Discrepant Phenotyping of Monocytes Based on CX3CR1 and CCR2 Using Fluorescent Reporters and Antibodies.

Monocytes, as well as downstream macrophages and dendritic cells, are essential players in the immune system, fulfilling key roles in homeostasis as well as in inflammatory conditions. Conventionally, driven by studies on reporter models, mouse monocytes are categorized into a classical and a non-classical subset based on their inversely correlated surface expression of Ly6C/CCR2 and CX3CR1. Here, we aimed to challenge this concept by antibody staining and reporter mouse models. Therefore, we took advantage of Cx3cr1GFP and Ccr2RFP reporter mice, in which the respective gene was replaced by a fluorescent reporter protein gene. We analyzed the expression of CX3CR1 and CCR2 by flow cytometry using several validated fluorochrome-coupled antibodies and compared them with the reporter gene signal in these reporter mouse strains. Although we were able to validate the specificity of the fluorochrome-coupled flow cytometry antibodies, mouse Ly6Chigh classical and Ly6Clow non-classical monocytes showed no differences in CX3CR1 expression levels in the peripheral blood and spleen when stained with these antibodies. On the contrary, in Cx3cr1GFP reporter mice, we were able to reproduce the inverse correlation of the CX3CR1 reporter gene signal and Ly6C surface expression. Furthermore, differential CCR2 surface expression correlating with the expression of Ly6C was observed by antibody staining, but not in Ccr2RFP reporter mice. In conclusion, our data suggest that phenotyping strategies for mouse monocyte subsets should be carefully selected. In accordance with the literature, the suitability of CX3CR1 antibody staining is limited, whereas for CCR2, caution should be applied when using reporter mice.

Etrolizumab-s fails to control E-Cadherin-dependent co-stimulation of highly activated cytotoxic T cells.

Despite promising preclinical and earlier clinical data, a recent phase III trial on the anti-ß7 integrin antibody etrolizumab in Crohn's disease (CD) did not reach its primary endpoint. The mechanisms leading to this outcome are not well understood. Here we characterize the ß7+ T cell compartment from patients with CD in comparison to cells from individuals without inflammatory bowel disease. By flow cytometric, transcriptomic and functional profiling of circulating T cells, we find that triple-integrin-expressing (α4+ß7+ß1hi) T cells have the potential to home to the gut despite α4ß7 blockade and have a specific cytotoxic signature. A subset of triple-integrin-expressing cells readily acquires αE expression and could be co-stimulated via E-Cadherin-αEß7 interactions in vitro. Etrolizumab-s fails to block such αEß7 signalling at high levels of T cell stimulation. Consistently, in CD patients treated with etrolizumab, T cell activation correlates with cytotoxic signatures. Collectively, our findings might add one important piece to the puzzle to explain phase III trial results with etrolizumab, while they also highlight that αEß7 remains an interesting target for future therapeutic approaches in inflammatory bowel disease.

Blocking GPR15 counteracts integrin-dependent T cell gut homing in vivo.

BACKGROUND AND AIMS: The G protein coupled receptor GPR15 is expressed on and functionally important for T cells homing to the large intestine. However, the precise mechanisms by which GPR15 controls gut homing have been unclear. Thus, we aimed to elucidate these mechanisms as well as to explore the potential of targeting GPR15 for interfering with T cell recruitment to the colon in IBD. METHODS: We used dynamic adhesion and transmigration assays as well as a humanized in vivo model of intestinal cell trafficking to study GPR15-dependent effects on gut homing. Moreover, we analysed GPR15 and integrin expression in patients with and without IBD cross-sectionally and longitudinally. RESULTS: GPR15 controlled T cell adhesion to MAdCAM-1 and VCAM-1 upstream of α4ß7 and α4ß1 integrin, respectively. Consistently, high co-expression of these integrins with GPR15 was found on T cells from patients with IBD and GPR15 also promoted T cell recruitment to the colon in humanized mice. Anti-GPR15 antibodies effectively blocked T cell gut homing in vitro and in vivo. In vitro data as well as observations in a cohort of patients treated with vedolizumab suggest that this might be more effective than inhibiting α4ß7. CONCLUSIONS: GPR15 seems to have a broad, but organ-selective impact on T cell trafficking and is therefore a promising target for future therapy of IBD. Further studies are needed.

