MHC Class I Masking to Prevent AMR in a Porcine Kidney Transplantation Model in Alloimmunized Recipients.

Presensitized patients awaiting a kidney transplant have a lower graft survival and a longer waiting time because of the limited number of potential donors and the higher risk of antibody-mediated rejection (AMR), particularly in the early posttransplant period, because of preformed donor-specific antibodies binding major histocompatibility complex (MHC) molecules expressed by the graft endothelium followed by the activation of the complement. Advances in kidney preservation techniques allow the development of ex vivo treatment of transplants. We hypothesized that masking MHC ex vivo before transplantation could help to prevent early AMR in presensitized recipients. We evaluated a strategy of MHC I masking by an antibody during ex vivo organ perfusion in a porcine model of kidney transplantation in alloimmunized recipients. Methods: Through the in vitro calcein-release assay and flow cytometry, we evaluated the protective effect of a monoclonal anti-swine leukocyte antigen class I antibody (clone JM1E3) against alloreactive IgG complement-dependent cytotoxicity toward donor endothelial cells. Kidneys perfused ex vivo with JM1E3 during hypothermic machine perfusion were transplanted to alloimmunized recipients. Results: In vitro incubation of endothelial cells with JM1E3 decreased alloreactive IgG cytotoxicity (mean complement-dependent cytotoxicity index [% of control condition] with 1 µg/mL 74.13% ± 35.26 [calcein assay] and 66.88% ± 33.46 [cytometry]), with high interindividual variability. After transplantation, acute AMR occurred in all recipients on day 1, with signs of complement activation (C5b-9 staining) as soon as 1 h after transplantation, despite effective JM1E3 binding on graft endothelium. Conclusions: Despite a partial protective effect of swine leukocyte antigen I masking with JM1E3 in vitro, ex vivo perfusion of the kidney with JM1E3 before transplantation was not sufficient alone at preventing or delaying AMR in highly sensitized recipients.

Successful pancreas allotransplantations after hypothermic machine perfusion in a novel diabetic porcine model: a controlled study.

The standard technique for pancreas preservation for transplantation is static cold storage (SCS). In this experimental study, we compare SCS to hypothermic machine perfusion (HMP) of the pancreas to assess if the latter could safely prolong the ischaemia period prior to transplantation. We worked in two phases, first with organ preservation for 24 h and second, preservation for either 2 or 6 h before allotransplantation. In phase 1, exocrine injury markers were found to be nonsignificantly lower, in the HMP group (n = 3) vs. SCS (n = 3) after 24 h of preservation; amylase (P = 0.2), lipase (P = 0.3) and lactate dehydrogenase (P = 0.1). In phase 2, 14 recipient diabetic pigs (after total pancreatectomy) received allotransplantations with n = 4 and n = 4 pancreases after HMP for 2 and 6 h vs. n = 3 and n = 3 pancreases after SCS for 2 and 6 h, respectively. There were no differences in recipient survival (P = 0.7), and mean survival was 14 days (0-53 days). All recipients had allograft function defined as detectable C-peptide and independent normoglycemia. We have not highlighted vascular thrombosis in all allotransplantations. This study reports the first successful pancreas allotransplantation after HMP preservation for up to 6 h with no evidence of graft thrombosis.

Total Pancreatectomy and Pancreatic Allotransplant in a Porcine Experimental Model.

