T cell clonal expansions are present in the inflamed mucosa of patients with Crohn’s disease (CD) and may be implicated in postoperative recurrence after ileocolonic resection. Methods T cell receptor (TCR) analysis was performed in 57 patients included in a prospective multicentre cohort. Endoscopic recurrence was defined by a Rutgeerts score >i0. DNA and mRNA were extracted from biopsies collected from the surgical specimen and endoscopy, and analysed by high throughput sequencing and microarray, respectively. Results TCR repertoire in the mucosa of patients with CD displayed diverse clonal expansions. Active smokers at time of surgery had a significantly increased proportion of clonal expansions as compared with non-smokers (25.9%vs17.9%, p=0.02). The percentage of high frequency clones in the surgical specimen was significantly higher in patients with recurrence and correlated with postoperative endoscopic recurrence (area under the curve (AUC) 0.69, 95% CI 0.54 to 0.83). All patients with clonality above 26.8% (18/57) had an endoscopic recurrence. These patients with a high clonality were more frequently smokers than patients with a low clonality (61% vs 23%, p=0.005). The persistence of a similar TCR repertoire at postoperative endoscopy was associated with smoking and disease recurrence. Patients with high clonality showed increased expression of genes associated with CD8 T cells and reduced expression of inflammation-related genes. Expanded clones were found predominantly in the CD8 T cell compartment. Conclusion Clonal T cell expansions are implicated in postoperative endoscopic recurrence. CD patients with increased proportion of clonal T cell expansions in the ileal mucosa represent a subgroup associated with smoking and where pathogenesis appears as T cell driven. Trial registration number NCT03458195.
Intestinal tissue-resident memory CD8 T cells (Trm) are non-recirculating effector cells ideally positioned to detect and react to microbial infections in the gut mucosa. There is an emerging understanding of Trm cell differentiation and functions, but their implication in inflammatory bowel diseases, such as Crohn's disease (CD), is still unknown. Here, we describe CD8 cells in the human intestine expressing KLRG1 or CD103, two receptors of E-cadherin. While CD103 CD8 T cells are present in high numbers in the mucosa of CD patients and controls, KLRG1 CD8 T cells are increased in inflammatory conditions. Mucosal CD103 CD8 T cells are more responsive to TCR restimulation, but KLRG1 CD8 T cells show increased cytotoxic and proliferative potential. CD103 CD8 T cells originate mostly from KLRG1 negative cells after TCR triggering and TGF stimulation. Interestingly, mucosal CD103 CD8 T cells from CD patients display major changes in their transcriptomic landscape compared to controls. They express Th17 related genes including CCL20, IL22 and IL26, which could contribute to the pathogenesis of CD. Overall, these findings suggest that CD103 CD8 T cells in CD induce a tissue-wide alert increasing innate immune responses and recruitment of effector cells such as KLRG1 CD8 T cells.
In Brief Background The incidence of cancer is increased after solid organ transplantation. Natural killer (NK) cells are key effectors of the tumor immune response. Methods We conducted a cross sectional multicentre matched case–control study including 42 kidney transplant recipients (KTRs) on diagnosis of cancer and 41 KTRs without cancer. Extensive phenotyping of NK cells populations and functional tests of NK cells were performed. Results Kidney transplant recipients with cancer had a higher incidence of acute rejection (P = 0.02) and cytomegalovirus (CMV) infection (P = 0.03) than controls. They had more lymphopenia than control KTRs (1020/mm3 ± 32 vs 1218/mm3 ± 34; P = 0.001) including a CD4+ lymphopenia (P = 0.01). Total CD3−/CD56+ NK cell counts were similar in both groups. However, KTRs with cancer had a lower frequency of the cytokine-enriched CD56bright NK cell subset (P = 0.001). The percentage of NK cells expressing NKp46 was decreased in KTRs with cancer (45% vs 53 %, P = 0.001). Furthermore, the ability of NK cells to degranulate CD107a+ cytolytic vesicles was reduced (11% vs 22%; P = 0.02), and the percentage of NK cells secreting IFNγ was decreased (7.5% vs 28.8%; P = 0.01) in KTRs with cancer. Conclusions These results reveal an imbalance between NK cell subpopulations and functional NK cell defects in KTRs at the diagnosis of malignancy, including a decreased expression of NKp46 and decreased numbers of NK cells producing INFγ. This study highlights the role of NKp46, a major activating NK cell receptor, which could be considered as a potential marker during immunological follow-up of KTRs. This exploratory cross sectional study shows that kidney transplant recipients (KTR) with malignancy have an imbalance between NK cell subpopulations and NK cell functional defects, despite similar NK cell counts, compared to matched KTR without malignancy.
