A Salam

Graft-versus-host reactivity and graft-versus-leukemia effect in murine allogeneic bone marrow chimeras conditioned with total body irradiation or total lymphoid irradiation.

To investigate whether graft-versus-host reactivity (GVHR) and the graft-versus-leukemia (GVL) effect can be differentiated, C3H-->AKR mixed bone marrow (BM) chimeras were prepared using two different conditioning regimens. Total body irradiation (TBI) chimeras were induced by infusing 5 x 1O(6) T cell depleted syngeneic AKR BM cells together with 15 x 1O(6) non T cell depleted allogeneic C3H BM cells 1 day after a single fraction of 10.5 Gy TBI. Total lymphoid irradiation (TLI) chimeras were prepared by injecting only 15 x 10(6) non T cell depleted C3H BM cells after 10 daily fractions of 2 Gy TLI Both groups of chimeras were healthy, without clinical or histological signs of graft-versus-host disease (GVHD). In both groups, clonal deletion of antihost-reactive donor type T lymphocytes was found. Whereas TBI chimeras did resist rejection by host type splenocytes injected 2 months after transplantation, TLI chimeras did not. As the latter phenomenon is believed to reflect remaining GVHR, TLI chimeras did have a lower remaining GVHD capacity than TBI chimeras. Nevertheless, TLI chimeras survived significantly longer after host type leukemia challenge (injection of 6 x 10(6) AKR lymphoma cells). The better survival of the TLI chimeras was not due to the radiation regimen, because TLI or TBI conditioned syngeneic AKR-->AKR BM recipients did succumb equally rapidly after AKR lymphoma injection. Donor type (CM) lymphokine-activated killer cell activity, however, was higher in TLI chimeras and may explain the better GVL activity in TLI mice. This model thus illustrates that GVHD and GVL effects can be dissociated and are differentially influenced by the conditioning regimen used for BM transplantation.

Marked mitigation of transplant vascular sclerosis in FasLgld (CD95L) mutant recipients. The role of alloantibodies in the development of chronic rejection.

BACKGROUND In the acute rejection of allografts, the interaction between Fas (CD95) and its ligand (FasL; CD95L) has been shown to be involved in mediating apoptotic cell death. The role, however, of these molecules in the pathogenesis of transplant vascular sclerosis is as yet undetermined. The present study was therefore designed to address this issue. MATERIAL C3H/HEJ FasLgld (FasL-; H2k) spontaneously mutant mice were used either as donors or recipients of aortic allografts; wild-type C57B1/6 (B6; H2b) were used as corresponding recipients or donors (n=6/group), respectively. Controls included aortas transplanted across appropriate allogeneic and syngeneic strain combinations. For histopathological evaluations, the grafts were harvested at day 40 after transplantation, at which time, splenocytes and sera were also obtained for mixed leukocyte reaction and complement-mediated microcytotoxicity assays, respectively. RESULTS Similar to aortas obtained from allogeneic controls, allografts harvested from FasL- -->B6 recipients had morphological evidence of chronic rejection characterized by circumferential intimal thickening with partial disruption of the elastic membranes. Correspondingly, heightened antidonor cellular reactivity was also witnessed in these recipients. On the contrary, B6 allografts harvested from the majority of C3H-->FasL- recipients exhibited marked preservation of aortic morphology. Although these recipients had diminished antidonor cellular proliferation, the titers of alloantibodies were markedly elevated. CONCLUSION The presence of FasL-expressing functional cytotoxic T cells is required for the pathogenesis of transplant vascular sclerosis. The significant reduction and/or absence of chronic rejection with the concomitant retention of antidonor humoral response in C3H FasL- recipients of B6 aortas prompt us to suggest that perhaps posttransplantation vasculopathy is initiated by cell-mediated cytotoxicity with its perpetuation facilitated by alloantibodies.

Flt3-L augments the engraftment of donor-derived bone marrow cells when combined with sublethal irradiation and costimulatory (CD28/B7 and CD40/CD40L) blockade.

