Lionel Couzi

Key Points: Normalization methods affected the performance of gene-based diagnostic classifiers for kidney transplant rejection across a large multicenter cohort. Complex normalization reduced classifier performance by overcorrecting signal, while simpler methods preserved rejection signature and key robustness. By identifying optimal normalization methods, this work advances a standardized preprocessing framework for molecular diagnostics in transplantation. Background: The Banff 2022 classification endorses intragraft gene expression profiling using the Banff Human Organ Transplant (B-HOT) consensus gene panel for rejection diagnosis. However, lack of standardized analytical pipelines, including data normalization, limits clinical implementation, with its effect on diagnostic performance yet to be determined. Methods: We evaluated ten normalization methods in 868 kidney allograft biopsies from nine European and North American centers, all Banff-graded and B-HOT–profiled on nCounter, comprising derivation ( n =441), internal ( n =186), and external ( n =241) validation cohorts. Each method was assessed through its downstream impact on ( 1 ) gene count stability, ( 2 ) differential expression and cross-platform concordance with RNA sequencing (RNA-seq) data, and ( 3 ) discrimination and calibration of predictive models for antibody-mediated rejection (AMR) and T -cell–mediated rejection (TCMR). Results: Most methods improved count stability and showed high concordance with RNA-seq for overall gene expression. They also produced robust differential expression signatures consistent with those detected by RNA-seq, except for RUVSeq and RCRNorm , which identified fewer differentially expressed genes and showed lower concordance. In the overall validation cohort ( n =427), diagnostic performance was consistently high across nSolver -based approaches, nanostringr , NanoStringDiff , MetaNorm , and RCRNormFast (AMR, area under the ROC curve [AUROC], 0.88–0.91; area under precision-recall curves [AUPRC], 0.86–0.89; TCMR AUROC, 0.90–0.92; AUPRC, 0.78–0.83). Performance declined with RCRNorm (AMR AUROC/AUPRC, 0.55/0.41; TCMR, 0.53/0.18) and, for TCMR, with RUVSeq (AUROC, 0.84–0.85; AUPRC, 0.64–0.65). Calibration was satisfactory for most methods, except for RCRNorm and for TCMR models after RUVSeq . Conclusions: Normalization choice significantly impacted gene expression profiles and diagnostic classifier performance. Most methods, including nSolver-based pipelines, achieved robust discrimination for both AMR and TCMR. Complex methods, including RCRNorm, and RUVSeq for TCMR, reduced performance, with simpler approaches consistently outperforming them for B-HOT–based molecular diagnostics.

