Primary nasal influenza infection rewires tissue-scale memory response dynamics.

The nasal mucosa is often the initial site of respiratory viral infection, replication, and transmission. Understanding how infection shapes tissue-scale primary and memory responses is critical for designing mucosal therapeutics and vaccines. We generated a single-cell RNA-sequencing atlas of the murine nasal mucosa, sampling three regions during primary influenza infection and rechallenge. Compositional analysis revealed restricted infection to the respiratory mucosa with stepwise changes in immune and epithelial cell subsets and states. We identified and characterized a rare subset of Krt13+ nasal immune-interacting floor epithelial (KNIIFE) cells, which concurrently increased with tissue-resident memory T (TRM)-like cells. Proportionality analysis, cell-cell communication inference, and microscopy underscored the CXCL16-CXCR6 axis between KNIIFE and TRM cells. Secondary influenza challenge induced accelerated and coordinated myeloid and lymphoid responses without epithelial proliferation. Together, this atlas serves as a reference for viral infection in the upper respiratory tract and highlights the efficacy of local coordinated memory responses.

CCR6 and CXCR6 Identify the Th17 Cells With Cytotoxicity in Experimental Autoimmune Encephalomyelitis.

Due to the plasticity of IL-17-producing CD4 T cells (Th17 cells), a long-standing challenge in studying Th17-driven autoimmune is the lack of specific surface marker to identify the pathogenic Th17 cells in vivo. Recently, we discovered that pathogenic CD4 T cells were CXCR6 positive in experimental autoimmune encephalomyelitis (EAE), a commonly used Th17-driven autoimmune model. Herein, we further revealed that peripheral CXCR6+CD4 T cells contain a functionally distinct subpopulation, which is CCR6 positive and enriched for conventional Th17 molecules (IL-23R and RORγt) and cytotoxic signatures. Additionally, spinal cord-infiltrating CD4 T cells were highly cytotoxic by expressing Granzyme(s) along with IFNγ and GM-CSF. Collectively, this study suggested that peripheral CCR6+CXCR6+CD4 T cells were Th17 cells with cytotoxic property in EAE model, and highlighted the cytotoxic granzymes for EAE pathology.

CXCR6+CD4+ T cells promote mortality during Trypanosoma brucei infection.

Liver macrophages internalize circulating bloodborne parasites. It remains poorly understood how this process affects the fate of the macrophages and T cell responses in the liver. Here, we report that infection by Trypanosoma brucei induced depletion of macrophages in the liver, leading to the repopulation of CXCL16-secreting intrahepatic macrophages, associated with substantial accumulation of CXCR6+CD4+ T cells in the liver. Interestingly, disruption of CXCR6 signaling did not affect control of the parasitemia, but significantly enhanced the survival of infected mice, associated with reduced inflammation and liver injury. Infected CXCR6 deficient mice displayed a reduced accumulation of CD4+ T cells in the liver; adoptive transfer experiments suggested that the reduction of CD4+ T cells in the liver was attributed to a cell intrinsic property of CXCR6 deficient CD4+ T cells. Importantly, infected CXCR6 deficient mice receiving wild-type CD4+ T cells survived significantly shorter than those receiving CXCR6 deficient CD4+ T cells, demonstrating that CXCR6+CD4+ T cells promote the mortality. We conclude that infection of T. brucei leads to depletion and repopulation of liver macrophages, associated with a substantial influx of CXCR6+CD4+ T cells that mediates mortality.

CXCR6 is required for antitumor efficacy of intratumoral CD8+ T cell.

