Acceptance of cardiac allografts by lethally irradiated rats repopulated with syngeneic bone marrow. Role of class I and class II alloantigens

LEW rats (RT1l) that are lethally irradiated and repopulated with syngenic bone marrow accept WF (RT1u) cardiac allografts. If bone marrow repopulation is delayed for two days after irradiation and operation, grafts containing passenger leukocytes (nonperfused grafts) are generally rejected, but perfused grafts, which have fewer passenger leukocytes, are accepted. If bone marrow is given on the same day as irradiation and surgery, then both perfused and nonperfused grafts are accepted. The difference in acceptance of grafts by recipients that are repopulated on day 0 as opposed to day 2 depends on class II alloantigens, because grafts that are similar to LEW for class II antigens are not rejected by day-2-repopulated recipients. Also, the acceptance of totally major histocompatibility complex (MHC)-mismatched cardiac allografts by day-0-repopulated recipients is influenced by a radiation-resistant host cell. Splenectomy of the irradiated and repopulated recipient prevents tolerance induction unless syngeneic irradiated spleen cells are returned to the recipient. Thus class II alloantigen disparities appear to be a major barrier to tolerance induction in the system of total body irradiation and syngeneic bone marrow reconstitution, although proper timing of bone marrow administration can minimize rejection of completely MHC-mismatched grafts.     

Long-term survival of skin allografts in rats treated with topical cyclosporine

The use of topical cyclosporine (CsA) was studied in skin allografts from Buffalo to Lewis rats. CsA prepared in olive oil and dimethyl sulfoxide was administered in various dosages topically on allografts. Untreated allografts were rejected in 7.4 +/- 1.1 days but survived for 18.6 +/- 0.9, 29.3 +/- 1.8, or 40.6 +/- 2.2 days after 10, 20, or 28 days of topical CsA treatment (10 mg/rat/day), respectively. Long-term graft survival (greater than 100 days) was seen with continuous CsA treatment at 10 mg/rat/day, 10 mg/rat/2 days, and 5 mg/rat/day, as compared with rejection in 13.1 +/- 2.3 and 8.9 +/- 0.9 days with CsA 10 mg/rat/3 days and 5 mg/rat/2 days, respectively. The therapeutic blood level of CsA ranged from 250 to 500 ng/ml. Most grafts were rejected when CsA blood levels fell below 200 ng/ml. Direct administration of topical CsA onto the allografts resulted in longer survival compared with those applied on the normal recipient skin 6 cm distal to the allografts, with both high and low doses. Locally high concentrations of CsA in allografts may play an important role in prolongation of graft survival. Minimal cell infiltration and loss of hair follicles were the main histological features in long-surviving allografts (greater than 120 days).     

Chimaerism of immunocompetent cells in allogeneic bone marrow-reconstituted lethally irradiated chickens.

Injection of parental bone marrow cells into 12-day-old lethally irradiated F1 hybrid chickens resulted in chimaerism of donor-type graft-versus-host (GVH)-reactive cells and suppression of antisheep red blood cell antibody response. These manifestations of a chronic graft-versus-host reaction were prevented by pretreatment of the donor marrow with specific anti-T cell globulin. In some chimaeras donor-type GVH-reactive cells developed gradually from T cells precursors of donor origin. Transplantation of spleen and marrow cells from sheep red blood cell-primed F1 hybrid donors into lethally irradiated parental recipients resulted in the loss of memory potential within 1-2 weeks of transfer, whereas donor-type IgG allotype synthesis was preserved. Injection of goat antichicken thymocyte serum to recipients 1 day before reconstitution enabled the antibody response of memory cells at 1-2 weeks, although it failed to prevent their rejection by 8-9 weeeks after transplantation. Split chimaerism of donor-type GVH-reactive cells was demonstrated in chickens which had previously rejected the B cells derived from the same graft.     

Fast lymphoid reconstitution after vascularized bone marrow transplantation in lethally irradiated rats.

