CXCR6-positive circulating mucosal-associated invariant T cells can identify patients with non-small cell lung cancer responding to anti-PD-1 immunotherapy.

BACKGROUND: Mucosal-associated invariant T (MAIT) cells have been reported to regulate tumor immunity. However, the immune characteristics of MAIT cells in non-small cell lung cancer (NSCLC) and their correlation with the treatment efficacy of immune checkpoint inhibitors (ICIs) remain unclear. PATIENTS AND METHODS: In this study, we performed single-cell RNA sequencing (scRNA-seq), flow cytometry, and multiplex immunofluorescence assays to determine the proportion and characteristics of CD8+MAIT cells in patients with metastatic NSCLC who did and did not respond to anti-PD-1 therapy. Survival analyses were employed to determine the effects of MAIT proportion and C-X-C chemokine receptor 6 (CXCR6) expression on the prognosis of patients with advanced NSCLC. RESULTS: The proportion of activated and proliferating CD8+MAIT cells were significantly higher in responders-derived peripheral blood mononuclear cells (PBMCs) and lung tissues before anti-PD-1 therapy, with enhanced expression of cytotoxicity-related genes including CCL4, KLRG1, PRF1, NCR3, NKG7, GZMB, and KLRK1. The responders' peripheral and tumor-infiltrating CD8+MAIT cells showed an upregulated CXCR6 expression. Similarly, CXCR6+CD8+MAIT cells from responders showed higher expression of cytotoxicity-related genes, such as CST7, GNLY, KLRG1, NKG7, and PRF1. Patients with ≥15.1% CD8+MAIT cells to CD8+T cells ratio and ≥35.9% CXCR6+CD8+MAIT cells to CD8+MAIT cells ratio in peripheral blood showed better progression-free survival (PFS) after immunotherapy. The role of CD8+MAIT cells in lung cancer immunotherapy was potentially mediated by classical/non-classical monocytes through the CXCL16-CXCR6 axis. CONCLUSION: CD8+MAIT cells are a potential predictive biomarker for patients with NSCLC responding to anti-PD-1 therapy. The correlation between CD8+MAIT cells and immunotherapy sensitivity may be ascribed to high CXCR6 expression.

CXCR6 defines therapeutic subtypes of CD4+ cytotoxic T cell lineage for adoptive cell transfer therapy in pediatric B cell acute lymphoblastic leukemia.

The potential of cytotoxic CD4+ T cells and tissue resident memory T cells (Trm) in achieving adult leukemia remission have been highlighted [1,2]. We hypothesized that CXCR6 could serve as a marker for cytotoxic CD4+ Trm cells in the bone marrow (BM) of pediatric B-ALL patients. Flow cytometry (FCM) and published single cell RNA sequencing (scRNA-seq) datasets were employed to characterize CXCR6+CD4+ T cells in the BM and peripheral blood (PB) of pediatric B-ALL patients and healthy donors. FCM, scRNA-seq and co-culture were utilized to explore the cytotoxicity of CXCR6+CD4+ T cells in vitro based on in vitro induction of CXCR6+CD4+ T cells using tumor antigens and peripheral blood mononuclear cells (PBMCs). The ssGSEA based on the cell markers identified according to the in vivo scRNA-seq data, the TARGET-ALL-P2 datasets, and integrated machine learning algorithm were employed to figure out the key cells with prognostic values, followed by simulation of adoptive cell transfer therapy (ACT). Integrated machine learning identified the high-risk cells for disease free survival, and overall survival, while simulation of ACT therapy using CXCR6+CD4+T cells indicated that CXCR6+CD4+ T cells could remodel the bone marrow microenvironments towards anti-tumor. Based on the expression of genes involved in formation of resident memory T cells, CXCR6 is not a marker of resident memory CD4+T cells but defines therapeutic subtypes of CD4+ cytotoxic T cell lineage for pediatric B-ALL.

The role of the CXCR6/CXCL16 axis in the pathogenesis of fibrotic disease.

