C-X-C Motif Chemokine Receptor 3 Research Articles

CXCR3-CXCL11 Signaling Restricts Angiogenesis and Promotes Pericyte Recruitment.

Endothelial cell (EC)-pericyte interactions are known to remodel in response to hemodynamic forces; yet there is a lack of mechanistic understanding of the signaling pathways that underlie these events. Here, we have identified a novel signaling network regulated by blood flow in ECs-the chemokine receptor CXCR3 (CXC motif chemokine receptor 3) and one of its ligands, CXCL11 (CXC motif chemokine ligand 11)-that delimits EC angiogenic potential and promotes pericyte recruitment to ECs during development. We investigated the role of CXCR3 on vascular development using both 2- and 3-dimensional in vitro assays, to study EC-pericyte interactions and EC behavioral responses to blood flow. Additionally, genetic mutants and pharmacological modulators were used in zebra fish in vivo to study the impacts of CXCR3 loss and gain of function on vascular development. In vitro modeling of EC-pericyte interactions demonstrates that suppression of EC-specific CXCR3 signaling leads to loss of pericyte association with EC tubes. In vivo, phenotypic defects are particularly noted in the cranial vasculature, where we see a loss of pericyte association with ECs and expansion of the vasculature in zebra fish treated with the Cxcr3 inhibitor AMG487 or in homozygous cxcr3.1/3.2/3.3 triple mutants. We also demonstrate that CXCR3-deficient ECs are more elongated, move more slowly, and have impaired EC-EC junctions compared with their control counterparts. Our results suggest that CXCR3 signaling in ECs helps promote vascular stabilization events during development by preventing EC overgrowth and promoting pericyte recruitment.

Dietary supplementation with N-acetylcysteine confers a protective effect on muscle and liver in lipopolysaccharide-challenged piglets.

N-acetylcysteine (NAC) is a well-established antioxidant that offers exciting opportunities for intestinal health in weaned piglets, while the effects of NAC on muscle and liver has not been fully characterized. Therefore, the present study was performed to investigate the effects of dietary supplementation with NAC on muscle and liver in weaned piglets challenged with lipopolysaccharide (LPS). Twenty-four piglets (24-day-old) were randomly assigned to three treatment groups, the piglets in the control (CTR) and LPS- challenged (LPS) groups were fed the basal diet and those in the LPS+ NAC group was fed the basal diet supplemented with 500 mg/kg NAC. The animal trial lasted for 21 days. At the end of the trial, piglets in the LPS and LPS+ NAC groups were injected intraperitoneally with LPS (100 μg/kg body weight) and piglets in the CTR group were administrated with an equal volume of normal saline. 3 h later, the blood was collected and tissue samples were obtained after 6 h of LPS or normal saline treatment. The results showed that the level of IL-1β, and the mRNA levels of C-X-C motif chemokine receptor 3 (CXCR3) and interferon-γ (IFN-γ) in the liver were up-regulated, and the mRNA levels of insulin-like growth factor 1 (IGF-1), total glutathione (T-GSH), and the ratio of total protein to DNA in the liver were decreased under LPS challenge (P < 0.05). At the same time, LPS increased the level of H2O2 and decreased the content of T-GSH and DNA in the longissimus dorsi and gastrocnemius muscles (P < 0.05). In addition, the percentage of monocytes and the level of epidermal growth factor (EGF) were down-regulated in the LPS treatment (P < 0.05). Interestingly, dietary NAC supplementation reversed the above changes induced by LPS (P < 0.05). Furthermore, NAC might alleviate the muscle and liver injury in LPS-challenged piglets by regulating the expression of genes related to the type I interferon signaling pathway, as well as hypoxia inducible factor 1 (HIF1) and nuclear factor erythroid-2 related factor 2 (Nrf-2). Our findings suggested that dietary supplementation with NAC could benefit the health of muscle and liver in LPS-challenged weaned piglets.

Age and sex-dependent gut alterations in mice induced by neonatal immune activation with lipopolysaccharide

