Background: HDIL-2 is associated with long-term remissions in stage IV melanoma, but in less than 10% of patients. Combination therapy with HDIL-2 and agents immunomodulating CTLA-4 have been undertaken. We investigated the effect of HDIL-2 on CTLA4 expression on T cell subsets in patients with melanoma. Methods: Peripheral blood was collected at baseline and serially post-treatment at 24, 48, 72, and 96 hours from 6 patients with advanced stage melanoma undergoing HDIL-2. All patients were treatment naïve with regard to anti-CTLA4 therapy. PBMCs were isolated and restimulated ex vivo for 4 hours using PMA/ionomycin. Fluorophore conjugated anti-CTLA4 antibody was added at time of restimulation and incubated with cells for 4 hours. Intracellular and extracellular cytokine staining was then performed and co-signaling molecule expression was quantified via flow cytometry and measured on a continuous scale. Statistical analysis was performed using paired t tests via Prism 6.0e software. Results: HDIL-2 resulted in increased frequencies of T regulatory cells (T_reg; CD25+FoxP3+CD4+) on day 4 of therapy (24% +/- 12% on day 4 compared to 12% +/- 7% at baseline; p = 0.03; 95% CI [1.5,22]). Importantly, CTLA4 expression on T_reg cells was increased on day 4 post-treatment relative to baseline (51% +/- 19% on day 4 compared to 35% +/- 19% at baseline; p = 0.02; 95% CI [2.5,28]). CTLA4 expression was not statistically different within other CD4+ T cell compartments or within the CD8+ T cell compartments. Conclusions: These results demonstrate that HDIL-2 is associated with an increase in the expression of CTLA4 onT_reg cells in patients with advanced melanoma. HDIL-2 effect on the T_reg population provides a potential rationale for the lack of efficacy of HDIL-2 in most patients and implies potential synergy between HDIL-2 and CTLA4 blockade. Together, these findings provide justification for testing the combination of HDIL-2 and anti-CTLA4 therapy in patients with advanced melanoma and that timing of anti-CTLA4 therapy with HDIL2 will be crucial.