Le cytomégalovirus (CMV) est l’agent responsable de l’infection opportuniste la plus fréquemment rencontrée en transplantation d’organe. Chez les receveurs séronégatifs qui reçoivent un rein provenant d’un donneur séropositif, 50 % de ces patients peuvent développer une virémie, et 30 % une maladie. A court terme, malgré les traitements anti-viraux, elle est responsable d’une morbidité non négligeable. A long terme, le CMV est associé à une augmentation de la fréquence des sténoses artérielles, plus d’infections associées, plus de rejet aigu, plus de lésions de fibrose interstitielle et d’atrophie tubulaire, une moins bonne survie des greffons et des patients. La cohabitation et la coévolution du CMV avec l’homme depuis des milliers d’années ont aboutie à un état d’équilibre entre le virus et son hôte. Le virus s’est profondément adapté à son hôte afin d’échapper à la réponse immune. En réponse à cela, la réponse immunitaire anti-CMV occupe une part unique et majeure au sein de la réponse immune de l’hôte. Les lymphocytes T CD8+ spécifiques du CMV représentent par exemple 10.2% des lymphocytes T CD8+ mémoires. Avec l’âge, ils s’accumulent et peuvent représenter jusqu’à 30% du pool total de lymphocyte T CD8+. Le système immunitaire sous la contrainte du virus s’est donc refaçonné de façon à garder le contrôle du virus. Depuis 1999, un nouvel acteur de cette réponse immunitaire a été identifié : les lymphocytes T gamma delta Vdelta2-negative. Ces cellules sont impliquées habituellement dans la lutte contre les différents stress d’origine microbien et non microbien (tumeur). Elles interviennent plutôt localement (dans les épithéliums) par différents mécanismes et sont désormais considérées comme des effecteurs intermédiaires entre l’immunité innée et l’immunité adaptative. Leur expansion dans le sang est associée à la guérison de la maladie et à la résolution de l’infection à CMV. Elles ont par ailleurs in vitro une réactivité croisée contre des cellules infectées par le CMV et des cellules tumorales. Les lymphocytes T gamma delta Vdelta2-negative sont donc une représentation supplémentaire de l’énorme impact du CMV sur le système immunitaire de l’hôte. Dans ce travail, nous avons pu étendre et approfondir leur rôle en transplantation d’organe. Nous avons tout d’abord décrit que les lymphocytes T gamma delta Vdelta2-negative avaient un phénotype et une cinétique d’expansion exactement superposable aux lymphocytes T CD8+ spécifiques du CMV in vivo. Nous avons ensuite observé que l’expansion des lymphocytes T gamma delta Vdelta2-negative induits pas l’infection à CMV s’associait à une survenue moindre de cancer à long terme chez les patients transplantés rénaux. Nous avons pu montrer que leur activation était sous la dépendance d’une interaction entre leur TCR et un ligand. Enfin, une autre voie d’activation dépendante du CD16, faisant intervenir les complexes immuns CMV-IgG anti-CMV a aussi été identifiée. Nos travaux depuis 10 ans ont donc démontré que les lymphocytes T gamma delta Vdelta2-negative occupaient une place majeure dans la réponse immune anti-CMV au même titre que les lymphocytes T CD8+. L’intégration de ces cellules à l’immunologie anti-CMV devrait permettre de mieux comprendre certains effets indirects induits par le virus, et pourrait être utile dans le suivi de la réponse immune anti-CMV en transplantation d’organe. L’identification de leur ligand pourrait permettre enfin de tester assez rapidement de nouveaux protocoles d’immunothérapie anti-virale ou anti-tumorale.

γδ T cells show promise for anti-tumoral therapies but have yet to be evaluated to treat infectious diseases. In this preclinical study, we assess a Vδ1 + γδ T cell-based adoptive cell therapy, named Delta One T cells, to treat cytomegalovirus (CMV) infection in high-risk transplant recipients. Even when expanded from CMV-naïve healthy donors, Delta One T cells efficiently control CMV dissemination in vitro. CMV recognition is independent of the γδTCR but requires LFA-1 co-stimulation. In an in vivo model, adoptive transfer of mouse γδ T cells recapitulating Delta One T cell features protects mice against lethal murine CMV infection. Importantly, CMV-reactive Delta One T cells can be successfully generated from kidney transplant recipients undergoing refractory CMV infections and maintain their functionality in the presence of immunosuppressive drugs. These findings broaden the scope of γδ T cell therapies to infectious diseases and uncover a universal adoptive T cell therapy to treat refractory CMV infections.

Background.: Posttransplant immunosuppression in kidney transplant recipients is associated with an increased risk of developing cutaneous squamous cell carcinoma (CSCC), contributing to significant morbidity and mortality. Various dermatological and immunosuppression modulation strategies have been identified that may reduce the risk of CSCC, both in primary and secondary prevention settings. Recent recommendations have provided consensus regarding dermatological approaches to prevent CSCC. Comparable transplant nephrology recommendations to guide immunosuppression modulation for CSCC prevention are currently lacking, leading to marked variation in practice. Methods.: To address this knowledge gap, 46 international transplant nephrology experts participated in a 3-round Delphi survey to develop consensus recommendations for CSCC secondary prevention based on the actinic damage and skin cancer index stages of CSCC. Results.: The panel of experts reached consensus to consider a change in immunosuppression after multiple low-risk invasive CSCC (stage 5a, 1/y >3 y) and encouraged collaboration with dermatology to optimize dermatologic preventative care after the first CSCC. There was also consensus to prioritize azathioprine modification where this is present in an immunosuppressive regimen. Conclusions.: This study provides the first international consensus recommendations for management of immunosuppression in kidney transplant recipients at discrete stages of CSCC. Additional prospective studies are necessary to determine the optimal management of immunosuppression in this patient population. These recommendations have been endorsed by the Board of the American Society of Transplantation.

