Prolonged survival of fetal pig islet xenografts in mice lacking the capacity for an indirect response

BACKGROUND: Xenografts of islets from organ-cultured fetal pig pancreases transplanted into non-immunosuppressed mice are rejected within 10 days. Immunosuppression with anti-T cell (anti-CD4) monoclonal antibody alone delays rejection of these xenografts for about 28 days, but rejection eventually occurs despite marked depletion of T cells. To determine if the critical CD4+ T cells responsible for xenograft islet rejection function through the direct or indirect pathway, selective class II-deficient mice that express class II antigens only on their thymic epithelium (not on peripheral cells) with normal numbers of CD4+ T cells, (class II-, CD4+), were used as recipients of xenograft islets to test if rejection occurs in the absence of an indirect response. METHODS: Control (C57BL/6) or class II-, CD4+ mice were transplanted under the kidney capsule with cultured fetal pig islets. Class II-, CD4+ mice have normal numbers of B cells, CD4+, gamma delta T cells, and slightly increased numbers of CD8+ T cells. Additional mice were thymectomized before receiving anti-CD4 or anti-CD8 monoclonal antibodies. Islet graft survival was determined histologically as fetal pig islets were too immature to secrete insulin. RESULTS: Xenograft survival in control animals was 7 to 14 days. In contrast, graft survival in class II-, CD4+ mice was significantly prolonged to greater than 35 days. Depletion of CD8+ T cells in class II-, CD4+ mice prolonged graft survival to about 70 days. Depletion of CD4+ T cells from these mice further prolonged xenograft survival to about 100 days. CONCLUSIONS: These results suggest that the rejection of pig islets by mice initially depends on a CD4 dependent indirect response. The CD4 direct response also contributes to graft destruction. CD8+ T cells also participate in graft destruction, albeit weakly.     

Long-term survival of hamster islet xenografts in mice under short-course treatment with nondepleting versus depleting anti-CD4 monoclonal antibodies

ABSTRACT: Xenogeneic grafts provide a potential alternative to the current shortage of human organs for transplantation. However, the prevention of rejection and tolerance induction of xenografts still remain to be further explored. Islet xenografts appear more promising than vascularized whole organ xenografts and additionally also more resistant to the recurrence of autoimmune disease than allografts. Recently, the nondepleting monoclonal antibody (mAb), which blocks the CD4 molecule on lymphocytes, was reported to be able to induce tolerance in allotransplantation and CD4 positive cells were further confirmed to be a major factor responsible for cellular xenograft rejection. Therefore, we hypothesize that anti-CD4 nondepleting mAb could also be effective in protecting cellular xenografts and inducing unresponsiveness of recipients. We studied the effect of the nondepleting anti-CD4 mAb YTS177.9 on islet xenograft survival by using the hamster-to-mouse islet transplantation model. Results were compared with that of the depleting anti-CD4 mAb GK1.5 that was shown to have similar binding sites on the CD4 molecule to mAb YTS 177.9. Our data show that mAb YTS 177.9 did effectively prolong the survival of islet xenografts and, in addition, also successfully did induce long-term acceptance of 40% grafts after only three penoperative injections of 0.5 mg mAb per mouse. The average survival of the graft was markedly prolonged to >66.8±37.1 days compared with controls (8.3±1.4 days) or with the depleting anti-CD4 mAb GK1.5 (25.7±5.5 days). However, the latter displayed a more profound inhibition in in vitro and ex vivo mixed lymphocyte xenoreaction than mAb YTS 177.9. Moreover, the activity of this nondepleting mAb was found to be dose-dependent and 80% of grafts survived permanently when the dose was increased to six injections of 0.5 mg mAb. Like mAb GK1.5, mAb YTS 177.9 also prevented rejection when given after a delay of two days posttransplant. In addition, we found that neither depleting nor nondepleting anti-CD8 mAb was effective in this model. Our results strongly suggest that an anti-CD4 nondepleting or blocking mAb alone is able to induce long-term acceptance of islet xenografts and that blocking the CD4 molecule is significantly superior to depleting CD4 positive cells for the protection of islet xenografts. This may indicate that CD4 cells play a major role in xenograft tolerance induction.     

