γδ T cells in human colon adenocarcinomas comprise mainly Vδ1, Vδ2, and Vδ3 cells with distinct phenotype and function.

Γδ T cell infiltration into tumours usually correlates with improved patient outcome, but both tumour-promoting and tumoricidal effects of γδ T cells have been documented. Human γδ T cells can be divided into functionally distinct subsets based on T cell receptor (TCR) Vδ usage. Still, the contribution of these different subsets to tumour immunity remains elusive. Here, we provide a detailed γδ T cell profiling in colon tumours, using mass and flow cytometry, mRNA quantification, and TCR sequencing. δ chain usage in both the macroscopically unaffected colon mucosa and tumours varied considerably between patients, with substantial fractions of Vδ1, Vδ2, and non-Vδ1 Vδ2 cells. Sequencing of the Vδ complementarity-determining region 3 showed that almost all non-Vδ1 Vδ2 cells used Vδ3 and that tumour-infiltrating γδ clonotypes were unique for every patient. Non-Vδ1Vδ2 cells from colon tumours expressed several activation markers but few NK cell receptors and exhaustion markers. In addition, mRNA analyses showed that non-Vδ1 Vδ2 cells expressed several genes for proteins with tumour-promoting functions, such as neutrophil-recruiting chemokines, Galectin 3, and transforming growth factor-beta induced. In summary, our results show a large variation in γδ T cell subsets between individual tumours, and that Vδ3 cells make up a substantial proportion of γδ T cells in colon tumours. We suggest that individual γδ T cell composition in colon tumours may contribute to the balance between favourable and adverse immune responses, and thereby also patient outcome.

Immune checkpoint blockade improves the activation and function of circulating mucosal-associated invariant T (MAIT) cells in patients with non-small cell lung cancer.

Mucosal-associated invariant T (MAIT) cells constitute one of the most numerous unconventional T cell subsets, and are characterized by rapid release of Th1- and Th17-associated cytokines and increased cytotoxic functions following activation. MAIT cells accumulate in tumor tissue but show an exhausted phenotype. Here, we investigated if immune checkpoint blockade (ICB) with antibodies to PD-1 or PD-L1 affects the function of circulating MAIT cells from non-small cell lung cancer patients. ICB increased the proliferation and co-expression of the activation markers HLA-DR and CD38 on MAIT cells in most patients after the first treatment cycle, irrespective of treatment outcome. Furthermore, production of cytokines, especially TNF and IL-2, also increased after treatment, as did MAIT cell polyfunctionality. These results indicate that MAIT cells respond to ICB, and that MAIT cell reinvigoration may contribute to tumor regression in patients undergoing immune checkpoint therapy.

Immune cell infiltrates in peritoneal metastases from colorectal cancer.

Background: The presence of peritoneal metastases (PMs) in patients with colorectal cancer (CRC) confers a poor prognosis and only a minority of patients will benefit from the available treatment options. In primary CRC tumors, it is well established that a high infiltration of CD8+ effector T cells correlates to a favorable patient outcome. In contrast, the immune response induced in PMs from CRC and how it relates to patient survival is still unknown. In this study, we characterized the immune infiltrates and the distribution of immune checkpoint receptors on T cells from PMs from CRC, in order to evaluate the potential benefit of checkpoint blockade immunotherapy for this patient group. Methods: Surgically resected PM tissue from CRC patients (n=22) and synchronous primary tumors (n=8) were processed fresh to single cell suspensions using enzymatic digestion. Surface markers and cytokine production were analyzed using flow cytometry. Results: T cells dominated the leukocyte infiltrate in the PM specimens analyzed, followed by monocytes and B cells. Comparing two different PMs from the same patient usually showed a similar distribution of immune cells in both samples. The T cell infiltrate was characterized by an activated phenotype and markers of exhaustion were enriched compared with matched circulating T cells, in particular the checkpoint receptors PD-1 and TIGIT. In functional assays most cytotoxic and helper T cells produced INF-γ and TNF following polyclonal stimulation, while few produced IL-17, indicating a dominance of Th1-type responses in the microenvironment of PMs. Conclusion: Immune cells were present in all PMs from CRC examined. Although infiltrating T cells express markers of exhaustion, they produce Th1-type cytokines when stimulated. These results indicate the possibility to augment tumor-specific immune responses within PMs using checkpoint blockade inhibitors.

