Magali Giral

The aim of our study was to determine whether granzyme B-expressing regulatory B cells (GZMB⁺ B cells) are enriched in the blood of transplant patients with renal graft tolerance. To achieve this goal, we analysed two single-cell RNA sequencing (scRNAseq) datasets: (1) peripheral blood mononuclear cells (PBMCs), including GZMB⁺ B cells from renal transplant patients, i.e., patients with stable graft function on conventional immunosuppressive treatment (STA, n = 3), drug-free tolerant patients (TOL, n = 3), and patients with antibody-mediated rejection (ABMR, n = 3), and (2) ex-vivo-induced GZMB⁺ B cells from these groups. In the patient PBMCs, we first showed that natural GZMB⁺ B cells were enriched in genes specific to Natural Killer (NK) cells (such as NKG7 and KLRD1) and regulatory B cells (such as GZMB, IL10, and CCL4). We performed a pseudotemporal trajectory analysis of natural GZMB⁺ B cells and showed that they were highly differentiated B cells with a trajectory that is very different from that of conventional memory B cells and linked to the transcription factor KLF13. By specifically analysing GZMB⁺ natural B cells in TOLs, we found that these cells had a very specific transcriptomic profile associated with a reduction in the expression of HLA molecules, apoptosis, and the inflammatory response (in general) in the blood and that this signature was conserved after ex vivo induction, with the induction of genes associated with migration processes, such as CCR7, CCL3, or CCL4. An analysis of receptor/ligand interactions between these GZMB^+/− natural B cells and all of the immune cells present in PBMCs also demonstrated that GZMB⁺ B cells were the B cells that carried the most ligands and had the most interactions with other immune cells, particularly in tolerant patients. Finally, we showed that these GZMB⁺ B cells were able to infiltrate the graft under inflammatory conditions, thus suggesting that they can act in locations where immune events occur.

Background.: Efficacy and safety of belatacept have not been specifically reported for kidney transplantations from donors after circulatory death. Methods.: In this retrospective multicenter paired kidney study, we compared the outcome of kidney transplantations with a belatacept-based to a calcineurin inhibitor (CNI)-based immunosuppression. We included all kidney transplant recipients from donors after uncontrolled or controlled circulatory death performed in our center between February 2015 and October 2020 and treated with belatacept (n = 31). The control group included the recipients of the contralateral kidney that were treated with CNI in 8 other centers (tacrolimus n = 29, cyclosporine n = 2). Results.: There was no difference in the rate of delayed graft function. A higher incidence of biopsy-proven rejections was noted in the belatacept group (24 versus 6 episodes). Estimated glomerular filtration rate (eGFR) was significantly higher in the belatacept group at 3-, 12-, and 36-mo posttransplant, but the slope of eGFR was similar in the 2 groups. During a mean follow-up of 4.1 y, 12 patients discontinued belatacept and 2 patients were switched from CNI to belatacept. For patients who remained on belatacept, eGFR mean value and slope were significantly higher during the whole follow-up. At 5 y, eGFR was 80.7 ± 18.5 with belatacept versus 56.3 ± 22.0 mL/min/1.73 m² with CNI (P = 0.003). No significant difference in graft and patient survival was observed. Conclusions.: The use of belatacept for kidney transplants from either uncontrolled or controlled donors after circulatory death resulted in a better medium-term renal function for patients remaining on belatacept despite similar rates of delayed graft function and higher rates of cellular rejection.

Introduction: The human immune system contains cells with either effector/memory or regulatory functions. Besides the well-established CD4+CD25hiCD127lo regulatory T cells (Tregs), we and others have shown that B cells can also have regulatory functions since their frequency and number are increased in kidney graft tolerance and B cell depletion as induction therapy may lead to acute rejection. On the other hand, we have shown that CD28-CD8+ T cells represent a subpopulation with potent effector/memory functions. In the current study, we tested the hypothesis that kidney allograft rejection may be linked to an imbalance of effector/memory and regulatory immune cells.Methods: Based on a large cohort of more than 1000 kidney graft biopsies with concomitant peripheral blood lymphocyte phenotyping, we investigated the association between kidney graft rejection and the percentage and absolute number of circulating B cells, Tregs, as well as the ratio of B cells to CD28-CD8+ T cells and the ratio of CD28-CD8+ T cells to Tregs. Kidney graft biopsies were interpreted according to the Banff classification and divided into 5 biopsies groups: 1) normal/subnormal, 2) interstitial fibrosis and tubular atrophy grade 2/3 (IFTA), 3) antibody-mediated rejection (ABMR), 4) T cell mediated-rejection (TCMR), and 5) borderline rejection. We compared group 1 with the other groups as well as with a combined group 3, 4, and 5 (rejection of all types) using multivariable linear mixed models.Results and discussion: We found that compared to normal/subnormal biopsies, rejection of all types was marginally associated with a decrease in the percentage of circulating B cells (p=0.06) and significantly associated with an increase in the ratio of CD28-CD8+ T cells to Tregs (p=0.01). Moreover, ABMR, TCMR (p=0.007), and rejection of all types (p=0.0003) were significantly associated with a decrease in the ratio of B cells to CD28-CD8+ T cells compared to normal/subnormal biopsies. Taken together, our results show that kidney allograft rejection is associated with an imbalance between immune cells with effector/memory functions and those with regulatory properties.

