Spatiotemporal immune atlas of a clinical-grade gene-edited pig-to-human kidney xenotransplant.

Pig-to-human xenotransplantation is rapidly approaching the clinical arena; however, it is unclear which immunomodulatory regimens will effectively control human immune responses to pig xenografts. Here, we transplant a gene-edited pig kidney into a brain-dead human recipient on pharmacologic immunosuppression and study the human immune response to the xenograft using spatial transcriptomics and single-cell RNA sequencing. Human immune cells are uncommon in the porcine kidney cortex early after xenotransplantation and consist of primarily myeloid cells. Both the porcine resident macrophages and human infiltrating macrophages express genes consistent with an alternatively activated, anti-inflammatory phenotype. No significant infiltration of human B or T cells into the porcine kidney xenograft is detectable. Altogether, these findings provide proof of concept that conventional pharmacologic immunosuppression may be able to restrict infiltration of human immune cells into the xenograft early after compatible pig-to-human kidney xenotransplantation.

Physiologic homeostasis after pig-to-human kidney xenotransplantation.

Demand for kidney grafts outpaces supply, limiting kidney transplantation as a treatment for kidney failure. Xenotransplantation has the potential to make kidney transplantation available to many more patients with kidney failure, but the ability of xenografts to support human physiologic homeostasis has not been established. A brain-dead adult decedent underwent bilateral native nephrectomies followed by 10 gene-edited (four gene knockouts, six human transgenes) pig-to-human xenotransplantation. Physiologic parameters and laboratory values were measured for seven days in a critical care setting. Data collection aimed to assess homeostasis by measuring components of the renin-angiotensin-aldosterone system, parathyroid hormone signaling, glomerular filtration rate, and markers of salt and water balance. Mean arterial blood pressure was maintained above 60 mmHg throughout. Pig kidneys secreted renin (post-operative day three to seven mean and standard deviation: 47.3 ± 9 pg/mL). Aldosterone and angiotensin II levels were present (post-operative day three to seven, 57.0 ± 8 pg/mL and 5.4 ± 4.3 pg/mL, respectively) despite plasma renin activity under 0.6 ng/mL/hr. Parathyroid hormone levels followed ionized calcium. Urine output down trended from 37 L to 6 L per day with 4.5 L of electrolyte free water loss on post-operative day six. Aquaporin 2 channels were detected in the apical surface of principal cells, supporting pig kidney response to human vasopressin. Serum creatinine down trended to 0.9 mg/dL by day seven. Glomerular filtration rate ranged 90-240 mL/min by creatinine clearance and single-dose inulin clearance. Thus, in a human decedent model, xenotransplantation of 10 gene-edited pig kidneys provided physiologic balance for seven days. Hence, our in-human study paves the way for future clinical study of pig-to-human kidney xenotransplantation in living persons.

Swine models in translational research and medicine.

Swine are increasingly studied as animal models of human disease. The anatomy, size, longevity, physiology, immune system, and metabolism of swine are more like humans than traditional rodent models. In addition, the size of swine is preferred for surgical placement and testing of medical devices destined for humans. These features make swine useful for biomedical, pharmacological, and toxicological research. With recent advances in gene-editing technologies, genetic modifications can readily and efficiently be made in swine to study genetic disorders. In addition, gene-edited swine tissues are necessary for studies testing and validating xenotransplantation into humans to meet the critical shortfall of viable organs versus need. Underlying all of these biomedical applications, the knowledge of husbandry, background diseases and lesions, and biosecurity needs are important for productive, efficient, and reproducible research when using swine as a human disease model for basic research, preclinical testing, and translational studies.

Regional Social Vulnerability is Associated With Geographic Disparity in Waitlist Outcomes for Patients With Non-Hepatocellular Carcinoma Model for End-stage Liver Disease Exceptions in the United States.

