Research Highlights

Prolonged Xenokidney Graft Survival in Sensitized NHP Recipients by Expression of Multiple Human Transgenes in a Triple Knockout Pig Manook M, Olaso D, Anwar I, et al. Sci Transl Med. 2024;16(751):eadk6152. The development of anti-HLA antibodies, through exposure to antigens during pregnancy, blood transfusions, or prior transplants, reduces the potential pool of organs available to prospective transplant recipients. For those who are highly sensitized, waiting times on the transplant list are long, and for some individuals, the number of donors for whom an HLA-compatible transplant would be possible is vanishingly small. Although desensitization strategies, such as plasmapheresis, intravenous immunoglobulin, and, more recently, imlifidase treatment, exist, their use is often precluded in deceased organ donation and reserved for very specific cases.1 To increase the pool of organs available to these highly sensitized individuals, the use of xenografts has been proposed. Progress in xenotransplantation, through genetic engineering of pig donors and modifications to immunosuppressive regimens aimed at ameliorating antibody-mediated rejection (AMR), has led to increased survival times in nonhuman primate (NHP) models. Based on these successes, short-duration studies have now been performed using decedent human recipients and the first pig kidney and heart transplants to living human recipients.2-4 AMR is an emerging challenge in xenotransplantation, although the precise antigens responsible for its development are unclear. Anti-swine leukocyte antigens have previously been shown to be problematic but do not explain the entire alloresponse.5 It is further not known whether prior sensitization impacts the survival of xenografts, whether this can be ameliorated through further donor genetic editing, or whether transplantation in this setting would increase the immunological risks to recipients. To examine this, Manook et al6 described the approach to transplantation of pig kidneys from 2 pig cohorts with differing genetic edits into presensitized NHPs.6 Before xenotransplantation, the NHP recipients underwent sequential skin transplants from swine leukocyte antigen–mismatched donors to induce allosensitization. Subsequently, recipient animals underwent bilateral native nephrectomies combined with porcine kidney transplantation. Xenografts were from 2 cohorts of pigs (1KO.1TG and 3KO.7TG) with genes knocked out and inserted using CRISPR-Cas9-mediated gene editing. 1KO.1TG pigs had a single knockout (GGTA1) and a single human transgene insertion (CD55), whereas the 3KO.7TG pigs had 3 knockouts (GGTA1, CMAH, and b4GNT2/b4GALNT2) and 7 human transgenes inserted (CD46, CD55, CD47, THBD, PROCR, TNFAIP3, and HMOX1). These knockouts targeted carbohydrates produced on porcine cells, which humans have preformed antibodies against; inserted human transgenes are involved in coagulation, complement regulation, and the amelioration of inflammatory responses. The induction immunosuppressive regimens used anti-CD4 and anti-CD8 monoclonal antibodies (mAbs), with maintenance therapy comprising anti-CD154 mAb, mycophenolate mofetil, and methylprednisolone. The authors found that sensitized recipients of 1KO.1TG grafts experienced early graft loss with features of AMR and thrombotic microangiopathy. This occurred later than previously published data on allograft transplantation to sensitized recipients but earlier than xenografts in nonsensitized recipients. Grafts from the 3KO.7TG donors survived significantly longer with reduced features of AMR. Analysis of gene expression suggested a milder rejection phenotype in the 3KO.7TG recipients. Further analysis of 1KO.1TG recipients showed hyperplasticity of germinal centers, induction of follicular helper T cells, and a shift of circulating T cells to an exhausted phenotype, suggesting that these factors play a role in developing antibody-mediated responses to xenografts. In contrast, 3KO.7TG recipients exhibited a relative lack of germinal center responses, reduced numbers of neutrophils and monocytes, and reduced T-cell repopulation, indicating a downregulation of innate and adaptive responses to the 3KO.7TG grafts. Anti-CD154 mAb therapy was discontinued on day 35, and all animals developed AMR shortly after, but there was no evidence of T cell–mediated rejection, confirming the prominent role of AMR and the necessity for targeted immunosuppression. The authors also demonstrated that allosensitization does not produce a permanent increase in antixenograft donor-specific antibodies, and xenotransplantation does not further allosensitize recipients. This study confirms the prominent role of AMR in porcine xenotransplantation and the increased risk in allosensitized recipients. However, this risk can be reduced by further gene