Skin Allograft Acceptance Research Articles

INDUCTION OF TOLERANCE TO SKIN ALLOGRAFTS BY INTRATHYMIC INJECTION OF DONOR SPLENOCYTES

The effect of donor-recipient strain combination and supplemental rapamycin (Rapa) on tolerance induction by intrathymic (IT) injection of donor splenocytes was examined in a mouse skin allograft model. In an MHC class I-mismatched C3H/He skin to (C57BL/6 x A)F1 (B6AF1) mouse combination, IT injection of 50 x 10(6) donor splenocytes with transient immunosuppression by rabbit anti-mouse lymphocyte serum (ALS) induced significant prolongation of skin allograft survival with a median survival time (MST) of 115 days versus an MST of 24.5 days in controls given ALS alone. With an additional short course of supplemental Rapa treatment at a dose of 1.5 mg/kg i.p. every other day from day 0 to 12, all C3H/He skin allografts survived indefinitely (> 350 days) in ALS-treated, donor splenocyte intrathymically injected B6AF1 recipient mice. Tolerance was antigen-specific, since the second donor-type skin allografts were accepted while third-party skin allografts were acutely rejected in these mice bearing long-term C3H/He skin allografts. In MHC class I- and II-disparate (DBA/2 to B6AF1) and fully MHC-incompatible (AKR to B6) strain combinations, IT injection of donor splenocytes and ALS treatment failed to prolong skin allograft survival over ALS controls. When supplemental Rapa was used, long-term skin allograft acceptance was observed with an MST of 127 days for the DBA/2 to B6AF1 combination and 70 days for the AKR to B6 combination. In contrast, supplemental treatment with cyclosporine was not effective in these combinations, which suggests that supplemental Rapa may have a unique effect in augmenting IT tolerance induction. Thymectomy within 7 days after IT injection significantly shortened the allograft survival, which suggests that interaction of the host thymus and the injected donor splenocytes, which takes place early after IT injection, plays an important role in the induction of allograft tolerance in this model.

Induction of donor-specific transplantation tolerance to skin and cardiac allografts using mixed chimerism in (A + B-->A) in rats.

Mixed allogeneic chimerism (A + B-->A) was induced in rats by reconstitution of lethally irradiated LEW recipients with a mixture of T-cell depleted (TCD) syngeneic and TCD allogeneic ACI bone marrow. Thirty-seven percent of animals repopulated as stable mixed lymphopoietic chimeras, while the remainder had no detectable allogeneic chimerism. When evaluated for evidence of donor-specific transplantation tolerance, only those recipients with detectable allogeneic lymphoid chimerism exhibited acceptance of donor-specific skin and cardiac allografts. Despite transplantation over a major histocompatibility complex (MHC)- and minor-disparate barrier, animals accepted donor-specific ACI skin and primarily vascularized cardiac allografts permanently, while rejecting third party Brown Norway (BN) grafts. The tolerance induced was also donor-specific in vitro as evidenced by specific hyporeactivity to the allogeneic donor lymphoid elements, yet normal reactivity to MHC-disparate third party rat lymphoid cells. This model for mixed chimerism in the rat will be advantageous to investigate specific transplantation tolerance to primarily vascularized solid organ grafts that can be performed with relative ease in the rat, but not in the mouse, and may provide a method to study the potential existence of organ- or tissue-specific alloantigens in primarily vascularized solid organ allografts.

Ontogeny of tolerogen-responsive lymphocytes following neonatal inoculation of class II disparate semiallogeneic cells

Spleens of adult mice of the A strain background that were rendered tolerant as neonates of class II alloantigens only (ATH tolerant of A.TL, A.TL tolerant of A.TH) contain large numbers of tolerogen-responsive T cells, many of which secrete IL-4, but not IL-2. Since these spleens also contain suppressor cells that can adoptively transfer skin allograft acceptance in vivo and can prevent generation of class II-specific cytotoxic T cells in vitro, it is important to determine the origins during postnatal development of these cells. Class II disparate, semiallogeneic haematopoietic cells were injected into newborn A.TH and A.TL mice. Periodically thereafter (1 to 60 days post-injection, but prior to challenge with a tolerogen-bearing skin graft), thymocytes and splenocytes from these mice were examined in vitro for tolerogen-specific reactivity in mixed lymphocyte reactions during which proliferation and IL-2 and IL-4 production were assayed. Within 24 hours of neonatal injection, the thymus and spleens of injected mice were profoundly depleted of tolerogen-responsive T cells. However, there was no commensurate loss of I-E-related Vβ5 + cells in the thymus of A.TH mice that received neonatal inoculations of I-E-bearing A.TL cells. During the ensuing weeks, tolerogenresponsive proliferative and IL-4-secreting T cells were detected in thymus and spleen. However, not until after the mice were more than 60 days of age were tolerogen-responsive cells able to secrete IL-2. Since physical clonal deletion of tolerogen-related Vβ5 + cells is a characteristic of neither neonatal nor adult A.TH and A.TL mice that received injections of semiallogeneic cells at birth, and since tolerogenresponsive IL 4 producing cells exist in adult mice that have permanently accepted (A.TH x A.TL)F 1 skin grafts, our results imply that the tolerogen-responsive T cells detected in adult tolerant mice are descendants of the novel IL-4-producing T cells that arise in the thymus almost immediately after the tolerance conferring inoculum of semiallogeneic cells. The possible mechanisms responsible for generation of IL-4-producing, tolerogen-responsive T cells and the role of these cells in maintenance of tolerance of class II alloantigens are discussed.