Oral delivery of xeno-antigen combined with non-depleting anti-CD4 monoclonal antibody induces significantly prolonged survival of concordant skin xenograft

Abstract: Oral administration can induce unresponsiveness to protein antigens. Therefore, we examined whether oral administration of xeno-antigen could induce the prolonged survival of xenogeneic skin grafts. CBA mice were given 1 × 10⁷ SD rat splenocytes orally, 7 days before transplantation of a SD rat skin in the presence or absence of a non-depleting anti-CD4 monoclonal antibody (mAb) (YTS177, 200 µg/dose, −8 and −7 days relative to transplantation). All skin grafts survived with a median survival time (MST) of 62 days when xeno-antigens were administered orally in combination with anti-CD4 mAb. Mice treated with anti-CD4 mAb alone or oral administration of xeno-antigen alone induced modest prolonged survival of rat skin grafts (MST = 18 and 19 days, respectively) while naive mice rejected rat skin acutely (MST = 12 days). Oral administration alone or combined with anti-CD4 mAb reduced the level of xeno-antibody production compared with that in untreated mice after transplantation. Xenogeneic mixed leukocyte response was reduced when splenocytes from mice pre-treated with oral administration of xenogeneic cells were used as the responder compared with that in untreated mice. Oral delivery of xeno-antigen plus non-depleting anti-CD4 mAb can induce prolongation of concordant xenogeneic skin grafts.     

Acute cell-mediated rejection in orthotopic pig-to-mouse corneal xenotransplantation

Abstract: Background:  To investigate the role of T cells and natural killer (NK) cells in mediating corneal xenograft rejection in a pig-to-mouse model.
Methods:  Pig corneas were orthotopically transplanted into BALB/c, C57BL/6, nude, severe combined immunodeficiency (SCID), and NOD/SCID/γc^null (NOG) mice. Graft survival was clinically assessed by slit-lamp biomicroscopy and median survival times (MST) were calculated. The rejected grafts were histologically evaluated using antibodies against CD4, CD8, NK1.1, and F4/80.
Results:  The pig corneal xenografts were acutely rejected by BALB/c and C57BL/6 mice (MST 9.0 days), while nude, SCID and NOG mice rejected pig corneas in a more delayed fashion (MST 16.0, 16.4, and 16.9 days, respectively). The majority of infiltrating cells found in rejected grafts in C57BL/6 mice were macrophages and CD4⁺ T cells, while CD8⁺ T cells and NK cells were rarely found. The grafts in nude mice had markedly decreased inflammatory infiltration with small numbers of macrophages and CD4⁺ T cells. Infiltration was even more modest in grafts in SCID and NOG mice.
Conclusions:  T cells play an important role in acute rejection of pig corneal xenografts in mice, although acute rejection is not solely the result of T-cell-mediated immunity. NK cells are less likely to be involved in the rejection process.     

Delayed rejection of fetal pig pancreas in CD4 cell deficient mice was correlated with residual helper activity

