Development of donor-specific immunoregulatory T-cells after local CTLA4Ig gene transfer to pancreatic allograft

BACKGROUND: CTLA4Ig gene transfer directly to graft tissue might have the potential to avoid the need for systemic immunosuppression. In our previous studies of bio-breeding (BB) rats, local adenovirus-mediated CTLA4Ig gene transfer protected the pancreas from autoimmune and alloimmune responses. This study investigated the potency of local CD28/B7 costimulatory blockade for induction of donor-specific tolerance and further examined the existing mechanisms. METHODS: Brown Norway (BN; RT1)-pancreaticoduodenal grafts transfected with Ad.CTLA4Ig via intraarterial ex vivo perfusion were transplanted into streptozotocin-induced diabetic Lewis (LEW; RT1) rats. RESULTS: Ad.CTLA4Ig transduced grafts combined with a short course of FK506 resulted in indefinitely prolonged survival (>156 days vs. 19.5 days with FK506 alone). CTLA4Ig was predominantly expressed in grafts on day 4. The expression was gradually diminished and was only slightly detectable at day >100. The proliferative responses against BN antigen were remarkably enhanced among recipients with rejected grafts, but the T-cells from tolerant recipients (>100 days) showed poor cytotoxic responses. On adoptive transfer assay, the splenic T-cells of tolerant recipients were able to suppress the rejection of BN, but not third-party Wistar Furth (WF; RT1) hearts in irradiated (480 cGy) LEW recipients. The percentage of CD4CD25 splenic T-cells was significantly increased in tolerant recipients (13.53 +/- 4.06% vs. 6.06 +/- 0.56% in naive rats). CONCLUSION: CTLA4Ig gene transfer to the pancreaticoduodenal allograft combined with a short course of FK506 induces donor-specific tolerance. The mechanism of maintaining tolerance could be explained by development of splenic T suppressor cells.     

Prevention of recurrent autoimmune diabetes in BB rats by anti-asialo-GM1 antibody

BB rats exhibit a syndrome of spontaneous diabetes that has clinical and pathological characteristics analogous to those found in human insulin-dependent diabetes mellitus (IDDM). Islet tissue transplanted into spontaneously diabetic BB rats is uniformly destroyed by a recurrence of the autoimmune response that destroyed the diabetic subject's native islets. To examine recurrent autoimmune destruction of transplanted islets, it is necessary to exclude islet damage that might result from allograft rejection. We utilized neonatal tolerance induction to prevent rejection of Wistar-Furth (WF) (RT1u) islet allografts by spontaneously diabetic BB recipients. We determined that islet-recipient treatment with anti-asialo-GM1 (anti-AGM1) antibody prevents recurrent autoimmune diabetes that would otherwise destroy transplanted WF islet grafts. Anti-AGM1 therapy significantly decreased peripheral blood natural killer (NK) cell activity. These data suggest a role for NK cells in the pathogenesis of recurrent diabetes in neonatally tolerant BB rats.     

Prevention of recurrent autoimmune diabetes in the BB rat by islet transplantation under the renal capsule.

Pancreatic islet grafts transplanted into subjects with spontaneous autoimmune diabetes are threatened by two immune responses, allograft rejection and the recurrence of autoimmune insulitis. To examine the recurrent autoimmune response to transplanted islets it is necessary to exclude islet allograft rejection. The BB rat is a unique model of spontaneous diabetes with clinical and pathological characteristics identical or similar to those found in human insulin dependent diabetes mellitus (IDDM). In this study we demonstrate permanent acceptance of histocompatible islet grafts in chemically induced diabetes and a lack of intracolony tissue antigen rejection in our BB rat colony. Therefore the vigorous destruction of transplanted BB islets in the liver of spontaneously diabetic BB rats is due to recurrence of diabetes. This recurrence can be prevented by transplantation of islets under the renal capsule. This may be important for clinical application in IDDM, particularly with regard to host and donor tissue matching.     

