We have succeeded in developing a practical procedure that consistently removes > 99% of the lymphocytes from bone marrow allografts while sparing 65-80% of clonogenic cells. Through the use of counterflow centrifugal elutriation, a physical separation technique that relies on differences in cell size and density, we have successfully eliminated acute GVHD as a cause of treatment failure in a high risk patient population without significantly compromising engraftment. The only major limitation that precludes its widespread application is the unexpected increase in incidence of leukemic relapse in patients transplanted for chronic myelocytic leukemia. This finding suggests the elimination of the cell population(s) responsible for the graft-versus-leukemia effect; it indicates that by eliminating the majority of T cells without a rational means for selection of those lymphocytes to be retained in the graft, we have fallen short of our objective of graft engineering. In the present application we propose to expand our graft engineering capabilities in by integrating CCE and monoclonal antibody based separation technologies. The combination of these methods offers advantages over the use of either method alone. We also propose to explore in detail the basis for the functional differences observed between lymphocyte subpopulations isolated by CCE. We hypothesize that these distinctions have as their basis parameters such as cell cycle stage, activation state, and prior history of antigen exposure (i.e. naive vs memory). As such they may bear on the fate of these cell populations when included in a bone marrow graft. In particular, differential activation requirements may bear on the ability of CCE separated lymphocyte fractions to mediate beneficial or detrimental effects. The capacity to separate large numbers of freshly isolated cells on the basis of size and density, while simultaneously selecting both positively and negatively on the basis of surface markers defined by monoclonal antibodies, furnishes a ready means for clinical application of the results. On this basis we have targeted our future studies toward integration of CCE and antibody mediated cell separation and toward understanding the functional significance of size/density heterogeneity among immunocompetent cells.
