Promotion of natural killer cell growth in vitro by bispecific (anti-CD3 x anti-CD16) antibodies

Bispecific heteroconjugated F(ab')2 fragments were prepared from pepsin-digested monoclonal OKT3 (anti-CD3) and 3G8 (anti-CD16) antibodies with 5,5'-dithiobis- (2-nitrobenzoic acid). When these bispecific antibodies (BsA) were added to peripheral blood lymphocyte (PBL) cultures with 100 U/ml human recombinant interleukin-2 (rIL-2), preferable growth of natural killer cells occurred. After 3 weeks the frequencies of CD56+ and CD56+3- cells in cultures with BsA were 74 +/- 7% and 65 +/- 7%, respectively, compared with 48 +/- 6% and 29 +/- 7% in control cultures. The frequencies of CD3+ lymphocytes in the presence of BsA, cells from 1-day cultures were labelled with fluorescein isothiocyanate (FITC)-conjugated anti-CD3, CD4 and CD8 monoclonal antibodies (mAb) and propidium iodide which stains dead cells. Flow cytometry revealed that more than 95% of the dead cells in cultures with BsA were CD3+. Thirty-seven per cent of CD3+, 43% of CD4+ and 17% of CD8+ cells were dead on day 1, and after 3 days the CD4+/CD8+ ratio among viable lymphocytes was 1.6 in the control and 0.5 in BsA cultures. Taken together, these results show that bispecific (anti-CD3 x anti-CD16) F(ab')2 fragments are strongly immunomodulatory by inducing the killing of T cells by CD16+ cells.