In vitro immunogenicity of cadaver donor bone marrow cells used for the induction of allograft acceptance in clinical transplantation.

Background. The cascade of immunological effects brought about by donor bone marrow cell (DBMC) infusions to induce allograft acceptance in clinical transplantation is not fully understood. Aside from acting as immune responding and regulatory cells, the infused DBMC also may sensitize the recipient to the donor antigens. Methods. To analyze this stimulatory activity of DBMC, in vitro mixed lymphocyte cultures (MLC) and cell-mediated lymphocytotoxicity (CML) culture systems analogous to the transplant model with DBMC infusion were used. Results. When responding peripheral blood lymphocytes (PBL) from normal volunteers were placed in culture with suspensions of Ficoll-purified, T cell-depleted, un-irradiated allogeneic DBMC (NT-DBMC), a reaction was seen in both MLC and CML. However, when compared to allogeneic spleen cells as stimulating cells, the responses to NT-DBMC were of markedly lower magnitude and were not seen when the NTDBMC was irradiated (3000 R). When responding PBL were stimulated with either NT-DBMC that had been previously cultured with irradiated cells from the responders for 1 week (activated NT-DBMC), NT-DBMC further depleted of CD15 1 and glycophorin A-positive cells (NT-LP/DBMC), or purified CD34 1 and CD2 1 DBMC subsets, stronger lymphoproliferative and cytotoxic responses were observed. Moreover, these responses were not abrogated by irradiation of the stimulating DBMC subpopulations. Depletion of antigenpresenting cells by positive selection of CD3 1 cells from the responding PBL abrogated MLC and CML reactivity, even when purified NT-LP/DBMC, the most stimulatory cells, were used. This latter observation was in contrast to the responses seen with cultures containing allogeneic stimulating spleen cell populations. This indicated the requirement for indirect alloantigen presentation, i.e., the failure of these DBMC to stimulate by direct alloantigen presentation. NTDBMC was able to stimulate responding PBL in secondary MLC and CML responses with an equivalent magnitude, irrespective of whether the stimulators were spleen cells or NT-DBMC. Finally, the MLC and CML responses were inhibited by tacrolimus (FK506), mycophenolic acid (MPA), and cyclosporine (CsA) in a dose dependent manner, in contrast to previously observed refractoriness of DBMC preparations to these agents if DBMC was tested as responder cells or in modulatory assays. Conclusions. These results indicate that DBMC are able to function as effective in vitro stimulators, but only by indirect antigen presentation, and that the immune responses mediated by them can be downregulated by their own inherent suppressive nature, an effect that can be enhanced by the presence of immunosuppressive drugs. Specific organ donor bone marrow cells (DBMC*) have been infused accompanying solid organ transplantation in an effort to establish a long-term chimeric state that would cause additional donor-specific immunoregulatory effects in recipients also conditioned with maintenance immunosuppression (1‐5). Although these chronic effects of DBMC chimerism are still open to question, the perioperative supplementation of DBMC, in addition to the transplanted organ could possibly cause a number of acute immunological effects, especially in the first several weeks after treatment. Aside from responding to the recipient with unique subpopulations indigenous to the bone marrow, as well as downregulating the responses of the recipient to the donor allogeneic stimulating cells, both of which effects we have described in in vitro assays (6, 7), the infused bone marrow cells may sensitize the recipient against the donor alloantigens. Clinically, this might favor rejection of the allograft. In a series of 67 cadaveric kidney transplant recipients aug