MIC treatment of subcutaneous B16.F10 melanoma induced melanoma-reactive CD8+ T cells and -NK cells <i>in vivo</i>.

<p>Splenocytes were tested for their <i>ex vivo</i> activation upon co-culture with B16.F10 melanoma or EL4 thymoma control cells (n = 5 mice per group). <b>A</b>, <i>Left panel</i>: CD8+ T cells from monobenzone- and MIC-treated mice showed significant TNF-α production upon co-culture with melanoma cells (<i>black bars</i>; p<0.05 and p<0.003 respectively). In contrast, CD8+ T cells from CI-treated mice did not display significant TNF-α production upon melanoma cell co-culture (non significant difference: ns). <i>Right panel</i>: To identify CD8+ T cell activation upon co-culture with immunogenic melanoma cells with high MHC class-I expression, co-cultures with IFN-γ primed B16.F10 cells were included (<i>dashed bars</i>). Under these conditions, CI-treated mice showed significant TNF-α production as compared to untreated mice (p<0.03). The MIC-treated mice showed even more TNF-α production as in the non-IFN-γ-primed stimulation shown in the left panel. Monobenzone-treated and untreated mice did not display this increased T cell activation upon splenocyte co-culture with IFN-γ primed melanoma cells. T cell activation upon splenocyte co-culture with syngeneic EL4 thymoma control cells showed comparable background levels in all groups (white bars). <b>B</b>, <i>Left panel</i>: Only NK cells from MIC-treated mice showed significantly increased TNF-α production upon co-culture with melanoma cells (p<0.008). <i>Right panel</i>: Elevated production of IFN-γ was found in NK cells from CI- and MIC-treated mice in response to co-culture with melanoma cells (p<0.02 and p<0.02 respectively). TNF-α and IFN-γ production by NK cells was comparable in all groups upon co-culture with EL4 control cells. For the statistical analysis of the <i>in vivo</i> tumor growth kinetics of the treatments depicted in this figure, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0010626#pone-0010626-t001" target="_blank">table 1</a> (“Exp. 2”). <b>C</b>, B16.F10 melanoma cells upregulate their surface MHC class-I expression upon IFN-γ exposure. While IFN-γ-unexposed melanoma cells express very low levels of surface MHC class-I (<i>dashed line</i>), priming of these cells with 1000 U/ml IFN-γ restores their surface expression of MHC class-I (<i>black line</i>). Control incubations of IFN-γ-primed melanoma cells with only the IgG2a-detecting secondary antibody were negative (<i>grey line</i>).