Immune Microenvironment in Sporadic Early-Onset versus Average-Onset Colorectal Cancer.

The incidence of left-sided colon and rectal cancer in young people are increasing worldwide, but its causes are poorly understood. It is not clear if the tumor microenvironment is dependent on age of onset, and little is known about the composition of tumor-infiltrating T cells in early-onset colorectal cancer (EOCRC). To address this, we investigated T-cell subsets and performed gene expression immune profiling in sporadic EOCRC tumors and matched average-onset colorectal cancer (AOCRC) tumors. Left-sided colon and rectal tumors from 40 cases were analyzed; 20 EOCRC (<45 years) patients were matched 1:1 to AOCRC (70-75 years) patients by gender, tumor location, and stage. Cases with germline pathogenic variants, inflammatory bowel disease or neoadjuvant-treated tumors were excluded. For T cells in tumors and stroma, a multiplex immunofluorescence assay combined with digital image analysis and machine learning algorithms was used. Immunological mediators in the tumor microenvironment were assessed by NanoString gene expression profiling of mRNA. Immunofluorescence revealed no significant difference between EOCRC and AOCRC with regard to infiltration of total T cells, conventional CD4+ and CD8+ T cells, regulatory T cells, or γδ T cells. Most T cells were located in the stroma in both EOCRC and AOCRC. Immune profiling by gene expression revealed higher expression in AOCRC of the immunoregulatory cytokine IL-10, the inhibitory NK cell receptors KIR3DL3 and KLRB1 (CD161), and IFN-a7 (IFNA7). In contrast, the interferon-induced gene IFIT2 was more highly expressed in EOCRC. However, in a global analysis of 770 tumor immunity genes, no significant differences could be detected. T-cell infiltration and expression of inflammatory mediators are similar in EOCRC and AOCRC. This may indicate that the immune response to cancer in left colon and rectum is not related to age of onset and that EOCRC is likely not driven by immune response deficiency.

Lipoxins reduce obesity-induced adipose tissue inflammation in 3D-cultured human adipocytes and explant cultures.

Adipose tissue inflammation drives obesity-related cardiometabolic diseases. Enhancing endogenous resolution mechanisms through administration of lipoxin A4, a specialized pro-resolving lipid mediator, was shown to reduce adipose inflammation and subsequently protects against obesity-induced systemic disease in mice. Here, we demonstrate that lipoxins reduce inflammation in 3D-cultured human adipocytes and adipose tissue explants from obese patients. Approximately 50% of patients responded particularly well to lipoxins by reducing inflammatory cytokines and promoting an anti-inflammatory M2 macrophage phenotype. Responding patients were characterized by elevated systemic levels of C-reactive protein, which causes inflammation in cultured human adipocytes. Responders appeared more prone to producing anti-inflammatory oxylipins and displayed elevated prostaglandin D2 levels, which has been interlinked with transcription of lipoxin-generating enzymes. Using explant cultures, this study provides the first proof-of-concept evidence supporting the therapeutic potential of lipoxins in reducing human adipose tissue inflammation. Our data further indicate that lipoxin treatment may require a tailored personalized-medicine approach.

Lipoxins modulate neutrophil oxidative burst, integrin expression and lymphatic transmigration differentially in human health and atherosclerosis.

