Anti-pig Antibody Levels Research Articles

Mixed xenogeneic porcine chimerism tolerizes human anti-pig natural antibody-producing cells in a humanized mouse model.

A major obstacle to the success of organ transplantation from pigs to humans, necessitated by the shortage of human organs, is robust humoral immune rejection by pig-reactive human antibodies. Mixed xenogeneic hematopoietic chimerism induces xenoreactive B cell tolerance in rodents, but whether mixed pig/human chimerism could induce tolerance of human B cells to pig xenoantigens is unknown. We investigated this question using a humanized mouse model in which durable mixed (pig-human) xenogeneic chimerism can be established. Human natural anti-pig cytotoxic antibodies, predominantly IgM, are detectable in non-chimeric humanized mouse serum, and pig-reactive antibodies were reduced in mixed chimeric versus non-chimeric humanized mice. This difference required persistent mixed chimerism and was not due to the adsorption of antibodies on pig cells in vivo. Furthermore, human B cells from spleens of mixed chimeric mice produced lower levels of anti-pig antibodies when stimulated in vitro compared with those from non-chimeric mice. Our findings demonstrate that mixed chimerism reduces human natural antibodies to pig xenoantigens, providing the first in vivo evidence of human B cell tolerance induction by mixed xenogeneic chimerism and supporting further evaluation of this approach for inducing human B cell tolerance to xenografts.

Intra-bone bone marrow transplantation from hCD47 transgenic pigs to baboons prolongs chimerism to >60 days and promotes increased porcine lung transplant survival.

We have recently demonstrated that human-CD47 (hCD47) expressed on endothelial cells of porcine lung xenografts extended median graft survival from 3.5days to 8.7days in baboons. Intra-bone bone marrow transplantation (IBBMTx) in a pig-to-baboon model was previously shown to markedly prolong the duration of macrochimerism up to 21days from 1 to 4days by intravenous BMTx. We now examined whether the use of hCD47 transgenic (Tg) BM further prolonged the duration of chimerism following IBBMTx. We then tested if lung xenograft survival was prolonged following IBBMTx. Baboons received GalTKO-hCD47/hCD55Tg (n=5) or -hCD55Tg (n=1) or -hCD46/HLA-E Tg (n=1) pig IBBMTx. Macrochimerism, anti-pig T cells and antibody responses were assessed. Animals received lung xenografts from either hCD47+ or hCD47- porcine lungs 1-3months later. All baboons that received hCD47Tg porcine IBBM maintained durable macrochimerism >30days, and two maintained chimerism for >8weeks. Notably, anti-pig antibody levels decreased over time and anti-pig cellular unresponsiveness developed following IBBMTx. Lungs from hCD47Tg IBBMTx matched pigs were transplanted at day 33 or day 49 after IBBMTx. These animals showed extended survival up to 13 and 14days, while animals that received lungs from hCD47 negative pigs displayed no prolonged survival (1-4days). This is the first report demonstrating durable macrochimerism beyond 8weeks, as well as evidence for B cell tolerance in large animal xenotransplantation. Using hCD47Tg pigs as both IBBMTx and lung donors prolongs lung xenograft survival. However, additional strategies are required to control the acute loss of lung xenografts.

Low anti-pig antibody levels are key to the success of solid organ xenotransplantation: But is this sufficient?

Low anti-pig antibody levels are key to the success of solid organ xenotransplantation: But is this sufficient?

Beneficial effects of the transgenic expression of human sTNF-αR-Fc and HO-1 on pig-to-mouse islet xenograft survival

Both human soluble tumor necrosis factor-α receptor-Fc (sTNF-αR-Fc) and heme oxygenase-1 (HO-1) transgenic pigs have been generated previously for xenotransplantation. Here, we investigated whether overexpression of sTNF-αR-Fc or HO-1 in pig islets prolongs islet xenograft survival. Adult porcine islets were isolated from human sTNF-αR-Fc or HO-1 transgenic and wild type pigs, and were transplanted into diabetic nude mice. Effects of the expression of both genes on islet apoptosis, chemokine expression, cellular infiltration, antibody production, and islet xenograft survival were analyzed. Human sTNF-αR-Fc transgenic pigs successfully expressed sTNF-αR-Fc in the islets; human HO-1 transgenic pigs expressed significant levels of HO-1 in the islets. Pig-to-mouse islet xenograft survival was significantly prolonged in both the sTNF-αR-Fc and HO-1 groups compared with that in the wild type group. Both the sTNF-αR-Fc and HO-1 groups exhibited suppressed intragraft expression of monocyte chemoattractant protein-1 (MCP-1) and decreased perigraft infiltration of immune cells. However, there was no difference in the anti-pig antibody levels between the groups. Apoptosis of islet cells during the early engraftment was suppressed only in the HO-1 group. Porcine islets from both sTNF-αR-Fc and HO-1 transgenic pigs prolonged xenograft survival by suppressing islet cell apoptosis or secondary inflammatory responses following islet death, indicating that these transgenic pigs might have applications in successful islet xenotransplantation.