Autoimmune destruction of islets transplanted into RT6-depleted diabetes-resistant BB/Wor rats

We report a novel animal model of islet transplantation that distinguishes recurrence of autoimmunity from allograft rejection. In this study, diabetes-resistant (DR) BB rats, less than 1% of which develop spontaneous diabetes, were made hyperglycemic by either a single injection of streptozocin (STZ) or in vivo immune elimination of a regulatory T-lymphocyte subset that expresses the RT6 alloantigen. DR islet grafts were then transplanted into both groups. DR transplants into STZ-induced diabetic DR rats produced long-term normoglycemia. In contrast, DR transplants into DR rats that had been treated with anti-RT6 monoclonal antibody were all destroyed within an average of 4 days. Allogeneic islets transplanted into both STZ-induced and RT6-depleted diabetic DR rats were rejected within a mean of 3 days. We conclude that failure of DR islet grafts in RT6-depleted diabetic DR BB rats represents recurrent autoimmunity.     

Major-histocompatibility-complex restricted and nonrestricted autoimmune effector mechanisms in BB rats

To investigate whether the immunologic mechanisms of autoimmune pancreatic beta-cell destruction are MHC restricted, we examined the relative vulnerability of islet allografts from a panel of MHC-compatible and -incompatible donors to autoimmune damage after transplantation to spontaneously diabetic BB recipients. To circumvent a potentially confounding allograft response to the foreign islet graft, we utilized two strategies: (1) pretransplant in vitro culture of islets to delete intraislet APCs; and (2) induction of islet donor-specific immunologic tolerance in diabetes-prone BB rats. Experiments employing organ culture to prevent rejection demonstrated that MHC-incompatible grafts were significantly less vulnerable to autoimmunity than MHC-compatible grafts. In contrast, when we used the model of immunologic tolerance to exclude rejection, both MHC-compatible and -incompatible islet grafts were equally susceptible to autoimmune damage. The reason for this discrepancy has not been defined fully but may be related to our observation that tolerant BB animals exhibit increased peripheral blood NK-cell activity. NK cells are known to be cytotoxic to islets in vitro and could play a role in a non-MHC-restricted diabetogenic response in vivo. We conclude that both MHC-restricted and nonrestricted mechanisms are capable of contributing to anti-beta-cell autoimmunity in BB rats.     

Ex vivo and systemic transfer of adenovirus-mediated CTLA4Ig gene combined with a short course of FK506 therapy prolongs islet graft survival

Adenovirus-mediated CTLA4Ig gene transfer has been reported to enhance graft survival in several rodent transplantation models. In this study, we investigated the efficacy of ex vivo and systemic transfer of the CTLA4Ig gene by adenoviral vectors in pancreatic islet allo-transplantation. Islet grafts from BN rats were transplanted to chemically induced diabetic LEW rats. First, ex vivo CTLA4Ig gene transfer into isolated islets was performed prior to transplantation. Survival of transduced grafts under the kidney capsule was slightly prolonged (8.6+/-1.3 days) compared with survival of untransduced grafts (6.7+/-1.2 days); when combined with a short course of FK506, graft survival was further extended (32.6+/-10.7 days vs. 13.7+/-1.0 days with FK506 alone). Secondly, systemic gene transfer was accomplished by intravenous administration immediately after the transplantation procedure. In these animals, islet grafts under the kidney capsule survived longer (15.2+/-3.3 days) than in controls (6.7+/-1.2 days), and when FK506 was administered perioperatively, all the islet grafts survived for more than 100 days. In systemically transduced recipients, the survival of islet grafts transplanted into the liver was not significantly different from that of the grafts placed under the kidney capsule. In order to examine organ-specific immunogenicity, heterotopic BN cardiac grafts were transplanted to LEW rats intra-abdominally, with the virus transferred systemically as in the islet model. In contrast to the islet grafts, all the cardiac grafts were accepted for longer than 100 days, even without FK506 therapy. Finally, the LEW recipients with long-surviving islet or cardiac grafts were re-transplanted with islet grafts from the same donor strain (BN) on day 100. The second islet grafts survived longer than 100 days in half of the cardiac recipients, but consistently failed in the islet recipients. We conclude that in this transplant model, CTLA4Ig gene transfer and FK506 treatment synergistically improved islet graft survival, systemic transfer of the gene was more effective than ex vivo transfer to the islets, and donor-specific tolerance could not be achieved for islet transplantation but was achieved for cardiac transplantation.     