knockouts, insertion of human transgenes, and the adoption of an anti-CD154-based immunosuppressive regimen. Further studies comparing the 3KO.7TG grafts in nonsensitized and sensitized recipients, in addition to longer observation periods (given all grafts were functioning in the 3KO.7TG group before anti-CD154 mAb withdrawal), will add further evidence for their use in sensitized human recipients. Despite the recent transplantation of porcine xenografts in living humans, the long-term viability and future widespread use within transplantation remain uncertain. Certainly, evidence for an antibody-mediated alloresponse has been identified in recent pig-to-human transplants.7 This remains an important area of research because xenotransplants have the potential to provide vital, life-saving treatment for a range of patients with limited alternatives. Fecal Metabolite Profiling Identifies Liver Transplant Recipients at Risk for Postoperative Infection Lehmann CJ, Dylla NP, Odenwald M, et al. Cell Host Microbe. 2024;10;32(1):117–130.e4. An imbalance in the intestinal microbiome, characterized by a decrease in diversity and an expansion of pathobionts, is associated with changes in fecal metabolite concentrations and an increased risk of systemic infections.1 Nutrients, whole microbes, microbial components, and metabolic byproducts from the intestine are transported directly to the liver. Recipients of liver transplantation (LT) frequently face complications because high infection rates, which are linked to the intestinal microbiome, leading to the expansion of multidrug-resistant organisms and subsequent invasive infections.2 In the highlighted study by Lehmann et al, fecal metabolites from 107 patients undergoing LT were profiled to assess microbiome diversity and to help identify individuals at higher risk of postoperative bacterial infections associated with pathobiont expansion.3 Microbiome diversity was analyzed using gas chromatography and liquid chromatography-tandem mass spectrometry to detect metabolites produced or modified by the gut microbiota in fecal samples collected at the time of LT.3 Recipients with low microbial diversity demonstrated lower concentrations of acetate, propionate, and butyrate in their fecal samples. Conversely, patients with high microbial diversity maintained short-chain fatty acids concentrations like those observed in healthy donors.3 Similar trends were found with conjugated bile acids, including glycocholic acid and taurocholic acid, which showed comparable levels among healthy donors and the medium- to high-diversity groups but were notably lower in the low microbial diversity group.3 These findings suggest that LT patients with the highest microbiota diversity maintained normal metabolite levels, whereas those with low diversity exhibited significantly reduced levels of beneficial metabolites. The study documented the incidence and types of postoperative infections in LT recipients to investigate the correlations between microbiome and metabolome profiles and clinical outcomes. Among the 107 LT recipients, bacterial infections were reported in 25 individuals. The predominant infection sites were the abdomen (50%), urinary tract (20%), and skin or surgical sites (13%), mainly attributed to Enterococcus (45%) and Enterobacterales (20%).3 Unidentified organisms accounted for 13% of infections. Moreover, by longitudinally comparing fecal samples from infected and noninfected patients, the study revealed that infections were often preceded by a decrease in microbiome diversity and an increase in Enterococci and Enterobacterales.3 Four representative timelines, 3 from infected patients and 1 from an uninfected patient, illustrated these associations with postoperative infections.3 The study highlighted the challenge of determining the roles played by commensal bacterial species and their metabolic products in disease resistance within clinical settings. Broad-spectrum antibiotics, commonly used in the cohort, frequently disrupted the microbiome, leading to the depletion of various microbial taxa and microbiota-derived metabolites. Furthermore, the work showcased that LT recipients with compromised microbiomes, identified through metagenomic analyses, can be distinguished by measuring specific microbially produced metabolites. These measurements identify patients with reduced microbial diversity and those with pathobiont expansion, heightening their vulnerability to postoperative infections. The rapid changes in microbiome compositions underline the importance of obtaining immediate results from fecal metabolite quantitation, providing essential real-time insights that could guide clinical strategies. Such strategies might involve probiotics or microbiome-modulating foods aimed at modulating the microbiome,4 potentially improving patient outcomes.