BACKGROUND: Increasing infiltration of CD8+ T cells within tumor tissue predicts a better prognosis and is essential for response to checkpoint blocking therapy. Furthermore, current clinical protocols use unfractioned T cell populations as the starting point for transduction of chimeric antigen receptors (CARs)-modified T cells, but the optimal T cell subtype of CAR-modified T cells remains unclear. Thus, accurately identifying a group of cytotoxic T lymphocytes with high antitumor efficacy is imperative. Inspired by the theory of yin and yang, we explored a subset of CD8+ T cell in cancer with the same phenotypic characteristics as highly activated inflammatory T cells in autoimmune diseases. METHODS: Combination of single-cell RNA sequencing, general transcriptome sequencing data and multiparametric cytometric techniques allowed us to map CXCR6 expression on specific cell type and tissue. We applied Cxcr6-/- mice, immune checkpoint therapies and bone marrow chimeras to identify the function of CXCR6+CD8+ T cells. Transgenic Cxcr6-/- OT-I mice were employed to explore the functional role of CXCR6 in antigen-specific antitumor response. RESULTS: We identified that CXCR6 was exclusively expressed on intratumoral CD8+ T cell. CXCR6+CD8+ T cells were more immunocompetent, and chimeras with specific deficiency on CD8+ T cells showed weaker antitumor activity. In addition, Cxcr6-/- mice could not respond to anti-PD-1 treatment effectively. High tumor expression of CXCR6 was not mainly caused by ligand-receptor chemotaxis of CXCL16/CXCR6 but induced by tumor tissue self. Induced CXCR6+CD8+ T cells possessed tumor antigen specificity and could enhance the effect of anti-PD-1 blockade to retard tumor progression. CONCLUSIONS: This study may contribute to the rational design of combined immunotherapy. Alternatively, CXCR6 may be used as a biomarker for effective CD8+ T cell state before adoptive cell therapy, providing a basis for tumor immunotherapy.

Overall survival in metastatic melanoma correlates with pembrolizumab exposure and T cell exhaustion markers.

Trial data support an absence of an exposure-survival relationship for pembrolizumab. As these relationships remain unexamined in a real-world setting, we determined them in metastatic melanoma prospectively in an observational study. Translational objectives included identifying biomarkers of progressive disease (PD). Checkpoint blockade naïve patients receiving 2 mg/kg Q3W pembrolizumab had pharmacokinetic and clinical outcome data collected. Trough, a valid surrogate for drug exposure, was assessed using ELISA. T-cell exhaustion and chemokine markers were determined using flow cytometry. Geometric means of exposures and biomarkers were tested against objective response groups using one-way ANOVA. The cohort was split by the median into high versus low pembrolizumab exposure groups. Kaplan-Meier progression-free survival (PFS) and overall survival (OS) curves were estimated for high versus low exposure, compared using the log rank test. The high pembrolizumab exposure group (n = 14) experienced substantially longer median OS (not reached vs. 48 months, p = .014), than the low exposure group (n = 14). A similar positive exposure PFS relationship was found (median not reached vs. 48 months, p = .045). The frequency of TIM-3 expression on CD4+ T cells was significantly higher in PD (mean 27.8%) than complete response (CR) (13.38%, p = .01) and partial response (12.4%, p = .05). There was a near doubling of CXCR6 and TIM-3 co-expression on CD4+ T cells in PD (mean 23.3%) versus CR (mean 11.4, p = .003) and partial response (9.8%, p = .0001). We describe positive exposure-PFS and exposure-OS relationships for pembrolizumab in metastatic melanoma. TIM-3, alongside co-expression of CXCR6 and TIM-3 on circulating CD4+ T cells are potential bio markers of treatment failure.

Single-cell dissection of cellular components and interactions shaping the tumor immune phenotypes in ovarian cancer.

