E Cybulska

Comparative analysis of immunological reconstitution induced by vascularized bone marrow versus bone marrow cell transplantation

Abstract  We have reported previously that vascularized bone marrow transplantation (VBMT) in an orthotopic hind limb graft brings about complete repopulation of bone marrow cavities in lethally irradiated syngeneic recipients within 10 days. Intravenous infusion of an equivalent volume of bone marrow cell suspension was evidently less effective. The purpose of this study was to investigate the reconstitution of immunocompetent compartments of lethally irradiated syngeneic rats after VBMT. Lewis rat hind limbs were transplanted orthotopically into irradiated recipients. Ten days after irradiation and bone marrow transplantation, bone marrow, mesenteric lymph nodes, and sera from rats were harvested. Mesenteric lymph node lymphocytes were analyzed. The responsiveness fomesenteric lymph node lymphocytes (MLNL) to mitogens and cell proliferation in the presence of sera and bone marrow cell (BMC) culture supernatants were measured. Our studies have shown that vascularized bone marrow transplantation brings about rapid replenishment of lymphoid organs of lethally irradiated syngeneic recipients. The re‐populating subsets are fully responsive to mitogens. Sera from reconstituting rats had no effect on the proliferation of mature lymphocytes. Intravenous infusion of a number of BMC in suspension equivalent to that grafted in hind limb transplant was less efficient in reconstitution of lymphoid tissue.

Repopulation of donor heart by recipient bone marrow–derived dendritic cells prior to transplantation causes acute rejection by both the allogeneic and syngeneic recipient

Experimental studies on allogeneic transplantation have shown that recipient dendritic cells (DC) play a role in peripheral tolerance as well as in rejection of allografts. It is not known whether DC exert their tolerogenic function in recipient lymphoid tissue, and whether they process shed alloantigen in the graft itself. To answer this question we created a chimeric heart model deprived of its own DC and repopulated by recipient DC. The rationale for this model was to observe whether recipient DC located in the graft attenuate recruitment and stimulation of recipient lymphocytes, subsequently prolonging graft survival. Vascularized bone marrow transplants (VBMTx) from the prospective recipient to the lethally irradiated heart donor, which function for a period of 14 days, were used to replace donor DC with prospective recipient DC. Hearts from chimeric LEW rats (with BN DC) were transplanted to untreated BN rats. Also, hearts from chimeric LEW rats (with BN DC) were returned to untreated LEW rats. Replacement of the donor heart with recipient DC did not prolong graft survival. Rather, it initiated a rejection reaction that was already present in the donor. Recipient DC retained their immunogenic properties also when the graft was returned back to a donor strain animal.

Donor DNA can be detected in recipient tissues during rejection of allograft

Abstract The main source of donor DNA in recipients of allograft are “passenger” cells. It is claimed that they are responsible for the posttransplantation microchimerism and prolongation of allograft survival. We have observed that besides cellular microchimerism, donor DNA can be found in the recipient tissues at the time of rejection of the allograft. In this study, we provide evidence for the presence in the recipient of both DNA in “passenger cells” and free DNA in tissues at the terminal stage of rejection. Male BN (RT1 n) rat heart or skin was transplanted to female LEW (RT1 1) rats followed by a vascularized bone marrow in a hind‐limb transplant. In another group, heart and skin were transplanted followed by immediate i. v. infusion of donor‐type bone marrow cells. CsA was given in a dose of 17 mg/kg body weight for 30 days, then the rats were followed up until day 100 unless rejection occurred earlier. LEW blood, spleen, mesenteric node and bone marrow cells were stained with moAb OX27 specific for BN but not LEW. Genomic male DNA was isolated and amplified with SRY oligonucleotide. At day 30 and day 100 cellular microchimerism was detected in blood, spleen, nodes and bone marrow cells. Donor DNA was detected in recipient skin, liver and heart extracts, as well as lymphoid organs, at the time of rejection of allograft, but not when the rats were maintained on CsA. Taken together, donor DNA was detected in recipient tissues at the time of heart or skin rejection. It appeared to be released from cells of rejecting grafts and not from “passenger” cells, representing only a minor cellular mass compared with the graft.

Tolerance induction following allogeneic vascularized bone marrow transplantation — the possible role of microchimerism

Abstract We have noticed that bone marrow transplanted in a vascularized limb graft, providing a continuous supply of donor bone marrow cells (BMC), may prolong the survival time of a skin graft from the same donor. The question arises whether the microchimerism raised plays a role in the prolonged survival of skin allografts. The aim of the study was to follow the development of microchimerism after allogeneic vascularized bone marrow transplantation (VBMTx) concomitantly with the rejection process of transplanted skin. Brown Norway (BN) rats served as donors and Lewis rats as recipients of VBMTx and free skin flap allografts. A hind limb was transplanted, followed by a full‐thickness skin graft on the dorsum. Cellular microchimerism was investigated in recipients of VBMTx and skin grafts in blood, spleen, mesenteric lymph node, and bone marrow with the monoclonal antibody OX27 directed against MHC class I polymorphic RT1 on BN cells and quantitatively analyzed in a FACStar. In the VBMTx group, the free skin flap survived 70 days after weaning off cyclosporine A (CsA). An intravenous infusion of BMC in suspension equivalent to that grafted in the hind limb did not prolong skin graft survival after cessation of CsA therapy. Donor‐derived cells could be detected in VBMTx recipients as long 70 days after weaning off CsA but not in recipients of i. v. suspension BMC grafting.