Differential Effects of Ontamalimab Versus Vedolizumab on Immune Cell Trafficking in Intestinal Inflammation and Inflammatory Bowel Disease.

BACKGROUND AND AIMS: The anti-MAdCAM-1 antibody ontamalimab demonstrated efficacy in a phase II trial in ulcerative colitis and results of early terminated phase III trials are pending, but its precise mechanisms of action are still unclear. Thus, we explored the mechanisms of action of ontamalimab and compared it to the anti-α4ß7 antibody vedolizumab. METHODS: We studied MAdCAM-1 expression with RNA sequencing and immunohistochemistry. The mechanisms of action of ontamalimab were assessed with fluorescence microscopy, dynamic adhesion and rolling assays. We performed in vivo cell trafficking studies in mice and compared ontamalimab and vedolizumab surrogate [-s] antibodies in experimental models of colitis and wound healing. We analysed immune cell infiltration under anti-MAdCAM-1 and anti-α4ß7 treatment by single-cell transcriptomics and studied compensatory trafficking pathways. RESULTS: MAdCAM-1 expression was increased in active inflammatory bowel disease. Binding of ontamalimab to MAdCAM-1 induced the internalization of the complex. Functionally, ontamalimab blocked T cell adhesion similar to vedolizumab, but also inhibited L-selectin-dependent rolling of innate and adaptive immune cells. Despite conserved mechanisms in mice, the impact of ontamalimab-s and vedolizumab-s on experimental colitis and wound healing was similar. Single-cell RNA sequencing demonstrated enrichment of ontamalimab-s-treated lamina propria cells in specific clusters, and in vitro experiments indicated that redundant adhesion pathways are active in these cells. CONCLUSIONS: Ontamalimab has unique and broader mechanisms of action compared to vedolizumab. However, this seems to be compensated for by redundant cell trafficking circuits and leads to similar preclinical efficacy of anti-α4ß7 and anti-MAdCAM-1 treatment. These results will be important for the interpretation of pending phase III data.

Manual cell selection in single cell transcriptomics using scSELpy supports the analysis of immune cell subsets.

Introduction: Single cell RNA sequencing plays an increasing and indispensable role in immunological research such as in the field of inflammatory bowel diseases (IBD). Professional pipelines are complex, but tools for the manual selection and further downstream analysis of single cell populations are missing so far. Methods: We developed a tool called scSELpy, which can easily be integrated into Scanpy-based pipelines, allowing the manual selection of cells on single cell transcriptomic datasets by drawing polygons on various data representations. The tool further supports the downstream analysis of the selected cells and the plotting of results. Results: Taking advantage of two previously published single cell RNA sequencing datasets we show that this tool is useful for the positive and negative selection of T cell subsets implicated in IBD beyond standard clustering. We further demonstrate the feasibility for subphenotyping T cell subsets and use scSELpy to corroborate earlier conclusions drawn from the dataset. Moreover, we also show its usefulness in the context of T cell receptor sequencing. Discussion: Collectively, scSELpy is a promising additive tool fulfilling a so far unmet need in the field of single cell transcriptomic analysis that might support future immunological research.

Anti-ß7 integrin treatment impedes the recruitment on non-classical monocytes to the gut and delays macrophage-mediated intestinal wound healing.

BACKGROUND: Closing mucosal defects to reach mucosal healing is an important goal of therapy in inflammatory bowel disease (IBD). Among other cells, monocyte-derived macrophages are centrally involved in such intestinal wound healing. We had previously demonstrated that the anti-α4ß7 integrin antibody vedolizumab blocks the recruitment of non-classical monocytes as biased progenitors of wound healing macrophages to the gut and delays wound healing. However, although important for the interpretation of disappointing results in recent phase III trials in IBD, the effects of the anti-ß7 antibody etrolizumab on wound healing are unclear so far. METHODS: We analyzed the expression of etrolizumab targets on human and mouse monocyte subsets by flow cytometry and assessed their function in adhesion and homing assays. We explored wound-associated monocyte recruitment dynamics with multi-photon microscopy and compared the effects of etrolizumab and vedolizumab surrogate (-s) antibodies on experimental wound healing and wound-associated macrophage abundance. Finally, we investigated wound healing macrophage signatures in the large intestinal transcriptome of patients with Crohn's disease treated with etrolizumab. RESULTS: Human and mouse non-classical monocytes expressed more αEß7 integrin than classical monocytes and were a target of etrolizumab-s, which blocked non-classical monocyte adhesion to MAdCAM-1 and E-Cadherin as well as gut homing in vivo. Intestinal wound healing was delayed on treatment with etrolizumab-s along with a reduction of peri-lesional wound healing macrophages. Wound healing macrophage signatures in the colon of patients with Crohn's disease were substantially down-regulated on treatment with etrolizumab, but not with placebo. CONCLUSIONS: Combined blockade of αEß7 and α4ß7 with etrolizumab seems to exceed the effect of anti-α4ß7 treatment on intestinal wound healing, which might help to inform further investigations to understand the recent observations in the etrolizumab phase III trial program.