CD4 cells have been shown to play a dominant role in the rejection of xenografts. Depletion of murine CD4 cells by injecting anti-CD4 antibody prolongs the graft survival, but does not prevent its rejection. For a more stable phenotype, we used genetically modified mice. To test whether the delayed rejection is caused by incomplete depletion of CD4 cells, we evaluated the response to fetal pig pancreas (FPP) xenografts in three types of CD4 cell deficient mice. They are MHC class II deficient mice (MHC II(o/o), CD4 deficient mice (CD4(o/o)) and a novel type of CD4 cell deficient mice (designated GK). GK mice were rendered permanently and completely CD4 deficient by transgenic expression of anti-CD4 antibody, whereas both MHC II(o/o) and CD4(o/o) mice have a residual helper cell population. FPP grafts in wild type mice were rejected within a week, whereas FPP grafts survived up to 4 weeks in MHC II(o/o) and CD4(o/o) mice. Survival of grafts in GK mice was even longer (8 weeks). Differences in histology were also noted. Rejecting grafts in MHC II(o/o) and wild-type mice were infiltrated with both eosinophils and mononuclear cells, whereas the infiltrates in CD4(o/o) and GK mice were exclusively mononuclear cells. Immunohistochemistry showed that they were primarily CD8 cells. The immune response to FPP was clearly different in the three types of CD4 cell deficient mice. Splenocytes of MHC II(o/o) 3 weeks post-transplant with FPP produced substantial amounts of IFN-gamma and IL-5, whereas splenocytes of CD4(o/o) mice produced low levels of IFN-gamma but no detectable IL-5. At similar times, these cytokines were not detected in GK mice. Furthermore, CD4(o/o) mice were capable of mounting helper dependent, although reduced, IgG responses to FPP antigens, while GK mice were not. The above results indicate that residual helper activity in some types of CD4 cell deficient mice could still contribute to xenograft rejection. Caution needs to be exercised where such mice are used as models of CD4 cell deficiency. Also, because there is eventual rejection of xenograft FPP in GK mice which lack detectable helper activity, we argue that these mice are a better model to investigate the involvement of CD4-independent rejection mechanisms.     

Islet Xenotransplantation: Are We Really Ready for Clinical Trials?

Four clinical trials of porcine islet transplantation have been reported, and there are verbal reports that clinical trials on much larger scales are continuing in centers in China and Russia. The four reported trials are briefly reviewed and, in the light of the present status of experimental islet xenotransplantation, consideration is given to whether such trials are currently justified. The Ethics Committee of the International Xenotransplantation Association has (1) emphasized the need for encouraging studies in non-human primates before clinical trials should be undertaken, (2) mandatory monitoring for the transfer of porcine microorganisms, and (3) careful regulation and oversight by recognized bodies. Other aspects of the topic, such as the need for informed consent, are briefly discussed. We conclude that, at the present time, more data documenting convincing efficacy, focused on clinically applicable immunosuppressive regimens, are needed to justify the initiation of closely monitored clinical trials. A clinical trial may then be justified even though the potential risk to the patients, and possibly for society, will not be zero.     

Long-term culture, low-temperature culture, and hyperoxic culture do not prolong fish-to-mouse islet xenograft survival

Abstract: Macroscopic, anatomically discrete islets called Brockmann bodies (BB) were harvested from tilapia with microscissors and then cultured under various conditions known to prolong islet allograft survival. BBs were cultured in 95% air/5% CO₂ at 37°C either overnight (groups 1 and 2) or for 1 week (group 3); in 95% O₂/5% CO₂ at 37°C for 1 week (group 4), or in 95% air/5% CO₂ at 24°C for 1 week (groups 5 and 6). Viability and function of cultured islets was confirmed by fluorescein diacetate/ethidium bromide staining and by transplantation under the left renal capsules of streptozotocin-diabetic balb/c mice. Recipient mice in groups 2 and 6 received CsA (50 mg/kg/d p.o.) × 4 days. All recipient mice became normoglycemic posttransplant. Mean graft survival times (± S.D.) for groups 1, 2, 3, 4, 5, and 6 were 7.6 (± 1.1), 6.2 (± 1.5), 6.4 (± 0.79), 8.2 (± 2.3), 7.0 (± 0.71), and 6.2 (± 0.45) days, respectively. Rejection (i.e., nonfasting plasma glucose levels > 200 mg/dl) was confirmed histologically in all instances; rejection was characterized by infiltrates composed of mononuclear cells, plasma cells, and eosinophils. In our study, neither short-term CsA nor any of the culture protocols significantly prolonged discordant fish-to-mouse islet xenograft survival.     