OBJECTIVES: The main objective of this experimental study was to evaluate the feasibility of diabetes induction by total pancreatectomy and pancreatic allotransplant after diabetes induction by total pancreatectomy. The secondary objective was to evaluate metabolic (C-peptide, glycemia) and inflammatory (lactate and platelet levels) parameters after diabetes induction by total pancreatectomy and pancreatic allotransplant after total pancreatectomy. MATERIALS AND METHODS: The study protocol was approved by the French Minister of Research (APAFiS no.18169). Insulin-dependent diabetes was induced by total pancreatectomy in one male Sus scrofa pig, and pancreatic allotransplant was performed, after total pancreatectomy, in 3 male Sus scrofa pigs. Total pancreatectomy was performed under general anesthesia,with meticulous dissection of the portal vein and the splenic vein to preserve the spleen. Concerning pancreas procurement, extensive pancreas preparation occurred during thewarm phase,before coldperfusion. Pancreatic allotransplant was performed using donor aorta (with superior mesenteric artery and celiac trunk). RESULTS: Diabetes induction was successful, with negative C-peptide values at 3 hours after total pancreatectomy. Glycemic control without hypoglycemic events was obtained with the use of long-acting insulin administered once per day. No rapid-acting insulin was used. In animals that received pancreatic allotransplant, after enteral feeding was started, glycemic control without hypoglycemic events and without insulin was obtained in 2 animals. CONCLUSIONS: In an experimental porcine model, diabetes induction by total pancreatectomy and pancreatic allotransplant after total pancreatectomy are feasible and effective. The development of these models offers the potential for new investigations into ischemia-reperfusion injuries, improvement of pancreas procurement methods, and preservation techniques.

Ex situ hypothermic perfusion of nonhuman primate pancreas: A feasibility study.

Pancreatic static cold storage (SCS) is the gold-standard method for pancreas preservation. Our main objective was to evaluate feasibility of hypothermic perfusion (HP) of nonhuman primates' pancreases for potential organ transplantation. Seven baboon pancreases were tested. Animals were included in a study approved by the French Research Ministry of Health. Two groups were compared: the control group (n = 2) was preserved using conventional SCS for 24-h and the perfusion group (n = 5) used HP for 24-h, with three different perfusion pressures (PP): 15 (n = 3), 20 (n = 1), and 25 mm Hg (n = 1). In the control group, focal congestion of islets was observed after 6-h. At 24-h, ischemic necrosis and multifocal congestion also occurred. In the HP group, at 15 mm Hg PP, multifocal congestion of islets was present at 24-h. At 20 mm Hg PP, no ischemic necrosis was found after 6-h. At 12-h and 24-h, focal congestion of islets was seen. At 25 mm Hg PP, focal congestion of islets appeared after 12-h. Immunostaining for insulin, glucagon, and somatostatin was normal and similar in controls and perfused pancreas transplants even after 24-h. Apoptosis index represented by cleaved caspase 3 activity, was less than 1% in perfusion and control groups, even after 24-h. HP of nonhuman primate pancreas is feasible and not deleterious as far as 24-h compared to SCS. SCS for more than 12-h was harmful for the transplants. Systolic perfusion pressure between 15-20 mm Hg did not cause any pathological injury of the tested organs.

Interleukin-7 receptor blockade by an anti-CD127 monoclonal antibody in nonhuman primate kidney transplantation.

IL-7 is an important cytokine for T cell lymphopoiesis. Blockade of the IL-7 signaling pathway has been shown to induce long-term graft survival or graft tolerance in murine transplant models through inhibiting T cell homeostasis and favoring immunoregulation. In this study, we assessed for the first time the effects of a blocking anti-human cluster of differentiation 127 (CD127) mAb administered in combination with low-dose tacrolimus or thymoglobulin in a life-sustaining kidney allograft model in baboons. Contrary to our expectation, the addition of an anti-CD127 mAb to the treatment protocols did not prolong graft survival compared to low-dose tacrolimus alone or thymoglobulin alone. Anti-CD127 mAb administration led to full CD127 receptor occupancy during the follow-up period. However, all treated animals lost their kidney graft between 1 week and 2 weeks after transplantation. Unlike in rodents, in nonhuman primates, anti-CD127 mAb treatment does not decrease the absolute numbers of lymphocyte and lymphocyte subsets and does not effectively inhibit postdepletional T cell proliferation and homeostasis, suggesting that IL-7 is not a limiting factor for T cell homeostasis in primates.

IL-7 receptor blockade blunts antigen-specific memory T cell responses and chronic inflammation in primates.