CXC chemokine receptor 6 (CXCR6), a seven-transmembrane domain G-protein-coupled receptor, plays a pivotal regulatory role in inflammation and tissue damage through its interaction with CXC chemokine ligand 16 (CXCL16). This axis is implicated in the pathogenesis of various fibrotic diseases and correlates with clinical parameters that indicate disease severity, activity, and prognosis in organ fibrosis, including afflictions of the liver, kidney, lung, cardiovascular system, skin, and intestines. Soluble CXCL16 (sCXCL16) serves as a chemokine, facilitating the migration and recruitment of CXCR6-expressing cells, while membrane-bound CXCL16 (mCXCL16) functions as a transmembrane protein with adhesion properties, facilitating intercellular interactions by binding to CXCR6. The CXCR6/CXCL16 axis is established to regulate the cycle of damage and repair during chronic inflammation, either through modulating immune cell-mediated intercellular communication or by independently influencing fibroblast homing, proliferation, and activation, with each pathway potentially culminating in the onset and progression of fibrotic diseases. However, clinically exploiting the targeting of the CXCR6/CXCL16 axis requires further elucidation of the intricate chemokine interactions within fibrosis pathogenesis. This review explores the biology of CXCR6/CXCL16, its multifaceted effects contributing to fibrosis in various organs, and the prospective clinical implications of these insights.

Sepsis-trained macrophages promote antitumoral tissue-resident T cells.

Sepsis induces immune alterations, which last for months after the resolution of illness. The effect of this immunological reprogramming on the risk of developing cancer is unclear. Here we use a national claims database to show that sepsis survivors had a lower cumulative incidence of cancers than matched nonsevere infection survivors. We identify a chemokine network released from sepsis-trained resident macrophages that triggers tissue residency of T cells via CCR2 and CXCR6 stimulations as the immune mechanism responsible for this decreased risk of de novo tumor development after sepsis cure. While nonseptic inflammation does not provoke this network, laminarin injection could therapeutically reproduce the protective sepsis effect. This chemokine network and CXCR6 tissue-resident T cell accumulation were detected in humans with sepsis and were associated with prolonged survival in humans with cancer. These findings identify a therapeutically relevant antitumor consequence of sepsis-induced trained immunity.

rs71327024 Associated with COVID-19 Hospitalization Reduces CXCR6 Promoter Activity in Human CD4+ T Cells via Disruption of c-Myb Binding.

Single-nucleotide polymorphism rs71327024 located in the human 3p21.31 locus has been associated with an elevated risk of hospitalization upon SARS-CoV-2 infection. The 3p21.31 locus contains several genes encoding chemokine receptors potentially relevant to severe COVID-19. In particular, CXCR6, which is prominently expressed in T lymphocytes, NK, and NKT cells, has been shown to be involved in the recruitment of immune cells to non-lymphoid organs in chronic inflammatory and respiratory diseases. In COVID-19, CXCR6 expression is reduced in lung resident memory T cells from patients with severe disease as compared to the control cohort with moderate symptoms. We demonstrate here that rs71327024 is located within an active enhancer that augments the activity of the CXCR6 promoter in human CD4+ T lymphocytes. The common rs71327024(G) variant makes a functional binding site for the c-Myb transcription factor, while the risk rs71327024(T) variant disrupts c-Myb binding and reduces the enhancer activity. Concordantly, c-Myb knockdown in PMA-treated Jurkat cells negates rs71327024's allele-specific effect on CXCR6 promoter activity. We conclude that a disrupted c-Myb binding site may decrease CXCR6 expression in T helper cells of individuals carrying the minor rs71327024(T) allele and thus may promote the progression of severe COVID-19 and other inflammatory pathologies.

Association of 3p21.31 Locus (CXCR6 and LZTFL1) with COVID-19 Outcomes in Brazilian Hospitalyzed Subjects.