Adult porcine islet transplantation in baboons treated with conventional immunosuppression or a non-myeloablative regimen and CD154 blockade

The aim of the present study was to assess the survival of adult porcine islets transplanted into baboons receiving either (I) conventional triple drug immunosuppressive therapy or (2) a non-myeloablative regimen and an anti-CD154 monoclonal antibody (mAb) aimed at tolerance-induction. Group 1 baboons (n = 3) were pancreatectomized prior to intraportal injection of 10,000 porcine islet equivalents (IE)/kg and immunosuppressed with anti-thymocyte globulin (ATG), cyclosporine and azathioprine. In Group 2 (n = 2), non-pancreatectomized baboons underwent induction therapy with whole body and thymic irradiation, and ATG. Extracorporeal immunoadsorption (EIA) of anti-Galalpha1,3Gal (Gal) antibody was carried out. Maintenance therapy was with cobra venom factor, cyclosporine. mycophenolate mofetil, methylprednisolone and anti-CD154 mAb. Porcine islets were injected intraportally (14,000 and 32,000 IE/kg, respectively) and high-dose pig mobilized peripheral blood progenitor cells (3 x 10(10) cells/kg) were infused into a systemic vein. Porcine islets were also implanted in the sternomastoid muscle to facilitate subsequent biopsies. In both groups. porcine C-peptide was measured, and histological examination of liver or sternomastoid muscle biopsies was performed at regular intervals. In Group 1, total pancreatectomy reduccd human C-peptide to < 0.1 ng/ml and induced insulin-requiring diabetes. The transplantation of porcine islets was followed by normalization of glycemia for 15-24 h. Porcine C-peptide was detected only transiently immediately after porcine islet injection (maximum 0.12 ng/ml). Histological examination of liver biopsies taken between days 2 and 19 did not reveal viable islets, but necrotic cell structures with mononuclear cell infiltrates were identified in portal venules. In Group 2, injection of porcine islets into non-pancreatectomized recipients induced a transient hypoglycemia (2-4 h) requiring concentrated intravenous dextrose administration. Porcine C-peptide was detectable for 5 and 3 days (maximum 2.8 and 1.0 ng/ml), respectively. Baboon #4 died on day 12 from small bowel intussusception. Liver and sternomastoid muscle biopsies showed well-preserved porcine islets, staining positive for insulin and glucacon, without signs of rejection. In baboon #5, viable islets were detected in the sternomastoid muscle biopsy on day 14, but not on day 28 or thereafter. A progressive mononuclear cell and macrophage infiltration was seen in the biopsies. In conclusion, conventional immunosuppression allowed survival of porcine islets in baboons for < 24 h. The non-myeloablative regimen prolonged survival of porcine islets for > 14 days. However, despite depletion of T cells, anti-Gal antibody and complement, and CD154-hlockade, porcine islets were rejected by day 28. These results suggest that powerful innate immune responses are involved in rejection of discordant xenogencic islets.     

Comparison of immunogenicity and porcine-to-rhesus lamellar corneal xenografts survival between fresh preserved and dehydrated porcine corneas

Li A, Pan Z, Jie Y, Sun Y, Luo F, Wang L. Comparison of immunogenicity and porcine‐to‐rhesus lamellar corneal xenografts survival between fresh preserved and dehydrated porcine corneas. Xenotransplantation 2011; 18: 46–55. © 2011 John Wiley & Sons A/S. Abstract: Background: To compare the immunogenicity of fresh preserved and dehydrated lamellar porcine corneas in porcine‐to‐mouse heterotopic transplantation and to investigate the survival of preserved porcine corneas as xenografts in porcine‐to‐rhesus lamellar corneal transplantation. Methods: Dehydrated and fresh preserved, endothelium deprived, porcine corneas were cut into fragments and grafted beneath the kidney capsule of BALB/c mice. Porcine‐specific delayed type hypersensitivity (DTH) and antibody (IgM, IgG) immune responses of the recipient mice were assessed. In addition, fresh preserved and dehydrated porcine corneas were used in porcine‐to‐rhesus lamellar corneal xenotransplantation. The rhesus recipients were divided into three groups. Dehydrated corneas were applied to Group 1 and 3, and fresh preserved corneas were applied to Group 2. Only Group 3 received subconjunctival injections with triamcinolone acetonide for 1 month. All xenografts were evaluated by slit‐lamp microscopy for 6 months. Two recipients in each group were examined by in vivo confocal microscopy and then killed for corneal histopathological staining 3 months after surgery. Results: Neither fresh preserved nor dehydrated corneal fragments evoked any measurable change in mice recipient humoral immune status, but both could induce porcine‐specific DTH at 1, 2, and 4 week after being grafted. However, the intensity of the DTH responses evoked by dehydrated corneas was lower than that evoked by fresh preserved corneas. In porcine‐to‐rhesus lamellar keratoplasty, with the exception one graft in Group 2 that developed characteristics of rejection, all the xenografts remained transparent or translucent up to 6 month after surgery. Histopathological examination of Group 1 showed that the infratemporal xenografts were much thicker and displayed some inflammatory cell infiltration in the peripheral portion of the graft and bed interface. However, in Group 3, which was treated with triamcinolone acetonide, there was an easily identified scar at the lamellar interface with no inflammatory cells present. In the rejected graft in Group 2, infiltrating cells included a few eosinophils and massive lymphocytes. Confocal microscopy examination showed that activated keratocytes localized in the anterior stroma and highly reflective tissue at the interface of the graft and bed. Conclusions: Porcine corneas might be an ideal source for clinical lamellar corneal xenotransplantation. In cases of tectonic lamellar transplantation, the possibility to use dehydrated pig material may become an option in the future.     

