Γδ Intraepithelial Lymphocytes Research Articles

γδ intraepithelial lymphocytes acquire the ability to produce IFN-γ in a different time course than αβ intraepithelial lymphocytes.

An age-dependent increase in IFN-γ expression by intestinal intraepithelial lymphocytes (IELs) contributes to the acquisition of resistance to infection by pathogens. However, how IELs acquire the ability to produce IFN-γ remains to be elucidated. Here, we report that IELs in the small intestine acquire the ability to rapidly produce IFN-γ at two distinct life stages. TCRαβ+ IELs (αβIELs) started producing IFN-γ at 4 weeks of age, within 1 week after weaning. In contrast, TCRγδ+ IELs (γδIELs) started producing IFN-γ at 7 weeks of age. In mice lacking Eγ4, an enhancer of the TCRγ locus (Eγ4-/- mice), Thy-1+ Vγ5+ γδIELs, a major subpopulation of γδIELs, were specifically reduced and their ability to produce IFN-γ was severely impaired, whereas Vγ2+ γδIELs normally produced IFN-γ. In Eγ4-/- mice, TCR expression levels were reduced in Vγ5+ γδIEL precursors in the thymus but unchanged in the Vγ5+ IELs. Nevertheless, TCR responsiveness in Vγ5+ γδIELs was impaired in Eγ4-/- mice, suggesting that the TCR signal received in the thymus may determine TCR responsiveness and the ability to produce IFN-γ in the gut. These results suggest that αβIELs and γδIELs start producing IFN-γ at different life stages and that the ability of Vγ5+ γδIELs to produce IFN-γ in the gut may be predetermined by TCR signaling in IEL precursors in the thymus.

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.

T cell receptor signaling mediates enhanced IFNγ production by γδ intraepithelial lymphocytes in response to type I interferon

Abstract Intraepithelial lymphocytes (IEL) expressing the γδ T cell receptor (TCR) provide a rapid response to limit enteric pathogen invasion. Despite constant γδTCR triggering in vivo, these IELs remain immunologically quiescent until their activation threshold is surpassed. Activated γδ IELs limit viral replication by producing type I interferons (IFN), such as IFNα, yet the extent to which IFNα activates γδ IELs remains unknown. To this end, murine small intestinal γδ IELs were stimulated ex vivowith 1 ug/mL αCD3, 10 ng/mL IFNα or both for 5 h. We observed a 26%±10 increase in IFNγ +γδ IELs treated with αCD3 and IFNα compared to αCD3 treatment alone (p<0.0001) and a 2-fold increase in IFNγ MFI (p<0.0001). Using Nur77-GFP mice, we found that only Nur77 +γδ IELs produced IFNγ (p<0.0001), indicating that TCR signaling is necessary for IFNα-induced effector function. Suboptimal αCD3 stimulation (0.1 ug/mL) was sufficient to increase the frequency of IFNγ +γδ IELs following IFNα exposure relative to αCD3 alone (p<0.001). To interrogate the molecular mechanism involved in IFNα co-stimulation, γδ IELs were treated with PMA and/or ionomycin in the presence or absence of IFNα. Ionomycin and IFNα increased the frequency of IFNγ +γδ IELs by 16%±7 compared to ionomycin alone (p<0.0001), whereas PMA had no effect. Pharmacological inhibition of NFAT signaling (10 uM INCA6) abrogated both TCR- or IFNα-induced γδ IEL IFNγ production (p<0.01 or p<0.0001, respectively). Lastly, we observed that the co-stimulatory effect of IFNα was lost in STAT4-deficient γδ IELs compared to WT (p<0.0001). Together, these data indicate that low level TCR signaling through NFAT is required for IFNα to rapidly enhance γδ IEL IFNγ production in a STAT4-dependent manner. This work was supported by the National Institutes of Health R01 DK119349, the New Jersey Commission on Cancer Research, Busch Biomedical Research grant and the RBHS Chancellor Scholar Award (KLE).