Abstract T lymphocytes play a major role in intestinal homeostasis, with a particular impact on the balance between self-renewal and differentiation of intestinal epithelial cells (IECs). In Crohn’s disease (CD) patients, the intestinal mucosa is inflamed, epithelium permeability is increased, and IECs present compositional and functional defects. The role of T lymphocytes interactions with IECs in the physiopathology of CD remains in question. Here, we use a three-dimensional human autologous coculture model between purified intestinal organoids derived from primary tissues of CD and non-inflammatory control patients, and mucosal T lymphocytes extracted from the same location. We show that while in homeostatic context T cells support the proliferation and differentiation balance of organoids, mucosal T lymphocytes from CD patients present a high cytotoxicity against IECs. Importantly, this cytotoxicity is a persistent defect overtime in culture. Organoids also show defective intestinal stem cells (ISCs) proliferation and morphological changes. Single cell RNA sequencing after coculture highlights a general response of T cells to the epithelial microenvironment, and more particularly, an increase activation of a pro-inflammatory CD8+ T cells effector population in CD patients compared to controls. Graphical abstract
Abstract Background T resident memory (Trm) cells in the intestinal mucosa and in particular subpopulations expressing phenotypic markers such as alphaE-beta7 or NKG2D have been associated with chronic inflammatory bowel disease (IBD) activity. We hypothesize that these populations may have a direct deleterious impact on the intestinal epithelium in IBD. Methods The phenotypic study of mucosal lymphocytes was performed in two prospective cohorts: ELYP including patients with active IBD before initiation of biotherapy and REMIND including patients with ileal Crohn’s disease (CD) requiring ileocaecal resection. These analyses were performed before initiation of treatment and one year after continuous therapy in the ELYP cohort and on the resection specimen in the REMIND cohort. An innovative ex vivo autologous organoid-mucosal T cell coculture model was developed using the REMIND cohort specimens for patients and healthy ileum from individuals without IBD for controls (Figure 1). T cell infiltration within the organoid and epithelial cell death were assessed by confocal microscopy. A panel of 30 cytokines was quantified in the supernatants of cocultures. Figure 1 Results In the ELYP cohort, before the start of treatment, IBD patients had lower expression of alphaE-beta7 and NKG2D on mucosal CD8 T cells. While alphaE-beta7 and NKG2D expression on mucosal CD8 T cells had returned to normal in endoscopic responders, it remained decreased in non-responders. Similarly, the inflammatory mucosa of the REMIND surgical specimens had lower levels of CD4 and CD8 T cells expressing alphaE-beta7 and NKG2D compared with controls and non-inflamed regions. We developed a coculture model between mucosal lymphocytes and organoids generated under autologous conditions. We showed an increase in apoptotic cell death in epithelial cells from CD patients which was not found in control cocultures. There was a significant correlation between the degree of epithelial cell death and T cell infiltration (Figure 2). Various proinflammatory cytokines such as IFNgamma, TNFalpha, IL-6 and IL- 17a were also increased in the supernatants. The use of antibodies blocking the alphaE-beta7 and NKG2D pathways of interest inhibited this effect by different mechanisms (Figure 3) While anti-beta7 and anti-NKG2D had comparable effects in terms of cell death inhibition, only anti-beta7 reduced T cell infiltration. Anti-NKG2D had no effect on cell infiltration but a reduction of perforin was observed in the supernatants. Figure 2 Figure 3 CC= Coculture Conclusion These data demonstrate for the first time the direct cytotoxic deleterious effect of mucosal T cells on the epithelium of CD patients using a novel coculture model. AlphaE-beta7 and NKG2D pathways appear to be relevant in this process.