Whilst calcineurin inhibitors (CNI) are the cornerstone of immunosuppressive maintenance therapy in kidney transplantation, several studies have investigated the safety of CNI withdrawal in order to avoid their numerous side effects. In this context, we performed several years ago a clinical randomized trial evaluating CNI weaning in stable kidney transplant recipients without anti-HLA immunization. The trial was interrupted prematurely due to a high number of de novo DSA (dnDSA) and biopsy proven acute rejection (BPAR) in patients who underwent tacrolimus weaning, resulting in treatment for rejection and resumption of tacrolimus. We report here the long-term outcomes of patients included in this clinical trial. Ten years after randomization, all patients are alive with a functional allograft. They all receive tacrolimus therapy except one with recurrent cutaneous neoplasia issues. Long-term eGFR was comparable between patients of the two randomized groups (46.4 ml/min vs 42.8 ml/min). All dnDSA that occurred during the study period became non-detectable and all rejections episodes were reversed. The retrospective assessment of HLA DQ single molecule epitope mismatching determined that a majority of patients who developed dnDSA after tacrolimus withdrawal would have been considered at high immunological risk. Minimization of immunosuppression remains a challenging objective, mainly because of the issues to properly select very low immunological risk patients. Valuable improvements have been made the last decade regarding evaluation of the allograft rejection notably through the determination of numerous at-risk biomarkers. However, even if the impact of such tools still need to be clarify in clinical routine, they may permit an improvement in patients’ selection for immunosuppression minimization without increasing the risk of allograft rejection.

There is extensive literature with comparisons between Anti-Thymocyte Globulin (ATG) and Basiliximab (BSX) as induction therapy in kidney transplant recipients. The purpose of our benchmarking study was to describe the consequences in terms of practices in 6 transplantation centers of a French prospective cohort. We included adult patients who received a first or second kidney graft between 2013 and 2019 (n = 4157). We used logistic regressions to identify characteristics associated with the use of ATG or BSX. Use of ATG between the centers ranged from 41% to 75%. We observed different factors associated with the treatment decision. Compared to a first transplant, performing a second graft was the only factor significantly associated with the choice of ATG in all centers. The AUC ranged from 0.67 to 0.91, indicating that the centers seemed to define their own rules. As a result, for patients with the same low immunological risk, the probability of receiving ATG varied from 7% to 36%. We stratified the analyses according to two periods, from 2013 to 2015 and from 2016 to 2019. A similar heterogeneity was observed, and in some cases ATG indications between the centers were inverted. The heterogeneity of induction therapy practices did not decrease in France, even if the reated literature is prolific. This illustrates the necessity to improve the literature by using meta-analyses of recent studies stratified by graft and patient profiles.

Background: Kidney transplantation is considered to be the treatment of choice for people with end-stage renal disease (ESRD). However, due to the shortage of available organs and the increase in the ESRD prevalence in Europe, it is essential to improve transplantation outcomes by studying the related prognostic factors. Today, there is no European registry collecting data to perform such clinical epidemiology studies. Main body: Entitled EKiTE, for European cohort for Kidney Transplantation Epidemiology, this prospective and multicentric cohort includes patients from Spanish (Barcelona), Belgian (Leuven), Norwegian (Oslo) and French (Paris Necker, Lyon, Nantes, Nancy, Montpellier, Nice and Paris Saint Louis) transplantation centers and currently contains 13,394 adult recipients of kidney (only) transplantation from 2005 and updated annually. A large set of parameters collected from transplantation until graft failure or death with numbers of post-transplantation outcomes. The long-term follow-up and the collected data enable a wide range of possible survival and longitudinal analyses. Conclusion: EKiTE is a multicentric cohort aiming to better assess the natural history of the ESRD in European kidney transplant recipients and perform benchmarking of clinical practices. The data are available for clinical epidemiology studies and open for external investigators upon request to the scientific council. Short-term perspectives are to extend EKITE network to other European countries and collect additional parameters in respect of the common thesaurus.

Introduction: Les rejets chroniques grèvent la survie au long terme des greffons rénaux ; prévenir la survenue de ces atteintes est un enjeu majeur, car elles sont irréversibles. Méthodes: L’objectif de notre travail était la recherche d’une corrélation entre un phénotype lymphocytaire sanguin périphérique, réalisable en routine clinique, et l’histologie de la biopsie de greffon. Nous avons analysé en cytométrie de flux les phénotypes lymphocytaires CD8+CD28− cytotoxiques, décrits associés aux rejets chroniques dans des études de petits effectifs, ainsi que les LT régulateurs CD4+CD25highCD127. Nous avons réalisé une cohorte prospective de patients transplantés rénaux ayant bénéficié d’un immunophénotypage lymphocytaire sanguin le jour de leur biopsie de greffon (réalisée en routine : surveillance à 3 et 12 mois ou devant une dégradation de la fonction du greffon). Les résultats histologiques ont été classés en 9 catégories basées sur la classification Banff 2015 : normale, subnormale, rejet humoral, suspicion de rejet humoral, rejet cellulaire, lésions borderlines, fibrose interstitielle avec et sans inflammation, autres diagnostics. Résultats obtenus ou attendus Entre 2008 et 2016, 1097 patients ont été inclus, 1640 biopsies associées à l’immunophénotypage des LT CD8+CD28− et des LT CD4+CD25highCD127 ont été analysées. En analyse multivariée nous avons démontré que toute augmentation de 10 % du taux de LT CD8 + CD28− sanguin périphérique était associée à des lésions histologiques borderlines (OR = 1,40 IC 95 [1,1–1,8] par rapport aux biopsies normales). Nous n’avons pas mis en évidence de différence concernant la population LT régulateurs. Conclusion: Nous avons démontré que le phénotype lymphocytaire circulant est associé au diagnostic histologique. L’objectif est maintenant d’établir si le phénotype LT CD8 + CD28− permettrait de différencier au sein des lésions borderline celles de mauvais pronostic nécessitant une intensification thérapeutique, des lésions histologiques non délétères. Le phénotype CD8 + CD28− réalisable en routine pourrait s’inscrire dans la surveillance des patients transplantés rénaux et constituer un signal d’alerte de risque de rejet.