Background.: Kidney transplant recipients (KTRs) are at elevated risk of malignancy. Data on the safety and efficacy of immune checkpoint inhibitors (ICIs) in this population remain limited. We aimed to reassess the benefit-risk profile of ICI therapy in KTRs using a multicenter cohort, in the recent era. Methods.: We conducted a retrospective cohort study of all KTRs treated with ICIs for advanced or metastatic cancer, across 6 transplant centers. We evaluated cancer response, acute rejection (AR) incidence, graft survival, and patient survival. Results.: From 2015 to 2024, 34 KTRs were analyzed. The most common cancers were cutaneous (56%) and non–small cell lung cancer (32%). Pembrolizumab was the most used ICI (53%). The objective response rate was 38%, with a median progression-free survival of 5.5 mo and an overall survival of 10.7 mo. Biopsy-proven AR occurred in 26.5% of patients, at a median time of 52 d after ICI start. All rejection episodes involved T cells, and one-third showed additional humoral features. No clinical predictors of AR were identified. Among all patients, 29% had a favorable outcome (tumor response without ICI-induced graft loss), 12% experienced a tumor response with graft loss, 59% had progression disease without graft loss, and 3% experienced the worst outcome (progression disease with graft loss). Conclusions.: Our study suggests that ICI therapy is a viable option for KTRs with poor-prognosis cancers, demonstrating a 38% tumor response rate and a lower incidence of graft loss (15%) compared with previously reported rates. Prospective studies are needed to optimize the use of ICI in KTRs.

Key Points: Urinary C-X-C motif ligand 9 demonstrated moderate clinical utility beyond standard-of-care monitoring in detecting overall allograft rejection. Urinary C-X-C motif ligand 10 did not show additional value in detecting overall allograft rejection beyond standard-of-care monitoring. In sensitivity analyses limited to acute/active rejection and single biopsies per patient, urinary C-X-C motif ligand 9 and C-X-C motif ligand 10 showed no added value. Background: Urinary chemokines C-X-C motif ligand 9 (CXCL9) and C-X-C motif ligand 10 (CXCL10) have shown promise for detecting kidney allograft rejection, but the demonstration of their added value beyond standard-of-care patient monitoring requires further study. Methods: We prospectively enrolled adult patients who underwent kidney transplantation in seven transplant referral centers between July 2018 and December 2019 (ClinicalTrials.gov, NCT03582436). We quantified urinary CXCL9 and CXCL10 protein levels at the time of kidney allograft biopsies in the first year post-transplantation using an automated immunoassay platform. The primary outcome was allograft rejection defined according to the international Banff 2019 classification. Results: Overall, 733 kidney transplant patients (64% male, 36% female) were included in the main analysis, with 1549 biopsies paired with a urine sample. The cumulative incidence of rejection was 10%. For detecting allograft rejection, urinary CXCL9 and CXCL10 demonstrated areas under the receiver operating characteristic curve (AUROC) of 0.70 (95% confidence interval [CI], 0.64 to 0.75) and 0.64 (95% CI, 0.58 to 0.71), respectively. Adding urinary CXCL9 to a standard-of-care model improved discrimination for allograft rejection (AUROC 0.75 [percentile bootstrap CI, 0.70 to 0.79] to 0.78 [percentile bootstrap CI, 0.73 to 0.83]), while urinary CXCL10 did not. There was no improvement of overall fit with the addition of urinary CXCL9 (Brier score changed from 0.056 [95% CI, 0.046 to 0.067] to 0.054 [95% CI, 0.045 to 0.064]), as this tended to overestimate the risk for allograft rejection. In sensitivity analyses restricting to only acute/active forms of rejection or to a single randomly selected biopsy per patient, urinary chemokines did not show additional value beyond the standard of care. In addition, existing chemokine-based models showed low-to-moderate performance for the detection of allograft rejection. Conclusions: Urinary CXCL9 demonstrated limited clinical utility, while urinary CXCL10 provided no additional value beyond standard-of-care monitoring for detecting allograft rejection within the first year after kidney transplantation. Clinical Trial registry name and registration number: ClinicalTrials.gov, NCT03582436.