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Antigen presentation to T cells results in their activation through T Cell Receptor (TCR) stimulation, resulting in sustained elevation of cytosolic Ca2+ concentration critical for T cell activation. Sustained Ca2+ signals are important for the activation of Nuclear Factor of Activated T cells (NFAT), which is a key regulator of T cell activation through its transcriptional control of genes in multiple process including cytokine production, proliferation and differentiation(Rao, Luo, & Hogan, 1997). Recently it was shown that Stromal Interaction Molecule 1 (STIM1) inhibits plasma membrane Ca2+/ATPase 4 (PMCA4) function during T cell activation resulting in sustained elevation of Ca2+ signals(Ritchie, Samakai, & Soboloff, 2012). This interaction requires upregulation of both STIM1 and PMCA4. In this thesis, I hypothesize that changes in Ca2+ signals arising from transcriptional changes of STIM1 and PMCA are important for the efficient activation of T cells. In the first part of this thesis, I assess the transcriptional regulation of STIM1 and PMCA4. My in vitro studies show that expression of both proteins is regulated by the EGR family members, EGR1 and EGR4. Additionally, transcriptional regulation of PMCA inhibition by EGR1 and EGR4 is required for efficient activation of T cells. Interestingly, whereas significant roles for EGR1, EGR2 and EGR3 in T cell development and function have been established, a role for EGR4 has not, hitherto been elucidated. In the second half of this thesis, using qPCR, I reveal that EGR4 expression is stimulated by TCR engagement in primary double positive, CD4 and CD8 positive murine T cells. Further, EGR4-null mice exhibit shifts in early thymic development, although this does not affect the relative number of double or single positive T cells in the thymus. Interestingly, EGR4-null primary T cells exhibit normal Ca2+ entry, but fail to exhibit activation-induced inhibition of Ca2+ clearance. Although not all subsets of EGR1 and EGR4 null primary T cells exhibited decreased STIM1 expression, significant defects in proliferation, migration and/or cytokine production were observed upon stimulation in all populations, albeit to different extents. These findings reveal a two-faceted role in which EGRs regulate T cell development and function through both Ca2+-dependent and independent methods. I believe that these findings have important implications towards the general understanding of transcriptional control of Ca2+ signaling, as well as having a possible impact in the quest to advance therapies targeting immunological disorders. / Biochemistry

CAR-T cell immunotherapy is a highly efficacious treatment for CD19-positive hematological malignancies, however, some patients are non-responsive for reasons that are not well understood. Clinical efficacy has been correlated with long-term persistence, a propensity that can be predicted by the differentiation state of transplanted cells. Despite this, decades-old methods for expanding T cells have not been updated to prevent the deleterious effects of excessive differentiation in CAR-T cells. Uncoupling proliferation and differentiation is a long-held goal in the field of immunotherapy with both cytokines and pharmacological approaches being implemented to dissociate these parallel processes. Histone methyltransferases rewire transcriptional programs in T cells and simultaneously regulate multitudes of genes, making them attractive targets for modifying the proliferation-differentiation axis. Despite this, only a handful of studies have examined their role in regulating the transcriptional programs of human CD8+ T cells. MLL4 (encoded by KMT2B) belongs to the six-member group of MLL histone methyltransferases. MLL1, a paralog of MLL4, has been implicated in regulating the maintenance of IL-4 and GATA-3 expression in TH2 CD4 memory T cell populations, however the function of MLL4 in human CD8+ T cells is unknown. We report a critical role for MLL4 in the proliferation and differentiation of CD8+ T cells. CRISPR-Cas9-editing of MLL4 uncoupled the processes of proliferation and differentiation, increasing proliferation but maintaining central memory T cell (TCM)-like populations, allowing for the production of increased numbers of TCM-like CD62L+CD45RO+ cells. Pharmacologically inhibiting the MLL4-Menin complex with MI-2 during T cell expansion enriched the frequency of minimally differentiated TCM-like CD8+ T cells. TCM-associated CD62L, CCR7, CD122 and CD127 surface markers were upregulated and early memory-associated transcription factor TCF7, LEF1, EOMES, and FOXP1 transcripts were increased. CD8+ CAR-T cells expanded in the presence of MI-2 responded earlier, while improving both tumor burden and survival in a NSG xenograft model of human leukemia. This finding has important translational impact in improving the persistence and proliferative capacity of CD8+ CAR-T cells. / Infectious Disease & Immunity