Dysregulated chronic inflammation plays a crucial role in the pathophysiology of atherosclerosis and may be a result of impaired resolution. Thus, restoring levels of specialized pro-resolving mediators (SPMs) to promote the resolution of inflammation has been proposed as a therapeutic strategy for patients with atherosclerosis, in addition to standard clinical care. Herein, we evaluated the effects of the SPM lipids, lipoxin A4 (LXA4 ) and lipoxin B4 (LXB4 ), on neutrophils isolated from patients with atherosclerosis compared with healthy controls. Patients displayed altered endogenous SPM production, and we demonstrated that lipoxin treatment in whole blood from atherosclerosis patients attenuates neutrophil oxidative burst, a key contributor to atherosclerotic development. We found the opposite effect in neutrophils from healthy controls, indicating a potential mechanism whereby lipoxins aid the endogenous neutrophil function in health but reduce its excessive activation in disease. We also demonstrated that lipoxins attenuated upregulation of the high-affinity conformation of the CD11b/CD18 integrin, which plays a central role in clot activation and atherosclerosis. Finally, LXB4 enhanced lymphatic transmigration of human neutrophils isolated from patients with atherosclerosis. This finding is noteworthy, as impaired lymphatic function is now recognized as an important contributor to atherosclerosis. Although both lipoxins modulated neutrophil function, LXB4 displayed more potent effects than LXA4 in humans. This study highlights the therapeutic potential of lipoxins in atherosclerotic disease and demonstrates that the effect of these SPMs may be specifically tailored to the need of the individual.

Antigen Presenting Cells from Tumor and Colon of Colorectal Cancer Patients Are Distinct in Activation and Functional Status, but Comparably Responsive to Activated T Cells.

Although mouse models of CRC treatments have demonstrated robust immune activation, it remains unclear to what extent CRC patients' APCs and TILs interact to fuel or quench treatment-induced immune responses. Our ex vivo characterization of tumor and adjacent colon cell suspensions suggest that contrasting environments in these tissues promoted inversed expression of T cell co-stimulatory CD80, and co-inhibitory programmed death (PD)-ligand1 (PD-L1) on intratumoral vs. colonic APCs. While putative tumor-specific CD103+CD39+CD8+ TILs expressed lower CD69 (early activation marker) and higher PD-1 (extended activation/exhaustion marker) than colonic counterparts, the latter had instead higher CD69 and lower PD-1 levels. Functional comparisons showed that intratumoral APCs were inferior to colonic APCs regarding protein uptake and upregulation of CD80 and PD-L1 after protein degradation. Our attempt to model CRC treatment-induced T cell activation in vitro showed less interferon (IFN)-γ production by TILs than colonic T cells. In this model, we also measured APCs' CD80 and PD-L1 expression in response to activated co-residing T cells. These markers were comparable in the two tissues, despite higher IFN- γ exposure for colonic APCs. Thus, APCs within distinct intratumoral and colonic milieus showed different activation and functional status, but were similarly responsive to signals from induced T cell activation.

Intratumoral regulatory T cells from colon cancer patients comprise several activated effector populations.

BACKGROUND: Intratumoral regulatory T cells (Treg) in colon cancer are a heterogeneous cell population, with potential impact on patient outcome. Generally, a high Treg infiltration has been correlated to a worse patient outcome, but it is still unclear how the composition of different Treg subsets affects patient relapse and survival. In this study, we used mass and flow cytometry to characterize Treg in colon tumors and corresponding unaffected tissue, followed by a correlation to clinical parameters and patient outcome. RESULTS: Using mass cytometry, we defined 13 clusters of intestinal Treg, three of which were enriched in the tumors. The two most enriched clusters were defined by their expression of the proliferation marker Ki67 and CD56, respectively. The Treg accumulating in the tumors expressed inducible T-cell co-stimulator (ICOS), OX-40, and CD39, indicating that they were effector Treg (eTreg). Intratumoral CD39+ Treg also had a higher expression of Foxp3, suggesting a higher suppressive activity, and we subsequently used CD39 as a marker for eTreg. Our further studies showed that colon tumors can be divided into two tumor groups, based on the proportion of CD39+ putative eTreg in the tumors. This property was independent of both tumor microsatellite status and tumor stage, which are important factors in predicting cancer disease progression. In a prospective study of forty-four colon cancer patients, we also showed that patients with a high CD39 expression on tumor-infiltrating Treg have a tendency towards a less favorable patient outcome in terms of cumulative cancer-specific survival. CONCLUSIONS: This study uncovers novel subsets of tumor-infiltrating Treg in colon cancer, and suggests that CD39 may be a potential therapeutic target in patients with microsatellite stable colon tumors, which are usually refractory to checkpoint blockade therapy.