Expansion of the intraepithelial lymphocyte (IEL) compartment results in an increased bioenergetic profile and reduced IFNγ production in γδ IELs.

Abstract Intestinal γδ intraepithelial lymphocytes (γδ IEL) serve as the first line of defense against pathogen invasion. We recently identified a transmissible hyperproliferative γδ IEL (γδ HYP) phenotype that protects against enteric infection, and these mice have no signs of overt intestinal pathology. Given that IELs are metabolically constrained to prevent aberrant activation, we hypothesized that the bioenergetics of γδ HYPIELs may regulate their effector function. Electron microscopy revealed a 70% increase in mitochondrial number (p=0.005) and 24% increase in mitochondrial area (p=0.04) in γδ HYPIELs relative to WT. Seahorse mitochondrial stress assays on γδ IELs showed a 50% increase in spare respiratory capacity in γδ HYPIELs compared to WT (p=0.014). Mitochondrial metabolism was shown to influence γδ T cell cytokine production, and similarly, we found that stimulation with PMA and ionomycin reduced the frequency of IFNγ +γδ IELs by 59% in γδ HYPrelative to WT (p<0.0001). We also observed that γδ HYPIELs exhibit higher CD39 expression compared to WT (p<0.0001), and a CD39 hisubset produces less IFNγ compared to CD39 negand CD39 intcells (p<0.0001) The addition of the ATP-synthase inhibitor oligomycin during IEL stimulation increased IFNγ production by both WT and γδ HYPIELs by 66% and 44%, respectively (p<0.0001). Together, our data demonstrate that the shift towards high CD39 expression and elevated mitochondrial metabolism contribute to decreased IFNγ production in γδ HYPIELs. Further understanding of the mechanisms regulating γδ IEL homeostasis and effector function may ultimately allow fine tuning of mucosal surveillance to confer protection against intestinal injury or infection while limiting aberrant cytotoxicity. Supported by NJCCR (COCR23PRF024)

Metabolic regulation of γδ intraepithelial lymphocytes.

Elucidating the relationship between cellular metabolism and T cell function has substantially advanced our understanding of how T cells are regulated in response to activation. The metabolic profiles of circulating or peripheral T cells have been well-described, yet less is known regarding how complex local microenvironments shape or modulate the bioenergetic profile of tissue-resident T lymphocytes. Intraepithelial lymphocytes expressing the γδ T cell receptor (γδ IEL) provide immunosurveillance of the intestinal epithelium to limit tissue injury and microbial invasion; however, their activation and effector responses occur independently of antigen recognition. In this review, we will summarize the current knowledge regarding γδ T cell and IEL metabolic profiles and how this informs our understanding of γδ IEL metabolism. We will also discuss the role of the gut microbiota in shaping the metabolic profile of these sentinel lymphocytes, and in turn, how these bioenergetics contribute to regulation of γδ IEL surveillance behavior and effector function. Improved understanding of the metabolic processes involved in γδ IEL homeostasis and function may yield novel strategies to amplify the protective functions of these cells in the context of intestinal health and disease.

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.

Type I interferon co‐stimulation enhances γδ intraepithelial lymphocyte IFNγ production

