and lineage commitment. To the contrary, we find that GATA3 promotes positive selection and TCR signal transduction in pre- and post-selected thymocytes. Our findings taken with those of others led us to propose that TCR signaling and GATA3 operate in a positive reinforcing loop with each amplifying the activity of the other. This model explains the preferential loss of CD4 T cells with GATA3-deficiency since MHC2 generates stronger/sustained TCR signals (and thus more GATA3) than MHC1 in thymocytes undergoing selection. We observed that decreasing levels of GATA3 can also re-direct MHC2-selected thymocytes from the CD4 to the CD8 T lineage. Collectively, these findings indicate that GATA3 participates both in TCR signaling during positive selection and in CD4/CD8 lineage commitment. We are exploring the possibility that high levels of GATA3 negatively regulate CD8 T cell development. Indeed, overexpression of GATA3 can re-direct MHC1-selected thymocytes from the CD8 to the CD4 T lineage. This was the case only with forced expression of both CD8 and GATA3 transgenes, consistent with our observation that the TCR requires continued CD8 expression for MHC1 recognition. These data suggested that the proposed TCR-GATA3 loop may regulate the expression of lineage specifying factors, ThPOK (for the CD4 lineage) and Runx3 (for the CD8 lineage). Although both factors are induced after the initiation of GATA3 upregulation by positive selection, we discovered that Runx3 was expressed at an earlier stage than previously reported and prior to ThPOK expression. In vitro, there was a positive correlation of TCR stimulation with GATA3 and Runx3 expression;however, at the highest levels of stimulation, GATA3 expression continued to rise while Runx3 expression was suppressed. This result suggested that TCR signaling may independently regulate the expression of both factors and/or high levels of GATA3 in response to strong TCR signals suppress Runx3 expression. The latter is consistent with our in vivo finding of enhanced Runx3 expression in MHC2-selected thymocyte precursors re-directed to the CD8 lineage as a consequence of GATA3-deficiency. Collectively, these data suggest a link between TCR signal strength, GATA3, and factors regulating the CD4/CD8 cell fate decision in which GATA3 could be the critical switch factor. Even though there is a greater impact on CD4 T cell development with GATA3-deficiency, we find this deficiency also attenuates maturation and TCR signal transduction of MHC1-selected CD8 thymocytes. We considered the possibility that GATA3 and TCR signaling might regulate each other also in mature T cells. We produced mice with Gata3 gene deletion targeted to peripheral T cells so that thymocyte development proceeded unperturbed. CD4 and CD8 T cells appeared in lymphoid organs with normal frequency and TCR expression;nevertheless, both T cell subsets exhibited defects in calcium mobilization and ERK activation in response to TCR stimulation. These results demonstrated that GATA3 facilitates TCR signaling in developing thymocytes and peripheral T cells of both CD4 and CD8 T lineages. The effects on TCR signal transduction suggest that GATA3 may operate at earlier stages of CD4 T helper (Th) differentiation than previously reported and warrant an investigation of GATA3 also in CD8 effector functions. In a second project, we continued to focus on the question of how Notch functions in the activation and differentiation of mature T cells. In this effort the lab has created a number of experimental tools for investigating Notch function in vitro and in vivo, including several unique mouse strains for manipulating Notch signaling exclusively in peripheral T cells, a mouse strain with Notch ligand (DLL4)-deficiency targeted to dendritic cells (DC), and antigen presenting cell (APC) lines engineered to express Notch ligands in combination with various co-stimulatory receptors that can be used to investigate Notch signaling in normal T cells. Since our previous studies revealed that fewer Th2 lineage cells were generated in vitro when T cells were deprived of Notch signals, we initiated a collaboration with T. Wynn (NIAID/LPD) to determine whether Notch signaling in T cells influences responses to the helminth parasite, S. mansoni, a strong inducer of Th2-dominated immune responses. When Notch signaling was abrogated in T cells;IL-4, IL-13, granuloma formation, fibrosis, and serum IgE were all diminished in response to acute challenge with egg antigen, exposure to intact eggs, or infection with live parasites. Notably, Th2 responses were diminished in three distinct lines of mice with Notch signaling-deficient T cells and in a line with Notch ligand DLL4-deficeint DC. These data demonstrated that Notch signaling in T cells plays an important role in regulating Th2-mediated pathology in vivo, including inflammation and fibrosis. Since it is difficult to dissect mechanisms underlying Notch function in complex in vivo responses, we designed a reductionist in vitro assay with T cells responding to antigen on APC in which Notch ligands could be manipulated on the APC or Notch signaling, in the T cells. These studies provided compelling evidence that Notch, together with signal 1 from the TCR and signal 2 provided by CD28, is a critical regulator of primary immune responses by influencing the initial stages of CD4 T cell activation and IL2 secretion. We reasoned if Notch is a costimulator of CD28-mediated costimulation, then DLL4 should enhance signaling pathways downstream of CD28. We found that the effects of DLL4/Notch did not influence the level of CD28 expression but were predicated on CD80 (CD28 ligand)-dependent phosphoinositide3-kinase (PI3K) signaling. In the absence of Notch signaling components, antigen, CD80, or PI3K activity;DLL4 had minimal effect on any of the parameters measured. DLL4 induced activation and metabolic changes also in IL2-deficient T cells, indicating that the dependence on CD28 was independent of IL2 secretion. By determining which pharmacological inhibitors could reverse the effects of DLL4, we excluded any role for the p38 MAPK pathway and established that inhibitors of PI3K, Akt, or mTOR negated DLL4 effects. Using a combination of traditional and image flow cytometry, we demonstrated that DLL4 enhances expression and activity of numerous PI3K-dependent genes. Notch signaling increased the frequency of T cells induced to express NFATc1 and translocate it to the nucleus, as well as the level of NFATc1, phospho-inactivated GSK3b, and IL-2 expression on a per cell basis. Although many aspects of Notch-induced changes we observe are consistent with a traditional view of Notch signaling, our data challenge the dogma that the role of Notch is to deliver an instructive signal that directs cell fate. Instead they suggest that Notch potentiates antigen-specific signals downstream of TCR/CD28 and thereby re-enforce antigen-specific responses in T cells. The discovery that Notch signaling can augment T cell activation, metabolism, and IL-2 secretion helps to reconcile a plethora of seemingly conflicting data regarding the role of Notch in CD4 T cells.
|Fowlkes, B J; Edison, Linette; Mathieson, Bonnie J et al. (2009) Early T lymphocytes. Differentiation in vivo of adult intrathymic precursor cells. 1985. J Immunol 183:5-25|
|Wang, Lie; Wildt, Kathryn F; Zhu, Jinfang et al. (2008) Distinct functions for the transcription factors GATA-3 and ThPOK during intrathymic differentiation of CD4(+) T cells. Nat Immunol 9:1122-30|
|Laky, Karen; Fowlkes, B J (2008) Notch signaling in CD4 and CD8 T cell development. Curr Opin Immunol 20:197-202|