Current desensitization and maintenance immunosuppression regimens for kidney transplantation in sensitized individuals show limited ability to control the posttransplant humoral response, resulting in high rates of antibody-mediated rejection (ABMR) and graft failure. Here, we showed that anti-CD154 monoclonal antibody (mAb)-based immunosuppression more effectively controlled allograft rejection and humoral rebound in a highly sensitized nonhuman primate kidney transplantation model compared with tacrolimus-based standard-of-care (SOC) immunosuppression. Desensitization with an anti-CD154 mAb (5C8) and a proteasome inhibitor led to decreased donor-specific antibodies (DSAs) and disruption of lymph node germinal centers with reduction of proliferating, memory, and class-switched B cells as well as T follicular helper cells. After transplant, the nonhuman primates maintained on 5C8-based immunosuppression had significantly better survival compared with those maintained on SOC immunosuppression (135.2 days versus 32.8 days, P = 0.013). The 5C8-treated group demonstrated better suppression of DSAs after transplant, more robust suppression of B cell populations, and better induction of regulatory T cells. Fewer infectious and welfare complications, including viral reactivation and weight loss, were also observed with 5C8-based immunosuppression compared with SOC immunosuppression. Therefore, anti-CD154 mAbs may improve kidney transplant outcomes through better control of posttransplant immune responses. The superior efficacy of anti-CD154 mAb-based immunosuppression over tacrolimus-based SOC seen in this highly sensitized NHP transplant model suggests that anti-CD154 mAbs could potentially be used to desensitize and treat highly sensitized patients receiving kidney transplantation.

BACKGROUND:Surgical bleeding is a risk in any solid organ transplant, and is commonly addressed with the transfusion of human blood products to replace or supplement coagulation factors. It is unknown if these blood products would harm xenotransplanted pig organs in human recipients demonstrating coagulopathy. The aim of this study was to investigate in vitro if blood products such as fresh frozen plasma (FFP) or cryoprecipitate (cryo) contain xenoantibodies capable of cytotoxicity to GTKO pig cells. METHODS:We obtained 12 individual single-donor (7 FFP and 5 cryo) blood products from our institution's blood bank for testing. Peripheral blood mononuclear cells (PBMCs) were obtained from a GTKO/hCD55 pig for use as target cells. We performed a series of flow cytometry crossmatch (FCXM) and complement-dependent cytotoxicity (CDC) assays. RESULTS:We found that all the tested blood products contained some degree of IgM and IgG xenoantibody. Tests using a 1:50 dilution revealed a significant decrease in IgM xenoantibody binding, but an increase in the detection of IgG binding. Multiple preparations were capable of GTKO PBMC cytotoxicity but the level of antibody binding and cell death varied by preparation. CONCLUSIONS:Both FFP and cryo contain IgM and IgG non-galactose-α-1,3-galactose (αGal) xenoantibodies capable of killing GTKO PBMCs, though the level varies by preparation. Although some centers utilize a genetic background with mutations in the three enzymes responsible for the known xenoantigens, others are investigating the GTKO pig as a potential option. These results suggest that a center pursuing a human xenotransplantation study with a GTKO genetic background should pre-screen blood products prior to administration.