Intraepithelial lymphocytes (IEL) expressing the γδ T cell receptor (TCR) constantly survey the intestinal epithelium, facilitating a localized effector response to limit pathogen invasion. Despite constant γδ TCR triggering in vivo, these IELs remain immunologically quiescent, suggesting that a co‐stimulatory signal is required to overcome their activation threshold. Type I interferon (IFN) is a family of pro‐inflammatory cytokines that are critical for host defense against bacterial and viral infection. Activated γδ IELs limit viral replication in intestinal epithelial cells by producing type I IFNs, such as IFNα; however, the role of IFNα in mediating γδ IEL antiviral effector function remains unknown. To determine whether IFNα directly activates these sentinel lymphocytes, murine small intestinal γδ IELs were isolated, sorted, and stimulated ex vivo with 1μg/mL αCD3, 10 ng/mL IFNα, or both for 5 h. Following treatment with αCD3 and IFNα, we observed a 27%±9.3 increase in IFNγ+ γδ IELs (p<0.0001) and a 2.5‐fold increase in IFNγ mean fluorescence intensity (MFI)(p<0.0001) compared to αCD3 stimulation. Interestingly, IFNα treatment alone was not sufficient to induce IFNγ production. To determine the threshold of TCR signaling necessary for this enhanced IFNγ production, we treated γδ IELs with suboptimal concentrations of αCD3 (0.01 or 0.1 μg/mL), to mimic constant TCR triggering in vitro.At 0.1 μg/mL αCD3, IFNα induced a 27%±7.2 increase in frequency of IFNγ+ γδ IELs (p<0.01) and a significant increase in MFI of γδ IEL IFNγ production over αCD3 treatment alone (p<0.001). With a low level of TCR priming (0.1 μg/mL αCD3), a low dose of IFNα (0.1 ng/mL) was able to stimulate a 22%±6.8 increase in IFNγ+ γδ IELs compared to αCD3 treatment alone. However, higher concentrations of IFNα failed to further enhance γδ IEL IFNγ production. Stimulation of γδ IELs with IFNα ex vivo was sufficient to induce STAT1 and STAT4 phosphorylation; therefore, we asked whether STAT1 or STAT4 is required for IFNα‐induced IFNγ production. To this end, we treated WT, STAT1 KO, or STAT4 KO γδ IELs with 1 μg/mL αCD3, 10 ng/mL IFNα, or both and found that the IFNα‐induced increase in IFNγ production was abrogated in STAT4‐, but not STAT1‐deficient γδ IELs (p<0.0001). To assess changes in STAT1 and STAT4 expression in γδ IELs during acute viral infection, WT mice were infected orally with 1.5x106 PFU murine gamma herpesvirus (MHV). We observed a 16%±3 increase in the frequency of γδ IELs 3 days post‐infection compared to controls, which corresponded with a decrease in total STAT1 (MFI: 905±95 vs 638±102, p<0.01). Taken together, our data demonstrate that γδTCR signaling is required for IFNα‐induced IFNγ production in a STAT4‐dependent manner. These findings suggest that IFNα signaling in conjunction with constant TCR triggering promotes the rapid activation and expansion of γδ IELs, which may enhance their effector response to microbial pathogens.

Loss of γδ intraepithelial lymphocytes and reduced immunosurveillance of the epithelial barrier precedes the onset of Crohn’s disease‐like ileitis