Expression and function of α4ß7 integrin predict the success of vedolizumab treatment in inflammatory bowel disease.

Inflammatory bowel diseases are medically intractable and require constant therapy in many cases. While a growing number of biologicals and small molecules is available for treatment, a substantial portion of patients experiences primary non-response to these compounds and head-to-head evidence for therapy selection is scarce. Thus, approaches to predict treatment success in individual patients are a huge unmet need. We had previously suggested that the expression and function of α4ß7 integrin on T cells in the peripheral blood correlate to outcomes of therapy with the anti-α4ß7 integrin antibody vedolizumab. Here, we conducted a translational multicenter trial to prospectively evaluate this hypothesis. In a cohort of 89 patients with inflammatory bowel disease undergoing regular therapy with vedolizumab, lower baseline expression of α4ß7 was associated with short-term clinical response. Consistently, low α4ß7 expression in patients achieving remission predicted sustained remission in week 30. Moreover, high dynamic adhesion of CD4+ T cells to MAdCAM-1 and high reduction of adhesion by vedolizumab in vitro at baseline were associated with clinical remission. These data substantiate the potential of α4ß7 integrin function and expression to forecast outcomes of vedolizumab therapy. Further translational efforts are necessary to improve the performance of the assays and to implement the concept in clinical practice.

Vedolizumab blocks α4ß7 integrin-mediated T cell adhesion to MAdCAM-1 in microscopic colitis.

Background: In Crohn's disease and ulcerative colitis, the anti-α4ß7 integrin antibody vedolizumab has demonstrated efficacy in phase III trials and has been successfully used under real-world conditions. Occasionally, it has also been used in other forms of inflammatory bowel disease (IBD) such as microscopic colitis (MC). However, the mechanisms of vedolizumab in MC have not been studied to date. Therefore, we aimed to investigate the expression and functional role of gut-homing integrins and in particular α4ß7 integrin in a cohort study in MC. Methods: We studied the expression of gut homing integrins on T cells from patients with MC and healthy controls by flow cytometry. To investigate the function of α4ß7 integrin in MC and the potential of vedolizumab to block it, we used dynamic adhesion assays and transmigrations assays. Moreover, we describe two clinical cases of MC patients treated with vedolizumab. Results: A specific profile of gut homing markers can be found on T cells from patients with MC. α4ß7 integrin functionally leads to firm adhesion to MAdCAM-1 and supports transmigration. Vedolizumab is able to block both processes. In two cases of MC, we observed reduced clinical symptoms and histologic improvement upon therapy with vedolizumab. Conclusion: Our data suggest that α4ß7 mediates gut homing of T cells also in MC and that, on single cell level, vedolizumab blocks the function of α4ß7 in MC. Thus, we provide mechanistic evidence supporting vedolizumab as promising therapeutic option for MC.

Etrolizumab-s Does Not Induce Residual Trafficking of Regulatory T Cells.

BACKGROUND: Blocking immune cell gut homing via α4ß7 integrin with the monoclonal antibody vedolizumab is an established therapeutic strategy in inflammatory bowel disease. However, despite promising preclinical and phase 2 clinical data, the anti-ß7 antibody etrolizumab yielded disappointing results in a large phase 3 trial program in UC. Mechanistic explanations are still lacking. We have recently shown that vedolizumab is associated with residual homing of regulatory T (Treg) cells in a certain exposure range and aimed to investigate whether a similar mechanism applies for etrolizumab. METHODS: We used flow cytometry, competitive dynamic adhesion, and transmigration assays to assess binding of the etrolizumab surrogate (etrolizumab-s) antibody FIB504 to Treg and effector T cells (Teff) and to explore the impact on cell trafficking. RESULTS: We observed only minimal differences in the binding of etrolizumab-s to Treg and Teff cells. Dynamic adhesion and transmigration of Treg and Teff cells was not substantially differentially affected at relevant concentrations. The ß1+ and PI16+ Treg cells were only resistant to etrolizumab-s at low concentrations. CONCLUSIONS: Etrolizumab does not seem to induce notable residual trafficking of Treg cells. Thus, the Teff overweight in the inflamed gut might persist despite reduced overall T cell recruitment. This might be one piece of the puzzle to explain recent clinical results in phase 3.

