RORγt-dependent antigen-presenting cells direct regulatory T cell-mediated tolerance to food antigen.

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 pTregs 5-7 , understanding of their characteristics remains incomplete, and the APC subset responsible for food tolerance has remained elusive. Here, we demonstrate that RORγt-APCs are similarly required for differentiation of food antigen-specific pTregs and establishment of oral tolerance. The ability of these cells to direct both food and microbiota-specific pTreg cell differentiation is contingent on expression of RORγt and on a unique cis-regulatory element within the Rorc gene locus ( Rorc(t) +7kb). Absent this +7kb element, there was a notable increase in food antigen-specific T helper 2 (Th2) cells in lieu of pTregs, leading to compromised tolerance in a mouse asthma model. By employing single-cell analyses across these models, as well as freshly resected mesenteric lymph nodes from a human organ donor, we identified a rare subset of evolutionarily conserved APCs that are dependent on RORγt, uniquely express the Prdm16 transcription factor, and are endowed with essential mediators for inducing pTreg cell differentiation. Our findings suggest that a better understanding of how RORγt-APCs 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.

Survival benefit of living donor liver transplant for patients with hepatocellular carcinoma.

With the increasing incidence of hepatocellular carcinoma (HCC) in both the United States and globally, the role of liver transplantation in management continues to be an area of active conversation as it is often considered the gold standard in the treatment of HCC. The use of living donor liver transplantation (LDLT) and the indications in the setting of malignancy, both generally and in HCC specifically, are frequently debated. In terms of both overall survival and recurrence-free survival, LDLT is at least equivalent to DDLT, especially when performed for disease within Milan criteria. Emerging and compelling evidence suggests that LDLT is superior to DDLT in treating HCC as there is a significant decrease in waitlist mortality. As the oncologic indications for liver transplantation continue to expand and the gap between organ demand and organ availability continues to worsen, high volumes centers should consider using LDLT to shrink the ever-expanding waitlist.

Dynamic establishment and maintenance of the human intestinal B cell population and repertoire following transplantation in a pediatric-dominated cohort.

Introduction: It is unknown how intestinal B cell populations and B cell receptor (BCR) repertoires are established and maintained over time in humans. Following intestinal transplantation (ITx), surveillance ileal mucosal biopsies provide a unique opportunity to map the dynamic establishment of recipient gut lymphocyte populations in immunosuppressed conditions. Methods: Using polychromatic flow cytometry that includes HLA allele group-specific antibodies distinguishing donor from recipient cells along with high throughput BCR sequencing, we tracked the establishment of recipient B cell populations and BCR repertoire in the allograft mucosa of ITx recipients. Results: We confirm the early presence of naïve donor B cells in the circulation (donor age range: 1-14 years, median: 3 years) and, for the first time, document the establishment of recipient B cell populations, including B resident memory cells, in the intestinal allograft mucosa (recipient age range at the time of transplant: 1-44 years, median: 3 years). Recipient B cell repopulation of the allograft was most rapid in infant (<1 year old)-derived allografts and, unlike T cell repopulation, did not correlate with rejection rates. While recipient memory B cell populations were increased in graft mucosa compared to circulation, naïve recipient B cells remained detectable in the graft mucosa for years. Comparisons of peripheral and intra-mucosal B cell repertoires in the absence of rejection (recipient age range at the time of transplant: 1-9 years, median: 2 years) revealed increased BCR mutation rates and clonal expansion in graft mucosa compared to circulating B cells, but these parameters did not increase markedly after the first year post-transplant. Furthermore, clonal mixing between the allograft mucosa and the circulation was significantly greater in ITx recipients, even years after transplantation, than in deceased adult donors. In available pan-scope biopsies from pediatric recipients, we observed higher percentages of naïve recipient B cells in colon allograft compared to small bowel allograft and increased BCR overlap between native colon vs colon allograft compared to that between native colon vs ileum allograft in most cases, suggesting differential clonal distribution in large intestine vs small intestine. Discussion: Collectively, our data demonstrate intestinal mucosal B cell repertoire establishment from a circulating pool, a process that continues for years without evidence of stabilization of the mucosal B cell repertoire in pediatric ITx patients.

Cellular dynamics in pig-to-human kidney xenotransplantation.

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: 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: This work was supported by NIH RM1HG009491 and DP5OD033430.

The decedent model: A new paradigm for de-risking high stakes clinical trials like xenotransplantation.

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.

Portable hypothermic oxygenated machine perfusion for organ preservation in liver transplantation: A randomized, open-label, clinical trial.