Inflammatory bowel disease, including Crohn’s disease (CD), affects over 3 million Americans. Recently, single cell sequencing of resected ileal tissue from CD patients revealed a loss of γδ intraepithelial lymphocytes (IELs). IELs expressing the γδ T cell receptor (γδ IEL) bridge innate and adaptive immunity and migrate dynamically within the intestinal epithelium as a means of immunosurveillance. However, the contribution of γδ IELs to the initiation of ileitis remains unknown. We and others have found that the onset of histopathology in TNFΔARE/+mice, which develop a chronic CD‐like ileitis, occurs at 8 weeks of age. We profiled the IEL populations of TNFΔARE/+mice prior to and during the initial stages of ileal inflammation (4, 6, 8 and 10 weeks). In agreement with CD patient data, we observed about a 2‐fold reduction in the γδ IEL population in ilea of 8‐week‐old TNFΔARE/+mice compared to TNF+/+ (WT) littermates (p=0.05). Seeding of γδ IELs within the epithelial compartment is regulated in part by expression of epithelial butyrophilin‐like genes Btnl1 and Btnl6. We find that the expression of both Btnl1 and Btnl6is reduced in 4‐ and 6‐week‐old TNFΔARE/+mice (p=0.05). Furthermore, the frequency of EdU+ γδ IELs is decreased in TNFΔARE/+mice compared to WT littermates at 6 weeks of age (p=0.05). γδ IEL motility and function contribute to maintenance of an intact epithelial barrier, as we have recently demonstrated that γδ IELs facilitate shedding of apoptotic epithelial cells. Cleaved caspase 3 staining in fixed ileal tissue revealed increased shedding of apoptotic enterocytes in 6‐week‐old TNFΔARE/+mice compared to WTlittermates (p=0.05). Since γδ IEL surveillance behavior is altered during the shedding of apoptotic enterocyte, we next assessed γδ IEL motility in 6‐week‐old WT and TNFΔARE/+ mice using intravital microscopy. γδ IEL migratory behavior is significantly impaired in TNFΔARE/+ mice, as reflected by reduced track speed (7.9 vs 2.8 μm/min, p=0.005) and increased arrest coefficient (p=0.001) relative to WT mice. To determine the contribution of γδ IELs in the development of CD‐like ileitis, we generated TNFΔARE/+ mice expressing diphtheria toxin (DT) receptor driven by a γδ T‐cell‐specific promoter (TNFΔARE/+ TcrdGDL). Our preliminary findings indicate that DT‐treated 3‐week‐old TNFΔARE/+ TcrdGDL mice exhibit reduced survival compared to TNFΔARE/+ littermates treated with vehicle control (Mantel‐Cox log‐rank test, p=0.155). In summary, we report that the onset of ileal inflammation coincides with the reduction of γδ IELs, which may be attributed to decreased epithelial Btnl expression and impaired γδ IEL proliferation. Reduced survival of TNFΔARE/+ mice following γδ T cell depletion suggests that γδ IEL‐mediated maintenance and surveillance of the barrier is critical to maintain mucosal tolerance prior to disease onset. These studies suggest that restoring γδ IEL number or migratory behavior may be effective therapeutic strategies to maintain remission in CD patients.

Cell-type-specific gene expression profile by laser capture microdissection on mirror sections

Immuno-laser capture microdissection (Immuno-LCM) has been used to analyze cell-specific gene expression profiles. However, the usefulness of such a technique is frequently limited by RNA degradation. We, therefore, developed a rapid protocol of LCM on mirror sections, which allows for preserving RNA integrity. With such a procedure, we investigated cell-type-specific gene expression of γδ intraepithelial lymphocytes (IELs) in untreated celiac disease (CD).An increase in TGF-β mRNA expression levels was observed in γδ + IELs compared to intestinal enterocytes (IEs), whereas anti-inflammatory IL-10 mRNA production from γδ + IELs was lower compared to IEs.In untreated CD patients, the production of anti-inflammatory cytokines by γδ + IELs is suggestive of a regulatory function, thus playing a critical role in limiting inflammation. This work underscores the importance of LCM on mirror sections as a valuable tool to perform cell-type-specific molecular analysis in tissue.

γδ Intraepithelial Lymphocytes Facilitate Pathological Epithelial Cell Shedding Via CD103-Mediated Granzyme Release

Excessive shedding of apoptotic enterocytes into the intestinal lumen is observed in inflammatory bowel disease and is correlated with disease relapse. Based on their cytolytic capacity and surveillance behavior, we investigated whether intraepithelial lymphocytes expressing the γδ T cell receptor (γδ IELs) are actively involved in the shedding of enterocytes into the lumen. Intravital microscopy was performed on GFP γδ T cell reporter mice treated with intraperitoneal lipopolysaccharide (10 mg/kg) for 90 minutes to induce tumor necrosis factor-mediated apoptosis. Cell shedding in various knockout or transgenic mice in the presence or absence of blocking antibody was quantified by immunostaining for ZO-1 funnels and cleaved caspase-3 (CC3). Granzyme A and granzyme B release from ex vivo-stimulated γδ IELs was quantified by enzyme-linked immunosorbent assay. Immunostaining for γδ T cell receptor and CC3 was performed on duodenal and ileal biopsies from controls and patients with Crohn's disease. Intravital microscopy of lipopolysaccharide-treated mice revealed that γδ IELs make extended contact with shedding enterocytes. These prolonged interactions require CD103 engagement by E-cadherin, and CD103 knockout or blockade significantly reduced lipopolysaccharide-induced shedding. Furthermore, we found that granzymes A and B, but not perforin, are required for cell shedding. These extracellular granzymes are released by γδ IELs both constitutively and after CD103/E-cadherin ligation. Moreover, we found that the frequency of γδ IEL localization to CC3-positive enterocytes is increased in Crohn's disease biopsies compared with healthy controls. Our results uncover a previously unrecognized role for γδ IELs in facilitating tumor necrosis factor-mediated shedding of apoptotic enterocytes via CD103-mediated extracellular granzyme release.

