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) TCRδ1 (TCRγ/δ-specific) or WT31 (TCR2, α/β-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 α and γ but not ,and γ 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. Tle 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 α 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-δ, 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.
Highlights
T-cell precursors arise in the bone marrow, from where they must migrate to the thymus via per-Present address: Department of Dermatology, Klinikum Steglitz, Berlin.ipheral blood
CD3-CD7+ bone marrow (BM) cells from two healthy donors were cloned by limiting dilution in the presence of allogeneic peripheral blood mononuclear cells (PBMC), PHA, and IL-2
CD7, a member of the Ig superfamily, is an early marker of the T-cell lineage, which is expressed on fetal lymphocytes prior to colonization of the thymus and prior to TCRfl-chain gene rearrangement or expression of CD1, 2, or 3 (Lobach et al, 1985; Haynes et al, 1988)
Summary
That the thymus is important for the generation of the mature T lymphocytes, for which both positive and negative selection of developing T cells is required, is demonstrated by their virtual absence in young congenitally athymic rats, mice, and di George Syndrome patients. It is not clear whether the thymus is absolutely necessary for the T-cell differentiation process, because older athymic rodents do possess limited numbers of both TCR1 Data suggested that peripheral blood mononuclear cells (PBMC) stringently depleted of T cells formed colonies that rapidly acquired surface CD3 in semisolid agar cultures (Triebel et al, 1981) and in liquid cultures under limiting dilution conditions (Moreau and Miller, 1983)
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