Regulatory T cells reduce endothelial neutral sphingomyelinase 2 to prevent T-cell migration into tumors.

Endothelial cells are key regulators of transendothelial migration and their secretion of chemokines and expression of adhesion molecules facilitates lymphocyte entry into tissues. Previously, we demonstrated that Tregs can reduce transendothelial migration of T cells into tumors by decreasing endothelial CXCL10 secretion, but the mechanism by which this occurs is still not known. In this study, we aimed to define how Tregs decrease transendothelial migration into tumors. mRNA sequencing of intestinal tumor endothelial cells from Treg depleted mice identified neutral sphingomyelinase 2 (nSMase2) as a gene downregulated in the presence of Tregs. nSMase2 is expressed in human umbilical vein endothelial cells (HUVECs) and was decreased after coculture with Tregs. Furthermore, blocking of nSMase2 activity in vitro decreased VCAM1, CX3CL1, and CXCL10 expression in HUVECs, mirroring the same decrease found in Treg cocultures. In the APCmin/+ mouse model of intestinal cancer, nSMase2 is lower in tumor endothelial cells than in unaffected small intestine and chronic treatment with a nSMase2 inhibitor suppressed the increased migration that is otherwise seen in the absence of Tregs. We conclude that nSMase2 is an important mediator in endothelial cells supporting transendothelial migration, which may be targeted by Tregs to reduce T-cell migration into tumors.

Exhaustion in tumor-infiltrating Mucosal-Associated Invariant T (MAIT) cells from colon cancer patients.

Mucosal-associated invariant T (MAIT) cells are unconventional T cells recognizing microbial metabolites, presented by the invariant MR1 protein. Upon activation, MAIT cells rapidly secrete cytokines and exert cytotoxic functions, and may thus be highly relevant also in tumor immunity. MAIT cells accumulate in colon tumors, but in contrast to other cytotoxic T cell subsets, their presence in tumors has been associated with worse patient outcome. Here we investigated if exhaustion may contribute to reduced anti-tumor immunity by MAIT cells. Freshly isolated lymphocytes from colon tumors, unaffected tissue and blood from the same patients were analyzed by flow cytometry to detect MAIT cells with effector functions that are relevant for tumor immunity, and their expression of inhibitory receptors and other exhaustion markers. Our studies show that MAIT cells with a PD-1highTim-3+CD39+ terminally exhausted phenotype and an increased proliferation accumulate in colon tumors. The exhausted MAIT cells have reduced polyfunctionality with regard to production of important anti-tumor effector molecules, and blocking antibodies to PD-1 partly improved activation of tumor-infiltrating MAIT cells in vitro. We conclude that the tumor microenvironment leads to exhaustion not only of conventional T cells, but also MAIT cells, and that checkpoint blockade therapy may be useful also to reinvigorate tumor-infiltrating MAIT cells.

Specialized Pro-Resolving Mediators and the Lymphatic System.

Diminished lymphatic function and abnormal morphology are common in chronic inflammatory diseases. Recent studies are investigating whether it is possible to target chronic inflammation by promoting resolution of inflammation, in order to enhance lymphatic function and attenuate disease. Resolution of inflammation is an active process regulated by bioactive lipids known as specialized pro-resolving mediators (SPMs). SPMs can modulate leukocyte migration and function, alter cytokine/chemokine release, modify autophagy, among other immune-related activities. Here, we summarize the role of the lymphatics in resolution of inflammation and lymphatic impairment in chronic inflammatory diseases. Furthermore, we discuss the current literature describing the connection between SPMs and the lymphatics, and the possibility of targeting the lymphatics with innovative SPM therapy to promote resolution of inflammation and mitigate disease.

Regulatory T cells specifically suppress conventional CD8αß T cells in intestinal tumors of APCMin/+ mice.