DOP02 αEβ7 positive γδ T cells are associated with mucosal healing in Ulcerative Colitis and have a homeostatic immunophenotype

Abstract Background α4β7 blockade is a well-established therapy in ulcerative colitis (UC), acting in part by preventing lymphocyte ingress into the mucosa. The β7 unit of the α4β7 heterodimer is shared by α Εβ7, which is expressed on both tissue resident memory cells and γδ intra-epithelial lymphocytes (IEL). It was hypothesised that targeting both α4- and α Εβ7 might be more efficacious; however mixed results from phase III studies of β7 blockade asks questions of the biological relevance of different α Eβ7 expressing cells. Methods Colonic biopsies were obtained during endoscopy from >40 subjects. Lymphocytes were isolated using short term culture or digested from whole tissue. RNA sequencing was performed on α Εβ7pos and α Εβ7neg colonic γδ T cells from 4 donors and findings were validated by flow cytometry. Results α Εβ7 is widely expressed on TCRαβ CD8 cells and γδ IEL in both non-IBD controls and the uninflamed mucosa in UC, but its expression is significantly reduced on analogous subsets harvested from inflamed UC. On further study, the capacity of TCRαβ CD8 T cells to make TNFα and IFNγ on stimulation is similar between α Εβ7pos and α Εβ7neg cells, whereas in the γδ T cell compartment α Εβ7neg cells produce significantly more pro-inflammatory cytokine than their homeostatic α Εβ7pos counterparts. To examine the cells’ biology further, γδ T cells were isolated according to their α Εβ7 status and RNAseq undertaken. This revealed a distinct signature with α Εβ7neg cells demonstrating an activated phenotype high in markers such as CD18, CD5 and lymphoid homing receptor CCR7 whereas α Εβ7pos cells demonstrate a homeostatic tissue-resident phenotype, expressing immune checkpoints TIGIT and CD101 and gut-homing marker CCR9. On culturing tissue from non-IBD controls in pro-inflammatory cytokines, IL-12 and IL-18, the γδ T cell compartment down-regulated α Εβ7 and TIGIT and upregulated CD18, in part recapitulating a disease phenotype. On examining previously affected mucosa of patients who have achieved mucosal healing, α Eβ7 expression of the γδ T cells returned to a profile resembling non-IBD controls whereas the expression levels in the inflamed mucosa remained predictably low. Conclusion This study demonstrates that α Εβ7 expression is low in active UC but restored in mucosal healing. α Εβ7neg cells are proinflammatory with a distinct phenotype which may in part be recapitulated by inflammatory cytokines in vitro; whereas α Εβ7pos cells demonstrate a homeostatic phenotype, which may both reflect and maintain steady state barrier integrity. Hence in terms of γδ T cells, pharmacological β7 blockade has potential to interfere with the homeostatic roles of α Εβ7 expressing cells while having little effect on a potentially pathogenic subset of tissue α Εβ7neg γδ cells.

