Subsets Of Intraepithelial Lymphocytes Research Articles

Natural intraepithelial lymphocytes are critical intestinal defense against necrotic enteritis in chickens

Abstract In chickens, the subsets of intraepithelial lymphocytes (IEL) and their responses to enteric diseases remain unclear. Therefore, our study aimed to elucidate the roles of IEL populations in necrotic enteritis (NE) in chickens. At 14 days of age, 63 SPF chicks were randomly divided into 3 groups: Control (sham challenge), Eimeria maxima (EM) challenge, and Clostridium Perfringens (CP) challenge. Birds in the EM and CP groups were orally infected with EM at day 14 of age, and while CP birds received additional oral infection with CP bacteria at days 18 and 19 of age. At 20 days of age (1 day post CP infection; dpi), and 27 days of age (7 dpi), 7 birds per group were euthanized, and jejunum was harvested for gross lesion scores, IEL isolation, and gene expression. Birds exhibited subclinical NE based on lesion scores, and CP-infected birds showed lower BWG and shorter colon length. The Most changes in the IEL populations were observed at 1 dpi. The CP group showed substantial increases in the total number of natural IEL subsets, including TCRαβ+CD4-CD8-, TCRαβ+CD8αα+, TCRγδ+, TCRneg and iCD8α IEL. By 7 dpi, only the number of TCRαβ+CD4-CD8- and TCRαβ+CD8αα+ IEL maintain their increase in the CP group. The CP group had significant higher expression of IL-1β and IFN-γ and osteopontin genes at 1 dpi. These findings suggest that natural IEL play a critical role in the host response during subclinical NE, potentially conferring protection against CP infection.

Intraepithelial Lymphogram in the Diagnosis of Celiac Disease in Adult Patients: A Validation Cohort.

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). 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. 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. 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. 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.

Myeloid Cells and Sphingosine-1-Phosphate Are Required for TCRαβ Intraepithelial Lymphocyte Recruitment to the Colon Epithelium.

Intraepithelial lymphocytes (IELs) are T cells important for the maintenance of barrier integrity in the intestine. Colon IELs are significantly reduced in both MyD88-deficient mice and those lacking an intact microbiota, suggesting that MyD88-mediated detection of bacterial products is important for the recruitment and/or retention of these cells. Here, using conditionally deficient MyD88 mice, we show that myeloid cells are the key mediators of TCRαβ+ IEL recruitment to the colon. Upon exposure to luminal bacteria, myeloid cells produce sphingosine-1-phosphate (S1P) in a MyD88-dependent fashion. TCRαβ+ IEL recruitment may be blocked using the S1P receptor antagonist FTY720, confirming the importance of S1P in the recruitment of TCRαβ+ IELs to the colon epithelium. Finally, using the TNFΔARE/+ model of Crohn's-like bowel inflammation, we show that disruption of colon IEL recruitment through myeloid-specific MyD88 deficiency results in reduced pathology. Our results illustrate one mechanism for recruitment of a subset of IELs to the colon.

Natural intraepithelial lymphocyte populations rise during necrotic enteritis in chickens.

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 CD8αβ T cells) and natural IEL (TCRαβCD8αα, 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 TCRαβ+CD4-CD8-, TCRαβ+CD8αα+, TCRγδ+, TCRneg and innate CD8α (iCD8α) cells by at least two-fold. However, by 7 dpi, only the number of TCRαβ+CD4-CD8- and TCRαβ+CD8αα+ IEL maintained their increase in the EM/CP group. The EM/CP group had significantly higher expression of proinflammatory cytokines (IL-1β 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.

Too Much of a Good Thing: Extended Duration of Gut Microbiota Depletion Reverses Protection From Experimental Autoimmune Uveitis.

