The goals of this project are to characterize the donor T cell response to host alloantigen occurring in unrelated donor marrow transplant recipients and to identify donor T cells responsible for GVHD. Host reactive T cells will be cloned from blood and tissue, and the specificity of these clones for HLA or HLA restricted peptides will be determined. The clonal diversity of this response will be further defined by analysis of Valpha and Vbeta encoded T cell receptor (TCR) CDR3 sequences. TCR V(D)J cDNA will be prepared from donor T cells isolated from blood or tissue culture, and size variations of CDR3 message will be compared by spectratyping. Sequence specific oligonucleotide probes homologous to CDR3 segments expressed by individual T cell clones will be used for clonotyping. Donor T cells responding in vitro to cells from the patient will be cloned and the TCR CDR3 segment sequenced. The TCR clonotypes of these in vitro generated T cells will be compared with the changes in TCR repertoire occurring among peripheral blood T cells after transplantation and with host reactive donor T cells cloned from the blood and tissues of these patients. The expansion or contraction of peripheral blood T cell clones will be monitored in relation to GVHD and treatment of GVHD, and TCRs expressed by T cells infiltrating GVHD positive skin biopsies will be compared with TCR expression in the patient's peripheral blood and with the TCR clonotypes of donor T cells cloned from the patient's blood and know to be host reactive. Host reactive T cell clones which expand in peripheral blood with onset of GVHD and which appear in GVHD positive skin biopsies are potentially responsible for GVHD, and the HLA bound peptides that they recognize may be the targets for GVHD. Host reactive T cell clones associated with GVHD will also be used to assist in the isolation and sequencing of these peptide antigens.
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