Transplantation of islets transduced with CTLA4-Ig and TGFbeta using adenovirus and lentivirus vectors

BACKGROUND: A major problem facing islet transplantation is immune destruction of grafts by transplant rejection and autoimmunity. Some success in prolonging graft rejection has been obtained by transducing islets prior to transplantation with adenoviral (Ad) vectors containing CTLA4-Ig and TGFbeta. The purpose of this study was to see if lentiviral (LV) vectors would provide superior results compared with adenoviral vectors. METHODS: Islets were isolated from Sprague-Dawley rats and transduced with Ad or LV vectors containing LacZ, CTLA4-Ig, CTLA4, and TGFbeta1 using various MOIs. Islets transduced with LV were healthy as judged by DNA and insulin content, and insulin secretion. Using the kidney capsule transplant site, 500 transduced rat islets were transplanted into streptozotocin diabetic B6AF1 mice. RESULTS: Maintenance of normoglycemia was prolonged in recipient mice carrying islets transduced with Ad vectors containing CTLA4-Ig, CTLA4, and TGFbeta1. Return of hyperglycemia in controls was 17-18 days while loss of function for the experimental groups occurred at 20-27 days. For the lentivirus transduced islets, rejection of controls was 20+/-1.6 days, for CTLA4-Ig was 42+/-21 days and for TGFbeta was 28+/-3.2 days. CONCLUSIONS: Although islets transduced with either adenovirus or lentivirus containing CTLA4-Ig, CTLA4, and TGFbeta1 could prolong graft survival in a rat to mouse transplantation model, with the conditions of this study lentivirus provided no advantage over adenovirus vectors.     

Intrathymic islet transplantation in the spontaneously diabetic BB rat.

Recently it was demonstrated that pancreatic islet allografts transplanted to the thymus of rats made diabetic chemically are not rejected and induce specific unresponsiveness to subsequent extrathymic transplants. The authors report that the thymus can also serve as an effective islet transplantation site in spontaneously diabetic BB rats, in which autoimmunity and rejection can destroy islets. Intrathymic Lewis islet grafts consistently reversed hyperglycemia for more than 120 days in these rats, and in three of four recipients the grafts promoted subsequent survival of intraportal islets. In contrast intraportal islet allografts in naive BB hosts all failed rapidly. The authors also show that the immunologically privileged status of the thymus cannot prevent rejection of islet allografts in Wistar Furth (WF) rats sensitized with donor strain skin and that suppressor cells are not likely to contribute to the unresponsive state because adoptive transfer of spleen cells from WF rats bearing established intrathymic Lewis islets fails to prolong islet allograft survival in secondary hosts.     

Transplanted MHC class I-deficient nonobese diabetic mouse islets are protected from autoimmune injury in diabetic nonobese recipients

The injury of transplanted islets may occur by both autoimmune and alloimmune processes directed against MHC targets. To examine the role of MHC class I in islet graft injury, we transplanted syngeneic and allogeneic beta2-microglobulin-deficient islets into diabetic nonobese diabetic (NOD) mice. Loss of graft function was observed within 14 days using allogeneic C57BL/6 and BALB/c MHC class I deficient as well as wild-type MHC class I-bearing NOD donor islets. However, islets isolated from MHC class I-deficient NOD mice (NOD-B2 m-/-) survived indefinitely when transplanted under the kidney capsule of diabetic NOD recipients. Transplanted NOD-B2 m-/- islets were surrounded by a nondestructive periinsular infiltrate that expressed interleukin-4 in addition to interferon-gamma. These studies demonstrate the primary role of MHC class I molecules in causing autoimmune destruction or recurrent diabetes in transplanted islets.     

