Mechanism and control of recombinant murine interleukin-12-induced immunosuppression

Interleukin 12 (IL-12) is an immunomodulatory cytokine with potent antitumor, antiviral and antimicrobial effects. Its activities are attributable to its ability to induce Th1 CD4+ T cell differentiation, CD8 + T cell cytotoxicity and NK cell activation. Through its ability to induce the production of IFN-γ by T and NK cells, IL-12 indirectly activates macrophages and induces the production of nitric oxide. IFN-γ also has a variety of effects on other host cells and, relevant to its antitumor effects, IFN-γ upregulates MHC expression, slows cell proliferation and inhibits tumor angiogenesis. In our efforts to determine the mechanisms underlying rmIL-12 efficacy, we chose to study IL-12 effects during vaccination with irradiated cancer cell vaccines to avoid confounding factors of tumor growth. We identified a transient immunosuppression of antitumor responses associated with rmIL-12 treatment. rmIL-12 given to A/J mice vaccinated with irradiated SCK mammary carcinoma cells engineered to secrete GM-CSF resulted in significantly better protection from tumor challenges 28 days after vaccination but, unexpectedly, severely compromised host protection 14 days after vaccination. Immune suppression was rmIL-12 dose-dependent and manifest as reduced splenic CTL activity, stimulated cytokine release and ability to reject SCK cells. The period of suppression coincided with transiently reduced splenic T cell mitogenic responses to Con A and IL-2, suggesting that they may be causally related. We showed that suppressed mitogenic responses associated with rmIL-12 therapy were not restricted to splenocytes from SCK.GM vaccinated, rmIL-12 treated A/J mice but were also found in rmIL-12 treated, vaccinated and naive mice of multiple strains. Suppression appears to be due to impaired immune effector mechanisms rather than impaired host immunization, as evidenced by the enhanced reaction to immunogens when hosts are challenged later after rmIL-12 administration. We therefore sought to determine the mechanism of this transient immunosuppression and used both DTH (in vivo) and mitogenic (in vitro) responses in C57BL/6 mice immunized with allogeneic HKB cells. Administration of neutralizing antibodies to HKB-vaccinated C57BL/6 mice showed a role for IFN-γ, known to mediate many of IL-12 effects, but not TNFa, a cytokine implicated in IL-12 induced suppression during LCMV infection, and these results were later confirmed in IFN-γ and TNFR knockout mice. Adherent cells from the spleens of rmIL-12 treated mice were identified as the subpopulation associated with suppressed T cell mitogenic and alloproliferative responses. Further investigation revealed an IFN-γ dependent induction of macrophage derived nitric oxide. Reversion of both in vitro and in vivo suppressed responses was possible by using chemical inhibitors to NOS. Further, these inhibitors are capable of preventing suppression of vaccine efficacy measured by tumor cell rejection and reveal rmIL-12 vaccine adjuvant effects

Mechanism and control of recombinant murine interleukin-12-induced immunosuppression

Interleukin 12 (IL-12) is an immunomodulatory cytokine with potent antitumor, antiviral and antimicrobial effects. Its activities are attributable to its ability to induce Th1 CD4+ T cell differentiation, CD8 + T cell cytotoxicity and NK cell activation. Through its ability to induce the production of IFN-γ by T and NK cells, IL-12 indirectly activates macrophages and induces the production of nitric oxide. IFN-γ also has a variety of effects on other host cells and, relevant to its antitumor effects, IFN-γ upregulates MHC expression, slows cell proliferation and inhibits tumor angiogenesis. In our efforts to determine the mechanisms underlying rmIL-12 efficacy, we chose to study IL-12 effects during vaccination with irradiated cancer cell vaccines to avoid confounding factors of tumor growth. We identified a transient immunosuppression of antitumor responses associated with rmIL-12 treatment. rmIL-12 given to A/J mice vaccinated with irradiated SCK mammary carcinoma cells engineered to secrete GM-CSF resulted in significantly better protection from tumor challenges 28 days after vaccination but, unexpectedly, severely compromised host protection 14 days after vaccination. Immune suppression was rmIL-12 dose-dependent and manifest as reduced splenic CTL activity, stimulated cytokine release and ability to reject SCK cells. The period of suppression coincided with transiently reduced splenic T cell mitogenic responses to Con A and IL-2, suggesting that they may be causally related. We showed that suppressed mitogenic responses associated with rmIL-12 therapy were not restricted to splenocytes from SCK.GM vaccinated, rmIL-12 treated A/J mice but were also found in rmIL-12 treated, vaccinated and naive mice of multiple strains. Suppression appears to be due to impaired immune effector mechanisms rather than impaired host immunization, as evidenced by the enhanced reaction to immunogens when hosts are challenged later after rmIL-12 administration. We therefore sought to determine the mechanism of this transient immunosuppression and used both DTH (in vivo) and mitogenic (in vitro) responses in C57BL/6 mice immunized with allogeneic HKB cells. Administration of neutralizing antibodies to HKB-vaccinated C57BL/6 mice showed a role for IFN-γ, known to mediate many of IL-12 effects, but not TNFa, a cytokine implicated in IL-12 induced suppression during LCMV infection, and these results were later confirmed in IFN-γ and TNFR knockout mice. Adherent cells from the spleens of rmIL-12 treated mice were identified as the subpopulation associated with suppressed T cell mitogenic and alloproliferative responses. Further investigation revealed an IFN-γ dependent induction of macrophage derived nitric oxide. Reversion of both in vitro and in vivo suppressed responses was possible by using chemical inhibitors to NOS. Further, these inhibitors are capable of preventing suppression of vaccine efficacy measured by tumor cell rejection and reveal rmIL-12 vaccine adjuvant effects