The 3p21.31 locus has been associated with severe COVID-19 prognosis in GWAS studies. Here, we evaluated whether three polymorphisms (LZTFL1 rs10490770, CXCR6 rs2234355 and rs2234358) in the reported locus were associated with the need for mechanical ventilation, hospitalization length and death in 102 COVID-19 hospitalized Brazilian subjects. No genetic association was found with the need for mechanical ventilation and hospitalization length. CXCR6 rs2234355 was associated with mortality under the codominance model, with carriers of the A/A genotype having a greater chance of death than A/G (OR: 10.5; 95% CI: 1.55-70.76). Our results further suggest that the CXCR6 genetic variant contributes to COVID-19 outcomes.

T cell receptor signaling strength establishes the chemotactic properties of effector CD8+ T cells that control tissue-residency.

Tissue-resident memory (TRM) CD8+ T cells are largely derived from recently activated effector T cells, but the mechanisms that control the extent of TRM differentiation within tissue microenvironments remain unresolved. Here, using an IFNγ-YFP reporter system to identify CD8+ T cells executing antigen-dependent effector functions, we define the transcriptional consequences and functional mechanisms controlled by TCR-signaling strength that occur within the skin during viral infection to promote TRM differentiation. TCR-signaling both enhances CXCR6-mediated migration and suppresses migration toward sphingosine-1-phosphate, indicating the programming of a 'chemotactic switch' following secondary antigen encounter within non-lymphoid tissues. Blimp1 was identified as the critical target of TCR re-stimulation that is necessary to establish this chemotactic switch and for TRM differentiation to efficiently occur. Collectively, our findings show that access to antigen presentation and strength of TCR-signaling required for Blimp1 expression establishes the chemotactic properties of effector CD8+ T cells to promote residency within non-lymphoid tissues.

Tumor-associated macrophages promote cisplatin resistance in ovarian cancer cells by enhancing WTAP-mediated N6-methyladenosine RNA methylation via the CXCL16/CXCR6 axis.

PURPOSE: Tumor-promotive tumor-associated macrophages (TAMs) and the CXCL16/CXCR6 axis have been reported to be correlated with the limited efficacy of chemotherapy in ovarian cancer (OC). However, the role of TAM-secreted CXCL16 and the mechanism by which it affects the cisplatin (DDP) resistance of OC cells remain elusive. METHODS: We induced human THP-1 monocytes to differentiate into macrophages. Next, SKOV3 and TOV-112D cells were co-cultured with the macrophages, followed by incubation with increasing concentrations of DDP. The effects of CXCL16, CXCR6, and WTAP on the DDP resistance of OC cells were investigated using the CCK-8 assay, colony formation assay, flow cytometry, and TUNEL staining. CXCL16 concentrations were determined by ELISA. Quantitative real-time PCR and western blotting were used to examine related markers. RESULTS: Our results showed that after being co-cultured with TAMs, the DDP resistance of OC cells was significantly enhanced and their CXCL16 levels were elevated. Acquired DDP resistance was characterized by an increased IC50 value for DDP, the formation of cell colonies, and decreased levels of cell apoptosis, which were accompanied by reduced levels of caspase-3 and Bax expression, and increased levels of Bcl-2, PARP1, BRCA1, and BRCA2 expression. Either CXCL16 knockdown in TAMs or CXCR6 knockdown in OC cells suppressed the DDP resistance of OC cells that had been co-cultured with TAMs. Knockdown of CXCL16 affected m6A RNA methylation in OC cells, as reflected by decreased YTHDF1/WTAP expression and increased ALKBH5 expression. WTAP overexpression and knockdown promoted and suppressed the DDP resistance of OC cells, respectively. CONCLUSION: Tumor-associated macrophages promote the cisplatin resistance of OC cells by enhancing WTAP-mediated N6-methyladenosine RNA methylation via the CXCL16/CXCR6 axis.

Role of the CXCR6/CXCL16 axis in autoimmune diseases.