Immunoprediction of diabetes and evaluation of pancreatic islet transplantation during the prediabetic period

The autoimmune diabetic syndrome of the biobreeding (BB) rat currently constitutes the best animal model for human type I diabetes. Since prediction of human beings at high risk for development of diabetes by immunologic and physiologic parameters is not possible at present, we investigated the feasibility of immunoprediction of diabetes in normoglycemic BB diabetes-prone (BBdp) rats. Peripheral blood lymphocyte counts (PBLC) accurately predicted future development of diabetes in a cohort of 50 BBdp rats (100% specificity, 95.8% sensitivity for PBLC less than 3800/mm3). We also assessed the fate of normal islets transplanted from MHC-compatible non-diabetes-prone Wistar Furth (WF) donors in the prediabetic milieu of BBdp hosts that were known on the basis of their PBLC destined either to become diabetic or to remain normoglycemic. In 13 BBdp rats in which diabetes was predicted the native pancreatic beta cells were eliminated at 35 days of age with streptozocin and replaced with transplanted WF islets. Neonatally induced tolerance of WF antigens prevented rejection. After successful islet transplantation, spontaneous diabetes occurred in 100% (5/5) of the rats in which the development of diabetes was predicted. The identical protocol in eight rats predicted not to have diabetes resulted in permanent normoglycemia in all. We conclude that normal islets are susceptible to anti-islet autoimmunity after transplantation in the prediabetic period.     

Blockade of OX40 signals enhance survival of xenoislet grafts in spontaneously diabetic NOD mice

Role of OX40 blockade was investigated in islet xenograft model in chemically diabetic C57B1/6 and spontaneously diabetic NOD mice. The effect of OX40/OX40L blockade on effector function of diabetogenic T cells was studied in an adoptive transfer model. 2000 IEQ of porcine islets were transplanted under the kidney capsule of diabetic C57B1/6 or NOD mice and were treated i.p. with control Ig, anti-OX40L, or CTLA4Ig. Graft survival was assessed using blood glucose (BG) measurement. Cells from diabetic NOD spleens and pancreatic lymphnodes were injected i.v. into NOD.scid mice. Recipients were treated i.p. with anti-OX40L, CTLA4Ig alone, or in combination; control mice received Ig. Diabetes incidence was assessed using BG measurement. Anti-OX40L treatment delayed xenoislet rejection significantly in chemically diabetic animals, although CTLA4Ig delayed rejection even further. Neither treatment completely prevented rejection. In spontaneously diabetic NOD mice, rejection of xenoislet graft was delayed by anti-OX40L treatment but not by CTLA4Ig. Anti-OX40L alone and in combination with CTLA4Ig significantly delayed diabetes transfer by activated diabetogenic T cells, compared with control and CTLA4Ig treatment. Preliminary mechanistic studies suggest that anti-OX40L treatment preserves Treg numbers, unlike CTLA4Ig that diminishes Tregs. Our studies show that OX40 blockade offers better xenoislet graft survival than CTLA4Ig in spontaneous autoimmune NOD model, suggesting that preactivated T cells use alternate costimulatory pathways, independent of CD28. Results from adoptive transfer studies further support the role of OX40 signaling in effector function of diabetogenic T cells. These data suggest that OX40/OX40L may offer novel therapeutic target for xenoislet graft protection in type 1 diabetic patients.     

Differences in suppressor of cytokine signaling-1 (SOCS-1) expressing islet allograft destruction in normal BALB/c and spontaneously-diabetic NOD recipient mice

BACKGROUND: The ability to block interferon signaling represents an important strategy in designing therapies to prevent beta-cell destruction during islet allograft rejection. METHODS: The SOCS proteins regulate cytokine signaling by blocking activation of JAK/STAT proteins. Using islets isolated from SOCS-1 transgenic mice (SOCS-1-Tg; these mice express SOCS-1 under the control of the human insulin promoter and are on the C57BL6/J background), we investigated whether SOCS proteins can prevent the destruction pancreatic islet cells transplanted beneath the kidney capsule of major histocompatibility complex mismatched normal BALB/c and spontaneously-diabetic NOD mouse recipients. RESULTS: Immunohistochemical staining for insulin confirmed the presence of donor SOCS-1-Tg islets in islet allografts harvested at 22 days posttransplant, whereas grafts of control non-Tg islets were destroyed by 14 days. In contrast, SOCS-1-Tg allogeneic islets were not protected from beta-cell destruction in clinically diabetic NOD mice. The islet allografts functioned for 1 week posttransplant; however, hyperglycemia returned after 2 weeks and the grafts were destroyed. Rejection of SOCS-1-Tg and non-Tg islets in autoimmune diabetic NOD mice was associated with an infiltrate of both CD4+ and CD8+ T cells and a T2-type cytokine response (IL-4) rather than the conventional T1-type cytokine response observed during islet allograft rejection. Self-antigen upregulation in response to IFN-gamma stimulation did not appear to be a factor in rejection of the islet allografts. CONCLUSIONS: These results demonstrate that expression of SOCS-1 in islets delays islet allograft rejection but cannot circumvent destruction of the islets by the recurrence of the tissue-specific autoimmune process of spontaneous diabetes.     

