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Intestinal intraepithelial T lymphocytes (IEL) constitutively express high amounts of the cytotoxic proteases Granzymes (Gzm) A and B and are therefore thought to protect the intestinal epithelium against infection by killing infected epithelial cells. However, the role of IEL granzymes in a protective immune response has yet to be demonstrated. We show that GzmA and GzmB are required to protect mice against oral, but not intravenous, infection with Salmonella enterica serovar Typhimurium, consistent with an intestine-specific role. IEL-intrinsic granzymes mediate the protective effects by controlling intracellular bacterial growth and aiding in cell-intrinsic pyroptotic cell death of epithelial cells. Surprisingly, we found that both granzymes play non-redundant roles. GzmB-/- mice carried significantly lower burdens of Salmonella, as predominant GzmA-mediated cell death effectively reduced bacterial translocation across the intestinal barrier. Conversely, in GzmA-/- mice, GzmB-driven apoptosis favored luminal Salmonella growth by providing nutrients, while still reducing translocation across the epithelial barrier. Together, the concerted actions of both GzmA and GzmB balance cell death mechanisms at the intestinal epithelium to provide optimal control that Salmonella cannot subvert.

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BACKGROUND: Intraepithelial lymphocytes are the first line of defence of the human intestinal immune system. Besides, their composition is altered on patients with coeliac disease (CD), so they are considered as biomarkers with utility on their diagnose and/or monitoring. Our aim is to address their variability through the human gastrointestinal tract in health and characterized them in further depth in the coeliac duodenum. METHODS: Intraepithelial lymphocytes were isolated from human gastric, duodenal, ileal and colonic biopsies, then stained with specific antibodies and acquired by flow cytometry. RESULTS: Our results confirmed that the profile of Intraepithelial lymphocytes change through the length of the human gastrointestinal tract. Besides and given the central role that Interleukin-15 (IL-15) elicits on CD pathogenesis; we also assessed the expression of its receptor revealing that there was virtually no functional IL-15 receptor on duodenal Intraepithelial lymphocytes. Nevertheless and contrary to our expectations, the active IL-15 receptor was not increased either on Intraepithelial lymphocytes from CD patients. CONCLUSIONS: IL-15 might require additional stimulus to activate intraepithelial lymphocytes. These findings may provide novel tools to aid on a CD diagnosis and/or monitoring, at the time that provide the bases to perform functional studies in order of getting a deeper insight in the specific function that Intraepithelial lymphocytes elicit on CD pathogenesis.

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The intestine represents the most complex cellular network in the whole body. It is constantly faced with multiple types of immunostimulatory agents encompassing from food antigen, gut microbiome, metabolic waste products, and dead cell debris. Within the intestine, most T cells are found in three primary compartments: the organized gut-associated lymphoid tissue, the lamina propria, and the epithelium. The well-orchestrated epithelial-immune-microbial interaction is critically important for the precise immune response. The main role of intestinal mesenchymal stromal cells is to support a structural framework within the gut wall. However, recent evidence from stromal cell studies indicates that they also possess significant immunomodulatory functions, such as maintaining intestinal tolerance via the expression of PDL1/2 and MHC-II molecules, and promoting the development of CD103+ dendritic cells, and IgA+ plasma cells, thereby enhancing intestinal homeostasis. In this review, we will summarize the current understanding of CD8+ T cells and stromal cells alongside the intestinal tract and discuss the reciprocal interactions between T subsets and mesenchymal stromal cell populations. We will focus on how the tissue residency, migration, and function of CD8+ T cells could be potentially regulated by mesenchymal stromal cell populations and explore the molecular mediators, such as TGF-ß, IL-33, and MHC-II molecules that might influence these processes. Finally, we discuss the potential pathophysiological impact of such interaction in intestine hemostasis as well as diseases of inflammation, infection, and malignancies.