Prolonged survival of rat orthotopic liver allografts after intrathymic inoculation of donor-strain cells.

Permanent donor-specific tolerance to tissue or organ allografts can be readily achieved without immunosuppression by administration of donor lymphohematopoietic cells to neonatal rodents. In adult recipients, however, induction of transplantation tolerance by this strategy generally requires intensive cytoablative conditioning of the recipient. We have now demonstrated that intrathymic inoculation of donor bone marrow or hepatic cells in conjunction with a single dose of antilymphocyte serum is effective in prolonging survival of DA rat orthotopic liver allografts in LEW strain recipients, which ordinarily rapidly reject such transplants. The unresponsive state achieved is donor-specific, as evidenced by the failure of intrathymic inocula of third-party WF cells to promote survival of LEW recipients of orthotopic DA liver allografts. Moreover, intravenous administration of the donor cells fails to extend liver allograft survival, demonstrating that the inoculum must be present in the thymus to promote unresponsiveness. Established DA liver allografts induced a state of systemic tolerance in LEW hosts, allowing their subsequent acceptance of donor-strain skin allografts. We hypothesize that the unresponsive state achieved by intrathymic inoculation of donor cells may result from the deletion or functional inactivation of alloreactive clones in a thymus bearing donor alloantigens. In this regard, cells of the macrophage/dendritic lineage (descendants of the bone marrow inoculum or hepatic Kupffer cells) may play a critical role by promoting thymic microchimerism and exerting modulatory effect on T cell development.

Management of subdural empyema: a series of 24 cases.

Twenty four cases of subdural empyema are reviewed. The overall mortality was 17%, 18 patients were managed by burr hole and five by craniotomy or craniectomy. Antibiotic therapy was commenced once pus had been evacuated. Infection of the paranasal and mastoid sinuses was the commonest aetiological factor. Aspiration of pus through burr holes is the recommended surgical procedure with low mortality and morbidity, when combined with early diagnosis and aggressive chemotherapy.

Thymocyte-dependent immunity to toxoplasmosis in the normal and immunocompromised guinea-pig host.

Guinea-pigs made T-cell deficient by thymectomy and irradiation, and protected with syngeneic bone marrow cells (TXB) have a greatly reduced capacity to express normal cell-mediated immune functions, based on their poor responses to T-cell mitogens, prolonged acceptance of skin allografts, and susceptibility to the lethal effects of graft-versus-host disease. Further evidence for impaired T-cell activity in TXB guinea pigs was based on their inability to be fully sensitized to mycobacterial antigens, and increased susceptibility to an intradermally induced infection with the intracellular protozoan parasite, Toxoplasma gondii (RH strain). After challenge at multiple sites with 10(6) or 10(5) parasites, toxoplasmosis in thymus-intact, fully immunocompetent guinea pigs is a self-limiting and survivable infection, whereas the disease takes an acutely lethal course in the majority of TXB guinea-pigs. The latter also had more parasites disseminating to various tissues sites than their euthymic counterparts. The reduced capacity of TXB guinea-pigs to respond to mycobacterial products, and to generate anti-Toxoplasma immunity can be restored by an intravenous infusion of normal syngeneic thymocytes. These findings provide substantial direct evidence strengthening the concept that protection against toxoplasmosis is heavily dependent upon an intact T-cell component of the host's immune response.

Reversal of cyclosporine-induced mortality with a synthetic polymeric immunostimulant in a murine model of fecal peritonitis.

