Thèse Impact du Microbiote Digestif sur le Risque de Rejet de Greffe Rénale H/F - 75

Établissement : Université Paris-Saclay GS Life Sciences and Health
École doctorale : Signalisations et Réseaux Intégratifs en Biologie
Laboratoire de recherche : Next-Generation Immuno-Oncology Research and Therapy in Pediatric and Adult cancer
Direction de la thèse : Antoine DÜRRBACH ORCID 0000000303858726
Début de la thèse : 2026-10-01
Date limite de candidature : 2026-05-05T23:59:59

La transplantation rénale est le meilleur traitement de l'insuffisance rénale chronique, mais les résultats sont limités par la toxicité rénale des médicaments immunosuppresseurs actuels. Aussi Belatacept, un nouvel inhibiteur de point de contrôle dérivé de CTLA4-Ig, a été développé pour inhiber l'activation des cellules T. Il permet une meilleure fonction rénale et une survie du greffon et du patient. Cependant, ses effets bénéfiques sont altérés par l'apparition d'un taux élevé de rejet aigu dans différentes populations avec d'importantes variations géographiques. Nous avons identifié une sous-population de lymphocytes T CD4 + CD57 + PD1- qui est associée à un risque plus élevé de rejet aigu. Cette population de cellules T est résistante au bélatacept in vitro et est associée au développement d'une forte proportion de cellules NKT lorsqu'elles sont stimulées. Les cellules NKT sont des lymphocytes qui migrent dans la muqueuse intestinale et qui peuvent être régulés par le microbiote digestif. Dans une étude préliminaire, nous avons mis en évidence une corrélation entre certaines bactéries, la présence de peptides liant des composés de ces bactéries, certaines cytokines et la survenue de rejets cellulaires. Dans le cancer, il a été démontré que l'efficacité de 'l'inhibiteur de point de contrôle' dépend de la composition du microbiote. Nous proposons d'étudier le lien entre le microbiote et le risque de rejet de greffe rénale en utilisant des échantillons de donneurs sains et de patients transplantés et d'identifier les mécanismes moléculaires impliqués dans la régulation de l'alloimmunité. Nous validerons ces résultats dans un modèle murin de greffe de rein. Nos résultats aideront à identifier les patients traités par Belatacept à haut risque de rejet aigu et à identifier de nouvelles stratégies pour manipuler le microbiote intestinal afin de réduire le risque de rejet aigu chez les patients traités par Belatacept.

