The effect of anti-CD3 on the induction of non-MHC restricted cytolytic activity

Abstract

The effects of an anti-CD3 mAb on induction of non-MHC restricted cytolysis was investigated. Peripheral blood mononuclear cells (PBMC) from normal donors (29) and cancer patients (18) were cultured in 100 U/ml of interleukin-2 (rIL-2) with and without anti-CD3 mAb (OKT3, 10 ng/ml) for the first 48 hours of incubation. Thereafter, both PBMC cultures were maintained on rIL-2 up to 20 days. PBMC proliferation was enhanced 17-fold in number by day 20 when anti-CD3 mAb and rIL-2 was present during the first 48 hours but only 3-fold by day 20 when rIL-2 alone was present. Concomitantly anti-CD3 mAb but not Lym-1, an isotype matched control, inhibited the induction of lytic activity against both NK sensitive (K562) and NK resistant (Raji) target cell lines. Thus the inhibitory effect is dependent on anti-CD3 mAb stimulating the CD3/TCR T-cell receptor complex. While lytic activity was dependent on the concentration of rIL-2, inhibition of the induction phase of non-MHC restricted lytic activity was independent of the concentration of rIL-2. Flow cytometry analysis indicated that treatment with the anti-CD3 mAb increased the percentage of CD3 positive cells, CD4 positive cells and especially CD25 positive cells, but decreased th percentage of CD56 positive cells. Supernatants from anti-CD3 mAb stimulated cultures also inhibited the induction of non-MHC restricted lytic activity. Lymphokine analysis showed that supernatants of anti-CD3 mAb stimulated cultures had higher levels of TNF-alpha and IFN-gamma. However, TNF-alpha and IFN-gamma alone or in combination could not mediate the inhibitory effect. The inhibitory factor(s) was partially purified by sequential chromatography on matrices of controlled pore glass and Sepharose CL-6B. The molecular weight of the inhibitory factor(s) was less than 67K. These studies have identified a novel regulatory pathway controlling non-MHC restricted cytolysis. Perturbation of the T-cell CD3/TCR complex with the anti-CD3 mAb results in the secretion of a soluble mediator that down-regulates the induction of rIL-2 dependent non-MHC restricted cytolysis.