Using the model of experimental autoimmune uveitis (EAU) induced by immunization with a retinal antigen, two studies have reported contradictory results on disease development following oral antibiotic treatment (ABX). We showed that long-term ABX did not affect EAU, but another study showed that short-term ABX was protective. We therefore studied the effects of ABX on EAU, gut microbiota, and host immune responses as a function of treatment duration. EAU-susceptible mice were treated orally with broad-spectrum antibiotics starting at least 10 weeks (long-term) or 1 week (short-term) before immunization until termination of the experiment. Gut microbiota were characterized by 16S amplicon sequencing, and host gut immune elements were studied phenotypically and functionally. Long-term ABX had no effect, whereas short-term ABX delayed EAU, as previously reported by us and others, respectively. Microbial sequencing revealed progressive reduction of gut microbiota that showed some differences in the two ABX groups. Interestingly, duration of ABX was associated with a gradual disappearance of the CD4+ and CD4+CD8+ subset of gut intraepithelial lymphocytes (IELs). This IEL subset is microbiota dependent and is absent in germ-free mice. Relative abundance of Lactobacillus reuteri correlated with the frequencies of CD4+CD8+ IELs. IELs suppressed antigen-specific activation of autoreactive T cells in culture. Gut microbiota may play dual roles in uveitis development: They promote EAU development but also help maintain IEL populations that have regulatory function against autoreactive T cells. We propose that the progressive loss of this population during long-term ABX reverses the EAU-ameliorating effects of microbiota depletion.

Phenotype characteristic of colonic intraepithelial lymphocytes in patients with Crohn's disease

Intraepithelial lymphocytes (IEL) play a critical role in maintaining the immune balance of the gut and provide the first line of mucosal defense against luminal antigens as well as rapidly respond to epithelial injury. Recently, IEL have received a lot of attention as key mediators of aberrant immune response resulted in persistent immune activation, inflammation and altered intestinal barrier function, seen in Crohn's disease (CD). This study describes for the first time subsets of colonic IEL in CD patients as compared to healthy controls aimed at characterization of altered IEL contribution to the pathogenesis of Crohn's disease.The peripheral venous blood and colon tissues were obtained from 10 CD patients and 6 donors. IEL were isolated from the mucosa by incubation the tissue in a predigesting solution. Lymphoid cells phenotype was investigated using monoclonal antibodies and flow cytometry.The majority of colonic IEL was identified as СD3+T lymphocytes and no significant differences were found in their numbers in investigated groups. However, changes in T cell subsets composition have been shown: the ratio of СD3+СD4+IEL and СD3+СD8+IEL was 1:1 in colon of CD patients and correlated with T cells in peripheral blood (R = 0.7; p < 0.05) while donor tissues were characterized by expected СD3+СD8+T killers prevalence and the ratio reached 1:2 (p < 0.05). The increase of unconventional γδIEL (mainly due to V81+T cells) and СD161+T cells in association with TNK cells decrease were revealed in colon (p < 0.01) as well as in peripheral blood (p < 0.05) of CD patients as compared to donors. Moreover, the number of colonic γδIEL was correlated with disease location (R = -0.6; p < 0.05), and disease behavior (R = 0.7; p < 0.01) according to Montreal classification.The observed data indicates changes in colonic IEL composition in CD patients that may provide valuable insight into the contribution of T helpers, γδT cells and mucosa-associated СD161+T cells in autoimmune intestinal inflammation but need further possible mechanisms discussion.

Combined RNAscope and immunohistochemistry staining on duodenal paraffin sections as a new tool to reveal cytolytic potential of intraepithelial lymphocytes

Immunohistochemistry (IHC) is a consolidated technique for the identification of surface and cytoplasmic antigens in cells or tissue sections using specific antibodies, yet simultaneous detection of two markers on the same cell may be difficult to achieve. Here we develop a protocol to perform a double staining using RNAscope, a new in-situ hybridization (ISH) technology, to visualize perforin transcripts, and classical IHC to visualize either CD8 or TcRγδ positive intraepithelial lymphocytes (IELs) in small intestinal paraffin sections of celiac disease (CD) patients. This double assay will allow to investigate the cytotoxic properties of two subsets of IELs in different stages of CD, thus contributing to understand the events leading to tissue destruction and healing.

Flow cytometric analysis of duodenal intraepithelial lymphocytes (celiac lymphogram): A diagnostic test for celiac disease.