BACKGROUND AND AIMS: In liver transplantation, cold preservation induces ischemia, resulting in significant reperfusion injury. Hypothermic oxygenated machine perfusion (HMP-O 2 ) has shown benefits compared to static cold storage (SCS) by limiting ischemia-reperfusion injury. This study reports outcomes using a novel portable HMP-O 2 device in the first US randomized control trial. APPROACH AND RESULTS: The PILOT trial (NCT03484455) was a multicenter, randomized, open-label, noninferiority trial, with participants randomized to HMP-O 2 or SCS. HMP-O 2 livers were preserved using the Lifeport Liver Transporter and Vasosol perfusion solution. The primary outcome was early allograft dysfunction. Noninferiority margin was 7.5%. From April 3, 2019, to July 12, 2022, 179 patients were randomized to HMP-O 2 (n=90) or SCS (n=89). The per-protocol cohort included 63 HMP-O 2 and 73 SCS. Early allograft dysfunction occurred in 11.1% HMP-O 2 (N=7) and 16.4% SCS (N=12). The risk difference between HMP-O 2 and SCS was -5.33% (one-sided 95% upper confidence limit of 5.81%), establishing noninferiority. The risk of graft failure as predicted by Liver Graft Assessment Following Transplant score at seven days (L-GrAFT 7 ) was lower with HMP-O 2 [median (IQR) 3.4% (2.4-6.5) vs. 4.5% (2.9-9.4), p =0.024]. Primary nonfunction occurred in 2.2% of all SCS (n=3, p =0.10). Biliary strictures occurred in 16.4% SCS (n=12) and 6.3% (n=4) HMP-O 2 ( p =0.18). Nonanastomotic biliary strictures occurred only in SCS (n=4). CONCLUSIONS: HMP-O 2 demonstrates safety and noninferior efficacy for liver graft preservation in comparison to SCS. Early allograft failure by L-GrAFT 7 was lower in HMP-O 2 , suggesting improved early clinical function. Recipients of HMP-O 2 livers also demonstrated a lower incidence of primary nonfunction and biliary strictures, although this difference did not reach significance.

Dynamic establishment and maintenance of the human intestinal B cell population and repertoire following transplantation.

It is unknown how intestinal B cell populations and B cell receptor (BCR) repertoires are established and maintained over time in humans. Following intestinal transplantation (ITx), surveillance ileal mucosal biopsies provide a unique opportunity to map the dynamic establishment of gut lymphocyte populations. Using polychromatic flow cytometry that includes HLA allele group-specific mAbs distinguishing donor from recipient cells along with high throughput BCR sequencing, we tracked the establishment of recipient B cell populations and BCR repertoire in the allograft mucosa of ITx recipients. We confirm the early presence of naïve donor B cells in the circulation and, for the first time, document the establishment of recipient B cell populations, including B resident memory cells, in the intestinal allograft mucosa. Recipient B cell repopulation of the allograft was most rapid in infant (<1 year old)-derived allografts and, unlike T cell repopulation, did not correlate with rejection rates. While recipient memory B cell populations were increased in graft mucosa compared to circulation, naïve recipient B cells remained detectable in the graft mucosa for years. Comparisons of peripheral and intra-mucosal B cell repertoires in the absence of rejection revealed increased BCR mutation rates and clonal expansion in graft mucosa compared to circulating B cells, but these parameters did not increase markedly after the first year post-transplant. Furthermore, clonal mixing between the allograft mucosa and the circulation was significantly greater in ITx recipients, even years after transplantation, than in healthy control adults. Collectively, our data demonstrate intestinal mucosal B cell repertoire establishment from a circulating pool, a process that continues for years without evidence of establishment of a stable mucosal B cell repertoire.

Immune response after pig-to-human kidney xenotransplantation: a multimodal phenotyping study.

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: The data collected from xenografts suggested early signs of antibody-mediated rejection, characterised by microvascular inflammation with immune deposits, endothelial cell activation, and positive xenoreactive crossmatches. Capillary inflammation was mainly composed of intravascular CD68+ and CD15+ innate immune cells, as well as NKp46+ 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.

Pig-to-human heart xenotransplantation in two recently deceased human recipients.

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.

Living donor liver transplant candidate and donor selection and engagement: Meeting report from the living donor liver transplant consensus conference.

