BACKGROUND:Xenotransplantation has emerged as a promising solution to the critical organ shortage, with encouraging results in preclinical nonhuman primate studies and recent first-in-human transplants. Our group previously performed pig-to-rhesus macaque renal xenotransplants using the clinically available costimulation blockade agent belatacept; however, graft survival was modest (>1 moh). Analysis of rejected xenografts identified natural killer (NK) cells as a predominant infiltrating population. METHODS:In this study, we investigated whether adding adjuvant αIL-15-an agent known to suppress T-cell subsets and deplete NK cells in rhesus macaques-could improve xenograft survival. αIL-15 was combined with a clinically relevant immunosuppressive regimen comprising T-cell depletion, belatacept, mycophenolate mofetil, and steroids. RESULTS:We found that the addition of αIL-15 significantly improved xenograft survival and function. Longitudinal analysis of NK cell subsets revealed a shift from a predominant cytotoxic CD16 + CD56 - population to a double-negative CD16 - CD56 - phenotype following αIL-15 treatment. CONCLUSIONS:These findings deepen our understanding of NK cell subsets and their potential contributions to xenograft injury and suggest that targeted modulation of NK cell populations can enhance xenograft outcomes in a preclinical pig-to-rhesus macaque model of renal xenotransplantation.

The risk of invasive fungal infection (IFI) in kidney transplant recipients (KTRs) receiving de novo belatacept-based immunosuppression remains incompletely understood. We conducted a single-center retrospective cohort study of adult KTRs receiving de novo belatacept- or tacrolimus-based immunosuppression between 2016 and 2025. Cumulative incidence of IFI was estimated using competing-risks methods with death as a competing event and compared using Gray's test. Associations between immunosuppression and IFI risk were evaluated using a cause-specific Cox model, with thymoglobulin and high-dose steroids modeled as time-dependent covariates. A prespecified propensity score-matched cohort was analyzed to address selection bias. Associations between IFI and mortality were assessed using time-dependent Cox proportional hazards models. Among 2729 KTRs, 1875 (68.7%) received belatacept and 854 (31.3%) tacrolimus. The 1-year cumulative incidence of IFI was 1.4% in belatacept-treated patients vs 1.8% in tacrolimus-treated patients (P = .47). Belatacept compared with tacrolimus was not associated with IFI (hazard ratio, 0.87; 95% confidence interval, 0.54-1.39). IFI was associated with mortality (hazard ratio, 5.03; 95% confidence interval, 3.12-8.09). Similar results were observed in the propensity score-matched cohort. De novo belatacept-based immunosuppression does not confer increased risk of IFI, and routine antifungal prophylaxis may not be necessary.

The inaugural Richard Slayman Clinical Xenotransplantation Workshop convened >140 participants from North America, Europe, and Asia to discuss emerging advances and challenges in translating xenotransplantation from bench to bedside. This report summarized key discussions spanning kidney, heart, and liver xenotransplantation, with an emphasis on clinical readiness and future directions. Core themes included the importance of patient selection, the role of genetic editing to reduce immune incompatibility, adaptive immunosuppressive strategies, novel molecular tools for immune and infectious surveillance, and the growing recognition of innate immune activation as a barrier to long-term graft survival. The workshop highlighted decedent models as a translational bridge, the use of machine perfusion in liver xenograft applications, and progress in living recipients. Notably, 1 patient achieved 9 mo of kidney xenograft function, underscoring the feasibility of extended survival in carefully selected candidates. Perspectives from patients and families, including a reflection honoring Richard Slayman, the first living recipient of a genetically edited pig kidney, framed the scientific dialogue within the broader human impact of this emerging field. The workshop marked a pivotal moment in aligning scientific, ethical, and regulatory efforts to advance safe and equitable access to xenotransplantation.

