THE EFFECT OF ANTI-CD3-IMMUNOTOXIN ON T-LYMPHOCYTE FUNCTION IN-VITRO

Recent advances in the design of immunotoxins (IT) have yielded signif icant improvements. FN18-CRM9, a construct of anti-CD3 epsilon mAb FN1 8 and mutated diphtheria toxin CRM9 has exhibited high specificity, lo w systemic toxicity and unusual efficacy compared to previous iteratio ns of immunotoxins. Others and we have examined this anti-CD3-IT for t he purpose of inducing immunological tolerance through selective ablat ion of T cells in rhesus macaques and have obtained encouraging result s. In order to characterize its mode of action, we have examined its e ffects on peripheral blood and lymph node T cell killing in vitro. We have studied the cytotoxic mechanism induced by this anti-CD3-IT as we ll as its effects on proliferation, phenotypic changes and cytokine pr oduction (IL2, IFN gamma and TNF alpha). The results indicate that ant i-CD3-IT was highly specific for T cell killing at doses as low as 1 x 10(6) mu g/ml and showed a maximal effect at 48 h after exposure. The toxicity was restricted to T cells, as B cells and other bystander ce lls were spared. This immunotoxin was shown to induce T cell apoptosis , as assessed by TUNEL assay, DNA content and cytotoxicity. Fas expres sion was upregulated on T cells within 24 h after in vitro exposure to anti-CD3-IT, suggesting an early T cell activation phase prior to T c ell death. T cell killing was manifest as an early cell cycle arrest a t the G(1)/S phase transition, which appeared to virtually eliminate t he production of cytokines. These findings corroborate the temporal, s pecificity and quantitative patterns for anti-CD3 immunotoxin administ ration previously observed in vivo.