Type I interferon enhances γδ intraepithelial lymphocyte migratory behavior via CD47 upregulation

Abstract γδ intraepithelial lymphocytes (IEL) migrate along the basement membrane and into the lateral intracellular space (LIS) between adjacent intestinal epithelial cells (IEC) and is critical to limit microbial translocation across the barrier. Although activated γδ IELs produce interferon (IFN)-α, whether IFNα directly influences γδ IEL migratory behavior remains unknown. To test this, intravital microscopy was performed on GFP γδ T cell reporter mice treated with PBS or IFNα (1 μg, i.v., 3h). We found that γδ IEL track speed was increased 30% and migration into the LIS was enhanced by 75% in IFNα-treated mice compared to controls. To identify candidate genes involved in regulating γδ IEL motility, we performed RNAseq on γδ IELs isolated from control and IFNα-treated mice. As expected, IFNα induced IFN-stimulated gene expression including CD47 (3.5-fold increase), a transmembrane protein involved in neutrophil transepithelial migration. Stimulation of ex vivo cultured γδ IELs with IFNα resulted in a 3-fold increase in CD47 expression compared to unstimulated controls. Next, to investigate whether IFNα enhances γδ IEL motility in vitro, GFP γδ IELs were co-cultured with enteroids expressing membrane tdTomato and treated with 10ng/ml IFNα for 3h. IFNα increased γδ IEL speed (4.5±0.2 vs 3.4±0.2 μm/min; p<0.05) and displacement (47±3.0 μm vs 34±3.1, p<0.05) relative to untreated controls. Addition of anti-CD47 blocking antibody (MIAP301) abrogated the IFNα-mediated increase in γδ IEL migratory behavior. Taken together, these data demonstrate that IFNα enhances γδ IEL motility via a CD47-mediated mechanism, and suggests that CD47 may represent a conserved mechanism of leukocyte migration within the epithelial barrier.

Inulin and Lycium barbarum polysaccharides ameliorate diabetes by enhancing gut barrier via modulating gut microbiota and activating gut mucosal TLR2+ intraepithelial γδ T cells in rats

Previously we demonstrated the anti-diabetic effect of dietary inulin (INU), but the underling mechanism still remains unclear. In this study, 8 weeks treatment of INU with or without Lycium barbarum polysaccharides (LBP) decreased blood glucose, plasma lipopolysaccharide (LPS) and inflammation in diabetic rats. Moreover, altered gut microbial community and short-chain fatty acids (SCFAs) were found after the intervention. Intriguingly, these changes in gut microbiota were closely correlated with improved mucosal Toll-like receptor (TLR) 2+γδ intraepithelial lymphocytes (IELs) and tight-junction proteins (including ZO-1 and occludin) in gut barrier. Taken together, our results highlighted that dietary INU combined with LBP possessed the ability to modify gut microbiota and improve diabetes-related biochemical abnormalities through improving gut barrier by activating gut mucosal γδ IELs via TLR2, which may potentially contribute to the control of the disease.

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.

Dietary Factors and Mucosal Immune Response in Celiac Disease Patients Having Persistent Symptoms Despite a Gluten-free Diet.

The aim of this study was to investigate the role of dietary factors, distinct small-bowel mucosal immune cell types, and epithelial integrity in the perpetuation of gastrointestinal symptoms in treated celiac disease patients. For unexplained reasons, many celiac disease patients suffer from persistent symptoms, despite a strict gluten-free diet (GFD) and recovered intestinal mucosa. We compared clinical and serological data and mucosal recovery in 22 asymptomatic and 25 symptomatic celiac patients on a long-term GFD. The density of CD3 and γδ intraepithelial lymphocytes (IELs), CD25 and FOXP3 regulatory T cells, and CD117 mast cells, and the expression of tight junction proteins claudin-3 and occludin, heat shock protein 60, interleukin 15, and Toll-like receptor 2 and 4 were evaluated in duodenal biopsies. All subjects kept a strict GFD and had negative celiac autoantibodies and recovered mucosal morphology. The asymptomatic patients had higher mean fiber intake (20.2 vs. 15.2 g/d, P=0.028) and density of CD3 IELs (59.3 vs. 45.0 cell/mm, P=0.045) than those with persistent symptoms. There was a similar but nonsignificant trend in γδ IELs (17.9 vs. 13.5, P=0.149). There were no differences between the groups in other parameters measured. Low fiber intake may predispose patients to persistent symptoms in celiac disease. There were no differences between the groups in the markers of innate immunity, epithelial stress or epithelial integrity. A higher number of IELs in asymptomatic subjects may indicate that the association between symptoms and mucosal inflammation is more complicated than previously thought.

