ABSTRACT
In order to study extrathymic differentiation in vitro, CD7+CD3- lymphocytes were sorted from normal human bone marrow and cultured under conditions of limiting dilution together with irradiated pooled allogeneic peripheral blood mononuclear cells (PBMC) and phytohemagglutinin (PHA) in the presence of 1000 U/ml of interleukin-2 (IL-2). One clone was obtained that failed to react with monoclonal antibody (mAb) TCR delta 1 (TCR1, gamma/delta-specific) or WT31 (TCR2, alpha/beta-specific). From day 35 through day 74 in culture, the surface phenotype of this clone evolved into CD3+, CD4+, CD8-, TCR2+, TCR1-, and was further characterized as CD2+, CD45RO+, CD16-, and CD56-. The presence of mRNA for TCR alpha and beta but not gamma and delta chains was confirmed by Northern blotting. Accessory cell-dependent autocrine proliferative responses to PHA (most likely driven by IL-2) were initially absent, but became 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 homozygous B-cell lines representing the majority of MHC class II alleles. Autoreactivity 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 stimulation, the cells secreted tumor necrosis factor alpha and granulocyte/macrophage colony-stimulating factor (GM-CSF) detected by immunoassays, and T-cell growth factors, most likely IL-2, as detected by bioassays. Polymerase chain-reaction methods demonstrated the presence of mRNA for IL-2, IL-3, IL-4, IL-9, interferon-gamma, and GM-CSF in these cells after stimulation with PHA and B-LCL. These results suggest that cells with the phenotype and some functional characteristics of mature T lymphocytes can evolve extrathymically in vitro from T-cell precursors sorted from normal human bone marrow.