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BACKGROUND: Oral lichen planus (OLP) is a common T cell-mediated oral mucosal immune inflammatory disease. Intraepithelial lymphocytes (IELs) are a unique subset of T cells that play an important role in regulating immune response. This study aims to investigate the phenotype and the differentiation mechanism of IELs in OLP. METHODS: The expression of CD4, CD8α, CD8ß, T-helper-inducing POZ/Krueppel-like factor (ThPOK), and RUNX family transcription factor 3 (Runx3) in the epithelium and peripheral blood mononuclear cells (PBMCs) of OLP was determined by immunofluorescence and immunohistochemistry. Then, the correlations among them were analyzed. Naïve CD4+ T cells were sorted from blood of OLP patients and stimulated with retinoic acid (RA) and transforming growth factor-ß1 (TGF-ß1). Then the expression of CD4, CD8α, CD8ß, ThPOK, and Runx3 was investigated by immunocytochemistry. RESULTS: CD8α expression and CD8αα+ cells were upregulated in the epithelium of OLP, whereas they were downregulated in PBMCs of OLP. CD8ß was not expressed in the epithelium of OLP. CD4, CD8α, and Runx3 expression and CD4+CD8α+ cells were increased, whereas ThPOK expression was decreased in the epithelium of OLP. CD8α expression was positively correlated with Runx3 expression, whereas ThPOK expression was negatively correlated with Runx3 expression. After RA and TGF-ß1 stimulation, CD8α and Runx3 expression was upregulated, and ThPOK expression was downregulated in naïve CD4+ T cells. CONCLUSION: CD4+CD8αα+ IELs may be the dominant phenotype of IELs in OLP, and the differentiation of CD4+CD8αα+ IELs in OLP is negatively regulated by ThPOK and positively regulated by Runx3.

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BACKGROUND: The diagnosis of coeliac disease (CD) in adults is based on clinical, serological and histological criteria. The inappropriate performance of intestinal biopsies, non-specificity of mild histological lesions and initiation of a gluten-free diet (GFD) before biopsy may hamper the diagnosis. In these situations, determining the intraepithelial lymphogram of the duodenum by flow cytometry (IEL-FC) can be helpful. OBJECTIVES: To describe the clinical scenarios in which the IEL-FC is used and its impact on the diagnosis of CD. METHODS: All adult patients with suspected CD at three tertiary centres for whom the duodenal histology and IEL-FC were available were identified. Catassi and Fasano's diagnostic criteria and changes to a CD diagnosis after the IEL-FCs were collected. RESULTS: A total of 348 patients were included. The following indications for an IEL-FC formed part of the initial study for CD (38%): negative conventional work-up (32%), already on a GFD before duodenal biopsies (29%) and refractoriness to a GFD (2%). The IEL-FC facilitated a definitive diagnosis in 93% of patients with an uncertain diagnosis who had had a conventional work-up for CD or who were on a GFD before histology. CONCLUSIONS: The IEL-FC facilitates the confirmation or rejection of a diagnosis of CD in clinical scenarios in which a conventional work-up may be insufficient.

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Background: The classical medicinal formula Huangqi Gancao Decoction (HQGCD), originating from the medical book" Yi Lin Gai Cuo". Up to now, the studies focusing on the immunoenhancement effects of HQGCD are few, and the actionpathway is not yet clear. Method: In this study, SPF male KM mice were utilized as a model for immunosuppression. Comprehensive observations were made regarding the general behavior and condition of the mice, in addition to monitoring fluctuations in body weight and food intake. The blood routine index was measured, and morphological changes in the ileum and colon tissues were examined. The level of secretory immunoglobulin A (sIgA), superoxide dismutase (SOD), and malondialdehyde (MDA) in ileum and colon tissues were quantified. Additionally, the bone marrow total DNA index was assessed. Flow cytometry analyzed the proportions of CD3⁺, CD4⁺, CD8⁺, and CD4+CD8+ double-positive (DP) T lymphocytes in small intestinal intraepithelial lymphocytes (IELs). Lastly, the composition and diversity of the cecal microbiota were evaluated using 16S rDNA sequencing technology. Results: After HQGCD intervention, there were no significant changes in the mice's feed intake and body weight. However, the tissue structures of the ileum and colon showed recovery. In the blood routine index, there was an increase in the total white blood cell count, lymphocyte count, red blood cell count, hematocrit, and hemoglobin content. Additionally, the bone marrow total DNA index was elevated. Level of SOD and sIgA in ileum and colon tissues increased, while the level of MDA decreased. The proportions of CD3⁺ and CD4⁺ T lymphocytes within IELs increased, along with an increase in DP T lymphocytes in IELs (DP IELs), whereas the proportion of CD8⁺ T lymphocytes decreased. The cecal microbiota underwent changes, with an increase in the variety and number of beneficial microbiota. Conclusion: HQGCD could restore the intestinal immune function of immunocompromised mice, and had a certain positive effect on cecal microbiota.

