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Regulatory T cells (Tregs) play a key role in suppressing systemic effector immune responses, thereby preventing autoimmune diseases but also potentially contributing to tumor progression. Thus, there is great interest in clinically manipulating Tregs, but the precise mechanisms governing in vitro-induced Treg (iTreg) differentiation are not yet fully understood. Here, we used multiparametric mass cytometry to phenotypically profile human iTregs during the early stages of in vitro differentiation at single-cell level. A panel of 25 metal-conjugated antibodies specific to markers associated with human Tregs was used to characterize these immunomodulatory cells. We found that iTregs highly express the transcription factor FOXP3, as well as characteristic Treg-associated surface markers (e.g. CD25, PD1, CD137, CCR4, CCR7, CXCR3, and CD103). Expression of co-inhibitory factors (e.g. TIM3, LAG3, and TIGIT) increased slightly at late stages of iTreg differentiation. Further, CD103 was upregulated on a subpopulation of iTregs with greater suppressive capacity than their CD103- counterparts. Using mass-spectrometry-based proteomics, we showed that sorted CD103+ iTregs express factors associated with immunosuppression. Overall, our study highlights that during early stages of differentiation, iTregs resemble memory-like Treg features with immunosuppressive activity, and provides opportunities for further investigation into the molecular mechanisms underlying Treg function.

Immune checkpoint blockade (ICB) with PD-1/PD-L1 inhibition has revolutionized the treatment of non-small cell lung cancer (NSCLC). Durable responses, however, are observed only in a subpopulation of patients. Defective antigen presentation and an immunosuppressive tumor microenvironment (TME) can lead to deficient T cell recruitment and ICB resistance. We evaluate intratumoral (IT) vaccination with CXCL9- and CXCL10-engineered dendritic cells (CXCL9/10-DC) as a strategy to overcome resistance. IT CXCL9/10-DC leads to enhanced T cell infiltration and activation in the TME and tumor inhibition in murine NSCLC models. The antitumor efficacy of IT CXCL9/10-DC is dependent on CD4+ and CD8+ T cells, as well as CXCR3-dependent T cell trafficking from the lymph node. IT CXCL9/10-DC, in combination with ICB, overcomes resistance and establishes systemic tumor-specific immunity in murine models. These studies provide a mechanistic understanding of CXCL9/10-DC-mediated host immune activation and support clinical translation of IT CXCL9/10-DC to augment ICB efficacy in NSCLC.

Background: Within the tumor microenvironment, altered lipid metabolism promotes cancer cell malignancy by activating oncogenic cascades; however, impact of lipid metabolism in CD4+ tumor-infiltrating lymphocytes (TILs) remains poorly understood. Here, we elucidated that role of stearoyl-CoA desaturase (SCD) increased by treatment with cancer-associated fibroblast (CAF) supernatant in CD4+ T cells on their subset differentiation and activity of CD8+ T cells. Results: In our study, we observed that CD4+ TILs had higher lipid droplet content than CD4+ splenic T cells. In tumor tissue, CAF-derived supernatant provided fatty acids to CD4+ TILs, which increased the expression of SCD and oleic acid (OA) content. Increased SCD expression by OA treatment enhanced the levels of Th1 cell markers TBX21, interleukin-2, and interferon-γ. However, SCD inhibition upregulated the expression of regulatory T (Treg) cell markers, FOXP3 and transforming growth factor-β. Comparative fatty acid analysis of genetically engineered Jurkat cells revealed that OA level was significantly higher in SCD-overexpressing cells. Overexpression of SCD increased expression of Th1 cell markers, while treatment with OA enhanced the transcriptional level of TBX21 in Jurkat cells. In contrast, palmitic acid which is higher in SCD-KO cells than other subclones enhanced the expression of Treg cell markers through upregulation of mitochondrial superoxide. Furthermore, SCD increased the secretion of the C–X–C motif chemokine ligand 11 (CXCL11) from CD4+ T cells. The binding of CXCL11 to CXCR3 on CD8+ T cells augmented their cytotoxic activity. In a mouse tumor model, the suppressive effect of CD8+ T cells on tumor growth was dependent on CXCR3 expression. Conclusion: These findings illustrate that SCD not only orchestrates the differentiation of T helper cells, but also promotes the antitumor activity of CD8+ T cells, suggesting its function in adverse tumor microenvironments. [ABSTRACT FROM AUTHOR]

CXCR3 is a chemokine receptor with three ligands: CXCL9, CXCL10 and CXCL11. We report that in addition to attracting CXCR3+ T cells to tumor sites a key role of CXCL9 and CXCL10 is in inducing a self-feeding feedback loop that accelerates effector/cytotoxic activities of both CD4+ and CD8+ T cells while downregulating immunoregulatory protein TIM3. CXCR3KO mice displayed a markedly reduced response to anti-PD-1 and anti-CTLA-4 therapy. Results from a panel of in vivo and ex vivo 3D tumor models imply that, beyond driving CD8+ T cells into T-cell exhaustion, a major role of PD-1 and CTLA-4 is in limiting the CXCR3-based self-feeding mechanism of T cell potentiation. This may explain why patients that are CXCL9/CXCL10high tend to respond well to anti-PD-1 therapy, as opposed to patients that are CXCL9/CXCL10low. It also suggests a therapeutic role for CXCL9-Fc or CXCL10-Fc therapy; herein we demonstrate significant anti-tumor activity in multiple murine tumor models with such agents. [ABSTRACT FROM AUTHOR]

