Activation of transplant immunity: effect of donor leukocytes on thyroid allograft rejection.

The survival of thyroid allografts in mice was prolonged by either holding the grafts in vitro culture for 20 to 27 days or by cobalt-60 irradiation of the donor 2 days before transplantation with or without the intravenous injection of colloidal carbon just before removing the thyroid from the donor. In both cases the rejection process was restored by an intravenous injection of recipients with living peritoneal exudate cells (50 to 80 percent macrophages) syngeneic to the thyroid donor.

Thyroid allograft immunogenicity is reduced after a period in organ culture.

The survival time of mouse thyroid, transplanted under the kidney capsule of an H-2 incompatible recipient, is extended by holding the thyroid in organ culture for 12 days prior to transplantation.

The Th1/Th2 balance in autoimmunity.

The study of autoimmune disease in the context of T-helper type 1 (Th1) and T-helper type 2 (Th2) CD4+ T-cell responses demonstrates that the relative contribution of either T-cell type to the development of a particular autoimmune response can influence whether or not this response leads to clinical disease. Moreover, this influence can be quite different depending on whether the particular disease process is cell mediated or antibody mediated. Recent studies have demonstrated that the development of Th1 and Th2 responses may be significantly influenced by the costimulatory molecules recognized by responding CD4 T cells, and by other undefined factors in the genetic background. It has also been demonstrated that autoreactive Th2 CD4+ cells can regulate the activity of disease-causing Th1 CD4+ T cells in vivo. Control of autoimmune disease may thus be achieved by procedures that regulate the relative contribution of Th1/Th2 CD4 T cells to an autoimmune response.

Successful allotransplantation of mouse pancreatic islets to nonimmunosuppressed recipients.

Pancreatic islets from BALB/c (H-2d) mice are rejected within 14 days of transplantation to the kidney capsule of allogeneic, CBA/H (H-2k) recipients. Cyclophosphamide pretreatment of the islet donor reduced the intensity of the allograft response, and these islets undergo a more chronic rejection process. Islets from cyclophosphamide-pretreated donors can be cultured in a gas phase of 95% O2 and 5% CO2, provided the islets are aggregated into clusters of about 50 islets. After a culture period of 7--12 days, the islet tissue was transplanted to normal allogeneic recipients, where the tissue elicited little or no allograft response over a 3-mo observation period.

Islet allografts are destroyed by disease occurrence in the spontaneously diabetic BB rat.

Transplantation of cultured islet and pituitary tissue from PVG (RT1c) donors to major histocompatibility complex-incompatible BB/D recipients (RT1u) results in tissue-specific destruction of the grafted islets but not of the pituitary. We interpret this response as disease occurrence in the MHC-incompatible islet graft. Islet damage is associated with eosinophil and mast cell accumulation in and around the grafted tissue. Antibody deposition is also present in tissues of the BB rat. This is suggestive of an antibody-mediated allergic reaction.

Combined treatment with nicotinamide and desferrioxamine prevents islet allograft destruction in NOD mice.

Nonobese diabetic (NOD) mice get spontaneous diabetes with clinical and pathological manifestations similar to those seen in human type I diabetes. NOD mice will destroy transplants of treated allogeneic islet tissue by a recurrence of the disease process that destroyed the original islet tissue. This may be prevented by treatment of the animals with combined desferrioxamine and nicotinamide. Transplanted animals become normoglycemic and remain so for the duration of the treatment. This suggests that oxygen-derived free radicals may be involved in islet damage in spontaneous diabetes.

Reversal of diabetes in outbred mice by islet allotransplantation.

The combination of donor pretreatment with cyclophosphamide, organ culture in 95% O2:5% CO2 for 7-10 days, and short-term immunosuppression of recipients with cyclosporin A (CsA) were necessary to obtain 100% survival of single-cluster BALB/c islet allografts in outbred mice. In vivo and in vitro pretreatment of the donor tissue alone resulted in the acceptance of 45% of the islet allografts in nonimmunosuppressed outbred mice. CsA treatment of recipients alone yielded 40% survival of the untreated allografts. CsA treatment played an important role in maintaining the capacity of islet allografts to function in outbred mice. During CsA treatment, 88% of streptozocin-treated mice showed graft-dependent reversal of diabetes; the remainder showed no evidence of graft function, and CsA treatment failed to prevent acute graft rejection. After withdrawal of CsA immunosuppression, 38% of this total group remained normoglycemic. These findings suggest that modulation of both donor-tissue immunogenicity and recipient responsiveness will be required for successful pancreatic islet transplantation in diabetic humans.