Neonatal immune activation (NIA) through exposure to lipopolysaccharide (LPS) induces adult behavioral changes in rodents that resemble symptoms of developmental disorders, such as autism spectrum disorder. The neonatal timing of LPS exposure appears to play a crucial role in determining the nature and extent of long-term changes. This study aims to explore whether a 3-day LPS-NIA triggers sex- and age-related changes in gut function, potentially linking LPS-NIA to gastrointestinal dysfunction. Male and female Swiss mice received intraperitoneal injections of LPS or saline on postnatal days (PN) 3, 5, and 7. At PN35 (juvenile) and PN70 (adult), gut inflammation and oxidative stress were evaluated in addition to assessments of working memory, depressive-like symptoms, sociability, and repetitive behavior. Gut examination showed elevated C-X-C motif chemokine receptor 3 (CXCR3) in LPS-NIA mice, while MyD88 and Zonulin expressions were significantly higher only in adult LPS-NIA females. Interleukin (IL)-23 expression increased in juvenile and adult male and juvenile female LPS-NIA mice. Oxidative changes included decreased duodenal reduced glutathione (GSH) in juvenile females and ileal GSH in adult females exposed to LPS-NIA. Regarding behavioral alterations, adult LPS-NIA females exhibited depressive-like behavior. Working memory deficits were observed across all LPS-NIA groups. Only juvenile LPS-NIA females increased grooming, while rearing was higher in adult LPS-NIA mice of both sexes. The findings imply that LPS-NIA impacts intestinal barrier function and causes gut inflammatory alterations that are sex- and age-specific. These findings pave the way for exploring potential mechanisms that could contribute to LPS-induced gastrointestinal disturbances among individuals with ASD.

Targeting MMP9 in CTNNB1 mutant hepatocellular carcinoma restores CD8+ T cell-mediated antitumour immunity and improves anti-PD-1 efficacy

ObjectiveThe gain of function (GOF) CTNNB1 mutations (CTNNB1GOF) in hepatocellular carcinoma (HCC) cause significant immune escape and resistance to anti-PD-1. Here, we aimed to investigate the mechanism of CTNNB1GOF HCC-mediated...

Abstract 6808: Investigating the role of CXCR3+ monocytes on CAR T cell function

Abstract Chimeric antigen receptor T cells (CAR-Ts) are an emerging immunotherapy that have remarkable efficacy against leukemias and lymphomas but limited success against solid tumors. We conducted a phase I clinical trial (NCT 02107963) of GD2 CAR-T (GD2-CAR.OX40.28.z.ICD9) administration to children and young adults with neuroblastoma and, for the first time, osteosarcoma. While all patients progressed and were switched to alternative therapy, we observed a significant association between good CAR-T expanders and expression of C-X-C Motif Chemokine receptor 3 (CXCR3) on monocytes detected in pretreatment apheresis. This study seeks to uncover the possible interactions between CXCR3 and CAR-Ts that may alter CAR-T function and ultimately lead to better CAR-T expansion. We hypothesize that CXCR3+ monocytes induce functional change in CAR-Ts resulting in improved expansion. To recapitulate the interaction between tumor, CAR-Ts and monocytes, we took 143Bs (osteosarcoma cell line) and GD2 CAR-Ts and co cultured them with THP-1s (a monocytic leukemia cell line) that were either untransduced (UTD) or transduced via lentivirus to express CXCR3 (CXCR3+). The ratio of UTD or CXCR3+ THP-1s to CAR-Ts and 143Bs was varied between co cultures. Enzyme-linked immunoassay (ELISA) for interferon gamma (IFNy) expression was run as a proxy measurement for CAR-T cell activity. Flow cytometry was used to assess for expression of activation and exhaustion markers. Differences between UTD and CXCR3+ co cultures were evaluated for statistical significance via unpaired t-test. We observed that CXCR3 was elevated on GD2 CAR-Ts co cultured with CXCR3+ monocytes. This pattern was seen most prominently in CD4+ CAR-Ts, but also subtly in CD8+ T-cells. Exhaustion markers CD39 and LAG3 were elevated in CAR-Ts co cultured with CXCR3+ monocytes. Additionally, CAR-Ts co cultured with CXCR3+ monocytes displayed greater activity via elevated IFNy expression. We conclude that GD-2 CAR-T cells co cultured with CXCR3+ monocytes express elevated exhaustion markers due to possible increased activity when in the presence of CXCR3+ monocytes. Additionally, we suspect that elevated CXCR3 expression on CAR-Ts co cultured with CXCR3+ monocytes may be helping CAR-Ts better target tumor cells, thus leading to improved expansion. If CXCR3 can improve CAR-T cell function, this could lead to better optimization of CAR-Ts against solid tumors and create a viable treatment option for neuroblastoma and osteosarcoma patients. Citation Format: Claire Victoria Ong, Sabina Kaczanowska, Wei Ju, James Cronk, Sneha Ramakrishna, Rosandra Kaplan. Investigating the role of CXCR3+ monocytes on CAR T cell function [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6808.

T cell specific deletion of Casitas B lineage lymphoma-b reduces atherosclerosis, but increases plaque T cell infiltration and systemic T cell activation.