Chemokines are cytokines that mediate leukocyte traffic between the lymphoid organs, the bloodstream, and the site of tissue damage, which is essential for an efficient immune response. In particular, the gamma interferon (IFN-g) inducible chemokines CXCL9, CXCL10, and CXCL11, and their receptor CXCR3, are involved in T cell and macrophage recruitment to the site of infection. The nature and function of these chemokines and their receptor are well-known in mammals, but further research is needed to achieve a similar level of understanding in fish immunity. Thus, in this study, we seek to identify the genes encoding the components of the Atlantic salmon (Salmo salar) CXCL9, CXCL10, CXCL11/CXCR3 axis (CXCL9-11/CXCR3), predict the protein structure from the amino acid sequence, and explore the regulation of gene expression as well as the response of these chemokines and their receptor to viral infections. The cxcl9, cxcl10, cxcl11, and cxcr3 gene sequences were retrieved from the databases, and the phylogenetic analysis was conducted to determine the evolutionary relationships. The study revealed an interesting pattern of clustering and conservation among fish and mammalian species. The salmon chemokine sequences clustered with orthologs from other fish species, while the mammalian sequences formed separate clades. This indicates a divergent evolution of chemokines between mammals and fish, possibly due to different evolutionary pressures. While the structural analysis of the chemokines and the CXCR3 receptor showed the conservation of critical motifs and domains, suggesting preserved functions and stability throughout evolution. Regarding the regulation of gene expression, some components of the CXCL9-11/CXCR3 axis are induced by recombinant gamma interferon (rIFN-γ) and by Infectious pancreatic necrosis virus (IPNV) infection in Atlantic salmon cells. Further studies are needed to explore the role of Atlantic salmon CXCL9-11 chemokines in regulating immune cell migration and endothelial activation, as seen in mammals. To the best of our knowledge, there have been no functional studies of chemokines to understand these effects in Atlantic salmon. [ABSTRACT FROM AUTHOR]

Extensive research has been conducted on the role of CXCR3 in immune responses and inflammation. However, the role of CXCR3 in the reproductive system, particularly in oocyte development, remains unknown. In this study, we present findings on the involvement of CXCR3 in the meiotic division process of mouse oocytes. We found CXCR3 was expressed consistently throughout the entire maturation process of mouse oocyte. Inhibition of CXCR3 impaired the asymmetric division of oocyte, while the injection of Cxcr3 mRNA was capable of restoring these defects. Further study showed that inhibition of CXCR3 perturbed spindle migration by affecting LIMK/cofilin pathway-mediated actin remodeling. Knockout of CXCR3 led to an upregulation of actin-binding protein and an increased ATP level in GV-stage oocytes, while maintaining normal actin dynamics during the process of meiosis. Additionally, we noticed the expression level of DYNLT1 is markedly elevated in CXCR3-null oocytes. DYNLT1 bound with the Arp2/3 complex, and knockdown of DYNLT1 in CXCR3-null oocytes impaired the organization of cytoplasmic actin, suggesting the regulatory role of DYNLT1 in actin organization, and the compensatory expression of DYNLT1 may contribute to maintain normal actin dynamics in CXCR3-knockout oocytes. In summary, our findings provide insights into the intricate network of actin dynamics associated with CXCR3 during oocyte meiosis. • CXCR3 is expressed consistently throughout the entire maturation process of mouse oocyte. • Inhibition of CXCR3 impaired the asymmetric division of oocyte. • Inhibition of CXCR3 perturbed spindle migration by affecting LIMK/cofilin pathway-mediated actin remodeling. • The compensatory increase in DYNLT1 contributes to the maintenance of normal actin dynamics in CXCR3-knockout oocytes. [ABSTRACT FROM AUTHOR]

Objective: To investigate the interactions between select flavonoids (Luteolin, Quercetin, Apigenin, Kaempferol, and Amorphine) and the CXCR3 receptor, evaluating their potential as novel therapeutic agents in cancer immunotherapy. Methods: Molecular docking simulations were employed to analyze flavonoid-CXCR3 receptor interactions. Comprehensive in silico ADMET analyses were conducted to assess pharmacokinetic properties and toxicity profiles of the compounds. Results: Flavonoids exhibited high-affinity binding to the CXCR3 receptor, with binding affinities ranging from -8.7 to -13.0 kcal/mol. Amorphine demonstrated the highest binding affinity (-13.0 kcal/mol), indicating superior inhibition potential. Luteolin showed optimal ADME characteristics, including favorable oral bioavailability (62%) and blood-brain barrier permeability (log BB -1.911). Molecular docking analyses identified critical amino acid residues (TYR205, TYR308, TRP109, PHE131, and ASN132) in flavonoid-CXCR3 interactions. In silico toxicity predictions suggested low risk profiles for all examined flavonoids. Conclusion: This study provides evidence for the potential of flavonoids as CXCR3 receptor antagonists in cancer immunotherapy. The elucidated molecular interactions and favorable ADMET profiles warrant further investigation of these compounds. Future research should focus on optimization of flavonoid-based CXCR3 inhibitors, preclinical and clinical evaluations, and assessment of their immunomodulatory effects within the tumor microenvironment. These findings contribute to the development of novel, flavonoid-derived therapeutic strategies in cancer treatment. [ABSTRACT FROM AUTHOR]

Abstract Background Within the tumor microenvironment, altered lipid metabolism promotes cancer cell malignancy by activating oncogenic cascades; however, impact of lipid metabolism in CD4+ tumor-infiltrating lymphocytes (TILs) remains poorly understood. Here, we elucidated that role of stearoyl-CoA desaturase (SCD) increased by treatment with cancer-associated fibroblast (CAF) supernatant in CD4+ T cells on their subset differentiation and activity of CD8+ T cells. Results In our study, we observed that CD4+ TILs had higher lipid droplet content than CD4+ splenic T cells. In tumor tissue, CAF-derived supernatant provided fatty acids to CD4+ TILs, which increased the expression of SCD and oleic acid (OA) content. Increased SCD expression by OA treatment enhanced the levels of Th1 cell markers TBX21, interleukin-2, and interferon-γ. However, SCD inhibition upregulated the expression of regulatory T (Treg) cell markers, FOXP3 and transforming growth factor-β. Comparative fatty acid analysis of genetically engineered Jurkat cells revealed that OA level was significantly higher in SCD-overexpressing cells. Overexpression of SCD increased expression of Th1 cell markers, while treatment with OA enhanced the transcriptional level of TBX21 in Jurkat cells. In contrast, palmitic acid which is higher in SCD-KO cells than other subclones enhanced the expression of Treg cell markers through upregulation of mitochondrial superoxide. Furthermore, SCD increased the secretion of the C–X–C motif chemokine ligand 11 (CXCL11) from CD4+ T cells. The binding of CXCL11 to CXCR3 on CD8+ T cells augmented their cytotoxic activity. In a mouse tumor model, the suppressive effect of CD8+ T cells on tumor growth was dependent on CXCR3 expression. Conclusion These findings illustrate that SCD not only orchestrates the differentiation of T helper cells, but also promotes the antitumor activity of CD8+ T cells, suggesting its function in adverse tumor microenvironments. Graphical abstract