Effects of ultraviolet B irradiation on fetal pig islet-like cell clusters

Abstract: Ultraviolet B (UV-B) irradiation of donor islets has previously been shown to result in the prolongation of their survival when transplanted into rodents. This study examined the in vitro and in vivo effects of UV-B irradiation on fetal pig islet-like cell clusters (ICCs), which like adult islets are being transplanted to reverse diabetes. Under control conditions, fetal pig ICCs were able to stimulate both human and pig peripheral blood mononuclear cells (PBMC) in mixed islet lymphocyte culture (MILC). Exposure of the ICCs to UV-B irradiation significantly reduced their ability to stimulate PBMC of both species in MILC when 600 J/m² but not lower doses (300 and 400 J/m²) of irradiation were applied. In contrast, all doses of UV-B irradiation were effective in inhibiting the ability of pig and human PBMC to stimulate human PBMC in a mixed lymphocytes culture (MLC). This demonstrates that UV-B irradiation is effective in reducing xeno immunogenicity of pig antigens. A toxic effect of all doses of UV-B irradiation on ICCs was demonstrated in vitro with a reduction in ³H-thymidine incorporation of 57, 71, 64, and 80% at 150, 300, 450, and 600 J/m², respectively. Toxicity of UV-B irradiation was also demonstrated when treated ICCs were transplanted beneath the renal capsule of SCID mice. The insulin content of the ICCs, 6 weeks after transplantation, was significantly reduced in the 600 J/m² group ( P <0.05). ICCs treated with UV-B irradiation (300 J/m²) in vitro and then transplanted beneath the renal capsule of BALB/c mice were rejected within 2 weeks as were untreated ICCs. Injection of cyclosporine (12.5 mg/kg/day) into these mice did not alter the results. It is concluded that UV-B irradiation is toxic to fetal pig ICCs and, in low dose, unable to prevent their rejection when transplanted into mice.     

Engraftment of Adult Porcine Islet Xenografts in Diabetic Nonhuman Primates Through Targeting of Costimulation Pathways

Recent advances in human allogeneic islet transplantation have established beta-cell replacement therapy as a potentially viable treatment option for individuals afflicted with Type 1 diabetes. Two recent successes, one involving neonatal porcine islet xenografts transplanted into diabetic rhesus macaques treated with a costimulation blockade-based regimen and the other involving diabetic cynomolgus monkeys transplanted with adult porcine islet xenografts treated with an alternative multidrug immunosuppressive regimen have demonstrated the feasibility of porcine islet xenotransplantation in nonhuman primate models. In the current study, we assessed whether transplantation of adult porcine islet xenografts into pancreatectomized macaques, under the cover of a costimulation blockade-based immunosuppressive regimen (CD28 and CD154 blockade), could correct hyperglycemia. Our findings suggest that the adult porcine islets transplanted into rhesus macaques receiving a costimulation blockade-based regimen are not uniformly subject to hyperacute rejection, can engraft (2/5 recipients), and have the potential to provide sustained normoglycemia. These results provide further evidence to suggest that porcine islet xenotransplantation may be an attainable strategy to alleviate the islet supply crisis that is one of the principal obstacles to large-scale application of islet replacement therapy in the treatment of Type 1 diabetes.     