Targeting the expansion of pathogenic memory immune cells is a promising therapeutic strategy to prevent chronic autoimmune attacks. Here we investigate the therapeutic efficacy and mechanism of new anti-human IL-7Rα monoclonal antibodies (mAb) in non-human primates and show that, depending on the target epitope, a single injection of antagonistic anti-IL-7Rα mAbs induces a long-term control of skin inflammation despite repeated antigen challenges in presensitized monkeys. No modification in T cell numbers, phenotype, function or metabolism is observed in the peripheral blood or in response to polyclonal stimulation ex vivo. However, long-term in vivo hyporesponsiveness is associated with a significant decrease in the frequency of antigen-specific T cells producing IFN-γ upon antigen restimulation ex vivo. These findings indicate that chronic antigen-specific memory T cell responses can be controlled by anti-IL-7Rα mAbs, promoting and maintaining remission in T-cell mediated chronic inflammatory diseases.

Neu5Gc and α1-3 GAL Xenoantigen Knockout Does Not Affect Glycemia Homeostasis and Insulin Secretion in Pigs.

Xenocell therapy from neonate or adult pig pancreatic islets is one of the most promising alternatives to allograft in type 1 diabetes for addressing organ shortage. In humans, however, natural and elicited antibodies specific for pig xenoantigens, α-(1,3)-galactose (GAL) and N-glycolylneuraminic acid (Neu5Gc), are likely to significantly contribute to xenoislet rejection. We obtained double-knockout (DKO) pigs lacking GAL and Neu5Gc. Because Neu5Gc-/- mice exhibit glycemic dysregulations and pancreatic ß-cell dysfunctions, we evaluated islet function and glucose metabolism regulation in DKO pigs. Isolation of islets from neonate piglets yielded identical islet equivalent quantities to quantities obtained from control wild-type pigs. In contrast to wild-type islets, DKO islets did not induce anti-Neu5Gc antibody when grafted in cytidine monophosphate-N-acetylneuraminic acid hydroxylase KO mice and exhibited in vitro normal insulin secretion stimulated by glucose and theophylline. Adult DKO pancreata showed no histological abnormalities, and immunostaining of insulin and glucagon was similar to that from wild-type pancreata. Blood glucose, insulin, C-peptide, the insulin-to-glucagon ratio, and HOMA-insulin resistance in fasted adult DKO pigs and blood glucose and C-peptide changes after intravenous glucose or insulin administration were similar to wild-type pigs. This first evaluation of glucose homeostasis in DKO pigs for two major xenoantigens paves the way to their use in (pre)clinical studies.

Anti-CD28 Antibody and Belatacept Exert Differential Effects on Mechanisms of Renal Allograft Rejection.

Belatacept is a biologic that targets CD80/86 and prevents its interaction with CD28 and its alternative ligand, cytotoxic T lymphocyte antigen 4 (CTLA-4). Clinical experience in kidney transplantation has revealed a high incidence of rejection with belatacept, especially with intensive regimens, suggesting that blocking CTLA-4 is deleterious. We performed a head to head assessment of FR104 (n=5), a selective pegylated Fab' antibody fragment antagonist of CD28 that does not block the CTLA-4 pathway, and belatacept (n=5) in kidney allotransplantation in baboons. The biologics were supplemented with an initial 1-month treatment with low-dose tacrolimus. In cases of acute rejection, animals also received steroids. In the belatacept group, four of five recipients developed severe, steroid-resistant acute cellular rejection, whereas FR104-treated animals did not. Assessment of regulatory T cell-specific demethylated region methylation status in 1-month biopsy samples revealed a nonsignificant trend for higher regulatory T cell frequencies in FR104-treated animals. Transcriptional analysis did not reveal significant differences in Th17 cytokines but did reveal higher levels of IL-21, the main cytokine secreted by CD4 T follicular helper (Tfh) cells, in belatacept-treated animals. In vitro, FR104 controlled the proliferative response of human preexisting Tfh cells more efficiently than belatacept. In mice, selective CD28 blockade also controlled Tfh memory cell responses to KLH stimulation more efficiently than CD80/86 blockade. Our data reveal that selective CD28 blockade and belatacept exert different effects on mechanisms of renal allograft rejection, particularly at the level of Tfh cell stimulation.