Abstract Purpose CXCR3 is expressed on activated T cells and plays a crucial role in T-cell recruitment to the tumor microenvironment (TME) during cell-based and immune checkpoint inhibitor (ICI) immunotherapy. This study utilized a 64Cu-labeled NOTA-α-CXCR3 antibody to assess CXCR3 expression in the TME and validate it as a potential T cell activation biomarker in vivo. Procedures CXCR3+ cells infiltrating MC38 tumors (B57BL/6 mice, untreated and treated with αPD-1/αCTLA-4 ICI) were quantified using fluorescence microscopy and flow cytometry. A commercial anti-mouse CXCR3 antibody (α-CXCR3) was site-specifically conjugated with 2,2,2-(1,4,7-triazacyclononane-1,4,7-triyl)triacetic acid (NOTA) and radiolabeled with 64Cu. Saturation binding of [64Cu]Cu-NOTA-α-CXCR3 was investigated using CHO cells stably transfected with murine CXCR3. Biodistribution and PET imaging studies both at baseline and after 1 to 3 cycles of ICI, respectively, were carried out using different molar activities (10 GBq/µmol to 300 GBq/µmol) of [64Cu]Cu-NOTA-α-CXCR3. Results Flow cytometry analysis at baseline confirmed the presence of CXCR3 + T-cells in MC38 tumors, which was significantly increased at day five after ICI (treated 33.8 ± 17.4 vs. control 8.8 ± 6.2 CD3+CXCR3+ cells/mg). These results were qualitatively and quantitatively confirmed by immunofluorescence of tumor cryoslices. In vivo PET imaging of MC38 tumor bearing mice before, during and after ICI using [64Cu]Cu-NOTA-α-CXCR3 (Kd = 3.3 nM) revealed a strong dependence of CXCR3-specificity of tracer accumulation in secondary lymphoid organs on molar activity. At 300 GBq/µmol (1.5 µg of antibody/mouse), a specific signal was observed in lymph nodes (6.33 ± 1.25 control vs. 3.95 ± 1.23%IA/g blocking) and the spleen (6.04 ± 1.02 control vs. 3.84 ± 0.79%IA/g blocking) at 48 h p.i. Spleen-to-liver ratios indicated a time dependent systemic immune response showing a steady increase from 1.08 ± 0.19 (untreated control) to 1.54 ± 0.14 (three ICI cycles). Conclusions This study demonstrates the feasibility of in vivo imaging of CXCR3 upregulation under immunotherapy using antibodies. However, high molar activities and low antibody doses are essential for sensitive detection in lymph nodes and spleen. Detecting therapy-induced changes in CXCR3+ T cell numbers in tumors was challenging due to secondary antibody-related effects. Nonetheless, CXCR3 remains a promising target for imaging T cell activation, with anticipated improvements in sensitivity using alternative tracers with high affinities and favorable pharmacokinetics.

Purpose: CXCR3 is expressed on activated T cells and plays a crucial role in T-cell recruitment to the tumor microenvironment (TME) during cell-based and immune checkpoint inhibitor (ICI) immunotherapy. This study utilized a 64Cu-labeled NOTA-α-CXCR3 antibody to assess CXCR3 expression in the TME and validate it as a potential T cell activation biomarker in vivo. Procedures: CXCR3+ cells infiltrating MC38 tumors (B57BL/6 mice, untreated and treated with αPD-1/αCTLA-4 ICI) were quantified using fluorescence microscopy and flow cytometry. A commercial anti-mouse CXCR3 antibody (α-CXCR3) was site-specifically conjugated with 2,2,2-(1,4,7-triazacyclononane-1,4,7-triyl)triacetic acid (NOTA) and radiolabeled with 64Cu. Saturation binding of [64Cu]Cu-NOTA-α-CXCR3 was investigated using CHO cells stably transfected with murine CXCR3. Biodistribution and PET imaging studies both at baseline and after 1 to 3 cycles of ICI, respectively, were carried out using different molar activities (10 GBq/µmol to 300 GBq/µmol) of [64Cu]Cu-NOTA-α-CXCR3. Results: Flow cytometry analysis at baseline confirmed the presence of CXCR3 + T-cells in MC38 tumors, which was significantly increased at day five after ICI (treated 33.8 ± 17.4 vs. control 8.8 ± 6.2 CD3+CXCR3+ cells/mg). These results were qualitatively and quantitatively confirmed by immunofluorescence of tumor cryoslices. In vivo PET imaging of MC38 tumor bearing mice before, during and after ICI using [64Cu]Cu-NOTA-α-CXCR3 (Kd = 3.3 nM) revealed a strong dependence of CXCR3-specificity of tracer accumulation in secondary lymphoid organs on molar activity. At 300 GBq/µmol (1.5 µg of antibody/mouse), a specific signal was observed in lymph nodes (6.33 ± 1.25 control vs. 3.95 ± 1.23%IA/g blocking) and the spleen (6.04 ± 1.02 control vs. 3.84 ± 0.79%IA/g blocking) at 48 h p.i. Spleen-to-liver ratios indicated a time dependent systemic immune response showing a steady increase from 1.08 ± 0.19 (untreated control) to 1.54 ± 0.14 (three ICI cycles). Conclusions: This study demonstrates the feasibility of in vivo imaging of CXCR3 upregulation under immunotherapy using antibodies. However, high molar activities and low antibody doses are essential for sensitive detection in lymph nodes and spleen. Detecting therapy-induced changes in CXCR3+ T cell numbers in tumors was challenging due to secondary antibody-related effects. Nonetheless, CXCR3 remains a promising target for imaging T cell activation, with anticipated improvements in sensitivity using alternative tracers with high affinities and favorable pharmacokinetics. [ABSTRACT FROM AUTHOR]

