MTORC1 promotes T-bet phosphorylation to regulate Th1 differentiation

CD4+ T cells lacking the mTORC1 activator Rheb fail to secrete IFN-g under Th1 polarizing conditions. We hypothesized that this phenotype is due to defects in regulation of the canonical Th1 transcription factor T-bet at the level of protein phosphorylation downstream of mTORC1. To test this hypothesis, we employed targeted mass-spectrometry proteomic analysis-multiple reaction monitoring mass spectrometry. We used this method to detect and quantify predicted phosphopeptides derived from T-bet. By analyzing activated murine wild-type and Rheb-deficient CD4+ T cells, as well as murine CD4+ T cells activated in the presence of rapamycin, a pharmacologic inhibitor of mTORC1, we were able to identify six T-bet phosphorylation sites. Five of these are novel, and four sites are consistently dephosphorylated in both Rheb-deficient CD4+ T cells and T cells treated with rapamycin, suggesting mTORC1 signaling controls their phosphorylation. Alanine mutagenesis of each of the six phosphorylation sites was tested for the ability to impair IFN-g expression. Single phosphorylation site mutants still support induction of IFN-g expression; however, simultaneous mutation of three of the mTORC1-dependent sites results in significantly reduced IFN-g expression. The reduced activity of the triple mutant T-bet is associated with its failure to recruit chromatin remodeling complexes to the Ifng gene promoter. These results establish a novel mechanism by which mTORC1 regulates Th1 differentiation, through control of T-bet phosphorylation

Proteomic Identification and Biological Validation of Novel Breast Cancer MHC II Peptide Vaccine Candidates and Immunosuppressive Mechanisms

Breast cancer is readily treatable at early stages; however few if any treatments exist for patients diagnosed with late stage metastatic disease. Because CD4+ T cells are crucial for long term immunologic memory and may prevent or eliminate dissemination of latent metastases, this thesis is focused on identifying novel immunogenic major histocompatibility class (MHC) II-restricted peptides that activate tumor-specific CD4+ T cells. MHC II cell-based vaccines (human breast cancer cells transduced with MHC II and costimulatory molecules) efficiently activate healthy donors' and breast cancer patients' CD4+ T cells, provided vaccines do not express chaperone protein Invariant chain (Ii). Therefore, it was hypothesized that in the absence of Ii, novel immunogenic MHC II-restricted peptides would be presented by tumor cells. To prove this hypothesis, MHC II-restricted peptides from Ii- vaccine cells and Ii+ tumor cells were sequenced using mass spectrometry-based peptidomics. Four hundred and thirty peptides were identified, 92 of which were uniquely presented by Ii- MHC II cell-based vaccines. Seven of these peptides efficiently activated tumor-specific T cells from healthy donors and breast cancer patients. Therefore, these studies established that Ii regulates the peptide repertoire presented by MHC II+ tumor cells and identified peptides that are potential candidates for breast cancer vaccines. Most breast cancer patients are immune suppressed, and therefore may be restricted in their ability to respond to vaccine immunotherapy. Among the most potent mediators of tumor-induced immune suppression are myeloid-derived suppressor cells (MDSC). Heightened inflammation exacerbates MDSC accumulation and immune suppressive potency. To understand the mechanisms by which inflammation regulates MDSC, the proteins and cellular pathways of MDSC induced in less inflammatory conditions (conventional MDSC) and in highly inflammatory conditions (inflammatory MDSC) were compared using mass spectrometry. Pathway analysis and biological experiments revealed that inflammation enhanced MDSC accumulation by making MDSC less susceptible to apoptosis. Collectively, these studies indicate that immunization with the immunogenic MHC II-restricted peptides while targeting inflammatory pathways that cause MDSC resistance to apoptosis may be a promising approach for breast cancer immunotherapy

Proteomic Identification and Biological Validation of Novel Breast Cancer MHC II Peptide Vaccine Candidates and Immunosuppressive Mechanisms

Breast cancer is readily treatable at early stages; however few if any treatments exist for patients diagnosed with late stage metastatic disease. Because CD4+ T cells are crucial for long term immunologic memory and may prevent or eliminate dissemination of latent metastases, this thesis is focused on identifying novel immunogenic major histocompatibility class (MHC) II-restricted peptides that activate tumor-specific CD4+ T cells. MHC II cell-based vaccines (human breast cancer cells transduced with MHC II and costimulatory molecules) efficiently activate healthy donors' and breast cancer patients' CD4+ T cells, provided vaccines do not express chaperone protein Invariant chain (Ii). Therefore, it was hypothesized that in the absence of Ii, novel immunogenic MHC II-restricted peptides would be presented by tumor cells. To prove this hypothesis, MHC II-restricted peptides from Ii- vaccine cells and Ii+ tumor cells were sequenced using mass spectrometry-based peptidomics. Four hundred and thirty peptides were identified, 92 of which were uniquely presented by Ii- MHC II cell-based vaccines. Seven of these peptides efficiently activated tumor-specific T cells from healthy donors and breast cancer patients. Therefore, these studies established that Ii regulates the peptide repertoire presented by MHC II+ tumor cells and identified peptides that are potential candidates for breast cancer vaccines. Most breast cancer patients are immune suppressed, and therefore may be restricted in their ability to respond to vaccine immunotherapy. Among the most potent mediators of tumor-induced immune suppression are myeloid-derived suppressor cells (MDSC). Heightened inflammation exacerbates MDSC accumulation and immune suppressive potency. To understand the mechanisms by which inflammation regulates MDSC, the proteins and cellular pathways of MDSC induced in less inflammatory conditions (conventional MDSC) and in highly inflammatory conditions (inflammatory MDSC) were compared using mass spectrometry. Pathway analysis and biological experiments revealed that inflammation enhanced MDSC accumulation by making MDSC less susceptible to apoptosis. Collectively, these studies indicate that immunization with the immunogenic MHC II-restricted peptides while targeting inflammatory pathways that cause MDSC resistance to apoptosis may be a promising approach for breast cancer immunotherapy