OBJECTIVE: This study was undertaken to evaluate the role of regional social vulnerability in geographic disparity for patients listed for liver transplantation with non-hepatocellular carcinoma (HCC) model for end-stage liver disease (MELD) exceptions. SUMMARY AND BACKGROUND: Prior work has demonstrated regional variability in the appropriateness of MELD exceptions for diagnoses other than HCC. METHODS: Adults listed at a single center for first-time liver-only transplantation without HCC after June 18, 2013 in the Scientific Registry of Transplant Recipients database as of March 2021 were examined. Candidates were mapped to hospital referral regions (HRRs). Adjusted likelihood of mortality and liver transplantation were modeled. Advantaged HRRs were defined as those where exception patients were more likely to be transplanted, yet no more likely to die in adjusted analysis. The Centers for Disease Control's Social Vulnerability Index (SVI) was used as the measure for community health. Higher SVIs indicate poorer community health. RESULTS: There were 49,494 candidates in the cohort, of whom 4337 (8.8%) had MELD exceptions. Among continental US HRRs, 27.3% (n = 78) were identified as advantaged. The mean SVI of advantaged HRRs was 0.42 versus 0.53 in nonadvantaged HRRs ( P = 0.002), indicating better community health in these areas. Only 25.3% of advantaged HRRs were in spatial clusters of high SVI versus 40.7% of nonadvantaged HRRs, whereas 44.6% of advantaged HRRs were in spatial clusters of low SVI versus 38.0% of nonadvantaged HRRs ( P = 0.037). CONCLUSIONS: An advantage for non-HCC MELD exception patients is associated with lower social vulnerability on a population level. These findings suggest assigning similar waitlist priority to all non-HCC exception candidates without considering geographic differences in social determinants of health may actually exacerbate rather than ameliorate disparity.

Spatiotemporal immune atlas of the first clinical-grade, gene-edited pig-to-human kidney xenotransplant.

Pig-to-human xenotransplantation is rapidly approaching the clinical arena; however, it is unclear which immunomodulatory regimens will effectively control human immune responses to pig xenografts. We transplanted a gene-edited pig kidney into a brain-dead human recipient on pharmacologic immunosuppression and studied the human immune response to the xenograft using spatial transcriptomics and single-cell RNA sequencing. Human immune cells were uncommon in the porcine kidney cortex early after xenotransplantation and consisted of primarily myeloid cells. Both the porcine resident macrophages and human infiltrating macrophages expressed genes consistent with an alternatively activated, anti-inflammatory phenotype. No significant infiltration of human B or T cells into the porcine kidney xenograft was detected. Altogether, these findings provide proof of concept that conventional pharmacologic immunosuppression is sufficient to restrict infiltration of human immune cells into the xenograft early after compatible pig-to-human kidney xenotransplantation.

Perpetuating Disparity: Failure of the Kidney Transplant System to Provide the Most Kidney Transplants to Communities With the Greatest Need.

BACKGROUND: The burden of end-stage kidney disease (ESKD) and kidney transplant rates vary significantly across the United States. This study aims to examine the mismatch between ESKD burden and kidney transplant rates from a perspective of spatial epidemiology. METHODS: US Renal Data System data from 2015 to 2017 on incident ESKD and kidney transplants per 1000 incident ESKD cases was analyzed. Clustering of ESKD burden and kidney transplant rates at the county level was determined using local Moran's I and correlated to county health scores. Higher percentile county health scores indicated worse overall community health. RESULTS: Significant clusters of high-ESKD burden tended to coincide with clusters of low kidney transplant rates, and vice versa. The most common cluster type had high incident ESKD with low transplant rates (377 counties). Counties in these clusters had the lowest overall mean transplant rate (61.1), highest overall mean ESKD incidence (61.3), and highest mean county health scores percentile (80.9%, P <0.001 vs all other cluster types). By comparison, counties in clusters with low ESKD incidence and high transplant rates (n=359) had the highest mean transplant rate (110.6), the lowest mean ESKD incidence (28.9), and the lowest county health scores (20.2%). All comparisons to high-ESKD/low-transplant clusters were significant at P value <0.001. CONCLUSION: There was a significant mismatch between kidney transplant rates and ESKD burden, where areas with the greatest need had the lowest transplant rates. This pattern exacerbates pre-existing disparities, as disadvantaged high-ESKD regions already suffer from worse access to care and overall community health, as evidenced by the highest county health scores in the study.

Donor-reported barriers to living kidney donor follow-up.