Macrolide antibiotic azithromycin is widely used in clinical practice to treat respiratory tract infections and inflammatory diseases. However, its mechanism of action is not fully understood. Given the involvement of the CD27 pathway in the pathophysiology of various T-lymphocyte-mediated inflammatory, autoimmune, and lymphoproliferative diseases, we examined the impact of AZM on CD27 regulation and potential consequences on CD4+ and CD8+ T-cell phenotypes. Using cellular immunology approaches on healthy donors' peripheral blood mononuclear cells, we demonstrate AZM-mediated downregulation of surface CD27 expression as well as its extracellular release as soluble CD27. Notably, AZM-exposed CD27high (hi) cells were defective in their ability to expand compared to CD27intermediate (Int) and CD27low (lo) subsets. The defective CD27hi subset expansion was found to be associated with impaired cell proliferation and cell division. At the molecular level, the CD27hi subset exhibited lower mTOR activity than other subsets. Functionally, AZM treatment resulted in marked depletion of helper CD4+ (Th1) and cytotoxic CD8+ T-lymphocyte (Tc1)-associated CXCR3+CD27hi effector cells and inhibition of inflammatory cytokine IFN-g production. These findings provide mechanistic insights on immunomodulatory features of AZM on T-lymphocyte by altering the CD27 pathway. From a clinical perspective, this study also sheds light on potential clinical benefits observed in patients on prophylactic AZM regimens against various respiratory diseases and opens avenues for future adjunct therapy against Th1- and Tc1-dominated inflammatory and autoimmune diseases. [ABSTRACT FROM AUTHOR]

Type 1 diabetes (T1D) is a chronic disease characterized by self‐destruction of insulin‐producing pancreatic β cells by cytotoxic T cell activity. However, the pathogenic mechanism of T cell infiltration remains obscure. Recently, tissue‐resident memory T (TRM) cells have been shown to contribute to cytotoxic T cell recruitment. TRM cells are found present in human pancreas and are suggested to modulate immune homeostasis. Here, the role of TRM cells in the development of T1D is investigated. The presence of TRM cells in pancreatic islets is observed in non‐obese diabetic (NOD) mice before T1D onset. Mechanistically, elevated fatty acid‐binding protein 4 (FABP4) potentiates the survival and alarming function of TRM cells by promoting fatty acid utilization and C‐X‐C motif chemokine 10 (CXCL10) secretion, respectively. In NOD mice, genetic deletion of FABP4 or depletion of TRM cells using CD69 neutralizing antibodies resulted in a similar reduction of pancreatic cytotoxic T cell recruitment, a delay in diabetic incidence, and a suppression of CXCL10 production. Thus, targeting FABP4 may represent a promising therapeutic strategy for T1D. [ABSTRACT FROM AUTHOR]

Chronic inflammation is believed as the main culprit of the link between cardiovascular disease (CVD) and rheumatoid arthritis (RA). Interleukin‐6 (IL‐6) is a pro‐inflammatory cytokine with a key role in RA pathophysiology and also correlates with joint destruction and disease activity. This study evaluates the association between IL‐6 plasma level and cardiac biomarker NT‐proBNP, HS‐CRP, CVD predictor algorithms, Framingham Risk Score (FRS) and Systematic Coronary Risk Evaluation (SCORE), as well as with CXCL9 and its receptor, CXCR3 in RA patients compared to the controls. Sixty RA patients (30 early and 30 late) and 30 healthy persons were included in this study. IL‐6 and NT‐proBNP plasma levels were measured by the ELISA. Also, HS‐CRP plasma levels were quantified using the immunoturbidimetric assay. The CVD risk was assessed by the FRS and SCORE. IL‐6 plasma levels were significantly higher in the early and late RA patients compared to the controls (p < 0.001). There was a positive correlation between IL‐6 with DAS‐28 (p = 0.007, r = 0.346), BPS (p = 0.002, r = 0.396), BPD (p = 0.046, r = 0.259), SCORE (p < 0.001, r = 0.472), and FRS (p < 0.001, r = 0.553), and a negative association with HDL (p = 0.037, r = −0.270), in the patients. Also, IL‐6 plasma level positively correlated with HS‐CRP (p = 0.021, r = 0.297) and NT‐proBNP (p = 0.045, r = 0.260) in the patients. Furthermore, a positive association was found between IL‐6 plasma levels and CXCL9 (p = 0.002, r = 0.386), and CXCR3 (p = 0.018, r = 0.304) in the patients. Given the interesting association between IL‐6 with various variables of CVD, IL‐6 may be considered a biomarker for assessing the risk for future cardiovascular events in RA patients. [ABSTRACT FROM AUTHOR]

Infiltration of regulatory T (Treg) cells, an immunosuppressive population of CD4+ T cells, into solid cancers represents a barrier to cancer immunotherapy. Chemokine receptors are critical for Treg cell recruitment and cell-cell interactions in inflamed tissues, including cancer, and thus are an ideal therapeutic target. Here, we show in multiple cancer models that CXCR3+ Treg cells were increased in tumors compared with lymphoid tissues, exhibited an activated phenotype, and interacted preferentially with CXCL9-producing BATF3+ dendritic cells (DCs). Genetic ablation of CXCR3 in Treg cells disrupted DC1-Treg cell interactions and concomitantly increased DC-CD8+ T cell interactions. Mechanistically, CXCR3 ablation in Treg cells increased tumor antigen-specific cross-presentation by DC1s, increasing CD8+ T cell priming and reactivation in tumors. This ultimately impaired tumor progression, especially in combination with anti-PD-1 checkpoint blockade immunotherapy. Overall, CXCR3 is shown to be a critical chemokine receptor for Treg cell accumulation and immune suppression in tumors.