Abstract Background BK polyoma virus (BKPyV) DNAemia imparts significant morbidity in kidney transplant recipients (KTR). It can lead to polyomavirus-associated nephropathy (PyVAN), impaired graft function, and graft failure. The effect of belatacept on BK polyoma virus (BKPyV) control remains largely unknown. Methods This is a propensity matched retrospective cohort study in adult kidney transplant recipients (KTR) transplanted between 2016-2020 who received a belatacept- versus tacrolimus-based immunosuppression regimen. A multi-state Markov model was used to evaluate BKPyV replication dynamics (BKPyV-dyn). Three BKPyV-dyn states were defined: BKPyV-dyn1 (viral load < 3 log10), BKPyV-dyn2 (viral load > 3 log10 and < 4 log10), and BKPyV-dyn3 (viral load > 4 log10). Estimated total length of stay in a BKPyV replication dynamic state after evolution of the entire Markov process Results 280 KTR on belatacept- and 280 KTR on tacrolimus-based regimens were compared. The probability of transitioning between BKPyV-dyn states and time spent in each state in both groups was comparable. Total duration in BKPyV-dyn-1 was 632.1 days (95%CI 612.1, 648.5) for belatacept vs 615.2 days (95% CI 592.5, 635.8) for tacrolimus, BKPyV-dyn-2 was 49.2 days (95% CI 41.3, 58.4) for belatacept vs 55.6 days (95% CI 46.5, 66.8) for tacrolimus, and BKPyV-dyn-3 was 48.7 days (95% CI 52.3, 79.5) for belatacept vs 60.9 days (95% CI 48.3, 76.4) for tacrolimus (Figure 1). Estimated mean sojourn time did (time spent in a given BKPyV-dyn state prior to transitioning to another state) did not differ by immunosuppression (Figure 2). BKPyV associated nephropathy (PyVAN) occurred in 3.9% in belatacept- and 3.9% tacrolimus-treated KRT (P >0.9). Estimated mean sojourn time by immunosuppression Average single occupancy time in a BKPyV replication dynamic state Conclusion Collectively, our results indicate that relative to patients on tacrolimus-based regimens, patients on belatacept-based immunosuppression regimens do not require more frequent BKPyV QNAT monitoring or more aggressive immunosuppression reduction when BKPyV-DNAemia 3 log10 or greater is detected. Disclosures Christian P. Larsen, MD, PhD, Bristol-Myers Squibb: Advisor/Consultant|CareDx: Advisor/Consultant|Eledon: Advisor/Consultant

Antibodies against the donor human leukocyte antigen (HLA) molecules drive late transplant failure, with HLA-DQ donor-specific antibodies (DSAs) posing the highest rejection risk. Here, we investigated the role of indirect CD4+ T cell epitopes-donor-derived peptides presented by recipient major histocompatibility complex (MHC) class II-in DSA formation. Antigen mapping of samples from HLA-DQ DSA-positive kidney and heart transplant recipients revealed two polymorphic hotspots in donor HLA-DQ that generated alloreactive peptides. Antigen mapping of indirect CD4+ T cell epitopes in a mouse model of fully MHC mismatched skin graft transplantation (BALB/c to C57BL/6) identified a similar epitope (amino acids 287-301) derived from the donor H2-Kd. Tetramer-binding Kd287+ CD4+ T cells were detected during rejection and their transfer into T cell-deficient mice induced DSA. Systemic delivery of high-dose donor H2-Kd peptides combined with CTLA4-Ig reduced the frequencies of Kd287+ CD4+ T cells and DSA formation. Thus, targeting a narrow range of donor antigens may prevent DSA formation and improve transplant outcomes.

INTRODUCTION:Kidney function at 1-year post-transplant is an indicator of long-term graft function. Using functional data analysis (FDA), we evaluate the relationship between early renal recovery trajectories and kidney function at 1 year. METHODS:We analyzed 1748 adults who underwent deceased-donor kidney transplantation between 2010 and 2021. Renal recovery trajectory functions were derived from longitudinal inverse creatinine values. Functional linear regression models were used to evaluate how well early (<90 days) renal recovery trajectory functions, and their rate of change explained 1-year eGFR. The explanatory power of the functional regression models was compared to results from ordinary least squares models, which used cross-sectional inverse creatinine values and linear slopes. Models were adjusted for age, sex, kidney donor profile index (KDPI), delayed graft function (DGF), race, body mass index (BMI), rejection, diabetes, hypertension, cytomegalovirus (CMV) serostatus risk, index admission length of stay, and immunosuppression agent. The R2 coefficient quantified the 1-year eGFR variation explained by model variables. RESULTS:Adjusted functional linear models with renal recovery trajectory and trajectory velocity functions as independent variables explained 68% (65, 71), 70% (67, 74), 70% (66, 74), 70% (66, 75), and 73% (69, 79) of the variation in 1-year eGFR by 7, 14, 30, 60, and 90 days, respectively. By comparison, the ordinary least squares linear models explained a maximum of 69% of the variation in 1-year eGFR at 90 days. CONCLUSION:Renal recovery patterns captured as continuous functions as early as 14 days are predictive of renal function at 1 year and may enable early personalized care of recipients at increased risk of poor graft function.

