Nitric Oxide Regulation during Anti-tumor Immunotherapy

Abstract: During tumor progression a strong immunosuppression is developed and is believed to be the major reason why immunotherapy fails. This suppression is induced in response to factors produced from tumor cells, but can also be induced after immunotherapy. NO has been shown to play a major role in this suppression both in patients with gliomas and colon carcinomas. The aim of this thesis is to clarify the role of the NO mediated immunosuppression in rats with intra-hepatic colon carcinomas and intra-cerebral gliomas, and to clarify whether inhibition of NO inducing enzyme, iNOS, would diminish this suppression and improve the anti-tumor immunotherapy. In study I-IV we investigated both unspecific and specific iNOS inhibitors, and we could show that the specific iNOS inhibitors were superior in enhancing immune responses, including lymphocyte proliferation and cytokine production, as well as nitrite reduction. The specific iNOS inhibitor mercaptoethylguanidine, MEG, was the most effective inhibitor. MEG in combination with IFN-? based immunotherapy prolonged the survival and increased the cure rate three fold of rats with intra-cerebral gliomas. Interestingly, the inhibition of NO by MEG was time dependent, and the prolonged survival was achieved only when MEG was administered after more than one immunization. The prolonged survival seemed to be dependent on enhanced local and systemic immune responses. MEG treatment significantly increased the lymphocyte numbers both in the spleen and in the tumor draining lymph nodes (the deep cervical lymph nodes). In the deep cervical lymph nodes, MEG treatment increased CD4+ T cells but not the B cells, suggesting a skewing toward a Th1 immunity, which is important during tumor eradication. Furthermore, MEG treatment decreased the auto-reactive and the suppressive CD8+CD25+ T cells. In the colon carcinoma model, we showed that additional suppressive mechanism exists, which is mediated by the enzyme cyclooxygenase (COX). The inhibition of both NOS and COX increased immune responses six times as compared to inhibition of each enzyme alone. Finally, treatment with MEG in combination IL-18/IFN-? reduced tumor volume by 50 % in rats with colon carcinomas. MEG has been shown to inhibit production of both iNOS and COX, which might explain its superior effects compared to the other NOS inhibitors. Thus we have been able to show that immunotherapy using tumor cells genetically modified to produce cytokines could be used to eradicate established tumors, however, only when combined with therapies that minimize the immune suppression developed during tumor progression.