CXCR3 is a chemokine receptor with three ligands: CXCL9, CXCL10 and CXCL11. We report that in addition to attracting CXCR3+ T cells to tumor sites a key role of CXCL9 and CXCL10 is in inducing a self-feeding feedback loop that accelerates effector/cytotoxic activities of both CD4+ and CD8+ T cells while downregulating immunoregulatory protein TIM3. CXCR3KO mice displayed a markedly reduced response to anti-PD-1 and anti-CTLA-4 therapy. Results from a panel of in vivo and ex vivo 3D tumor models imply that, beyond driving CD8+ T cells into T-cell exhaustion, a major role of PD-1 and CTLA-4 is in limiting the CXCR3-based self-feeding mechanism of T cell potentiation. This may explain why patients that are CXCL9/CXCL10high tend to respond well to anti-PD-1 therapy, as opposed to patients that are CXCL9/CXCL10low. It also suggests a therapeutic role for CXCL9-Fc or CXCL10-Fc therapy; herein we demonstrate significant anti-tumor activity in multiple murine tumor models with such agents.

Chimeric antigen receptor-engineered T (CAR-T) cell therapy of cancer has been a hotspot and promising. However, due to rapid exhaustion, CAR-T cells are less effective in solid tumors than in hematological ones. CD122+CXCR3+ memory T cells are characterized with longevity, self-renewal and great antitumoral capacity. Thus, it’s compelling to induce memory CAR-T cells to enhance their efficacy on solid tumors. Astragalus polysaccharide (APS) has reportedly exhibited antitumoral effects. However, it’s unclear if APS has an impact on CD8+ memory T cell generation or persistence. Using two human cancer cell lines, here we found that APS significantly improved the persistence of GPC3-targeted CAR-T cells and enhanced their suppression of tumor growth in both Huh7 and HepG2 xenograft models of hepatocellular carcinoma. APS increased CD122+/CXCR3+ memory T cells, but decreased their PD-1+ subset within CD8+ CAR-T cells in tumor-bearing mice, while these effects of APS were also confirmed with in vitro experiments. Moreover, APS augmented the expression of chemokines CXCL9/CXCL10 by the tumor in vivo and in vitro. It also enhanced the proliferation and chemotaxis/migration of CAR-T cells in vitro. Finally, APS promoted the phosphorylation of STAT5 in CD8+ CAR-T cells, whereas inhibition of STAT5 activation reversed these in vitro effects of APS. Therefore, APS enhanced the antitumoral effects of CD8+ CAR-T cells by promoting formation/persistence of CD122+/CXCR3+/PD-1- memory T cells and their migration to the tumor.

Chemokines are cytokines that mediate leukocyte traffic between the lymphoid organs, the bloodstream, and the site of tissue damage, which is essential for an efficient immune response. In particular, the gamma interferon (IFN- γ) inducible chemokines CXCL9, CXCL10, and CXCL11, and their receptor CXCR3, are involved in T cell and macrophage recruitment to the site of infection. The nature and function of these chemokines and their receptor are well-known in mammals, but further research is needed to achieve a similar level of understanding in fish immunity. Thus, in this study, we seek to identify the genes encoding the components of the Atlantic salmon (Salmo salar) CXCL9, CXCL10, CXCL11/CXCR3 axis (CXCL9-11/CXCR3), predict the protein structure from the amino acid sequence, and explore the regulation of gene expression as well as the response of these chemokines and their receptor to viral infections. The cxcl9, cxcl10, cxcl11, and cxcr3 gene sequences were retrieved from the databases, and the phylogenetic analysis was conducted to determine the evolutionary relationships. The study revealed an interesting pattern of clustering and conservation among fish and mammalian species. The salmon chemokine sequences clustered with orthologs from other fish species, while the mammalian sequences formed separate clades. This indicates a divergent evolution of chemokines between mammals and fish, possibly due to different evolutionary pressures. While the structural analysis of the chemokines and the CXCR3 receptor showed the conservation of critical motifs and domains, suggesting preserved functions and stability throughout evolution. Regarding the regulation of gene expression, some components of the CXCL9-11/CXCR3 axis are induced by recombinant gamma interferon (rIFN-γ) and by Infectious pancreatic necrosis virus (IPNV) infection in Atlantic salmon cells. Further studies are needed to explore the role of Atlantic salmon CXCL9-11 chemokines in regulating immune cell migration and endothelial activation, as seen in mammals. To the best of our knowledge, there have been no functional studies of chemokines to understand these effects in Atlantic salmon.

Introduction: Ovarian cancer is the most malignant gynecological tumor. Previous studies have demonstrated that chimeric antigen receptor (CAR)-engineered NK-92 cells targeting folate receptor α (αFR) (NK-92-αFR-CAR) can specifically kill αFR-positive ovarian cancer cells. However, the migration barrier restricts antitumor effects of CAR-engineered cells. Objectives: To elucidate the mechanism by which NK-92-αFR-CAR cells induce the secretion of chemokine CXCL10 during killing ovarian cancer cells. It is speculated that NK-92-αFR-CAR-CXCR3A can target αFR and have chemotaxis of CXCL10, and they may have stronger killing effect of ovarian cancer. Methods: Study the mechanism of CXCL10 expression strongly induced by TNF-α and IFN-γ combined stimulation in ovarian cancer cells. Construct the fourth generation of NK-92-αFR-CAR-CXCR3A cells, which were co-expressed CXCR3A and αFR-CAR. Evaluate the killing and migration effects of NK-92-αFR-CAR-CXCR3A in vitro and in vivo. Results: RNA sequencing (RNA-seq) first revealed that the expression level of the chemokine CXCL10 was most significantly increased in ovarian cancer cells co-cultured with NK-92-αFR-CAR. Secondly, cytokine stimulation experiments confirmed that IFN-γ and TNF-α secreted by NK-92-αFR-CAR synergistically induced high CXCL10 expression in ovarian cancer cells. Further signaling pathway experiments showed that IFN-γ and TNF-α enhanced the activation level of the IFN-γ-IFNGR-JAK1/2-STAT1-CXCL10 signaling axis. Cytotoxicity experiments showed that NK-92-αFR-CAR-CXCR3A cells could not only efficiently kill αFR-positive ovarian cancer cells in vitro but also secrete IFN-γ and TNF-α. Higher migration than that of NK-92-αFR-CAR was detected in NK-92-αFR-CAR-CXCR3A using transwell assay. NK-92-αFR-CAR-CXCR3A effectively killed tumor cells in different mouse xenograft models of ovarian cancer and increased infiltration into tumor tissue. Conclusion: This study confirmed that IFN-γ and TNF-α secreted by αFR-CAR-engineered NK cells can synergistically induce high expression of CXCL10 in ovarian cancer cells and constructed self-driving αFR-CAR-engineered NK cells that can break through migration barriers based on CXCL10, which may provide a new therapeutic weapon for ovarian cancer.