The C-X-C motif ligand 16, or CXCL16, is a chemokine that belongs to the ELR - CXC subfamily. Its function is to bind to the chemokine receptor CXCR6, which is a G protein-coupled receptor with 7 transmembrane domains. The CXCR6/CXCL16 axis has been linked to the development of numerous autoimmune diseases and is connected to clinical parameters that reflect disease severity, activity, and prognosis in conditions such as multiple sclerosis, autoimmune hepatitis, rheumatoid arthritis, Crohn's disease, and psoriasis. CXCL16 is expressed in various immune cells, such as dendritic cells, monocytes, macrophages, and B cells. During autoimmune diseases, CXCL16 can facilitate the adhesion of immune cells like monocytes, T cells, NKT cells, and others to endothelial cells and dendritic cells. Additionally, sCXCL16 can regulate the migration of CXCR6-expressing leukocytes, which includes CD8+ T cells, CD4+ T cells, NK cells, constant natural killer T cells, plasma cells, and monocytes. Further investigation is required to comprehend the intricate interactions between chemokines and the pathogenesis of autoimmune diseases. It remains to be seen whether the CXCR6/CXCL16 axis represents a new target for the treatment of these conditions.

Hepatic C-X-C chemokine receptor type 6-expressing innate lymphocytes limit detrimental myeloid hyperactivation in acute liver injury.

BACKGROUND: Acute liver failure (ALF) is characterized by rapid clinical deterioration and high mortality. Acetaminophen (APAP or paracetamol) overdose is a leading cause of ALF, resulting in hepatocellular necrosis with subsequent inflammation, inflicting further liver damage. Infiltrating myeloid cells are early drivers of liver inflammation. However, the role of the abundant population of liver-resident innate lymphocytes, which commonly express the chemokine receptor CXCR6, is incompletely understood in ALF. METHODS: We investigated the role of CXCR6-expressing innate lymphocytes using the model of acute APAP toxicity in mice deficient in CXCR6 (Cxcr6gfp/gfp). RESULTS: APAP-induced liver injury was strongly aggravated in Cxcr6gfp/gfp mice compared with wild-type counterparts. Immunophenotyping using flow cytometry revealed a reduction in liver CD4+T cells, natural killer (NK) cells, and most prominently, NKT cells, whereas CXCR6 was dispensable for CD8+ T-cell accumulation. CXCR6-deficient mice exhibited excessive neutrophil and inflammatory macrophage infiltration. Intravital microscopy revealed dense cellular clusters of neutrophils in necrotic liver tissue, with higher numbers of clustering neutrophils in Cxcr6gfp/gfp mice. Gene expression analysis linked hyperinflammation in CXCR6 deficiency to increased IL-17 signaling. Although reduced in overall numbers, CXCR6-deficient mice had a shift in NKT cell subsets with increased RORγt-expressing NKT17 cells as a likely source of IL-17. In patients with ALF, we found a prominent accumulation of IL-17-expressing cells. Accordingly, CXCR6-deficient mice lacking IL-17 (Cxcr6gfp/gfpx Il17-/-) had ameliorated liver damage and reduced inflammatory myeloid infiltrates. CONCLUSIONS: Our study identifies a crucial role of CXCR6-expressing liver innate lymphocytes as orchestrators in acute liver injury containing IL-17-mediated myeloid cell infiltration. Hence, strengthening the CXCR6-axis or downstream inhibition of IL-17 could yield novel therapeutics in ALF.

Monocyte migration profiles define disease severity in acute COVID-19 and unique features of long COVID.