The presence of activated T cells in colorectal cancer tissues is a strong predictor of patient survival. Our previous studies have shown that regulatory T cells (Treg) are able to reduce T cell transendothelial migration in vitro and accumulation of effector T cells in intestinal tumors in vivo in the murine APCMin/+ model for microsatellite stable intestinal tumors. In this study, we investigated the effect of Treg depletion on the density and effector functions of different TCRαß+ and TCRγδ+ T cell populations in intestinal tumors. We used the APCMin/+\DEREG mouse model, which harbor a diphtheria toxin receptor under the control of the FOXP3 promoter, to deplete Treg in tumor bearing mice. We found that the density of conventional TCRαß+CD8αß+ T cells was significantly increased in Treg-depleted tumors in comparison with Treg-proficient tumors. Furthermore, TCRαß+CD8αß+ T cells showed increased proliferation and activation as well as increased Granzyme B and IFN-γ production in Treg-depleted tumors. In sharp contrast, the densities and effector functions of TCRαß+CD8αα+ T cells and TCRγδ+ T cells remained unchanged by Treg depletion. We also documented a distinct population of IL-17A+TNF+ TCRγδ+CD8- T cells in tumors, which were not affected by Treg depletion. We conclude that Treg depletion affects only conventional TCRαß+CD8αß+ T cells in intestinal tumors, while unconventional T cells and T cells in unaffected tissue are not altered. Immunotherapies aimed at depleting Treg from tumors may thus be a viable option for reinvigoration of conventional cytotoxic T cells with a Th1 cytokine profile.

Isolation and Characterization of MAIT Cells from Tumor Tissues.

Mucosal-associated invariant T (MAIT) cell infiltration has been demonstrated in colorectal and hepatocellular carcinoma, and their ability to produce Th1- and Th17-associated cytokines, as well as their cytotoxic function, suggests that MAIT cells may have important functions in both reducing and promoting protective tumor immunity. Here, we describe enzymatic methods to isolate intraepithelial and lamina propria lymphocyte single cell suspensions from colon tissue and tumors containing viable MAIT cells, which can be used for further purification, flow cytometry analysis, or culture.

Tumor-infiltrating mucosal-associated invariant T (MAIT) cells retain expression of cytotoxic effector molecules.

Mucosal-associated invariant T (MAIT) cells all express a semi-invariable T cell receptor recognizing microbial metabolites presented on the MHC class I-like molecule MR1. Upon activation, they rapidly secrete cytokines and increase their cytotoxic potential. We showed recently that MAIT cells with Th1 phenotype accumulate in human colon adenocarcinomas. Here, we investigated the cytotoxic potential of tumor-infiltrating MAIT cells in colon adenocarcinomas, and to what extent it may be affected by the tumor microenvironment. Activation of MAIT cells from tumors induced increased Granzyme B, and to a lesser extent, perforin expression. Degranulation was assessed by surface expression of CD107a, and was also seen in response to cognate antigen recognition. The cytotoxic potential of tumor-associated MAIT cells was very similar to that of MAIT cells from unaffected colon. MAIT cells were also identified by immunofluorescence in direct contact with tumor cells in sections from colon cancer specimens. To summarize, tumor-associated MAIT cells from colon tumors have strong cytotoxic potential and are not compromised in this regard compared to MAIT cells from the unaffected colon. We conclude that MAIT cells may contribute significantly to the protective immune response to tumors, both by secretion of Th1-associated cytokines and by direct killing of tumor cells.

ß7 integrins contribute to intestinal tumor growth in mice.

The gut homing receptor integrin α4ß7 is essential for the migration of pro-inflammatory T cells into the gut mucosa. Since intestinal neoplasia has been associated with chronic inflammation, we investigated whether interfering with gut-homing affects intestinal tumorigenesis. Using chemically induced and spontaneous intestinal tumor models we showed that lack of ß7 integrin significantly impairs tumor growth without affecting tumor frequencies, with a mild translatable effect on overall survival. This correlates with human data showing lower MAdCAM-1 expression and disease-free survival in colorectal cancer patients. Thus, paradoxically in contrast to extra-intestinal tumors, blocking migration of immune cells into the gut might have a positive therapeutic effect on intestinal neoplasia.

Regulatory T cells control endothelial chemokine production and migration of T cells into intestinal tumors of APCmin/+ mice.