The precise pathogenesis of Kawasaki disease remains unknown. In an attempt to elucidate the pathogenesis of KD through the analysis of acquired immunity, we comprehensively examined the immunophenotypic changes in immune cells such as lymphocytes and monocytes along with various cytokines, focusing on differences between pre- and post- treatment samples. We found high levels of CXCL9 and CXCL10 chemokines that decreased with treatment, which coincided with a post-treatment expansion of Th1 cells expressing CXCR3. Our results show that the CXCL10-CXCR3 axis plays an important role in the pathogenesis of KD. In an attempt to elucidate the pathogenesis of Kawasaki disease, we comprehensively examined the changes in immune cells and various cytokines. We found high levels of CXCL9 and CXCL10 chemokines that decreased with treatment, which coincided with a post-treatment expansion of cells expressing CXCR3. Our results show that the CXCL10-CXCR3 axis plays an important role in the pathogenesis of KD. Graphical Abstract [ABSTRACT FROM AUTHOR]

Aim: To clarify the role of Eomesodermin (EOMES) to serve as a disease‐relevant biomarker and the intracellular molecules underlying the immunophenotype shifting of CD4+T subsets in amyotrophic lateral sclerosis (ALS). Methods: The derivation and validation cohorts included a total of 148 ALS patients and 101 healthy controls (HCs). Clinical data and peripheral blood were collected. T‐cell subsets and the EOMES expression were quantified using multicolor flow cytometry. Serum neurofilament light chain (NFL) was measured. In 1‐year longitudinal follow‐ups, the ALSFRS‐R scores and primary endpoint events were further recorded in the ALS patients of the validation cohort. Results: In the derivation cohort, the CD4+EOMES+T‐cell subsets were significantly increased (p < 0.001). EOMES+ subset was positively correlated with increased serum NFL levels in patients with onset longer than 12 months. In the validation cohort, the elevated CD4+EOMES+T‐cell proportions and their association with NFL levels were also identified. The longitudinal study revealed that ALS patients with higher EOMES expression were associated with higher progression rates (p =.010) and worse prognosis (p =.003). Conclusions: We demonstrated that increased CD4+EOMES+T‐cell subsets in ALS were associated with disease progression and poor prognosis. Identifying these associations may contribute to a better understanding of the immunopathological mechanism of ALS. [ABSTRACT FROM AUTHOR]

Inflammation and immunity play important roles in the pathogenesis of ischemic stroke. This study aimed to explore key regulatory genes in acute ischemic stroke (AIS) and their underlying mechanisms to provide new research targets for the diagnosis and treatment of ischemic stroke. We searched for differentially expressed mRNAs and miRNAs in patients with AIS and healthy populations in GEO databases, constructed a miRNA–mRNA network, and screened key miRNAs using least absolute shrinkage and selection operator regression and the support vector machine-recursive feature elimination model. Correlations between key miRNAs and infiltrating immune cells and inflammatory factors were analyzed using CIBERSORT and immunoassays and verified using clinical experiments. Bioinformatics analysis identified hsa-miR-877-5p as a key regulatory miRNA in AIS that can modulate immune and inflammatory responses. In clinical studies, it was verified by quantitative PCR analysis that the expression of hsa-miR-877-5p in the blood of AIS patients was higher than that of the healthy group. Then, enzyme-linked immunosorbent assay revealed that the expression of IL-23 and TNF-α related to inflammation in AIS patients was higher than that of the healthy. Quantitative PCR further found that the relative mRNA expression of IL-23, CXCR3, and TNF-α in AIS group was higher than that of the healthy group. This study may provide a basis for a more comprehensive understanding of the potential mechanism of the occurrence and development of AIS, and hsa-miR-877-5p and its downstream effectors IL-23, CXCR3, and TNF-α may be potential intervention targets in AIS. [ABSTRACT FROM AUTHOR]

Abstract Type 1 diabetes (T1D) is a chronic disease characterized by self‐destruction of insulin‐producing pancreatic β cells by cytotoxic T cell activity. However, the pathogenic mechanism of T cell infiltration remains obscure. Recently, tissue‐resident memory T (TRM) cells have been shown to contribute to cytotoxic T cell recruitment. TRM cells are found present in human pancreas and are suggested to modulate immune homeostasis. Here, the role of TRM cells in the development of T1D is investigated. The presence of TRM cells in pancreatic islets is observed in non‐obese diabetic (NOD) mice before T1D onset. Mechanistically, elevated fatty acid‐binding protein 4 (FABP4) potentiates the survival and alarming function of TRM cells by promoting fatty acid utilization and C‐X‐C motif chemokine 10 (CXCL10) secretion, respectively. In NOD mice, genetic deletion of FABP4 or depletion of TRM cells using CD69 neutralizing antibodies resulted in a similar reduction of pancreatic cytotoxic T cell recruitment, a delay in diabetic incidence, and a suppression of CXCL10 production. Thus, targeting FABP4 may represent a promising therapeutic strategy for T1D.