Celiac disease (CD) diagnosis in adults and certain cases of children mainly relies on the assessment of histopathological features in duodenal biopsies. However, none of the histological findings that characterize CD are pathognomonic. This, in addition to the clinical heterogeneity of the disease and the presence of seronegative forms, makes the diagnosis of CD still a challenge. A hallmark of the celiac mucosa is the elevated number of TCRγδ intraepithelial lymphocytes (IEL) in the epithelium, which may remain increased even long after gluten withdrawal. Active disease is also characterized by the decreased CD3- IEL subset. The use of flow cytometry enables a precise cell counting and phenotyping, allowing the ascertainment of both TCRγδ+ and CD3- IEL subsets, what is known as the "IEL lymphogram." Although determination of this lymphogram has become a routine evaluation tool in numerous hospitals, standardization of the technical method will guarantee an accurate performance in order to become a pivotal technique for CD diagnosis. Here we describe the protocol to process duodenal biopsies in order to obtain the IELs from the mucosa and to characterize lymphocyte populations by flow cytometry to obtain the IEL lymphogram.

The γδ IEL effector API5 masks genetic susceptibility to Paneth cell death.

Loss of Paneth cells and their antimicrobial granules compromises the intestinal epithelial barrier and is associated with Crohn's disease, a major type of inflammatory bowel disease1-7. Non-classical lymphoid cells, broadly referred to as intraepithelial lymphocytes (IELs), intercalate the intestinal epithelium8,9. This anatomical position has implicated them as first-line defenders in resistance to infections, but their role in inflammatory disease pathogenesis requires clarification. The identification of mediators that coordinate crosstalk between specific IEL and epithelial subsets could provide insight into intestinal barrier mechanisms in health and disease. Here we show that the subset of IELs that express γ and δ T cell receptor subunits (γδ IELs) promotes the viability of Paneth cells deficient in the Crohn's disease susceptibility gene ATG16L1. Using an ex vivo lymphocyte-epithelium co-culture system, we identified apoptosis inhibitor 5 (API5) as a Paneth cell-protective factor secreted by γδ IELs. In the Atg16l1-mutant mouse model, viral infection induced a loss of Paneth cells and enhanced susceptibility to intestinal injury by inhibiting the secretion of API5 from γδ IELs. Therapeutic administration of recombinant API5 protected Paneth cells in vivo in mice and ex vivo in human organoids with the ATG16L1 risk allele. Thus, we identify API5 as a protective γδ IEL effector that masks genetic susceptibility to Paneth cell death.

More Active Intestinal Immunity Developed by Obese Mice Than Non-Obese Mice After Challenged by Escherichia coli.

Obese mice presented lower mortality to non-fatal pneumonia induced by Escherichia coli (E. coli) than the non-obese mice. However, it remained obscure whether the intestine contributed to the protective effect of obese mice with infection. The 64 non-obese (NOB) mice were divided into NOB-uninfected and NOB-E. coli groups, while 64 high-fat diet-induced obesity (DIO) mice were divided into DIO-uninfected and DIO-E. coli groups. Mice in E. coli groups were intranasally instilled with 40 μl E. coli (4.0 ×109 colony-forming units [CFUs]), while uninfected groups with the same volume of phosphate buffer saline (PBS). The T subsets of Intraepithelial lymphocytes (IELs) and lamina propria lymphocytes (LPLs) in the intestine were collected for flow cytometry analysis at 0, 12, 24, and 72 h post-infection, also the duodenum and colon were harvested to survey histopathological change. The results showed that the percentage of CD3+T cells in LPLs in DIO-E. coli group was significantly lower than that in the DIO-uninfected group after infection (p < 0.05). The percentage of CD4+T cells in IELs in NOB-E. coli was significantly lower than that in DIO-E. coli after infection (p < 0.05). The percentage of CD8+T cells in LPLs in NOB-E. coli was significantly lower than that in DIO-E. coli at 12 and 24 h (p < 0.05). The immunoglobulin A (IgA)+ cells in DIO-uninfected were higher than that in NOB-uninfected at all time points (p < 0.05). The IgA+ cells in DIO-E. coli were higher than that in DIO-uninfected at 12, 24, and 72 h (p < 0.05). The results revealed that the level of intestinal mucosal immunity in obese mice was more active than that in non-obese mice.

