Prolonged xenokidney graft survival in sensitized NHP recipients by expression of multiple human transgenes in a triple knockout pig.

Genetic modification of porcine donors, combined with optimized immunosuppression, has been shown to improve outcomes of experimental xenotransplant. However, little is known about outcomes in sensitized recipients, a population that could potentially benefit the most from the clinical implementation of xenotransplantation. Here, five highly allosensitized rhesus macaques received a porcine kidney from GGTA1 (α1,3-galactosyltransferase) knockout pigs expressing the human CD55 transgene (1KO.1TG) and were maintained on an anti-CD154 monoclonal antibody (mAb)-based immunosuppressive regimen. These recipients developed de novo xenoreactive antibodies and experienced xenograft rejection with evidence of thrombotic microangiopathy and antibody-mediated rejection (AMR). In comparison, three highly allosensitized rhesus macaques receiving a kidney from GGTA1, CMAH (cytidine monophospho-N-acetylneuraminic acid hydroxylase), and b4GNT2/b4GALNT2 (ß-1,4-N-acetyl-galactosaminyltransferase 2) knockout pigs expressing seven human transgenes including human CD46, CD55, CD47, THBD (thrombomodulin), PROCR (protein C receptor), TNFAIP3 (tumor necrosis factor-α-induced protein 3), and HMOX1 (heme oxygenase 1) (3KO.7TG) experienced significantly prolonged graft survival and reduced AMR, associated with dampened post-transplant humoral responses, early monocyte and neutrophil activation, and T cell repopulation. After withdrawal of all immunosuppression, recipients who received kidneys from 3KO.7TG pigs rejected the xenografts via AMR. These data suggest that allosensitized recipients may be suitable candidates for xenografts from genetically modified porcine donors and could benefit from an optimized immunosuppression regimen designed to target the post-transplant humoral response, thereby avoiding AMR.

A pig kidney supporting human physiology.

Because of the global shortage of donor kidneys, xenotransplantation emerges as a potential solution for individuals with kidney failure who face challenges in securing a suitable donor kidney. A study featured in this month's issue of Kidney International assesses the kidney physiology of a porcine kidney transplanted into a brain-dead human with kidney failure, demonstrating life-sustaining physiological function for 7 days. Together with preclinical nonhuman primate studies, decedent models provide complementary data for development of clinical kidney xenotransplantation.

Banff 2022 Liver Group Meeting report: Monitoring long-term allograft health.

The Banff Working Group on Liver Allograft Pathology met in September 2022. Participants included hepatologists, surgeons, pathologists, immunologists, and histocompatibility specialists. Presentations and discussions focused on the evaluation of long-term allograft health, including noninvasive and tissue monitoring, immunosuppression optimization, and long-term structural changes. Potential revision of the rejection classification scheme to better accommodate and communicate late T cell-mediated rejection patterns and related structural changes, such as nodular regenerative hyperplasia, were discussed. Improved stratification of long-term maintenance immunosuppression to match the heterogeneity of patient settings will be central to improving long-term patient survival. Such personalized therapeutics are in turn contingent on a better understanding and monitoring of allograft status within a rational decision-making approach, likely to be facilitated in implementation with emerging decision-support tools. Proposed revisions to rejection classification emerging from the meeting include the incorporation of interface hepatitis and fibrosis staging. These will be opened to online testing, modified accordingly, and subject to consensus discussion leading up to the next Banff conference.

The T-cell environment: may the regulatory force be with you.

In the study by Sasaki et al. in this issue, the authors studied infusions of ex vivo-expanded regulatory T cells in a highly clinically relevant nonhuman primate kidney transplant model. This commentary will aim to discuss the use of regulatory T cells in the wider context of transplantation, with particular emphasis on the milieu and various engineering potentials to enhance their function, as well as their relationship to other cell populations with regulatory capacity.

The impact of IdeS (imlifidase) on allo-specific, xeno-reactive, and protective antibodies in a sensitized rhesus macaque model.