ABSTRACTBackgroundThe removal of preformed antibodies with cleaving enzymes like IdeS (imlifidase) has demonstrated therapeutic potential in organ transplantation for sensitized recipients. However, preformed xenoreactive antibodies (XAbs) against porcine glycans are predominantly IgM and considered detrimental in pig‐to‐human xenotransplantation.MethodsRecombinant IceM, an endopeptidase cleaving IgM, was generated in Escherichia coli (E. coli). Four maximally MHC‐mismatched rhesus macaques underwent two serial skin transplantations to model allosensitized patients awaiting xenotransplantation. IceM was administered IV in allosensitized animals at 28 and 56 days after the first skin transplantation to assess in vivo IgM cleavage. Total IgG and IgM were quantified with western blot, and anti‐pig (xenoreactive) IgG/IgM were evaluated using flow crossmatch. B cells and their subpopulations were assessed using flow cytometry.ResultsIceM selectively cleaved human IgM, while showing no cleavage activity toward other isotypes, including IgG, IgA, IgD, and IgE. Additionally, IceM cleaves only human and nonhuman primate IgM in vitro, but not in sera from other species. At a dose of 0.5 mg/kg, IceM reduced xenoreactive IgM levels to 13.76% ± 4.98% of baseline (B cell flow crossmatch) at 24 h postadministration, with baseline levels restored approximately 2 weeks after treatment. Additionally, animals showed similar kinetics of xenoreactive IgM degradation with the repeated dose of IceM.ConclusionIn this study, we report a recombinant bacterial enzyme that selectively cleaves IgM in human and nonhuman primate sera. Repeat administration of IceM in macaques enables selective, robust clearance of circulating xenoreactive IgM. This approach will be useful in treating preformed natural and rebound IgM in xenotransplantation.

Xenotransplantation is a potential option for individuals for whom an acceptable human allograft is unavailable. Individuals with broadly reactive HLA antibodies due to prior exposure to foreign HLA are potential candidates for a clinical xenotransplant trial. It remains controversial if allosensitisation results in the development of cross-reactive antibodies against SLA. This may require increased histocompatibility scrutiny for highly sensitised individuals prior to enrollment in a clinical trial. Serum samples were obtained from non-human primates sensitised via serial skin transplantation from maximally MHC-mismatched donor, as reported. Sera from pre- and post-allosensitisation timepoints were assessed in a flow crossmatch (FXM) for IgM and IgG binding to pig splenocytes with or without red blood cell adsorption. Xenoreactive antibodies were eluted from pig splenocytes and screened on a single antigen HLA bead assay. A MHC Matchmaker algorithm was developed to predict potential conserved amino acid motifs among the pig, NHP, and human. Our sensitised NHP model was used to demonstrate that allosensitisation does not result in an appreciable difference in xenoreactive antibody binding in a cell-based FXM. However, antibody elution and screening on single antigen HLA beads suggest the existence of potential cross-reactive antibodies against SLA. The cross-reactive IgG after allosensitisation were predicted by comparing the recipient Mamu alleles against its previous allograft donor Mamu alleles and the donor pig SLA alleles. Our study suggests that allosensitisation could elevate cross-reactive antibodies, but a more sensitive assay than a cell-based FXM is required to detect them. The MHC Matchmaker algorithm was developed as a potential tool to help determine amino acid motif conservation and reactivity pattern.

This report summarizes the content of a debate sponsored by eGenesis Bio, organized by the International Xenotransplantation Association (IXA), and attended by more than 150 delegates in the context of the IPITA-IXA-CTRMS Joint Congress held in San Diego in October 2023. The debate centered around two important immunological topics relating to xenotransplantation. The first was a debate relating to the statement that "HLA-sensitized patients are at higher risk for rejecting a pig xenograft." Stuart Knechtle provided evidence to support this statement and Massimo Mangiola opposed it. Before the debate, a majority (>80%) of the audience agreed with this statement. After listening to the debate, this percentage was reduced to approximately 60%. The second debated statement was "Recipients of pig xenografts who develop anti-pig antibodies are at higher risk for rejecting a subsequent allograft." This was proposed by A. Joseph Tector and opposed by L & eacute;o H. B & uuml;hler. Before the debate, once again a majority of the audience (approximately 60%) believed that prior sensitization to a pig xenograft would be detrimental to the survival of a subsequent allograft. However, after listening to the debate, only about 40% believed this statement to be correct. The topics discussed remain complex and answers are not yet conclusive. However, the present evidence suggests that allosensitization may prove detrimental to subsequent xenotransplantation, whilst sensitization to pig antigens may not be detrimental to subsequent allotransplantation.