Epithelial HNF4A shapes the intraepithelial lymphocyte compartment via direct regulation of immune signaling molecules

Abstract Hepatocyte Nuclear Factor 4A (HNF4A) is a highly conserved orphan nuclear receptor that has been associated with ulcerative colitis. Mice lacking Hnf4a specifically in the intestinal epithelial cells (IECs) are more susceptible to DSS-induced colitis and develop spontaneous colonic inflammation with aging. However, the mechanisms how HNF4A maintains intestinal homeostasis and protects against colitis remain unclear. Here we demonstrate that epithelial HNF4A shapes the intraepithelial lymphocyte (IEL) compartment by selectively promoting the development of natural IEL subsets that are TCRgd+ or TCRab+CD8aa+. Mechanistically, HNF4A serves as a transcription factor that directly regulates epithelial expression of immune signaling molecules involved in the IEC-IEL crosstalk. Those molecules include the butyrophilin-like (Btnl) molecules Btnl1 and Btnl6. Further, while HNF4A is stably expressed along the intestinal tract, these molecules are more abundant in the small intestine, which correlates with the distribution of natural IELs. We propose HNF4A paralog, HNF4G, which is restricted to the small intestine, cooperates with HNF4A in driving expression of signaling molecules and accounts for the geographic specificity. Moreover, HNF4A-BTNL regulatory axis is conserved in Caco2 cells of human colorectal origin. Collectively, our findings underscore the importance of HNF4A as a conserved transcription factor controlling IEC-IEL crosstalk and suggest that HNF4A maintains intestinal homeostasis through regulation of the IEL compartment.

Alteration in the Population of Intraepithelial Lymphocytes and Virus Shedding in Specific-Pathogen-Free Chickens Following Inoculation with Lentogenic and Velogenic Newcastle Disease Virus Strains.

Intraepithelial lymphocytes (IELs) provide the first line of immunological defense after the invasion of the intestine by a pathogen. To understand the changes of IEL response in chickens, we measured the population of different subsets of avian IELs at different time points after primary inoculation of Newcastle disease virus (NDV) lentogenic strain (LaSota) and subsequent challenge with NDV velogenic strain- genotypes VII and VIII. Furthermore, NDV shed after each treatment was quantified. Specific-pathogen-free chickens were randomly divided into six groups of chickens, one to six, inoculated with phosphate buffered saline; NDV lentogenic strain (LaSota); genotype VII (GVII); LaSota and challenged with GVII (LSGVII); genotype VIII (GVIII); and group of LaSota and challenged with GVIII (LSGVIII). The chickens were euthanized at 12, 36, and 60 h postchallenge. Immunophenotyping of CD25+ IEL, CD3+ cells, CD4+ cells, and CD8+ cells was conducted using flow cytometer. Furthermore, virus shedding was measured using reverse transcriptase-quantitative polymerase chain reaction. Data were analyzed using a two-way analysis of variance (ANOVA). The results showed that the percentage population of IEL subsets was generally lower in the chickens inoculated with GVII or GVIII when compared with LaSota, LSGVII and LSGVIII inoculated groups. The NDV copy number was significantly higher in chickens challenged with NDV GVII or GVIII when compared with chickens inoculated with LaSota, LSGVII or LSGVIII. Taking together, NDV velogenic strain caused decrease in the population of subsets of chickens' IEL. However, inoculation of NDV LaSota may increase the population of avian IEL subsets and decrease shedding of virulent NDV.

Assessment of Duodenal Intraepithelial Lymphocyte Composition (Lymphogram) for Accurate and Prompt Diagnosis of Celiac Disease in Pediatric Patients.