BACKGROUND: Highly sensitized patients face many barriers to kidney transplantation, including higher rates of antibody-mediated rejection after HLA-incompatible transplant. IdeS, an endopeptidase that cleaves IgG nonspecifically, has been trialed as desensitization prior to kidney transplant, and successfully cleaves donor-specific antibody (DSA), albeit with rebound. METHODS: IdeS was generated and tested (2 mg/kg, IV) in two naïve and four allosensitized nonhuman primates (NHP). Peripheral blood samples were collected at regular intervals following IdeS administration. Total IgG, total IgM, and anti-CMV antibodies were quantified with ELISA, and donor-specific antibody (DSA) and anti-pig antibodies were evaluated using flow cytometric crossmatch. B cell populations were assessed using flow cytometry. RESULTS: IdeS successfully cleaved rhesus IgG in vitro. In allosensitized NHP, robust reduction of total, DSA, anti-pig, and anti-CMV IgG was observed within one day following IdeS administration. Rapid rebound of all IgG antibody populations was observed, with antibody levels returning to baseline around day 14 post-infusion. Total IgM level was not affected by IdeS. Interestingly, a comparable reduction in antibody populations was observed after the second dose of IdeS. However, we have not observed any significant modulation of B cell subpopulations after IdeS. CONCLUSIONS: This study evaluated efficacy of IdeS in the allosensitized NHP in IgG with various specificities, mirroring antibody kinetics in human patients. The efficacy of IdeS on preexisting anti-pig antibodies may be useful in clinical xenotransplantation. However, given the limitation of IdeS on its durability as a monotherapy, optimization of IdeS with other agents targeting the humoral response is further needed.

HRSA's Patient-Centric Approach to Modernizing the US Transplant System.

This Viewpoint discusses the benefits of modernization efforts for the US transplant system.

Detailed Analysis of Simultaneous Renal and Liver Allografts in the Presence of DSA.

Liver allografts protect renal allografts from the same donor from some, but not all, preformed donor specific alloantibodies (DSA). However, the precise mechanisms of protection and the potential for more subtle alterations/injuries within the grafts resulting from DSA interactions require further study. Methods: We reevaluated allograft biopsies from simultaneous liver-kidney transplant recipients who had both allografts biopsied within 60 d of one another and within 30 d of DSA being positive in serum (positive: mean florescence intensity ≥5000). Routine histology, C4d staining, and specialized immunohistochemistry for Kupffer cells (KCs; CD163) and a C4d receptor immunoglobulin-like transcript-4 were carried out in 4 patients with 6 paired biopsies. Results: Overt antibody-mediated rejection was found in 3 of 4 renal and liver allografts. One patient had biopsy-confirmed renal and liver allograft antibody-mediated rejection despite serum clearance of DSA. All biopsies showed KC hypertrophy (minimal: 1; mild: 2; moderate: 1; severe: 2) and cytoplasmic C4d KC staining was easily detected in 2 biopsies from 2 patients; minimal and negative in 2 biopsies each. Implications of which are discussed. Control 1-y protocol liver allograft biopsies from DSA- recipients showed neither KC hypertrophy nor KC C4d staining (n = 6). Conclusions: Partial renal allograft protection by a liver allograft from the same donor may be partially mediated by phagocytosis/elimination of antibody and complement split products by KCs, as shown decades ago in controlled sensitized experimental animal experiments.

Normothermic Machine Perfusion of Donor Livers for Transplantation in the United States: A Randomized Controlled Trial.

OBJECTIVE: To compare conventional low-temperature storage of transplant donor livers [static cold storage (SCS)] with storage of the organs at physiological body temperature [normothermic machine perfusion (NMP)]. BACKGROUND: The high success rate of liver transplantation is constrained by the shortage of transplantable organs (eg, waiting list mortality >20% in many centers). NMP maintains the liver in a functioning state to improve preservation quality and enable testing of the organ before transplantation. This is of greatest potential value with organs from brain-dead donor organs (DBD) with risk factors (age and comorbidities), and those from donors declared dead by cardiovascular criteria (donation after circulatory death). METHODS: Three hundred eighty-three donor organs were randomized by 15 US liver transplant centers to undergo NMP (n = 192) or SCS (n = 191). Two hundred sixty-six donor livers proceeded to transplantation (NMP: n = 136; SCS: n = 130). The primary endpoint of the study was "early allograft dysfunction" (EAD), a marker of early posttransplant liver injury and function. RESULTS: The difference in the incidence of EAD did not achieve significance, with 20.6% (NMP) versus 23.7% (SCS). Using exploratory, "as-treated" rather than "intent-to-treat," subgroup analyses, there was a greater effect size in donation after circulatory death donor livers (22.8% NMP vs 44.6% SCS) and in organs in the highest risk quartile by donor risk (19.2% NMP vs 33.3% SCS). The incidence of acute cardiovascular decompensation at organ reperfusion, "postreperfusion syndrome," as a secondary outcome was reduced in the NMP arm (5.9% vs 14.6%). CONCLUSIONS: NMP did not lower EAD, perhaps related to the inclusion of lower-risk liver donors, as higher-risk donor livers seemed to benefit more. The technology is safe in standard organ recovery and seems to have the greatest benefit for marginal donors.