The interaction between the commensal microbiota and the immune system has grown recently in the scientific and medical communities.This interest has coincided with the development of 'checkpoint inhibitors'. These new immunotherapies specifically target more selectively immune cells by inhibiting co-signals of T-cell activation. Checkpoint inhibitors (CPI) have transformed the management of cancers and inflammatory diseases, and other molecules have been used to prevent transplant rejection. However, the results obtained with these immunotherapies have been highly variable, suggesting that exogenous factors may influence the immune response. Recent studies of CPI for cancer treatment have revealed that intestinal microbiota composition affects the immune response to melanoma via the PD-1/PDL-1 axis (1,2). Sivan et al. [1] showed that the microbiota affects tumor growth and the number of tumor-specific T cells (LT). Another study analyzed the impact of microbiota composition on the efficacy of PD1 inhibitors in a murine model of sarcoma and in patients with kidney or lung cancers [2]. The patients with favorable responses to inhibition of the PD-1/PD-L1 axis had an intestinal flora enriched in A. muciniphila. Subsequent studies showed that the inhibition of tumor progression in the presence of a PD1 inhibitor required a favorable bacterial flora. Similarly, the anti-tumor effect of CTLA4 inhibitors is dependent on the microbiota and, more precisely, on the presence of B. fragilis, in a murine model [3]. Conversely, other bacteria species favor the tolerance of tumor cells. Overall, these studies have demonstrated a link between microbiota composition and the activation of dendritic cells (DCs) and lymphocytes that might influence the response to these immunotherapies. Studies of interactions between microbiota and immunotherapies have, to date, focused on CPI-inducing LT activation. However, the checkpoint inhibitors used to prevent transplant rejection have not been studied in the context of the microbiota composition of patients. The most widely used of these molecules, belatacept, is a modified CTLA4-Ig fusion protein that acts as a highly specific inhibitor of the CD28-CD80/86 axis. CTLA4-Ig inhibits LT activation by disrupting the interaction of the CD28 on LT with the CD80/86 on antigen-presenting cells, thereby inhibiting the CD28 activation pathway in LT, the predominant co-signal for LT activation. This molecule (Belatacept) has been developed for the prevention of acute rejection in kidney transplantation. Kidney transplantation is the best treatment for patients with chronic kidney disease (CKD) because it improves patient survival and quality of life, and is less expensive than dialysis. CKD is a major public health issue, as its frequency is increasing steadily (+ 11% between 2012 and 2016 in France) and it causes significant morbidity and mortality. Graft acceptance by the immune system of the recipient is possible thanks to a set of immunosuppressive molecules, including calcineurin inhibitors (CNI), which alter lymphocyte activation. However, CNI has several major adverse effects, such as increasing cardiovascular risk, nephrotoxicity, and increasing the general immunosuppression responsible for infectious and tumor complications. A need for T cell-specific immunosuppression has thus emerged [4]. The development of the CTLA4-Ig fusion protein, acting principally by impairing the activation of naive LT, has been evaluated in EBV-positive kidney transplant recipients. This treatment has fewer side effects than cyclosporine A, a CNI. It preserves the function of the transplanted kidney and reduces the occurrence of the donor-specific anti-HLA antibodies implicated in humoral immunity-mediated rejection, a major cause of graft loss [5]. However, patients treated with CTLA4-Ig have a higher risk of acute cellular rejection [6, 7]. Like other checkpoint inhibitors, CTLA4-Ig has given variable results in the context of solid organ transplantation. For example, graft rejection rates depend on the area in which the patient lives, suggesting a probable effect of the microenvironment. The risk of rejection is four times higher in North America than in South America and twice as high in Europe, even for patients of the same ethnic origin. The identification of the factors impairing CTLA4-Ig efficacy is urgently required to improve the management of patients treated with this inhibitor. Our previous study in patients treated with belatacept suggested a relationship between the pretransplant T-cell phenotype, including, in particular, the presence of a large percentage of CD4+CD57+PD1- T cells, in the blood of patients and the risk of cellular rejection [8]whereas others have observed a relation with the presence of CD8+CD28- cells, or CD4+CD28+TEM and more recently a subset of 5 markers expressed on CD8 and NK cells [9, 10]. The expression of CD57 on circulating T cells could be modulated by the presence of latent viruses or by prior immunization and is currently associated with clonal exhaustion, mostly for CD8 T cells after chronic viral stimulation, but CD57 expression is not restricted to memory T cells and can also be observed on non-senescent LT [11]. We then characterized the CD4+CD57+PD1- population. In the mixed leukocyte reaction (MLR) in vitro, CD57+ cell proliferation was not inhibited by CTLA4-Ig, whereas CTLA4-Ig inhibited the proliferation of CD4+CD57- cells [8]. We also observed that the resistance ofCD4+CD57+PD1- LT to CTLA4-Ig was not abolished by inhibitors of other co-stimulation pathways (CD40, CD28, ICOS) or cytokines (IL-2, IL-15), suggesting that these molecules are not required for the expansion and differentiation of these CD4+CD57+PD1- LT. RNA sequencing RNA analysis of initial events occurring during the activation of this population in the presence of belatacept has shown that they require IL6, IFN alpha and mTOR pathways to proliferate and for their cytotoxic functions. Furthermore, a previous microarray analysis demonstrated the existence of an NK signature in this population. We also found that this LT population was heterogeneous, with a significant proportion of cells having an NKT phenotype (NKT), this proportion increased after in vitro stimulation in the presence or absence of CTLA4-Ig. NKT cells express the V24 TCR, which is present on intestine-resident cells and to be involved in the modulation of immune responses in intestinal inflammatory diseases. An interaction has been demonstrated between the commensal flora and the function and development of NKT cells. Animals reared in aseptic conditions have larger numbers of NKT cells in the intestinal mucosa, and the splenic NKT cells are less mature in microbiota-free animals [12, 13]. Thus, some of the bacteria of microbiota may have inhibitory effects on NKT cells in the intestine, whilst increasing the number of these cells in the periphery [14]. Moreover, the number of NKT cells is regulated by CD1d, a non-classical molecule of the major histocompatibility complex that is responsible for presenting lipids to LT, including the alpha-galactosylceramide produced by some bacterial strains [15, 16]. Several studies in animals have also highlighted a role of the microbiota in the allogeneic cellular response in transplantation. The absence of a microbiota (animals raised in aseptic conditions) or a particularly high abundance of certain intestinal bacteria (Clostridium species,etc.) favors the development of regulatory LT, whereas a preponderance of filamentous bacteria or Lactobacillus johnsonii induces an expansion of the TH17 lymphocyte population in rats [17, 18].Thus, the microbiota has been shown to affect immune responses in the treatment of cancers with CPI, and a strong link has been demonstrated between the composition of the commensal microbiota and the efficacy of these therapies. Several studies of transplantation in animals have demonstrated the influence of the microbiota on the risk of rejection.
The hypothesis of this work is to analyze the interaction between microbiota, the risk of kidney rejection, and T cell phenotype in renal transplant patients treated with Belatacept. Therefore the study is designed to analyze the microbiota or it blood signature (RNA16S or metabolomic) in patients who will receive a first renal transplantation for end-stage renal disease and treated with belatacept to identify whether some bacterial strains could be associated with a higher risk of acute rejection or at the opposite a better graft outcome. Preliminary results on a cohort of 60 kidney transplant patients treated with belatacept showed that circulating 16S DNA from gram-positive bacterial strains detected in the blood of patients was associated with a low risk of acute cellular rejection. We also found that some peptides synthesized by these bacteria, when added to intestinal cells before incubation with allogeneic T lymphocytes, stimulated, induced a strong reduction in T cell proliferation and activation. A transcriptomic analysis suggested that a non-canonical activation pathway regulates different chemokines involved in the modulation of the immune system. Altogether, our results strongly suggest that microbiota can modulate the risk of acute cellular rejection in kidney transplant recipients receiving belatacept based regimen.

