Increase of hybridoma formation by human lymphocytes after stimulation in vitro; effect of antigen, endothelial cells, and PWM.

Peripheral blood lymphocytes from antibody-producing Rh- donors were fused with mouse myeloma cells, and the hybrids were screened for anti-Rh antibody production. Although the resulting hybrids were not stable in long-term culture, they provided a useful test system for an investigation of ways to stimulate human lymphocytes in vitro before fusion in order to maximize the recovery of hybrids producing antibodies with a desired specificity. Although 6-day culture with PWM or with antigen (Rh+ erythrocytes) increased slightly the yield of desired hybrids, exposure of the PBL to antigen in the presence of human endothelial cells was dramatically more effective. This effect was not produced with endothelial cell culture supernatants (HECS) that had been shown previously to enhance hybridoma growth. Culture of PBL before fusion with both PWM and antigen also improved the proportion of active hybridomas, and incubation with antigen plus endothelial cells plus PWM resulted in a very high number of hybridomas, with a very high proportion producing antibody to the immunizing antigen. There are several alternatives to explain the mechanisms involved, but in any case, the technique should be particularly useful when the possibility of fusion of human PBL with a human myeloma line becomes more widely available.