Selective CD28 Antagonist Blunts Memory Immune Responses and Promotes Long-Term Control of Skin Inflammation in Nonhuman Primates.

Novel therapies that specifically target activation and expansion of pathogenic immune cell subsets responsible for autoimmune attacks are needed to confer long-term remission. Pathogenic cells in autoimmunity include memory T lymphocytes that are long-lived and present rapid recall effector functions with reduced activation requirements. Whereas the CD28 costimulation pathway predominantly controls priming of naive T cells and hence generation of adaptive memory cells, the roles of CD28 costimulation on established memory T lymphocytes and the recall of memory responses remain controversial. In contrast to CD80/86 antagonists (CTLA4-Ig), selective CD28 antagonists blunt T cell costimulation while sparing CTLA-4 and PD-L1-dependent coinhibitory signals. Using a new selective CD28 antagonist, we showed that Ag-specific reactivation of human memory T lymphocytes was prevented. Selective CD28 blockade controlled both cellular and humoral memory recall in nonhuman primates and induced long-term Ag-specific unresponsiveness in a memory T cell-mediated inflammatory skin model. No modification of memory T lymphocytes subsets or numbers was observed in the periphery, and importantly no significant reactivation of quiescent viruses was noticed. These findings indicate that pathogenic memory T cell responses are controlled by both CD28 and CTLA-4/PD-L1 cosignals in vivo and that selectively targeting CD28 would help to promote remission of autoimmune diseases and control chronic inflammation.

hCTLA4-Ig transgene expression in keratocytes modulates rejection of corneal xenografts in a pig to non-human primate anterior lamellar keratoplasty model.

BACKGROUND: Human corneal allografting is an established procedure to cure corneal blindness. However, a shortage of human donor corneas as well as compounding economic, cultural, and organizational reasons in many countries limit its widespread use. Artificial corneas as well as porcine corneal xenografts have been considered as possible alternatives. To date, all preclinical studies using de-cellularized pig corneas have shown encouraging graft survival results; however, relatively few studies have been conducted in pig to non-human primate (NHP) models, and particularly using genetically engineered donors. METHODS: In this study, we assessed the potential benefit of using either hCTLA4-Ig transgenic or α1,3-Galactosyl Transferase (GT) Knock-Out (KO) plus transgenic hCD39/hCD55/hCD59/fucosyl-transferase pig lines in an anterior lamellar keratoplasty pig to NHP model. RESULTS: Corneas from transgenic animals expressing hCTLA4-Ig under the transcriptional control of a neuron-specific enolase promoter showed transgene expression in corneal keratocytes of the stroma and expression was maintained after transplantation. Although a first acute rejection episode occurred in all animals during the second week post-keratoplasty, the median final rejection time was 70 days in the hCTLA4-Ig group vs. 21 days in the wild-type (WT) control group. In contrast, no benefit for corneal xenograft survival from the GTKO/transgenic pig line was found. At rejection, cell infiltration in hCTLA4Ig transgenic grafts was mainly composed of macrophages with fewer CD3+ CD4+ and CD79+ cells than in other types of grafts. Anti-donor xenoantibodies increased dramatically between days 9 and 14 post-surgery in all animals. CONCLUSIONS: Local expression of the hCTLA4-Ig transgene dampens rejection of xenogeneic corneal grafts in this pig-to-NHP lamellar keratoplasty model. The hCTLA4-Ig transgene seems to target T-cell responses without impacting humoral responses, the control of which would presumably require additional peripheral immunosuppression.

Advantages of Papio anubis for preclinical testing of immunotoxicity of candidate therapeutic antagonist antibodies targeting CD28.