Pre-transplant antibody screening and anti-CD154 costimulation blockade promote long-term xenograft survival in a pig-to-primate kidney transplant model.

Xenotransplantation has the potential to alleviate the organ shortage that prevents many patients with end-stage renal disease from enjoying the benefits of kidney transplantation. Despite significant advances in other models, pig-to-primate kidney xenotransplantation has met limited success. Preformed anti-pig antibodies are an important component of the xenogeneic immune response. To address this, we screened a cohort of 34 rhesus macaques for anti-pig antibody levels. We then selected animals with both low and high titers of anti-pig antibodies to proceed with kidney transplant from galactose-α1,3-galactose knockout/CD55 transgenic pig donors. All animals received T-cell depletion followed by maintenance therapy with costimulation blockade (either anti-CD154 mAb or belatacept), mycophenolate mofetil, and steroid. The animal with the high titer of anti-pig antibody rejected the kidney xenograft within the first week. Low-titer animals treated with anti-CD154 antibody, but not belatacept exhibited prolonged kidney xenograft survival (>133 and >126 vs. 14 and 21days, respectively). Long-term surviving animals treated with the anti-CD154-based regimen continue to have normal kidney function and preserved renal architecture without evidence of rejection on biopsies sampled at day 100. This description of the longest reported survival of pig-to-non-human primate kidney xenotransplantation, now >125days, provides promise for further study and potential clinical translation.

Initial Evidence for Functional Immune Modulation in Primate Recipients of Porcine Skin Grafts Following Conditioning With Human CD47 Transgenic Pig Hematopoietic Stem Cells.

Background: To inhibit phagocytosis of porcine cells by primate macrophages in tolerization protocols including mixed chimerism, we generated human CD47 transgenic swine on an α-1,3-galactosyltransferase null background (hCD47/GalT-KO). We report the initial results of hematopoietic stem (HSC) transplantation from this swine into baboons with respect to cell survival and immune response. Methods: Two baboons underwent a conditioning regimen consisting of non-myeloablative total body and thymic irradiation, T and B cell depletion, and infusion of HSC obtained by cytokine mobilization and leukapheresis of a CD47 transgenic GalT-KO swine. Immunosuppression consisted of clinical level administration of FK506 for the first 30 days and anti-CD154 antibody for the first 7 weeks following cell transplant. Recipients received challenge infusions of HSPC from the same donor swine 7 and 10 weeks following the initial infusion. Both baboons received split thickness skin grafts from the porcine donor on full thickness wound beds at 14 weeks without any additional immunosuppression. Results: Peripheral macrochimerism was detected following three cell transplants, with >10% levels observed at 24 hours declining over the next 3-7 days. Despite detectable macrochimerism no increase in anti-pig antibody levels or T-cell sensitization was observed prior to skin grafting. Both xenogeneic skin grafts developed extensive areas of bruising during the first week, while maintaining islands of apparently healthy tissue. These islands underwent expansion, reaching confluence over the entire wound bed in one recipient 6 weeks after graft placement and undergoing slow rejection over the following 5 weeks. In vitro measures of T cell responsiveness showed no evidence of sensitization to the xenografts prior to rejection. Conclusions: Assessment of the definitive role of hCD47 transgenic HSC transplantation in these results must await further ongoing studies of non-transgenic cell transplantation in similarly conditioned recipients. Nevertheless, the level and duration of chimerism using hCD47/GalT-KO mobilized HSC is very encouraging and may have been responsible for the greatly attenuated response against nonvascular components of xenogeneic skin grafts that we have observed in the absence of additional immunosuppression.

Enhanced PD-1 Signalling Downregulated B-Cell Activation and Antibody Responses to Porcine Xenoantigen.