INTRODUCTION:Quantitative and phenotypic analyses of duodenal intraepithelial lymphocytes (IELs) by flow cytometry (IEL lymphogram) confer specificity and enable the diagnosis even in unconventional presentations of celiac disease (CD). To evaluate the validity of the IEL lymphograms in the pediatric population for new insights into their use as biomarkers in the natural history of CD.METHODS:We retrospectively included 1,211 children (602 with active CD, 92 on a gluten-free diet, 47 with potential CD, and 470 nonceliac controls) who required duodenal biopsies in this study. The cutoff values for IEL subsets were established to calculate the probability of disease according to the lymphogram.RESULTS:A celiac lymphogram (a ≥15% increase in gamma-delta T-cell receptor IELs and a simultaneous ≤6% decrease in CD3 surface-negative [sCD3−]) IELs was strongly associated with the diagnosis of active CD, which was present in 89.7% of the confirmed patients. The remaining 10% of the celiac patients had a partial celiac lymphogram (≥15% increase gamma-delta T-cell receptor IELs or ≤6% decrease in sCD3− IELs), with lower diagnostic certainty. On a gluten-free diet, nearly 20% of the patients were indistinguishable from nonceliac subjects based on the lymphogram. In potential CD, a decrease in sCD3− IELs was a risk marker of progression to villous atrophy and a diagnosis of active CD.DISCUSSION:If a biopsy is clinically indicated, the IEL lymphogram adds specificity to the histological findings, reducing diagnostic delays and misdiagnoses. The lymphogram is useful for monitoring the natural progression of the disease and predicting the transition from potential celiac to overt CD.

Gut epithelial IL-27 confers intestinal immunity through the induction of intraepithelial lymphocytes.

IL-27 controls a diverse range of immune responses in many disease settings. Here, we identify intestinal epithelial cells (IECs) as one of the major IL-27 cellular sources in the gut-associated tissue. Unlike IL-27 secreted by innate immune cells, gut epithelial IL-27 is dispensable for T-bet+ regulatory T cell (T reg cell) differentiation or IL-10 induction. Rather, IEC-derived IL-27 specifically promotes a distinct CD8αα+CD4+ intraepithelial lymphocyte (IEL) population that acquires their functional differentiation at the intestinal epithelium. Loss of IL-27 in IECs leads to a selective defect in CD8αα+CD4+ IELs over time. Consequently, mice with IEC-specific IL-27 ablation exhibited elevated pathogen burden during parasitic infection, and this could be rescued by transfer of exogenous CD8αα+CD4+ IELs. Collectively, our data reveal that in addition to its known regulatory properties in preventing immune hyperactivity, gut epithelial IL-27 confers barrier immunity by inducing a specific IEL subset and further suggest that IL-27 produced by different cell types plays distinct roles in maintaining intestinal homeostasis.

Oral immunization induces a novel CXCR6+ β7+ intraepithelial lymphocyte subset predominating in the small intestine.

Intestinal T cells form a central part of the front-line defence against foreign organisms and need to be situated in the mucosa where infection occurs. It is well accepted that immunization by a mucosal route favours localization of antigen-specific effector T cells in the mucosal epithelium, while systemic immunization does not. The aim of the study is to determine how homing receptors are specifically involved in retaining effector T cells in the small intestine after oral immunization. We here demonstrate that the chemokine receptor CXCR6, integrins β7 and CD29 contribute differentially to the epithelial retention phenotype of CD8+ T cells in the small intestine of mice. CD8+ intraepithelial lymphocytes (IELs) of unvaccinated mice are predominantly β7 single positives, and subcutaneous immunization-induced antigen-specific CD8+ effector IELs are mainly composed of CXCR6+ , CD29+ and CXCR6+ CD29+ cells. Strikingly, the majority of oral immunization-induced antigen-specific CD8+ effector IELs exhibit a distinct, tissue-specific CXCR6+ β7+ double-positive phenotype, cytotoxic potential and enhanced intraepithelial localization. Transfer of antigen-specific CD8+ T cells preactivated with certain immuno-stimuli (such as monophosphoryl lipid A) results in increased accumulation of donor IELs with the CXCR6+ β7+ phenotype. As β7 exclusively paired with αE on IELs, our results strongly suggest that CXCR6 may cooperate with the heterodimer αEβ7 to preferentially retain intestinally induced effector IELs in the epithelium. The identification of this novel IEL phenotype has significant implications for the development of vaccines and therapeutic strategies to enhance gut immunity.

