Evidence that indefinite survival of small bowel allografts achieved by a brief course of cyclosporine or FK506 is not due to systemic hyporesponsiveness.

The immunological status of Lewis (LEW) recipients of indefinitely surviving (greater than 400 days) orthotopic Brown-Norway (BN) small bowel allografts was investigated 1 to 1 1/2 years after cessation of immunosuppressive therapy with either cyclosporine or FK506 and compared with recipients of syngeneic grafts. A normal proliferative response (as measured by a mixed lymphocyte culture) of recipient peripheral lymph node lymphocytes in response to the donor-specific (BN) and the third-party (ACI) antigen, was observed in all experimental groups. Cytolytic T cell generation (as measured by a standard 51Cr-release cytotoxicity assay) in response to the donor-specific (BN) and the third-party (ACI) antigen was observed also in all groups. A FACS analysis of allograft-recipient splenocytes showed no evidence for systemic lymphoid chimerism. BN or ACI skin grafts transplanted onto recipients of allogeneic and syngeneic small bowel grafts were rejected completely in 12-17 days, while the intestinal grafts remained functional. Immunohistologic evaluation of the allografts, using anti-BN class I and anti-Lewis class II monoclonal antibodies showed anti-BN staining on the epithelial and endothelial structures, whereas the mononuclear cells in the lamina propria stained positively with the anti-LEW monoclonal antibody. However, lymphoid depletion and scarring of Peyer's patches and mesenteric lymph nodes as well as focal obliterative mesenteric arteriopathy, indicative of an indolent chronic rejection, were observed. These data demonstrate that recipients of indefinitely surviving small bowel allografts remain immune competent and do not retain the intestinal graft on the basis of specific hyporesponsiveness to the donor antigens.     

Graft-versus-host reaction in small bowel transplantation and possibilities for its circumvention

To describe GVHR in small bowel transplantation and its underlying mechanisms and to find methods for circumventing that response, accessory small bowel transplantation was carried out in the rat model. Animals not treated with cyclosporine, irradiation, or removal of the mesenteric lymph nodes of the graft died within 22 days postoperatively due to graft versus host disease. Mesenteric lymph nodes of the graft and recipient spleen and peripheral lymph nodes showed strong immunologic stimulation histologically and high antihost T-cell-mediated cytotoxic antihost reactivity. Seventy-one percent of the animals that had received 15 mg of cyclosporine per kilogram body weight orally survived 150 days after transplantation. After donor irradiation with 50 rads, 77 percent of the recipients survived 120 days. After microsurgical removal of the mesenteric lymph nodes of the graft, 89 percent survived 120 days. We conclude that GVHR plays an important role in small bowel transplantation and that the experimental regimens of donor, graft, and recipient treatment described herein have proved their efficacy for circumventing GVHR.     

Graft-versus-host disease in fully allogeneic small bowel transplantation in the rat

Small bowel and its mesentery contain considerable amounts of lymphoid tissue that can mediate graft-versus-host disease in small bowel transplant (SBT) recipients. Present studies determined the existence of GVHD in a fully allogeneic SBT model and examined the effect of donor pretreatment with ALS in eliminating GVHD. Adult male Lewis (Lew) rats received orthotopic small bowel transplants from untreated (LewxBN)F1 (LBNF1) donors (group 1) or Brown Norway (BN) donors that were untreated (group 2) or pretreated with ALS (days -2 and -1) (group 3). All recipients were treated with cyclosporine 15 mg/kg/day i.m. on days 0-6 postoperatively. Animals were weighed and examined daily for signs of rejection and GVHD. No animals in groups 1 or 3 showed any physical signs of GVHD, but all of those in group 2 had characteristic weight loss, diarrhea, and dermatitis between 4 and 6 weeks postoperatively, from which they all recovered. Histologic examination of skin and spleen at this time confirmed the presence of GVHD. The relative spleen weight [( spleen weight/body weight] x 100) of group 2 animals was also significantly greater than that of unoperated control Lew animals. Spleen cells obtained from group 2 animals at the time of subclinical GVHD, but not cells from group 1 or 3 animals, caused enlargement of popliteal lymph nodes when they were injected into the footpads of Lew rats. This study shows that GVHD can manifest itself in recipients of a fully allogeneic small bowel transplant even when rejection is prevented by effective immunosuppression with CsA. However, combined use of recipient treatment with CsA and pretreatment of donor animals with ALS eliminates all manifestations of GVHD.     