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BACKGROUND: Celiac disease is a gluten-related pathology, highly prevalent and heterogeneous in its clinical presentation, which leads to delays in diagnosis and misdiagnosis. The analysis of duodenal intraepithelial lymphocytes (IELs) by flow cytometry (lymphogram) is emerging as a discriminative tool in the diagnosis of various forms of celiac disease (CD). AIMS: The aim of this study was to validate IEL lymphogram performance in the largest adult series to our knowledge, in support of its use as a diagnostic tool and as a biomarker of the dynamic celiac process. METHODS: This was a retrospective study including 768 adult patients (217 with active CD, 195 on a gluten-free diet, 15 potential CD patients, and 411 non-celiac controls). The IEL subset cut-off values were established to calculate the diagnostic accuracy of the lymphogram. RESULTS: A complete celiac lymphogram profile (≥14% increase in T cell receptor [TCR]γδ IELs and simultaneous ≤4% decrease in surface-negative CD3 [sCD3-] IELs) was strongly associated with active and potential forms in over 80% of the confirmed patients with CD, whereas the remaining patients with CD had partial lymphogram profiles (≥14% increase in TCRγδ or ≤4% decrease in sCD3- IELs), with lower diagnostic certainty. None of these patients had a non-celiac lymphogram. Quantifying the TCRγδ versus sCD3- imbalance as a ratio (≥5) is a discriminative index to discard or suspect CD at diagnosis. CONCLUSIONS: We have validated the IEL lymphogram's diagnostic efficiency (79% sensitivity, 98% specificity), with an LR+ accuracy of 36.2. As expected, the increase in TCRγδ IELs is a reliable marker for celiac enteropathy, while changes in sCD3- IEL levels throughout the dynamic CD process are useful biomarkers of mucosal lesions.

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Nonresponsive celiac disease (CeD) is relatively common. It is generally attributed to persistent gluten exposure and resolves after correction of diet errors. However, other complications of CeD and disorders clinically mimicking CeD need to be excluded. Novel therapies are being evaluated to facilitate mucosal recovery, which might benefit patients with nonresponsive CeD. Refractory CeD (RCeD) is rare and is divided into 2 types. The etiology of type I RCeD is unclear. A switch to gluten-independent autoimmunity is suspected in some patients. In contrast, type II RCeD represents a low-grade intraepithelial lymphoma. Type I RCeD remains a diagnosis of exclusion, requiring ruling out gluten intake and other nonmalignant causes of villous atrophy. Diagnosis of type II RCeD relies on the demonstration of a clonal population of neoplastic intraepithelial lymphocytes with an atypical immunophenotype. Type I RCeD and type II RCeD generally respond to open-capsule budesonide, but the latter has a dismal prognosis due to severe malnutrition and frequent progression to enteropathy-associated T-cell lymphoma; more efficient therapy is needed.

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Glucagon-like peptide 1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP) are peptide hormones produced by enteroendocrine cells in the small intestine. Despite being produced in the gut, the leveraging of their role in potentiating glucose-stimulated insulin secretion, also known as the incretin effect, has distracted from discernment of direct intestinal signaling circuits. Both preclinical and clinical evidence have highlighted a role for the incretins in inflammation. In this review, we highlight the discoveries of GLP-1 receptor (GLP-1R)+ natural (TCRαß and TCRγδ) and induced (TCRαß+CD4+ cells and TCRαß+CD8αß+) intraepithelial lymphocytes. Both endogenous signaling and pharmacological activation of GLP-1R impact local and systemic inflammation, the gut microbiota, whole-body metabolism, as well as the control of GLP-1 bioavailability. While GIPR signaling has been documented to impact hematopoiesis, the impact of these bone marrow-derived cells in gut immunology is not well understood. We uncover gaps in the literature of the evaluation of the impact of sex in these GLP-1R and GIP receptor (GIPR) signaling circuits and provide speculations of the maintenance roles these hormones play within the gut in the fasting-refeeding cycles. GLP-1R agonists and GLP-1R/GIPR agonists are widely used as treatments for diabetes and weight loss, respectively; however, their impact on gut homeostasis has not been fully explored. Advancing our understanding of the roles of GLP-1R and GIPR signaling within the gut at homeostasis as well as metabolic and inflammatory diseases may provide targets to improve disease management.