Butyrophilin-like proteins display combinatorial diversity in selecting and maintaining signature intraepithelial \u03b3\u03b4 T cell compartments

Butyrophilin-like (Btnl) genes are emerging as major epithelial determinants of tissue-associated γδ T cell compartments. Thus, the development of signature, murine TCRγδ+ intraepithelial lymphocytes (IEL) in gut and skin depends on Btnl family members, Btnl1 and Skint1, respectively. In seeking mechanisms underlying these profound effects, we now show that normal gut and skin γδ IEL development additionally requires Btnl6 and Skint2, respectively, and furthermore that different Btnl heteromers can seemingly shape different intestinal γδ+ IEL repertoires. This formal genetic evidence for the importance of Btnl heteromers also applied to the steady-state, since sustained Btnl expression is required to maintain the signature TCR.Vγ7+ IEL phenotype, including specific responsiveness to Btnl proteins. In sum, Btnl proteins are required to select and to maintain the phenotypes of tissue-protective γδ IEL compartments, with combinatorially diverse heteromers having differential impacts on different IEL subsets.

Exposures to uranium and arsenic alter intraepithelial and innate immune cells in the small intestine of male and female mice

Human exposures to environmental metals, including uranium (U) and arsenic (As) are a global public health concern. Chronic exposures to U and As are linked to many adverse health effects including, immune suppression and autoimmunity. The gastrointestinal (GI) tract is home to many immune cells vital in the maintenance of systemic immune health. However, very little is known about the immunotoxicity of U and As at this site. The present study examined the burden of U and As exposure in the GI tract as well as the resultant immunotoxicity to intraepithelial lymphocytes (IELs) and innate immune cells of the small intestine following chronic drinking water exposures of male and female mice to U (in the form of uranyl acetate, UA) and As (in the form of sodium arsenite, As3+). Exposure to U or As3+ resulted in high levels of U or As in the GI tract of male and female mice, respectively. A reduction of small intestinal CD4+ IELs (TCRαβ+, CD8αα+) was found following As3+ exposure, whereas U produced widespread suppression of CD4− IEL subsets (TCRαβ+ and TCRγδ+). Evaluation of innate immune cell subsets in the small intestinal lamina propria revealed a decrease in mature macrophages, along with a corresponding increase in immature/proinflammatory macrophages following As3+ exposures. These data show that exposures to two prevalent environmental contaminants, U and As produce significant immunotoxicity in the GI tract. Collectively, these findings provide a critical framework for understanding the underlying immune health issues reported in human populations chronically exposed to environmental metals.

The Intracellular Intensity of CD3 on Aberrant Intraepithelial Lymphocytes Is a Prognostic Factor of the Progression to Overt Lymphoma in Refractory Celiac Disease Type II (Pre-Enteropathy-Associated T Cell Lymphoma)

Background: Refractory celiac disease type II (RCD-II) is a very rare yet severe complication of celiac disease (CD) with a 50% rate of progression to Enteropathy-associated T-cell lymphoma (EATL). Timely diagnosis and treatment of RCD-II is of the essence and requires the identification of a population of frequently clonal, phenotypically aberrant intraepithelial lymphocytes (IELs). Flow Cytometry of intestinal IELs is the recommended method to identify the aberrant surface CD3-negative (sCD3^–) intracytoplasmic CD3-positive (icCD3ε⁺) IELs, and a proportion of >20% is diagnostic of RCD-II. There is substantial heterogeneity in the clinical course of RCD-II, and insufficient information on prognostic factors. Aim: To establish flow cytometric predictors of the clinical evolution of RCD-II, to help guide treatment approaches. Patients and Methods: Retrospective single-center study of clinical and immunological features of 6 RCD-II patients and a control group, both identified from a 2,000-patient cohort over 16 years. IEL subset frequencies and the intensity of staining for surface (s) and intracytoplasmic (ic) CD3ε+ on IEL subsets were quantified and correlated with the clinical outcome. Results: Unexpectedly, the frequency of aberrant sCD3^– icCD3ε⁺ cells at diagnosis did not correlate with histological or clinical affection. However, a higher intensity of icCD3ε⁺ staining in the aberrant IELs relative to expression on normal IELs correlated with monoclonality and with worse clinical outcomes. Conclusion: The ratio of icCD3ε⁺ on aberrant IELs vs. normal IELs appears to be a useful indicator of prognosis at the time of diagnosis, and may represent a novel tool in the follow-up of RCD-II patients after therapy.