Antagonist anti-CD28 antibodies prevent T-cell costimulation and are functionally different from CTLA4Ig since they cannot block CTLA-4 and PDL-1 co-inhibitory signals. They demonstrated preclinical efficacy in suppressing effector T cells while enhancing immunoregulatory mechanisms. Because a severe cytokine release syndrome was observed during the Phase 1 study with the superagonist anti-CD28 TGN1412, development of other anti-CD28 antibodies requires careful preclinical evaluation to exclude any potential immunotoxicity side-effects. The failure to identify immunological toxicity of TGN1412 using macaques led us to investigate more relevant preclinical models. We report here that contrary to macaques, and like in man, all baboon CD4-positive T lymphocytes express CD28 in their effector memory cells compartment, a lymphocyte subtype that is the most prone to releasing cytokines after reactivation. Baboon lymphocytes are able to release pro-inflammatory cytokines in vitro in response to agonist or superagonist anti-CD28 antibodies. Furthermore, we compared the reactivity of human and baboon lymphocytes after transfer into non obese diabetic/severe combined immunodeficiency (NOD/SCID) interleukin-2rγ knockout mice and confirmed that both cell types could release inflammatory cytokines in situ after injection of agonistic anti-CD28 antibodies. In contrast, FR104, a monovalent antagonistic anti-CD28 antibody, did not elicit T cell activation in these assays, even in the presence of anti-drug antibodies. Infusion to baboons also resulted in an absence of cytokine release. In conclusion, the baboon represents a suitable species for preclinical immunotoxicity evaluation of anti-CD28 antibodies because their effector memory T cells do express CD28 and because cytokine release can be assessed in vitro and trans vivo.

Gene transfer of human CD40Ig does not prevent rejection in a non-human primate kidney allotransplantation model.

BACKGROUND: Blockade of costimulation signaling required for immune response, such as CD40/CD40L and CD28/B7, is a reasonable strategy to prevent rejection and in defined combinations may allow donor specific tolerance. Indeed, in rodents, costimulation blockade with CD28/B7 antagonists or with CD40Ig was able to induce regulatory T cells and transplant tolerance whereas in primates, anti-CD40 antibodies, anti-CD40L antibodies or CTLA4Ig, used as monotherapy, significantly delayed graft rejection. METHODS: Using an adeno-associated virus (AAV) vector mediated gene transfer of a human CD40Ig fusion protein (hCD40Ig) in primates, we evaluated the capacity of this costimulation blockade molecule interfering with CD40/CD40L signaling in prolonging kidney transplants in cynomolgus monkeys. RESULTS: This gene transfer strategy allowed for maintaining a plateau of hCD40Ig production within two months and avoided a high-scale production phase of this molecule. Although the hCD40Ig was able to bind efficiently to human and macaque CD40L and high (>200 µg/ml) transgene expression was obtained, no effect on graft survival was observed. In addition, there was no inhibition of humoral response to vaccination. In vitro, hCD40Ig strongly increased mixed lymphocyte reaction, and when compared to the anti-CD40L antibody h5C8, was not as potent to induce complement-dependent cytotoxicity. CONCLUSION: These data suggest that CD40/CD40L blockade using a non-depleting CD40Ig fusion protein, a therapeutic strategy that showed efficacy in rodents, is not able to modulate the immune response in primates. These data highlight important biological differences between rodent and primate models to evaluate therapeutic strategies at the preclinical level.

Recombinant human C1-inhibitor prevents acute antibody-mediated rejection in alloimmunized baboons.

Acute antibody-mediated rejection is an unsolved issue in transplantation, especially in the context of pretransplant immunization. The deleterious effect of preformed cytotoxic anti-HLA antibodies through complement activation is well proven, but very little is known concerning complement blockade to prevent/cure this rejection. Here, we used a baboon model of preimmunization to explore the prevention of acute antibody-mediated rejection by an early inhibition of the classical complement pathway using human recombinant C1-inhibitor. Baboons were immunized against peripheral blood mononuclear cells from allogeneic donors and, once a specific and stable immunization had been established, they received a kidney from the same donor. Rejection occurred at day 2 posttransplant in untreated presensitized recipients, with characteristic histological lesions and complement deposition. As recombinant human C1-inhibitor blocks in vitro cytotoxicity induced by donor-specific antibodies, other alloimmunized baboons received the drug thrice daily intravenously during the first 5 days after transplant. Rejection was prevented during this treatment but occurred after discontinuation of treatment. We show here that early blockade of complement activation by recombinant human C1-inhibitor can prevent acute antibody-mediated rejection in presensitized recipients. This treatment could also be useful in other forms of acute antibody-mediated rejection caused by induced antibodies.