BACKGROUND: COVID-19 is associated with a dysregulated immune response but it is unclear how immune dysfunction contributes to the chronic morbidity persisting in many COVID-19 patients during convalescence (long COVID). METHODS: We assessed phenotypical and functional changes of monocytes in COVID-19 patients during hospitalisation and up to 9 months of convalescence following COVID-19, respiratory syncytial virus or influenza A. Patients with progressive fibrosing interstitial lung disease were included as a positive control for severe, ongoing lung injury. RESULTS: Monocyte alterations in acute COVID-19 patients included aberrant expression of leukocyte migration molecules, continuing into convalescence (n=142) and corresponding with specific symptoms of long COVID. Long COVID patients with unresolved lung injury, indicated by sustained shortness of breath and abnormal chest radiology, were defined by high monocyte expression of C-X-C motif chemokine receptor 6 (CXCR6) (p<0.0001) and adhesion molecule P-selectin glycoprotein ligand 1 (p<0.01), alongside preferential migration of monocytes towards the CXCR6 ligand C-X-C motif chemokine ligand 16 (CXCL16) (p<0.05), which is abundantly expressed in the lung. Monocyte CXCR6 and lung CXCL16 were heightened in patients with progressive fibrosing interstitial lung disease (p<0.001), confirming a role for the CXCR6-CXCL16 axis in ongoing lung injury. Conversely, monocytes from long COVID patients with ongoing fatigue exhibited a sustained reduction of the prostaglandin-generating enzyme cyclooxygenase 2 (p<0.01) and CXCR2 expression (p<0.05). These monocyte changes were not present in respiratory syncytial virus or influenza A convalescence. CONCLUSIONS: Our data define unique monocyte signatures that define subgroups of long COVID patients, indicating a key role for monocyte migration in COVID-19 pathophysiology. Targeting these pathways may provide novel therapeutic opportunities in COVID-19 patients with persistent morbidity.

Possible involvement of CXCR3-CXCR6 + CD4 + T cells in Langerhans cell histiocytosis.

INTRODUCTION: Langerhans cell histiocytosis (LCH) is a condition characterized by proliferation of Langerhans cells and wide-range pathologies, ranging from single granulomatous lesions to multi-organ involvement, associated with tissue destruction. LCH pathogenesis remains obscure although association with interleukin (IL)-17A has been reported. We report here a case that illustrates the potential pathogenic role of helper T17 (Th17) cells in LCH-related bone destruction. MATERIALS AND METHODS: The patient was a 66-year-old woman. The clinical course included craniectomy and bone mass excision in X-9, diagnosis of LCH confirmed by histopathology, followed by 26-month chemotherapy. In August X, the patient was diagnosed with complete central diabetes insipidus. Symptoms improved after treatment with desmopressin. Pituitary magnetic resonance imaging showed swelling extending from the suprasellar region to the pituitary stalk, suggestive of LCH recurrence. This was followed by chemotherapy combined with mercaptopurine hydrate.  RESULTS: Subsequent peripheral blood lymphocyte analysis showed marked increase in activated Th17 cells (CXCR3-CXCR6+ CD4+ T cells). Double staining for CD4 and IL-17 by immunofluorescence of pathological tissue samples obtained during temporal bone mass excision, which confirmed the diagnosis of LCH in X-9, showed areas of combined presence of CD4-positive cells and IL-17-positive cells. Chemotherapy resulted in size reduction of the pituitary lesion and decrease in peripheral blood-activated Th17 cells. CONCLUSIONS: We found abundant peripheral blood-activated Th17 cells and high percentages of IL-17-producing cells in osteolytic bone lesions in LCH. This finding, together with the decrease in peripheral blood-activated Th17 cells following chemotherapy, suggests the potential involvement of activated Th17 cells in LCH-related osteolysis.

Reduced expression of CXCL16/CXCR6 is involved in the pathogenesis of hidradenitis suppurativa.

Mutations in the γ-secretase complex have been well-described in familial hidradenitis suppurativa (HS). No gene mutations have been identified in sporadic HS, which comprises 60%-70% of all HS cases. Obesity and smoking are risk factors for HS and are closely related to DNA methylation, an essential epigenetic phenomenon. Hence, we hypothesized that epigenetic modifications might be involved in sporadic HS. To investigate genes with aberrant methylation in sporadic HS cases and assess their expression in skin lesions and blood from patients with HS. Skin lesion samples and corresponding normal skin were obtained from three patients with HS and subjected to whole-genome DNA methylation sequencing. Blood samples were collected from 20 patients with HS and 20 healthy controls (HCs). The HS mouse model was established by applying tamoxifen to NcstnΔKC mice. Target gene expression was analysed by immunohistochemistry, immunofluorescence, western blotting, enzyme-linked immunosorbent assay (ELISA) and semiquantitative real-time polymerase chain reaction (RT-qPCR). Among 10 807 differentially methylated genes, we filtered 2101 genes with hypermethylated promoter regions, and following bioinformatics analyses, we focused on CXC chemokine ligand 16 (CXCL16). Subsequent functional experiments confirmed the downregulation of CXCL16 and its receptor, CXC chemokine receptor (CXCR) 6, in skin tissue from HS patients and NcstnΔKC mice. Serum CXCL16 concentrations were also significantly decreased in patients with HS. Our data revealed the downregulation of CXCL16 and CXCR6 in HS.