Understanding heterogeneity of human bone marrow plasma cell maturation and survival pathways by single-cell analyses.

Human bone marrow (BM) plasma cells are heterogeneous, ranging from newly arrived antibody-secreting cells (ASCs) to long-lived plasma cells (LLPCs). We provide single-cell transcriptional resolution of 17,347 BM ASCs from five healthy adults. Fifteen clusters are identified ranging from newly minted ASCs (cluster 1) expressing MKI67 and high major histocompatibility complex (MHC) class II that progress to late clusters 5-8 through intermediate clusters 2-4. Additional ASC clusters include the following: immunoglobulin (Ig) M predominant (likely of extra-follicular origin), interferon responsive, and high mitochondrial activity. Late ASCs are distinguished by G2M checkpoints, mammalian target of rapamycin (mTOR) signaling, distinct metabolic pathways, CD38 expression, utilization of tumor necrosis factor (TNF)-receptor superfamily members, and two distinct maturation pathways involving TNF signaling through nuclear factor κB (NF-κB). This study provides a single-cell atlas and molecular roadmap of LLPC maturation trajectories essential in the BM microniche. Altogether, understanding BM ASC heterogeneity in health and disease enables development of new strategies to enhance protective ASCs and to deplete pathogenic ones.

Doing it right: Caring for and protecting patient information for US organ donors and transplant recipients.

In the current US organ transplantation system, there are no regulations defining how organ procurement organizations must manage personal data and protect the privacy of donors and recipients. In response to the recent announcement of a major overhaul of the US transplantation system, we describe a practical approach to improving transplant data quality and protecting the autonomy of patients interacting with the system.

Desensitization and belatacept-based maintenance therapy in pregnancy-sensitized monkeys receiving a kidney transplant.

Among sensitized patients awaiting a transplant, females are disproportionately represented, partly because of pregnancy-induced sensitization. Using female NHPs sensitized by pregnancy alone, we examined the efficacy of costimulation blockade and proteasome inhibition for desensitization. Three animals received no desensitization (control), and seven animals received weekly carfilzomib (27 mg/m2) and belatacept (20 mg/kg) before kidney transplantation. All animals received renal allografts from crossmatch-positive/maximally MHC-mismatched donors. Controls and three desensitized animals received tacrolimus-based immunosuppression. Four desensitized animals received additional belatacept with tacrolimus-based immunosuppression. Multiparous females had less circulating donor-specific antibody when compared to skin-sensitized males before transplantation. While females receiving desensitization showed only a marginal survival benefit over control females (MST = 11 days versus 63 days), additional belatacept to posttransplant maintenance significantly prolonged graft survival (MST > 164 days) and suppressed posttransplant DSA and circulating follicular helper T-like cells. This combination of therapies demonstrates great potential to reduce antibody-mediated rejection in sensitized recipients.

Multicenter Validation of a Urine CXCL10 Assay for Noninvasive Monitoring of Renal Transplants.

