4ACB, two tailed unpaired test, p=0.7863 and 0.5525, respectively). individual window Physique 2 Vemurafenib does not significantly effect KU-55933 recovery of activated transgenic T cells. B10.D2 BRAF WT mice were immunized and Thy1.1+ Clone 1 cells and CD45.1+ SFE T cells were injected via the intravenous route on day 1. Mice were treated with 200mg/kg vemurafenib in 2% aqueous Klucel LF or vehicle control daily via gavage for 5 days. Following stimulation with appropriate peptide, splenic lymphocytes were analyzed by FACS on day 6. Mice not receiving stimulation with peptide had significantly fewer T cells recovered on FACS analysis (data not shown). Data are representative of 4 impartial experiments with 3C5 mice per group. 2A, no significant difference in % CD8+Thy1.1+ splenic lymphocytes recovered on day 6 in mice treated with vemurafenib or control (two tailed unpaired test, p=0.4535). 2B, no significant difference in %CD4+CD45.1+ splenic lymphocytes recovered on day 6 in mice treated with vemurafenib or control (two tailed unpaired test, p=0.7535). We further tested the activated T cells for cytokine production by intracellular staining on day 6. No significant difference was noted in the percent KU-55933 of Clone 1 CD8+ cells of treated and non-treated mice producing IFN-gamma, IL2, or TNF-alpha (Fig. 3ACC, two tailed unpaired test, p=1.00, 0.4155, and 0.4257, respectively). Similarly, no significant difference was noted in the percent of SFE CD4+ cells producing IFN gamma and IL2 (Fig. 4ACB, two tailed unpaired test, p=0.7863 and 0.5525, respectively). KU-55933 Collectively, these data demonstrate that treatment with vemurafenib does not affect antigen specific immune responses. Open in a separate window Physique 3 No significant effect of vemurafenib on cytokine production by of activated transgenic CD8+ T cells. B10.D2 BRAF WT mice were immunized and Thy1.1+ Clone 1 cells and CD45.1+ SFE T cells were injected on day 1. Mice were treated with 200mg/kg vemurafenib in 2% aqueous Klucel LF or vehicle control daily via gavage for 5 days. Following Rabbit Polyclonal to MYOM1 stimulation with appropriate peptide, cytokine production by isolated splenic lymphocytes was analyzed by FACS on day 6. Data are representative of 4 impartial experiments with 3C5 mice per group. 3A, no significant difference in % of CD8/Thy1.1+ splenic lymphocytes producing IFN- in mice treated with vemurafenib or control (two tailed unpaired test, p=1.00); 3B, no significant difference in % of CD8/Thy1.1+ splenic lymphocytes producing IL2 in mice treated with vemurafenib or control (two tailed unpaired test, p=0.4155); 3C, no significant difference in % of CD8/Thy1.1+ splenic lymphocytes producing TNF- in mice treated with vemurafenib or control (two tailed unpaired test, p=0.4257). Open in a separate window Physique 4 No significant effect of vemurafenib on cytokine production by of activated transgenic CD4+ T cells. B10.D2 BRAF WT mice were immunized and Thy1.1+ Clone 1 cells and CD45.1+ SFE T cells were injected on day 1. Mice were treated with 200mg/kg vemurafenib in 2% aqueous Klucel LF or vehicle control daily via gavage for 5 days. Following stimulation with appropriate peptide, cytokine production by isolated splenic lymphocytes was analyzed by FACS on day 6. Data are representative of 4 impartial experiments with 3C5 mice per group. 4A, no significant difference in % of CD4+/SFE splenic lymphocytes producing IFN- in mice treated with vemurafenib or control (two tailed unpaired test, p=0.7863); 4B, no significant difference in % of CD4+/SFE splenic lymphocytes producing IL2 in mice treated with vemurafenib or control (two tailed unpaired test, p=0.5525). Vemurafenib does not affect the CD4 and CD8 dependent anti-tumor response in Ins-HA tumor bearing mice To determine whether vemurafenib treatment affects CD4 and CD8 T cell effector function, we used a tumor model in which tumor eradiation is dependent on CD4 and CD8 T cells.9 RIP-Tag2 HA mice spontaneously develop insulinoma and carry an endogenous T-cell repertoire which is tolerant for this self-antigen.9 Previous studies have shown that a combination of.