Atherosclerosis is a lipid-driven inflammatory disease of the arterial wall, and the underlying cause of the majority of cardiovascular diseases. Recent advances in high-parametric immunophenotyping of immune cells indicate that T cells constitute the major leukocyte population in the atherosclerotic plaque. The E3 ubiquitin ligase Casitas B-lymphoma proto-oncogene-B (CBL-B) is a critical intracellular regulator that sets the threshold for T cell activation, making CBL-B a potential therapeutic target to modulate inflammation in atherosclerosis. We previously demonstrated that complete knock-out of CBL-B aggravated atherosclerosis in Apoe-/- mice, which was attributed to increased macrophage recruitment and increased CD8+ T cell activation in the plaque. To further study the T cell specific role of CBL-B in atherosclerosis, Apoe-/- CD4cre Cblb fl/fl (Cbl-bcKO) mice and Apoe-/-CD4WTCblbfl/fl littermates (Cbl-bfl/fl) were fed a high cholesterol diet for ten weeks. Cbl-bcKO mice had smaller atherosclerotic lesions in the aortic arch and root compared to Cbl-bfl/fl, and a substantial increase in CD3+ T cells in the plaque. Collagen content in the plaque was decreased, while other plaque characteristics including plaque necrotic core, macrophage content, and smooth muscle cell content, remained unchanged. Mice lacking T cell CBL-B had a 1.4-fold increase in CD8+ T cells and a 1.8-fold increase in regulatory T cells in the spleen. Splenic CD4+ and CD8+ T cells had increased expression of C-X-C Motif Chemokine Receptor 3 (CXCR3) and interferon-γ (IFN-γ), indicating a T helper 1 (Th1)-like/effector CD8+ T cell-like phenotype. In conclusion, Cbl-bcKO mice have reduced atherosclerosis but show increased T cell accumulation in the plaque accompanied by systemic T cell activation.

Targeting Fli‐1 and chemokine axis CXCL10/CXCL13/CXCR3 in systemic lupus erythematosus and lupus nephritis

AbstractFli‐1 is a critical factor involved in hematopoietic stem cells, vascular endothelial cells apoptosis, and differentiation. Similarly, Fli‐1 regulates immune cell proliferation and differentiation, such as T cells, B cells, and monocytes. Regarding T/B cell dysfunction, SLE including LN is recognized to be associated with abnormal expression and function of Fli‐1. Moreover, three ligands of Fli‐1 including CXCL10 (C‐X‐C motif chemokine ligand 10), CXCR3 (C‐X‐C motif chemokine receptor 3), CXCL13 are widely discussed in SLE/LN. In this comprehensive review, we not only discussed Fli‐1 within SLE/LN pathogenesis, but also discussed the effects of CXCL10, CXCR3, and CXCL13 on SLE/LN development. Furthermore, we discussed how Fli‐1 regulates CXCL10, CXCR3, and CXCL13, and then involved in lupus development. It is possible that the Fli‐1 axis might be a potential target in SLE and LN.

Effect of IP-10/CXCR3 signaling pathway on rats with diabetic retinopathy.

This study aimed to investigate the effect of the interferon-inducible protein-10 (IP-10)/C-X-C motif chemokine receptor 3 (CXCR3) signaling pathway on rats with diabetic retinopathy. A total of 21 Sprague-Dawley rats were selected as the objects and divided into control (n=7), model (n=7) and inhibitor (n=7) groups. The rats in control group did not receive any treatment. The diabetic retinopathy model was established using streptozotocin and vascular endothelial growth factor in model group, while the rats in inhibitor group were treated with AMG 487, an inhibitor of the IP-10/CXCR3 signaling pathway, based on the treatment in model group. The changes in gene expression patterns in rats with diabetic retinopathy were screened by sequencing. After the differential genes were determined, the pathways mainly related to the complication were obtained via enrichment analysis. The expression of the IP-10/CXCR3 signaling pathway, the apoptotic cells and the expression of inflammatory molecules (IL-6, IL-12, TNF-α and IL-1β) in each group of rats were detected. It was shown in volcano plot that there were some differentially expressed genes (fold change >1.2, P<0.01) in retinal tissues of rats in control and model groups. Meanwhile, the heatmap displayed that there were great differences in the gene expression patterns between control and model groups, and the gene expressions of IP-10 and CXCR3 in model group were higher than those in control group (P<0.05). The differential genes in control and model groupwere enriched in such processes as the cAMP metabolic regulatory pathway, chemotaxis of immunocytes, proliferation of endothelial cells, response of cytokine receptors, apoptosis, mTOR signaling pathway and TGF-β-related signaling pathway. The mRNA expressions of IP-10 and CXCR3 in model group were higher than those in control group (P<0.05), while they were notably lower in inhibitor group than those in model group (P<0.05). Besides, the protein levels of IP-10 and CXCR3 were identical to the mRNA levels. The apoptotic cells were increased markedly in model group compared with those in control group (P<0.05) and inhibitor group (P<0.05). Model group exhibited higher expression levels of IL-6, TNF-α and IL-1β in retinal tissues than control group (P<0.05), while inhibitor group had distinctly lower expression levels of IL-6, TNF-α and IL-1β in retinal tissues than model group (P<0.05). The IP-10/CXCR3 signaling pathway can affect rats with diabetic retinopathy.