INTRODUCTION: Living donor liver transplantation (LDLT) is a promising option for mitigating the deceased donor organ shortage and reducing waitlist mortality. Despite excellent outcomes and data supporting expanding candidate indications for LDLT, broader uptake throughout the United States has yet to occur. METHODS: In response to this, the American Society of Transplantation hosted a virtual consensus conference (October 18-19, 2021), bringing together relevant experts with the aim of identifying barriers to broader implementation and making recommendations regarding strategies to address these barriers. In this report, we summarize the findings relevant to the selection and engagement of both the LDLT candidate and living donor. Utilizing a modified Delphi approach, barrier and strategy statements were developed, refined, and voted on for overall barrier importance and potential impact and feasibility of the strategy to address said barrier. RESULTS: Barriers identified fell into three general categories: 1) awareness, acceptance, and engagement across patients (potential candidates and donors), providers, and institutions, 2) data gaps and lack of standardization in candidate and donor selection, and 3) data gaps regarding post-living liver donation outcomes and resource needs. CONCLUSIONS: Strategies to address barriers included efforts toward education and engagement across populations, rigorous and collaborative research, and institutional commitment and resources.

Center use of technical variant grafts varies widely and impacts pediatric liver transplant waitlist and recipient outcomes in the United States.

To assess the impact of technical variant grafts (TVGs) [including living donor (LD) and deceased donor split/partial grafts] on waitlist (WL) and transplant outcomes for pediatric liver transplant (LT) candidates, we performed a retrospective analysis of Organ Procurement and Transplantation Network (OPTN) data on first-time LT or liver-kidney pediatric candidates listed at centers that performed >10 LTs during the study period, 2004-2020. Center variance was plotted for LT volume, TVG usage, and survival. A composite center metric of TVG usage and WL mortality was developed to demonstrate the existing variation and potential for improvement. Sixty-four centers performed 7842 LTs; 657 children died on the WL. Proportions of WL mortality by center ranged from 0% to 31% and those of TVG usage from 0% to 76%. Higher TVG usage, from deceased donor or LD, independently or in combination, significantly correlated with lower WL mortality. In multivariable analyses, death from listing was significantly lower with increased center TVG usage (HR = 0.611, CI: 0.40-0.92) and LT volume (HR = 0.995, CI: 0.99-1.0). Recipients of LD transplants (HR = 0.637, CI: 0.51-0.79) had significantly increased survival from transplant compared with other graft types, and recipients of deceased donor TVGs (HR = 1.066, CI: 0.93-1.22) had statistically similar outcomes compared with whole graft recipients. Increased TVG utilization may decrease WL mortality in the US. Hence, policy and training to increase TVG usage, availability, and expertise are critical.

The road to xenotransplantation.

PURPOSE OF REVIEW: The aim of this study was to highlight recent progress in xenotransplantation and discuss the remaining obstacles/steps before the FDA is likely to approve a clinical trial. RECENT FINDINGS: Long-term survival of life-supporting xenografts in preclinical models has led to discussion of clinical trials of xenotransplantation. The reports of clinical cardiac xenotransplant based on compassionate use FDA approval and renal xenotransplants to brain-dead humans have led to further considerations of clinical trials. Discussions between the transplant community and the FDA have established critical next steps before a clinical trial of xenotransplants is likely to be approved. These steps include testing the clinical immunosuppression protocol and the organ from a genetically modified source animal in nonhuman primates with reproducible survival of at least 6 months. In addition, appropriate viral surveillance protocols and confirmation that the xenografts support appropriate human physiology are likely to be critical elements for FDA-approval. Finally, further studies in the human decedent model are likely to provide critical information about human immune and physiologic responses to xenografts. SUMMARY: This review highlights the current progress in nonhuman primate models and recent reports of human xenotransplantation. It also describes the remaining hurdles and currently understood FDA requirements that remain to be achieved before a clinical trial of xenotransplantation can be approved.

Physiologic considerations of pig-to-human kidney xenotransplantation.

PURPOSE OF REVIEW: The greatest challenge facing end-stage kidney disease (ESKD) patients is the scarcity of transplantable organs. Advances in genetic engineering that mitigate xenogeneic immune responses have made transplantation across species a potentially viable solution to this unmet need. Preclinical studies and recent reports of pig-to-human decedent renal xenotransplantation signify that clinical trials are on the horizon. Here, we review the physiologic differences between porcine and human kidneys that could impede xenograft survival. Topics addressed include porcine renin and sodium handling, xenograft water handling, calcium, phosphate and acid-base balance, responses to porcine erythropoietin and xenograft growth. RECENT FINDINGS: Studies in nonhuman primates (NHPs) have demonstrated that genetically modified pig kidneys can survive for an extended period when transplanted into baboons. In recent studies conducted by our group and others, hyperacute rejection did not occur in pig kidneys lacking the α1,3Gal epitope transplanted into brain-dead human recipients. These experimental trials did not study potential clinical abnormalities arising from idiosyncratic xenograft responses to human physiologic stimuli due to the brief duration of observation this model entails. SUMMARY: Progress in biotechnology is heralding an era of xenotransplantation. We highlight the physiologic considerations for xenogeneic grafts to succeed.