P038 A single-cell transcriptomics approach reveals high-resolution cellular signatures in Crohn’s disease

Abstract Background Single-cell RNA sequencing (sc-RNAseq) provides high-resolution analysis of individual cells and cell clusters within complex tissues like the intestinal mucosa. No study to date has applied this novel technology to understand the complexity of intestinal inflammation in Crohn’s disease (CD) patients. Our aim is to identify the cellular subsets present in healthy and inflamed colon, and to describe how individual subsets are regulated in active CD. Methods Colon biopsies from a healthy control (HC) and two patients with endoscopically active colonic CD were collected and processed by enzymatic digestion and cell sorting to discard dead cells. Single-cell suspensions were processed by droplet-based protocol 10× chromium system followed by library preparation and sequencing. Sequencing data were aligned and quantified using Cell Ranger. Downstream analysis was performed using the R package Seurat. Results scRNA-seq analysis identified 14 transcriptionally different cell subsets in the healthy colon, including diverse types of T cells, plasma cells, mesenchymal cells, epithelial cells and myeloid populations, as well as a subset of MHCII+ CD20+ B cells. Overall, the number of transcriptionally differentiated cellular subsets was greater (16 and 17 subsets per sample) in the two CD colonic samples revealing higher cellular heterogeneity in active CD. In particular, in the HC we identified three T-cell subsets including a CD4+, CD8+ and a smaller subset of intraepithelial lymphocytes. In contrast, samples from CD patients presented four to six clearly differentiated T-cell clusters depending on the patient, with a marked enrichment of cytotoxicity genes (GZMA, GZMB, NKG7, CCL5 and AOAH) and T-regulatory genes (FOXP3, CTLA4, BATF and IL2RA). In addition, the HC sample presented three differentiated subsets of antibody-secreting plasma cells with predominant IgA and IgM production. In contrast, a clear expansion of IgG-producing plasma cells was observed in both CD patients. Moreover, the profiles of specific subsets significantly changed revealing transcriptional regulation within the individual subsets. Specifically, macrophages within the HC mucosa expressed the M2 markers CD163 and CD209 together with a whole signature of 163 additional genes, while in CD, they lost expression of these markers and upregulated CXCL8 and IL1B which were co-expressed with 268 additional genes common to macrophage populations in both CD patients. Conclusion We show that sc-RNAseq can be applied to study at a high cellular resolution human intestinal biopsies. Understanding the transcriptomic profile of the CD-related subsets will help dissect disease pathophysiology and provide knowledge for therapeutic targets.

CD8αα+ T cells show amoeboid shape and frequent morphological change in vitro, and localize to small intestinal intraepithelial region in vivo

Intraepithelial lymphocytes (IELs) are very unique in the intestinal immune system. They include γδT cells and CD4–CD8–TCRαβ+T cells (double negative: DNT), both of which are specific for the intestine, in addition to CD4+ and CD8+ T cells. IELs exist within the monolayer of the intestinal epithelial cells and dynamically move between lamina propria (LP) and intraepithelial (IE) region. The localization and movement patterns of IEL subsets and the regulatory factors have been unknown. Here, we developed a novel in vitro live imaging system and quantified the motility and morphological changes among subsets of IELs. We identified CD8αα as the key regulatory factor. IELs, especially γδ and DNT cells, showed amoeboid shape and frequent morphological change, while most T cells in MLN or SP showed round shape in vitro. TCR signal, IL-15, gut microbes, CCL25, and integrin αEβ7 expression were non-essential for IEL movement in vitro. CD8αα+ cells showed higher motility and larger morphological changes than CD8αα– cells. Adoptive transferred CD8αα+CD4-IELs localized to IE region of recipient NSG mice, while CD8αα–CD4-IELs localized to the LP. Our results showed that the CD8αα/TL signal is essential for the localization of IELs to IE region in vivo. CD8αα/TL may be an effective target to increase the number of IELs, which protects against intestinal infection, allergy, tumorigenesis or inflammation.