Autoantibodies against the chemokine receptor 3 predict cardiovascular risk.

Chronic inflammation and autoimmunity contribute to cardiovascular (CV) disease. Recently, autoantibodies (aAbs) against the CXC-motif-chemokine receptor 3 (CXCR3), a G protein-coupled receptor with a key role in atherosclerosis, have been identified. The role of anti-CXCR3 aAbs for CV risk and disease is unclear. Anti-CXCR3 aAbs were quantified by a commercially available enzyme-linked immunosorbent assay in 5000 participants (availability: 97.1%) of the population-based Gutenberg Health Study with extensive clinical phenotyping. Regression analyses were carried out to identify determinants of anti-CXCR3 aAbs and relevance for clinical outcome (i.e. all-cause mortality, cardiac death, heart failure, and major adverse cardiac events comprising incident coronary artery disease, myocardial infarction, and cardiac death). Last, immunization with CXCR3 and passive transfer of aAbs were performed in ApoE(-/-) mice for preclinical validation. The analysis sample included 4195 individuals (48% female, mean age 55.5 ± 11 years) after exclusion of individuals with autoimmune disease, immunomodulatory medication, acute infection, and history of cancer. Independent of age, sex, renal function, and traditional CV risk factors, increasing concentrations of anti-CXCR3 aAbs translated into higher intima-media thickness, left ventricular mass, and N-terminal pro-B-type natriuretic peptide. Adjusted for age and sex, anti-CXCR3 aAbs above the 75th percentile predicted all-cause death [hazard ratio (HR) (95% confidence interval) 1.25 (1.02, 1.52), P = .029], driven by excess cardiac mortality [HR 2.51 (1.21, 5.22), P = .014]. A trend towards a higher risk for major adverse cardiac events [HR 1.42 (1.0, 2.0), P = .05] along with increased risk of incident heart failure [HR per standard deviation increase of anti-CXCR3 aAbs: 1.26 (1.02, 1.56), P = .03] may contribute to this observation. Targeted proteomics revealed a molecular signature of anti-CXCR3 aAbs reflecting immune cell activation and cytokine-cytokine receptor interactions associated with an ongoing T helper cell 1 response. Finally, ApoE(-/-) mice immunized against CXCR3 displayed increased anti-CXCR3 aAbs and exhibited a higher burden of atherosclerosis compared to non-immunized controls, correlating with concentrations of anti-CXCR3 aAbs in the passive transfer model. In individuals free of autoimmune disease, anti-CXCR3 aAbs were abundant, related to CV end-organ damage, and predicted all-cause death as well as cardiac morbidity and mortality in conjunction with the acceleration of experimental atherosclerosis.

Systematic Pan-Cancer Analysis Reveals X-C Motif Chemokine Receptor 1 as a Prognostic and Immunological Biomarker.

Chemokines and their receptors play an important role in immune monitoring and immune defense during tumor growth and metastasis. However, their prognostic roles in pan-cancer have not been elucidated. In this work, we screened all chemokine receptors in pan-cancer and discovered X-C Motif Chemokine Receptor 1 (XCR1) as a reliable immunological and prognostic biomarker in pan-cancer using bioinformation. The TCGA database served as the foundation for the primary research database analysis in this work. XCR1 was downregulated in tumors. Patients with reduced XCR1 showed worse prognoses and a concomitant decrease in immune cell infiltration (DCs and CD8+ T cells). According to a gene enrichment study, XCR1 enhanced immune system performance by promoting T-cell infiltration through the C-X-C Motif Chemokine Ligand 9 (CXCL9)- C-X-C Motif Chemokine Receptor 3 (CXCR3) axis. In addition, XCR1 is mainly expressed in infiltrated DCs and some malignant cells in tumor tissues. Our data revealed the important role of XCR1 in remodeling the tumor microenvironment and predicting the survival prognosis, which could also be used as a sensitive biomarker for tumor immunotherapy.

