Enrichment of human marrow T-cell precursors by complement-dependent cytotoxicity with monoclonal antibodies and Percoll Gradient centrifugation

Abstract

Attempts to enrich and characterize human marrow T-cell precursors have been performed using discontinuous Percoll gradient centrifugation, phenotypic analysis of cells with monoclonal antibodies (Mabs), and T-cell colony-forming capacity. Marrow cells were extensively depleted of T cells and separated into seven fractions. The depletion was performed with the following Mabs: CD6 (MBG6 or RFT12) + CD8 (RFT8), CD2 (D66) + CD8 (RFT8), and CD6 (RFT12) + CD8 (RFT8) + CD7 (RFT2). A peak of cells with the capacity to differentiate into mature CD2+CD4+ T-cell agar colonies (TL-CFU) was obtained in a fraction with a density 1.063 less than d less than 1.069 g/ml. This peak was associated with the presence of cells expressing RFB1 and OKT10, two markers shared by hemopoietic precursors. Cells in this fraction were negative for CD3, CD4, CD1, CD8, and CD2 antigens. Their treatment by complement-dependent cytotoxicity with the CD7 Mab resulted in a loss of T-cell colony-forming capacity together with a reduction of T10-, RFB1-, and My10-positive cells.