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Coeliac disease is a complex, polygenic inflammatory enteropathy caused by exposure to dietary gluten that occurs in a subset of genetically susceptible individuals who express either the HLA-DQ8 or HLA-DQ2 haplotypes 1,2 . The need to develop non-dietary treatments is now widely recognized 3 , but no pathophysiologically relevant gluten- and HLA-dependent preclinical model exists. Furthermore, although studies in humans have led to major advances in our understanding of the pathogenesis of coeliac disease 4 , the respective roles of disease-predisposing HLA molecules, and of adaptive and innate immunity in the development of tissue damage, have not been directly demonstrated. Here we describe a mouse model that reproduces the overexpression of interleukin-15 (IL-15) in the gut epithelium and lamina propria that is characteristic of active coeliac disease, expresses the predisposing HLA-DQ8 molecule, and develops villous atrophy after ingestion of gluten. Overexpression of IL-15 in both the epithelium and the lamina propria is required for the development of villous atrophy, which demonstrates the location-dependent central role of IL-15 in the pathogenesis of coeliac disease. In addition, CD4 + T cells and HLA-DQ8 have a crucial role in the licensing of cytotoxic T cells to mediate intestinal epithelial cell lysis. We also demonstrate a role for the cytokine interferon-γ (IFNγ) and the enzyme transglutaminase 2 (TG2) in tissue destruction. By reflecting the complex interaction between gluten, genetics and IL-15-driven tissue inflammation, this mouse model provides the opportunity to both increase our understanding of coeliac disease, and develop new therapeutic strategies.

Background & Aims: Gamma chain (γc) cytokines (interleukin [IL]2, IL4, IL7, IL9, IL15, and IL21) signal via a common γc receptor. IL2 regulates the immune response, whereas IL21 and IL15 contribute to development of autoimmune disorders, including celiac disease. We investigated whether BNZ-2, a peptide designed to inhibit IL15 and IL21, blocks these cytokines selectively and its effects on intraepithelial cytotoxic T cells., Methods: We obtained duodenal biopsies from 9 patients with potential celiac disease (positive results from tests for anti-TG2 but no villous atrophy), 30 patients with untreated celiac disease (with villous atrophy), and 5 patients with treated celiac disease (on a gluten-free diet), as well as 43 individuals without celiac disease (controls). We stimulated primary intestinal intraepithelial CD8 + T-cell lines, or CD8 + T cells directly isolated from intestinal biopsies, with γc cytokines in presence or absence of BNZ-2. Cells were analyzed by immunoblots, flow cytometry, or RNA-sequencing analysis for phosphorylation of signaling molecules, gene expression profiles, proliferation, and levels of granzyme B., Results: Duodenal tissues from patients with untreated celiac disease had increased levels of messenger RNAs encoding IL15 receptor subunit alpha (IL15RA) and IL21 compared with tissues from patients with potential celiac disease and controls. Activation of intraepithelial cytotoxic T cells with IL15 or IL21 induced separate signaling pathways; incubation of the cells with IL15 and IL21 cooperatively increased their transcriptional activity, proliferation, and cytolytic properties. BNZ-2 specifically inhibited the effects of IL15 and IL21, but not of other γc cytokines., Conclusions: We found increased expression of IL15RA and IL21 in duodenal tissues from patients with untreated celiac disease compared with controls. IL15 and IL21 cooperatively activated intestinal intraepithelial cytotoxic T cells. In particular, they increased their transcriptional activity, proliferation, and cytolytic activity. The peptide BNZ-2 blocked these effects, but not those of other γc cytokines, including IL2. BNZ-2 might be used to prevent cytotoxic T-cell-mediated tissue damage in complex immune disorders exhibiting upregulation of IL15 and IL21., (Copyright © 2020 AGA Institute. Published by Elsevier Inc. All rights reserved.)

Intraepithelial lymphocytes (IELs) expressing the γδ TCR (γδ IELs) provide continuous surveillance of the intestinal epithelium. However, the mechanisms regulating the basal motility of these cells within the epithelial compartment have not been well defined. We investigated whether IL-15 contributes to γδ IEL localization and migratory behavior in addition to its role in IEL differentiation and survival. Using advanced live cell imaging techniques in mice, we find that compartmentalized overexpression of IL-15 in the lamina propria shifts the distribution of γδ T cells from the epithelial compartment to the lamina propria. This mislocalization could be rescued by epithelial IL-15 overexpression, indicating that epithelial IL-15 is essential for γδ IEL migration into the epithelium. Furthermore, in vitro analyses demonstrated that exogenous IL-15 stimulates γδ IEL migration into cultured epithelial monolayers, and inhibition of IL-2Rβ significantly attenuates the basal motility of these cells. Intravital microscopy showed that impaired IL-2Rβ signaling induced γδ IEL idling within the lateral intercellular space, which resulted in increased early pathogen invasion. Similarly, the redistribution of γδ T cells to the lamina propria due to local IL-15 overproduction also enhanced bacterial translocation. These findings thus reveal a novel role for IL-15 in mediating γδ T cell localization within the intestinal mucosa and regulating γδ IEL motility and patrolling behavior as a critical component of host defense., (Copyright © 2018 by The American Association of Immunologists, Inc.)

