The effect of anti-CD3-immunotoxin on T lymphocyte function in vitro.
Recent advances in the design of immunotoxins (IT) have yielded significant improvements. FN18-CRM9, a construct of anti-CD3 epsilon mAb FN18 and mutated diphtheria toxin CRM9 has exhibited high specificity, low systemic toxicity and unusual efficacy compared to previous iterations of immunotoxins. Others and we have examined this anti-CD3-IT for the purpose of inducing immunological tolerance through selective ablation of T cells in rhesus macaques and have obtained encouraging results. In order to characterize its mode of action, we have examined its effects on peripheral blood and lymph node T cell killing in vitro. We have studied the cytotoxic mechanism induced by this anti-CD3-IT as well as its effects on proliferation, phenotypic changes and cytokine production (IL2, IFN gamma and TNF alpha). The results indicate that anti-CD3-IT was highly specific for T cell killing at doses as low as 1 x 10(6) micrograms/ml and showed a maximal effect at 48 h after exposure. The toxicity was restricted to T cells, as B cells and other bystander cells were spared. This immunotoxin was shown to induce T cell apoptosis, as assessed by TUNEL assay, DNA content and cytotoxicity. Fas expression 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 cell death. T cell killing was manifest as an early cell cycle arrest at the G1/S phase transition, which appeared to virtually eliminate the production of cytokines. These findings corroborate the temporal, specificity and quantitative patterns for anti-CD3 immunotoxin administration previously observed in vivo.
Meng
G
G
University of Alabama at Birmingham, USA.
Jiang
Y
Y
Hubbard
W
W
Thomas
J M
JM
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D017209:Apoptosis; D017252:CD3 Complex / Q000276:immunology*; D002455:Cell Division; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D004167:Diphtheria Toxin / Q000276:immunology / Q000494:pharmacology*; D000922:Immunotoxins / Q000276:immunology / Q000494:pharmacology*; D007371:Interferon-gamma / Q000096:biosynthesis; D007376:Interleukin-2 / Q000096:biosynthesis; D008253:Macaca mulatta; D008934:Mitogens / Q000494:pharmacology; D010835:Phytohemagglutinins / Q000494:pharmacology; D013601:T-Lymphocytes / Q000187:drug effects* / Q000276:immunology / Q000378:metabolism; D014409:Tumor Necrosis Factor-alpha / Q000096:biosynthesis; D015854:Up-Regulation; D019014:fas Receptor / Q000096:biosynthesis
D000911:Antibodies, Monoclonal; D017252:CD3 Complex; D004167:Diphtheria Toxin; D000922:Immunotoxins; D007376:Interleukin-2; D008934:Mitogens; D010835:Phytohemagglutinins; D014409:Tumor Necrosis Factor-alpha; D019014:fas Receptor; D007371:Interferon-gamma