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 progen itors of T-lymphocytes, CD7(+) CD3(-) cells were sorted (purity was es timated at >99.9%) and cultured under limiting dilution conditions wit h 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 re ceptor (TCR2). Fifty percent of the clones were CD4(+) and 50% were CD 8(+), with no double positives, whereas almost all clones obtained und er 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 I L 4 and IL 7 as well as IL 2, but not to IL 3 or GM-CSF and only two r esponded 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 mRN A, and all were negative for IL 6 mRNA. Sixty-two percent of CD4(+) an d 85% of CD8(+) clones (total 70% of all clones) mediated lectin-depen dent cell lysis; but whereas 35% of CD4(+) and 65% of CD8(+) clones (t otal 46% of all clones) lysed K562 natural killer (NK)susceptible targ ets, only 24% of CD4(+) and 5% of CD8(+) clones (total 17% of all clon es) killed lymphokine-activated killer (LAK)-susceptible Daudi cells. Only three clones lysed allogeneic LCL targets and none lysed autologo us targets. Furthermore, none of the clones proliferated when stimulat ed by autologous cells, neither did they suppress proliferative respon ses of autologous cells. These results suggest that CD3(-) cells from the bone marrow can acquire functional cytotoxic and proliferative pro grams 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, (C) 1 994 Academic Press, Inc.