Dysbiosis resulting in gut-microbiome alterations with reduced butyrate production are thought to disrupt intestinal immune homeostasis and promote complex immune disorders. However, whether and how dysbiosis develops before the onset of overt pathology remains poorly defined. Interleukin-15 (IL-15) is upregulated in distressed tissue and its overexpression is thought to predispose susceptible individuals to and have a role in the pathogenesis of celiac disease and inflammatory bowel disease (IBD). Although the immunological roles of IL-15 have been largely studied, its potential impact on the microbiota remains unexplored. Analysis of 16S ribosomal RNA-based inventories of bacterial communities in mice overexpressing IL-15 in the intestinal epithelium (villin-IL-15 transgenic (v-IL-15tg) mice) shows distinct changes in the composition of the intestinal bacteria. Although some alterations are specific to individual intestinal compartments, others are found across the ileum, cecum and feces. In particular, IL-15 overexpression restructures the composition of the microbiota with a decrease in butyrate-producing bacteria that is associated with a reduction in luminal butyrate levels across all intestinal compartments. Fecal microbiota transplant experiments of wild-type and v-IL-15tg microbiota into germ-free mice further indicate that diminishing butyrate concentration observed in the intestinal lumen of v-IL-15tg mice is the result of intrinsic alterations in the microbiota induced by IL-15. This reconfiguration of the microbiota is associated with increased susceptibility to dextran sodium sulfate-induced colitis. Altogether, this study reveals that IL-15 impacts butyrate-producing bacteria and lowers butyrate levels in the absence of overt pathology, which represent events that precede and promote intestinal inflammatory diseases.

Eicosanoids are inflammatory mediators that play a key but incompletely understood role in linking the innate and adaptive immune systems. Here, we show that cytotoxic effector T cells (CTLs) are capable of both producing and responding to cysteinyl leukotrienes (CystLTs), allowing for the killing of target cells in a T cell receptor-independent manner. This process is dependent on the natural killer receptor NKG2D and exposure to IL-15, a cytokine induced in distressed tissues. IL-15 and NKG2D signaling drives the up-regulation of key enzymes implicated in the synthesis of CystLTs, as well as the expression of CystLT receptors, suggesting a positive feedback loop. Finally, although the CystLT pathway has been previously linked to various allergic disorders, we provide unexpected evidence for its involvement in the pathogenesis of celiac disease (CD), a T helper 1 cell-mediated enteropathy induced by gluten. These findings provide new insights into the cytolytic signaling pathway of NKG2D and the pathogenesis of organ-specific immune disorders. Furthermore, they suggest that the blockade of CystLT receptors may represent a potent therapeutic target for CD or potentially other autoimmune disorders in which NKG2D has been implicated., (© 2015 Tang et al.)

Interleukin-15 (IL-15) exerts many biological functions essential for the maintenance and function of multiple cell types. Although its expression is tightly regulated, IL-15 upregulation has been reported in many organ-specific autoimmune disorders. In celiac disease, an intestinal inflammatory disorder driven by gluten exposure, the upregulation of IL-15 expression in the intestinal mucosa has become a hallmark of the disease. Interestingly, because it is overexpressed both in the gut epithelium and in the lamina propria, IL-15 acts on distinct cell types and impacts distinct immune components and pathways to disrupt intestinal immune homeostasis. In this article, we review our current knowledge of the multifaceted roles of IL-15 with regard to the main immunological processes involved in the pathogenesis of celiac disease., (© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