Acute humoral rejection of renal transplants in alloimmunized pigs.

BACKGROUND: Despite progress in immunosuppression, acute humoral rejection (AHR) remains an unsolved issue in transplantation, with a possible reversibility in only about 50% of cases. AHR is characterized by antidonor antibodies in the recipient circulation and characteristic histological lesions within the graft itself, as well as complement degradation products and immunoglobulins (IgM and IgG) deposition in vascular zones. The lack of a large animal models of AHR in experimental allotransplantation led us to establish such a model in the pig. MATERIALS AND METHODS: Pigs were immunized with peripheral blood mononuclear cells from allogeneic donors and subsequently received a kidney from the same donor, once antidonor antibodies (Ab) had reached a plateau. The efficiency of the alloimmunization, the nature of the induced antibodies and rejection were studied. RESULTS: Six out of seven recipients developed specific antidonor Ab. Three days post-transplantation, characteristic lesions of AHR were observed together with intragraft IgG, IgM, and complement deposition. The AlloAb were found to be cytotoxic and directed against donor MHC class I molecules. CONCLUSIONS: We described, for the first time, a large animal model of AHR, which will enable us to more extensively study phenomena implicated in AHR and to test new strategies aimed at its prevention or cure, as well as improve transplant protocols in the case of presensitized or hyperimmunized patients.

Association of rapamycin and co-stimulation blockade using anti-B7 antibodies in renal allotransplantation in baboons.

BACKGROUND: Co-stimulation blockade has already been shown to induce transplantation tolerance in rodents, but until now has failed in large animal models. We therefore sought to investigate whether the addition of rapamycin to a co-stimulation blockade regimen could induce tolerance in baboon recipients of a renal allograft and to characterize the immunological characteristics of rejection. METHODS: Two baboons were used for a pharmacological and toxicological analysis and received anti-B7.1 and anti-B7 antibodies every other day for 60 days. Three groups of baboons underwent classical heterotopic renal allotransplantation; the first group received no treatment (control group; n = 2), the second received a combination of anti-CD80 and anti-CD86 monoclonal antibodies (mAbs) (B7 group; n = 4), and the third received the anti-B7 antibody treatment as above with an additional treatment of rapamycin (B7-Rapa; n = 4). Graft survival as well as immunological analyses were performed. RESULTS: Anti-B7 mAb monotherapy prolonged allograft survival in three out of four of the animals, one of whom survived rejection free for 87 days but died from a pulmonary embolism; the fourth animal died without rejection. The addition of rapamycin to the regimen did not prolong survival further; three of the four animals underwent early rejection whereas the fourth survived long term but eventually rejected at day 114. Whereas alloimmunization only occurred in this latter animal, rejection was always characterized by a substantial lymphocyte and monocyte infiltration, associated with a strong pro-inflammatory/cytotoxic mRNA accumulation in the anti-B7-treated animals, but to a lesser extent in the B7-Rapa group. T cells extracted and cloned from a biopsy taken at a stable post-transplant time showed a lower frequency of anti-donor alloreactivity in vitro than those extracted from a rejected tissue. Nevertheless, these non-responding clones failed to show regulatory activity in vitro. CONCLUSIONS: We thus confirm that blocking the CD28/B7 pathway by anti-B7 mAbs could prolong graft survival in baboons, but the addition of rapamycin was insufficient to induce tolerance.

The study of mitoxantrone as a potential immunosuppressor in transgenic pig renal xenotransplantation in baboons: comparison with cyclophosphamide.