Influence of postdiabetic onset time and immunosuppressive treatment on islet grafts in the spontaneous diabetic BB/W rat

1000 islets from one donor, isolated with a modified neutral red method, were intraportally transplanted from BB/Wistar or Lewis (RT1) donors to short- and long-term diabetic BB/Wistar recipients. Recipients received immunosuppression with cyclosporine i.m.--either postoperative 4 X 25 mg/kg body weight or pre- and postoperative 3 X 10 mg and 4 X 25 mg/kg body weight. Graft survival in short-term diabetic recipients was significantly shorter than in long-term diabetics, whether they were immunosuppressed or not. Especially in recent onset recipients, the additional preoperative immunosuppression with cyclosporine provided longer graft survival than postoperative cyclosporine alone. Histological examinations of islet grafts showed eosinophilic cells in all transplanted islets, in both functioning and clinically rejected grafts of allogenic and "pseudoisogenic" transplanted recipients. a coincidence of eosinophilic cells with the reenactment of the original autoimmune disease in islet grafts seems possible.     

Protective effect of CTLA4Ig secreted by transgenic fetal pancreas allografts

BACKGROUND: Pancreas allotransplantation offers a cure for insulin-dependent diabetes mellitus. Systemic immunosuppression used to prevent immune destruction of the graft has side-effects, including increased susceptibility to infection and neoplasia. These unwanted effects may be limited by engineering the graft to secrete immunomodulatory molecules, to achieve local immunosuppression. Several studies have shown that transient local CTLA4Ig results in partial protection of allogeneic grafts. Our intent has been to determine whether sustained secretion of transgenic CTLA4Ig from pancreatic islets is able to protect against allograft rejection. METHODS AND RESULTS: Mouse CTLA4 (test=CTLA4Ig) or CD5 leader sequence (control=CD5LIg) was fused to the Fc of mouse IgG2c, and expressed transgenically under the control of the rat insulin promoter in C57BL/6 mice carrying the bml mutation of H-2K(b) (B6.C-H-2(bm1)). This resulted in expression in pancreatic islets. We used ELISA quantification of transgene products secreted into the supernatants of cultured fetal pancreata to select high (CTLA4Ig(hi)) and low (CTLA4Ig(lo)) expresser transgenic mice. Cultured fetal pancreata were transplanted under the kidney capsule of wholly allogeneic CBA recipient mice. CTLA4Ig(hi) but not CTLA4Ig(lo) expresser grafts showed enhanced survival compared with control CD5LIg grafts at 6 weeks posttransplant, provided the recipient mice were transiently depleted of CD4 T cells (by a single low-dose injection of GK1.5) before transplantation. CONCLUSIONS: Sustained local secretion of CTLA4Ig from transgenic grafts in combination with transient systemic CD4 T-cell depletion can enhance allograft acceptance.     

Prevention of diabetes in rats by bone marrow transplantation.

Hyperglycemia, hypoinsulinemia and ketonemia often develop abruptly in previously normal young "BB" rats. The syndrome mimics human juvenile diabetes closely and is, thus, appropriate for assessing pancreatic transplantation. Transplantation of islet cells from closely histocompatible Wistar Furth (WF) donor resulted in permanent normoglycemia when immunosuppression with ALS was given. However, when islet cells from nondiabetic "BB" donors were transplanted to nonimmunosuppressed diabetic "BB" recipients, only transient normoglycemia followed. Transplantation of WF islets cells also failed in diabetic "BB" rats which were tolerant of WF antigens, again suggesting destruction of transplanted islet cells by the original disease process-possibly autoimmunity. Evidence for autoimmunity was strengthened by the finding that newly diabetic "BB" rats could be rendered normoglycemic by immunosuppression. Since genetic susceptibility to spontaneous autoimmune diabetes is unique to some members of the "BB" stock, an attempt was made to alter their vulnerability by modifying their cellular immune system. Accordingly, 50 million bone marrow cells from WF donors were inoculated into half the newborn members of "BB" litters, leaving the littermates as unmodified controls. Most bone marrow recipients were protected, only four of 37 (10.8%) ever becoming diabetic, while the incidence of diabetes in noninoculated littermates was 22 of 39 (56.4%). The ultimate goal in human diabetes, which also seems very likely to be an autoimmune disease, may not be replacement of destroyed islet cells but identification of potentially susceptible children and prevention of islet destruction by immunologic manipulation.     