NK cells are large granular lymphocytes that form a critical component of the innate immune system, whose functions include the killing of cells expressing stress-induced molecules. It is increasingly accepted that despite being considered prototypical effector cells, NK cells require signals to reach their full cytotoxic potential. We previously showed that IL-15 is capable of arming CD8 effector T cells to kill independently of their TCR via NKG2D in a cPLA2-dependent process. As NK cells also express NKG2D, we wanted to investigate whether this pathway functioned in an analogous manner and if resting NK cells could be primed to the effector phase by IL-15. Furthermore, to establish relevance to human disease we studied a possible role for this pathway in the pathogenesis of psoriatic arthritis, since there are aspects of this disease that suggest a potential effector role for the innate immune system. We found that PsA patients had upregulated IL-15 and MIC in their affected synovial tissues, and that this unique inflammatory environment enabled NK cell activation and killing via NKG2D and cPLA2. Moreover, we were able to reproduce the phenotype of joint NK cells from blood NK cells by incubating them with IL-15. Altogether, these findings suggest a destructive role for NK cells when activated by environmental stress signals during the pathogenesis of PsA and demonstrate that IL-15 is capable of priming resting NK cells in tissues to the effector phase.

Increased serum levels of IL-15 are reported in type 1 diabetes (T1D). Here we report elevated serum soluble IL-15Rα levels in human T1D. To investigate the role of IL-15/IL-15Rα in the pathogenesis of T1D, we generated double transgenic mice with pancreatic β-cell expression of IL-15 and IL-15Rα. The mice developed hyperglycemia, marked mononuclear cell infiltration, β-cell destruction, and anti-insulin autoantibodies that mimic early human T1D. The diabetes in this model was reversed by inhibiting IL-15 signaling with anti-IL2/IL15Rβ (anti-CD122), which blocks IL-15 transpresentation. Furthermore, the diabetes could be reversed by administration of the Janus kinase 2/3 inhibitor tofacitinib, which blocks IL-15 signaling. In an alternative diabetes model, nonobese diabetic mice, IL15/IL-15Rα expression was increased in islet cells in the prediabetic stage, and inhibition of IL-15 signaling with anti-CD122 at the prediabetic stage delayed diabetes development. In support of the view that these observations reflect the conditions in humans, we demonstrated pancreatic islet expression of both IL-15 and IL-15Rα in human T1D. Taken together our data suggest that disordered IL-15 and IL-15Rα may be involved in T1D pathogenesis and the IL-15/IL15Rα system and its signaling pathway may be rational therapeutic targets for early T1D.

A major challenge of cancer immunotherapy is the persistence and outgrowth of subpopulations that lose expression of the target antigen. IL-15 is a potent cytokine that can promote organ-specific autoimmunity when up-regulated on tissue cells. Here we report that T cells eradicated 2-wk-old solid tumors that expressed IL-15, eliminating antigen-negative cells. In contrast, control tumors that lacked IL-15 expression consistently relapsed. Interestingly, even tumors lacking expression of cognate antigen were rejected when expressing IL-15, indicating that rejection after adoptive T-cell transfer was independent of cognate antigen expression. Nevertheless, the T-cell receptor of the transferred T cells influenced the outcome, consistent with the notion that T-cell receptor activation and effector status determine whether IL-15 can confer lymphokine killer activity-like properties to T cells. The effect was limited to the microenvironment of tumors expressing IL-15; there were no noticeable effects on contralateral tumors lacking IL-15. Taken together, these results indicate that expression of IL-15 in the tumor microenvironment may prevent the escape of antigen loss variants and subsequent tumor recurrence by enabling T cells to eliminate cancer cells lacking cognate antigen expression in a locally restricted manner.

Natural killer (NK) cells inhibit early stages of tumor formation, recurrence, and metastasis. Here, we show that NK cells can also eradicate large solid tumors. Eradication depended on the massive infiltration of proliferating NK cells due to interleukin 15 (IL-15) released and presented by the cancer cells in the tumor microenvironment. Infiltrating NK cells had the striking morphologic feature of being densely loaded with periodic acid-Schiff-positive, diastase-resistant granules, resembling uterine NK cells. Perforin-mediated killing by these densely granulated NK cells was essential for tumor eradication. Expression of the IL-15 receptor α on cancer cells was needed to efficiently induce granulated NK cells, and expression on host stromal cells was essential to prevent tumor relapse after near complete destruction. These results indicate that IL-15 released at the cancer site induces highly activated NK cells that lead to eradication of large solid tumors.