Background: Human T-cells express several receptors like programmed death-1 receptor (PD-1), which is known to negatively regulate T-cell activation. Thus, transgenic overexpression of corresponding ligands on porcine cells and tissues could be a strategy to prevent human immune responses to xenografts. We have previously shown that in vitro human T-cell responses to porcine transfectants overexpressing human PD-Ligand 1 (L23-PD-L1) are reduced. In this study we assessed the role of enhanced PD-1 signalling on B-cell activation and antibody responses to xenoantigen. Methods: Polyclonal activation of isolated human B-cells was induced by CpG-ODN and the effect of a dimeric PD-L1 immunoglobulin (Ig) fusion protein was determined by [3H]-thymidine cell proliferation assay. A xenoreactive response was induced by coculturing human peripheral blood mononuclear cells (hPBMC) with L23-PD-L1 cells or mock-transfected controls in vitro. To characterize the xenospecific antibody responsein vivo, PD-L1.Ig was applied to rats immunized with L23-control cells. Antibody titers in the culture supernatant or the sera of rats and the activation status of B-cells in draining lymph nodes were determined by flow cytometry or ELISA. Results: Stimulation of isolated B-cells with CpG-ODN resulted in a high proliferation rate and upregulation of PD-1. Addition of PD-L1.Ig significantly reduced B-cell proliferation in dose-dependent manner. Moreover, cocultivation of hPBMCs with porcine cells resulted in high levels of anti-pig antibodies, whereas only low titers were observed in the presence of membrane bound PD-L1 (L23-PD-L1 cells) or soluble PD-L1.Ig. Data of rat immunization experiments confirmed that triggering of the PD-1 pathway reduced serum anti-pig antibody levels. Further analysis of B-cell populations in draining lymph nodes revealed a lower frequency of activated B-cells (Ig+CD44hi) after application of PD-L1-Ig. Conclusion: These data suggest a direct inhibitory role of the PD-1 pathway on B-cell activation, as reported for T-cells. Thus, antibody production to porcine xenoantigen can be markedly lower in the presence of high concentrations of the inhibitory ligand PD-L1. Altogether, treatment with PD-L1 might be a strategy to diminish not only T-cell activation, but also the elicited antibody response to xenoantigen, which is a major hurdle to successful xenotransplantation.

Hyperacute rejection of hDAF-transgenic pig organ xenografts in cynomolgus monkeys: influence of pre-existing anti-pig antibodies and prevention by the alpha GAL glycoconjugate GAS914.

Our introductory pig-to-cynomolgus monkey heart or kidney transplantation using organs from pigs transgenic for human decay-accelerating factor (hDAF), showed a high incidence of hyperacute rejection (HAR), which was ascribed to extraordinary high levels of anti-pig antibodies. We evaluated the efficacy of GAS914, a Gal alpha 1-3Gal trisaccharide linked to a poly-l-lysine backbone, in inhibition of HAR. hDAF transgenic heterotopic heart (n = 15) or life-supporting kidney (n = 8) transplantation included induction with cyclophosphamide or anti-thymocyte globulin, and maintenance with cyclosporine or tacrolimus, steroids and mycophenolate sodium/mofetil. Four doses of GAS914 were given before transplantation. Rejection was confirmed by graft histology, and anti-pig antibody levels were determined in various assays. Four of six heart transplants without GAS914 treatment showed HAR. Nine subsequent transplants with GAS914 pre-treatment, did not show HAR (chi-square, P < 0.05). Two of four kidney transplants without GAS914 treatment ended with HAR. Four subsequent transplants with GAS914 did not show HAR. Animals with HAR showed extremely high antibody levels. Samples just before transplantation showed significantly higher antibody levels in recipients presenting with HAR. In all assays antibody levels were significantly lowered by GAS914 pre-treatment. HAR of hDAF solid organs could be ascribed to high levels of anti-pig antibodies. It is hypothesized that the hDAF transgene shows a threshold in efficacy, above which an overwhelming attack by antibodies and complement activation cannot be modulated to prevent HAR. HAR does not occur when animals with lower levels are used, or when antibodies are effectively depleted from the circulation by GAS914 treatment.

