Thèse Impact de Cytokines Épithéliales sur les Lymphocytes t Non Conventionnels et sur le Rejet Cellulaire chez les Receveurs de Greffes d'Organes Solides H/F Doctorat.Gouv.Fr

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 : 2026-09-30T23:59:59 La transplantation rénale constitue le traitement optimal de l'insuffisance rénale terminale, mais son bénéfice à long terme reste limité par la toxicité des traitements immunosuppresseurs et le risque de rejet cellulaire aigu (ACR). Le belatacept, protéine de fusion CTLA4-Ig bloquant la costimulation des lymphocytes T via CD28, représente une alternative attractive aux inhibiteurs de la calcineurine et améliore la fonction du greffon à long terme. Cependant, il est associé à une incidence plus élevée et très variable d'ACR, suggérant que des facteurs environnementaux, notamment le microbiote intestinal, modulent la réponse immunitaire au belatacept.

Nos travaux ont identifié une population pré-transplantation de lymphocytes T CD4+CD57+PD1 associée au rejet résistant au belatacept. Ces cellules fortement cytotoxiques présentent des caractéristiques de lymphocytes T non conventionnels/NK et leur activation dépend notamment des interférons de type I et de l'IL-6. Parallèlement, l'analyse des produits bactériens circulants et des cytokines a mis en évidence une signature pré-transplantation associée à la protection contre l'ACR. Les patients ne développant pas de rejet sous belatacept présentent notamment des concentrations plus élevées de CYTOKINE-A et CYTOKINE-B, cette dernière étant principalement produite par les cellules épithéliales. Des résultats préliminaires chez la souris montrent par ailleurs que l'invalidation du récepteur de CYTOKINE-B modifie profondément le rejet de greffe. Ces données suggèrent que les interactions microbiote-épithélium-système immunitaire contrôlent les lymphocytes T non conventionnels et la susceptibilité au rejet sous blocage de la costimulation.

Le projet associe approches clinique, mécanistique et translationnelle autour de quatre axes. Premièrement, la cohorte prospective multicentrique MERRLIN, comprenant 70 patients issus de neuf centres français de transplantation, permettra de valider les biomarqueurs associés à l'ACR. Les marqueurs circulants de perméabilité intestinale, de translocation bactérienne et d'inflammation seront analysés conjointement aux cytokines et au profil OLINK.

Deuxièmement, afin de pallier la courte demi-vie de CYTOKINE-B, une molécule recombinante de fusion sera développée pour améliorer sa pharmacocinétique tout en conservant sa capacité à se fixer à son récepteur et à induire une signalisation spécifique. Troisièmement, ses effets sur l'immunité intestinale et systémique seront caractérisés par cytométrie multiparamétrique, analyses multiplex et histologie, notamment sur les populations lymphocytaires non conventionnelles et en association avec l'abatacept.