Under physiological conditions the gut-associated lymphoid tissues not only prevent the induction of a local inflammatory immune response, but also induce systemic tolerance to fed antigens. A notable exception is coeliac disease, where genetically susceptible individuals expressing human leukocyte antigen (HLA) HLA-DQ2 or HLA-DQ8 molecules develop inflammatory T-cell and antibody responses against dietary gluten, a protein present in wheat. The mechanisms underlying this dysregulated mucosal immune response to a soluble antigen have not been identified. Retinoic acid, a metabolite of vitamin A, has been shown to have a critical role in the induction of intestinal regulatory responses. Here we find in mice that in conjunction with IL-15, a cytokine greatly upregulated in the gut of coeliac disease patients, retinoic acid rapidly activates dendritic cells to induce JNK (also known as MAPK8) phosphorylation and release the proinflammatory cytokines IL-12p70 and IL-23. As a result, in a stressed intestinal environment, retinoic acid acted as an adjuvant that promoted rather than prevented inflammatory cellular and humoral responses to fed antigen. Altogether, these findings reveal an unexpected role for retinoic acid and IL-15 in the abrogation of tolerance to dietary antigens.

IL-15 and NKG2D promote autoimmunity and celiac disease by arming cytotoxic T lymphocytes (CTLs) to cause tissue destruction. However, the downstream signaling events underlying these functional properties remain unclear. Here, we identify cytosolic phospholipase A(2) (cPLA(2)) as a central molecule in NKG2D-mediated cytolysis in CTLs. Furthermore, we report that NKG2D induces, upon recognition of MIC(+) target cells, the release of arachidonic acid (AA) by CTLs to promote tissue inflammation in association with target killing. Interestingly, IL-15, which licenses NKG2D-mediated lymphokine killer activity in CTLs, cooperates with NKG2D to induce cPLA(2) activation and AA release. Finally, cPLA(2) activation in intraepithelial CTLs of celiac patients provides an in vivo pathophysiological dimension to cPLA(2) activation in CTLs. These results reveal an unrecognized link between NKG2D and tissue inflammation, which may underlie the emerging role of NKG2D in various immunopathological conditions and define new therapeutic targets.

The intestinal mucosal immune response must differentiate between harmless foreign antigens and pathogens, a distinction that may depend upon changes in the cytokine milieu. A key cytokine in the adaptive immune response is interleukin-12 (IL-12), secreted by antigen-presenting cells (APC) immediately after encounter with a pathogen. IL-12 is important in the priming and polarization of naïve T cells. Here, we show that IL-12 and IL-15 direct human intestinal lamina propria lymphocytes (LPL) in the absence of T-cell receptor engagement to secrete extremely high amounts of interferon-gamma (IFN-gamma), greater than with any other stimulus. The functional synergy of IL-12 with IL-15 surprisingly operates independently of signal transducer and activator of transcription 1 (STAT1), STAT3, STAT4, or STAT5 phosphorylation and occurs during transcription. Four-colour immunofluorescence showed that IL-12 receptor beta1 is found on the CD4+ T cells expressing intracytoplasmic IFN-gamma. Importantly, IL-12 receptors beta1 and beta2 are not up-regulated by IL-12, unlike findings using antigen-specific T cells, and are lost over time. This study demonstrates the early and massive IFN-gamma response of LPL to IL-12 and IL-15, providing the tools to deal with a pathogen. The down-regulation of IL-12 receptors may curtail any excess damaging inflammation.

A major function of NKG2D linking innate and adaptive immunity is to upregulate antigen-specific CTL-mediated cytotoxicity in tissues expressing stress-induced NKG2D ligands, such as MIC, by coactivating TCR signaling. Here, we show that, under conditions of dysregulated IL15 expression in vivo in patients with celiac disease and in vitro in healthy individuals, multiple steps of the NKG2D/DAP10 signaling pathway leading to ERK and JNK activation are coordinately primed to activate direct cytolytic function independent of TCR specificity in effector CD8 T cells. These findings may not only explain previous reports of transformation of CTL into NK-like "lymphokine-activated killers" (LAK cells) under high doses of IL2 (a substitute for IL15) but may also have significant implications for understanding and treating immunopathological diseases.

Unlike primary T cells in lymph nodes, effector CD8(+) CTL in tissues do not express the costimulatory receptor CD28. We report that NKG2D, the receptor for stress-induced MICA and MICB molecules expressed in the intestine, serves as a potent costimulatory receptor for CTL freshly isolated from the human intestinal epithelium. Expression and function of NKG2D are selectively up-regulated by the cytokine IL-15, which is released by the inflamed intestinal epithelium. These findings identify a novel CTL costimulatory pathway regulated by IL-15 and suggest that tissues can fine-tune the activation of effector T cells based on the presence or absence of stress and inflammation. Uncontrolled secretion of IL-15 could lead to excessive induction of NKG2D and thus contribute to the development of autoimmune disease by facilitating the activation of autoreactive T cells.