Early biological and immune response to semi-identical liver or kidney allograft in miniature swine

In inbred miniature swine, semi-identical liver allograft recipients survive up to 3 months without immunosuppression, whereas similarly mismatched kidney allografts are uniformly rejected within 2 weeks. The early biological and immunological events were assessed in this unique model. SLA(d/d) pigs (MGH, Harvard Medical School, Boston, MA, USA) received liver or kidney allograft from heterozygous SLA(c/d) miniature swine. Survival, graft function, histology, intragraft cytokines, peripheral lymphocyte and platelet count, plasma cortisol level and cellular/humoral anti-donor immune response were assessed. Kidney allografts were uniformly rejected within 2 weeks, whereas liver allografts survived for up to 87 days. After both liver and kidney transplantation, the peripheral lymphocyte count decreased during the first week concomitantly to a significant elevation of plasma cortisol level. Early decrease of peripheral platelet count was observed after liver but not renal transplantation. Up-regulation of transforming growth factor beta1 (TGF-beta1) and interferon-gamma (IFN-gamma) was observed during the first postoperative week in semi-identical liver allografts and IFN-gamma as well as IL-10 in kidney allografts. In liver recipients, labelled autologous lymphocytes accumulated in the liver graft and native spleen, whereas after renal allograft, lymphocytes accumulated in the native spleen and liver but never in the kidney allograft. Specific cellular anti-donor unresponsiveness was observed from the first post-transplant day in both liver and kidney recipients, while the humoral anti-donor response remained intact. In semi-identical liver allograft, recipient rejection is milder and slower than in similarly matched kidney allograft. The intragraft up-regulation of TGF-beta1 in semi-identical liver allograft might be one mediator to explain the modulation of rejection after liver transplant. The rapid, nonspecific accumulation of recipient lymphocytes in the liver allograft but not in kidney allograft might also play a role in the different survival time in this model.     

Indefinite survival of rat islet allografts following infusion of donor bone marrow without cytoablation

We have tested the effect of donor bone marrow cell (DBMC) infusion on the survival of pancreatic islet allografts in the rat, without the use of cytoablative recipient conditioning. Lewis and diabetic Brown Norway rats were used as donors and recipients, respectively. Donor islets were placed beneath the left renal capsule. Infusion of DBMC and temporary immunosuppression followed by delayed islet transplantation resulted in indefinite survival of all islet grafts (MST >180 days). Control animals demonstrated recurrent hyperglycemia (islet allografts rejection). Donor bone marrow derived cells were detected in the spleen and cervical lymph nodes of BN recipients of LEW bone marrow but not in the recipients of islet transplants alone. Second set full thickness skin grafts were performed in normal BN and in recipients of a previously successful ITX. Donor specific skin grafts were accepted in the animals that had received DBMC 40 days before the islet allograft, while animals receiving DBMC at the time of the islet allograft rejected the donor specific skin graft similarly to the controls. However, these animals did not reject a second set donor-specific islet transplant. The results indicate that radiation conditioning of the recipients was not necessary to induce microchimerism and graft acceptance in this rodent model of islet allotransplantation.     

Detection of chimerism following vascularized bone allotransplantation by polymerase chain reaction using a Y-chromosome specific primer.

Chimerism following allogeneic organ transplantation is a phenomenon known to occur and be associated with development of immunologic tolerance in allotransplantation. However, little is known about graft cell migration following vascularized bone allografting. In this study, chimerism was assessed following vascularized tibia transplantation from male DA or PVG donors to female PVG rat recipients using a semi-quantitative polymerase chain reaction for the Y-chromosome. FK-506 (Tacrolimus) was administered after transplantation for immunosuppression. All immunosuppresssed PVG rat recipients of PVG bone grafts showed a high level of chimerism (1%) in the thymus, spleen, liver and cervical lymph nodes at 18 weeks post-transplant. Donor cells were also detected in the contralateral tibia and humerus. In non-immunosuppressed PVG rat recipients of DA bone grafts, donor cells were detected in the spleen in three of five rats within 2 weeks post-transplant. In these animals the bone grafts were severely rejected. In immunosuppressed PVG rat recipients of DA bone grafts, two of five, four of eight and eight of 10 rats showed low level chimerism (0.1%) in peripheral blood at 1, 12, and 18 weeks post-transplant. Six rats showed a high level of chimerism in the spleen and thymus. Histological studies revealed no rejection findings through 18 weeks post-transplant. Our results indicate that chimerism, or the presence of graft cells in host tissue, may occur in the face of acute rejection and be demonstrable following vascularized isograft and allograft living bone transplantation when chronic immunosuppression is maintained. Graft vascular patency during the short-term likely allows cellular migration, even in the face of acute rejection. Long-term survival and proliferation of graft marrow elements in host tissue may be possible with adequate immunosuppression.     