Tumor-infiltrating lymphocytes are crucial for anti-tumor immunity. We have previously shown that regulatory T cells (Treg) are able to reduce T-cell transendothelial migration in vitro and accumulation of effector T cells in intestinal tumors in vivo. Treg depletion also resulted in increased levels of the chemokines CXCL9 and CXCL10 specifically in the tumors. In this study, we investigated the mechanisms for Treg mediated suppression of T-cell migration into intestinal tumors in the APCmin/+ mouse model. By breeding APCmin/+ mice with DEREG mice, which harbour a high affinity diphtheria toxin receptor under the control of the FOXP3 promoter, we were able to deplete Treg in tumor-bearing mice. Using adoptive transfer experiments, we could document a markedly increased migration of T cells specifically into Treg depleted tumors, and that Treg depletion results in increased production of the CXCR3 ligand CXCL10 from endothelial cells in the tumors. Furthermore, we were able to demonstrate that T cells use CXCR3 to migrate into intestinal tumors. In addition, human colon adenocarcinomas express high levels of mRNA CXCR3 ligands and tumor endothelial cells produce CXCL9 and CXCL10 ex vivo. In conclusion, this study demonstrates that Treg reduce endothelial CXCL10 production, inhibit T-cell migration into tumors and that CXCR3 mediated signalling is crucial for lymphocyte accumulation in intestinal tumors. Thus, immunotherapy aimed at Treg depletion may be effective by increasing not only T effector cell activity, but also their accumulation in tumors.

Activated T follicular helper-like cells are released into blood after oral vaccination and correlate with vaccine specific mucosal B-cell memory.

T follicular helper (Tfh)-like cells with potent B-cell helping ability are mobilized into human circulation after parenteral vaccination and are generally held to reflect ongoing germinal center reactions. However, whether mucosal vaccination induces systemic Tfh responses and how such responses may relate to IgA production are unknown. We investigated the frequencies, phenotype and function of circulating Tfh-like CD4+CXCR5+ T cells (cTfh) in adults receiving an oral inactivated enterotoxigenic Escherichia coli vaccine. Subjects were classified as vaccine responders or weak/non-responders based on their intestine-derived antibody-secreting cell (ASC) IgA responses to major vaccine antigens. Oral immunization induced significantly increased proportions of cTfh cells expressing the cTfh activation marker inducible costimulator (ICOS) in ASC responders, but not in weak/non-responders. Vaccination also enhanced the expression of IL-21, Th17 markers and integrin ß7 by activated cTfh cells, supporting functionality and gut homing potential. cTfh cells promoted total and vaccine specific IgA production from cocultured B cells. Magnitudes of cTfh responses assessed within a week after primary vaccinations correlated with memory intestine-derived vaccine specific IgA responses 1-2 years later. We conclude that activated ICOS+ Tfh-like cells are mobilized into blood after oral vaccination and may be used as biomarkers of vaccine specific mucosal memory in humans.

CD39+ regulatory T cells accumulate in colon adenocarcinomas and display markers of increased suppressive function.

Increasing knowledge of the function and regulation of tumor-infiltrating lymphocytes has led to new insights in cancer immunotherapy. Regulatory T cells (Treg) accumulate in colon tumors, and we recently showed that CD39+ Treg from cancer patients inhibit transendothelial migration of conventional T cells. CD39 mediates the hydrolysis of ATP to immunosuppressive adenosine and adds to the immunosuppressive effects of Treg. Here, we further investigated the regulatory features of intratumoral CD39+ Treg in colon cancer. Using flow cytometry analyses of cells from 46 colon cancer patients, we confirm the accumulation of CD39+ Treg in the tumor tissue compared to unaffected colon tissue, and also show that tumor-infiltrating Treg express more CD39 and Foxp3 on a per cell basis. Furthermore, CD39+ Treg in tumors express markers indicating increased turnover and suppressive ability. In particular, tumor-infiltrating CD39+ Treg have high expression of surface molecules related to immunosuppression, such as ICOS, PD-L1 and CTLA-4. Functional suppression assays also indicate potent suppressive capacity of CD39+ Treg on proliferation and IFN-γ secretion by conventional T cells. In conclusion, our results identify tumor-infiltrating CD39+ Treg as a numerous and potentially important immunosuppressive subset, and suggest that immunotherapy aimed at reducing the activity of CD39+ Treg may be particularly useful in the setting of colon cancer.