Distinct T cell infiltration patterns, i.e., immune infiltrated, excluded, and desert, result in different responses to cancer immunotherapies. However, the key determinants and biology underpinning these tumor immune phenotypes remain elusive. Here, we provide a high-resolution dissection of the entire tumor ecosystem through single-cell RNA-sequencing analysis of 15 ovarian tumors. Immune-desert tumors are characterized by unique tumor cell-intrinsic features, including metabolic pathways and low antigen presentation, and an enrichment of monocytes and immature macrophages. Immune-infiltrated and -excluded tumors differ markedly in their T cell composition and fibroblast subsets. Furthermore, our study reveals chemokine receptor-ligand interactions within and across compartments as potential mechanisms mediating immune cell infiltration, exemplified by the tumor cell-T cell cross talk via CXCL16-CXCR6 and stromal-immune cell cross talk via CXCL12/14-CXCR4. Our data highlight potential molecular mechanisms that shape the tumor immune phenotypes and may inform therapeutic strategies to improve clinical benefit from cancer immunotherapies.

Auto-aggressive CXCR6+ CD8 T cells cause liver immune pathology in NASH.

Nonalcoholic steatohepatitis (NASH) is a manifestation of systemic metabolic disease related to obesity, and causes liver disease and cancer1,2. The accumulation of metabolites leads to cell stress and inflammation in the liver3, but mechanistic understandings of liver damage in NASH are incomplete. Here, using a preclinical mouse model that displays key features of human NASH (hereafter, NASH mice), we found an indispensable role for T cells in liver immunopathology. We detected the hepatic accumulation of CD8 T cells with phenotypes that combined tissue residency (CXCR6) with effector (granzyme) and exhaustion (PD1) characteristics. Liver CXCR6+ CD8 T cells were characterized by low activity of the FOXO1 transcription factor, and were abundant in NASH mice and in patients with NASH. Mechanistically, IL-15 induced FOXO1 downregulation and CXCR6 upregulation, which together rendered liver-resident CXCR6+ CD8 T cells susceptible to metabolic stimuli (including acetate and extracellular ATP) and collectively triggered auto-aggression. CXCR6+ CD8 T cells from the livers of NASH mice or of patients with NASH had similar transcriptional signatures, and showed auto-aggressive killing of cells in an MHC-class-I-independent fashion after signalling through P2X7 purinergic receptors. This killing by auto-aggressive CD8 T cells fundamentally differed from that by antigen-specific cells, which mechanistically distinguishes auto-aggressive and protective T cell immunity.

The impact of genetic adaptation on chimpanzee subspecies differentiation.

Chimpanzees, humans' closest relatives, are in danger of extinction. Aside from direct human impacts such as hunting and habitat destruction, a key threat is transmissible disease. As humans continue to encroach upon their habitats, which shrink in size and grow in density, the risk of inter-population and cross-species viral transmission increases, a point dramatically made in the reverse with the global HIV/AIDS pandemic. Inhabiting central Africa, the four subspecies of chimpanzees differ in demographic history and geographical range, and are likely differentially adapted to their particular local environments. To quantitatively explore genetic adaptation, we investigated the genic enrichment for SNPs highly differentiated between chimpanzee subspecies. Previous analyses of such patterns in human populations exhibited limited evidence of adaptation. In contrast, chimpanzees show evidence of recent positive selection, with differences among subspecies. Specifically, we observe strong evidence of recent selection in eastern chimpanzees, with highly differentiated SNPs being uniquely enriched in genic sites in a way that is expected under recent adaptation but not under neutral evolution or background selection. These sites are enriched for genes involved in immune responses to pathogens, and for genes inferred to differentiate the immune response to infection by simian immunodeficiency virus (SIV) in natural vs. non-natural host species. Conversely, central chimpanzees exhibit an enrichment of signatures of positive selection only at cytokine receptors, due to selective sweeps in CCR3, CCR9 and CXCR6 -paralogs of CCR5 and CXCR4, the two major receptors utilized by HIV to enter human cells. Thus, our results suggest that positive selection has contributed to the genetic and phenotypic differentiation of chimpanzee subspecies, and that viruses likely play a predominate role in this differentiation, with SIV being a likely selective agent. Interestingly, our results suggest that SIV has elicited distinctive adaptive responses in these two chimpanzee subspecies.

CXCR6+ NK Cells in Human Fetal Liver and Spleen Possess Unique Phenotypic and Functional Capabilities.