BACKGROUND: Urine CXCL10 (C-X-C motif chemokine ligand 10, interferon gamma-induced protein 10 [IP10]) outperforms standard-of-care monitoring for detecting subclinical and early clinical T-cell-mediated rejection (TCMR) and may advance TCMR therapy development through biomarker-enriched trials. The goal was to perform an international multicenter validation of a CXCL10 bead-based immunoassay (Luminex) for transplant surveillance and compare with an electrochemiluminescence-based (Meso Scale Discovery [MSD]) assay used in transplant trials. METHODS: Four laboratories participated in the Luminex assay development and evaluation. Urine CXCL10 was measured by Luminex and MSD in 2 independent adult kidney transplant trial cohorts (Basel and TMCT04). In an independent test and validation set, a linear mixed-effects model to predict (log 10 -transformed) MSD CXCL10 from Luminex CXCL10 was developed to determine the conversion between assays. Net reclassification was determined after mathematical conversion. RESULTS: The Luminex assay was precise, with an intra- and interassay coefficient of variation 8.1% and 9.3%; showed modest agreement between 4 laboratories (R 0.96 to 0.99, P < 0.001); and correlated with known CXCL10 in a single- (n = 100 urines, R 0.94 to 0.98, P < 0.001) and multicenter cohort (n = 468 urines, R 0.92, P < 0.001) but the 2 assays were not equivalent by Passing-Bablok regression. Linear mixed-effects modeling demonstrated an intercept of -0.490 and coefficient of 1.028, showing Luminex CXCL10 are slightly higher than MSD CXCL10, but the agreement is close to 1.0. After conversion of the biopsy thresholds, the decision to biopsy would be changed for only 6% (5/85) patients showing acceptable reclassification. CONCLUSIONS: These data demonstrate this urine CXCL10 Luminex immunoassay is robust, reproducible, and accurate, indicating it can be readily translated into clinical HLA laboratories for serial posttransplant surveillance.

Information design to support growth, quality, and equity of the US transplant system.

The Organ Procurement and Transplantation Network, an arm of the Health Resources and Services Administration, has a contract with the United Network for Organ Sharing since 1986 to provide central oversight of organ donation and transplants in the United States. The United Network for Organ Sharing has recently come under scrutiny, prompting a review by the National Academies of Sciences, Engineering, and Medicine as summarized in its recent report and also by the US Senate Finance Committee. The national news services have opined about organ donation ethics, access to transplantation particularly for medically underserved populations, and management of organ transplantation data. These critiques raise important concerns that deserve our best response as a transplant community. Broadly, we suggest that the data management approach of the Organ Procurement and Transplantation Network be replaced with a patient-centric omnichannel network in which all donor and recipient data exist in a single longitudinal record that can be used by all applications. A more comprehensive and standardized approach to donor data collection would drive quality improvement across organ procurement organizations and help address inequities in transplantation. Finally, a substantial increase in organ donation would be prompted by considering organ donors as a public health resource, meriting transparent publicly available data collection with respect to organ donor referral, screening, and management.

Human Bone Marrow Plasma Cell Atlas: Maturation and Survival Pathways Unraveled by Single Cell Analyses.

Human bone marrow (BM) plasma cells are heterogeneous, ranging from newly arrived antibody-secreting cells (ASC) to long-lived plasma cells (LLPC). We provide single cell transcriptional resolution of 17,347 BM ASC from 5 healthy adults. Fifteen clusters were identified ranging from newly minted ASC (cluster 1) expressing MKI67 and high MHC Class II that progressed to late clusters 5-8 through intermediate clusters 2-4. Additional clusters included early and late IgM-predominant ASC of likely extra-follicular origin; IFN-responsive; and high mitochondrial activity ASC. Late ASCs were distinguished by differences in G2M checkpoints, MTOR signaling, distinct metabolic pathways, CD38 expression, and utilization of TNF-receptor superfamily members. They mature through two distinct paths differentiated by the degree of TNF signaling through NFKB. This study provides the first single cell resolution atlas and molecular roadmap of LLPC maturation, thereby providing insight into differentiation trajectories and molecular regulation of these essential processes in the human BM microniche. This information enables investigation of the origin of protective and pathogenic antibodies in multiple diseases and development of new strategies targeted to the enhancement or depletion of the corresponding ASC. One Sentence Summary: The single cell transcriptomic atlas of human bone marrow plasma cell heterogeneity shows maturation of class-switched early and late subsets, specific IgM and Interferon-driven clusters, and unique heterogeneity of the late subsets which encompass the long-lived plasma cells.

Daratumumab Use Prior to Kidney Transplant and T Cell-Mediated Rejection: A Case Report.

There is growing interest in daratumumab in the solid organ transplant realm owing to the potential immunomodulatory effects on CD38-expressing cells, primarily plasma cells, as they have a key role in antibody production. In particular there is interest in use of daratumumab for desensitization and potential treatment for antibody-mediated rejection. However, ongoing investigation with daratumumab has shown potential immunologic concerns in vitro, with a significant increase in populations of CD4-positive cytotoxic T cells and CD8-positive helper T cells in both peripheral blood and bone marrow that could lead to acute T cell-mediated rejection in the solid organ transplant patient. To date, there are no published reports of an association with daratumumab use and T cell-mediated rejection in vivo. In this case report we present what is to our knowledge the first documented case of an early severe T cell-mediated rejection in a low-immunologic-risk living-donor kidney transplant recipient who received daratumumab for multiple myeloma maintenance prior to transplant.