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BACKGROUND: Dysregulated T lymphocyte response is thought to play a key role in chronic intestinal inflammation (CIE). OBJECTIVES: To evaluate the presence of changes in peripheral and intestinal T lymphocyte subsets and to describe potential immune and inflammatory biomarkers in dogs with CIE. ANIMALS: Sixteen healthy dogs and 26 dogs were diagnosed with CIE. METHODS: Prospective case-control study evaluating peripheral and intestinal T lymphocytes using flow cytometry and inflammatory markers obtained from complete blood cell counts. RESULTS: Dogs with CIE had higher peripheral activated T helper (Th) lymphocytes (87/µL [18-273] CIE, 44/µL [16-162] healthy control (HC, P = .013) and regulatory T cells (Treg; 108/µL [2-257] CIE, 34/µL [1-114] HC, P = .004). In the intestinal epithelium, CIE dogs presented lower percentages of Th (4.55% [1.75-18.67] CIE, 8.77% [3.79-25.03] HC, P = .002), activated Th cells (0.16% [0.02-0.83] CIE, 0.33% [0.05-0.57] HC, P = .03) and CD4/CD8 ratio (0.08 [0.02-0.39] CIE, 0.21 [0.07-0.85] HC, P = .003). Conversely, higher percentage of activated T cytotoxic cells (20.24% [3.12-77.12] CIE, 12.32% [1.21-39.22] HC, P = .04) and interferon-gamma (IFN-γ) producing T lymphocytes (7.36% [0.63-55.83] CIE, 1.44% [0.00-10.56] HC, P = .01) within the epithelium was observed. In the lamina propria the percentage of Treg lymphocytes was higher (6.02% [1.00-21.48] CIE, 3.52% [0.18-10.52] HC, P = .02). CONCLUSIONS AND CLINICAL IMPORTANCE: Systemic and intestinal immune alterations occur in dogs with CIE suggesting that blood IFN-γ producing T lymphocytes and the systemic immune-inflamation index (SII) could potentially serve as biomarkers for the disease.

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Resident epidermal T cells of murine skin, called dendritic epidermal T cells (DETCs), express an invariant γδ TCR that recognizes an unidentified self-ligand expressed on epidermal keratinocytes. Although their fetal thymic precursors are preprogrammed to produce IFN-γ, DETCs in the adult epidermis rapidly produce IL-13 but not IFN-γ early after activation. Here, we show that preprogrammed IFN-γ-producing DETC precursors differentiate into rapid IL-13 producers in the perinatal epidermis. The addition of various inhibitors of signaling pathways downstream of TCR to the in vitro differentiation model of neonatal DETCs revealed that TCR signaling through the p38 MAPK pathway is essential for the functional differentiation of neonatal DETCs. Constitutive TCR signaling at steady state was also shown to be needed for the maintenance of the rapid IL-13-producing capacity of adult DETCs because in vivo treatment with the p38 MAPK inhibitor decreased adult DETCs with the rapid IL-13-producing capacity. Adult DETCs under steady-state conditions had lower glycolytic capacity than proliferating neonatal DETCs. TCR stimulation of adult DETCs induced high glycolytic capacity and IFN-γ production during the late phase of activation. Inhibition of glycolysis decreased IFN-γ but not IL-13 production by adult DETCs during the late phase of activation. These results demonstrate that TCR signaling promotes the differentiation of IL-13-producing DETCs in the perinatal epidermis and is needed for maintaining the rapid IL-13-producing capacity of adult DETCs. The low glycolytic capacity of adult DETCs at steady state also regulates the rapid IL-13 response and delayed IFN-γ production after activation.