CXCL16 inhibits epithelial regeneration and promotes fibrosis during the progression of radiation enteritis.

Radiation enteritis (RE) is a prevalent complication of radiotherapy for pelvic malignant tumors, characterized by severe intestinal epithelial destruction and progressive submucosal fibrosis. However, little is known about the pathogenesis of this disease, and so far, there is no specific targeted therapy. Here, we report that CXCL16 is upregulated in the injured intestinal tissues of RE patients and in a mouse model. Genetic deletion of Cxcl16 mitigates fibrosis and promotes intestinal stem cell-mediated epithelial regeneration after radiation injury in mice. Mechanistically, CXCL16 functions on myofibroblasts through its receptor CXCR6 and activates JAK3/STAT3 signaling to promote fibrosis and, at the same time, to transcriptionally modulate the levels of BMP4 and hepatocyte growth factor (HGF) in myofibroblasts. Moreover, we find that CXCL16 and CXCR6 auto- and cross-regulate themselves in positive feedback loops. Treatment with CXCL16 neutralizing monoclonal antibody attenuates fibrosis and improves the epithelial repair in RE mouse model. Our findings emphasize the important role of CXCL16 in the progression of RE and suggest that CXCL16 signaling could be a potential therapeutic target for RE. © 2022 The Pathological Society of Great Britain and Ireland.

The Small-Molecule compound baicalein alleviates experimental autoimmune encephalomyelitis by suppressing pathogenetic CXCR6+ CD4 cells.

CXC chemokine receptor6 (CXCR6)-based immunotherapy plays a significant role in autoimmune diseases, however, little is known about possible small compounds that inhibit pathogenic CXCR6+ T cells for treating multiple sclerosis (MS). Baicalein, a flavonoid isolated from Scutellarin baicalensis (Huang Qin), was shown to exert therapeutic effects on MS, but the underlying mechanisms are largely unknown. In the current study, we found that baicalein inhibited Th1 and Th17 differentiation in vitro. Oral administration of baicalein (25 mg/kg) significantly reduced the disease severity and the infiltration process, decreased the extent of demyelination in EAE, and selectively blocked IL-17A production and specific antibodies (IgG and IgG3) in MOG35-55-induced specific immune responses. In addition, the expression of CD4 cell effectors (CD44hiCD62Llow) and pathogenic Th17 cells was decreased by baicalein treatment. Furthermore, baicalein treatment largely decreased CXCR6+ CD4 and CD8 cells and prominently inhibited CXCR6+ Th17 cells in EAE. Taken together, the findings of this study suggest for the first time that baicalein may ameliorate EAE by suppressing pathogenetic CXCR6+ CD4 cells.

CXCR6 Mediates Pressure Overload-Induced Aortic Stiffness by Increasing Macrophage Recruitment and Reducing Exosome-miRNA29b.