Enfin, l'efficacité de cette molécule sera évaluée dans des modèles murins de greffe cutanée avec incompatibilité mineure ou complètement allogénique, puis dans un modèle d'allogreffe rénale sous-capsulaire, seule ou associée à l'abatacept. L'objectif final est d'identifier des biomarqueurs prédictifs du rejet sous belatacept, de caractériser un nouvel axe microbiote-épithélium-cytokines-lymphocytes T non conventionnels et de développer une stratégie immunomodulatrice favorisant l'acceptation du greffon tout en limitant l'immunosuppression globale. Context: Chronic kidney diseases (CKD) are becoming increasingly frequent worldwide and represent a growing health and socioeconomic burden. It is now estimated to affect 10-13% of the global population (640 million people). CKD not only reduces patients' quality of life but also significantly impacts their long-term survival and is projected to become the 5th leading cause of death by 2040 1. Kidney transplantation remains the most effective treatment to restore renal function and improve life expectancy. However, it requires lifelong immunosuppressive therapy, which causes severe toxicity and increases the risk of infections and malignancies.
In recent years, the emergence of innovative biotherapies has considerably improved graft and patient survival, offering new perspectives for personalized and safer immunomodulation. However, these treatments are associated with a highly variable risk of acute rejection depending on the region and clinical context, suggesting some influence of the micro-environment. Understanding and addressing these disparities is therefore essential to optimize patient outcomes and ensure equitable access to effective therapies.
The scientific and medical community is showing increasing interest in deciphering interactions between the commensal microbiota and the immune system. The development of 'checkpoint inhibitors' (CPIs) has transformed the management of patients suffering from cancers, inflammatory diseases, or renal transplant patients by targeting immune cells more selectively. However, these immunotherapies have yielded heterogeneous results, suggesting a role for environmental factors or personal factors in the immune response variability to CPIs. Recent studies have shown that the composition of the intestinal microbiota influences the immune response associated with the inhibition of the PD-1/PDL-1 axis via CPI for the treatment of melanoma, renal and lung cancer 2,3.
The role of the microbiota on transplant outcome has been demonstrated in a mouse model of skin transplantation by preemptive treatment with broad-spectrum antibiotics 4. In humans, two monocentric studies have reported preliminary results concerning the microbiota composition of patients at and shortly after transplantation(Tx) 5, which are correlated with adverse effects in patients treated with mTOR inhibitors. However, so far, studies of the interaction between the microbiota and CPIs have mostly focused on CPIs inducing T-lymphocyte (LT) activation in cancer but not those used in transplantation.
We are currently studying the interaction between the gut microbiota and the outcome of treatment with belatacept, used to prevent graft rejection, in the context of renal transplantation.
Belatacept (a modified CTLA4-Ig) is a CPI that inhibits LT activation by disrupting the interaction between CD28 on LT and CD80/86 on presenting cells, thereby inhibiting the CD28 activation pathway. In the context of renal Tx, it improves both graft and patient survival 6. However, those patients have a higher risk of acute cellular rejection (ACR) 7. The frequency of ACR depends on the geographic location of the patient, suggesting that environmental factors are involved, notably interactions between the microbiota and immunosuppressive treatment 8.
We have recently identified the nature of the cells involved in ACR. We have shown, in patients treated with belatacept to prevent transplant rejection, a relationship between a high percentage of pre-transplant CD4+CD57+PD1- LT and ACR (6). We have already established that CD4+CD57+PD1-LTs are highly cytotoxic and synthesize IL-6 9,10. We subsequently showed that these cells are activated by a non-conventional pathway having an IFN signature and requiring type I interferon (IFN-I) signal and/or IL-6 to proliferate 10. Microarray analysis of CD4+CD57+PD1- LT identified an NK signature in this population of cells. Further analysis also showed that, during allogeneic stimulation of these cells, 2 important cell populations expanded and/or had a higher survival rate. Part of these cells had an iNKT phenotype expressing a V24 J18 TCR, and the others expressed NKGD2, a marker of unconventional LTs (not shown). This suggests the presence of type II NKT, expressing heterogeneous TCRs or other unconventional LTs 11 12,13.
Interestingly, we have also identified via a multiplex approach involving 16 different cytokines or growth factors, a specific blood profile before transplantation in patients who do not experience ACR when receiving belatacept maintenance therapy, compared to those on CNI-based therapy. Patients treated with belatacept who do not develop ACR show an increase in circulating CYTOKINE-A and CYTOKINE-B, produced by unconventional T cells and Intestinal epithelial cells, respectively. CYTOKINE-B plays a dual role in controlling infection and promoting mucosal healing. Additionally, CYTOKINE-B has been shown to promote the differentiation of Treg-inducing DCs, and administration of CYTOKINE-B also prevents gastrointestinal graft-versus-host disease in a mouse model. Moreover, recent preliminary results from our group showed that ACR is increased in a model of skin graft in mouse invalidated for the receptor of CYTOKINE-B (rCYTOKINE-B KO mice), suggesting an important role in the immunomodulation of Cytokine-B.