Fetal porcine thymus engraftment, survival and CD4 reconstitution in αGal‐KO mice is impaired in the presence of high levels of antibodies against αGal

Xenospecific T-cell tolerance can be induced among murine and human T-cells by porcine thymic grafting. However, anti-alpha 1,3-galactosyltranserase (alphaGal) (Galalpha1-3Galbeta1-4GlcNAc-R) natural antibodies (NAbs) pose a major barrier to porcine xenografts in humans. We used alphaGal knockout (KO) and muchain KO mice to explore the effect of natural anti-alphaGal and other xenoantibodies on porcine thymic engraftment and to examine the potential of thymic tissue to tolerize anti-alphaGal antibody-producing cells. Thymectomized [adult thymectomy (ATX)] non-immunized and rabbit red blood cell (RRBC) pre-transplant immunized alphaGal-KO (knockout), wild-type (WT) and mu chain KO B6 mice were treated with 3Gy total body irradiation (TBI), and T and natural killer (NK) cell depleting monoclonal antibodies (mAbs). These conditioned mice were grafted with fetal porcine thymus and liver (FP THY/LIV) tissue under the kidney capsule. Flow cytometric analysis was performed to follow CD4 reconstitution as a measure of FP THY engraftment and function. Only mice with >10% CD4+ peripheral blood lymphocytes (PBL) were considered successfully engrafted. Enzyme-linked immunosorbent assay (ELISA) was used to assess the kinetics of immunoglobulin M (IgM) and IgG anti-alphaGal antibodies. Anti-pig antibodies were monitored by flow cytometry (FCM). FP THY engrafted successfully in most of the immunoglobulin deficient mice (11 out of 12, 92%) and the outcome was similar in WT B6 controls (8 out of 12, 67%). Non-immunized alphaGal-KO mice grafted with FP THY had a similar success rate (7 out of 11) to that observed in non-immunized alphaGal-WT controls (2 out of 4). In contrast, alphaGal-KO mice immunized pre-transplant with RRBC, then grafted with FP THY/LIV, showed a significant reduction in the success of thymic grafting (2 out of 9, 22%) compared with pre-transplant immunized WT controls (4 out of 7; 57%) and non-immunized alphaGal-KO mice (7 out of 11, 64%). Anti-Gal and anti-pig antibody levels were not markedly augmented by porcine thymus grafts in mice with successful thymus grafts. FP THY engraftment is impaired in the presence of high levels of anti-alphaGal xenoantibodies. However, low levels of anti-alphaGal antibodies and other mouse anti-pig NAbs appear not to play a major role in the rejection of FP THY. Although grafting FP THY expressing the alphaGal epitope did not tolerize B cells producing anti-alphaGal antibodies in a T-cell independent manner, it prevented T-cell dependent sensitization by inducing T-cell tolerance to porcine antigens.

Anti-pig antibody levels in naïve baboons and cynomolgus monkeys.

Anti-pig antibodies (APA) were analysed in serum from 28 naïve wild-caught baboons (originating from Kenya) and 31 naïve captive-bred cynomolgus monkeys (13 from the Philippines and 18 from Mauritius), using a haemolytic assay with pig erythrocytes (APA), flow cytometry on the porcine lymphoma T-cell cell line L35, and enzyme linked immunosorbent assay (ELISA) using alpha-Gal type II and type VI antigen. This was extended in baboon samples by the evaluation in two laboratories (Imutran, Cambridge, UK and Immerge, Boston, USA), and by antibody absorption using either immobilized alpha-Gal type II or alpha-Gal type VI. Anti-porcine antibodies were demonstrated in all assays with substantial variability within and between the three non-human primate groups. Immunoglobulin (Ig)M antibody levels tended to be similar to or higher than those in a pooled normal human standard serum while IgG levels tended to be lower. Highest antibody levels were recorded in Mauritius cynomolgus monkeys. There were statistically significant correlations between assays for IgM or IgG class anti-Gal antibodies using either alpha-Gal type II or alpha-Gal type VI as antigen, both for different assays and two laboratories involved. Also, significant correlations were observed between the anti-Gal and L35 binding assays. Baboon sera before and after absorption to immobilized alpha-Gal type II or type VI were analysed for anti-Gal type VI or type II antibody: levels were almost undetectable indicating that most anti-Gal antibodies react to epitopes shared between alpha-Gal type II and type VI oligosaccharides. Finally, the relation between APA and outcome of porcine heart xenotransplantation in cynomolgus monkeys and baboons showed no apparent relation between pre-transplant APA levels and the occurrence of hyperacute rejection (HAR) when compared with non-immunological cause of organ/recipient dysfunction or acute humoral xenograft rejection during the first 4 days post-transplantation or survival exceeding 4 days post-transplantation.