Core--Chimerism and Immunologic Recovery

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The purpose of this core is to provide a centralized laboratory resource to monitor the effect of GVHD prophylaxis on the degree of donor/host chimerism and the kinetics of immune reconstitution post transplant. This core is also responsible for cryopreservation of peripheral blood cells, marrow cells and serum or plasma from all patients undergoing allogeneic hematopoietic stem cell transplantation at DFCI/BWH. Prior to transplantation, peripheral blood mononuclear cells (PBMC) and serum will be obtained from all patients and normal donors. Following allogeneic hematopoietic stem cell transplantation (HSCT), PBMC, bone marrow and serum will be cryopreserved at regular intervals. The regenerating marrow and circulating lymphoid cells will be assessed for chimerism by PCR analysis of VNTR or STR as well as by rapid pyrosequencing of single nucleotide polymorphisms (SNP). Immune reconstitution after HSCT will be assessed by quantitative measurement of circulating lymphocytes with defined phenotype by flow cytometry. In addition, several new assays of T cell function and differentiation have been adapted to examine T cell reconstitution. Specifically, reconstitution of T cell receptor repertoire will be examined by the method of TCR Vbeta spectratyping. Reconstitution of thymic function will be examined by quantitative PCR measurement of T-cell receptor excision circles (TREC) in PBMC. This will provide a novel measurement of T cell neogenesis. This core will also be able to provide assessment of T cell responses to specific antigens using either ELISPOT or HLA-peptide tetamers. SNP-based chimerism analysis will also provide genotyping for known minor histocompatibility antigen disparity. Results of these assays will be available for correlation with other parameters of immune function as well as with clinical outcomes. Cryopreserved samples of viable cells and serum will also be available to investigators in each of the research projects within this program.