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MUC1, a protein of epithelial and carcinoma cells, has recently been shown on activated T cells where it inhibits CD3-stimulated proliferation. Two immunoregulatory domains similar to ITAM and ITIMs are present on its cytoplasmic tail, suggesting that MUC1 can act as both a costimulatory and coinhibitory molecule of T cells. In my work, I have examined immunoregulatory function of MUC1 on human T cells. We first showed that MUC1, when ligated in a population of unpurified T cells with an anti-CD3 and a crosslinking antibody, enhances proliferative and cytokine responses in a NF-AT-dependent manner by recruiting the AP-1 transcription factor and translocating it into the nucleus. With purified CD3+ T cells, we instead observed inhibition after MUC1/CD3 coligation and crosslinking. Reconstituting with irradiated CD3- cells, we discovered that MUC1 costimulation is dependent on the amount of accessory cells. These data imply a novel role for MUC1 in T cell immunoregulation. / Experimental Surgery

The interrogation of T cell signaling over the past fifty years has led to the discovery of amazingly intricate cascade networks and elaborate descriptions of individual proteins' domains and functions. A complex landscape has been rendered in which proteins relay messages from the extracellular ligation of the TCR by a cognate peptide loaded MHC via changes in sub-cellular location, phosphorylation, and binding affinities and partners to enact nuclear localization of three key transcription factors required for cellular effector function and proliferation: AP-1, NF-AT, and NF-κB. Dogma has favored activation of each of these transcription regulating elements to be a linear and parallel activity, thus very little interaction between pathways has been highlighted by previous findings in the molecular immunology community. The focus of this dissertation explores the role of NF-κB in T cell signaling with emphasis on subunits p50, cRel, IκBα, and IKKβ, and with respect to NF-κB’s ability to modulate calcium and NF-AT signaling, proximal TCR phosphorylation, and CRAC and purinergic calcium channel proteins. The role of NF-κB in T cells can be a difficult thing to establish, as this thirteen member family innervates almost every cellular process from homeostasis to activation, and even functions in the opposing processes of survival and apoptosis. To convolute the investigation further, many family members also fulfill redundant tasks, as a result of their high evolutionarily conserved sequence homology. To this end, we discovered the best way to evaluate the function of NF-κB in the activation of T cells was to knockdown two family members: p50 and cRel. In doing this, we rendered mice that were viable (unlike knockdown of RelA) and fertile, but possessed T cells that were highly unresponsive to strong stimulation (anti CD3/CD28) or foreign antigen (OVA) presented to mice bearing the correct transgenic TCRs (OT-1) by professional antigen presenting cells (APC). Through in vitro assays, we discovered that in addition to the specific defects in NF-κB activation, NF-AT signaling was also greatly disrupted in these cells, sequela to retarded calcium influx and signaling. This was of great interest, as while several studies have shown that calcium signaling has the ability to amplify and fine tune NF-κB activation, there is a dearth of studies and publications highlighting the effect of an activated NF-κB pathway on calcium influx and signaling leading to the activation of NF- AT. Another fascinating discovery, that explicated the calcium reduction and NF-AT inhibition, was that ablation of p50 and cRel led to decreases in mRNA and protein levels of two additional NF-κB family members: IKKβ and IKKγ. The results presented here suggest that it is the reduction in IKKβ and IKKγ that leads to impaired phosphorylation of the key TCR proximal proteins: Zap70 and PLC&gamma1, and it is the decrease in activated PLCγ1 that renders less IP3 and ultimately abrogates calcium signaling. Overall, this thesis highlights the ability of IKKβ to enhance general proximal TCR protein phosphorylation (and specifically Zap70) leading to a greater influx of calcium (perhaps aided by IKKβ also augmenting the function of the CRAC protein, STIM1) which leads to superior activation of NF- AT, and amplifies downstream cellular effector functions such as IL-2 production and proliferation. Moreover, this work demonstrates that NF-κB subunits likely form supermolecular clusters, and ablation of certain subunits (i.e. p50 and cRel) can lead to instability and decreased levels of other family members (i.e. IKKβ and IKKγ.)