Faculty Bibliography

The gastrointestinal tract is continuously exposed to foreign antigens in food and commensal microbes with potential to induce adaptive immune responses. Peripherally induced T regulatory (pTreg) cells are essential for mitigating inflammatory responses to these agents^1-4. While RORγt⁺ antigen-presenting cells (RORγt-APCs) were shown to program gut microbiota-specific pTreg^5-7, their definition remains incomplete, and the APC responsible for food tolerance has remained elusive. Here, we identify an APC subset required for differentiation of both food- and microbiota-specific pTreg cells and for establishment of oral tolerance. Development and function of these APCs require expression of the transcription factors Prdm16 and RORγt, as well as a unique Rorc(t) cis-regulatory element. Gene expression, chromatin accessibility, and surface marker analysis establish the pTreg-inducing APCs as myeloid in origin, distinct from ILC3, and sharing epigenetic profiles with classical dendritic cells (cDC), and designate them Prdm16⁺ RORγt⁺ tolerizing DC (tolDC). Upon genetic perturbation of tolDC, we observe a substantial increase in food antigen-specific T helper 2 (Th2) cells in lieu of pTreg, leading to compromised tolerance in mouse models of asthma and food allergy. Single-cell analyses of freshly resected mesenteric lymph nodes from a human organ donor, as well as multiple specimens of human intestine and tonsil, reveal candidate tolDC with co-expression of PRDM16 and RORC and an extensive transcriptome shared with mice, highlighting an evolutionarily conserved role across species. Our findings suggest that a better understanding of how tolDC develop and how they regulate T cell responses to food and microbial antigens could offer new insights into developing therapeutic strategies for autoimmune and allergic diseases as well as organ transplant tolerance.

OBJECTIVES/OBJECTIVE:To systematically review the safety and efficacy of nonbiological (NBAL) or biological artificial liver support systems (BAL) and whole-organ extracorporeal liver perfusion (W-ECLP) systems, in adults with acute liver failure (ALF) and acute-on-chronic liver failure (ACLF). DATA SOURCES/METHODS:Eligible NBAL/BAL studies from PubMed/Embase searches were randomized controlled trials (RCTs) in adult patients with ALF/ACLF, greater than or equal to ten patients per group, reporting outcomes related to survival, adverse events, transplantation rate, and hepatic encephalopathy, and published in English from January 2000 to July 2023. Separately, we searched for studies evaluating W-ECLP in adult patients with ALF or ACLF published between January1990 and July 2023. STUDY SELECTION AND DATA EXTRACTION/METHODS:Two researchers independently screened citations for eligibility and, of eligible studies, retrieved data related to study characteristics, patients and interventions, outcomes definition, and intervention effects. The Cochrane Risk of Bias 2 tool and Joanna Briggs Institute checklists were used to assess individual study risk of bias. Meta-analysis of mortality at 28-30 days post-support system initiation and frequency of at least one serious adverse event (SAE) generated pooled risk ratios (RRs), based on random (mortality) or fixed (SAE) effects models. DATA SYNTHESIS/RESULTS:Of 17 trials evaluating NBAL/BAL systems, 11 reported 28-30 days mortality and five reported frequency of at least one SAE. Overall, NBAL/BAL was not statistically associated with mortality at 28-30 days (RR, 0.85; 95% CI, 0.67-1.07; p = 0.169) or frequency of at least one SAE (RR, 1.15; 95% CI, 0.99-1.33; p = 0.059), compared with standard medical treatment. Subgroup results on ALF patients suggest possible benefit for mortality (RR, 0.67; 95% CI, 0.44-1.03; p = 0.069). From six reports of W-ECLP (12 patients), more than half (58%) of severe patients were bridged to transplantation and survived without transmission of porcine retroviruses. CONCLUSIONS:Despite no significant pooled effects of NBAL/BAL devices, the available evidence calls for further research and development of extracorporeal liver support systems, with larger RCTs and optimization of patient selection, perfusion durability, and treatment protocols.

