Studies on human prothymocytes by means of an agar T-cell colony assay

The identity of bone marrow (BM) T colony-forming cells has led to controversy because of BM contamination with mature T cells having clonogenic potential. To circumvent this problem, extensive purging of BM cells was achieved, using E-rosetting and complement-dependent cytotoxicity, with a cocktail of monoclonal antibodies (Mabs): CD6 + CD8 + CD4 + CD2 and two successive rabbit Complement (C) treatments. This resulted in a 2-log elimination of mature T cells, as assessed by marker studies. T-cell-depleted marrow (TDBM) was plated in agar in the presence of PHA- and B+ null cell-derived prothymocyte differentiating activity (PTDA). Two peaks of colony formation were observed on days 7 and 10 of incubation. Both seven- and ten-day colonies contained mainly T4+ and only a few T8+ cells, which also expressed T3, T11, HLA-DR, and Tac antigens. These colony-forming cells were enriched (three- to fourfold) by discontinuous Percoll gradient centrifugation. These results point to the existence of marrow T-cell progenitors, which differ in their kinetics of colony generation and surface markers.