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The murine bone marrow (BM) cells having a certain phenotypic similarity to null natural suppressor (NS) cells have been previously established to be able to inhibit in vitro leukemic cell growth in a genetically unrestricted manner. In this study we found that the treatment of normal (C57BL/6 x DBA)F1 BM cells with a lysosomotropic agent, L-leucine methyl ester (LME), largely abrogated their ability to reduce both P815 mastocytoma and L1210 lymphoma cell proliferation, as well as their NS activity tested for suppression of mitogen (Con A or LPS)-driven spleen cell proliferation. However, after being depleted of the cells binding wheat germ agglutinin (WGA), the BM cells maintained tumor growth-inhibitory activity, while demonstrating no significant NS activity. Moreover, in contrast to T-cell blastogenesis-inhibitory NS activity of BM cells, that was greatly reduced by the addition into the culture of either neutralizing anti-interferon (IFN)-gamma antibody (Ab) or NG-monomethyl-L-arginine, a competitive inhibitor of NO synthase, natural antitumor cytostatic activity of BM cells was not found to be dependent on the presence in medium of IFN-gamma and to be associated with NO production. When incubated at suboptimal numbers with tumor cells on conic, round, and flat well bottoms for 7 h, BM cells provided the most, middle, and least (or no) tumor growth inhibition, respectively, suggesting, thereby, a significance of cell density in cytostatic process. It was also found that the BM cells cultured for 20 h with the medium conditioned by mitogen-preactivated T or B lymphocytes were significantly more suppressive to tumor cell proliferation than the BM cells cultured in medium alone. The potentiation of BM-cell cytostatic activity by T-cell-conditioned medium (CM), but not that by B-cell-CM, was found to be partially reversed by anti-IFN-gamma Ab. Finally, a noticeable tumor growth-inhibitory activity, which could be significantly enhanced upon T-cell-CM, was shown to be also attributable to BM cells from athymic BALB/c mice. Taken together, the results suggest that (1) the tumor growth inhibitory BM cells and the NS BM cells are not identical in their cell compositions, but also differ in their mechanisms of antiproliferative action; (2) a contact cell-to-cell interaction may play a significant role in BM-cell-mediated tumor growth inhibition; (3) the activated lymphocytes, through both IFN-gamma-mediated and IFN-gamma-independent pathways, are able to operatively up-regulate the cytostatic activity of BM cells; and (4) the tumor growth-inhibitory activity exhibited by the normal unmanipulated BM cells, at least in its significant part, may not be a consequence of thymus-dependent immune processes occurring normally in the body.

When normal murine bone marrow (BM) cells were cultured with either L1210 lymphoma cells or P815 mastocytoma cells for 24 h, considerable tumor growth suppression without substantial tumor cell lysis was found. Under the same conditions, normal spleen cells also demonstrated the antitumor cytostatic activity, but not as significant as that characteristic of BM cells, whereas both normal thymus and lymph node cells had not any suppressive effect on tumor cell proliferation. The comparable cytostatic effects occurred in both syngeneic and allogeneic BM-tumor cell combinations. The cytostatic BM-effectors were distinct from T and B lymphocytes or mature macrophages. After being separated on a discontinuous Percoll density gradient, the cells active in suppressing tumor growth were recovered predominantly in 1.075 and 1.060 density fractions. The cytostatic BM effectors, at least in their part, were resistant to x-irradiation up to 2000 rad included. Collectively these results suggest that normal BM, being deficient in cell-mediated antitumor cytolytic activity, has a significant leukemia growth inhibitory potential; and that cytostatic BM effectors are similar in their characteristics to natural suppressor cells.

When bone marrow (BM) cells, isolated from normal (C57BL/6 x DBA/2)F1 mice (H-2b/H-2d), were cultured with leukemic cells for 24 hours, a significant tumor growth suppression, without noticeable tumor cell killing, was found. The level of BM cell-mediated cytostasis of both P815 mastocytoma (H-2d) and L1210 lymphoma (H-2d) cells was dependent on BM-to-tumor cell ratio; 100% growth inhibition was obtained at a ratio of 480/1. In addition, BM cells were found to be able to synergize in suppressing P815 cell growth with lymphoid cells. The synergistic suppressive effects on tumor cell proliferation were observed in BM-spleen, BM-thymus and BM-lymphnode cell co-cultures. The analysis of cytostatic activity of the cell culture supernatants showed that the synergistic leukemia growth suppression could be mediated, at least in part, by cell-derived soluble cytostatic molecules. The data presented herein also indicated that culturing BM cells with either crude supernatant (25%) from allogeneic mixed lymphocyte culture (MLC) or recombinant human interleukin(IL)-2 (20 U/ml) for 20 hours led to a 2-fold increase in their cytostatic activity against both P815 and L1210 cells. Taken together, the results suggest that although normal BM cells are ineffective in tumor cell killing, they may play an important role in cell-mediated effector mechanisms responsible for suppressing leukemia development; and that activated T lymphocytes, through producing cytokine(s), may rapidly upregulate leukemia growth inhibitory activity of BM cells.