Tumour-associated changes in intestinal epithelial cells cause local accumulation of KLRG1+ GATA3+ regulatory T cells in mice.

CD4+ Foxp3+ regulatory T (Treg) cells include differentiated populations of effector Treg cells characterized by the expression of specific transcription factors. Tumours, including intestinal malignancies, often present with local accumulation of Treg cells that can prevent tumour clearance, but how tumour progression leads to Treg cell accumulation is incompletely understood. Here using genetically modified mouse models we show that ablation of E-cadherin, a process associated with epithelial to mesenchymal transition and tumour progression, promotes the accumulation of intestinal Treg cells by the specific accumulation of the KLRG1+ GATA3+ Treg subset. Epithelial E-cadherin ablation activates the ß-catenin pathway, and we find that increasing ß-catenin signals in intestinal epithelial cells also boosts Treg cell frequencies through local accumulation of KLRG1+ GATA3+ Treg cells. Both E-cadherin ablation and increased ß-catenin signals resulted in epithelial cells with higher levels of interleukin-33, a cytokine that preferentially expands KLRG1+ GATA3+ Treg cells. Tumours often present reduced E-cadherin expression and increased ß-catenin signalling and interleukin-33 production. Accordingly, Treg cell accumulation in intestinal tumours from APCmin/+ mice was exclusively due to the increase in KLRG1+ GATA3+ Treg cells. Our data identify a novel axis through which epithelial cells control local Treg cell subsets, which may be activated during intestinal tumorigenesis.

AICAR ameliorates high-fat diet-associated pathophysiology in mouse and ex vivo models, independent of adiponectin.

AIMS/HYPOTHESIS: In this study, we aimed to evaluate the therapeutic potential of 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR), an activator of AMP-activated protein kinase, for ameliorating high-fat diet (HFD)-induced pathophysiology in mice. We also aimed to determine whether the beneficial effects of AICAR were dependent on adiponectin. Furthermore, human adipose tissue was used to examine the effect of AICAR ex vivo. METHODS: Six-week-old male C57BL/6J wild-type and Adipoq -/- mice were fed a standard-fat diet (10% fat) or an HFD (60% fat) for 12 weeks and given vehicle or AICAR (500 µg/g) three times/week from weeks 4-12. Diet-induced pathophysiology was examined in mice after 11 weeks by IPGTT and after 12 weeks by flow cytometry and western blotting. Human adipose tissue biopsies from obese (BMI 35-50 kg/m2) individuals were incubated with vehicle or AICAR (1 mmol/l) for 6 h at 37°C, after which inflammation was characterised by ELISA (TNF-α) and flow cytometry. RESULTS: AICAR attenuated adipose inflammation in mice fed an HFD, promoting an M1-to-M2 macrophage phenotype switch, while reducing infiltration of CD8+ T cells. AICAR treatment of mice fed an HFD partially restored glucose tolerance and attenuated hepatic steatosis and kidney disease, as evidenced by reduced albuminuria (p < 0.05), urinary H2O2 (p < 0.05) and renal superoxide levels (p < 0.01) in both wild-type and Adipoq -/- mice. AICAR-mediated protection occurred independently of adiponectin, as similar protection was observed in wild-type and Adipoq -/- mice. In addition, AICAR promoted an M1-to-M2 macrophage phenotype switch and reduced TNF-α production in tissue explants from obese human patients. CONCLUSIONS/INTERPRETATION: AICAR may promote metabolic health and protect against obesity-induced systemic diseases in an adiponectin-independent manner. Furthermore, AICAR reduced inflammation in human adipose tissue explants, suggesting by proof-of-principle that the drug may reduce obesity-induced complications in humans. TRIAL REGISTRATION: ClinicalTrials.gov NCT02322073.