Immunophenotyping and Cellular Immune Responses of Cadaveric Donor Bone Marrow Cells M.R. Carreno, V. Esquenazi, C. Gomez, R. Garcia-Morales, J. Mathew, R. Cirocco, A. Alamo, H. Gharagozloo, K. Zucker, C. Ricordi, L. Fuller, A. Tzakis, and J. Miller H UMAN vertebral bone marrow cells have been shown to be a source of hematopoietic progenitors and cells with regulatory function. 1 These cells have also been used clinically in perioperative infusions in organ transplantation to induce a microchimeric state and possibly tolerance. 2,3 We have found that donor bone marrow cells (DBMC) contain several phenotypically distinct cell populations that negatively regulate CTL and MLC immune responses of recipient cells toward donor cells. 4 The present study further extends these in vitro observations on the respond- ing and regulatory functions of purified subpopulations of DBMC analyzed by flow cytometry, also addressing the question of whether immunosuppressive agents would alter these functions. MATERIALS AND METHODS The analysis of DBMC preparations 5 included cluster analysis and epitope quantitation using molecules of equivalent soluble fluoro- chrome (MESF) to discriminate between dimly and brightly stain- ing cells. Murine monoclonal antibodies (MAb) to human CD3, CD4, CD8, TCR (), CD19, CD34, CD38, CD33, CD45, CD56/ 16, and HLA-Drw in three or four fluorochrome combinations were used to analyze samples of 15,000 gated cells in a FACS- caliber flow cytometer. In vitro culture systems comparing cadaveric organ DBMC and individual bone marrow cell subsets vs spleen cells were tested in the presence or absence of pharmacologic immunosuppressive agents. This was done to analyze the possible immunologic re- sponding and regulatory properties of human bone marrow cell infusions in clinical organ transplantation. Bone marrow cells were isolated from human cadaveric donor vertebral bodies as described previously. 6 Briefly, after thorough washing, the vertebral bodies were crushed and single cell suspen- sions were made. The Macs cell separation system (Miltenyi Biotech Inc, Auburn, Calif) was used. The CD34+ and CD34- cells were prepared using the CD34 isolation kit according to the manufacturer’s method. Similarly, T cells were depleted or enriched by E-rosetting, de- scribed previously, 7 or by using anti-CD3 coated microbeads with the Macs system, following the instructions for positive and nega- tive selection. Flow cytometry using appropriate MAbs showed that the negatively or positively selected populations were depleted to 0.0% to 0.2% or enriched to 49.2% to 98.0% of cells expressing the target epitope, respectively (vide infra). These cells were then used in the in vitro assays. RESULTS The CD3+ cells made up 19% of the total cells, and 6% were CD34+. The CD3 cells were distributed into different subpopulations, predominated by CD8+ cells. The relative number of CD3 molecules on DBMC, as assessed by MESF, were significantly lower when compared to autolo- gous peripheral blood T cells. The double CD4-CD8- and the double CD4+CD8+ cells, composed of about one third of the total bone marrow T cells, had lower numbers of CD3 molecules than the single CD4+ or CD8+ cells. Thus, in their phenotypic characterization, the predominant pro- portion of T cells in the marrow appeared to be different from those of the peripheral circulation. In 7-day cultures of CD34+ cells, the predominant cell populations remaining were CD33+ when unstimulated and CD38+ lymphoid precursors when stimulated with irradiated allogenic cells. In the latter cultures, there was also a fivefold increase in the number of CD3+ cells. CONCLUSIONS Taken together, these results support the notion that in vitro DBMC subpopulations proliferate as responding cells in coculture with x-irradiated allogenic cells and cause regulatory effects when added as a third component to MLC reactions. They appear to be culture-generated lym- phoid cell lineage(s) progeny of CD34+ cells, possibly including unique CD3+ “primitive” (dimly staining) T cells, which are not as inhibited in their function by tacrolimus/ cyclosporine (CyA) and MPA as are postthymic (splenic) T cells. REFERENCES 1. Wood ML, Orosz CG, et al: Transplantation 54:665, 1992 From the Department of Surgery, University of Miami, Miami, Florida. Address reprint requests to Dr V. Esquenazi, Medical Sci Bldg Rm 8160, 1600 N.W. 10th Ave, Miami, FL 33136. © 1998 by Elsevier Science Inc. 0041-1345/98/$19.00 655 Avenue of the Americas, New York, NY 10010 PII S0041-1345(98)00024-4 Transplantation Proceedings, 30, 727–728 (1998) 727