Induction of transplantation tolerance in rats by spleen allografts. III. The role of T suppressor cells in the induction of specific unresponsiveness

Heterotopic (WAG x AGUS)F1 spleen allografts survive indefinitely when transplanted to normal AGUS recipients and induce long-term donor-specific unresponsiveness. In this report, we have examined the immune reactivity of spleen graft recipients soon after transplantation, in an attempt to define the immunological mechanisms responsible for the induction of donor-specific unresponsiveness. Unresponsiveness develops as early as one week after splenic transplantation. T cells obtained from the recipient lymph node and spleen exhibit reduced mixed lymphocyte reaction responses to donor (WAG) but respond normally to third-party (PVG) stimulators. In contrast, T cells obtained from the spleen graft are unresponsive to both donor and third-party stimulators. Donor specific T suppressor cells (Ts) appear in the recipient's lymph node and spleen by one week posttransplantation--however, at this time antigen nonspecific suppressor cells predominate in the spleen graft. Only minimal cytotoxic T cell activity could be detected in the spleen graft, with the host spleen and lymph nodes being devoid of cytotoxic T lymphocytes. Sera obtained one or two weeks following splenic transplantation did not contain cytotoxic alloantibodies, and only a very weak response could be detected at one month. These data demonstrate that the unresponsiveness associated with the spontaneous acceptance of spleen allografts is correlated with the early induction of antigen specific Ts in recipient lymphoid tissue and the presence of nonspecific suppressor cells at the graft site.     

Intestinal permeability and bacterial translocation following small bowel transplantation in the rat

In addition to its role in absorbing nutrients, the intestinal mucosa provides an important barrier against toxins and bacteria in the bowel lumen. The present study evaluated gut barrier function following orthotopic (in continuity) intestinal grafting in rats. Graft histology, intestinal permeability, and bacterial translocation to the grafted mesenteric lymph nodes, the host's liver, and the host's spleen were assessed on the 3rd, 5th, and 7th postoperative days. The study group received no immunosuppression after allotransplantation. The two control groups included rats with isografts and rats with cyclosporine-treated allografts. On the 7th POD, the study animals had moderate transmural inflammation due to rejection, with normal histology in the isografts and CsA-treated allografts; increased intestinal permeability, measured by urinary excretion of oral 51Cr-EDTA (P less than 0.01); and increased number of bacteria in the MLN and spleen (P less than 0.05). The number of bacteria in the MLN and spleen of the study group positively correlated with the changes in intestinal permeability (P less than 0.05). Rejection of the orthotopic intestinal graft leads to increased intestinal permeability and bacterial translocation from the lumen of the graft to the host's reticuloendothelial system. Measures to improve gut barrier function and antibiotic therapy during rejection episodes may help reduce the incidence of septic complications after intestinal grafting.     

Dissociation of antigenicity and immunogenicity of neonatal epidermal allografts in the mouse

Cultured neonatal epidermal cells or sheets have been grafted successfully onto allogeneic recipients across an MHC barrier, while genetically identical skin allografts were rejected. Induction of specific allosensitization by prior priming with allogeneic spleen cells or allogeneic skin grafts did not prejudice the survival of subsequent cultured epidermal allografts. When cultured epidermis and adult skin of the same genotype were grafted simultaneously, as double grafts, cultured epidermis survived despite the presence of an ongoing allograft rejection reaction in the host. Furthermore, pretreatment of the recipients with cultured epidermis failed to protect against rejection of subsequent allogeneic adult skin grafts of the same genetic origin. These data indicate that cultured epidermis is neither immunogenic nor antigenic in allogeneic host. In companion experiments it was determined that neonatal (noncultured) epidermal allografts also survived indefinitely, implying that neonatal epidermis lacks antigenicity. However, in contrast to cultured epidermis, neonatal epidermal allografts evoked specific systemic immunity in their recipients, since they rejected a subsequent allogeneic adult skin grafts in accelerated fashion. These data demonstrate that in neonatal epidermis antigenicity can be dissociated from immunogenicity.     

