Background: Patients (pts) with regionally advanced melanoma were treated with neoadjuvant ipilimumab (ipi) (previously reported: Tarhini. PLOS One 2014). Significant changes in circulating Treg, MDSC and peptide specific type I CD4+ and CD8+ T cells were observed at 6 weeks that correlated with clinical outcome. Characterization of effector T cell secreted cytokines may shed insights into ipi associated T cell activation and function. Methods: Pts were treated with ipi (10mg/kg IV q3 weeks x2) bracketing surgery. Blood specimens were collected throughout treatment. Samples from baseline and week 6 were used for this analysis. Each sample was divided into 5 groups and stimulated with shared melanoma antigen overlapping peptide pools (NY-ESO 1, gp-100, MART-1), and 2 controls. Secreted cytokines, chemokines and growth factors were assessed using Luminex and flow cytometry. We used the Wilcox rank-sum test to compare the cytokine expression levels between the 3 antigen groups. Results: At baseline, 17 cytokines were differentially expressed with stimulation by each antigen (p-value < 0.05): IL1β, MIP1β, IL1RA, VEGF, IL13, IL17, MIP1α, GM-CSF, MCP1, IL5, IL2R, IL4, IL10, IFNγ, TNFa, IL8 and IL2. At week 6 (after ipi), 15 cytokines were differentially expressed (p < 0.05): IL1β, VEGF, G-CSF, HGF, IL13, IL17, GM-CSF, MCP1, IL5, IL7, IL4, IL10, IFNγ, IL8 and IL2. Cytokine expression levels at baseline and at week 6 were used to cluster pts and recurrence free survival (RFS) was compared. We observed clear differences in RFS based on cytokine level clustering both at baseline (p = NS) and at week 6 (p = 0.07), more notably in the NY-ESO-1 stimulated subgroup. Conclusions: PBMCs of pts treated with ipi had more detectable T cells specific to NY-ESO-1 than to gp-100 or MART-1. Cytokines differentially expressed in the supernatants of PBMCs exposed to these antigens belong to a broad functional range, including inflammatory, type 1, type 2 and regulatory, that warrant further study. Patients whose PBMCs secreted more cytokines (more notably in response to NY-ESO-1) tended to have better RFS, supporting further exploration in terms of therapeutic predictive value.