Prevention of recurrence of IDDM in islet-transplanted diabetic NOD mice by adjuvant immunotherapy.

Insulin-dependent diabetes mellitus (IDDM) involves the destruction of the insulin-producing cells in the islets of Langerhans. One possible cure is by transplanting the islet cells; however, transplanted islets, even between identical twins, are subject to autoimmune destruction by the disease process, resulting in diabetes recurrence. We recently reported that complete Freund's adjuvant (CFA), an immunomodulating agent, prevented development of autoimmune diabetes in the NOD mouse. In this study, we evaluated adjuvant therapy in prevention of autoimmune destruction and rejection of transplanted islets in diabetic NOD mice. After transplantation, untreated syngeneic islet recipients (n = 16) initially became normoglycemic and then hyperglycemic, with a median survival time (MST) of the graft of 17 days. When CFA was administered at the time of transplantation, 11 of 13 CFA-treated syngeneic islet recipients remained normoglycemic long term (greater than 100 days) with an MST greater than 107 days. Ten of 11 mice maintained indefinite normoglycemia until the conclusion of follow-up (101 to 172 days). When adjuvant therapy was used in conjunction with allogeneic islet transplantation, graft survival was not extended, with MST being similar to the untreated allogeneic islet recipients (12 [n = 5] and 13 [n = 5] days, respectively). The extended acceptance of second syngeneic islet grafts by CFA-treated mice indicates that the persistent autoimmunity against the transplanted islets can be reversed in the diabetic NOD mice after CFA treatment.     

Recurrent Autoimmunity Accelerates Destruction of Minor and Major Histoincompatible Islet Grafts in Nonobese Diabetic (NOD) Mice

Recurrent autoimmunity destroys nonobese diabetic (NOD) islet isografts, but whether recurrent autoimmunity contributes to islet graft destruction in immunocompetent allogeneic recipients is unknown. In the NOD, a single dose of streptozocin prevents or delays primary autoimmunity, allowing the detection of alloimmunity alone in chemically diabetic hosts (streptozocin-NOD) to be compared to the combined effects of autoimmunity and alloimmunity in spontaneously diabetic NODs (autoimmune-NOD). Islets were isolated from prediabetic NOD (H-2KdDb), nonobese resistant (NOR) (H-2KdDb), Balb/cByJ (H-2d) and B10.BR (H-2k) donors and transplanted to either the renal subcapsule or the intraportal site in autoimmune-NODs or streptozocin-NODs. MHC-matched NOR islets had in definite graft survival in streptozocin-NODs. However, NOR islets showed graft loss at 12.6 +/- 3.2 days in renal subcapsule and at 6.8 +/- 0.1 days in intraportal site of autoimmune-NODs. Partially MHC-matched Balb/cByJ islet grafts failed significantly sooner in autoimmune-NODs than in streptozocin-NODs (p < 0.005). Fully MHC-mismatched B10.BR islet grafts also failed sooner in autoimmune-NODs, but the difference did not reach significance (p < 0.06). Although the streptozocin-NOD was functionally tolerant of MHC-matched NOR islets, NOR islets transplanted into autoimmune-NODs failed sooner than NOD islets in both renal subcapsule (12.6 +/- 3.2 days vs. 26.4 +/- 10.5 days, p = 0.009) and intraportal sites (6.8 +/- 0.1 days vs. 11.5 +/- 1.7 days, p = 0.014). In the autoimmune-NODs, the intraportal site consistently showed shorter graft survival than the renal subcapsule site (NOD: p = 0.009, NOR: p = 0.014, Balb/cByJ: p = 0.008, B10.BR: p = 0.032). In conclusion, autoimmune processes facilitate the alloimmune response to minor and major histocompatibility antigens and accelerate graft destruction. The same autoimmune processes are more pronounced in the intraportal site.     

