Natural killer cell precursors in the CD44(neg/dim) T-cell receptor(neg) population of mouse bone marrow

Natural kilter (NK) cells develop from the nonadherent cell component of NK long-term bone marrow (BM) cultures (NK-LTBMC). Because these nonadherent cells are depleted of mature NK cells and T cells, but appear to be enriched for NK precursors, they were used as a starting population to begin to define the NK precursors that function in NK-LTBMC. As the stromal cell component of NK-LTBMC has been shown to support interleukin (IL)-2-induced, CD44 dependent, NK cell development from nonadherent NK precursors, NK-LTBMC stroma was used in a limiting dilution assay (LDA) to quantitate the precursors. NK-LTBMC in 96-well plates were irradiated (20 Gy) to kill hematopoietic cells (including the NK precursors), seeded with limiting dilutions of the cells to be quantitated, cultured with 500 U/mL IL-2 for 13 days and assayed for development of NK activity by adding Cr-51-labeled YAC-1 cells to the wells and evaluating the release of Cr-51 after 4 hours. Flow cytometric analysis, sorting, and quantitation of the nonadherent cell component of NK-LTBMC showed that NK precursors were concentrated in the CD44(nag/dim) subset that comprised 10% of the ''lymphoid'' gated cells. When the CD44(nag/dim) subset was sorted from BM of mice treated with 5-fluorouracil (5-FU) 1 day before (-1FUBM), there were about 30% T cells, but no NK-1.1(+) cells. When the T cells were removed by sorting and the CD44(nag/dim), alpha beta, gamma delta T-cell receptor(nag) (TCR(similar to)) subpopulation was seeded onto irradiated stroma with IL-2, they proliferated, developed NK activity, became NK-1.1(+) and CD44(bright) and remained alpha beta, gamma delta TCR(-). The frequency of NK precursors in this population as estimated from the LDA was about 1/500. (C) 1996 by The American Society of Hematology.