Characterization of Novel Surface Molecules on the Facilitating Cell

Abstract

489 Bone marrow (BM) chimerism is a promising technique in the induction of donor-specific transplantation tolerance. The clinical application of BM chimerism, however, continues to be hampered by the morbidity and mortality associated with bone marrow transplantation, including Graft vs. Host Disease(GVHD) and engraftment failure. The Facilitating Cell (FC) is a unique marrow-derived cell population that promotes allogeneic stem cell (SC) engraftment without inducing GVHD. Recipients demonstrate donor-specific transplantation tolerance to solid organ and cellular grafts. The FC comprises 0.4% of the BM population, and has been phenotypically characterized and isolated via flow cytometric cell sorting as CD8+/CD3+/αβTCRdim/-. Although previously thought to require conventional TCR heterodimers for expression, functional CD3 complexes have been firmly established to occur on immature thymocytes in the flow cytometric absence of αβ- or γδTCR heterodimers. In all described cases, such CD3 expression is associated with TCRβ and a TCRα surrogate or chaperone protein. Therefore, the primary aim of this study was to determine if similar TCRβ-associated molecules were present on the FC cell surface. The FC population was isolated via multiparameter sterile live cell sorting from the bone marrow of B10.BR mice, using BM-derived T-cells(CD8+/CD3+/TCRbright) as controls. Surface protein biotinylation was performed, followed by immunoprecipitation with an anti-βTCR monoclonal antibody (H57-597). SDS-PAGE and Western blotting of the immunoprecipitate allowed visualization of the individual proteins associated with TCRβ. Such analysis demonstrated a 33kD protein on the FC (FCp33) that is not present on bone marrow-derived T-Cells[Figure]. This protein is similar in size to the Pre-T-Cell receptor alpha (pTα) expressed as part of the TCRβ-pTα heterodimer involved in regulation of T-Cell maturation in TCRα deficient mice. These findings suggest that a similar TCRβ-FCp33 complex may be important in regulating the ability of the FC to promote SC engraftment and survival, while establishing donor-specific transplantation tolerance.