Islet transplantation in spontaneously diabetic BB/Wor rats

We investigated the effectiveness of islet transplantation as therapy in an animal model of spontaneous type I (insulin-dependent) diabetes mellitus. Grafting MHC-matched and -mismatched islets with the spontaneously diabetic BB rat as a model has been previously reported to result in recurrence of the disease in the grafted tissue. When transplanted with nonimmunogenic islets isolated by nonenzymatic culture, we found that MHC-matched grafts proved to be susceptible to disease recurrence when allowed to remain in situ until ketosis developed in the host. Conversely, the MHC-mismatched grafts did not succumb to the disease process despite the destruction of the beta-cell population of the endogenous pancreas. Four manifestly hyperglycemic BB/Wor rats received sufficient islet mass by allotransplantation to reverse this state. All four animals had ameliorated conditions, and three of the four were restored to a normoglycemic state. Recurrence of diabetes in the BB rat was not observed.     

Impact of donor immune cells in pancreatic islet transplantation

Autoimmune-mediated cytotoxicity may cause pancreatic islet transplant failure, leading to recurrent diabetes. Protection of islet grafts depends on immunosuppressive control, which may also prevent autoimmune recurrence of diabetes. In this study, we compared the survival of syngeneic islet transplants using different strains of donor mice. We observed extended functional survival in the islet grafts from donors lacking the genetic background and potential of autoimmunity. Without immunosuppression, the islet grafts of NOR and immune-deficient NOD. Scid donors functioned up to 3 weeks in syngeneic islet transplants compared to 3-day survivals with the grafts from NOD donors. T-cell proliferation and activation markers, CD44 and CD69, were upregulated in NOD donors, suggesting that T-cell activation had occurred prior to pancreas procurement. Systemic delivery of a recombinant adenoassociated viral vector (AAV) encoding the viral (vIL-10) IL-10 gene (AAV vIL-10) in NOD recipients protected syngeneic islets from autoimmune destruction. Alternatively, pretreatment of NOD donor mice with AAV vIL-10 prolonged islet graft survival in untreated NOD recipients. Both studies indicate the effectiveness of vIL-10 gene therapy in autoimmune regulation. These results suggest that a donor factor may exist in autoimmune-prone donors. Therefore, autoimmune recurrence of diabetes may result from donor immune cells transferred during islet transplantation. The AAV vIL-10 gene therapy suppressed previously activated donor T cells and protected the grafted islets from autoimmune-mediated destruction.     

外用环孢素A联合CTLA4Ig延长异体移植鼠耳存活的研究

目的　探讨局部外用环孢素 A(Cs A)联合细胞毒性淋巴细胞相关抗原 4融合蛋白 (CTL A4 Ig)对异体复合组织移植的免疫抑制及诱导免疫耐受的作用。方法　建立吻合血管的同种异体大鼠耳廓移植模型 ,术后在移植耳皮肤表面外涂 Cs A并联合 CTL A4 Ig腹腔注射治疗 ,观察移植物的排斥反应及存活时间 ,检测移植后受体血清白细胞介素 - 2 (IL- 2 )含量变化。结果　对照组平均存活时间为 (7.8± 1.7)天 ;单纯用 Cs A治疗组为 (15 .2± 1.9)天 ,单纯CTL A4 Ig治疗组为 (16 .6± 2 .1)天 ;Cs A +CTL A4 Ig联合治疗组为 (2 8.8± 3.5 )天 ,与其它各组相比均有统计学意义 (P<0 .0 1) ;且联合治疗组的受体血清 IL - 2含量最低 ,尤以第 5、7天为著 ,与其它各组相比有统计学意义 (P<0 .0 1)。结论　局部外用 Cs A联合 CTL A4 Ig能有效抑制异体复合组织移植排斥反应 ,显著延长移植物存活时间。