Initial experience with the human anti-human CD154 monoclonal antibody, ABI793, in pig-to-baboon xenotransplantation

BACKGROUND: ABI793 (ABI) is a human monoclonal antibody (mAb) specific for human CD154. To assess the suitability of ABI for baboon transplantation studies, we carried out in vitro studies to determine ABI's reactivity with baboon cells expressing CD154, performed in vivo pharmacokinetic studies in two baboons, and tested the effect of ABI administration on elicited antibody production in two baboons undergoing either pig hematopoietic progenitor cell (PBPC) or heterotopic heart transplantation. METHODS: In vitro: Baboon peripheral blood mononuclear cells were activated in vitro to upregulate CD154, and binding of ABI to CD154 was measured by flow cytometry. In vivo: Serum levels of ABI were measured immediately before and 15 min after the intravenous administration of ABI (20 mg/kg) to two baboons over 28 days. Subsequently, ABI (25 mg/kg on days 0, 1, 4 and 7, and then 20 mg/kg every 5 days) was included in the immunosuppressive regimen in two pig-to-baboon transplants (PBPC or heart transplantation). RESULTS: In vitro: ABI was almost non-reactive to baboon T cells before stimulation, but bound to activated T cells. In vivo: In the pharmacokinetic study, trough levels of ABI (before the next dose) ranged between 190 and 580 microg/ml, and the estimated half-life was 10-15 days. There was no apparent toxicity. Following pig PBPC or heart transplantation, no elicited antibody was detected while ABI was being administered or during several weeks of follow-up. CONCLUSIONS: ABI functions in baboons, is well-tolerated, and prevents an elicited antibody response to pig antigens.     

Biological activity of pig islet-cell reactive IgG antibodies in xenotransplanted diabetic patients

BACKGROUND: The IgG antibody response in type I diabetic patients, transplanted with fetal pig islet-like cell-clusters, was investigated using purified immunoglobulin G (IgG) fractions from sera collected 7 to 9 yr after transplantation. From our earlier studies, we knew that the immunological specificities of xenoreactive IgG1 and IgG2 antibodies are different, and that IgG1 antibodies, in contrast to the IgG2 population, are mainly directed against non-Galalpha1,3Gal epitopes. In this study our goal was to establish whether xenoreactive IgG1 and IgG2 antibodies react with pig islet cells and, if so, to identify the target cell type, the biological function as well as the specificity of such antibodies for islet cell antigens. Sera from xenotransplanted patients were compared with those of patients with diabetes, selected for high titres of islet-cell specific autoantibodies. METHODS: IgG antibody fractions from patient sera were purified on a protein G column. Surface expression of target antigens was studied using flow cytometry as well as immunofluorescence microscopy. The biological function of islet-cell reactive sera was tested using antibody dependent cellular cytotoxicity with both xenogeneic adult pig islet cells and allogeneic human islet cells as targets. Antibody specificity was assessed using 2D Western blots with both fetal and adult pig islet as well as human islet cell antigenic preparations. RESULTS: Some of the diabetic patients, who have been transplanted with xenogeneic fetal pig islet cells, continue to produce xenospecific IgG1 and IgG2 antibodies for 7 to 9 yr post-transplantation. A separate analysis of IgG1 and IgG2 antibodies showed that IgG1 antibodies react with pig islet beta cells, whereas IgG2 antibodies mainly react with non-endocrine pig cells. Such antibodies are xenospecific, as they were found to mediate antibody-dependent cellular cytotoxicity of adult pig, but not human islet target cells. The reverse was true for antibodies from non-transplanted diabetic patients with high titres of autoantibodies against beta cells. Fluorescence analysis as well as 2D gel Western blots revealed that the reactivity was variable between patient samples, indicating that target antigens for non-Galalpha1,3Gal-specific antibodies are heterogeneous. CONCLUSION: Thus, xenotransplantation of diabetic patients induces islet-beta cell reactive xenospecific IgG1 antibodies, which are biologically active and can mediate antibody-dependent cellular cytotoxicity of pig islet cells.     

