B-CELL DYSFUNCTION FOLLOWING BONE MARROW TRANSPLANTATION

Immune reconstitution following allogeneic (allo) or autologous (auto) bone marrow transplantation (BMT) has been, and continues to be, a serious clinical problem. In the early post-BMT period deficiencies in humoral immunity have been attributed to B cell dysfunction. Unfortunately, no consensus has emerged that can explain the mechanism underlying the early B cell dysfunction in most transplant patients; nor has there been an analysis of the emerging B cell repertoires. During the next grant period, new experimental approaches to address the problem of B cell dysfunction post-BMT will be employed. We will test the hypothesis that B cell dysfunction post-BMT reflects a defect in BM microenvironmental adherent cell/B-cell precursor interactions required for normal self-renewal and differentiation. Adherent cells established from the BM of post-BMT patients will be characterized for patterns of gene expression using histochemistry, immunofluorescence and the polymerase chain reaction (PCR). Adherent cells deriVed from normal donor BM will be similarly characterized. The post-BMT and normal donor adherent cells will then be compared for their ability to support adhesion, IL-7 driven proliferation in long-term culture, and differentiation of BCP. We will test the hypothesis that an ordered re-establishment of antibody repertoires occurs post-BMT which may contribute to early, restricted B cell responses. Peripheral blood B cells taken at various time points from post-BMT recipients will be used to generate heavy and light chain Ig repertoire DNA and RNA libraries by PCR amplification. Libraries will be screened with heavy and light chain Ig probes specific for V gene families to evaluate expression frequency. Restriction mapping and DNA sequencing will be used to define specific V gene usage. A chronologic analysis of individual V gene repertoires post-BMT will be compared among patients, as well as with repertoire patterns existing in normal individuals. A better understanding of B cell differentiation post-BMT may stimulate the development of corrective strategies that could reverse or inhibit humoral immunodeficiency in these patients.