Induction of allogeneic islet tolerance in a large-animal model

For islet allotransplantation to become a therapy widely applicable to patients with insulin-dependent diabetes, it will be important to avoid conventional immunosuppression and yet maintain long-term rejection-free islet survival. This possibility was tested in a large-animal model using mixed allogeneic chimeras established using total lymphoid irradiation (TLI) and donor-specific bone marrow transplantation (BMTX). Four recipient sex-mismatched and DLA class II-matched English springer spaniels became chimeric after TLI and donor-specific BMTX. Subsequent donor-specific renal allografts survived for more than a year. Acceptance of a donor-specific skin graft and rejection of a third-party graft demonstrated tolerance with maintenance of immunocompetence. Pancreatic microfragments containing islets were refluxed into the splenic vein of the recipient. Purified islets were placed under the capsule of spleen and liver. After 75 days, recipients underwent total native pancreatectomy. All four chimeric pancreatectomized dogs had functioning islet grafts 75 days after transplantation, evidenced by a prompt rise in serum insulin levels following an IVGTT and histological demonstration of islet tissue at the site of transplantation. After removal of the transplanted islet tissue, no insulin was released after IVGTT. In summary, intrasplenic allogeneic canine islets transplanted into chimeric dogs rendered tolerant to donor MHC survive and function for greater than 75 days in the absence of immunosuppression. This study represents proof of the concept that allogeneic islet transplants have the potential to reverse diabetes without the use of conventional immunosuppression.     

Donor Lymphoid Organs Are a Major Site of Alloreactive T-Cell Priming Following Intestinal Transplantation

We hypothesized that lymphoid organs within intestinal allografts contribute to their immunogenicity. Consistent with this hypothesis recipient T cells rapidly migrated to the lymph nodes and Peyer's patches of syngeneic and allogeneic intestinal grafts such that at 24 h approximately 50% of the lymphocytes isolated from donor lymphoid organs were of recipient origin. However, only in the lymphoid organs of allografts did recipient T cells display an activated phenotype, proliferate and produce IFNgamma. Rejection of allogeneic intestines lacking lymphoid organs was dramatically impaired in splenectomized, lymph node-deficient recipients compared to lymph node bearing, wild-type allogeneic intestines. This demonstrates the important role of donor lymphoid organs in the rejection process. Furthermore, recipient T cells proliferated more extensively and produced more IFNgamma in donor lymphoid organs than in recipient lymphoid organs, indicating that donor lymphoid organs play a dominant role in initiating the recipient anti-donor immune response following intestinal transplantation.     

Effect of mixed hematopoietic chimerism on cardiac allograft survival in cynomolgus monkeys

BACKGROUND: We have previously reported the successful induction of mixed chimerism and long-term acceptance of renal allografts in MHC-mismatched nonhuman primates after nonmyeloablative conditioning and donor bone marrow transplantation. In this study, we extended our regimen to cardiac allotransplantation and compared the immunological responses of heart and kidney allograft recipients. METHODS: Five cynomolgus monkeys were conditioned with low-dose total body irradiation (1.5 Gy on days -6 and -5), supplemental thymic irradiation (7 Gy on day -1), antithymocyte globulin (50 mg/kg on days -2, -1, and 0), splenectomy (day 0), donor bone marrow transplantation (day 0), and a 4-week posttransplant course of cyclosporine. Heart allografts from MHC-mismatched donors were transplanted heterotopically on day 0. RESULTS: Two monkeys failed to develop multilineage chimerism and rejected their allografts soon after cyclosporine was stopped (postoperative days [PODs] 43 and 56). Three monkeys developed multilineage chimerism, which persisted 20 to 43 days posttransplant by flow cytometric analysis and to POD 124 by polymerase chain reaction analysis. Allograft survival in these recipients was prolonged to 138, 428, and 509 days, and in vitro mixed leukocyte reaction and cell-mediated lympholysis (CML) assays demonstrated donor-specific hyporesponsiveness. However, in contrast to kidney allograft recipients, long-term heart allograft recipients eventually developed humoral and cellular immunity against the donor and rejected the grafts. At the time of rejection, 1.3% to 9.5% of donor coronary arteries exhibited intimal proliferation. CONCLUSIONS: The induction of transient mixed hematopoietic chimerism leads to long-term heart allograft survival in MHC disparate monkeys without chronic immunosuppression. However, unlike kidney allografts, full tolerance to cardiac allografts was not achieved. Organ-specific modifications of the preparative regimen may be necessary to prevent the chronic cellular and humoral immune responses elicited by cardiac allografts.     