Aortic stiffness is an independent risk factor for aortic diseases such as aortic dissection which commonly occurred with aging and hypertension. Chemokine receptor CXCR6 is critically involved in vascular inflammation and remodeling. Here, we investigated whether and how CXCR6 plays a role in aortic stiffness caused by pressure overload. CXCR6-/- and WT mice underwent transverse aortic constriction (TAC) surgery for 8 weeks. CXCR6 deficiency significantly improved TAC-induced aortic remodeling and endothelial dysfunction by decreasing CD11c+ macrophage infiltration, suppressing VCAM-1 and ICAM-1, reducing collagen deposition, and downregulating MMP12 and osteopontin in the aorta. Consistently, blocking the CXCL16/CXCR6 axis also reduced aortic accumulation of CD11c+ macrophages and vascular stiffness but without affecting the release of TNF-α and IL-6 from the aorta. Furthermore, pressure overload inhibited aortic release of exosomes, which could be reversed by suppressing CXCR6 or CXCL16. Inhibition of exosome release by GW4869 significantly aggravated TAC-induced aortic calcification and stiffness. By exosomal microRNA microarray analysis, we found that microRNA-29b was significantly reduced in aortic endothelial cells (AECs) receiving TAC. Intriguingly, blocking the CXCL16/CXCR6 axis restored the expression of miR-29b in AECs. Finally, overexpression of miR-29b significantly increased eNOS and reduced MMPs and collagen in AECs. By contrast, antagonizing miR-29b in vivo further enhanced TAC-induced expressions of MMP12 and osteopontin, aggravated aortic fibrosis, calcification, and stiffness. Our study demonstrated a key role of the CXCL16/CXCR6 axis in macrophage recruitment and macrophage-mediated aortic stiffness under pressure overload through an exosome-miRNAs-dependent manner.

The CXCL16-CXCR6 axis in glioblastoma modulates T-cell activity in a spatiotemporal context.

Introduction: Glioblastoma multiforme (GBM) pathobiology is characterized by its significant induction of immunosuppression within the tumor microenvironment, predominantly mediated by immunosuppressive tumor-associated myeloid cells (TAMCs). Myeloid cells play a pivotal role in shaping the GBM microenvironment and influencing immune responses, with direct interactions with effector immune cells critically impacting these processes. Methods: Our study investigates the role of the CXCR6/CXCL16 axis in T-cell myeloid interactions within GBM tissues. We examined the surface expression of CXCL16, revealing its limitation to TAMCs, while microglia release CXCL16 as a cytokine. The study explores how these distinct expression patterns affect T-cell engagement, focusing on the consequences for T-cell function within the tumor environment. Additionally, we assessed the significance of CXCR6 expression in T-cell activation and the initial migration to tumor tissues. Results: Our data demonstrates that CXCL16 surface expression on TAMCs results in predominant T-cell engagement with these cells, leading to impaired T-cell function within the tumor environment. Conversely, our findings highlight the essential role of CXCR6 expression in facilitating T-cell activation and initial migration to tumor tissues. The CXCL16-CXCR6 axis exhibits dualistic characteristics, facilitating the early stages of the T-cell immune response and promoting T-cell infiltration into tumors. However, once inside the tumor, this axis contributes to immunosuppression. Discussion: The dual nature of the CXCL16-CXCR6 axis underscores its potential as a therapeutic target in GBM. However, our results emphasize the importance of carefully considering the timing and context of intervention. While targeting this axis holds promise in combating GBM, the complex interplay between TAMCs, microglia, and T cells suggests that intervention strategies need to be tailored to optimize the balance between promoting antitumor immunity and preventing immunosuppression within the dynamic tumor microenvironment.

Akkermansia muciniphila suppressing nonalcoholic steatohepatitis associated tumorigenesis through CXCR6+ natural killer T cells.

Introduction: Gut microbiota plays a crucial role in the development and progression of nonalcoholic steatohepatitis (NASH) and associated hepatocellular carcinoma (HCC). Akkermansia muciniphila was reported to inhibit inflammation-associated cancer in the intestine. The anti-NASH ability of A. muciniphila has recently been found. Thus, we were to investigate whether supplementation of A. muciniphila could prevent NASH-associated HCC. Methods: In a model we called STAM, male C57BL/6J mice were subcutaneously injected with 200 µg streptozotocin at 4 days after birth, and fed with high-fat diet at 4 weeks of age to induce NASH-associated HCC. Faeces from mice and patients with NASH-related HCC were collected for 16S rRNA sequencing. STAM mice were orally administered either saline or A. muciniphila twice a day starting at 4 or 10 weeks of age. The effects of A. muciniphila on the immune responses were also evaluated. Results: Patients and mice with NASH-related HCC showed significantly reduced gut A. muciniphila in comparison to healthy controls. Administration of breast milk-isolated A. muciniphila (AM06) but not feces-isolated A. muciniphila (AM02) could improve NASH severity. Interestingly, breast milk-isolated A. muciniphila treatment suppressed the progression of NASH to HCC, accompanied with an increased hepatic CXCR6+ natural killer T (NKT) cell and decreased macrophage infiltration. The antitumor ability of A. muciniphila was not evident in NKT cell-deficient mice (CD1d-/- and CXCR6-/-). In vitro, A. muciniphila promoted the killing of hepG2 cells by NKT cells. Discussion: Our study will provide the rationale for the application of A. muciniphila to treat NASH and for the prevention of its progression to HCC.