Although maintenance immunosuppression with calcineurin inhibitors (CNIs) has greatly reduced rejection rates in renal transplant recipients, long-term use can contribute to eventual nephrotoxicity, potentially leading to allograft injury and loss. Several clinical trials have shown that, compared with CNIs, belatacept-based maintenance immunosuppression can improve renal function, reduce the incidence of de novo donor-specific antibodies, and improve long-term patient/graft survival. However, the US Food and Drug Administration-approved belatacept-based regimen is also associated with higher acute rejection (AR) rates than CNI-based immunosuppression. Recent data from clinical trials and real-world studies suggest that initial posttransplant treatment with CNI-based immunosuppression followed by conversion to a belatacept-based regimen can lower the AR risk while preserving patient and renal health. This review article summarizes the available data pertaining to belatacept treatment protocols, with a focus on conversion to belatacept. Also discussed are studies of protocol modifications intended to further mitigate AR risks and belatacept-related outcomes in special populations, such as patients receiving marginal kidneys and those at risk of new-onset diabetes. Overall, the available data suggest that conversion from CNI- to belatacept-based immunosuppression ≥6 mo posttransplant appears to be effective in lowering the AR risk compared with belatacept use in the de novo setting or conversion <6 mo posttransplant. The addition of an extended transient or low-dose CNI treatment to de novo belatacept or a prolonged CNI taper in the conversion setting may also help lower the AR risk. However, additional studies will be needed to optimize the many variables applicable to belatacept treatment, particularly for different patient subgroups.

Donor-specific T cell responses, particularly against human leukocyte antigen (HLA)-DQ antigens, are critical in transplant immunology as they influence graft survival and rejection. This study investigated HLA-DQ-specific CD4 T cell responses in naive individuals using HLA-DQ monomers, representing common donor HLA-DQ antigens (DQ2.5, DQ5, DQ6, DQ8), as defined alloantigen sources to enrich and detect HLA-DQ-specific T cells. Using a repeated stimulation protocol with HLA-DQ monomers, we achieved an enrichment of HLA-DQ-specific CD4 T cells, evidenced by proliferation and activation marker upregulation. Additionally, in silico epitope prediction identified 15-mer HLA-DQ peptides capable of binding to self-HLA-DRB1 alleles. TCR sequencing demonstrated an enriched, oligoclonal repertoire of HLA-DQ-specific CD4 T cells post-stimulation, with specific TCR clonotypes expanding in response to HLA-DQ monomer stimulation. Together, our study presents an in vitro method for enriching HLA-DQ-specific CD4 T cells using HLA-DQ monomers as an alloantigen source, thereby improving the detection of alloreactive, donor-specific T cells in naive individuals with the potential to mediate immune responses, including graft rejection and donor-specific antibody (DSA) production.

BACKGROUND:Approximately 13% of African Americans have a combination of risk variants of the APOL1 gene, often termed the APOL1 "high-risk genotype", which predisposes individuals to serious kidney diseases such as focal segmental glomerulosclerosis. Despite the potential severity of APOL1-linked kidney disease, genetic testing for APOL1 variants is uncommonly performed. Point-of-care testing has the potential to be transformative in patient care to immediately identify risk status, particularly for the evaluation of deceased donor organs in the setting of transplantation, but no assays are available to date. METHODS:To begin to address this unmet need, we developed a CRISPR-Cas12a-mediated genotyping assay for the detection of pathological APOL1 variants from patient samples for potential use in a point-of-care setting. For each variant, crRNA guides were identified that could readily discriminate between either the 'wild-type' or variant sequence in a Cas12a- trans -cleavage assay using both fluorescence- and lateral flow-based detection methods. RESULTS:Interestingly, no protospacer-adjacent-motif (PAM) sequence was required for the CRISPR-Cas12a-based detection of any of the variants. CRISPR-Cas12a fluorescence-based genotyping assays performed on patient samples achieved 100% sensitivity and specificity compared to genotypes obtained using a clinically-validated TaqMan assay. Similar results were also obtained in lateral flow assay format with a FAM- and biotin-labeled reporter. CONCLUSIONS:The successful deployment in this study of Cas12a-based assays to generate APOL1 genotypes for individuals with high fidelity provides promise for a genotyping method potentially amenable to point-of-care use.