BACKGROUND:Xenotransplantation of genetically engineered porcine organs has the potential to address the challenge of organ donor shortage. Two cases of porcine-to-human kidney xenotransplantation were performed, yet the physiological effects on the xenografts and the recipients' immune responses remain largely uncharacterized. METHODS:We performed single-cell RNA sequencing (scRNA-seq) and longitudinal RNA-seq analyses of the porcine kidneys to dissect xenotransplantation-associated cellular dynamics and xenograft-recipient interactions. We additionally performed longitudinal scRNA-seq of the peripheral blood mononuclear cells (PBMCs) to detect recipient immune responses across time. FINDINGS/RESULTS:Although no hyperacute rejection signals were detected, scRNA-seq analyses of the xenografts found evidence of endothelial cell and immune response activation, indicating early signs of antibody-mediated rejection. Tracing the cells' species origin, we found human immune cell infiltration in both xenografts. Human transcripts in the longitudinal bulk RNA-seq revealed that human immune cell infiltration and the activation of interferon-gamma-induced chemokine expression occurred by 12 and 48 h post-xenotransplantation, respectively. Concordantly, longitudinal scRNA-seq of PBMCs also revealed two phases of the recipients' immune responses at 12 and 48-53 h. Lastly, we observed global expression signatures of xenotransplantation-associated kidney tissue damage in the xenografts. Surprisingly, we detected a rapid increase of proliferative cells in both xenografts, indicating the activation of the porcine tissue repair program. CONCLUSIONS:Longitudinal and single-cell transcriptomic analyses of porcine kidneys and the recipient's PBMCs revealed time-resolved cellular dynamics of xenograft-recipient interactions during xenotransplantation. These cues can be leveraged for designing gene edits and immunosuppression regimens to optimize xenotransplantation outcomes. FUNDING/BACKGROUND:This work was supported by NIH RM1HG009491 and DP5OD033430.

BACKGROUND AIMS/UNASSIGNED:In liver transplantation, cold preservation induces ischemia, resulting in significant reperfusion injury. Hypothermic Oxygenated Machine Perfusion (HMP-O2) has shown benefit compared to static cold storage (SCS) by limiting ischemia-reperfusion injury. This study reports outcomes using a novel portable HMP-O2 device in the first US randomized control trial. APPROACH RESULTS/UNASSIGNED:The PILOT™ trial (NCT03484455) was a multicenter, randomized, open-label, non-inferiority trial, with participants randomized to HMP-O2 or SCS. HMP-O2 livers were preserved using the Lifeport® Liver Transporter and Vasosol® perfusion solution. Primary outcome was early allograft dysfunction (EAD). Non-inferiority margin was 7.5%. From 4/3/19-7/12/22, 179 patients were randomized to HMP-O2 (n=90) or SCS (n=89). Per protocol cohort included 63 HMP-O2 and 73 SCS. EAD occurred in 11.1% HMP-O2 (N=7) and 16.4% SCS (N=12). The risk difference between HMP-O2 and SCS was -5.33% (one-sided 95% upper confidence limit of 5.81%), establishing noninferiority. Risk of graft failure as predicted by L-GrAFT7 was lower with HMP-O2 (median [IQR] 3.4% [2.4-6.5] vs. 4.5% [2.9-9.4], p=0.024). Primary nonfunction occurred in 2.2%, all SCS (n=3, p=0.10). Biliary strictures occurred in 16.4% SCS (n=12) and 6.3% (n=4) HMP-O2 (p=0.18). Non-anastomotic biliary strictures occurred only in SCS (n=4). CONCLUSIONS:HMP-O2 demonstrates safety and noninferior efficacy for liver graft preservation in comparison to SCS. EAD by L-GrAFT7 was lower in HMP-O2, suggesting improved early clinical function. Recipients of HMP-O2 livers also demonstrated a lower incidence PNF and biliary strictures, although this difference did not reach significance.

In a previous study, heart xenografts from 10-gene-edited pigs transplanted into two human decedents did not show evidence of acute-onset cellular- or antibody-mediated rejection. Here, to better understand the detailed molecular landscape following xenotransplantation, we carried out bulk and single-cell transcriptomics, lipidomics, proteomics and metabolomics on blood samples obtained from the transplanted decedents every 6 h, as well as histological and transcriptomic tissue profiling. We observed substantial early immune responses in peripheral blood mononuclear cells and xenograft tissue obtained from decedent 1 (male), associated with downstream T cell and natural killer cell activity. Longitudinal analyses indicated the presence of ischemia reperfusion injury, exacerbated by inadequate immunosuppression of T cells, consistent with previous findings of perioperative cardiac xenograft dysfunction in pig-to-nonhuman primate studies. Moreover, at 42 h after transplantation, substantial alterations in cellular metabolism and liver-damage pathways occurred, correlating with profound organ-wide physiological dysfunction. By contrast, relatively minor changes in RNA, protein, lipid and metabolism profiles were observed in decedent 2 (female) as compared to decedent 1. Overall, these multi-omics analyses delineate distinct responses to cardiac xenotransplantation in the two human decedents and reveal new insights into early molecular and immune responses after xenotransplantation. These findings may aid in the development of targeted therapeutic approaches to limit ischemia reperfusion injury-related phenotypes and improve outcomes.