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Intraepithelial lymphocytes (IEL) reside in the epithelium at the interface between the contents of the intestinal lumen and the sterile environment of the lamina propria. Because of this strategic location, IEL play a crucial role in various immunological processes, ranging from pathogen control to tissue stability. In mice and humans, IEL exhibit high diversity, categorized into induced IEL (conventional CD4 and CD8αß T cells) and natural IEL (TCRαßCD8αα, TCRγδ, and TCRneg IEL). In chickens, however, the subpopulations of IEL and their functions in enteric diseases remain unclear. Thus, we conducted this study to investigate the role of IEL populations during necrotic enteritis (NE) in chickens. At 14 days of age, sixty-three Specific-pathogen-free (SPF) birds were randomly assigned to three treatments: Control (sham challenge), Eimeria maxima challenge (EM), and Eimeria maxima + Clostridium Perfringens (C. Perfringens) co-challenge (EM/CP). The EM and EM/CP birds were infected with Eimeria maxima at day 14 of age, and EM/CP birds were additionally orally inoculated with C. perfringens at days 18 and 19 of age. Birds were weighed at days 18, 20, and 26 of age to assess body weight gain (BWG). At 20 days of age (1 day-post C. perfringens infection; dpi), and 26 days of age (7 dpi), 7 birds per treatment were euthanized, and jejunum was harvested for gross lesion scores, IEL isolation, and gene expression. The EM/CP birds exhibited subclinical NE disease, lower BWG and shorter colon length. The Most changes in the IEL populations were observed at 1 dpi. The EM/CP group showed substantial increases in the total number of natural IEL subsets, including TCRαß+CD4-CD8-, TCRαß+CD8αα+, TCRγδ+, TCRneg and innate CD8α (iCD8α) cells by at least two-fold. However, by 7 dpi, only the number of TCRαß+CD4-CD8- and TCRαß+CD8αα+ IEL maintained their increase in the EM/CP group. The EM/CP group had significantly higher expression of proinflammatory cytokines (IL-1ß and IFN-γ) and Osteopontin (OPN) in the jejunum at 1 dpi. These findings suggest that natural IEL with innate and innate-like functions might play a critical role in the host response during subclinical NE, potentially conferring protection against C. perfringens infection.

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The accumulating data regarding a non-biopsy diagnosis of celiac disease has led to its adoption in certain scenarios, although debate on whether and when to use non-biopsy criteria in clinical practice is ongoing. Despite the growing popularity and evidence basis for a biopsy-free approach to diagnosis in the context of highly elevated serologies, there will continue to be a role for a biopsy in some groups. This review summarizes the current evidence supporting a non-biopsy approach and arguments supporting continued reliance on biopsy, and focuses on opportunities to improve both approaches.

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The intestinal epithelium, which segregates the highly stimulatory lumen from the underlying tissue, harbors one of the largest lymphocyte populations in the body, intestinal intraepithelial lymphocytes (IELs). IELs must balance tolerance, resistance, and tissue protection to maintain epithelial homeostasis and barrier integrity. This review discusses the ontogeny, environmental imprinting, T cell receptor (TCR) repertoire, and function of intestinal IELs. Despite distinct developmental pathways, IEL subsets share core traits including an epithelium-adapted profile, innate-like properties, cytotoxic potential, and limited TCR diversity. IELs also receive important developmental and functional cues through interactions with epithelial cells, microbiota, and dietary components. The restricted TCR diversity of IELs suggests that a limited set of intestinal antigens drives IEL responses, with potential functional consequences. Finally, IELs play a key role in promoting homeostatic immunity and epithelial barrier integrity but can become pathogenic upon dysregulation. Therefore, IELs represent intriguing but underexamined therapeutic targets for inflammatory diseases and cancer.

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BACKGROUND: Inflammatory Bowel Diseases (IBD), an autoimmune disease characterised by abnormal intestinal immunity, are related to vital morbidity around the world. However, therapeutic agents for IBD have not achieved desired benefit. Exploring new therapeutic targets for IBD, especially based on its abnormally intestinal immunity, could alleviate the flare-up and worsening of IBD. Tissue resident memory T cells (TRM) are core of multiple autoimmune diseases, including IBD. However, the mechanism of TRM differentiation remains to be investigated. METHODS: The alterations in mRNA and lncRNA profile of intestinal intraepithelial lymphocytes (IELs), the largest component of intestinal TRM, were analyzed in DSS-induced chronic colitis. Based on it, we examined the function of rectal insulin instillation in a dextran sodium sulfate (DSS) induced chronic colitis. Furthermore, we investigated the downstream-target of the insulin pathway-EZH2 and the crucial role of EZH2 in intestinal tissue resident memory T cell differentiation by utilizing EZH2fl/flCD4cre mice. RESULTS: Insulin receptor (INSR) expression was found to be significantly reduced. Activation of mucosal insulin pathway by rectal insulin instillation exacerbated colitis by disrupting IELs subgroups and up-regulating TNF-ɑ and IL-17 expression. Rectal insulin instillation promoted EZH2 expression and EZH2 inhibition alleviated chronic colitis. EZH2fl/flCD4cre mice restored the normal IEL subgroups and suppressed TNF-ɑ and IL-17 expression, exhibiting alleviated colitis. IELs from EZH2fl/flCD4cre mice exhibit significant changes in TRM related phenotype. CD4+TRM was significantly increased in chronic colitis and decreased in EZH2fl/flCD4cre mice. CONCLUSION: Insulin receptor of intestinal mucosal T-cells could promote intestinal TRM differentiation via EZH2. Our discoveries suggest that therapies targeting colonic INSR and EZH2 could be potential treatment for IBD based on its regulatory effects on TRM. Insulin receptor inhibitors rather than insulin should be applied during colitis-active phase. In addition, EZH2 shows to be a downstream signal of the insulin pathway and EZH2 inhibitor could alleviating intestinal inflammation. However, the critical role of EZH2 in TRM differentiation restricts the anti-tumor effects of EZH2 inhibitor in vivo.