CD8 T cells are not required for islet destruction induced by a CD4+ islet-specific T-cell clone.

A panel of CD4+ T-cell clones has been isolated from the spleen and lymph nodes of diabetic NOD mice. These clones have been shown to be islet-specific both in vivo and in vitro. One of the clones, BDC-6.9, initiates extensive damage to islet tissue when placed adjacent to an NOD islet graft that has been used to reverse diabetes in (CBA x NOD)F1 recipients or when injected intraperitoneally into such animals. In this study, we show that BDC-6.9 T cells can initiate islet destruction in the absence of detectable CD8 T cells either in the periphery or in the lesion that develops after the transfer of the cloned islet-reactive T cells.

Autoimmune diabetes in NOD mouse is L3T4 T-lymphocyte dependent.

Cultured BALB/c islets fail to function when transplanted into diabetic nonobese diabetic (NOD) mice; such grafted tissue is rapidly destroyed by disease recurrence. The cellular requirements for this graft damage are unclear. This study was designed to investigate the role of the L3T4+ T-lymphocyte subset in disease recurrence in the NOD mouse. L3T4+ T-lymphocytes were depleted by the in vivo administration of the L3T4-specific monoclonal antibody GK1.5. This treatment reduced the level of L3T4+ T-lymphocytes from an initial 43% of the peripheral blood lymphocytes to less than 4%. L3T4 levels remained at this low level for approximately 2 wk after withdrawal of GK1.5 treatment, after which the L3T4 levels slowly began to increase in the periphery. Grafting of cultured BALB/c islet tissue into GK1.5-treated diabetic NOD mice resulted in a rapid return to normoglycemia that persisted for 2-4 wk. The gradual return to the hyperglycemic condition roughly correlated with the reappearance of L3T4+ T-lymphocytes in the peripheral circulation. From these findings we conclude that the disease process in the NOD mouse is L3T4 T-lymphocyte dependent.

Syngeneic transplantation of cryopreserved fetal mouse proislets.

Proislets, derived from fetal mouse pancreata by collagenase digestion and subsequent organ culture, can be frozen to -196 degrees C and stored in a viable condition before successful syngeneic transplantation. Cryopreserved proislets are relatively undifferentiated morphologically, but continue to differentiate into mature islets after transplantation.

Desferrioxamine treatment prevents chronic islet allograft damage.

BALB/cByJ islet allografts are acutely rejected when transplanted into allogeneic mice (CBA/J). Culture of the tissue for 7 days in 95% O2 before grafting is a suboptimal treatment for the reduction of immunogenicity in this strain combination. Approximately half the animals reject these transplants in a chronic fashion. Chronic islet rejection differs from acute rejection of uncultured allogeneic islets. During chronic rejection, beta cells within the transplanted tissue degranulate but remain intact when the animal returns to the diabetic condition. Acute islet rejection is characterized by the destruction of beta cells that remain heavily granulated as long as they remain intact. We examined the effect of the iron chelating agent, desferrioxamine, on chronic islet allograft damage. Desferrioxamine inhibited chronic islet allograft damage but did not influence the process of rejection of uncultured islet tissue. This effect of desferrioxamine could not be attributed to a direct immunosuppressive effect of this agent.

The reversal of diabetes by pancreatic islet transplantation.

In this paper the theoretical basis of alloreactivity and its relevance to transplantation biology is discussed prior to a review of work showing that culture of adult mouse pancreatic islets for 7 days in 95% O2 and 5% CO2 facilitates successful grafting to nonimmunosuppressed allogeneic recipients. These allografts function by reversing both chemically induced and spontaneous diabetes. The fetal mouse pancreas is more immunogenic than adult islets, and even after a culture period of 10 days in 95% O2 and 5% CO2, BALB/c allografts are consistently rejected by nonimmunosuppressed recipient mice. The immunogenicity of fetal pancreas is thought to be due to the presence of contaminating lymphoreticular cells in the mesentery surrounding the fetal pancreas. Digestion of the fetal pancreas with collagenase allows the isolation of proislets that develop into functional islet tissue on transplantation. Fetal proislets are less immunogeneic than the whole fetal pancreas and may provide a source of tissue for clinical transplantation. Established islet allografts are relatively stable and are not rejected following nonspecific stimulation of the recipient's immune system or following passive transfer of either antibody or antibody and complement. After prolonged residence in the recipient a state of allograft tolerance develops and such grafts resist rejection by specific stimulation of the recipient. The administration of donor antigen in the form of uv-irradiated cells enforces this state of allograft tolerance.