Zinc finger protein 189 promotes the differentiation of lamina propria T helper 17.1 cells in dextran sulfate sodium-induced colitis

The factors regulating the heterogeneity of interleukin-17A (IL-17A)-expressing CD4+ T cells in inflammatory bowel diseases remain unclear. In the current study, we characterised the expression and function of zinc finger protein 189 (ZFP189) in a murine colitis model. Mice were given dextran sulphate sodium to induce acute colitis. Flow cytometry was applied to recognise and enrich Th17 and Th17.1 cells based on the expression of IL-17A, interferon-γ (IFN-γ), C-X-C motif chemokine receptor 3 (CXCR3), and C-C motif chemokine receptor 4 (CCR4). The expression of ZFP189 in Th17 and Th17.1 cells was determined by Immunoblotting. Lentivirus-mediated ZFP189 knockdown was conducted to evaluate the effect of ZFP189 on the differentiation of Th17 and Th17.1 cells. The adoptive transfer was performed to analyse the pathogenicity of Th17.1 cells in vivo. We found that ZFP189 was mildly up-regulated in IL-17A-expressing CD4+ T cells in colonic lamina propria. Lamina propria Th17.1 cells expressed higher ZFP189 than Th17 cells. In vitro ZFP189 knockdown in CD4+ T cells did not impact Th17 cell differentiation but suppressed Th17.1 cell differentiation, as evidenced by lower T-box expressed in T cells (T-bet) and IFN-γ. When adoptively transferred into mice, ZFP189-deficient Th17.1 cells produced fewer IFN-γ, tumour necrosis factor-alpha (TNF-α), and granulocyte-macrophage colony-stimulating factor (GM-CSF) than ZFP189-expressing Th17.1 cells. Moreover, ZFP189-deficient Th17.1 cells induced less severe colitis than ZFP189-expressing Th17.1 cells, as evidenced by less body weight loss, a lower disease activity index, and a lower colon histological score. In summary, ZFP189 acts as a positive regulator of the differentiation and pathogenicity of lamina propria Th17.1 cells in colitis.

CD8+ T Cells Promote Pathological Angiogenesis in Ocular Neovascular Disease.

CD4+ (cluster of differentation) and CD8+ T cells are increased in the ocular fluids of patients with neovascular retinopathy, yet their role in the disease process is unknown. We describe how CD8+ T cells migrate into the retina and contribute to pathological angiogenesis by releasing cytokines and cytotoxic factors. In oxygen-induced retinopathy, flow cytometry revealed the numbers of CD4+ and CD8+ T cells were increased in blood, lymphoid organs, and retina throughout the development of neovascular retinopathy. Interestingly, the depletion of CD8+ T cells but not CD4+ T cells reduced retinal neovascularization and vascular leakage. Using reporter mice expressing gfp (green fluorescence protein) in CD8+ T cells, these cells were localized near neovascular tufts in the retina, confirming that CD8+ T cells contribute to the disease. Furthermore, the adoptive transfer of CD8+ T cells deficient in TNF (tumor necrosis factor), IFNγ (interferon gamma), Prf (perforin), or GzmA/B (granzymes A/B) into immunocompetent Rag1-/- mice revealed that CD8+ T cells mediate retinal vascular disease via these factors, with TNF influencing all aspects of vascular pathology. The pathway by which CD8+ T cells migrate into the retina was identified as CXCR3 (C-X-C motif chemokine receptor 3) with the CXCR3 blockade reducing the number of CD8+ T cells within the retina and retinal vascular disease. We discovered that CXCR3 is central to the migration of CD8+ T cells into the retina as the CXCR3 blockade reduced the number of CD8+ T cells within the retina and vasculopathy. This research identified an unappreciated role for CD8+ T cells in retinal inflammation and vascular disease. Reducing CD8+ T cells via their inflammatory and recruitment pathways is a potential treatment for neovascular retinopathies.

Involvement of chemokine receptor CXCR3 in the defense mechanism against Neospora caninum infection in C57BL/6 mice.

C-X-C motif chemokine receptor 3 (CXCR3) is an important receptor controlling the migration of leukocytes, although there is no report regarding its role in Neospora caninum infection. Herein, we investigated the relevance of CXCR3 in the resistance mechanism to N. caninum infection in mice. Wild-type (WT) C57BL/6 mice and CXCR3-knockout (CXCR3KO) mice were used in all experiments. WT mice displayed a high survival rate (100%), while 80% of CXCR3KO mice succumbed to N. caninum infection within 50 days. Compared with WT mice, CXCR3KO mice exhibited significantly lower body weights and higher clinical scores at the subacute stage of infection. Flow cytometric analysis revealed CXCR3KO mice as having significantly increased proportions and numbers of CD11c-positive cells compared with WT mice at 5 days post infection (dpi). However, levels of interleukin-6 and interferon-γ in serum and ascites were similar in all groups at 5 dpi. Furthermore, no differences in parasite load were detected in brain, spleen, lungs or liver tissue of CXCR3KO and WT mice at 5 and 21 dpi. mRNA analysis of brain tissue collected from infected mice at 30 dpi revealed no changes in expression levels of inflammatory response genes. Nevertheless, the brain tissue of infected CXCR3KO mice displayed significant necrosis and microglial activation compared with that of WT mice at 21 dpi. Interestingly, the brain tissue of CXCR3KO mice displayed significantly lower numbers of FoxP3+ cells compared with the brain tissue of WT mice at 30 dpi. Accordingly, our study suggests that the lack of active regulatory T cells in brain tissue of infected CXCR3KO mice is the main cause of these mice having severe necrosis and lower survival compared with WT mice. Thus, CXCR3+ regulatory T cells may play a crucial role in control of neosporosis.

