Characterization and Blockade of Tumor-Mediated Immunosuppression

The war on cancer was initiated in order to improve prevention, detection, and treatment of malignancies. One weapon available in this fight is the immune system. Immunotherapy of cancer originated in the 1890s when William Coley injected dead bacteria into tumors with dramatic results. Since then, much progress has been made in our knowledge of the immune system's ability to detect and destroy cancers. Tumors have a remarkable capability to repurpose normal cellular proteins and signaling pathways to promote tumor growth and survival. In particular, cancers evolve mechanisms that result in tumor immune evasion. This dissertation work has sought to further our understanding of tumor-derived immunosuppressive proteins and to develop strategies to overcome them and achieve effective anti-tumor immunity.;Many clinical trials have been conducted using cancer vaccines to induce immunity to poorly immunogenic tumors, but they have generally met with limited success despite detection of tumor-specific cytotoxic T lymphocytes (CTLs) both in the peripheral blood and tumors of treated patients. One reason for poor clinical outcomes is the direct suppression of anti-tumor immunity by tumors. Although tumor-specific CTLs are generated, their cytotoxic function is often inhibited in the tumor microenvironment so that they are unable to effectively destroy tumor cells. Previous studies in our laboratory found that conditioned supernatant from AGN2a neuroblastoma cells is highly immunosuppressive and likely contributes to the immunosuppressive tumor microenvironment in vivo. In aim 1, we sought to identify and characterize the factor responsible for this immunosuppression. The suppressive activity was not due to a soluble protein as we originally assumed, but rather to a membrane-associated protein present in the supernatant within tumor-derived membrane particles. Candidate proteins were identified by proteomic analysis.;The second aim of this dissertation work was to investigate the use of monoclonal antibodies to block tumor-mediated immunosuppression. Recent phase I clinical trials have used reagents to target the programmed death receptor-1/ligand-1 (PD-1/PD-L1) pathway with very promising results. This pathway is involved in many cancers and its blockade could have broad clinical applications in oncology. In a murine model of multiple myeloma that highly expresses PD-L1, we found that T cells from myelomainfiltrated organs showed increased PD-1 expression. However, expression of PD-1 was not increased on non-tumor-specific T cells in myeloma-bearing mice suggesting that blockade of the pathway could specifically target tumor-reactive immune cells. Prior studies in our lab and elsewhere demonstrated an effect of PD-1/PD-L1 blockade in myeloma only when active immunotherapies, such as cancer vaccines, were concurrently administered. In contrast, we recently found that anti-PD-L1 therapy alone rejected established tumor when administered to lymphodepleted mice. Mice given either myeloablative or non-myeloablative radiation in conjunction with anti-PD-L1 therapy had ∼50% or greater survival while mice treated with control antibody or no radiation almost universally succumbed to tumor progression. Effective rejection of myeloma induced by lymphodepletion and PD-L1 antibody treatment was dependent on the presence of CD4 and CD8 T cells, but not NK cells. Unexpectedly, tumor cells were eliminated relatively quickly following anti-PD-L1 therapy suggesting that radioresistant pre-activated T cells are responsible for the anti-myeloma immune responses, and not newly-generated tumor-reactive T cells. Anti-PD-L1 treatment plus lymphodepletion did not improve survival in two solid tumor models, suggesting that hematologic malignancies may be more susceptible to this combined treatment.;In summary, these studies improve our understanding of tumor-mediated immunosuppression. Results from aim 1 show that immune suppressive activity in culture supernatants from a neuroblastoma cell line are due to the production of suppressive microparticles. Aim 2 supports the clinical testing of lymphodepletion and PD-1/PD-L1 blockade as a novel combinatorial immunotherapeutic approach for improving the survival of patients with multiple myeloma.