Summary Project 2 Immune Checkpoint blockade (CPB) has revolutionized the care for many cancers, but clinical trials using anti- PD-1 ICB have not had significant efficacy in GBM. GBM has a profoundly immunosuppressive tumor microenvironment (TME) promoted by immunosuppressive cell types, pathways and mediators. Thus, it is not surprising that single CPB did not meet therapeutic expectations. The goal of Project 2 is to improve patient outcomes following anti-PD-1/ CTLA-4 therapy in GBM by identifying rational combination therapy approaches. We hypothesize that combining therapies to improve T cell priming and activation with anti-PD-1/ CTLA-4 therapy will significantly increase response rates in GBM, converting these immunologically “cold” tumors into “hot” tumors. Based on our preliminary data showing that selective loss of PD-1 at different stages of T cell differentiation can either promote or antagonize effector and memory differentiation, we further hypothesize that the timing of PD-1/ CTLA-4 blockade in combinatorial regimens may critically influence protective anti-tumor T cell responses. Project 2 will test these hypotheses using three complementary approaches that evaluate timing of PD-1 blockade combined with vaccination, oncolytic virus vaccine therapy, or targeted therapies in the following aims: 1- Determine the effect of peptide vaccination combined with anti-PD-1/ CTLA-4 therapy on the establishment of anti-tumor immune responses and development of immunological memory; 2- Test if addition of an oncolytic virus boost to GBM cell vaccine further increases sensitivity to anti-PD- 1/ CTLA-4; and 3- Evaluate the ability of small molecule CDK4/6 targeted therapies to enhance therapeutic benefit of anti-PD-1/ CTLA-4 in GBM models. Interactions with all Projects and utilization of all Cores are described in the Proposal. Our findings will provide optimal ways to combine therapies and insights into the immunosuppressive GBM microenvironment. These data will directly inform the rationale design of combination therapies for future clinical trials for GBM patients.