BACKGROUND: Bone marrow (BM) transplantation for treatment of hematological and solid malignancies is routinely carried out in conjunction with radio- and chemotherapy. Many patients achieve complete remission of the malignant process; however, their lymphohematopoietic recovery remains in most cases incomplete. This is probably due to the functional changes in the recipient BM stromal cells subsequent to myeloablative therapy. Transplantation of BM hematopoietic cells in a spatial relationship with stromal cells would give an insight into the kinetics of hematological repopulation of the recipient. The aim of this study was to investigate the lymphopoietic reconstitution of irradiated rats after vascularized bone marrow transplantation (VBMT) in comparison with i.v. bone marrow cell (BMC) infusion. METHODS: Lewis rats were totally irradiated with 8Gy and repopulated with syngeneic BMC introduced i.v. or in orthotopic hind limb graft. Ten days after irradiation and BMC graft BM, peripheral blood (PB) and mesenteric lymph nodes (MLN) were collected. The yield and the phenotype of cells were analyzed. RESULTS: VBMT brings much higher cell repopulation of BM cavities of lethally irradiated rats than BMC infusion. Orthotopic hind limb graft promotes also rapid lymphocyte replenishment of PB and MLN of lethally irradiated syngeneic recipients. The population rate of BMC, PB lymphocytes, and MLN lymphocytes was higher after VBMT than BMC injection in suspension. The percentage of T and B lymphocytes in PB and MLN on day 10 after VBMT was comparable with control values. Reconstituted PB lymphocytes showed two subsets of CD4+ cells: "bright" and "dull." All CD4+ cells in PB lymphocytes of i.v. BMC infused recipients expressed low level of these molecules ("dull" subset). CONCLUSIONS: The results of our studies indicate that the presence of stromal cells in their close relationship with stem cells is essential for the fast lyphohematopoietic repopulation of irradiated recipients. The population of CD4+dull cells may represent immature cells. These cells were not found in MLN of VBMT rats. All MLN CD4+ cells represented the "bright" subset, what suggests that the process of cell maturation may occur in the lymphoid organs.     

Improved survival of heterotopic cardiac allografts in rats with dietary n-3 polyunsaturated fatty acids

A heterotopic cardiac transplant model, with male Fischer 344 rats as donors and Long Evans rats as recipients, was utilized to investigate the effect of dietary n-3 polyunsaturated fatty acids on acute rejection. Both donor and recipient rats were fed purified diets high in either n-3 polyunsaturated fatty acids (from concentrated n-3 ethyl esters [EE] or fish oil [FO]) or n-6 polyunsaturated fatty acids (from corn oil [CO]) for either 2-3 or 3-4 weeks before transplant. The recipient rats continued on their diets until rejection. The AIN-76A-based diets (with 30% of calories as fat) had adequate essential fatty acids and were balanced for sterols and antioxidants. Allograft survival was significantly increased by 45% when recipient rats were fed EE as compared to the control (CO diet fed to both donor and recipient), regardless of the diet fed to the donor. There was a slight but significant increase in allograft survival when only donor rats were fed the EE diet 2-3 weeks before transplant. With the FO diet (containing one third of the n-3 fatty acids in the EE diet), only the group fed FO to both donor and recipient (starting 2-3 weeks before transplant) showed a significant increase in allograft survival over the control. However, if the FO diets were fed for 3-4 weeks before transplant, increased survival was seen in groups fed FO to either the donor or recipient alone. In this case, allograft survival with FO feeding to both donor and recipient was not different from recipient treatment alone. In all the studies there was a significant and direct correlation between allograft survival and the donor heart phospholipid n-3/n-6 fatty acid ratio and the n-3 fatty acid content (at rejection). There was an indirect relationship with the n-6 fatty acid content. There was no detectable 20:3 (n-9) in the cardiac phospholipids, indicating the absence of essential fatty acid deficiency. Recipient diets were the strongest determinant of the fatty acid composition in the transplanted donor heart. The data indicate that providing dietary n-3 polyunsaturated fatty acids before and after cardiac transplant to recipient animals provides a significant protection against acute rejection.     

Mitigation of graft-versus-host disease in lethally irradiated mice grafted with spleen cells adherent to glass beads.

