Vaccination of patients with ovarian cancer with overlapping long peptides (OLP) from cancer-testis antigen NY-ESO-1 and poly-ICLC in Montanide-ISA-51 (Montanide) was found to consistently induce integrated immune responses (antibody, CD4⁺, and CD8⁺ T cells). Using detailed methods, we investigated the respective effects of poly-ICLC and Montanide adjuvant on pre- and postvaccine NY-ESO-1–specific CD4⁺ T cells, because of their central function for induction and maintenance of both antibody and CD8⁺ T cells. Polyclonal NY-ESO-1–specific CD4⁺ T-cell lines were generated from 12 patients using CD154-based selection of precursors before and after vaccination with (i) OLP alone, (ii) OLP in Montanide, or (iii) OLP and poly-ICLC in Montanide. Kinetics, quantification, fine specificity, avidity, and cytokine-producing pattern were analyzed in depth and compared between vaccine cohorts. Vaccination with OLP alone did not elicit CD4⁺ T-cell responses; it suppressed high-avidity CD4⁺ T-cell precursors that recognized naturally processed NY-ESO-1 protein before vaccination. Emulsification of OLP in Montanide was required for the expansion of high-avidity NY-ESO-1–specific CD4⁺ T-cell precursors. Poly-ICLC significantly enhanced CD4⁺ Th1 responses while suppressing the induction of interleukin (IL)-4–producing Th2 and IL-9–producing Th9 cells. In summary, Montanide and poly-ICLC had distinct and cooperative effects for the induction of NY-ESO-1–specific Th1 cells and integrated immune responses by OLP vaccination. These results support the use of admixing poly-ICLC in Montanide adjuvant to rapidly induce antitumor type I immune responses by OLP from self/tumor antigens in human cancer vaccines. Cancer Immunol Res; 1(5); 340–50. ©2013 AACR.