BACKGROUND: Despite regulations mandating follow-up laboratory testing for living kidney donors, less than half of transplant centers are in compliance. We sought to understand barriers to follow-up testing from the donors' perspective. METHODS: We surveyed our center's living kidney donors. Binary logistic regression was used to assess factors associated with follow-up testing completion. RESULTS: Of 185 living kidney donors, 110 (59.4%) participated. Among them, 82 (74.5%) completed 6-month laboratory testing, 76 (69.1%) completed 12-month testing, 68 (61.8%) completed both, and 21 (19.0%) completed neither. Six-month testing completion was strongly associated with 12-month testing completion (OR 9.74, 95%CI: 2.23-42.50; p = .002). Those who disagreed with the statements, "Getting labs checked wasn't a priority for me," (OR for completing 6-month testing: 15.05, 95%CI: 3.70-61.18; p < .001; OR for completing 12-month testing: 5.85, 95%CI: 1.94-17.63; p = .002); and, "I forgot to get labs drawn [until I was reminded]" (OR for completing 6-month testing: 6.93, 95%CI: 1.59-30.08; p = .01; OR for completing 12-month testing: 6.55, 95%CI: 1.98-21.63; p = .002) were more likely to complete testing. CONCLUSIONS: To our knowledge, this is the only study providing perspective on donor insights regarding the need for follow-up testing post donation. Interventions to influence living donor attitudes toward follow-up testing may improve follow-up.

T and B lymphocyte dynamics after genetically-modified pig-to-baboon kidney xenotransplantation with an anti-CD40mAb-based immunosuppressive regimen.

BACKGROUND: The aim was to monitor recovery of T/B lymphocytes in baboons after depletion by anti-thymocyte globulin (ATG) and anti-CD20mAb (Rituximab), followed by pig kidney transplantation and maintenance therapy with an anti-CD40mAb-based regimen. METHODS: In baboons (n = 14), induction was with ATG and anti-CD20mAb, and maintenance with (i) anti-CD40mAb, (ii) rapamycin, and (iii) methylprednisolone. Follow-up was for 6 months, or until rejection or other complication developed. Baboon blood was collected at intervals to measure T/B cells and subsets by flow cytometry. In a separate study in baboons receiving the same immunosuppressive regimen (n = 10), the populations of T/B lymphocytes in PBMCs, lymph nodes, and spleen were examined. RESULTS: After induction therapy, the total lymphocyte count and the absolute numbers of CD3+, CD4+, and CD8+T cells fell by >80%, and no CD22+B cells remained (all p < 0.001). T cell numbers began to recover early, but no CD22+B cells were present in the blood for 2 months. Recovery of both T and B cells remained at <30% of baseline (p < 0.001), even if rejection developed. At 6 months, effector memory CD8+T cells had increased more than other T cell subsets, but a greater percentage of B cells were naïve. In contrast to blood and spleen, T and B cells were not depleted in lymph nodes. CONCLUSIONS: ATG and anti-CD20mAb effectively decreased T and B lymphocytes in the blood and, in the presence of anti-CD40mAb maintenance therapy, recovery of these cells was inhibited. The recovery of effector memory CD8+T cells may be detrimental to long-term graft survival.

Histopathology of pig kidney grafts with/without expression of the carbohydrate Neu5Gc in immunosuppressed baboons.

INTRODUCTION: Pigs deficient in three glycosyltransferase enzymes (triple-knockout [TKO] pigs, that is, not expressing the three known carbohydrate xenoantigens) and expressing 'protective' human transgenes are considered a likely source of organs for transplantation into human recipients. Some human sera have no or minimal natural antibody binding to red blood cells (RBCs) and peripheral blood mononuclear cells (PBMCs) from TKO pigs. However, all Old World monkeys exhibit natural antibody binding to TKO pig cells. The xenoantigen targets of Old World monkey natural antibodies are postulated to be carbohydrate moieties exposed when the expression of the carbohydrate N-glycolylneuraminic acid (Neu5Gc) is deleted. The aim of this study was to compare the survival in baboons and histopathology of renal grafts from pigs that either (a) expressed Neu5Gc (GTKO pigs; Group 1) or (b) did not express Neu5Gc (GTKO/CMAHKO [DKO] or TKO pigs; Group 2). METHODS: Life-supporting renal transplants were carried out using GTKO (n = 5) or DKO/TKO (n = 5) pig kidneys under an anti-CD40mAb-based immunosuppressive regimen. RESULTS: Group 1 baboons survived longer than Group 2 baboons (median 237 vs. 35 days; mean 196 vs. 57 days; p < 0.07) and exhibited histopathological features of antibody-mediated rejection in only two kidneys. Group 2 exhibited histopathological features of antibody-mediated rejection in all five grafts, with IgM and IgG binding to renal interstitial arteries and peritubular capillaries. Rejection-free survival was significantly longer in Group 1 (p < 0.05). CONCLUSIONS: The absence of expression of Neu5Gc on pig kidney grafts is associated with increased binding of baboon antibodies to pig endothelium and reduced graft survival.