CD8+ T cells mediate antiviral response in severe fever with thrombocytopenia syndrome.

Severe fever with thrombocytopenia syndrome (SFTS), which is caused by a novel Bunyavirus, has gradually become a threatening infectious disease in rural areas of Asia. Studies have identified a severe cytokine storm and impaired humoral immune response in SFTS. However, the cellular immune response to SFTS virus (SFTSV) infection remains largely unknown. Here we report that SFTS patients had a cytokine storm accompanied by high levels of chemokines. CD8+ T cells in peripheral blood mononuclear cells of SFTS patients exhibited a more activated phenotype and enhanced the antiviral responses. They increased the expression of CD69 and CD25, secreted a higher level of IFN-γ and granzyme, and had a stronger proliferative ability than in healthy controls. In convalescent SFTS patients, the expression of CD69 and CD25 on CD8+ T cells was reduced. In addition, we found the ratio and cellularity of CD14+ CD16+ intermediate monocytes were increased in peripheral blood of SFTS patients. Both the expression of C-X-C motif chemokine ligand 10 (CXCL10) on CD14+ CD16+ intermediate monocytes and the expression of C-X-C motif chemokine receptor 3 (CXCR3) on CD8+ T cells increased dramatically in SFTS patients. Our studies reveal a potential pathway that CD8+ T cells rapidly activate and are mostly recruited by intermediate monocytes through CXCL10 in SFTSV infection. Our results may be of clinical relevance for further treatment and discharge instructions in SFTSV infections.

IL-2/GM-CSF enhances CXCR3 expression in CAR-T cells via the PI3K/AKT and ERK1/2 pathways.

To investigate the effects of cytokines IL-2 and GM-CSF on CXCR3 expression and chemotaxis of CAR-T cells. High lymphocyte infiltration within the tumor is a basic requirement for good results in tumor immunotherapy; C-X-C motif chemokine receptor 3 (CXCR3) is an important factor for the chemotaxis of lymphocytes to tumor tissues. The tumor microenvironment can exhibit diverse cytokine suppression or promote antitumor immunity. Both interleukin (IL)-2 and granulocyte macrophage colony-stimulating factor (GM-CSF) contribute to the regulation of immunosuppression in the tumor microenvironment. However, the effects of IL-2 and GM-CSF on CXCR3 expression on the T cell surface and its mechanisms are not well understood. Here, we explored the effects of polycytokines on CXCR3 expression in chimeric antigen receptor T cells (CAR-T cells) and on HuH-7 in situ hepatocellular carcinoma. Peripheral blood mononuclear cells (PBMCs) were isolated, followed by purifying using CD3 immunomagnetic beads. Cells were divided into three groups. After 24h of activation using CD3/CD28 antibody, T cells were transfected using lentiviral vector, pGC-SV40-EGFP-GPC3-CAR. Three culture methods were used to amplify the transfected T cells. Method 'A' was to incubate T cells with CD3/CD28 antibody; method 'B' was with CD3/CD28 antibody and IL-2 at a final concentration of 1000 U/ml; method 'C' was with method B in addition of GM-CSF at a final concentration of 1000 U/ml. The phosphorylation of MAPK and PI3K/AKT was determined by western blot. The chemotaxis effect of CAR-T cells on Huh-7 HCCIA in situ was assayed by immunofluorescence and immunohistochemistry. The CD3/CD28/IL-2/GM-CSF combination is the most potent for stimulating activated CAR-T cell proliferation and CXCR3 expression in vitro; CD3/CD28/IL-2 induces CAR-T cell expression of CXCR3 through the activation of the PI3K/APK pathway and GM-CSF induces CXCR3 expression in CAR-T cells through the activation of ERK1/2 rather than the p38 MAPK signaling pathway. CAR-GPC3-T cells with high CXCR3 expression showed increased chemotaxis ability to HuH in situ hepatocellular carcinoma, and considerably inhibited the growth of in situ tumors in nude mouse livers. A multi-factorial amplification protocol can effectively improve CXCR3 expression on the surface of activated CAR-T cells in vitro, as well as enhance the chemotaxis ability of CAR-T cells in vivo, which significantly inhibit the growth of liver cancer.

