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A Thesis submitted to The University of Arizona College of Medicine - Phoenix in partial fulfillment of the requirements for the Degree of Doctor of Medicine. / Recent advances in cancer immunology, highlighted by immune checkpoint inhibitors, have demonstrated that immunotherapy is a viable option in the oncologist’s armamentarium. Despite these advances, many patients are nonresponders. Preliminary studies have suggested that non-responders lack a de-novo anti-tumor antigen immune response that can be unmasked by checkpoint blockade; thus, strategies to induce anti-tumor immune responses are needed. We hypothesized that many tumor associated antigens (Ag) are readily susceptible to immune attack, but only in the context of identifying the tumor antigen epitopes that can reliably initiate an immune response, regardless of individual patient human leukocyte antigen (HLA) haplotype restrictions. We further hypothesized that epitope prediction strategies which seek to identify pan- or highly promiscuous-HLA binding epitopes would reduce the number of potential candidates and be more likely to accurately identify high-priority tumor Ag epitopes. Utilizing known HLA-serotype frequencies and setting a threshold of ninety percent of population coverage, regardless of race or ethnicity, twenty-nine different HLA-DRB1 haplotypes were chosen for antigen prediction utilizing the open source epitope prediction algorithm netMHCIIpan. Predictions were also performed for HLA-A serotypes utilizing the open source algorithm netMHCpan. Predicted epitopes were synthesized in the form of synthetic long peptides and tested in immune system sensitization assays involving unfractionated peripheral blood mononuclear cells (PBMC). Briefly, PBMC were subjected to a two-step culture, first synchronizing their exposure to the long peptides with aggressive surrogate activation of innate immunity, followed by IL-7-modulated T-cell hyperexpansion. Predictions resulted in identification of highly promiscuous-HLA binding epitopes. Unexpectedly, these epitopes clustered together forming high priority regions: unique “hot spots” with high densities of promiscuous HLA-binding epitopes from the widely expressed oncoproteins MUC1, HER2/neu and CMV-pp65 (p<0.0001, for predicted HLA-DRB1 binding affinities, compared to non-hot spot regions). Added synthetic long peptides (>20aa) derived from “hot spot” regions of MUC1, HER2/neu, and CMVpp65 reliably produced selective and sustained expansion of both CD4+ and CD8+ peptide-specific, interferon-γ (IFNγ)-producing Tcells when synchronized with step 2 exposure to exogenous IL-7 (p<0.0001 and p=0.0048, for CD4+ and CD8+ Ag-specific T-cells, respectively, compared to T-cells directed against peptides from non-hot spot regions). “Hot spot” peptide Ag-specific T-cells preferentially recognized endogenous tumor derived MUC1, either in MUC1 expressing tumor cell killing assays (p=0.038, compared to non-peptide Ag-specific T-cells) or as MUC1 tumor lysate when pulsed onto restimulatory PBMC (p=0.022 and 0.025, for CD4+ and CD8+ T-cells, respectively, compared to T-cells directed against peptides from non-hot spot regions). This mechanistically rational antigen selection sequence, effective even for unvaccinated donors, regardless of HLA-haplotype, enables rapid identification of tumor protein regions relevant for cancer immunology, including adoptive immunotherapy, vaccines, and even identification of tumor neo-antigens unique to each patient.

The prevailing view of signal transduction is that it proceeds through the linear relay of information via sequential bimolecular interactions, involving, for example, Src homology (SH) 2 domains. It has been assumed that such interactions are highly selective, i.e. that the affinities of these interactions are several orders of magnitude higher than that for non-specific interactions. However, recent studies have suggested that the difference in affinities between so-called specific and non-specific interactions is not sufficient to support such a proposal. This therefore raises the question of how signalling pathway specificity is generated at all. To address this, we have taken a systems approach by expressing and purifying >90% of the SH2 domains identified in a T cell line using a next-generation sequencing-based transcriptomic analysis, and performed a systematic survey of the interaction of these SH2 domains with a set of potential phosphorylated peptides derived from the key signalling receptors of the T cell (including CD28, CTLA-4, PD-1, ICOS, BTLA, LAT and the CD3 subunits of the TCR complex), using surface plasmon resonance-based binding assays. Our results show that, instead of being highly selective for certain SH2 domains, the T cell-expressed receptors are very cross-reactive, such that each receptor is found to interact with ~50 different SH2 domains on average. In silico analysis based on these results confirms the expectation that affinity itself is not the sole determining factor for receptor specificity. Further exploration of the system using in silico simulations incorporating the absolute concentrations of SH2 domain-containing proteins measured in T cells using a proteomics-based approach, suggests instead that the specificity of SH2 domain recruitment by T-cell receptors is the result of systems effects, with expression levels of the signalling proteins being a major factor. Surprisingly, LCK, the most highly expressed SH2 domain in resting Jurkat, is predicted to dominate the binding of most receptors, suggesting a novel mechanism of Src kinase activation and function.

This thesis will concern itself with the development of the religious thought of Eliot as it is expressed in his poetry and plays.

T-cell activation is a complex process crucial for a proper function of immune system. It has been extensively studied and its main features are well understood. However, some of the events involved in T-cell signalling are still unclear. After T-cell receptor stimulation, Src-family kinase Lck drives the initiation of signalling by tyrosine phosphorylation. Phosphorylation of several downstream targets is dependent on the redistribution of Lck to the different compartment of the plasma membrane, called lipid rafts. In lipid rafts, active Lck is juxtaposed and activates raft-resident substrates which then trigger downstream signalling. The critical in this process is the mechanism of Lck translocation to lipid rafts which has not been studied so far and represents the topic of great academic and clinical interests. Previously, we identified the adaptor protein RACK1 as a candidate protein mediating the redistribution of Lck to lipid rafts by linking it to the microtubular network. In this thesis, we analysed the structural features and functional role of RACK1 in its interaction with Lck. We show here, using the SYF cell lines expressing the wild type and various mutated forms of Lck, that intact SH3 or SH2 domains of Lck are required for an effective RACK1-Lck complex formation. We also documented...