Activation of T cells by toxin-conjugated anti-CD3 antibodies

Toxin-conjugated anti-CD3 antibodies have been shown to markedly prolong allograft survival in monkeys. Because these antibodies are directed to the anti-CD3 domain of the T cell receptor, it is possible that these antibodies could activate T cells before killing them. To examine this, we incubated Ficol-purified monkey PBL with anti-CD3 (FN18)1 or with diphtheria toxin-conjugated anti-CD3 (FN18-CRM9). Anti-CD3 antibodies treatment of monkey cells induced rapid tyrosine phosphorylation of several proteins; similar to that reported for human T-cells. Thus, in whole cell lysates there was tyrosine phosphorylation of 115, 80, 70, and 40 kDa proteins. Importantly, immunotoxin-treated cells showed a similar pattern of tyrosine phosphorylation that was dependent on the concentration of the immunotoxin and that persisted for at least 3 hr. To examine if immunotoxin-treated cells could respond to subsequent stimulation, cells were treated for 15 min with the immunotoxin, washed, resuspended in media, allowed to rest for three days and then restimulated with anti-CD3 antibodies. As reported previously, immunotoxin treatment (> 0.01 mg/ml) lead to cell death within 48 h; however, cells treated with low concentration of the immunotoxin responded to anti-CD3 stimulation in a manner similar to the untreated cells. These data show that the immunotoxin can activate T cells before killing them and suggest that immunotoxin-activated cells may initiate an immune response that could influence graft survival. (1) FN 18 and FN 18-CRM9 were generously provided by Dr. David Neville, NIH, Bethesda, MD.

Duke Scholars

Author Stuart Johnston Knechtle Surgery, Abdominal Transplant Surgery