- To identify bacterial signatures in the blood or in stools that can be associated with the risk of acute rejection in patients treated with Belatacept
- To identify the relation between the bacterial signature and the phenotype of T cells at the date of transplantation. More precisely the relation between CD4+CD57+Pd1- or with memory CD8+CD28low CD45R0+ T cells and different bacterial strains.
- To decipher the molecular mechanisms involved in the modulation of the mucosal and systemic immune systems
- To identify a particular digestive microbiota or a plasmatic bacterial signature (RNA or metabolomic) associated with a good outcome of renal transplantation and to develop Probiotic to reduce the risk of graft rejection.

1.Clinical study: We have developed a prospective trial with patients treated with belatacept as a cornerstone immunosuppressive treatment. Stools and blood samples have been collected prior to transplantation and during the first 6 months of transplantation. We will compare the bacterial strains in the stools (metagenomic) with their signatures (circulating 16S DNA, cytokines, metabolomic) and the risk of developing an acute rejection. Plasma samples will be analyzed by a targeted/untargeted metabolomics approach of combined direct flow injection and liquid chromatography MS/MS coupled to tandem mass spectrometry (Gustave Roussy Institute). In addition, Phenotypes of lymphocytes will be evaluated (proportion of CD4+CD57+PD1- T cells, CD4+CD28+TEM or CD8+CD28- T cells or NKT cells) and compared to the occurrence of acute rejection. We will also analyze the routine biopsy specimens collected from these patients and stored at Henri Mondor Hospital. These biopsy specimens will be used to check for the presence of NKT cells in the kidney at the time of rejection. The results will be subjected to a bioinformatics analysis performed in collaboration with the bioinformatics platform of CentraleSupelect.
2.We will test the in vitro effect of the different bacterial strains on epithelial cells (Caco2 or HT29) and the effect of these cells on APC maturation, T cells phenotype, and activation. Molecules produced by bacteria and epithelial cells will be characterized by biochemistry and mass spectroscopy and validated in in vitro models.
3.We will develop an animal model of renal graft on a mouse with a well-characterized microbiota (OMM12 in collaboration with Pasteur Institute). Animals will be gavaged with the different bacteria strains. The phenotype of circulating lymphocytes, as well as mucosal and spleen immune cells will be performed by flow cytometry and microscopy. In addition allogeneic skin graft will be performed with or without CTLA4-Ig (homologous to belatacept for animals) to determine the effect of gut microbiota on graft survival.