Hong Di,&ast; Xinxin Han,&ast; Yue Yin, Yun Zhang, Xuejun Zeng Department of Family Medicine & Division of General Internal Medicine, Department of Medicine, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences, State Key Laboratory of Complex Severe and Rare Diseases (Peking Union Medical College Hospital), Beijing, People’s Republic of China&ast;These authors contributed equally to this workCorrespondence: Yun Zhang; Xuejun Zeng, Department of Family Medicine & Division of General Internal Medicine, Department of Medicine, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences, State Key Laboratory of Complex Severe and Rare Diseases (Peking Union Medical College Hospital), Beijing, 100730, People’s Republic of China, Tel/Fax +8610-69156098, Email zhangyun10806@pumch.cn; zxjpumch@126.comBackground: This study aimed to explore the potential roles of Vδ 2 T cells in the pathogenesis of acute gouty arthritis.Methods: Patients with gout (n=86), rheumatoid arthritis (n=7), osteoarthritis (n=9), and healthy controls (n=40) were enrolled. γδT cell frequency, chemokine receptor expression and production of pro-inflammatory cytokines in cells from peripheral blood and synovial fluid samples were quantified by flow cytometry. The chemotaxis ability of Vδ 2 T cells was determined by transwell migration assay. The levels of chemokines were also detected by enzyme-linked immunosorbent assay.Results: Peripheral Vδ 2 T cells had significantly lower frequencies in acute gout patients than that in healthy controls (P< 0.001). These peripheral Vδ 2 T cells were negatively correlated with inflammatory markers. Vδ 2 T cells from acute gout patients accumulated in synovial fluid, as evidenced by a higher abundance of Vδ 2 T cells in it than that in the serum (P< 0.01). And Vδ 2 T cells expressed a high level of C-X-C chemokine receptor 3 (P=0.035), and its corresponding chemokine C-X-C motif chemokine ligand 10 showed a high concentration in synovial fluid (P< 0.05). Vδ 2 T cells from synovial fluid of acute gout patients produced a high level of interleukin-17 (P=0.033).Conclusion: The up-regulated expression of C-X-C chemokine receptor 3 on Vδ 2 T cells potentially facilitates their infiltration into synovial fluid during acute gouty arthritis. Further production of interleukin-17 by Vδ 2 T cells may contribute to the pathogenesis of gout. This study sheds new light on developing novel Vδ 2 T cells-based therapeutic strategies for gout treatment. Keywords: acute gouty arthritis, γδT cells, Vδ 2 T cells, Interleukin-17, CXCR3, CXCL10

Abstract Background Unlike conventional T cells, innate and virtual-memory CD8 T cells in naïve mice acquire their memory phenotypes and functions in the absence of antigenic encounters in a cytokine-dependent manner. The relevant cytokines include interleukin-4 (IL-4), type I interferon, and interleukin-15 (IL-15). Moreover, exogenous IL-4 can also induce de novo generation and/or expansion of the virtual-memory CD8 T cell population. In this study, we investigated whether exogenous IL-4 could enhance the immune response to a viral infection. Results In vivo administration of IL-4 and an anti-IL-4 antibody complex (IL-4C) increased CXCR3 expression in both memory and naïve phenotype CD8 T cells in the absence of antigenic stimulation, and protected mice from lethal influenza infection. Flow cytometric analysis of lung-infiltrating immune cells on day 5 after virus infection revealed higher numbers of antigen-specific and bystander CD8 T cells in IL-4C-treated mice than in control mice. In particular, the bystander CD8 T cells were a naïve or evident memory phenotypes. Crucially, an anti-CXCR3 blocking antibody abrogated this IL-4C effect, reflecting that the increased accumulation of CD8 T cells in the lungs after IL-4C treatment is dependent on CXCR3. Conclusions These data demonstrate that exogenous IL-4C plays a protective role by enhancing CXCR3-dependent migration of CD8 T cells into influenza-infected lungs.

Clinical studies have demonstrated that local expression of the cytokine IL-12 drives interferon-gamma expression and recruits T cells to the tumor microenvironment, ultimately yielding durable systemic T cell responses. Interrogation of longitudinal biomarker data from our late-stage melanoma trials identified a significant on-treatment increase of intratumoral CXCR3 transcripts that was restricted to responding patients, underscoring the clinical relevance of tumor-infiltrating CXCR3+ immune cells. In this study, we sought to understand if the addition of DNA-encodable CXCL9 could augment the anti-tumor immune responses driven by intratumoral IL-12. We show that localized IL-12 and CXCL9 treatment reshapes the tumor microenvironment to promote dendritic cell licensing and CD8+ T cell activation. Additionally, this combination treatment results in a significant abscopal anti-tumor response and provides a concomitant benefit to anti-PD-1 therapies. Collectively, these data demonstrate that a functional tumoral CXCR3/CXCL9 axis is critical for IL-12 anti-tumor efficacy. Furthermore, restoring or amplifying the CXCL9 gradient in the tumors via intratumoral electroporation of plasmid CXCL9 can not only result in efficient trafficking of cytotoxic CD8+ T cells into the tumor but can also reshape the microenvironment to promote systemic immune response.

Lipoxins are small lipids that are potent endogenous mediators of systemic inflammation resolution in a variety of diseases. We previously reported that Lipoxins A4 and B4 (LXA4 and LXB4) have protective activities against neurodegenerative injury. Yet, lipoxin activities and downstream signaling in neuroinflammatory processes are not well understood. Here, we utilized a model of posterior uveitis induced by lipopolysaccharide endotoxin (LPS), which results in rapid retinal neuroinflammation primarily characterized by activation of resident macroglia (astrocytes and Müller glia), and microglia. Using this model, we observed that each lipoxin reduces acute inner retinal inflammation by affecting endogenous glial responses in a cascading sequence beginning with astrocytes and then microglia, depending on the timing of exposure; prophylactic or therapeutic. Subsequent analyses of retinal cytokines and chemokines revealed inhibition of both CXCL9 (MIG) and CXCL10 (IP10) by each lipoxin, compared to controls, following LPS injection. CXCL9 and CXCL10 are common ligands for the CXCR3 chemokine receptor, which is prominently expressed in inner retinal astrocytes and ganglion cells. We found that CXCR3 inhibition reduces LPS-induced neuroinflammation, while CXCR3 agonism alone induces astrocyte reactivity. Together, these data uncover a novel lipoxin–CXCR3 pathway to promote distinct anti-inflammatory and proresolution cascades in endogenous retinal glia. [ABSTRACT FROM AUTHOR]