Tissue-resident Natural Killer (NK) cells vary in phenotype according to tissue origin, but are typically CD56bright, CXCR6+, and CD69+. NK cells appear very early in fetal development, but little is known about when markers of tissue residency appear during gestation and whether the expression of these markers, most notably the chemokine receptor CXCR6, are associated with differences in functional capability. Using multi-parametric flow cytometry, we interrogated fetal liver and spleen NK cells for the expression of a multitude of extracellular markers associated with NK cell maturation, differentiation, and migration. We analyzed total NK cells from fetal liver and spleen and compared them to their adult liver and spleen counterparts, and peripheral blood (PB) NK. We found that fetal NK cells resemble each other and their adult counterparts more than PB NK. Maturity markers including CD16, CD57, and KIR are lower in fetal NK cells than PB, and markers associated with an immature phenotype are higher in fetal liver and spleen NK cells (NKG2A, CD94, and CD27). However, T-bet/EOMES transcription factor profiles are similar amongst fetal and adult liver and spleen NK cells (T-bet-/EOMES+) but differ from PB NK cells (T-bet+EOMES-). Further, donor-matched fetal liver and spleen NK cells share similar patterns of expression for most markers as a function of gestational age. We also performed functional studies including degranulation, cytotoxicity, and antibody-dependent cellular cytotoxicity (ADCC) assays. Fetal liver and spleen NK cells displayed limited cytotoxic effector function in chromium release assays but produced copious amounts of TNFα and IFNγ, and degranulated efficiently in response to stimulation with PMA/ionomycin. Further, CXCR6+ NK cells in fetal liver and spleen produce more cytokines and degranulate more robustly than their CXCR6- counterparts, even though CXCR6+ NK cells in fetal liver and spleen possess an immature phenotype. Major differences between CXCR6- and + NK cell subsets appear to occur later in development, as a distinct CXCR6+ NK cell phenotype is much more clearly defined in PB. In conclusion, fetal liver and spleen NK cells share similar phenotypes, resemble their adult counterparts, and already possess a distinct CXCR6+ NK cell population with discrete functional capabilities.

Prostate cancer cells hyper-activate CXCR6 signaling by cleaving CXCL16 to overcome effect of docetaxel.

Molecular reprogramming in response to chemotherapeutics leads to poor therapeutic outcomes for prostate cancer (PCa). In this study, we demonstrated that CXCR6-CXCL16 axis promotes DTX resistance and acts as a counter-defense mechanism. After CXCR6 activation, cell death in response to DTX was inhibited, and blocking of CXCR6 potentiated DTX cytotoxicity. Moreover, in response to DTX, PCa cells expressed higher CXCR6, CXCL16, and ADAM-10. Furthermore, ADAM-10-mediated release of CXCL16 hyper-activated CXCR6 signaling in response to DTX. Activation of CXCR6 resulted in increased GSK-3ß, NF-κB, ERK1/2 phosphorylation, and survivin expression, which reduce DTX response. Finally, treatment of PCa cells with anti-CXCR6 monoclonal antibody synergistically or additively induced cell death with ∼1.5-4.5 fold reduction in the effective concentration of DTX. In sum, our data imply that co-targeting of CXCR6 would lead to therapeutic enhancement of DTX, leading to better clinical outcomes for PCa patients.

Axl kinase drives immune checkpoint and chemokine signalling pathways in lung adenocarcinomas.