Duodenal mucosal immune cells in treatment-naive adult patients with celiac disease having different histological grades and controls.

It is hypothesized that the duodenal mucosal damage in patients with celiac disease (CeD) is caused by the mucosa-infiltrating lymphoid cells. This study aimed to analyze the immune effective and regulatory T (Treg) cells in duodenal biopsies from treatment-naive adult patients with CeD having different histological grades and controls. Dual-color immunohistochemical staining was done in a total of 234 duodenal biopsies, including 132 controls and 102 adult patients with CeD using CD20, CD3:CD4, CD3:CD8, CD4:FoxP3, CD8:FoxP3, and TCRαβ:TCRγδ antibodies. The density of these lymphoid cells in lamina propria and mucosal epithelium was compared between controls and CeD, with different modified Marsh grades. Densities of CD4+ T cells in lamina propria and CD8+γδ intraepithelial lymphocytes (IELs) were significantly more in biopsies from patients with CeD, than in controls. An increasing linear pattern of IELs, CD3+ T cells, and CD20+ B cells was observed with increasing grades of villous abnormalities. Although CD8+ FoxP3+ Treg cells were significantly more in biopsies from patients with CeD, there was no significant difference in CD4+ FoxP3+ Treg cell infiltrate between both the groups. Our finding in this observational study generates interest to study the local intestinal mucosal immunity in CeD in detail. A study to prove the failure of CD4+ FoxP3+ Treg cell recruitment in CeD and its direct functional impact may yield valuable information regarding loss of mucosal tolerance.

GPR55 regulates intraepithelial lymphocyte migration dynamics and susceptibility to intestinal damage.

Intraepithelial lymphocytes (IELs) of the small intestine are intimately associated with the epithelial cells. Yet, the factors controlling their migration and interaction dynamics are poorly understood. We demonstrate that GPR55, a receptor that mediates migration inhibition in response to lysophosphatidylinositol (LPI), negatively regulates T cell receptor γδ (TCRγδ) IEL accumulation in the small intestine. Intravital imaging studies show that GPR55-deficient IELs migrate faster and interact more extensively with epithelial cells. GPR55 also negatively regulates T cell homing to the small intestine and γδT cell egress from Peyer's patches. GPR55 deficiency or short-term antagonist treatment protects from nonsteroidal anti-inflammatory drug-induced increases in intestinal permeability. These findings identify a migration-inhibitory receptor that restrains IEL-epithelial cell cross-talk and show that antagonism of this receptor can protect from intestinal barrier dysfunction.

Flow cytometry of duodenal intraepithelial lymphocytes improves diagnosis of celiac disease in difficult cases

Diagnosis of celiac disease is difficult when the combined results of serology and histology are inconclusive. Studies using flow cytometry of intraepithelial lymphocytes (IELs) have found that celiac patients have increased numbers of γδ IELs, along with a decrease in CD3-CD103 + IELs. The objective of this article is to assess the role of flow cytometric analysis of IELs in the diagnosis of celiac disease in difficult cases. A total of 312 patients with suspicion of celiac disease were included in the study. Duodenal biopsy samples were used for histological assessment and for flow cytometric analysis of IELs. In 46 out of 312 cases (14.7%) the combination of serology and histology did not allow the confirmation or exclusion of celiac disease. HLA typing had been performed in 42 of these difficult cases. Taking into account HLA typing and the response to a gluten-free diet, celiac disease was excluded in 30 of these cases and confirmed in the remaining 12. Flow cytometric analysis of IELs allowed a correct diagnosis in 39 out of 42 difficult cases (92.8%) and had a sensitivity of 91.7% (95% CI: 61.5% to 99.8%) and a specificity of 93.3% (95% CI: 77.9% to 99.2%) for the diagnosis of celiac disease in this setting. Flow cytometric analysis of IELs is useful for the diagnosis of celiac disease in difficult cases.