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Our previous study indicated that cytotoxicity of intraepithelial lymphocytes is a poor prognostic factor in feline intestinal T-cell lymphoma (FITL), but the effect of cytotoxic lymphocytes on mucosal epithelium is still unknown. Thus, we investigated the association between cytotoxic lymphocytes and mucosal epithelium in 71 cases of feline intestinal T-cell lymphoma (FITL): epithelial injury, basement membrane injury, cleaved-caspase-3 positivity of epithelial cells, and the number and Ki67 positivity of intraepithelial lymphocytes in granzyme B (GRB)+ and GRB- FITLs were evaluated. Epithelial injury score and the number of intraepithelial lymphocytes in granzyme B (GRB)+ FITL were significantly higher than those of GRB- FITL (P<0.05, P<0.05), but no significant differences were found in the basement membrane injury score, the percentage of cleaved-caspase-3+ epithelial cells, and the percentage of Ki67+ intraepithelial lymphocytes. There was a significant correlation between the epithelial injury score and the number of intraepithelial lymphocytes (P<0.05), but no significant correlation was observed between the epithelial injury score and Ki67+ percentage of intraepithelial lymphocytes. Because epithelial cell cleaved-caspase-3 positivity was observed in FITL, regardless of GRB expression in lymphocytes, GRB-mediated apoptosis may not contribute to epithelial injury in FITL. The association between increased number of intraepithelial lymphocytes and epithelial injury suggests that intraepithelial lymphocytes infiltration may contribute to epithelial injury in FITL.

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Cryptosporidium is a leading cause of diarrheal-related deaths in children, especially in resource-poor settings. It also targets the immunocompromised, chronically infecting people living with HIV and primary immunodeficiencies. There is no vaccine or effective treatment. Although it is known from human cases and animal models that CD4+ T cells play a role in curbing Cryptosporidium, the role of CD8+ T cells remains to be defined. Using a Cryptosporidium tyzzeri mouse model, we show that gut-resident CD8+ intraepithelial lymphocytes (IELs) confer resistance to parasite growth. CD8+ IELs express and depend on the ligand-dependent transcription factor aryl hydrocarbon receptor (AHR). AHR deficiency reduces CD8+ IELs, decreases their cytotoxicity, and worsens infection. Transfer of CD8+ IELs rescues severely immunodeficient mice from death following Cryptosporidium challenge. Finally, dietary supplementation of the AHR pro-ligand indole-3-carbinol in newborn mice promotes resistance to infection. Therefore, common dietary metabolites augment the host immune response to cryptosporidiosis, protecting against disease.

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BACKGROUND: Few data are available on flow cytometry (FC) for monitoring intraepithelial lymphocytes (IELs) in refractory celiac disease (RCD), non-responsive celiac disease (NRCD), and non-celiac enteropathies (NCEs). AIMS: 1) To investigate the significance of monitoring IELs immunophenotype with FC in patients with NRCD, RCD and NCEs; 2) to evaluate FC concordance with immunohistochemistry (IHC) and γ-TCR clonality analysis. METHODS: Patients investigated between January-2012 and February-2023 were divided into two groups: 1)confirmed RCD or NRCD being investigated for persistent symptoms and suspected complications of celiac disease (CD); 2)NCEs lacking clinical/histological response. Clinical/molecular features and outcomes were retrospectively collected and analysed according to presence/absence of aberrant IELs on FC (cut-off≥20 % CD103+sCD3-CD8-iCD3+ IELs). RESULTS: 52 patients (18 RCD,21 NRCD,13 NCEs; 38F, 55±13 years; median follow-up 30 months, IQR 2-58) underwent 100 FC IELs determinations. 22/52 had ≥2 FC determinations and IEL phenotype remained unchanged over time in all them (κ=1.00). Aberrant IEL phenotype in CD was associated with increased mortality (HR 4.2, 95 % CI 1.5-11.9, p < 0.01). No patients with NCEs had an aberrant IEL phenotype at FC, although 3/13 developed lymphoma and 4/13 died. Concordance of FC was fair with both IHC (κ=0.40) and γ-TCR clonality analysis (κ=0.22). CONCLUSION: FC is accurate for assessing and monitoring IEL phenotype and providing important prognostic information in celiac patients. Further study is needed on its role in NCEs.

