Human CD3+4-8-WT31-T lymphocyte populations expressing the putative T cell receptor γ-gene product. A limiting dilution and clonal analysis

The small peripheral blood CD3⁺ T cell population lacking both CD4 and CD8 surface antigens has been analyzed in the present study. Enriched CD3⁺4^-8^- populations were obtained by depletion with anti-CD4 or anti-CD8 monoclonal antibodies (mAb) and complement. The resulting populations contained >99% CD2⁺ cells, whereas CD3⁺ represented approximately 50%. Virtually all of the cells were CD4^-8^- and did not react with the WT31 mAb, specific for a framework determinant of the α/β T cell receptor (TCR). In order to analyze the molecular nature of CD3-associated molecules in CD3⁺4^-8^-WT31^- populations, cells were stimulated with 0.5% phytohemagglutinin (PHA) for 24 h and expanded for an additional 7-14 days in interleukin 2 (IL 2). The resulting cells were >95% CD3⁺ and expressed neither CD4/CD8 nor WT31 antigen. Cell surface iodination followed by cross-linking and immunoprecipitation with anti-CD3 mAb showed that CD3-associated molecules consisted of a major 45-kDa band and a minor band of 43 kDa. Thus, whereas CD3-associated molecules isolated from polyclonal CD3⁺WT31⁺ populations (expanded in IL 2 under the same culture conditions) appeared as diffuse bands, CD3-associated molecules isolated from CD3⁺WT31^- populations displayed a homogeneous molecular mass. Northern blot analysis revealed the presence of mRNA for the TCR γ chain whereas the mRNA for the α chain was mostly represented by a truncated (1.2 kb) form. Also small amounts of a nonproductive mRNA for the β chain were detected. Freshly isolated CD3⁺WT31^--enriched populations proliferated in response to PHA and concanavalin A, moreover, IL 2 was detected in the culture supernatants after cell stimulation. By applying culture conditions which allow virtually all T cells to undergo clonal expansion, approximately 1/3 CD3⁺WT31^- were clonogenic. In addition, the large majority of proliferating microcultures lysed the K562 cell line and about half the natural killer (NK)-resistant fresh melanoma target cells. A large number of clones derived from CD3⁺WT31^- enriched populations by limiting dilution has been further analyzed. More than 95% of the clones were CD3⁺4^-8^-WT31^-; 12/15 clones analyzed in more detail displayed NK activity and 6/15 lysed melanoma cells; in addition, all lysed P815 target cells in the presence of PHA, thus indicating that all the clonogenic CD3⁺WT31^- cells have a cytolytic potential.