Immune checkpoint blocking antibodies are currently being tested in ovarian cancer (OC) patients and have shown some responses in early clinical trials. However, it remains unclear how human OC cancer cells regulate lymphocyte activation in response to therapy. In this study, we have established and optimised an in vitro tumour-immune co-culture system (TICS), which is specifically designed to quantify the activation of multiple primary human lymphocyte subsets and human cancer cell killing in response to PD-1/L1 blockade. Human OC cell lines and treatment naïve patient ascites show differential effects on lymphocyte activation and respond differently to PD-1 blocking antibody nivolumab in TICS. Using paired OC cell lines established prior to and after chemotherapy relapse, our data reveal that the resistant cells express low levels of HLA and respond poorly to nivolumab, relative to the treatment naïve cells. In accordance, knockdown of IFNγ receptor expression compromises response to nivolumab in the treatment naïve OC cell line, while enhanced HLA expression induced by a DNA methyltransferase inhibitor promotes lymphocyte activation in TICS. Altogether, our results suggest a ‘cross resistance’ model, where the acquired chemotherapy resistance in cancer cells may confer resistance to immune checkpoint blockade therapy through down-regulation of antigen presentation machinery. As such, agents that can restore HLA expression may be a suitable combination partner for immunotherapy in chemotherapy-relapsed human ovarian cancer patients.