TRANSFER OF PORCINE MHC DR-ALPHA INTO IE-ALPHA-DEFICIENT MURINE BONE-MARROW RESULTS IN REDUCED IE-RESTRICTED V-BETA USAGE

Background Allogeneic bone marrow transplantation has proven effective for inducing specific tolerance to subsequent solid organ allografts, although the clinical applicability of this approach is limited by th e morbidity and mortality associated with this procedure. As an altern ative, we are investigating the transfer of allogeneic MHC class II ge nes into recipient bone marrow cells (BMC), using the miniature swine as a model. Methods. To understand the mechanism of tolerance inductio n achieved through class II gene transfer, BMC from C57BL/10 mice, whi ch lack expression of the MHC class II DR alpha equivalent (H-2 IE alp ha), were transduced with a retrovirus vector for swine DR alpha. Resu lts. Expression of the DRA-vector in bone marrow-derived cells was dem onstrated by Northern analysis of colonies grown in vitro from transdu ced myeloid progenitors. Taking advantage of the fact that the introdu ced DR alpha chain was able to form heterodimers with endogenous IE be ta, surface expression of the transgene was demonstrated on splenocyte s harvested 1, 17, and 28 weeks after bone marrow transplantation. Tra nsgene expression was confirmed by reverse transcriptase-polymerase ch ain reaction in the thymus of those animals killed at weeks 17 and 28, Finally, the effects of bone marrow transduction on central tolerance induction was demonstrated by the progressive decrease of IE-reactive T-cell clones bearing V beta 5 and V beta 11 T cell receptors in the peripheral blood cells of engineered recipients. Conclusions. Our resu lts support the notion that transplantation tolerance, induced by clas s II gene transfer into syngeneic BMC, results in part from durable de letional unresponsiveness of graft-specific alloreactive T cells.