State of pediatric liver transplantation in the United States and achieving zero wait list mortality with ideal outcomes: A statement from the Starzl Network for Excellence in Pediatric Transplant Surgeon's Working Group.

BACKGROUND: Liver transplant is a life-saving therapy that can restore quality life for several pediatric liver diseases. However, it is not available to all children who need one. Expertise in medical and surgical management is heterogeneous, and allocation policies are not optimally serving children. Technical variant grafts from both living and deceased donors are underutilized. METHODS: Several national efforts in pediatric liver transplant to improve access to and outcomes from liver transplant for children have been instituted and include adjustments to allocation policies, UNOS-sponsored collaborative improvement projects, and the emergence of national learning networks to study ongoing challenges in the field the Surgical Working group of the Starzl Network for Excellence in Pediatric Transplantation (SNEPT) discusses key issues and proposes potential solutions to eliminate the persistent wait list mortality that pediatric patients face. RESULTS: A discussion of the factors impacting pediatric patients' access to liver transplant is undertaken, along with a proposal of several measures to ensure equitable access to life-saving liver transplant. CONCLUSIONS: Pediatric liver transplant wait list mortality can and should be eliminated. Several measures, including collaborative efforts among centers, could be leveraged to acheive this goal.

Ex Vivo Liver Resection and Autotransplantation: Should It be Used More Frequently?

OBJECTIVE: We herein advocate for more extensive utilization of ex vivo resection techniques for otherwise unresectable liver tumors by presenting the largest collective American experience. BACKGROUND: Advanced in situ resection and vascular reconstruction techniques have made R0 resection possible for otherwise unresectable liver tumors. Ex vivo liver resection may further expand the limits of resectability but remains underutilized due to concerns about technical complexity and vascular thrombosis. However, we believe that the skillset required for ex vivo liver resection is more widespread and the complications less severe than widely assumed, making ex vivo resection a more attractive option in selected case. METHODS: We retrospectively analyzed 35 cases performed by surgical teams experienced with ex vivo liver resections (at least 4 cases) between 1997 and 2021. RESULTS: We categorized malignancies as highly aggressive (n=18), moderately aggressive (n=14), and low grade (n=3). All patients underwent total hepatectomy, vascular reconstruction and resection in hypothermia on the backtable, and partial liver autotransplantation. Overall survival was 67%/39%/28%, at 1/3/5 years, respectively, with a median survival of 710 days (range: 22-4824). Patient survival for highly aggressive, moderately aggressive, and low-grade tumors was 61%/33%/23%, 67%/40%/22%, and 100%/100%/100% at 1/3/5 years, respectively, with median survival 577 days (range: 22-3873), 444 days (range: 22-4824), and 1825 days (range: 868-3549). CONCLUSIONS: Ex vivo resection utilizes techniques commonly practiced in partial liver transplantation, and we demonstrate relatively favorable outcomes in our large collective experience. Therefore, we propose that more liberal use of this technique may benefit selected patients in centers experienced with partial liver transplantation.

Immuno-PET Monitoring of Lymphocytes Using the CD8-Specific Antibody REGN5054.

Assessment of immune-cell subsets within the tumor immune microenvironment is a powerful approach to better understand cancer immunotherapy responses. However, the use of biopsies to assess the tumor immune microenvironment poses challenges, including the potential for sampling error, restricted sampling over time, and inaccessibility of some tissues/organs, as well as the fact that single biopsy analyses do not reflect discordance across multiple intrapatient tumor lesions. Immuno-positron emission tomography (PET) presents a promising translational imaging approach to address the limitations and assess changes in the tumor microenvironment. We have developed 89Zr-DFO-REGN5054, a fully human CD8A-specific antibody conjugate, to assess CD8+ tumor-infiltrating lymphocytes (TIL) pre- and posttherapy. We used multiple assays, including in vitro T-cell activation, proliferation, and cytokine production, and in vivo viral clearance and CD8 receptor occupancy, to demonstrate that REGN5054 has minimal impact on T-cell activity. Preclinical immuno-PET studies demonstrated that 89Zr-DFO-REGN5054 specifically detected CD8+ T cells in lymphoid tissues of CD8-genetically humanized immunocompetent mice (VelociT mice) and discerned therapy-induced changes in CD8+ TILs in two models of response to a CD20xCD3 T-cell activating bispecific antibody (REGN1979, odronextamab). Toxicology studies in cynomolgus monkeys showed no overt toxicity, and immuno-PET imaging in cynomolgus monkeys demonstrated dose-dependent clearance and specific targeting to lymphoid tissues. This work supports the clinical investigation of 89Zr-DFO-REGN5054 to monitor T-cell responses in patients undergoing cancer immunotherapy.