The first 2 living recipients of pig hearts died unexpectedly within 2 months, despite both recipients receiving what over 30 years of nonhuman primate (NHP) research would suggest were the optimal gene edits and immunosuppression to ensure success. These results prompt us to question how faithfully data from the NHP model translate into human outcomes. Before attempting any further heart xenotransplants in living humans, it is highly advisable to gain a more comprehensive understanding of why the promising preclinical NHP data did not accurately predict outcomes in humans. It is also unlikely that additional NHP data will provide more information that would de-risk a xenoheart clinical trial because these cases were based on the best practices from the most successful NHP results to date. Although imperfect, the decedent model offers a complementary avenue to determine appropriate treatment regimens to control the human immune response to xenografts and better understand the biologic differences between humans and NHP that could lead to such starkly contrasting outcomes. Herein, we explore the potential benefits and drawbacks of the decedent model and contrast it to the advantages and disadvantages of the extensive body of data generated in the NHP xenoheart transplantation model.

BACKGROUND:Cross-species immunological incompatibilities have hampered pig-to-human xenotransplantation, but porcine genome engineering recently enabled the first successful experiments. However, little is known about the immune response after the transplantation of pig kidneys to human recipients. We aimed to precisely characterise the early immune responses to the xenotransplantation using a multimodal deep phenotyping approach. METHODS:We did a complete phenotyping of two pig kidney xenografts transplanted to decedent humans. We used a multimodal strategy combining morphological evaluation, immunophenotyping (IgM, IgG, C4d, CD68, CD15, NKp46, CD3, CD20, and von Willebrand factor), gene expression profiling, and whole-transcriptome digital spatial profiling and cell deconvolution. Xenografts before implantation, wild-type pig kidney autografts, as well as wild-type, non-transplanted pig kidneys with and without ischaemia-reperfusion were used as controls. FINDINGS:cells. Both xenografts showed increased expression of genes biologically related to a humoral response, including monocyte and macrophage activation, natural killer cell burden, endothelial activation, complement activation, and T-cell development. Whole-transcriptome digital spatial profiling showed that antibody-mediated injury was mainly located in the glomeruli of the xenografts, with significant enrichment of transcripts associated with monocytes, macrophages, neutrophils, and natural killer cells. This phenotype was not observed in control pig kidney autografts or in ischaemia-reperfusion models. INTERPRETATION:Despite favourable short-term outcomes and absence of hyperacute injuries, our findings suggest that antibody-mediated rejection in pig-to-human kidney xenografts might be occurring. Our results suggest specific therapeutic targets towards the humoral arm of rejection to improve xenotransplantation results. FUNDING:OrganX and MSD Avenir.

Genetically modified xenografts are one of the most promising solutions to the discrepancy between the numbers of available human organs for transplantation and potential recipients. To date, a porcine heart has been implanted into only one human recipient. Here, using 10-gene-edited pigs, we transplanted porcine hearts into two brain-dead human recipients and monitored xenograft function, hemodynamics and systemic responses over the course of 66 hours. Although both xenografts demonstrated excellent cardiac function immediately after transplantation and continued to function for the duration of the study, cardiac function declined postoperatively in one case, attributed to a size mismatch between the donor pig and the recipient. For both hearts, we confirmed transgene expression and found no evidence of cellular or antibody-mediated rejection, as assessed using histology, flow cytometry and a cytotoxic crossmatch assay. Moreover, we found no evidence of zoonotic transmission from the donor pigs to the human recipients. While substantial additional work will be needed to advance this technology to human trials, these results indicate that pig-to-human heart xenotransplantation can be performed successfully without hyperacute rejection or zoonosis.