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Acute graft-versus-host disease (GvHD) remains the biggest clinical challenge and prognosis-determining complication after allogeneic hematopoietic stem cell transplantation (allo-HSCT). Donor T cells are acceptedly key mediators of alloreactivity against host tissues and here especially the gut. In support of previous studies, we found that the intestinal intra-epithelial lymphocyte (IEL) compartment was dynamically regulated in the course of MHC class I full mismatch allo-HSCT. However, while intestinal epithelial cell (IEC) damage endangers the integrity of the intestinal barrier and is a core signature of intestinal GvHD, the question whether and to what degree IELs are contributing to IEC dysregulation is poorly understood. To study lymphoepithelial interaction, we employed a novel ex vivo T cell/organoid co-culture model system. Here, allogeneic intra-epithelial T cells were superior in inducing IEC death compared to syngeneic IEL and allogeneic non-IEL T cells. The ability to induce IEC death was predominately confined to TCRß+ T cells and was executed in a largely IFNγ-dependent manner. Alloreactivity required a diverse T cell receptor (TCR) repertoire since IELs genetically modified to express a TCR restricted to a single, non-endogenous antigen failed to mediate IEC pathology. Interestingly, minor histocompatibility antigen (miHA) mismatch was sufficient to elicit IEL-driven IEC damage. Finally, advanced live cell imaging analyses uncovered that alloreactive IELs patrolled smaller areas within intestinal organoids compared to syngeneic controls, indicating their unique migratory properties within allogeneic IECs. Together, we provide here experimental evidence for the utility of a co-culture system to model the cellular and molecular characteristics of the crosstalk between IELs and IEC in an allogeneic setting ex vivo. In the light of the emerging concept of dysregulated immune-epithelial homeostasis as a core aspect of intestinal GvHD, this approach represents a novel experimental system to e.g. screen therapeutic strategies for their potential to normalize T cell/IEC- interaction. Hence, analyses in pre-clinical in vivo allo-HSCT model systems may be restricted to hereby positively selected, promising approaches.

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Background: Intraepithelial lymphocytes (IELs) are the first immune cells to contact and fight intestinal pathogens such as Cryptosporidium, a widespread parasite which infects the gut epithelium. IFN-γ producing CD4+ T IELs provide an efficient and a long-term protection against cryptosporidiosis while intraepithelial type 1 innate lymphoid cells limits pathogen spreading during early stages of infection in immunodeficient individuals. Yet, the role of T-cell like innate IELs, the most frequent subset of innate lymphocytes in the gut, remains unknown. Methods: To better define functions of innate IELs in cryptosporidiosis, we developed a co-culture model with innate IELs isolated from Rag2-/- mice and 3D intestinal organoids infected with C. parvum using microinjection. Results: Thanks to this original model, we demonstrated that innate IELs control parasite proliferation. We further showed that although innate IELs secrete IFN-γ in response to C. parvum, the cytokine was not sufficient to inhibit parasite proliferation at early stages of the infection. The rapid protective effect of innate IELs was in fact mediated by a cytotoxic, granzyme-dependent mechanism. Moreover, transcriptomic analysis of the Cryptosporidium-infected organoids revealed that epithelial cells down regulated Serpinb9b, a granzyme inhibitor, which may increase their sensitivity to cytolytic attack by innate IELs. Conclusion: Based on these data we conclude that innate IELs, most likely T-cell-like innate IELs, provide a rapid protection against C. parvum infection through a perforin/granzymes-dependent mechanism. C. parvum infection. The infection may also increase the sensitivity of intestinal epithelial cells to the innate IEL-mediated cytotoxic attack by decreasing the expression of Serpin genes.

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