CD4⁺ T cells provide help to enhance and sustain cytotoxic CD8⁺ T-cell responses. A direct lytic role for this cell population in mouse models further supports the use of tumor-reactive CD4⁺ T cells for cancer immunotherapy. CTLA-4 blockade has been shown to expand antigen-specific cytotoxic CD4⁺ T cells in mouse models. We took advantage of spontaneous immunity to the NY-ESO-1 cancer-testis antigen to investigate quantitative and qualitative changes in antigen-specific CD4⁺ T-cell responses after ipilimumab (anti-CTLA-4 monoclonal antibody) treatment in patients with advanced melanoma. Four patients with NY-ESO-1 seropositive melanoma were chosen upon the availability of suitable blood specimens for characterizing the functions of NY-ESO-1 antigen-specific CD4⁺ T-cell response by enzyme-linked immunospot (ELISPOT), intracellular cytokine staining (ICS), and cytotoxicity assays. Multiple NY-ESO-1 antigen-specific CD4⁺ T-cell responses with T_H1 dominance were induced or enhanced after ipilimumab treatment in peripheral blood in all four patients. NY-ESO-1 antigen–specific CD4⁺ T-cell lines established from all four patients after ipilimumab treatment recognized naturally processed NY-ESO-1 protein in antigen-presenting cells, expressed master transcription factor Eomesodermin (Eomes), and secreted perforin and Granzyme B. Finally, we showed that these NY-ESO-1 antigen-specific CD4⁺ T-cell lines directly lysed autologous melanoma cell lines expressing NY-ESO-1 in an MHC class II restricted manner. Our results show that antigen-specific cytotoxic CD4⁺ T-cell responses are induced after ipilimumab therapy in human cancer patients. Ipilimumab may induce the expression of lytic granules on antigen-specific cytotoxic CD4⁺ T cells via Eomes, revealing a novel consequence of immunologic checkpoint blockade. Cancer Immunol Res; 1(4); 235–44. ©2013 AACR.