Donor cell engraftment in recipient lymphoid tissues after rat limb allograft

BACKGROUND: The movement of cells from a transplanted tissue into the host organs, the so-called systemic chimerism, is a phenomenon known to occur and be associated with the development of immunologic tolerance in allotransplantation cases. The purpose of this study was to identify donor cell engraftment in recipient lymphoid tissues after performing rat hind limb allograft. MATERIALS AND METHODS: Fifty-five whole-limb allotransplantations were performed in sex-mismatched pairs of rats. Syngeneic male Lewis and allogeneic Dark Agouti donors were transplanted to female Lewis recipients. FK506 was used for immunosuppression. Donor male cells could be identified in the recipient female tissues by semiquantitative polymerase chain reaction analysis for a Y chromosome-specific DNA sequence. Chimerism was assessed at 1, 24, and 48 weeks after transplantation. RESULTS: There was no rejection episode in any of the limb grafts. Although levels of chimerism were highly variable in each lymphoid tissue, a gradual increase was noticed in all during the course of time. At 1 week after the transplant period, only intrasplenic chimerism was at high level (1%) in three groups. At 48 weeks after the transplant, all recipients with allografts showed very high level (10%) of chimerism in the bone marrow. Two, two, and two of six recipients showed very high levels in the spleen, lymph node, and liver, respectively, at 48 weeks. Intrathymic chimerism was higher at 24 weeks after transplant rather than at 48 weeks. CONCLUSION: We demonstrated donor cell engraftment into recipient lymphoid tissues after successful whole limb transplantation. We conclude that limb allograft can work as a vascularized carrier for the bone marrow transplantation, provide a continuous source of donor cells and contribute to chimerism in the recipient.     

Total orthotopic small bowel allotransplantation in the dog. Features of atypical rejection and graft-versus-host reaction

The critical histologic review of our experience with small bowel allotransplantation in the dog is presented. While "classical" rejection with dense small cell infiltration and mucosal destruction does occur, more common is the "atypical" rejection reaction in which cellular infiltration is sparse. This "atypical" rejection was characterized by a significant decrease of mucosal epithelial structures with increased mitotic figures in crypt cells and frequent vascular changes, including segmental fibrinoid necrosis and thrombosis with or without overlying mucosal destruction. Substantial regenerating capacity of bowel mucosa tends to compensate for the destruction, complicating the histology. The host lymph nodes and spleen present a histologic picture highly suggestive of GVH reaction. The reduction in host lymphocytes in these organs from karyorrhexis with replacement by histiocytes and subsequently by plasma cells is described. It is emphasized that rejection and GVH are not mutually exclusive and occur simultaneously. The decrease of functional mucosal mass could well account for the nutritional malsequelae. Also, the weakening of immune structures on the host and the graft side may predispose to catastrophic enterogenous infections, perhaps explaining the large number of animals that die without overt signs of rejection following small bowel transplantation.     

Tolerance induction permits the development of graft-versus-host disease: donor-mediated attack following small bowel transplantation in mixed chimeras.