Obesity as an isolated contraindication to kidney transplantation in the end-stage renal disease population: A cohort study.

OBJECTIVE: The aim of this study was to characterize end-stage renal disease (ESRD) patients with obesity as their only contraindication to listing and to quantify wait-list and transplant access. METHODS: Using the US Renal Data System, a retrospective cohort study of incident dialysis cases (2012 to 2014) was performed. The primary outcomes were time to wait-listing and time to transplantation. RESULTS: Of 157,572 dialysis patients not already listed, 39,844 had BMI as their only demonstrable transplant contraindication. They tended to be younger, female, and Black. Compared with patients with BMI < 35, those with BMI 35 to 39.9, 40 to 44.9, and ≥45 were, respectively, 15% (adjusted hazard ratio [aHR] 0.85; 95% CI: 0.83-0.88; p < 0.001), 45% (aHR 0.55; 95% CI: 0.52-0.57; p < 0.001), and 71% (aHR 0.29; 95% CI: 0.27-0.31; p < 0.001) less likely to be wait-listed. Wait-listed patients with BMI 35 to 39.9 were 24% less likely to achieve transplant (aHR 0.76; 95% CI: 0.72-0.80; p < 0.0001), BMI 40 to 44.9 were 21% less likely (aHR 0.79; 95% CI: 0.72-0.86; p < 0.0001), and BMI ≥ 45 were 15% less likely (aHR 0.85; 95% CI: 0.75-0.95; p = 0.004) compared with patients with BMI < 35. CONCLUSIONS: Obesity was the sole contraindication to wait-listing for 40,000 dialysis patients. They were less likely to be wait-listed. For those who were, they had a lower likelihood of transplant. Aggressive weight-loss interventions may help this population achieve wait-listing and transplant.

Evidence suggesting that deletion of expression of N-glycolylneuraminic acid (Neu5Gc) in the organ-source pig is associated with increased antibody-mediated rejection of kidney transplants in baboons.

Pigs deficient in three glycosyltransferase enzymes (triple-knockout [TKO] pigs) and expressing "protective" human transgenes are likely sources of organs for transplantation into human recipients. Testing of human sera against red blood cells (RBCs) and peripheral blood mononuclear cells (PBMCs) from TKO pigs has revealed minimal evidence of natural antibody binding. However, unlike humans, baboons exhibit natural antibody binding to TKO pig cells. The xenoantigen specificities of these natural antibodies are postulated to be one or more carbohydrate moieties exposed when N-glycolylneuraminic acid (Neu5Gc) is deleted. The aim of this study was to compare the survival of renal grafts in baboons from pigs that either expressed Neu5Gc (GTKO pigs; Group1, n = 5) or did not express Neu5Gc (GTKO/CMAHKO [DKO] or TKO pigs; Group2, n = 5). An anti-CD40mAb-based immunosuppressive regimen was administered in both groups. Group1 kidneys functioned for 90-260 days (median 237, mean 196 days), with histopathological features of antibody-mediated rejection in two kidneys. Group2 kidneys functioned for 0-183 days (median 35, mean 57), with all of the grafts exhibiting histologic features of antibody-mediated rejection. These findings suggest that the absence of expression of Neu5Gc on pig kidneys impacts graft survival in baboon recipients.

Early Outcomes With the Liver-kidney Safety Net.