Cellular changes in cultured mouse thyroid glands and islets of Langerhans.

Islets of Langerhans cultured 7 days in vitro no longer contained any capillary endothelial cells, but their endocrine cells remained ultrastructurally normal up to 14 days. Vascular endothelial cells were also lost from cultured thyroid lobes, but more slowly. Thyroid endothelium was readily identified after 7 days of culture, although many cells appeared to be degenerating, and a few degenerating endothelial cells were still present after 14 days in culture. Erythrocytes and leukocytes in the lumina of thyroid vessels were observed to degenerate at about the same rate as the endothelial cells, while those in islet capillary lumina were largely washed out during isolation of the islets. Thyroid lymphatic endothelium and the numerous adipose cells present in this tissue also degenerated during the culture period. Follicle epithelial cells remained viable throughout the culture period, but the number of colloid droplets and endocytic vesicles they contained was markedly, decreased. Thyroid fibroblasts remained viable and appeared to enlarge and accumulate dense granules during culture. These cells were a prominent feature of thyroid lobes after 14 days of culture. Parathyroid tissue associated with the thyroid lobes showed viable endocrine cells but a loss of vascular endothelium after 14 days in culture. The loss of blood leukocytes and vascular endothelial cells in probably the major factor in the altered behavior of thyroid and islet allografts after culture in vitro.

Effect of organ culture on the survival of thyroid allografts in mice.

Mouse thyroid can be maintained in organ culture for 4 weeks. Uncultured BALB/c thyroid is rejected 10-15 days after transplantation under the kidney capsule of H-2 disparate recipients (C57BL, CBA). Organ culture of thyroid tissue prior to transplantation prolongs allograft survival. This prolongation of graft survival increases with increasing time in culture and 80-90% of BALB/c thyroids maintained in culture for 26 days survive in allogeneic CBA recipients for a 60- to 70-day test period. These allografts show normal function as measured by 125I uptake, and show no histological evidence of chronic rejection. Cultured allografts can be rejected if the host's immune system is stimulated with viable leukocytes of donor origin. Host animals carrying a functioning allograft are not tolerant of donor tissues and will reject a second uncultured allograft from the same donor strain.

Modulation of tissue immunogenicity by organ culture. Comparison of adult islets and fetal pancreas.

Uncultured mouse islet allografts (BALB/c to CBA) are rejected 2 to 4 weeks after transplantation. Allografts, cultured in 95% O2 and 5% CO2 for 7 days before transplantation, show no sign of rejection up to 3 months post-transplantation. However, the cultured allografts are rejected if the CBA recipient is given an i.v. injection of 10(5) peritoneal cells at the time of transplantation. Organ culture of BALB/c fetal pancreas (16 to 17 days gestation) under the same conditions failed to prevent allograft rejection. The immunogenicity of fetal pancreas is reduced if this tissue is cultured in 95% O2 and 5% CO2 for 17 days before transplantation.

Evidence of recurrent autoimmunity in human allogeneic islet transplantation.

We transplanted 10,000 isolated, handpicked human pancreatic islets into the subfascial compartment of the forearm muscle of a type I diabetic recipient who had received a successful renal transplant one year prior. The recipient was maintained on his usual immunosuppressive therapy of cyclosporine, azathioprine, and prednisone. A biopsy performed 7 days after transplantation showed normal islets with both insulin- and glucagon-staining cells present and no lymphocytic infiltration. A second biopsy performed 14 days after transplantation showed a dense mononuclear cell infiltrate with a preferential loss of insulin-staining cells relative to glucagon-staining cells in the islets. These data are consistent with recurrent autoimmune diabetes in an isolated islet allograft in an immunosuppressed type I diabetic recipient. In addition, this forearm subfascial site may be a useful means to monitor islet rejection and autoimmune recurrence in therapeutic intraportal islet allografts.

Facilitation of specific tolerance induction in adult mice by RS-61443.

RS-61443 is an immunosuppressive agent that facilitates pancreatic islet allograft acceptance in two mouse strain combinations (BALB/c----CBA and C57Bl/6J----BALB/c). A remarkable feature of this agent is its ability to facilitate long-term graft acceptance after a short (30 days) period of treatment; following withdrawal of the agent 40-70% of islet allografts are maintained for an indefinite period. This long-term graft acceptance has been shown to result from specific tolerance induction in the recipient animal. The state of specific tolerance is an active rather than a passive form of tolerance, such as clonal deletion or anergy. Active tolerance induction is cyclosporine-sensitive, although cyclosporine enhances graft acceptance when used in combination therapy with RS-61443, this agent inhibits tolerance development under the influence of RS-61443.

