Extrathymic development and function of human T-lymphocytes from bone marrow cells in vitro.

To enrich low-density human bone marrow (BM) cells for putative progenitors of T-lymphocytes, CD7+ CD3- cells were sorted (purity was estimated at > 99.9%) and cultured under limiting dilution conditions with irradiated allogeneic stimulator cells, interleukin (IL) 2, and PHA. Clonal populations were available for analysis from Day 25 onward. By this time, all clones (n = 54) expressed CD3 and alpha/beta-T cell receptor (TCR2). Fifty percent of the clones were CD4+ and 50% were CD8+, with no double positives, whereas almost all clones obtained under identical conditions from peripheral blood (PB) cells were CD4+. All clones were capable of autocrine proliferation, which was blocked by CD25 or CD71 mAb. Most or all clones tested (n = 15) responded to IL 4 and IL 7 as well as IL 2, but not to IL 3 or GM-CSF and only two responded to IL 9. Most clones accumulated mRNA for GM-CSF, IL 2, IL 3, IL 4, IL 5 and also IL 9, but 6 of 11 were negative for IFN-gamma mRNA, and all were negative for IL 6 mRNA. Sixty-two percent of CD4+ and 85% of CD8+ clones (total 70% of all clones) mediated lectin-dependent cell lysis; but whereas 35% of CD4+ and 65% of CD8+ clones (total 46% of all clones) lysed K562 natural killer (NK)-susceptible targets, only 24% of CD4+ and 5% of CD8+ clones (total 17% of all clones) killed lymphokine-activated killer (LAK)-susceptible Daudi cells. Only three clones lysed allogeneic LCL targets and none lysed autologous targets. Furthermore, none of the clones proliferated when stimulated by autologous cells, neither did they suppress proliferative responses of autologous cells. These results suggest that CD3- cells from the bone marrow can acquire functional cytotoxic and proliferative programs extrathymically during in vitro culture with IL 2, mitogens and allogeneic cells, but do not manifest autoreactivity in the three test systems, cytotoxicity, suppression, or autocrine proliferation.

Evolution of a CD3+CD4+ alpha/beta T-cell receptor+ mature T-cell clone from CD3-CD7+ sorted human bone marrow cells.

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.

Cytotoxic and noncytotoxic mechanisms involved in the in vitro anti-leukaemia effects of T cell clones established from a chronic myelogenous leukaemia patient during treatment in vivo with interferon alpha.

T cell clones derived from a chronic myelogenous leukaemia (CML) patient during interferon alpha (IFN alpha, Wellferon) biotherapy preferentially lysed autologous rather than allogeneic CML target cells in an apparently MHC-unrestricted fashion, but also lysed bone marrow cells from certain normal donors regardless of whether or not they shared HLA antigens with the patient. Although T cell clones inhibited both CML and normal bone marrow in the colony-forming assay, they blocked proliferation of CML cells more efficiently than bone marrow cells. This inhibitory effect was mediated at least in part by the tumour necrosis factor alpha (TNF alpha) and IFN gamma secreted by the clones. Antisera to these cytokines partially prevented inhibition. Involvement of additional factors is also suggested in blocking CML cell proliferation because this was not 100% inhibited even by a combination of TNF alpha and IFN gamma. In addition, most clones failed strongly to block the proliferation of normal bone marrow cells, which were susceptible to inhibition by these cytokines.