Abstract Background A dysregulated T-cell response is involved in the pathophysiology of inflammatory bowel diseases (IBD). T cells expressing the NKG2D activation receptor are implicated. Here, we investigated changes in intestinal T lymphocyte subsets overtime in patients treated by biotherapy for active disease. Methods In this prospective single-centre study, patients with IBD who have started a biotherapy (Infliximab, Adalimumab, Golimumab, Vedolizumab, Ustekinumab) for active disease were included. Mucosal biopsies in the inflamed segment were taken during endoscopies before treatment initiation (W0) and in the same location 14 weeks (W14) and 52 weeks (W52) after inclusion. Control biopsies were taken from 16 patients without IBD (7 ileal and 9 colonic). Immunophenotyping of isolated lymphocytes was performed by flow cytometry at each timepoint. Correlation with endoscopic response was evaluated. For Crohn’s disease (CD), the response was defined by a 50% decrease of the CDEIS score. For ulcerative colitis (UC), the response was defined by a 2 points decrease of the UCEIS Results Between February 2016 and November 2018, 85 patients were included. Forty-four were male (52%). Forty had an active CD with the most inflamed segment located for 28 in the colon and 12 in the ileum. Thirty-nine patients had active UC and 6 pouchitis. An anti-TNF was started for 51 patients (60%), Vedolizumab for 16 patients (19%) and Ustekinumab for 18 (21%). Lymphocytes phenotyping was performed at inclusion for the whole cohort, at W14 for 67 patients (79%) and at W52 for 49 patients (58%). Data concerning endoscopic response was available for all patients. At W0, IBD patients displayed higher rates of CD4 T cells and lower rates of CD8 T cells than controls (medians: 31% vs. 48% for CD4, p < 0.001 and 52% vs. 37% for CD8, p < 0.001 respectively). Endoscopic responders experienced a normalisation of these mucosal populations as compared with non-responders (W14: 58% vs. 45% for CD4, p = 0.005 and 40% vs. 28% for CD8, p = 0.03; W52 56% vs. 43% for CD4, p = 0.01 and 27 vs. 43% for CD8, p = 0.003). Results remained significant when splitting the cohort in CD and UC patients. Before treatment initiation, IBD patients displayed lower expression of NKG2D on CD8 T cells (78% vs. 94%, p < 0.001). At W52, endoscopic responders presented significantly higher expression of this marker on CD8 T cells compared with non-responders (85% vs. 68%, p < 0.001). Results remained significant in UC and CD patients. Conclusion The intestinal T cells of IBD patients with active disease present an increase of the CD4 to CD8 T-cell ratio and lower expression of NKG2D on CD8 T cells reflecting engagement of this receptor in inflammation. These T-cell parameters are normalised in endoscopic responders but not in non-responders
Objective T cells are major effectors of the antitumoural immune response. Their activation by tumour-associated antigens can unleash their proliferation and cytotoxic functions, leading to tumour cell elimination. However, tumour-related immunosuppressive mechanisms including the overexpression of immune checkpoints like programmed cell death protein-1 (PD-1), are also engaged, promoting immune escape. Current immunotherapies targeting these pathways have demonstrated weak efficacy in colorectal cancer (CRC). It is thus crucial to find new targets for immunotherapy in this cancer type. Design In a prospective cohort of patients with CRC, we investigated the phenotype of tumour-related and non-tumour related intestinal T cells (n=44), particularly the adenosinergic pathway, correlating with clinical phenotype. An autologous coculture model was developed between patient-derived primary tumour spheroids and their autologous tumour-associated lymphocytes. We used this relevant model to assess the effects of CD39 blockade on the antitumour T cell response. Results We show the increased expression of CD39, and its co-expression with PD-1, on tumour infiltrating T cells compared with mucosal lymphocytes. CD39 expression was higher in the right colon and early-stage tumours, thus defining a subset of patients potentially responsive to CD39 blockade. Finally, we demonstrate in autologous conditions that CD39 blockade triggers T cell infiltration and tumour spheroid destruction in cocultures. Conclusion In CRC, CD39 is strongly expressed on tumour infiltrating lymphocytes and its inhibition represents a promising therapeutic strategy for treating patients.
Patients with Type 2 diabetes (T2D) are highly susceptible to infection and have an increased incidence of some tumors, possibly due to immune system dysfunction. In the innate cellular immune system, Natural Killer (NK) lymphocytes are important effectors responsible for controlling infections and combating tumor development. We analyzed NK cell subsets in 51 patients with long-standing T2D. Compared with healthy blood donors, diabetic patients showed a profound decrease in both NKG2D-positive NK cells (44% vs. 55.5%, P<0.01) and NKp46-positive cells (26% vs. 50%, P<0.01). Decreased expression of these receptors was associated with functional defects, such as reduced NK degranulation capacity when challenged with the tumor target cell line K562 (10.3 vs. 15.8%, P<0.05). This defect could be restored in vitro by stimulating NK cells from T2D patients with IL-15 (P<0.05). NKG2D expression was found to be negatively correlated with HBA1c level (r = −0.50; P = 0.009), suggesting that sustained hyperglycemia could directly influence NK cell defects. We demonstrated that endoplasmic reticulum (ER) stress, an important mediator in diabetes-associated complications, was inducible in vitro in normal NK cells and that tunicamycin treatment resulted in a significant decrease in NKG2D expression (P<0.05). Furthermore, markers of the Unfolded Protein Response (UPR) BiP, PDI and sXBP1 mRNAs were significantly increased in NK cells from T2D patients (P<0.05, P<0.01, P<0.05, respectively), indicating that ER stress is activated in vivo through both PERK and IRE1 sensors. These results demonstate for the first time defects in NK cell-activating receptors NKG2D and NKp46 in T2D patients, and implicate the UPR pathway as a potential mechanism. These defects may contribute to susceptibility to infections and malignancies and could be targetted therapeutically.