Specific Loss of ABCA1 (ATP-Binding Cassette Transporter A1) Suppresses TCR (T-Cell Receptor) Signaling and Provides Protection Against Atherosclerosis.

ABCA1 (ATP-binding cassette transporter A1) mediates cholesterol efflux to apo AI to maintain cellular cholesterol homeostasis. The current study aims to investigate whether T-cell-specific deletion of ABCA1 modulates the phenotype/function of T cells and the development of atherosclerosis. Mice with T-cell-specific deletion of ABCA1 on low-density lipoprotein receptor knockout (Ldlr-/-) background (Abca1CD4-/CD4-Ldlr-/-) were generated by multiple steps of (cross)-breedings among Abca1flox/flox, CD4-Cre, and Ldlr-/- mice. Deletions of ABCA1 greatly suppressed cholesterol efflux to apo AI but slightly reduced membrane lipid rafts on T cells probably due to the upregulation of ABCG1. Moreover, ABCA1 deficiency impaired TCR (T-cell receptor) signaling and inhibited the survival and proliferation of T cells as well as the formation of effector memory T cells. Despite the comparable levels of plasma total cholesterol after Western-type diet feeding, Abca1CD4-/CD4-Ldlr-/- mice showed significantly attenuated arterial accumulations of T cells and smaller atherosclerotic lesions than Abca1+/+Ldlr-/-controls, which were associated with reduced surface CCR5 (CC motif chemokine receptor 5) and CXCR3 (CXC motif chemokine receptor 3), decreased antiapoptotic Bcl-2 (B-cell lymphoma 2) and Bcl-xL (B-cell lymphoma extra-large), and hampered abilities to produce IL (interleukin)-2 and IFN (interferon)-γ by ABCA1-deficient T cells. ABCA1 is essential for T-cell cholesterol homeostasis. Deletion of ABCA1 in T cells impairs TCR signaling, suppresses the survival, proliferation, differentiation, and function of T cells, thereby providing atheroprotection in vivo.

Selective targeting of GARP-LTGFβ axis in the tumor microenvironment augments PD-1 blockade via enhancing CD8+ T cell antitumor immunity

BackgroundImmune checkpoint blockade (ICB) has revolutionized cancer immunotherapy. However, most patients with cancer fail to respond clinically. One potential reason is the accumulation of immunosuppressive transforming growth factor β (TGFβ)...

Inducible Costimulator-C-X-C Motif Chemokine Receptor 3 Signaling is Involved in Chronic Obstructive Pulmonary Disease Pathogenesis.

BackgroundThe role of inducible costimulator (ICOS) signaling in chronic obstructive pulmonary disease (COPD) has not been fully elucidated.MethodsWe compared the percentages of ICOS+ T cells and ICOS+ regulatory T (Treg) cells in CD4+ T cells and CD4+CD25+FOXP3+ Tregs, respectively, in the peripheral blood of smokers with or without COPD to those in healthy controls. We further characterized their phenotypes using flow cytometry. To investigate the influence of ICOS signaling on C-X-C motif chemokine receptor 3 (CXCR3) expression in COPD, we evaluated the expression levels of ICOS and CXCR3 in vivo and in vitro.ResultsICOS expression was elevated on peripheral CD4+ T cells and CD4+ Tregs of COPD patients, which positively correlated with the severity of lung function impairment in patients with stable COPD (SCOPD), but not in patients with acute exacerbation of COPD (AECOPD). ICOS+CD4+ Tregs in patients with SCOPD expressed higher levels of coinhibitors, programmed cell death protein 1 (PD-1) and T-cell immunoreceptor with Ig and ITIM domains (TIGIT), than ICOS−CD4+ Tregs, whereas ICOS+CD4+ T cells mostly exhibited a central memory (CD45RA−CCR7+) or effector memory (CD45RA−CCR7−) phenotype, ensuring their superior potential to respond potently and quickly to pathogen invasion. Furthermore, increased percentages of CXCR3+CD4+ T cells and CXCR3+CD4+ Tregs were observed in the peripheral blood of patients with SCOPD, and the expression level of CXCR3 was higher in ICOS+CD4+ T cells than in ICOS−CD4+ T cells. The percentage of CXCR3+CD4+ T cells was even higher in the bronchoalveolar lavage fluid than in matched peripheral blood in SCOPD group. Lastly, in vitro experiments showed that ICOS induced CXCR3 expression on CD4+ T cells.ConclusionsICOS signaling is upregulated in COPD, which induces CXCR3 expression. This may contribute to increased numbers of CXCR3+ Th1 cells in the lungs of patients with COPD, causing inflammation and tissue damage.