The efficacy of etrolizumab in phase 3 was disappointing. Our data suggest that, unlike vedolizumab, etrolizumab does not induce relevant residual trafficking of regulatory T cells. This might be one part of the explanation for recent observations in clinical trials.

Limited Dose-Dependent Effects of Vedolizumab on Various Leukocyte Subsets.

OBJECTIVES: The anti-α4ß7 integrin antibody vedolizumab (VDZ) is successfully used for the treatment of inflammatory bowel diseases. However, only a subgroup of patients respond to therapy. VDZ is administered at a fixed dose, leading to a wide range of serum concentrations in patients. Previous work from our group showed a dose-dependent preferential binding of VDZ to effector compared with regulatory CD4 + T cells. Therefore, we aimed to determine the dose-dependent binding profile of VDZ to other leukocyte subsets. METHODS: We characterized α4ß7 integrin expression on CD8 + T cells, CD19 + B cells, CD14 + monocytes, natural killer cells, and eosinophils from patients with inflammatory bowel disease and healthy controls. We studied the binding of VDZ to these cells at different concentrations and investigated the functional consequences for dynamic adhesion and transmigration in vitro . RESULTS: The expression of α4ß7 differed between the analyzed leukocyte subsets and was significantly higher on eosinophils from inflammatory bowel disease patients compared with controls. Almost all α4ß7-expressing cells from these subsets were bound by VDZ at a concentration of 10 µg/mL. Dynamic cell adhesion was significantly impaired in all subsets, but there were no dose-dependent differences in the inhibition of adhesion. DISCUSSION: Our data suggest that α4ß7-expressing CD8 + T cells, CD19 + B cells, CD14 + monocytes, natural killer cells, and eosinophils are a target of VDZ. However, there do not seem to be concentration-dependent differences, regarding the effects on these cells in the clinically relevant range. Thus, the reported exposure-efficacy characteristic of VDZ can probably mainly be attributed to CD4 + T-cell subsets.

Nr4a1-dependent non-classical monocytes are important for macrophage-mediated wound healing in the large intestine.

Introduction: Macrophages play an important role in intestinal wound healing. However, the trajectories from circulating monocytes to gut macrophages are incompletely understood. Methods: Taking advantage of mice depleted for non-classical monocytes due to deficiency for the transcription factor Nr4a1, we addressed the relevance of non-classical monocytes for large intestinal wound healing using flow cytometry, in vivo wound healing assays and immunofluorescence. Results: We show that wound healing in Nr4a1-deficient mice is substantially delayed and associated with reduced peri-lesional presence of macrophages with a wound healing phenotype. Discussion: Our data suggest that non-classical monocytes are biased towards wound healing macrophages. These insights might help to understand, how targeting monocyte recruitment to the intestine can be used to modulate intestinal macrophage functions.

α4ß7 integrin-dependent adhesion of T cells to MAdCAM-1 is blocked by vedolizumab in patients with chronic refractory pouchitis.

BACKGROUND: The anti-α4ß7 integrin antibody vedolizumab is an established therapeutic option for the treatment of inflammatory bowel disease (IBD). It has also been successfully used in patients with chronic antibiotic-refractory pouchitis following proctocolectomey with ileal pouch-anal anastomosis. However, the expression and function of gut-homing markers as well as strategies to predict the response to vedolizumab in pouchitis are understudied so far. METHODS: We used flow cytometry and dynamic adhesion assays to study the expression and function of gut-homing integrins on T cells from patients with pouchitis and controls as well as longitudinally during therapy of pouchitis with vedolizumab. Moreover, we describe clinical effects of vedolizumab in a cohort of patients with pouchitis. RESULTS: T cells from patients with pouchitis express a specific profile of gut-homing integrins. Integrin α4ß7 on T cells from patients with pouchitis mediates adhesion to mucosal addressin cell adhesion molecule (MAdCAM)-1, which can be blocked by vedolizumab in vitro. Vedolizumab efficiently treats pouchitis in a portion of patients and response correlates with dynamic adhesion profiles to MAdCAM-1. CONCLUSION: Our data suggest that T cell trafficking seems to be important for the pathogenesis of pouchitis and support the therapeutic use of vedolizumab. Integrin function might serve as a biomarker to predict response to vedolizumab.