Mice deficient in intestinal γδ intraepithelial lymphocytes display an altered intestinal O-glycan profile compared with wild-type littermates.

Intestinal γδ T-cell receptor-bearing intraepithelial lymphocytes (γδ IELs) play a multifaceted role in maintaining mucosal homeostasis. In order to investigate the relationship between O-glycosylation and inflammation, we carried out an in-depth mass spectrometric comparison of the intestinal O-glycosylation profile of mice lacking γδ IELs (TCRδ(-/-)) and of their wild-type (WT) littermates. A total of 69 nonsulfated and 59 sulfated compositional types of O-glycans were identified in the small intestine and colon of TCRδ(-/-) and WT mice. Our results demonstrated structural differences in intestinal glycosylation in TCRδ(-/-) mice compared with WT littermates. TCRδ(-/-) colons contained a lower proportion of core-2 structures and an increased proportion of core-1 structures whereas TCRδ(-/-) small intestines had a decreased percentage of core-3 structures. The glycan antennae in TCRδ(-/-) colon and small intestine showed altered structural diversity compared with WT mice. There were significant differences in the sialylated species between the TCRδ(-/-) and WT mice with the sialylated Tn antigen found exclusively in the TCRδ(-/-)small intestine, whereas the sulfation pattern remained mostly unchanged. These findings provide novel molecular insights underpinning the role of γδ IELs in maintaining gut homeostasis.

Small intestinal intraepithelial TCRγδ+ T lymphocytes are present in the premature intestine but selectively reduced in surgical necrotizing enterocolitis.

BackgroundGastrointestinal barrier immaturity predisposes preterm infants to necrotizing enterocolitis (NEC). Intraepithelial lymphocytes (IEL) bearing the unconventional T cell receptor (TCR) γδ (γδ IEL) maintain intestinal integrity and prevent bacterial translocation in part through production of interleukin (IL) 17.ObjectiveWe sought to study the development of γδ IEL in the ileum of human infants and examine their role in NEC pathogenesis. We defined the ontogeny of γδ IEL proportions in murine and human intestine and subjected tcrδ−/− mice to experimental gut injury. In addition, we used polychromatic flow cytometry to calculate percentages of viable IEL (defined as CD3+ CD8+ CD103+ lymphocytes) and the fraction of γδ IEL in surgically resected tissue from infants with NEC and gestational age matched non-NEC surgical controls.ResultsIn human preterm infants, the proportion of IEL was reduced by 66% in 11 NEC ileum resections compared to 30 non-NEC controls (p<0.001). While γδ IEL dominated over conventional αβ IEL early in gestation in mice and in humans, γδ IEL were preferential decreased in the ileum of surgical NEC patients compared to non-NEC controls (50% reduction, p<0.05). Loss of IEL in human NEC was associated with downregulation of the Th17 transcription factor retinoic acid-related orphan nuclear hormone receptor C (RORC, p<0.001). TCRδ-deficient mice showed increased severity of experimental gut injury (p<0.05) with higher TNFα expression but downregulation of IL17A.ConclusionComplimentary mouse and human data suggest a role of γδ IEL in IL17 production and intestinal barrier production early in life. Specific loss of the γδ IEL fraction may contribute to NEC pathogenesis. Nutritional or pharmacological interventions to support γδ IEL maintenance in the developing small intestine could serve as novel strategies for NEC prevention.