Therapeutic effect of multiple functional minicircle vector encoding anti-CD25/IL-10/CXCR3 in allograft rejection model.

Background/AimsWe previously proposed minicircle vector technology as the potential platform for the development and production of new biologics. In this study, we have designed a novel target molecule for the treatment of allograft rejection and evaluated its feasibility as the therapeutic agent in this disease using the minicircle vector system.MethodsWe engineered vectors to carry cassette sequences for anti-CD25, interleukin-10 (IL-10), and C-X-C motif chemokine receptor 3 (CXCR3) fusion protein, and then isolated minicircle vectors from the parent vectors. We verified the substantial production of anti-CD25/IL-10/CXCR3 fusion protein from minicircles and their duration in HEK293T cells and mice models. We also evaluated whether minicircle-derived anti-CD25/IL-10/CXCR3 has therapeutic effects in a skin allograft in mice model.ResultsWe confirmed the production of anti-CD25/IL-10/CXCR3 from minicircle by its significant availability in cells transfected with the minicircle and in its conditioned media. After a single injection of minicircle by hydrodynamic injection via mouse tail vein, luminescence or red fluorescence was maintained until 40 days in the liver tissue, suggesting the production of anti-CD25/IL-10/CXCR3 protein from minicircles via protein synthesis machinery in the liver. Mice treated with the minicircle encoding anti-CD25/IL-10/CXCR3 showed prolonged skin allograft survival times accompanied by improved immunologic regulation e.g., reduction of the lymphocyte population of Th1, Th2, and Th17 and an induction of regulatory T cells.ConclusionsThese findings implied that self-generated anti-CD25/IL-10/CXCR3 protein drug by minicircle technology is functionally active and relevant for reducing allograft rejection. The minicircle vector system may be useful for developing new biological drugs, avoiding manufacturing or practical problems.

Abstract 6349: Targeting the CXCR3 pathway with a novel peptide drug candidate mobilizes the immune system to enhance anti-tumor immunity

Abstract Immune-checkpoint inhibitor (ICI) therapy releases the molecular “brakes” on the immune system thereby promoting robust anti-tumor immune responses. However, many patients do not respond to ICI therapy due to development of primary and secondary resistance, and this population represents a large unmet medical need. The critical role of C-X-C motif chemokine receptor 3 (CXCR3) signaling in eliciting an effective response to anti-PD-1 therapy has been recently demonstrated. The CXCR3 chemokine system is instrumental in effector cell recruitment to the tumor and augments intratumoral CD8+ T cell proliferation and function, which are key mechanisms driving anti-tumor immunity and responses to ICI therapy. We used our proprietary peptide discovery platform to identify a unique microbiome-derived peptide, SG-3-00802, from bacterial strains associated with response to anti-PD1 inhibitors in patients with melanoma. We subsequently determined that CXCR3 is the target receptor for SG-3-00802 and demonstrated that SG-3-00802 enhanced the activity of CXCR3 in the presence of its endogenous ligands CXCL9/CXCL10/CXCL11. Optimization of SG-3-00802 pharmacological properties led to the selection of a novel drug development candidate SG-3-00802DC with improved potency and PK properties. Mechanistically, it increases CXCR3 activation by CXCL11 by greater than 10-fold from a nM to a pM range. As a positive allosteric modulator, SG-3-00802DC can potentially alter the conformation of the primary orthosteric binding site of CXCR3 and enhance the binding affinity of CXCL11, causing increased CXCR3 signaling activity, which is known to drive TIL infiltration that improves overall survival in mouse tumor models and cancer patients. Supporting this concept, we demonstrated that SG-3-00802DC showed anti-tumor activity in pre-clinical mouse tumor models, alone and in combination with anti-PD-1, improved overall survival and increased recruitment of CXCR3+ effector cells into the tumor microenvironment. Numerous strategies are currently undergoing clinical evaluation to improve long-lasting disease control in broader patient populations by combining ICIs with approved and novel therapeutic agents and procedures. SG-3-00802DC, with its well validated and unique mechanism of action to safely target the CXCR3-driven anti-tumor immune response offers a novel orthogonal approach complementary not only to immunotherapies, but also as a combination strategy with chemotherapy or radiotherapy. Citation Format: Dhwani Haria, Jina Lee, Justy Guagua, Archana Nagaraja, Kyle Roskamp, Bum-Yeol Hwang, Divya Ravichandar, Michi Willcoxon, Todd DeSantis, Karim Dabbagh, Helena Kiefel. Targeting the CXCR3 pathway with a novel peptide drug candidate mobilizes the immune system to enhance anti-tumor immunity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6349.