EVOLUTION OF A CD3-BETA-T-CELL RECEPTOR PLUS MATURE T-CELL CLONE FROMCD3-CD7+ SORTED HUMAN BONE-MARROW CELLS(CD4+ ALPHA)

In order to study extrathymic differentiation in vitro, CD7+CD3- lymph ocytes were sorted from normal human bone marrow and cultured under co nditions of limiting dilution together with irradiated pooled allogene ic peripheral blood mononuclear cells (PBMC) and phytohemagglutinin (P HA) in the presence of 1000 U/ml of interleukin-2 (IL-2). One clone wa s obtained that failed to react with monoclonal antibody (mAb) TCRdelt a1 (TCR1, gamma/delta-specific) or WT31 (TCR2, alpha/beta-specific). F rom day 35 through day 74 in culture, the surface phenotype of this cl one evolved into CD3+, CD4+, CD8-, TCR2+, TCR1-, and was further chara cterized as CD2+, CD45RO+, CD16-, and CD56-. The presence of mRNA for TCR alpha and beta but not gamma and delta chains was confirmed by Nor thern blotting. Accessory cell-dependent autocrine proliferative respo nses to PHA (most likely driven by IL-2) were initially absent, but be came measurable at the same time as the TCR was acquired. However, in the absence of PHA, the clone failed to respond to a panel of homozygo us B-cell lines representing the majority of MHC class II alleles. Aut oreactivity was also not demonstrable. Cytotoxicity was limited to MHC unrestricted ''natural killer (NK)-like'' lysis of K562 target cells, with no autocytotoxicity detected. The NK-like lysis diminished over time in parallel with the acquisition of surface TCR. The cloned cells were not suppressive for mature lymphocyte proliferation. After stimu lation, the cells secreted tumor necrosis factor alpha and granulocyte /macrophage colony-stimulating factor (GM-CSF) detected by immunoassay s, and T-cell growth factors, most likely IL-2, as detected by bioassa ys. Polymerase chain-reaction methods demonstrated the presence of mRN A for IL-2, IL-3, IL4, IL-9, interferon-gamma, and GM-CSF in these cel ls after stimulation with PHA and B-LCL. These results suggest that ce lls with the phenotype and some functional characteristics of mature T lymphocytes can evolve extrathymically in vitro from T-cell precursor s sorted from normal human bone marrow.