Immunosuppression can be withdrawn from selected liver transplant recipients, although robust clinical predictors of tolerance remain elusive. The Immune Tolerance Network ITN056ST study (OPTIMAL; NCT02533180) assessed clinical outcomes and mechanistic correlates of phased immunosuppression withdrawal (ISW) in nonautoimmune, nonviral adult liver transplant recipients. Enrolled subjects were >3 years posttransplant with minimal/absent inflammation or fibrosis on a screening liver biopsy. The primary end point was operational tolerance at 52 weeks following complete ISW. Of 61 subjects who initiated ISW, 34 failed during ISW and 10 restarted immunosuppression after completing ISW due to clinically manifest acute rejection. Only 10 of 17 clinically stable subjects remaining off immunosuppression at 1 year were ultimately deemed tolerant by biopsy. There were no cases of chronic rejection or graft loss; 28.3% developed de novo donor-specific antibody during ISW, which persisted in 11.3%. The majority of subjects (78.6%), including those who experienced rejection, ended the study on same or less calcineurin inhibitor than at baseline. A minority (16.4%) of histologically and clinically stable long-term adult liver transplant recipients can successfully discontinue and remain off immunosuppression. Increased frequency of donor-specific T cell senescence, C4d deposition, and higher density of immune synapses on the screening liver biopsy emerged as potential candidate biomarkers for operational tolerance.

Abstract Background BK polyomavirus (BKPyV) is the leading cause of polyomavirus associated nephropathy (PyVAN) in kidney transplant recipients (KRT). The goal of this study was to determine risk factors and outcomes associated with severe persistent BKPyV (SPBK).Figure 1A:Total estimated length of stay in a lymphocyte state: SPBK vs LBK Methods This is a single center, matched retrospective case control study of KTR with SPBK compared to 1) KTR with low/no BKPyV-DNAemia (LBK) and 2) KTR with high, transient BKPyV-DNAemia (HTBK). Definitions were as follows, SPBK: BKPyV load > 6 log10 for >90 consecutive days. LBK: BKPyV load remaining < 3 log10. HTBK: BKPyV load >4 log10 for < 90 consecutive days, all within the first 2 years post-transplant. SPBK were exact matched 1:3 to LBK controls on race, sex, donor type immunosuppression protocol, and genetic matched with robust Mahalanobis distance on age. SPBK cases were exact matched 1:2 with HTBK controls on race, gender, immunosuppression protocol, and genetic matched with robust Mahalanobis distance on age and donor type. Differences between case and control groups were analyzed using Wilcoxon-rank sum test and Fisher’s exact test. A multistate Markov model was used to evaluate whether time spent in a state of lymphopenia (< 1,000 cells/µL) was associated with SPBK.Figure 1A:Total estimated length of stay in a lymphocyte state: SPBK vs HTBK Results 20 KTR with SPBK were matched to 60 LBK and 40 HTBK. Significantly more SPBK experienced acute cellular rejection within the first 2 years of transplant relative to LBK (75% vs 27%, p< 0.001) and HTBK (22%, p< 0.001). SPBK had a longer estimated duration of lymphopenia in the first 200 days post-transplant relative to LBK and HTBK (SPBK: 107.5 days, 95% CI 92.1, 122.7 vs LBK: 68.5 days, 95% CI 55.3, 84.8, vs HTBK 70.3 days 95% CI 58.8, 82.4, Figure 1A-B). Median eGFR at 6 months post-transplant for SPBK was lower than patients with LBK and HTBK (44 vs 56, p=0.016; 44 vs 60, p=0.004, respectively). Burden of lab draws was significantly higher for SPBK vs LBK and HTBK (69 days vs 55, p< 0.001; 69 days vs 56, p=0.006, respectively). Total cost of labs was $7836 for SPBK vs $4,691 for LBK (p< 0.001) and $5922 for HTBK (p< 0.001). Conclusion Patients with SPBK spent more time in a state of lymphopenia in the first 200 days post-transplant vs LBK and HTBK. SPBK is associated with worse renal function at 6 months post-transplant and is accompanied by a higher burden of lab draws and cost. Disclosures Christian P. Larsen, MD, PhD, Bristol-Myers Squibb: Advisor/Consultant|CareDx: Advisor/Consultant|Eledon: Advisor/Consultant