BACKGROUND: A safety net policy was implemented in August 2017 giving liver transplant alone (LTA) recipients with significant renal dysfunction posttransplant priority for subsequent kidney transplantation (KT). This study was undertaken to evaluate early outcomes under this policy. METHODS: Adults undergoing LTA after implementation of the safety net policy and were subsequently listed for KT between 60 and 365 days after liver transplantation contained in United Network for Organ Sharing data were examined. Outcomes of interest were receipt of a kidney transplant and postliver transplant survival. Safety net patients were compared with LTA recipients not subsequently listed for KT as well as to patients listed for simultaneous liver-kidney (SLK) transplant yet underwent LTA and were not subsequently listed for KT. RESULTS: There were 100 patients listed for safety net KT versus 9458 patients undergoing LTA without subsequent KT listing. The cumulative incidence of KT following listing was 32.5% at 180 days. The safety net patients had similar 1-year unadjusted patient survival (96.4% versus 93.4%; P = 0.234) but superior adjusted survival (hazard ratio0.133, 0.3570.960; P = 0.041) versus LTA recipients not subsequently listed for KT. Safety net patients had superior 1-year unadjusted (96.4% versus 75.0%; P < 0.001) and adjusted (hazard ratio0.039, 0.1260.406; P < 0.001) survival versus SLK listed patients undergoing LTA without subsequent KT listing. CONCLUSIONS: The safety net appears to provide rapid access to KT with good early survival for those able to take advantage of it. Survival of patients unable to qualify for KT listing after LTA needs to be better understood before further limitation of SLK, however.

What Therapeutic Regimen Will Be Optimal for Initial Clinical Trials of Pig Organ Transplantation?

We discuss what therapeutic regimen might be acceptable/successful in the first clinical trial of genetically engineered pig kidney or heart transplantation. As regimens based on a calcineurin inhibitor or CTLA4-Ig have proved unsuccessful, the regimen we administer to baboons is based on induction therapy with antithymocyte globulin, an anti-CD20 mAb (Rituximab), and cobra venom factor, with maintenance therapy based on blockade of the CD40/CD154 costimulation pathway (with an anti-CD40 mAb), with rapamycin, and a corticosteroid. An anti-inflammatory agent (etanercept) is administered for the first 2 wk, and adjuvant therapy includes prophylaxis against thrombotic complications, anemia, cytomegalovirus, and pneumocystis. Using this regimen, although antibody-mediated rejection certainly can occur, we have documented no definite evidence of an adaptive immune response to the pig xenograft. This regimen could also form the basis for the first clinical trial, except that cobra venom factor will be replaced by a clinically approved agent, for example, a C1-esterase inhibitor. However, none of the agents that block the CD40/CD154 pathway are yet approved for clinical use, and so this hurdle remains to be overcome. The role of anti-inflammatory agents remains unproven. The major difference between this suggested regimen and those used in allotransplantation is the replacement of a calcineurin inhibitor with a costimulation blockade agent, but this does not appear to increase the complications of the regimen.

Pig kidney xenotransplantation: Progress toward clinical trials.

Pig organ xenotransplantation offers a solution to the shortage of deceased human organs for transplantation. The pathobiological response to a pig xenograft is complex, involving antibody, complement, coagulation, inflammatory, and cellular responses. To overcome these barriers, genetic manipulation of the organ-source pigs has largely been directed to two major aims-(a) deletion of expression of the known carbohydrate xenoantigens against which humans have natural (preformed) antibodies, and (b) transgenic expression of human protective proteins, for example, complement- and coagulation-regulatory proteins. Conventional (FDA-approved) immunosuppressive therapy is unsuccessful in preventing an adaptive immune response to pig cells, but blockade of the CD40:CD154 costimulation pathway is successful. Survival of genetically engineered pig kidneys in immunosuppressed nonhuman primates can now be measured in months. Non-immunological aspects, for example, pig renal function, a hypovolemia syndrome, and rapid growth of the pig kidney after transplantation, are briefly discussed. We suggest that patients on the wait-list for a deceased human kidney graft who are unlikely to receive one due to long waiting times are those for whom kidney xenotransplantation might first be considered. The potential risk of infection, public attitudes to xenotransplantation, and ethical, regulatory, and financial aspects are briefly addressed.

The final obstacle to successful pre-clinical xenotransplantation?