Phenotypic and functional characterization of human T cell clones indirectly activated against adult pig islet cells

BACKGROUND: Xenotransplanted patients produce xenospecific IgG1 antibodies directed against epitopes other than Galalpha1,3Gal. IgG1 antibody production is believed to be dependent upon T cell help. Therefore, as a natural continuation of our work aimed at characterizing the xenoimmune antibody response against pig islet cells, we have also examined the T cell response. T cell reactivity against islet cells is believed to result from indirect antigen presentation, and our in vitro study was designed to mimic the response in vivo. The main purpose of this study was to characterize the phenotype, the immunological specificity and the functional capacity of indirectly activated T cell clones, reactive against pig islet cell antigens. MATERIALS AND METHODS: Human T cell clones, activated against pig islet cells in the presence of autologous antigen-presenting cells, were produced from limiting dilutions of bulk cultures. Clonality was investigated by T cell receptor Vbeta (TcRVbeta) expression analysis. Clonal specificity was studied in proliferation assays using different pig cells as stimulators. ELISpot experiments were performed to detect cytokine production patterns. The cytotoxic capacity of the clones was assessed using standard cell-mediated lysis tests and different porcine and human target cells. Several long-term bulk cultures of human lymphocytes, indirectly activated against pig islet cells, maintained for up to 60 days, were used as a control for possible bias in the selection of the clones. RESULTS: Nineteen CD4+ TcRValphabeta+ T cell clones were recovered. No activation of natural killer T cells or gammadelta-T cells was recorded. There was no bias in the TcRVbeta-usage. The immunological specificity differed between clones; some were specifically reactive against pig islet cell antigens, while others were reactive with antigens present on a variety of pig cells. All clones produced a broad spectrum of cytokines, e.g. interferon (IFN)-gamma, tumor necrosis factor (TNF)-alpha, interleukin (IL)5, IL10 and IL13, with no evidence of bias for a particular phenotype. None of the T cell clones were cytotoxic against pig islet cells, but two clones were cytotoxic against pig phytohemagglutinin (PHA)-blasts. CONCLUSION: The analysis of several, indirectly activated, human CD4+ T cell clones shows that the response against pig islet cells is heterogeneous both with regard to immunological specificity and functional characteristics. This heterogeneity was further confirmed by analysis of the long-term bulk cultures of human lymphocytes, indirectly activated against pig islet cells.     

The long-term survival of baboon-to-monkey kidney and liver xenografts

The present study was undertaken to develop an optimum immunosuppressive regimen in baboon-to-monkey life-supporting kidney xenografts. Baseline therapy for all groups include cyclosporine (CsA) and steroids. We compared adding (1) cyclophosphamide (CyP) at dose of 20 mg/kg/day given on post-operative day (POD) 0, 2, 5 and 7; (2) mycophenolic mofetil (MMF) at a dose of 40 mg/kg/day by daily gavage; or (3) CyP + rapamycin (Rap). The latter group was divided into high and low dose subgroups. Untreated xenografts were rejected on POD 6, CsA alone treated xenografts survived for 35 days and CsA + CyP treated xenografts survived for 45 days. Adding MMF significantly prolonged mean survival to 111 +/- 53 days, but the xenografts eventually developed rejection. Combination therapy including CsA, CyP and Rap reliably prevented xenogenic rejection and achieved a mean survival of 290 +/- 30 days. However, high dose CyP + Rap led to high incidence of post-transplant lymphoproliferation disorders (PTLD), while the incidence of PTLD was significantly less in the low dose subgroup (P < 0.01). Four animals in this subgroup survived for more than 300 days with normal renal function and histology. In addition, two liver recipients treated with CsA + CyP survived for 91 and 1,076 days. We conclude that long-term survival of kidney or liver xenografts can be achieved in a non-human concordant xenograft model using currently available immunosuppressive agents.