T-cell costimulatory blockade as a constituent for recipient conditioning prior to bone marrow transplantation has led to the development of less toxic protocols for the establishment of donor cell chimerism. We therefore hypothesized that the addition of the hematopoietic growth factor, Flt3-ligand (Flt3-L), to the perioperative inhibition of the CD28/B7 and CD40/CD40 ligand costimulatory pathways would enhance the engraftment of allogeneic bone marrow. Recipient BALB/c ByJ (H-2d, Mlsc, Vβ6+/Vβ8+ TCR) received a single sublethal dose of total body irradiation (300 rad) 6 h prior to transplantation IV with unfractionated donor CBA/J (H-2k, Mlsd, Vβ6-/Vβ8+ TCR) bone marrow cells. CTLA4-Ig and/or MR1 were administered at 500 μg IP on days 0, 2, 4, and 6 posttransplantation. Flt3-L was administered at 10 μg IP on days 0–6. Donor cell chimerism was determined on days 30–90 by flow cytometric analysis. Donor-specific tolerance was assessed by skin grafting. In vitro TCR cross-linking assays and flow cytometry were utilized to explore the deletion of donor-reactive T cells. Recipients receiving CTLA4-Ig and MR1 engrafted allogeneic bone marrow cells in the peripheral blood (3/6; 50%) with chimerism being detected at 2–31%. Addition of Flt3-L to this preconditioning regimen enhanced the incidence of engraftment of donor bone marrow cells (10/13; 3–70%). Long-term survival of donor but not third-party-specific skin grafts demonstrated that donor-specific tolerance had been achieved in the chimeric recipients. Deletion of the donor-reactive T cells within the chimeric recipients was also observed. The addition of hematopoietic growth factors and cytokines to the nonmyeloablative regimen of sublethal irradiation and T-cell costimulatory blockade provides a novel strategy for the establishment of donor cell chimerism and for the induction of stable and robust donor-specific tolerance. The deletion of donor-reactive T cells using this protocol suggests the reliability and feasibility of this protocol for clinical transplantation.

Endothelin-1 receptor blockade and its effect on chronic rejection

ENDOTHELIN-l (ET-l), a 21 amino acid peptide with potent vasoconstrictive properties has been implicated in the development of acute allograft rejection.) It is primarily produced by endothelial cells and its secretion, during acute rejection episodes, has been shown to be upregulated by TGFf3 and other proinflammatory cytokines released by graft infiltrating cells.) Additionally, ET-l has also been shown to facilitate proliferation of a-smooth muscle actin-positive (a-smA +) cells? Given that proliferation and/or accumulation of a-smA + cells is a prominent feature in neointimal thickening encountered in posttransplant vasculopathy, it is rational to propose that ET-l may play an important role in the pathogenesis of this lesion. To test this tenet, we proceeded to block ET-receptors (both ETA and ET B) by bosentan (a non peptide antagonist of ET -1) and to study its influence on the evolvement of chronic rejection (CR) in an established mouse model of aortic allotransplantation.3

The role of Fas/FasL apoptotic pathway in the development of chronic rejection.

FAS (CD95), a member of the tumor necrosis factor-receptor family is universallv expressed on various tissues of the body.1 On the contrary. its ligand (Fas L) has a more restricted pattern of expression being found predominantly on activated T cells and on discrete cells in the eye and in the testis.2 It has been recently shown that the interaction between Fas and FasL plays an important role in inducing apoptotic cell death.3 Furthermore. interactions of these molecules have also been shown to mediate tissue destruction witnessed during acute cellular rejection (ACR) of organ allografts.4 This latter finding has prompted many to evolve novel therapeutic strategies aimed at interrupting Fas/FasL binding to mitigate or abrogate AC R. However. the role of Fas system in the pathogenesis of chronic rejection (CR) is as yet undetermined. To address this issue, FasL-deficient mice were used as donor and/or recipients of aortic allografts: reported herein is the out-come of this study.

Presence of intrinsic B lymphocyte tolerance in mixed but not in complete semiallogeneic bone marrow chimeras

The existence of intrinsic B lymphocyte transplantation tolerance was investigated in murine semiallogeneic complete and mixed bone marrow chimeras. Complete chimeras (CC), which were obtained by infusing 20x10(6) C57BL/10 (P1) bone marrow (BM) cells into irradiated (10.5 Gy) (C57BL/10xBALB/c) F1 recipients, were repopulated for 100% with P1 lymphohematopoietic cells. In mixed chimeras (MC), which were obtained by injection of 15 x 10(6) P1 together with 5 x 10(6) F1 BM cells, between 15% and 40% of F1 lymphohematopoietic cells persisted after BM transplantation. Neither MC nor CC were able to develop significant T cell immunity (mixed lymphocyte reaction) or B cell immunity (IgG alloantibodies) against the mismatched host antigens (BALB/c), despite repetitive immunizations. However, after immunization with third-party cells (C3H), the IgG alloantibodies raised cross-reacted with the host-type (BALB/c) antigens in the CC but not in the MC. This suggested that intrinsic B lymphocyte tolerance for host antigens had occurred in MC but not in CC. This was further evidenced in transfer experiments using lethally irradiated C57BL/10 mice reconstituted with purified control C57BL/10 T lymphocytes together with purified C57BL/10 B lymphocytes isolated from CC or MC. Only the recipients reconstituted with B lymphocytes from CC, and not those from MC, produced anti-BALB/c IgG alloantibodies after immunization. These results show that intrinsic B lymphocyte tolerance can be achieved after transplantation and that this depends on the presence of lymphohemopoietic cells expressing the tolerogens.