Mounting evidence suggests that delayed xenograft rejection (DXR) of discordant xenografts has a strong humoral component. To explore the possibility of targeting this humoral response more efficiently, we performed a preliminary study in baboons immunized against pig blood cells using the immunosuppressor mitoxantrone (Mx). The results from this study showed that, in comparison with cyclophosphamide (CyP), Mx induced a long-lasting depletion of circulating B cells within 6 days of its administration and delayed secondary anti-Gal antibody (Ab) responses to pig blood cell immunizations. Given these results, we next evaluated Mx in an in vivo model of pig to baboon renal xenotransplantation. We performed a series of renal xenotransplantations in baboons using human CD55-CD59 transgenic donor pigs. In the first group of baboons (Mx group; n = 4) Mx was administered 6 days prior to the day of transplantation, the objective being to perform the xenotransplantation in a context where the recipient would have few remaining circulating B cells and thus have an impaired capacity to mount an Ab response to the xenograft. We compared this group to a second group of baboons treated with CyP starting 1 day prior to transplantation (CyP group; n = 2). All baboons receiving Mx or CyP received an additional immunosuppression of cyclosporin A, mycophenolate mofetil and steroids. No hyperacute rejection was observed in either group but all xenografts underwent DXR. Mx did not show superiority to CyP in terms of graft survival with a mean survival time of 8 +/- 2 days compared with 9 days for both CyP-treated baboons. Neither CyP nor Mx decreased serum levels of pre-existing anti-Gal Abs but levels of these Abs decreased dramatically within 1 day of transplantation, likely reflecting their immediate trapping within the xenograft. Interestingly however, in contrast to CyP, Mx inhibited the return of anti-Gal immunoglobulin M (IgM) to the circulation, even at the time of rejection. Nevertheless, strong intragraft deposits of IgM, IgG and the activated complement complex C5b-9 were observed in biopsies at rejection. Furthermore, despite the expected profound depletion of circulating B cells by Mx within 6 days of its administration, biopsies from both groups at rejection displayed a mild B cell infiltrate accompanied by a strong macrophage and intermediate T-cell infiltration, the latter tending to be more abundant in Mx-treated animals. Our data show that in this particular model of pig to baboon xenotransplantation and at the dose used, Mx was not superior to CyP in conferring protection against rejection, despite its capacity to profoundly deplete circulating B cells and to inhibit anti-Gal Ab responses to xenografts. DXR was thus possible without the return of anti-Gal Abs and may have been mediated by the early fixation of pre-existing Abs with secondary complement activation. However, although Mx was not more efficient than CyP in controlling DXR, its capacity to deplete B cells and delay Ab recovery may be beneficial in the context of Gal knockout organ transplantation where the induced Ab response is likely to take precedence over the preformed response.

The effect of mitoxantrone on anti-pig immunization in baboons.

Besides virological and physiological concerns, the success of xenotransplantation (Xt) is still dependent on the prevention of delayed xenograft rejection (DXR). Although multifactorial, DXR is mainly due to xenonatural antibody (Ab) recognizing their xenogenic antigen (Ag) followed by complement activation. Despite the use of intensive treatments capable of inhibiting the humoral response, DXR can still not be avoided and always occurs within weeks following transplantation. Moreover, these latter treatments currently used in Xt could not be used clinically in humans because of their high risk of over-immunosuppressing the patients. Mitoxantrone (Mx) is a drug well known for its antiproliferative properties and is used clinically in oncology and in the treatment of relapsing multiple sclerosis. In models of arthritis in rats, it has been shown to be 10 to 20 times more powerful than cyclophosphamide (CyP) at blocking both inflammatory and B-cell responses. Because of its B-cell inhibitory capacity and considering the implication of the humoral response in xenograft rejection, we have compared Mx with CyP for its ability to block in vivo anti-pig immunization induced via subcutaneous injections of pig red blood cells into baboons. Neither drug was able to inhibit the anti-pig responses following the first and second immunizations, emphasizing the particularity of preformed Ab responses. However, the rise in Ab in the Mx treated animals was significantly delayed as compared with the non-treated as well as the CyP treated animals and was mainly because of a profound depletion of circulating B-cells. Mx displays an interesting antihumoral effect that we now intend to test in a pig kidney to baboon Xt model, with anticipated administration of the drug allowing an early B-cell depletion.