Axl receptor tyrosine kinase is involved in the growth and metastasis and is an indicator of poor prognosis in several cancers including lung cancers. Although a mitogen-activated protein kinase (MAPK) pathway and an epithelial-to-mesenchymal transition (EMT) program are critical, molecular mechanisms underlying the Axl-driven cancer progression have not been fully elucidated. We aimed to identify molecules up-regulated by Axl kinase in lung adenocarcinomas. Through the global gene expression analysis and the functional annotation clustering, we found that AXL expression positively correlated with mRNA expressions of immune checkpoint molecules and chemokine receptors in non-small-cell lung cancers. Validation cohorts including our biobank confirmed that the AXL expression significantly correlated with expression of genes encoding programmed death-ligand1 (PD-L1) and CXC chemokine receptor 6 (CXCR6) in lung adenocarcinoma, especially in epidermal growth factor receptor (EGFR) mutation-positive adenocarcinoma. Pharmacological inhibition of Axl kinase activity decreased mRNA expressions of PD-L1 and CXCR6 in EGFR mutation-positive cell lines. Our data indicates the novel role of Axl kinase as a driver of immune checkpoint molecules and chemokine signalling pathways in the progression of lung adenocarcinomas. This study also highlights the necessity of clinical trials in order to test the efficacy of Axl kinase inhibition in the Axl-highly expressing subset of lung adenocarcinomas. .

CXCR6 Inhibits Hepatocarcinogenesis by Promoting Natural Killer T- and CD4+ T-Cell-Dependent Control of Senescence.

BACKGROUND & AIMS: Inflammation in the liver provokes fibrosis, but inflammation is also important for tumor surveillance. Inhibitors of chemokine pathways, such as CXCL16 and CXCR6 regulation of lymphocyte trafficking, are being tested as antifibrotic agents, but their effects on the development of hepatocellular carcinoma (HCC) are unclear. We assessed the roles of CXCR6-dependent immune mechanisms in hepatocarcinogenesis. METHODS: C57BL/6J wild-type (WT) mice and CXCR6-deficient mice (Cxcr6eGfp/eGfp) were given injections of diethylnitrosamine (DEN) to induce liver cancer and α-galactosylceramide to activate natural killer T (NKT) cells. We also performed studies in mice with conditional, hepatocyte-specific deletion of NEMO, which develop inflammation-associated liver tumors (NemoLPC-KO and NemoLPC-KOCxcr6eGfp/eGfp mice). We collected liver tissues from patients with cirrhosis (n = 43), HCC (n = 35), and neither of these diseases (control individuals, n = 25). Human and mouse liver tissues were analyzed by histology, immunohistochemistry, flow cytometry, RNA expression arrays (from sorted hepatic lymphocytes), and matrix-assisted laser desorption/ionization imaging. Bone marrow was transferred from Cxcr6eGfp/eGfp or WT mice to irradiated C57BL/6J mice, and spleen and liver cells were analyzed by flow cytometry. CD4+ T cells or NKT cells were isolated from the spleen and liver of CD45.1+ WT mice and transferred into CXCR6-deficient mice after DEN injection. RESULTS: After DEN injection, CXCR6-deficient mice had a significantly higher tumor burden than WT mice and increased tumor progression, characterized by reduced intrahepatic numbers of invariant NKT and CD4+ T cells that express tumor necrosis factor and interferon gamma. Livers of NemoLPC-KOCxcr6eGfp/eGfp mice had significantly more senescent hepatocytes than livers of NemoLPC-KO mice. In studies of bone-marrow chimeras, adoptive cell transfer experiments, and analyses of NemoLPC-KO mice, we found that NKT and CD4 T cells promote the removal of senescent hepatocytes to prevent hepatocarcinogenesis, and that this process required CXCR6. Injection of WT with α-galactosylceramide increased removal of senescent hepatocytes by NKT cells. We observed peritumoral accumulation of CXCR6-associated lymphocytes in human HCC, which appeared reduced compared with cirrhosis tissues. CONCLUSIONS: In studies of mice with liver tumors, we found that CXCR6 mediated NKT-cell and CD4+ T-cell removal of senescent hepatocytes. Antifibrotic strategies to reduce CXCR6 activity in liver, or to reduce inflammation or modulate the immune response, should be tested for their effects on hepatocarcinogenesis.

Human liver-derived CXCR6+ NK cells are predominantly educated through NKG2A and show reduced cytokine production.