E-type prostanoid receptor 4 drives resolution of intestinal inflammation by blocking epithelial necroptosis.

Inflammatory bowel diseases present with elevated levels of intestinal epithelial cell (IEC) death, which compromises the gut barrier, activating immune cells and triggering more IEC death. The endogenous signals that prevent IEC death and break this vicious cycle, allowing resolution of intestinal inflammation, remain largely unknown. Here we show that prostaglandin E2 signalling via the E-type prostanoid receptor 4 (EP4) on IECs represses epithelial necroptosis and induces resolution of colitis. We found that EP4 expression correlates with an improved IBD outcome and that EP4 activation induces a transcriptional signature consistent with resolution of intestinal inflammation. We further show that dysregulated necroptosis prevents resolution, and EP4 agonism suppresses necroptosis in human and mouse IECs. Mechanistically, EP4 signalling on IECs converges on receptor-interacting protein kinase 1 to suppress tumour necrosis factor-induced activation and membrane translocation of the necroptosis effector mixed-lineage kinase domain-like pseudokinase. In summary, our study indicates that EP4 promotes the resolution of colitis by suppressing IEC necroptosis.

Circulating Adaptive Immune Cells Expressing the Gut Homing Marker α4ß7 Integrin Are Decreased in COVID-19.

Background: Infection with the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes a wide range of symptoms including gastrointestinal manifestations, and intestinal epithelial cells are a target of the virus. However, it is unknown how the intestinal immune system contributes to systemic immune responses in coronavirus disease 2019 (COVID-19). Methods: We characterized peripheral blood lymphocytes from patients with active COVID-19 and convalescent patients as well as healthy controls by flow cytometry. Results: The frequency and absolute number of circulating memory T and B cells expressing the gut homing integrin α4ß7 integrin was reduced during COVID-19, whether gastrointestinal symptoms were present or not. While total IgA-expressing B cells were increased, gut-imprinted B cells with IgA expression were stable. Conclusion: COVID-19 is associated with a decrease in circulating adaptive immune cells expressing the key gut homing marker α4ß7 suggesting that these cells are preferentially recruited to extra-intestinal tissues independently of α4ß7 or that the systemic immune response against SARS-CoV-2 is at least numerically dominated by extraintestinal, particularly pulmonary, immune cell priming.

Targeting Immune Cell Trafficking - Insights From Research Models and Implications for Future IBD Therapy.

Inflammatory bowel diseases (IBDs), including Crohn's disease (CD) and ulcerative colitis (UC) are multifactorial diseases with still unknown aetiology and an increasing prevalence and incidence worldwide. Despite plentiful therapeutic options for IBDs, the lack or loss of response in certain patients demands the development of further treatments to tackle this unmet medical need. In recent years, the success of the anti-α4ß7 antibody vedolizumab highlighted the potential of targeting the homing of immune cells, which is now an important pillar of IBD therapy. Due to its complexity, leukocyte trafficking and the involved molecules offer a largely untapped resource for a plethora of potential therapeutic interventions. In this review, we aim to summarise current and future directions of specifically interfering with immune cell trafficking. We will comment on concepts of homing, retention and recirculation and particularly focus on the role of tissue-derived chemokines. Moreover, we will give an overview of the mode of action of drugs currently in use or still in the pipeline, highlighting their mechanisms and potential to reduce disease burden.

Intestinal Mucosal Wound Healing and Barrier Integrity in IBD-Crosstalk and Trafficking of Cellular Players.