We have been investigating the effects of a synthetic immunostimulative polymer known as copovithane (Cpv). This agent appears to enhance humoral immunity in untreated and cyclosporine-immunosuppressed mice and is nontoxic in rodents and man. The purpose of this study was to determine whether cyclosporine (CsA) is deleterious to survival in a murine cecal ligation, puncture, and excision (CLPE) model of fecal peritonitis, and--if so--whether this effect could be ameliorated by Cpv without interfering with skin allograft acceptance. Cpv significantly prolongs survival in the CLPE model; the optimal dose for this effect was found to be 100 mg/kg. CsA was found to have a significant and deleterious effect on survival at several dosage levels when administrated 48 and 24 hr before cecal ligation, and immediately before and 16 hr after cecal ligation. Using a dose of CsA sufficient for skin allograft acceptance and the same schedule of administration outlined above, Cpv 100 mg/kg was administered 48 hr prior to cecal ligation. Mice treated with CsA plus Cpv had significantly longer survival than mice treated with CsA alone; furthermore, the survival of CsA-plus-Cpv-treated animals was not significantly different from that of saline-treated controls. Acceptance and survival of H-2 incompatible skin allografts in mice treated with CsA were not affected by Cpv 100 mg/kg/week. We conclude that CsA-induced mortality in the CLPE model can be abrogated by Cpv without adversely affecting skin allograft survival. It may eventually be possible to reduce the incidence of septic complications in clinical allotransplantation by prophylactically administering Cpv to patients on CsA immunosuppression.

DRUG-INDUCED TOLERANCE TO ALLOGRAFTS IN MICE

When AKR mice were primed with viable C57BL/6(B6) spleen cells and treated with cyclophosphamide 1-3 days later, a profound tolerance to B6 tumor allografts was induced. The tolerant state was maintained completely for as long as 8 weeks. Although tumor allografts grew progressively even when inoculated after complete rejection of skin grafts, B6 skin grafts were rejected by tolerant mice. In mice tolerant of B6 tumors, production of cytotoxic antibody and cytotoxic activity was reduced profoundly, but the delayed-type hypersensitivity level decreased only slightly. We therefore presume that the decrease in cytotoxic activity may allow progressive growth of tumor allografts, but the maintenance of delayed-type hypersensitivity or a low level of cytotoxicity, or both, precludes acceptance of skin allografts.

Humoral and cell-mediated immune responses in chickens with infectious bursal disease

Primary and secondary immune responses to Newcastle disease virus (NDV) was evaluated in chickens infected with infectious bursal disease virus (IBDV) at one and 28 days of age. The geometric mean primary hemagglutination-inhibition antibody titers (GMT) of chickens infected with IBDV at one day of age was significantly lower ( P ⩽ 0.01) than those infected at 28 days of age. Infection with IBDV had no influence on secondary immune response to NDV. The effect of IBDV infection at one day of age on the cell-mediated immunity of chickens was evaluated by skin allograft acceptance or survival time. There was no significant difference between the percentage of grafts accepted in IBDV infected and noninfected control chickens. However, the mean graft survival time in the IBDV infected chickens was significantly longer ( P ⩽ 0.05) than those in the control group. This suggested a suppression of cell-mediated immunity due to IBDV infection.

Immune Response to Immunization Via the Anterior Chamber of the Eye

Abstract The immunizing abilities of alloantigens placed within the anterior chamber of the eye have been studied in inbred rats. Although intracameral inoculation of F1 hybrid lymphocytes into parental strain recipients elicited both cell- and antibody-mediated immunity, a delimited interval was identified postinoculation during which the systemic cell-mediated immune response was suppressed as indicated by prolonged acceptance of orthotopic skin allografts. The prompt appearance of hemagglutinating antibodies in the serum of immunized rats followed a time course which coincided with the suppression of cell-mediated immunity and suggested that the two events are causally related. Since exposure to allogeneic antigens on lymphoid cells via the anterior chamber elicits a transient suppression in cell-mediated immunity, whereas humoral immunity is preserved, the phenomenon resembles immune deviation.

Characteristics of Disparate Histocompatibility Barriers in Congenic Strains of Mice

Abstract Characteristics of specific immunologic suppression were investigated in C57BL/10 congenic mouse strain combinations disparate at non-H-2 loci as a function of strong, moderate, and weak incompatibilities. These immunogenetic barriers were defined by both graft-vs-host reactivity and distinctive skin allograft survival times. Intraperitoneal injection of neonates with varying doses (2 × 106 to 20 × 106) of allogeneic spleen cells led to degrees of subsequent donor skin allograft acceptance which were closely dependent upon the interallelic combination. However, long-term skin allograft acceptors consistently produced serum alloantibodies against donor cells detectable in vitro by both cytotoxicity and hemagglutination-migration assays. The effectiveness of this specific antibody-associated suppression of transplantation immunity suggestive of enhancement was inversely proportional to the strength of the immunogenetic barrier and directly proportional to donor cell dosage. Increased immunogenetic incompatibility required increased dosage of allogeneic cells to induce specific suppression. The weaker the incompatibility the higher the incidence of effective allogeneic suppression and of concomitant alloantibody production obtained at lower antigen dosage. Coexistence of suppression of transplantation immunity (thymus-derived cell pathway blocked) with continuing anti-donor alloantibody production (bone marrow-derived cell pathway open) was found to be characteristic of the allograft-tolerant state.