Addition of interleukin-6 receptor blockade to carfilzomib-based desensitization in a highly sensitized nonhuman primate model.

Sensitized patients, those who had prior exposure to foreign human leukocyte antigens, are transplanted at lower rates due to challenges in finding suitable organs. Desensitization strategies have permitted highly sensitized patients to undergo kidney transplantation, albeit with higher rates of rejection. This study assesses targeting plasma cell and interleukin (IL)-6 receptor for desensitization in a sensitized nonhuman primate kidney transplantation model. All animals were sensitized using two sequential skin transplants from maximally major histocompatibility complex-mismatched donors. Carfilzomib (CFZ)/tocilizumab (TCZ) desensitization (N = 6) successfully decreased donor-specific antibody (DSA) titers and prevented the expansion of B cells compared to CFZ monotherapy (N = 3). Dual desensitization further delayed, but did not prevent humoral rebound, as evidenced by a delayed increase in post-kidney transplant DSA titers. Accordingly, CFZ/TCZ desensitization conferred a significant survival advantage over CFZ monotherapy. A trend toward increased T follicular helper cells was also observed in the dual therapy group along the same timeline as an increase in DSA and subsequent graft loss. Cytomegalovirus reactivation also occurred in the CFZ/TCZ group but was prevented with ganciclovir prophylaxis. In accordance with prior studies of CFZ-based dual desensitization strategies, the addition of IL-6 receptor blockade resulted in desensitization with further suppression of posttransplant humoral response compared to CFZ monotherapy.

Complement-targeted therapies in kidney transplantation-insights from preclinical studies.

Aberrant activation of the complement system contributes to solid-organ graft dysfunction and failure. In kidney transplantation, the complement system is implicated in the pathogenesis of antibody- and cell-mediated rejection, ischemia-reperfusion injury, and vascular injury. This has led to the evaluation of select complement inhibitors (e.g., C1 and C5 inhibitors) in clinical trials with mixed results. However, the complement system is highly complex: it is composed of more than 50 fluid-phase and surface-bound elements, including several complement-activated receptors-all potential therapeutic targets in kidney transplantation. Generation of targeted pharmaceuticals and use of gene editing tools have led to an improved understanding of the intricacies of the complement system in allo- and xeno-transplantation. This review summarizes our current knowledge of the role of the complement system as it relates to rejection in kidney transplantation, specifically reviewing evidence gained from pre-clinical models (rodent and nonhuman primate) that may potentially be translated to clinical trials.

A novel method for in vitro culture and expansion of nonhuman primate B cells.

BACKGROUND: Given the role of B cells in sensitization and antibody-mediated rejection pathogenesis, the ability to identify, isolate, and study B cells in vitro is critical for understanding these processes and developing novel therapeutics. While in vivo nonhuman primate models have been used to this end, an in vitro nonhuman primate model of B cell activation and proliferation has not been developed. METHODS: CD20+ B cells and CD3+ T cells were isolated using magnetic bead separation from the peripheral blood of naive and skin allograft sensitized nonhuman primates. Allogeneic B and T cells were co-cultured in plates pre-coated with murine stromal cells engineered to express human CD40L and stimulated with cytokines. Cells and supernatants were harvested every 2 days for immune phenotyping and donor specific antibody quantification by flow cytometry. RESULTS: The optimized culture system consisted of MS40L cells co-cultured with B and allogenic T cells and stimulated with cytokines. This culture system resulted in increased memory cells and plasmablasts over time compared to other culture systems. Comparison of culture of naïve and sensitized nonhuman primate samples revealed faster B cell exhaustion and marginally increased plasmablast differentiation in sensitized culture. Donor-specific antibody production was not observed in either culture group. CONCLUSIONS: This study describes the first in vitro nonhuman primate model of B cell activation and proliferation using both naïve and allosensitized samples. This model provides an opportunity for exploration of B cell mechanisms and novel therapeutics and is a preliminary step in the development of an in vitro germinal center model.