The induction of tolerance to organ allografts would eliminate acute and chronic rejection as well as the need for nonspecific immunosuppression. We have shown that tolerance induced through the creation of mixed allogeneic bone marrow chimeras allows for the long-term engraftment of cardiac and small bowel allografts across strong multiple major histocompatibility barriers. The possibility that tolerance might render the host susceptible to graft-versus-host disease (GVHD) has not been investigated in this or other models of tolerance. To test this possibility chimeras were created by transplantation of T-cell depleted ACI and Lewis bone marrow into lethally irradiated Lewis rats. Chimerism was determined post bone marrow transplant (BMTx) by flow cytometry of lymphocytes from reconstituted animals. ACI/Lew chimeras (ALC), Lewis/ACI F1 (LACF1), and Lewis (LEW) rats all received heterotopic ACI vascularized small bowel grafts. A second group of chimeras received small bowel grafts from ACI rats pretreated with low dose irradiation to eliminate T-cells from the graft. LEW-->LEW small bowel isografts were also performed. Animals were examined for evidence of GVHD by clinical signs and histologic examination of biopsied tissues. GVHD was quantified using the popliteal lymph node enlargement assay. All LACF1 rats developed severe lethal GVHD following ACI small bowel transplant. Bone marrow chimeras, ALC (n = 6), developed fatal GVHD in a similar fashion after receiving a small bowel transplant. LEW-->LEW isografts and chimeras receiving bowel from irradiated ACI rats survived long term without GVHD while ACI-->LEW allogeneic transplants all underwent acute rejection. GVHD or its absence was confirmed histologically. Popliteal lymph node enlargement indices reflected the presence of GVHD in the chimeras (1.87) and LACF1 (5.4) receiving allografts, but not in isografts or chimeras receiving irradiated allogeneic transplants. Analysis of cytokines in the tongues of rats undergoing GVHD showed elaboration of Th1 type proinflammatory cytokines which was not seen in isografted rats or rats receiving preirradiated small bowel. These results demonstrate that tolerance induction through mixed chimerism results in susceptibility to small bowel induced GVHD. Preirradiating the donor bowel prior to SBTx can prevent GVHD.     

Graft-versus-host reactivity and renal allograft survival in rats given allogeneic spleen cells or spleen allografts.

Selective recruitment of antigen-sensitive cells (ASC) into the spleen as a method of inducing specific suppression was attempted by intravenous injection of either DA or Lewis spleen cells 24 hr before a (DA X Lewis)F1 renal allograft into a Lewis or DA recipient, either with or without a splenectomy. This led to suppression of rejection in the DA recipient and delayed rejection in the Lewis recipient. Splenectomy produced a minimal augmentation effect. Assay of graft-versus-host (GVH) reactions in (DA X Lewis)F1 rats by a popliteal node assay showed that injection of allogeneic DA or Lewis spleen cells 48 hr before the assay significantly reduced the reaction produced by node lymphocytes but not spleen lymphocytes, suggesting a loss of ASC from the lymph nodes. Lewis spleen allografts did not produce such a significant reduction in the GVH reactivity of DA node lymphocytes as intravenous Lewis cells, whereas DA spleen allografts led to an increased GVH reactivity of Lewis node lymphocytes. From these studies, it is not possible to attribute the suppression produced by the intravenous injection of allogeneic cells to selective recruitment of antigen-sensitive cells to the spleen.     

Prolongation of allograft survival in ccr7-deficient mice

BACKGROUND: Lymphocyte homing to secondary lymphoid organs is thought to be required for initiation of the alloreactive immune response. Because CCR7 is the essential chemokine receptor responsible for lymphocyte and dendritic cell homing to secondary lymphoid organs, allograft survival was analyzed in CCR7-deficient (CCR7) mice. METHODS: Heterotopic heart and skin allotransplantation was performed in CCR7 and wild-type (WT) recipients. Graft survival was monitored daily. Grafts and draining lymph nodes were analyzed by immunohistology and flow cytometry at different time points. Groups of mice were splenectomized at the day of allotransplantation. RESULTS: A significant though modest prolongation of allograft survival in CCR7 recipients was observed for heart grafts (WT, 7.3 +/- 0.5 days; CCR7, 10.7 +/- 2.8 days) and skin grafts (WT, 8.9 +/- 0.9 days; CCR7, 12.3 +/- 0.9 days). This was accompanied by a delay in the cellular infiltration of allografts. T-cell accumulation and expansion in the draining lymph nodes in CCR7 recipients was severely impaired. Splenectomy had only a moderate prolongation effect on allograft survival in CCR7 mice. CONCLUSIONS: These results suggest that CCR7-dependent processes support allograft rejection yet are dispensable for the rejection response.     

