Involvement of multiple subpopulations of human bone marrow cells in the regulation of allogeneic cellular immune responses

Background. The identity of the cells in the human bone marrow that function as effective regulators of in vitro and possibly in vivo cellular immune responses is not well established. Methods. Cell subpopulations were isolated from cadaver donor vertebral-body bone marrow cells (DBMC) by using immuno-magnetic microbeads and were tested as inhibitors (modulators) in cell-mediated lympholysis (CML) and mixed lymphocyte reaction (MLR) responses of normal peripheral blood lymphocytes stimulated with irradiated cadaver donor spleen cells. Results. Compared with spleen cells as controls, unirradiated T-cell depleted DBMC inhibited both the MLR and CML responses of allogeneic responder cells in a dose dependent manner (as in our previous reports). The inhibition was also mediated by a number of purified subpopulations including pluripotent CD34⁺ stem cells, and their CD34 negative early progeny of both lymphoid and myeloid lineages. These included DBMC enriched for non-T-cell lymphoid precursors (NT-LP/DBMC; i.e., DBMC depleted of CD3, CD15, and glycophorin-A positive cells) and DBMC positively selected for CD38⁺, CD2⁺, CD5⁺, and CD1⁺ lymphoid cells (all were depleted of CD3⁺ cells) as well as CD33⁺ (but CD15 negative) myeloid precursors. However, positively selected CDI9⁺ B-cells and CDI5⁺ myeloid cells did not inhibit the MLR and CML responses. The NT-LP/DBMC that had been repeatedly stimulated with irradiated allogeneic peripheral blood lymphocytes caused the strongest inhibition of the MLR and CML responses of the same allogeneic cells with 200 times fewer modulator cells needed than uncultured DBMC (P<0.001). Flow cytometric analysis vealed that majority of cells in these cell lines had become CD3⁺? TcR-αβ⁺ CD4⁺ and CD28⁺ cells. Conclusion: A variety of less differentiated cells of various lineages residing in the human bone marrow are immunoregulatory in vitro. Among them, there is at least one subset that can undergo differentiation in vitro into regulatory T cells that can be maintained in long-term cultures.