Effect of cyclosporine on immunologically mediated diabetes in nonobese diabetic mice.

Spontaneous diabetes in NOD mice has an immunologically mediated cause and is a T cell-dependent process. When diabetic NOD mice are grafted with cultured BALB/c islet tissue, the islet graft is destroyed by disease recurrence in the graft. Disease recurrence is a CD4 T cell-dependent process as determined by in vivo administration of anti-CD4 or anti-CD8 monoclonal antibody prior to the grafting of islet tissue. Cyclosporine functions in the early sequence of T cell activation by regulating the production of messenger RNA for lymphokines synthesis. Cyclosporine does not inhibit the synthesis of lymphokine once the lymphokine message is present in the cell. Thus, we might expect cyclosporine to be relatively inefficient as an agent for the regulation of disease recurrence following transplantation to actively diabetic recipients, and we would expect cyclosporine to be more effective when administered before the onset of the disease. Low-dose cyclosporine treatment can prevent development of the disease when the drug is administered before the onset of disease. Data presented here show that cyclosporine is ineffective in controlling disease recurrence in the islet graft transplanted to actively diabetic animals. Also, when we eliminate CD4 T cells from the diseased animals and graft islet tissue prior to the administration of cyclosporine, we are unable to maintain a graft with low-dose cyclosporine therapy. This result leads us to conclude that, although anti-CD4 treatment controls the expression of the disease process and allows the survival and function of the islet graft, this treatment does not return diseased animals to the prediabetic condition in which the development of diabetes can be controlled by low-dose cyclosporine therapy.

Blocking of delivery of the antigen-mediated signal to the nucleus of T cells by cyclosporine.

Cyclosporine (CsA) inhibits release of interleukin 2 (IL-2) and hemopoietic growth activities such as interleukin 3 (IL-3) from major histocompatibility complex (MHC)-antigen-activated T cells. Production of both lymphokines appears to be coordinately regulated; the antigen dose response, T cell dose response, and time course of lymphokine appearance are similar. The triggering of lymphokine production by these cells is solely dependent on T cell-target cell interaction, as the T cell dose response curve indicates that no cooperation occurs between T cells, and any metabolic contribution by the target cell was eliminated by ultraviolet irradiation. This interaction triggers the transcription of lymphokine-encoding mRNA. The process of lymphokine release can be divided into 4 steps: Antigen binds to the T cell; a signal is transferred to the cell nucleus; transcription of lymphokine-encoding mRNA occurs; and intact lymphokine is synthesized and secreted. CsA inhibits antigen triggered lymphokine production. However, it does not inhibit lymphokine release from the constitutively producing tumor cell lines WEHI-3 (which releases IL-3) and MLA 144 (which produces IL-2). Thus CsA has no effects on the lymphokine secretion process or any direct action upon lymphokine-coding mRNA. CsA does not affect antigen recognition during cell-mediated cytotoxicity. Therefore, CsA acts after antigen binding and before transcription of lymphokine-encoding mRNA. That is CsA blocks the transmission of the antigen signal. This information is used to show that this CsA-sensitive signal is required continuously to maintain the T cell in a lymphokine-secreting state.

Cultured thyroid allografts induce a state of partial tolerance in adult recipient mice.

Thyroid allografts (BALB/c) prepared for transplantation by cyclophosphamide pretreatment of the donor, followed by organ culture of donor tissues for 3 weeks in a gas phase of 95% O2-5% CO2 function in normal CBA recipient mice for greater than or equal to 350 days. Up to 100 days post-transplantation, the tissue can be rejected by challenge of the recipient with 10(5) BALB/c peritoneal cells. After prolonged residence in the recipient (greater than 100, less than or equal to 350 days), only a proportion of allografts are rejected when the recipient is challenged with 10(5) followed by 10(6) peritoneal cells of donor origin. Recipients of long-term allografts are partially tolerant of BALB/c tissues. They are hyporesponsive to in vivo challenge with BALB/c spleen cells. However, lymph node cells from these animals respond essentially normally to stimulation with BALB/c spleen cells in vitro. Tolerant recipients will accept a second uncultured BALB/c allograft after a transitory rejection crisis. This crisis is not observed in the primary allograft.