Induction of CD4+ T and NK cell-mediated antitumoral response by immunization with breast cancer cells transfected with a dominant negative vector of Stat3

TKACH M; CORIA L; ROSEMBLIT C; RIVAS MA; BEGUELIN W; PROIETTI CJ; DIAZ FALQUE MC; CHARREAU EH ; ELIZALDE PV; CASSATARO J; SCHILLACI R

Buenos Aires

Congreso; Primer Congreso Franco-Argentino de Inmunología; 2010

Sociedad Argentina de Inmunología

Based on the evidence that disruption of Stat3 signaling in cancer cells can overcome tumor immune evasion we have designed an immunotherapy which consists in the administration of irradiated breast cancer cells that express a dominant negative (DN) form of Stat3. We have already shown that this immunization provides protection against the murine hormone-dependent C4HD tumor in a prophylactic protocol and also in a therapeutic approach. The aim of this work was to investigate the role of lymphocyte subsets in the prophylactic vaccination model. To achieve this, immunized BALB/c mice were antibody-depleted of CD4+, CD8+, or Natural Killer (NK) cells in vivo before tumor challenge. Depletion of CD4+ T cells or NK cells completely abrogated resistance to tumor challenge induced by immunization with DNStat3 C4HD cells (**p<0,001 and *p<0.01 for depletion of NK cells and CD4 T cells, respectively, in DNStat3 immunized mice versus control IgG). On the other hand, depletion of CD8+ T cells did not affect C4HD tumor rejection. Considering that we have already demonstrated that immunization with DNStat3-transfected cells in nude mice, with increased NK activity, did not protect against C4HD tumor development, these results support the concept that the generation of the antitumor activity observed requires participation of both CD4+ and NK+ cell subsets. As we have already demonstrated that splenocytes isolated from mice injected with DNStat3-transfected C4HD cells were effective in lysing C4HD in vitro, we asked whether NK cells were responsible for that cytotoxic effect. We isolated NK cells from spleens of immunized mice and performed a 4 hs 51Cr release assay against C4HD cells or the NK-sensitive YAC-1 cell line. Mice immunized with DNStat3-transfected C4HD cells showed an increased NK cytotoxicity against YAC-1 and C4HD cells compared to control group (27,5+2,5% vs 5,2+1,9% and 15,7+2,4 vs 5,3+2,7, respectively, 5:1 effector-to-target ratio, p<0,05). In conclusion, our results demonstrate that breast cancer growth can be inhibited through induction of a CD4+-NK cell dependent protective immune response in vivo+, CD8+, or Natural Killer (NK) cells in vivo before tumor challenge. Depletion of CD4+ T cells or NK cells completely abrogated resistance to tumor challenge induced by immunization with DNStat3 C4HD cells (**p<0,001 and *p<0.01 for depletion of NK cells and CD4 T cells, respectively, in DNStat3 immunized mice versus control IgG). On the other hand, depletion of CD8+ T cells did not affect C4HD tumor rejection. Considering that we have already demonstrated that immunization with DNStat3-transfected cells in nude mice, with increased NK activity, did not protect against C4HD tumor development, these results support the concept that the generation of the antitumor activity observed requires participation of both CD4+ and NK+ cell subsets. As we have already demonstrated that splenocytes isolated from mice injected with DNStat3-transfected C4HD cells were effective in lysing C4HD in vitro, we asked whether NK cells were responsible for that cytotoxic effect. We isolated NK cells from spleens of immunized mice and performed a 4 hs 51Cr release assay against C4HD cells or the NK-sensitive YAC-1 cell line. Mice immunized with DNStat3-transfected C4HD cells showed an increased NK cytotoxicity against YAC-1 and C4HD cells compared to control group (27,5+2,5% vs 5,2+1,9% and 15,7+2,4 vs 5,3+2,7, respectively, 5:1 effector-to-target ratio, p<0,05). In conclusion, our results demonstrate that breast cancer growth can be inhibited through induction of a CD4+-NK cell dependent protective immune response in vivobefore tumor challenge. Depletion of CD4+ T cells or NK cells completely abrogated resistance to tumor challenge induced by immunization with DNStat3 C4HD cells (**p<0,001 and *p<0.01 for depletion of NK cells and CD4 T cells, respectively, in DNStat3 immunized mice versus control IgG). On the other hand, depletion of CD8+ T cells did not affect C4HD tumor rejection. Considering that we have already demonstrated that immunization with DNStat3-transfected cells in nude mice, with increased NK activity, did not protect against C4HD tumor development, these results support the concept that the generation of the antitumor activity observed requires participation of both CD4+ and NK+ cell subsets. As we have already demonstrated that splenocytes isolated from mice injected with DNStat3-transfected C4HD cells were effective in lysing C4HD in vitro, we asked whether NK cells were responsible for that cytotoxic effect. We isolated NK cells from spleens of immunized mice and performed a 4 hs 51Cr release assay against C4HD cells or the NK-sensitive YAC-1 cell line. Mice immunized with DNStat3-transfected C4HD cells showed an increased NK cytotoxicity against YAC-1 and C4HD cells compared to control group (27,5+2,5% vs 5,2+1,9% and 15,7+2,4 vs 5,3+2,7, respectively, 5:1 effector-to-target ratio, p<0,05). In conclusion, our results demonstrate that breast cancer growth can be inhibited through induction of a CD4+-NK cell dependent protective immune response in vivo+ and NK+ cell subsets. As we have already demonstrated that splenocytes isolated from mice injected with DNStat3-transfected C4HD cells were effective in lysing C4HD in vitro, we asked whether NK cells were responsible for that cytotoxic effect. We isolated NK cells from spleens of immunized mice and performed a 4 hs 51Cr release assay against C4HD cells or the NK-sensitive YAC-1 cell line. Mice immunized with DNStat3-transfected C4HD cells showed an increased NK cytotoxicity against YAC-1 and C4HD cells compared to control group (27,5+2,5% vs 5,2+1,9% and 15,7+2,4 vs 5,3+2,7, respectively, 5:1 effector-to-target ratio, p<0,05). In conclusion, our results demonstrate that breast cancer growth can be inhibited through induction of a CD4+-NK cell dependent protective immune response in vivoin vitro, we asked whether NK cells were responsible for that cytotoxic effect. We isolated NK cells from spleens of immunized mice and performed a 4 hs 51Cr release assay against C4HD cells or the NK-sensitive YAC-1 cell line. Mice immunized with DNStat3-transfected C4HD cells showed an increased NK cytotoxicity against YAC-1 and C4HD cells compared to control group (27,5+2,5% vs 5,2+1,9% and 15,7+2,4 vs 5,3+2,7, respectively, 5:1 effector-to-target ratio, p<0,05). In conclusion, our results demonstrate that breast cancer growth can be inhibited through induction of a CD4+-NK cell dependent protective immune response in vivo51Cr release assay against C4HD cells or the NK-sensitive YAC-1 cell line. Mice immunized with DNStat3-transfected C4HD cells showed an increased NK cytotoxicity against YAC-1 and C4HD cells compared to control group (27,5+2,5% vs 5,2+1,9% and 15,7+2,4 vs 5,3+2,7, respectively, 5:1 effector-to-target ratio, p<0,05). In conclusion, our results demonstrate that breast cancer growth can be inhibited through induction of a CD4+-NK cell dependent protective immune response in vivo+-NK cell dependent protective immune response in vivo with tumor immunogens derived from DNStat3-transfected breast cancer cells.