In vitro proliferation and cloning of CD3-CD161 cells from human thymocyte precursors

Purified CD3^-4^- thymocytes were obtained by depletion of CD3⁺ and CD4⁺ cells from fresh thymocyte suspensions. 5-15% of these cells were found to express CD16 antigen, while other natural killer (NK) cell markers were virtually absent. Double fluorescence analysis revealed that 20-40% of thymic CD16⁺ cells coexpressed CD1, while approximately half were cyCD3⁺. When cultured in the presence of peripheral blood lymphocytes and H9 leukemia cell line as a source of irradiated feeder cells and interleukin 2 (IL-2), CD3^-4^- thymocytes underwent extensive proliferation. In addition, after 1-2 wk of culture, 30-50% of these cells were found to express CD16 surface antigen. Cloning under limiting dilution conditions of either CD3^-4^- or CD3^-4^-16^- thymocytes in the presence of irradiated H9 cells resulted in large proportions (∼50%) of CD16⁺ clones. On the basis of the expression of surface CD16 and/or cyCD3 antigen, clones could be grouped in the following subsets: CD16⁺ cyCD3⁺ ; CD16⁺ cyCD3⁺; CD16^-cyCD3⁺ ; and CD16^-cyCD3^-. All clones expressed CD56 surface antigen, displayed a strong cytolytic activity against NK sensitive (K562) and NK-resistant (M14) target cells, and produced IFN-γ and tumor necrosis factor, but not IL-2. Similar to peripheral NK cells, thymic CD16⁺ cells expressed transcripts for CD16 and for CD3 ε (Biassoni, R., S. Ferrini, I. Prigione, A. Moretta, and E.O. Long. 1988. J. Im̀munol 140:1685.) and ζ chains (Anderson, P., M. Caligiuri, J. Ritz, and S.F. Schlossman. 1989. Nature [Land.]. 341:159). Therefore, it appears that cells that are phenotypically and functionally similar to CD3^- CD16⁺ NK cells may arise from immature thymocytes.