Tissue-resident CD69+ CXCR6+ Natural Killer cells with exhausted phenotype accumulate in human non-small cell lung cancer.

Natural killer (NK) cells with tissue-residency features (trNK cells) are a new subpopulation of NK cells, which plays an important role in tissue homeostasis. However, the characteristics of trNK cells in the tumor microenvironment (TME) of human cancers remain unclear. Using multicolor flow cytometry, we investigated the quantity, phenotype, and function of trNK cells in biospecimens freshly resected from 60 non-small cell lung cancer (NSCLC) patients. We successfully identified a new CD69+ CXCR6+ trNK subset with an immunomodulatory-like and exhausted phenotype, specifically accumulated in the TME of NSCLC. In vitro experiments showed that CD69+ CXCR6+ trNK cells more readily secreted IFN-γ and TNF-α spontaneously. Furthermore, the production of IFN-γ and TNF-α by tumor-infiltrating CD69+ CXCR6+ trNK cells was not induced by their reactivation in vitro, which is analogous to T-cell exhaustion. Finally, we demonstrated that the dysfunction of CD69+ CXCR6+ trNK cells could be partly ameliorated by PD-1 and CTLA-4 blockade. In summary, we identified a new dysfunctional CD69+ CXCR6+ trNK cell subset that specifically accumulates in the TME of NSCLC. Our findings suggest that CD69+ CXCR6+ trNK cells are a promising target for immune checkpoint inhibitors in the treatment of NSCLC.

Epitope Mapping of an Anti-Mouse CXCR6 Monoclonal Antibody (Cx6Mab-1) Using the 2 × Alanine Scanning Method.

The CXC chemokine receptor 6 (CXCR6) is a member of the G protein-coupled receptor family that is highly expressed in helper T type 1 cells, cytotoxic T lymphocytes (CTLs), and natural killer cells. CXCR6 plays critical roles in local expansion of effector-like CTLs in tumor microenvironment to potentiate the antitumor response. Therefore, the development of anti-CXCR6 monoclonal antibodies (mAbs) is essential to evaluate the immune microenvironment of tumors. Using N-terminal peptide immunization, we previously developed an anti-mouse CXCR6 (mCXCR6) mAb, Cx6Mab-1 (rat IgG1, kappa) , which is useful for flow cytometry and western blotting. In this study, we determined the critical epitope of Cx6Mab-1 by enzyme-linked immunosorbent assay (ELISA) using the 1 × alanine scanning (1 × Ala-scan) method or the 2 × alanine scanning (2 × Ala-scan) method. Although we first performed ELISA by 1 × Ala-scan using one alanine-substituted peptides of mCXCR6 N-terminal domain (amino acids 1-20), we could not identify the Cx6Mab-1 epitope. We next performed ELISA by 2 × Ala-scan using two alanine (or glycine) residues-substituted peptides of mCXCR6 N-terminal domain, and found that Cx6Mab-1 did not recognize S8A-A9G, A9G-L10A, L10A-Y11A, and G13A-H14A of the mCXCR6 N-terminal peptide. The results indicate that the binding epitope of Cx6Mab-1 includes Ser8, Ala9, Leu10, Tyr11, Gly13, and His14 of mCXCR6. Therefore, we could demonstrate that the 2 × Ala scan method is useful for determining the critical epitope of mAbs.