Human gamma delta T cells are resistant to induction of anergy but not to induction of cell death in vitro.

A condition of hyporesponsiveness can be induced in certain mature human alpha beta (TCR2) cells relatively easily by their stimulation in the absence of costimulatory signals (signal 1 alone). This state of "anergy" has been implicated in tolerance to self and transplanted organs as well as tumors and may represent an important regulatory component of immune responsiveness. Little is known about whether the same condition applies to gamma delta (TCR1) cells. We therefore undertook to investigate anergy induction in TCR1 cell clones using several approaches known to induce this state in TCR2 cells. First, TCR1 clones were found not to be anergized by culture on immobilized CD3 monoclonal antibody (mAb), while the majority of TCR2 clones were anergized. Second, blocking of autocrine proliferation (stimulated in TCR1 or TCR2 clones by mitogen in the presence of accessory cells) using CTLA-4-lg, a soluble B7 family counterreceptor resulted in anergy induction in TCR2 cells but not TCR1 cells, although experiments with CHO cells transfected with B7-1 (CD80) genes confirmed that these TCR1 clones were responsive to costimulation with B7. Third, blocking mitogen-induced proliferation with anti-IL 2 receptor antibodies and anti-IL 2 antisera resulted in anergy induction in TCR2 but not TCR1 cells. Fourth, stimulation with the calcium ionophore ionomycin also anergized TCR2 but not TCR1 cells. In all four systems, but especially in the latter, stimulation by signal 1 alone resulted in high levels of cell death (> 50%) which was similar for both TCR1 and TCR2 cells. Therefore, these data may reflect a high level of resistance to tolerance induction (manifested as proliferative anergy) but not to clonal deletion (manifested as stimulation-dependent cell death) on the part of TCR1 cells.

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.