The intestinal epithelial barrier is carrying out two major functions: restricting the entry of potentially harmful substances while on the other hand allowing the selective passage of nutrients. Thus, an intact epithelial barrier is vital to preserve the integrity of the host and to prevent development of disease. Vice versa, an impaired intestinal epithelial barrier function is a hallmark in the development and perpetuation of inflammatory bowel disease (IBD). Besides a multitude of genetic, molecular and cellular alterations predisposing for or driving barrier dysintegrity in IBD, the appearance of intestinal mucosal wounds is a characteristic event of intestinal inflammation apparently inducing breakdown of the intestinal epithelial barrier. Upon injury, the intestinal mucosa undergoes a wound healing process counteracting this breakdown, which is controlled by complex mechanisms such as epithelial restitution, proliferation and differentiation, but also immune cells like macrophages, granulocytes and lymphocytes. Consequently, the repair of mucosal wounds is dependent on a series of events including coordinated trafficking of immune cells to dedicated sites and complex interactions among the cellular players and other mediators involved. Therefore, a better understanding of the crosstalk between epithelial and immune cells as well as cell trafficking during intestinal wound repair is necessary for the development of improved future therapies. In this review, we summarize current concepts on intestinal mucosal wound healing introducing the main cellular mediators and their interplay as well as their trafficking characteristics, before finally discussing the clinical relevance and translational approaches to therapeutically target this process in a clinical setting.

Clinical experiences and predictors of success of treatment with vedolizumab in IBD patients: a cohort study.

BACKGROUND: Vedolizumab has become a standard treatment for the inflammatory bowel diseases ulcerative colitis (UC) and Crohn's disease (CD). However, there is an ongoing debate on the ideal individual treatment algorithms and means to predict treatment response are not routinely established. AIMS: We aimed to describe our experiences with vedolizumab at a large German tertiary referral center and to identify clinical predictors of success of vedolizumab treatment. METHODS: We performed a retrospective single-center cohort study employing univariable and multivariable analyses as well as Kaplan-Meier analyses of persistence on treatment. RESULTS: 36% and 35% of the patients with UC and CD, respectively, reached clinical remission after 17 weeks. Patients with lower clinical disease activity were more likely to achieve remission. The median persistence on treatment was 33 months for UC and 29 months for CD. CONCLUSION: Our study confirms that vedolizumab is an efficient option for the treatment of UC and CD. Clinical parameters of disease activity may help to predict the success of treatment.

Immunology of IL-12: An update on functional activities and implications for disease.

As its first identified member, Interleukin-12 (IL-12) named a whole family of cytokines. In response to pathogens, the heterodimeric protein, consisting of the two subunits p35 and p40, is secreted by phagocytic cells. Binding of IL-12 to the IL-12 receptor (IL-12R) on T and natural killer (NK) cells leads to signaling via signal transducer and activator of transcription 4 (STAT4) and subsequent interferon gamma (IFN-γ) production and secretion. Signaling downstream of IFN-γ includes activation of T-box transcription factor TBX21 (Tbet) and induces pro-inflammatory functions of T helper 1 (TH1) cells, thereby linking innate and adaptive immune responses. Initial views on the role of IL-12 and clinical efforts to translate them into therapeutic approaches had to be re-interpreted following the discovery of other members of the IL-12 family, such as IL-23, sharing a subunit with IL-12. However, the importance of IL-12 with regard to immune processes in the context of infection and (auto-) inflammation is still beyond doubt. In this review, we will provide an update on functional activities of IL-12 and their implications for disease. We will begin with a summary on structure and function of the cytokine itself as well as its receptor and outline the signal transduction and the transcriptional regulation of IL-12 secretion. In the second part of the review, we will depict the involvement of IL-12 in immune-mediated diseases and relevant experimental disease models, while also providing an outlook on potential translational approaches.

Total Recall: Intestinal TRM Cells in Health and Disease.

Tissue-resident memory T cells (TRM cells) have crucial functions in host defense in mucosal tissues. They provide local adaptive immune surveillance and allow the fast initiation of targeted adaptive immune responses in case of antigen re-exposure. Recently, an aberrant activation in the case of immunologically mediated diseases has been increasingly acknowledged. As the organ with the largest interface to the environment, the gastrointestinal tract faces billions of antigens every day. Tightly balanced processes are necessary to ensure tolerance towards non-hazardous antigens, but to set up a powerful immune response against potentially dangerous ones. In this complex nexus of immune cells and their mediators, TRM cells play a central role and have been shown to promote both physiological and pathological events. In this review, we will summarize the current knowledge on the homeostatic functions of TRM cells and delineate their implication in infection control in the gut. Moreover, we will outline their commitment in immune dysregulation in gastrointestinal chronic inflammatory conditions and shed light on TRM cells as current and potential future therapeutic targets.