Monoclonal antibody against beta7 integrins,but not beta7 deficiency,attenuates intestinal allograft rejection in mice

BACKGROUND: beta7 integrins mediate homing and retention of lymphocytes to the normal and inflamed small bowel in a tissue-selective fashion. In the present study, we investigated the expression of beta7 integrins after small bowel transplantation (SBT) and tested the effects of blocking beta7-mediated pathways by using monoclonal antibody (mAb) or knockout mice. METHODS: Heterotopic SBT from BALB/c to C57BL/6 (B6) was used as a surgical model. Expression of beta7 integrins was measured on recipient lymphocytes (CD4 and CD8 ) in spleen, blood, and mesenteric lymph nodes (MLN) using flow cytometry. To analyze the effects of blocking beta7 integrins on graft survival, either beta7-deficient B6 or wild-type B6 mice that were treated with mAb against beta7 were studied. RESULTS: After allogeneic SBT, there were markedly increased levels of alpha4beta7 recipient CD8 lymphocytes in MLN, blood, and spleen as early as 3 days postoperatively. In contrast, alpha4beta7 integrin levels in isograft recipients were similar to those of normal mice. Mean survival time of intestinal allografts was not affected by beta7 deficiency (7.3+/-1 days) compared with wild-type mice (7.5+/-0.8 days). However, mAb against beta7 integrins significantly prolonged graft survival (12.8+/-1 days) compared with treatment with control mAb (7.3+/-1 days, <0.001). Histologic changes of SBT rejection were significantly attenuated when mice were given mAb against beta7. CONCLUSION: As indicated by the increased levels of alpha4beta7 CD8 lymphocytes, activation of this integrin contributes to the immune response in SBT rejection. Furthermore, blocking beta7 integrins with mAb provides a suitable target for immunosuppressive therapy. The discrepancy in survival data obtained by mAb and beta7 deficiency may be a result of the more rapid activation of compensatory mechanisms in the knockout mice.     

UV-B modulation of haplo-identical bone marrow cells in the prevention of GVHD and induction of specific transplantation tolerance to intestinal allografts

Ultraviolet B (UV-B) irradiation of BM cells (BMC) prior to transplantation into lethally gamma irradiated allogeneic rats prevents graft-versus-host disease (GVHD), induces stable allogeneic chimerism and specific tolerance to donor-type allografts. This study evaluated the role of UV-B modulation of bone marrow transplant (BMT) in the prevention of GVHD in the parent (P)-to-F1 hybrid rat combination of Lewis-to-Lewis × Brown Norway F1 (LBNF1) and of subsequent tolerance to small bowel transplantation (SBT). Lethally gamma irradiated (10.5 Gy) LBNF1 recipients of unmodified Lewis BMT (admixture of 10⁸ BMC and 5 × 10⁶ spleen cells) developed lethal GVHD and died within 55 days. Similarly, groups of lethally irradiated LBNF1 recipients of Lewis BMT treated with 100–300 J/m² UV-B developed GVHD and died within 47 days. All F1 hybrid recipients of Lewis BMT treated with 700 or 900 J/m² of UV-B permanently accepted their BM grafts, gained weight, showed no clinical evidence of GVHD and survived for > 300 days. These stable chimeras expressed 94–98% donor T-cells in their lymph nodes and became tolerant to BM donor-type (Lewis) SBT when grafted 180 days after BMT. In contrast, similarly prepared F1 recipients rejected BN SBT, thus demonstrating donor specificity. The chimeric rats were specifically unresponsive to donor (Lewis) Ag in MLR while they maintained normal alloreactivity to BN stimulators. These results suggest that UV-B irradiation of BMT offers a promising approach to overcoming the limitations of T-cell depletion of BMT and may offer a useful approach for recipient conditioning for induction of transplantation tolerance.     

Long-term tolerance to kidney allografts in a preclinical canine model

Durable immune tolerance supporting vascularized allotransplantation offers the possibility of extending graft survival and avoiding harmful complications of chronic immunosuppression. Immune tolerance to renal allografts was induced in a preclinical canine model through engraftment of donor hematopoietic cells using a combination of low-dose total body irradiation and a short course of immunosuppression. Subsequently, donor renal allografts were transplanted accompanied by bilateral native nephrectomies. With 5-year follow up, we found normal renal function in all recipients and no histological evidence of acute or chronic rejection. This tolerance does not extend universally to donor skin grafts, however, with two of four animals rejecting delayed donor skin grafts. Hematopoietic chimerism produces durable and robust immune tolerance to kidney allografts, although incomplete tolerance to donor skin grafting.