Overall, these results lead us to hypothesize that CYTOKINE-B could modulate intestinal and systemic immune cells toward a status of immunotolerance that reduces ACR.
The main objectives are to (i) confirm the link between biomarkers and the risk of ACR under belatacept treatment in patients, (ii and iii) understand the immunotolerance mechanisms based on epithelial cytokine release involved at the local and systemic level, (iv) and evaluate the protective effect on a mouse graft model. - WP1: Prospective analysis of blood markers in belatacept-treated patients.
Obj: To assess the link between biomarkers and ACR in belatacept treated patients: Our primary data showed a biological signature in the serum before transplantation that protected patients from ACR when treated with a belatacept-based regimen. To confirm these results, we have developed a multicenter prospective study (MERRLIN), which includes patients waiting for renal transplantation and those who have been transplanted and are receiving a de novo belatacept-based regimen. Blood and feces of the 70 patients have been collected at different time points. We will evaluate several blood markers for intestinal permeability (soluble CD14), translocation of bacterial-derived molecules (PGRP, LBP, LPS), and cytokines previously determined by multiplex assay. In addition, new biomarkers involved in inflammation will be determined by next-generation ELISA (OLINK). Their association with ACR will be determined. This longitudinal multicenter clinical study involves nine French Tx centers, and it was approved by the CPP Ouest (6 - CPP 1349 HPS3). All the samples have been collected.

- WP2: Development of a new recombinant protein including CYTOKINE-B to modulate the immune response.
Obj: To develop a fusion molecule including CYTOKINE-B to improve in vivo pharmacological efficiency. We have previously identified 2 cytokines associated with a lower risk of ACR in belatacept-treated patients which are involved in intestinal integrity and immunity. Because CYTOKINE-B has been suggested to be involved in immunoregulation, we initiated a preliminary study to understand its role in graft acceptance. CYTOKINE-B is mainly produced by epithelial cells and binds to its receptor (rCYTOKINE-B), which is expressed on immune cells. Therefore, we hypothesized that the administration of CYTOKINE-B could modulate the occurrence of allogeneic graft rejection. In a skin graft model, we observed that mice invalidated for rCYTOKINE-B have a significant delay of ACR in a minor mismatch model of skin graft, reinforcing the current hypothesis. However, CYTOKINE-B has a short half-life when injected in vivo and can not modulate the immune response. The objective of this WP is to develop a recombinant molecule to increase the half-life of CYTOKINE-B. Different constructs will be tested in vitro to demonstrate the capability of the fusion molecule to bind to its receptor and to induce specific intracellular signaling. The pharmacokinetics of these fusion molecules will be determined in vivo, as well as their route of administration.

- WP3: In vivo study of the Cytokine A and Cytokine B on intestinal and systemic immunity :
Obj: To assess, in vivo, the pharmacodynamic of CYTOKINE-B on intestinal and systemic immunity: The phenotype and functions of different immune populations of Colon/intestin, mesenteric lymph nodes, spleen, and skin graft by using multiparametric flow cytometry (24 different markers), multiplex ELISA, and histology in wild type animals or in rCYTOKINE-B KO animals or in those receiving the fusion molecule obtained in WP2. In addition, WT and rCYTOKINE-B KO animals will receive abatacept, the belatacept homologue active in mice. We will evaluate the role of the immunosuppressive treatment in maturation and differentiation of T cells in these strains.

- WP4: Role of Fusion molecule in the allogeneic response and graft rejection:
Obj: To assess the effect of CYTOKINE-B on allograft acceptance: To assess the effect of CYTOKINE-B of graft acceptance, we will develop two independent skin graft models: First by using a minor mismatch model (C57BL/6J transgenic mice expressing GFP as donor and WT C57BL/6J as recipient) and secondly by using fully allogeneic skin (BALB/c donor on WT C57BL/6J recipient) model. Animals will receive, or not, the fusion molecule before skin transplantation and during the follow-up. Control groups will receive albumin or isotype control antibodies at the same concentration as the fusion molecule. In addition, the animals will be treated or not with abatacept (the mouse homolog of belatacept) to assess the synergy of abatacept and the fusion molecule. We will determine the graft survival time and the phenotype of infiltrating cells.
To assess the fusion molecule in a solid organ model, we will also develop a mouse model of subcapsular kidney transplantation into C57BL/6-Ptprca mice (CD45.1)15. A section of fully allogeneic kidney from BALB/c mice will be subcapsularly implanted in the native kidney of C57BL/6J animals. This procedure is simpler and faster than kidney Tx in mice and requires less learning time. Circulating LT migration to the kidney will be monitored as previously described16 and the trafficking of transplanted section-resident LTs to draining lymph nodes will be followed by CD45.2 staining. Before Tx procedures, mice will be treated with CYTOKINE-B fusion molecule, as described above, with or without abatacept to evaluate the synergy of these molecules.