Inhibitors of immune checkpoint proteins have shown great promise in melanoma and other tumors, but response rates to antibodies against CTLA-4 and PD-1 have been modest, with no clear indication of what pre-treatment or pharmacodynamic biomarkers are associated with their efficacy. In work done at our institutions, a number of biomarkers of the efficacy of PD-1 antibody BMS 936558 and CTLA-4 antibody ipilimumab have been identified on T cells. Pre-treatment markers on tumors from patients treated with those antibodies, particularly PD-L1, may also have promise for prediction of outcome with anti-PD-1. Anti-PD-1 can up-regulate expression of CTLA-4 on T cells in vivo and vice-versa, and increased CTLA-4 expression on CD4 T cells appears to be associated with lack of response to PD-1 antibody, suggesting a rational basis for sequencing those two antibodies. In an ongoing NCI RO1- supported trial at Moffitt, P-7997, patients treated with ipilimumab that show progression of disease may subsequently respond to PD-1 antibody, and at both institutions, PD-1 antibody failures have been shown to respond to ipilimumab, so the two antibodies may be therapeutically non-cross resistant. In order to study biomarkers on PBMC and within the tumor and assess if they are associated with anti- tumor activity with sequential therapy we will perform a two-center phase II randomized study of PD-1 antibody BMS 936558 followed by CTLA-4 antibody ipilimumab or vice versa. In the first aim of the proposal, we will collect peripheral blood by leukapheresis prior to, after the first cycle of eithr ipilimumab or PD-1 antibody, and after the second cycle of treatment and measure a variety of markers on and in circulating T cells prior to and after the reciprocally sequenced regimens of the two antibodies based on prior work in the PI's laboratory at Moffitt. In order to explore what factors in T cells infiltrating the tumor that are targeted by these antibodies or within the tumormicroenvironment which may be important biomarkers for their efficacy, we will analyze tumor PD-L1 expression, calculate an "immunoscore" of infiltrating T cells as well as phenotypically characterize those cells within the tumor prior to treatment in the work of the second aim, based on work performed at Dana Farber. In the third aim we will then attempt to determine what factors in the tumor microenvironment and on the host effector T cells are associated with clinical outcome with the combined antibody therapy in either arm. If successful, this proposal will establish the tolerability and potential efficacy of a new sequential immunotherapy regimen and suggest novel predictive and pharmacodynamic biomarkers to be validated in larger phase III trials of the combination and allow a more rational selection of patents for treatment with these drugs.