Immunosuppressive regimens in kidney transplant are largely still reliant on steroids and calcineurin inhibitors (CNIs) despite long-term side effects and nephrotoxicity. Belatacept is a costimulatory blockade molecule which has been shown to improve GFR and metabolic parameters though has been associated with increased rates of acute rejection, limiting its widespread use. This review details the historical development of belatacept, describes the iterative refinement of de novo and conversion protocols utilizing belatacept, summarizes current trends in its use, and discusses its ongoing development in future applications. Belatacept use continues to widen. Gradual refinements to protocols have reduced the incidence of acute rejection to rates comparable to those seen in CNI-based regimens. Increasingly varied combinations with mTOR inhibition, depletional induction, low-dose CNIs, and emerging targeted monoclonal antibody therapies continues to expand the possible uses for belatacept as safe, effective, and durable immunosuppression. Belatacept provides significant advantages when used as immunosuppression in kidney transplantation, allowing minimization of the negative effects of steroids and CNIs; increasingly creative combinations with standard and emerging medications continues to widen its use and allow increasing access.

Background:We aim to describe the effect of belatacept on de novo donor-specific antibodies (DSA) formation, rejection, and renal function in heart transplant recipients. Methods:The cohort comprises 60 adult heart or heart-kidney recipients transplanted between 2005 and 2022. Twelve recipients initialized at ∼90 days post-transplantation on a belatacept-based immunosuppression regimen with tapered tacrolimus trough levels were matched to 48 standard tacrolimus-based regimen controls. Differences in the distribution of recipients with emergent de novo DSA and rejection were assessed over the first 85 days baseline period, the average duration pre-belatacept. Survival analysis assessed regimen group differences in the probability of remaining de novo DSA and rejection free over the follow-up period spanning 86 to 540 days. Renal function and cytomegalovirus viremia were examined as secondary outcomes. Results:There were no statistically significant regimen group differences in the distribution of recipients with de novo DSA or rejection during the baseline period. Furthermore, differences in the probability of remaining de novo DSA and rejection free during the follow-up period remained insignificant (log-rank test, p = 0.12). Belatacept-treated recipients, at follow-up, had no incidence of developing de novo DSA, unlike 19% of the controls. Additionally, there were no statistically significant differences in acute cellular and antibody mediated rejection events, renal function, and CMV viremia by regimen group. Conclusion:Recipients treated with belatacept-based regimen exhibited a trend of reduced de novo DSA development compared to standard tacrolimus-based regimen controls. Larger studies are needed to evaluate the benefit of belatacept use in heart transplant populations.

The cancer-intrinsic factors that reduce CD8 T cell responses in advanced tumors are unclear. Using a novel strategy to isolate tumor cells from 83 late stage ccRCC patients combined with genomic, transcriptomic and phenotypic analyses, we identified two developmentally related cancer subsets: ENPP3lo and an aggressive ENPP3hi cell. While ENPP3hi cells correlated with more invasive cancer, their presence alone did not dictate immune evasion. Instead, elevated oxidative phosphorylation (OXPHOS) within these cells associated with reduced CD8 T cell activity and increased rate of metastasis. Mechanistically, metabolomics on tumor interstitial fluids from mice and human revealed that high OXPHOS cancer cells create an immunosuppressive tumor microenvironment, preventing TCF1+ stem-like CD8 T cells from differentiating into anti-tumor effectors. Succinate was a key metabolite in this process, and knockout of its transporter, MCT1, within highly oxidative cancer cells restored the immune responses and improved tumor control. Furthermore, treating mice with an MCT1 inhibitor (AZD3965) rescued responsiveness to anti-PD-L1 therapy, identifying a novel clinical axis to bolster cancer treatments. In conclusion, this study uncovers a previously underappreciated role for cancer-oxidative metabolism in driving immune evasion and disease progression, identifies its underlying biological mechanisms, and proposes MCT1 inhibition as a promising therapeutic avenue for high risk ccRCC patients. Supported by NCI 1R01CA280069 and DOD W81XWH-20-1-0525 Tumor Immunology: Cellular Responses and Tumor Microevironment (TIME)