As with our observation for CD45+ cells, CD8+ T cells, and Tregs, the increase in the proportion of NK cells was not observed in the tumor-draining lymph nodes (in all groups NK cells constituted approximately 2% of CD45+ cells, p? ?0.5 for all comparisons) (Figure?5C) or spleen (data not shown). Open in a separate window Figure 5 Combination IL-2 and CTLA-4 blockade immunotherapy increases the proportion and changes the differentiation of tumor-infiltrating NK cells. either monotherapy alone. The therapeutic effect of combination immunotherapy was dependent on both CD8+ T and NK cells and co-depletion of these subsets (but Gentamycin sulfate (Gentacycol) not either one alone) abrogated the therapeutic effect. CTLA-4 blockade increased immune cell infiltration (including CD8+ T cells and NK cells) in the tumor and IL-2 reduced the proportion of highly differentiated/exhausted tumor-infiltrating NK cells. Conclusions These results have implications for the design of clinical trials in patients with metastatic melanoma and provide new insights into how the immune system may be mediating anti-tumor activity with combination IL-2 and CTLA-4 blockade in melanoma. Electronic supplementary material The online version of this article (doi:10.1186/s40425-015-0063-3) contains supplementary material, which is available to authorized users. with anti-CD45 conjugated to a different fluorophore than the one used for intravenous staining. This permitted the identification of tumor-resident leukocytes by flow cytometry (Figure?2A). CTLA-4 blockade alone and in Gentamycin sulfate (Gentacycol) combination with IL-2 promoted CD45+ immune infiltration (among live cells) compared to the control treatment (mean %CD45+ cells in the tumor: 48% and 28% versus 11%, p? ?0.001 and p? ?0.05, respectively) (Figure?2A,B). No significant difference in immune infiltration was observed between the combination group and the CTLA-4 blockade treatment group (48% versus 28%, respectively, p? ?0.05) or between IL-2 alone and the control group (18% versus 11%, respectively, p? ?0.05) (Figure?2B). Further, to confirm that the combination immunotherapy enhanced immune infiltration, we used epifluorescence microscopy. This demonstrated that IL-2 Gentamycin sulfate (Gentacycol) and CTLA-4 blockade combination immunotherapy results in augmented CD45+ immune cell infiltration within the tumor (1×105 versus 4×104 CD45+ cells/106 nuclei, respectively, p? ?0.05) (Figure?2C). These data demonstrate that CTLA-4 blockade, individually and when combined with IL-2, promotes immune cell infiltration within the tumor microenvironment. Open in a separate window Figure 2 Combination IL-2 and CTLA-4 blockade immunotherapy increases tumor immune infiltration. (A) Flow cytometry plots of tumors dissected at day 14 and analyzed by flow cytometry for CD45 expression from the experiment Gentamycin sulfate (Gentacycol) described in Figure?1A. Only tumor-infiltrating lymphocytes were analyzed (as determined by comparison of intravenous CD45 staining compared to CD45 staining). (B) Cumulative graph showing mean percent CD45+ T cell infiltration (of live cells) in the tumor from three independent experiments described in (A). (C) Representative immunofluorescence microscopy images of CD45 (red) and DAPI (4,6-diamidino-2-phenylindole; blue) staining of tumors from experiment in (A). Scale bars = 20 microns. Three to five mice were included in each group per experiment. *P? ?0.05, ***P? ?0.001, ns?=?not significant. CTLA-4 blockade results in increased CD8+ T cells in the tumor microenvironment CD8+ T cells are vital mediators of anti-tumor responses. Therefore, we characterized the tumor immune infiltrate to determine the effect of combination IL-2 and CTLA-4 blockade on CD8+ T cells. CTLA-4 blockade alone and in combination with IL-2 resulted in an increased proportion of CD8+ T cells among the immune cell infiltrate (CD45+ cells) of the tumor compared to the control group (35% and 19% versus 7%, p? ?0.01 and p? ?0.05, respectively) (Figure?3A,B). No significant difference in immune infiltration was observed between the combination group and the CTLA-4 blockade only treatment group (19% versus 34%, respectively, p? ?0.05) or between IL-2 alone and the control group (6% versus 7%, respectively, p? ?0.05) (Figure?3A, B). These data suggest that CTLA-4 blockade may be the primary driver of CD8+ T cell tumor infiltration after CTLA-4 blockade with or without IL-2 immunotherapy. Open in a separate window Figure 3 Combination IL-2 and CTLA-4 blockade immunotherapy increases the proportion of tumor-infiltrating CD8+ T cells. (A) Flow cytometry Rabbit Polyclonal to BL-CAM (phospho-Tyr807) plots of tumors dissected at day 14 and analyzed for CD3?+?CD8+ T cells (of CD45+ cells within the tumor) by flow cytometry from the experiment described in Figure?1A. (B) Cumulative graph showing the mean percent of CD3?+?CD8+ T cells from experiment in (A). (C) Cumulative graph showing the mean percent of CD3?+?CD8+ T cells in the tumor-draining lymph nodes from experiment in (A). (D) Representative histograms showing expression of cell markers from CD3?+?CD8+ T cells in the tumor and from a representative tumor-draining lymph node (LN). Numbers represent mean fluorescence intensity.