Team ALLEZ & LE BOURHIS

Intestinal Immunity in Inflammation and Cancer (3IC)

NKG2D is a C-type lectin expressed by cytotoxic immune cells such as NK cells, CD8 T lymphocytes, and γδ T lymphocytes. This activating receptor recognizes stress-induced ligands, such as MHC-like molecules, MICA, and MICB. Previous work from the laboratory showed that this receptor is expressed on a population of effector CD4 T lymphocytes, whose numbers are increased in the blood and mucosa of IBD patients and which may contribute to intestinal inflammation (Allez M. et al., Gastroenterology, 2007; Pariente B. et al., Gastroenterology, 2011). Building on this work, we demonstrated that this receptor is differentially expressed in inflamed mucosa, on both CD4 and CD8 T lymphocytes, in patients with Crohn’s disease. These observations led to the evaluation of a blocking antibody for the treatment of Crohn’s disease (Allez et al., Gut, 2017; Allez et al., JCC, 2023).

Our hypothesis is that T lymphocytes acquire specific effector functions during disease, thereby contributing to local inflammation. One of the mechanisms potentially involved, linked to the role of NKG2D, is a cytotoxic T lymphocyte response against intestinal epithelial cells (IECs), leading to increased intestinal barrier permeability. We observed a shift in the CD8 T lymphocyte populations resident in the intestinal mucosa, with a decrease in cells expressing the CD103 integrin in favor of cells expressing KLRG1 (Bottois et al., Front Immunol., 2020). Both molecules participate in the dialogue between T lymphocytes and the epithelium, their ligand being E-cadherin expressed by IECs. To better understand these lympho-epithelial interactions in a relevant system, we developed co-culture models of human intestinal organoids with mucosal lymphocytes. These organoids needed to be autologous in order to faithfully reproduce the functions of effector T lymphocytes that may be altered in IBD. Using this innovative and complex system, we show that ileal T lymphocytes from Crohn’s disease patients interact with autologous intestinal organoids from the same site and induce their cell death. This phenomenon is not observed with T lymphocytes and ileal epithelium from control tissues. Interestingly, these interactions and cytotoxic functions are blocked by antibodies specific for CD103 or NKG2D (Hammoudi et al., Front Immunol., 2022).

Crohn’s disease results from genetic and environmental predisposition and leads to destruction of the intestinal epithelium with ulcer formation, potentially at all levels of the digestive tract, but mainly at the terminal ileum. Significant damage to the terminal ileal mucosa requires surgical intervention during the course of the disease in more than two thirds of patients. However, surgery is not curative, as more than half of patients experience clinical recurrence within five years of the operation. Signs of endoscopic recurrence at the neo-terminal ileum are present in the majority of patients as early as six months after surgery. Understanding the pathophysiology of this recurrence and identifying factors capable of predicting it would have a significant impact on the management of Crohn’s disease patients. To study post-operative recurrence (POR) in Crohn’s disease, the REMIND group, which we co-founded in 2008, has established an international cohort of Crohn’s disease patients who underwent ileo-cecal resection. This cohort now includes more than 1,000 patients. Numerous clinical and biological parameters are collected, and an extensive biobank is shared among the various groups that have contributed to this cohort. This study has allowed us to contribute to important research on the role of the microbiota and certain bacterial species in the physiology of post-operative recurrence in Crohn’s disease (Sokol H. et al., Gut, 2020; Buisson A. et al., Gut, 2023; Chervy M. et al., Gut Microbes, 2022). Using this model of Crohn’s disease pathophysiology, we have studied clinical parameters and their association with the immune system in recurrence and long-term prognosis. We identified clinical parameters such as smoking and prior resection history as being associated with recurrence six months after surgery (Auzolle C. et al., APT, 2018). At the biological level, histological signs of inflammation at the proximal ileal resection margin of the surgical specimen and activation of the JAK/STAT pathways are associated with recurrence (Hammoudi H. et al., CGH, 2020; Ngollo M. et al., JCC, 2022).

Our main hypothesis posits that T cell clones can proliferate in the intestinal mucosa and acquire specific functions involved in disease. Previous work conducted in the laboratory identified the presence of oligoclonal expansions in mucosal lymphocytes from Crohn’s disease patients, including NKG2D-expressing CD4 T lymphocyte clones (Camus M. et al., Mucosal Immunol., 2014). To better characterize these clonal expansions, we study the TCR repertoire by next-generation sequencing and have identified persistent sequences in the same patient associated with disease (Allez M. et al., Gut, 2019), as well as shared sequences across patient subgroups (Pesesky M. et al., JCC, 2025).

We previously demonstrated the involvement of the NKG2D pathway in colon tumors (Chirica et al., Oncoimmunol., 2015), which led to our collaboration with Innate Pharma to study and test in our laboratory the potential modulation of this pathway using antibodies specific for the MICA and MICB ligands. We therefore established a cohort of patients who underwent surgery for colorectal cancer (CRC) at Saint-Louis Hospital, following the same model as the REMIND-POP cohort: a large clinical and biological database associated with an extensive biobank. This cohort includes 400 patients for whom we have samples and clinical data that we exploit across various projects and collaborations.

This cohort was used for phenotypic analysis of tumor-infiltrating T lymphocytes, compared to those from normal mucosa collected at the resection margins. In the IMCO cohort, we observed decreased NKG2D expression on NK cells and CD8+ T lymphocytes within the tumor, while NKG2A expression was increased (Courau T. et al., JITC, 2019). We transposed our expertise in primary epithelial and immune cell culture to colon cancers. We first established allogeneic co-cultures between cell line-derived spheroids and immune cells from donors or patients, in order to create the conditions necessary to progress toward an autologous system allowing study of the intratumoral response ex vivo. We used this model to study the involvement of NKG2D and NKG2A in the immune response to CRC. Indeed, using an antibody targeting these ligands in a spheroid-lymphocyte co-culture model, we demonstrated that NK and T cells exhibited enhanced cytotoxic responses against colon cancer cell lines. Similarly, modulation of NKG2A, an inhibitory receptor of the same family, improved antitumor responses (Courau T. et al., JITC, 2019).

In the IMCO cohort, other potential markers such as PD1 and CTLA-4 were overexpressed in tumors compared to normal mucosa, and additional potential modulators of the immune response were identified. The ectonucleotidase CD39 is more expressed on the surface of T lymphocytes within the tumor than in normal mucosa. These enzymes modulate the immune system by converting immunostimulatory extracellular ATP into adenosine, an inhibitor. By blocking this molecule in a co-culture model between a primary tumor-derived spheroid and autologous tumor-infiltrating T cells, we reveal the potential for enhancing the anticancer activity of this pathway (Bonnereau J. et al., Gut, 2023).

Surgical treatment of CRC is highly effective for early-stage disease. Immunotherapies would therefore be all the more important for metastatic forms. The I-CoMET cohort (Immunology of Metastatic Colorectal Cancers) was therefore created to provide access to samples from primary tumors as well as peritoneal and hepatic metastases. Launched in early 2025, the cohort currently includes approximately fifty patients.