Murine spleen cells were separated on the basis of adherence to glass beads into distinct subpopulations that differ in their ability to produce acute graft-versus-host disease (GVHD). Nonadherent CBA spleen cells produce acute GVHD in 6-10 days in lethally irradiated (C57BL/6 X CBA)F1 mice as do unfractionated spleen cells. Spleen cells which are adherent to glass beads, however, enable 71% of the mice to survive without symptomatology of acute GVHD. The low proliferative response of these cells to phytohemagglutinin (PHA) correlated with the mitigated GVHD seen in animals grafted with this fraction. Proliferative cells as determined by the spleen colony assay and the in vitro agar colony-forming assay are present in this fraction as are cells responsive to mitogenic stimulation with lipopolysaccharide (LPS). B6CBF1 mice grafted with CBA adherent cells exhibit a gradual return over a period of 5 months to normal PHA and LPS stimulation levels as shown by splenic cell responses of these mice to mitogens. Surviving mice grafted with adherent cells were chimeric as determined by electrophoretic hemoglobin pattern analysis and serial bone marrow transplantation.     

Microchimerism does not correlate with survival of murine cardiac allografts

The development of microchimerism was evaluated at different time points after infusion of a mixed population of bone marrow and spleen cells from (BALB/c x C57Bl/6)F1 mice in the presence or absence of a cardiac transplant. Microchimerism was observed in the spleen, bone marrow and thymus of transplanted BALB/c mice even after graft rejection. In the absence of transplantation, donor cells persisted especially in the thymus. The results show that despite augmentation of graft survival after donor cell infusion compared to nontreated controls, the development of microchimerism did not sustain cardiac semihistocompatible grafts. Moreover, the persistence of donor cells in the thymus in both situations suggests a role for this organ in the increased graft survival in our model.     

Specificity of cellular migration into cardiac allografts in rats.

The question has not been previously investigated as to whether host lymphocytes infiltrate vascularized organ allografts in indiscriminate fashion, or whether they are retained selectively because of specific antigenic recognition sites on their surfaces. By using a dual cardiac allograft model in LEW rats, we have examined this problem by two methods: (1) detection of preferentially accumulating cells selectively cytotoxic to allografts bearing specific transplantation antigens as compared with "third-party" allografts; and (2) examination of trafficking patterns of separate radiolabeled populations of sensitized cells adoptively transferred into double heart-grafted recipients. In the first series of experiments, using BN and BUF rats as donors, differences in specific cytotoxicity mounted by infiltrating lymphocytes harvested from the appropriate and inappropriate graft were moderately significant (P less than 0.05). Because the question of cross-reactivity betweeen BUF and BN antigen was raised, lymphocytes sensitized to BN and WF donors were differentially labeled in vitro with 3H- or 14C-thymidine. After mixture and adoptive transfer, the ratio of specific to third-party labels was measured in each graft. In this second series of experiments, significant (P less than 0.001) preferential accumulation of specifically sensitized cells were found in the appropriate vascularized organ allograft. These experiments confirm the results of other experimental models, and demonstrate that sensitized lymphocytes accumulate selectively in specific vascularized organ allografts.     

Long-term survival of cardiac allografts induced by cyclophosphamide combined with CTLA4Ig-gene transfer mediated by adenoviral vector

There is a need to achieve donor-specific tolerance in clinical organ transplantation, where potential benefits remain overshadowed by chronic rejection and the side-effects of long-term immunosuppressive therapy. It is known that the mature immune system in mice can be reprogrammed to accept a foreign graft as if it was "self". The AdCTLA4Ig-mediated gene transfer (SC) + cyclophosphamide (CP) treatment alone prolongs allograft survival but does not induce tolerance. However, in our study, the AdCTLA4Ig-mediated gene transfer combined with SC + CP treatment yielded significantly prolonged mean survival times (149.7 +/- 18.0 days), while those in the untreated or AdLacZ treated mice were rejected in normal fashion (5.3 +/- 0.5 and 5.2 +/- 0.4 days, respectively), and survival in the AdCTLA4Ig or SC + CP treated groups were 45.7 +/- 9.6 or 50.2 +/- 5.3 days, respectively. In conclusion, a protocol of AdCTLA4Ig + SC + CP improved the survival of DA-->LEW cardiac allografts.