Cellular participation in delayed xenograft rejection of hCD55 transgenic pig hearts by baboons.

Delayed xenograft rejection (DXR) of pig organs by baboons currently represents the major obstacle to successful xenotransplantation. Although antibodies (Abs) are believed to play a fundamental role in this form of rejection, so far little is known concerning the potential cellular component. Biopsies taken during DXR of human CD55 transgenic pig hearts by non-treated (n = 2), alpha-Gal immunoadsorbed (n = 2), or immunosuppressed (n = 9) baboons were studied. The cellular element was explored by determining not only its phenotype by classical immunohistochemical techniques but also its activity in terms of cytokines, cytolytic enzymes and other mediators using quantitative reverse transcription polymerase chain reaction. All porcine xenografts underwent DXR; within 5 days in non-treated and immunoadsorbed animals but significantly delayed (6 to 29 days) in immunosuppressed animals. Cellular infiltration in non-immunosuppressed grafts consisted predominantly of monocytes/macrophages, CD8 cells and a few CD4 T-cells. The predominant baboon transcripts detectable were the proinflammatory cytokines interleukin1-alpha and tumor necrosis factor-alpha, the lymphokine interferon-gamma and the cytotoxic enzyme granzyme B. However, these cellular components were lacking in the immunosuppressed animals. Despite these differences, strong immunoglobulin M (IgM) and C5b-9 complement deposition was observed in all animals at rejection. Together our findings suggest that although the humoral response plays a predominant role in DXR through IgM Abs and complement activation, there is a clear cellular infiltrate in DXR in this model that is likely to contribute to rejection through a strong pro-inflammatory and cytotoxic environment, necessitating substantial immunosuppression for a prolonged graft survival.

The effect of immunoglobulin immunadsorptions on delayed xenograft rejection of human CD55 transgenic pig kidneys in baboons.

Delayed xenograft rejection (DXR) remains a major obstacle in discordant xenotransplantation. As strategies of complement inhibition and xenogeneic natural antibody (Ab) removal have been shown to give prolonged xenograft survival, we endeavored to determine whether combining these two strategies would lead to an additive effect in terms of graft survival. The study was initiated with two groups, A and B, where group A received normal kidneys and group B received hCD55 transgenic kidneys. Both groups underwent pre-transplant (day-1) total immunoglobulin (Ig) immunoadsorption (IA) and received an immunosuppression of cyclophosphamide, cyclosporine A, mycophenolate mofetil and corticosteroids. Two subsequent groups (C and D) receiving hCD55 transgenic pig kidneys were then performed with an 'optimized' immunosuppression (Cyclophosphamide starting 1 day earlier) but only group D recipients were immunoadsorbed. Biopsies taken during the post-transplantation period were analyzed for Ab deposition, compliment activation and cellular infiltration. No hyperacute rejection was observed. In the initial immunoadsorbed groups A and B, all baboons underwent DXR, which started surprisingly early (day 5 in most cases. In the subsequent two groups, the immunoadsorbed group D baboons also underwent DXR, again as early as day 5. In contrast, group C baboons did not show any signs of DXR on their day 6 biopsy or at their time of death. Analysis of graft biopsies from the kidneys undergoing rejection or with stable function showed strong deposition of anti-Gal IgM in all cases whereas strong complement C5b-9 deposits were only observed in biopsies at rejection. Cellular infiltration consisted mostly of monocytes/macrophages, was more pronounced in biopsies taken at rejection and was associated with a pro-inflammatory environment involving interleukins 1alpha, 6 and 8. Our findings suggest non-specific Ig (anti-Gal and non-Gal Ig of all isotypes) IA or even incomplete IA in immunosuppressed baboon recipients of transgenic pig kidneys is detrimental to graft survival by being associated with an Ab and compliment driven rejection. We speculate that the IA were insufficient in terms of Ig depletion or frequency inducing an Ab rebound or that this total Ig depletion also removed components facilitating graft survival.