NK cells have been implicated to affect the outcome of numerous liver diseases. In particular, members of the killer-cell Ig-like receptor (KIR) family, predominantly expressed by NK cells, have been associated with the outcome of hepatitis C virus infection and clearance of hepatocellular carcinoma. Inhibitory KIRs tune NK cell function through interaction with HLA class I, a process termed education. Nevertheless, the impact of the hepatic environment on NK cell education is incompletely understood. Therefore, we investigated the composition and function of hepatic KIR-expressing NK cells. Matched PBMC and hepatic lymphocytes were isolated from 20 individuals undergoing liver surgery and subsequently phenotypically analyzed for expression of KIRs and markers for tissue residency using flow cytometry. NK cell function was determined by co-culturing NK cells with the target cell line 721.221 and subsequent assessment of CD107a, IFN-γ, and TNF-α expression. Liver-resident CXCR6+ /CD56Bright NK cells lacked KIRs and were predominantly educated through NKG2A, while CXCR6- /CD16+ NK cells expressed KIRs and resembled peripheral blood NK cells. Hepatic NK cells showed lower response rates compared to peripheral blood NK cells; in particular, CXCR6+ NK cells were hyporesponsive to stimulation with target cells. The high proportion of educated NK cells in both subsets indicates the importance of self-inhibitory receptors for the balance between maintenance of self-tolerance and functional readiness. However, the reduced functionality of hepatic NK cells may reflect the impact of the tolerogenic hepatic environment on NK cells irrespective of NK cell education.

Expression of CCR6 and CXCR6 by Gut-Derived CD4+/CD8α+ T-Regulatory Cells, Which Are Decreased in Blood Samples From Patients With Inflammatory Bowel Diseases.

BACKGROUND & AIMS: Faecalibacterium prausnitzii, a member of the Clostridium IV group of the Firmicutes phylum that is abundant in the intestinal microbiota, has anti-inflammatory effects. The relative level of F prausnitzii is decreased in fecal samples from patients with inflammatory bowel diseases (IBDs) compared with healthy individuals. Reduced F prausnitzii was correlated with relapse of Crohn's disease after surgery. We identified, in human colonic mucosa and blood, a population of T regulatory type 1-like T regulatory (TREG) cells that express CD4 and CD8α (DP8α T cells) and are specific for F prausnitzii. We aimed to determine whether they are altered in patients with IBD. METHODS: We isolated DP8α T cells from human colon lamina propria and blood samples and used flow cytometry to detect markers of cells that are of colon origin. We quantified DP8α cells that express colon-specific markers in blood samples from 106 patients with IBD, 12 patients with infectious colitis, and 35 healthy donors (controls). We identified cells that respond to F prausnitzii. Cells were stimulated with anti-CD3, and their production of interleukin 10 was measured by enzyme-linked immunosorbent assay. We compared the frequency and reactivity of cells from patients vs controls using the 2-sided Student t test or 1-way analysis of variance. RESULTS: Circulating DP8α T cells that proliferate in response to F prausnitzii express the C-C motif chemokine receptor 6 (CCR6) and C-X-C motif chemokine receptor 6 (CXCR6). These cells also have features of TREG cells, including production of IL-10 and inhibition of T-cell proliferation via CD39 activity. The proportion of circulating CCR6+/CXCR6+ DP8α T cells was significantly reduced (P < .0001) within the total population of CD3+ T cells from patients with IBD compared with patients with infectious colitis or controls. A threshold of <7.875 CCR6+/CXCR6+ DP8α T cells/10,000 CD3+ cells discriminated patients with IBD from those with infectious colitis with 100% specificity and 72.2% sensitivity. CONCLUSIONS: We identified a population of gut-derived TREG cells that are reduced in blood samples from patients with IBD compared with patients with infectious colitis or controls. These cells should be studied further to determine the mechanisms of this reduction and how it might contribute to the pathogenesis of IBD and their prognostic or diagnostic value.