Genetically engineered pigs are now available for xenotransplantation in which all three known carbohydrate xenoantigens, against which humans have natural antibodies, have been deleted (triple-knockout [TKO] pigs). Furthermore, multiple human transgenes have been expressed in the TKO pigs, all of which are aimed at protecting the cells from the human immune response. Many human sera demonstrate no or minimal antibody binding to, and little or no cytotoxicity of, cells from these pigs, and this is associated with a relatively low T-cell proliferative response. Unfortunately, baboons and other Old World NHPs have antibodies against TKO pig cells, apparently directed to a fourth xenoantigen that appears to be exposed after TKO. In our experience, most, if not all, humans do not have natural antibodies against this fourth xenoantigen. This discrepancy between NHPs and humans is providing a hurdle to successful translation of pig organ transplantation into the clinic, and making it difficult to provide pre-clinical data that support initiation of a clinical trial. The potential methods by which this obstacle might be overcome are discussed. We conclude that, whatever currently available genetically engineered pig is selected for the final pre-clinical studies, this may not be the optimal pig for clinical trials.

De Novo Belatacept in a Kidney-After-Heart Transplant Recipient.

Renal injury almost always accompanies the multisystem organ failure that precedes cardiac transplantation and renal function is further compromised by the nephrotoxicity of calcineurin inhibitors posttransplant. Renal dysfunction in turn causes significant morbidity and mortality. The development of belatacept was motivated by need for an alternative to calcineurin-based immunosuppression, particularly in renal transplantation where the nephrotoxicity of calcineurin inhibitors reduce graft longevity and adverse cardiovascular effects of calcineurin inhibitors increase overall mortality. In 2011, the FDA approved belatacept for use in renal transplantation. Seven-year data from the multicenter randomized phase III BENEFIT trial, which compared belatacept with cyclosporine in renal transplant recipients, show belatacept therapy offers both improved renal function and 43% risk reduction for the combined endpoint of graft loss and death. At present, belatacept use is predominantly confined to renal transplant recipients; however, reports of belatacept use in other transplant settings are emerging. Here, we describe successful long-term use of belatacept in a kidney-after-heart transplant recipient and review use of belatacept in cardiothoracic and other nonrenal transplant settings.

Impact of Donor Hepatitis C Virus on Kidney Transplant Outcomes for Hepatitis C-positive Recipients in the Direct-acting Antiviral Era: Time to Revise the Kidney Donor Risk Index?

BACKGROUND: Kidneys from donors with hepatitis C virus (HCV) infection are traditionally considered to be at risk for poorer survival outcomes, as reflected in the kidney donor profile index (KDPI). Modern direct-acting antivirals may modify this risk. METHODS: Using United Network for Organ Sharing data, HCV-infected adult first-time kidney transplant recipients from 2014 to 2017 were examined. Graft and patient survival were compared in a propensity-matched cohort of recipients of HCV antibody (Ab)(+) kidneys versus Ab(-) kidneys. Subsequent analysis was performed in a propensity-matched cohort of recipients of HCV-viremic (RNA positive) versus HCV-naïve kidneys. RESULTS: There were 379 recipients each in the matched cohort of recipients of HCV Ab(+) versus HCV Ab(-) kidneys. Despite a higher KDPI (58.2% for HCV Ab[+] versus 38.8% for HCV Ab[-]), 1-year patient and graft survival were similar in the HCV(+) and HCV(-) groups (95.4% and 94.9% versus 97.9% and 96.0%, P = 0.543 and P = 0.834, respectively). There were 200 recipients each in the cohort of recipients of HCV-viremic versus HCV-naïve kidneys, with the KDPI again higher in the HCV-viremic group (56.8% versus 35.2%). Baseline hazard ratios (HRs) for graft failure (HR, 4.69; P = 0.009) and death (HR, 7.60; P = 0.003) were significantly elevated in the viremic group, but crossed 1 at 21 and 24 months, respectively. CONCLUSIONS: In the modern direct-acting antiviral era, calculated likely KDPI overestimates risk kidneys from HCV (+) donors. Donor viremia conveys an early risk which appears to subside over time. These results suggest that it may be time to revise the kidney donor risk index.