Background: Unlike conventional T cells, innate and virtual-memory CD8 T cells in naïve mice acquire their memory phenotypes and functions in the absence of antigenic encounters in a cytokine-dependent manner. The relevant cytokines include interleukin-4 (IL-4), type I interferon, and interleukin-15 (IL-15). Moreover, exogenous IL-4 can also induce de novo generation and/or expansion of the virtual-memory CD8 T cell population. In this study, we investigated whether exogenous IL-4 could enhance the immune response to a viral infection. Results: In vivo administration of IL-4 and an anti-IL-4 antibody complex (IL-4C) increased CXCR3 expression in both memory and naïve phenotype CD8 T cells in the absence of antigenic stimulation, and protected mice from lethal influenza infection. Flow cytometric analysis of lung-infiltrating immune cells on day 5 after virus infection revealed higher numbers of antigen-specific and bystander CD8 T cells in IL-4C-treated mice than in control mice. In particular, the bystander CD8 T cells were a naïve or evident memory phenotypes. Crucially, an anti-CXCR3 blocking antibody abrogated this IL-4C effect, reflecting that the increased accumulation of CD8 T cells in the lungs after IL-4C treatment is dependent on CXCR3. Conclusions: These data demonstrate that exogenous IL-4C plays a protective role by enhancing CXCR3-dependent migration of CD8 T cells into influenza-infected lungs. [ABSTRACT FROM AUTHOR]

Various regulatory CD8+ T‐cell subsets have been proposed for immune tolerance and have been implicated in controlling autoimmune diseases. However, their phenotypic identities and suppression mechanisms are not yet understood. This study found that coculture of T‐cell receptor (TCR)‐ or interferon (IFN)‐β‐activated CD8+ T cells significantly suppressed the cytokine production of Th1 and Th17 cells. By experimenting with the experimental autoimmune uveitis (EAU), we found that adoptive transfer of TCR or IFN‐β‐activated CD8+ T cells significantly lessened disease development in an IFN‐γ‐dependent manner with a decreased uveitogenic Th1 and Th17 response. Interestingly, after adoptive transfer into the EAU mice, the IFN‐γ+CD8+ T cells were recruited more efficiently into the secondary lymphoid organs during the disease‐priming phase. This recruitment depends on the IFN‐γ‐inducible chemokine receptor CXCR3; knocking out CXCR3 abolishes the protective effect of CD8+ T cells in EAU. In conclusion, we identified the critical role of IFN‐γ for CD8+ T cells to inhibit Th1 and Th17 responses and ameliorate EAU. CXCR3 is necessary to recruit IFN‐γ+CD8+ T cells to the secondary lymphoid organ for the regulation of autoreactive Th1 and Th17 cells. [ABSTRACT FROM AUTHOR]

Production of effector CD8+ T cells during persistent infection requires a stable pool of stem-like cells that can give rise to effector cells via a proliferative intermediate population. In infection models marked by T cell exhaustion, this process can be transiently induced by checkpoint blockade but occurs spontaneously in mice chronically infected with the protozoan intracellular parasite Toxoplasma gondii. We observe distinct locations for parasite-specific T cell subsets, implying a link between differentiation and anatomical niches in the spleen. Loss of the chemokine receptor CXCR3 on T cells does not prevent white pulp-to-red pulp migration but reduces interactions with CXCR3 ligand-producing dendritic cells (DCs) and impairs memory-to-intermediate transition, leading to a buildup of memory T cells in the red pulp. Thus, CXCR3 increases T cell exposure to differentiation-inducing signals during red pulp migration, providing a dynamic mechanism for modulating effector differentiation in response to environmental signals.

Background and purpose: The interaction of CXC motif chemokine receptor (CXCR) on the cell surface of multiple myeloma (MM) with chemokines in the bone marrow microenvironment is involved in proliferation, survival and extramedullary invasion of MM cells. Sinomenine derivative YL064 exerts its biological effects by targeting intracellular signaling regulators in MM cells. This study aimed to explore possible modulating and biological effects of sinomenine derivative YL064 on CXCR3 in MM cells. Methods: The MM cell lines H929 and MM1.S were used as in vitro models. H929-OE and MM1.S-OE cells with stable overexpression of CXCR3 were constructed with lentivirus vector. The effects of CXCR3 on clonal formation and migration of MM cells were detected by clone spot formation and transwell migration assay. Meanwhile, flow cytometry was used to analyze apoptotic rates of H929, H929-OE, MM1.S and MM1.S-OE cells treated with different concentrations of YL064. Furthermore, real-time fluorescence quantitative polymerase chain reaction (RTFQ-PCR) and Western blot assay were applied to detect expression levels of CXCR3 and its downstream signal regulators extracellular regulated protein kinase (ERK) and p-protein kinase B (p-AKT) in H929 and MM1.S cells before and after the treatment with YL064. Results: The clonal formation rates of H929-OE and MM1.S-OE cells with CXCR3 overexpression reached to 81.33%±5.79% and 73.00%±4.90%, which were significantly higher compared with H929 (58.33%±3.30%) and MM1.S cells (41.00%±3.14%). The migration proportion of H929-OE and MM1.S-OE cells were 7.90%±0.81% and 23.00%±1.63%, which were significantly higher compared with H929 (4.63%±0.37%) and MM1.S cells (14.63%±1.04%). After treatment with YL064, the apoptotic rates of H929-OE and MM1.S-OE cells were 29.80%±0.30% and 14.20%±0.26%, which were lower than those of H929 (33.40%±0.25%) and MM1.S cells (21.60%±0.21%). It was also shown that YL064 could reduce clonal formation and migration, inhibit CXCR3 gene transcription and downregulate expressions of CXCR3, ERK and AKT in H929 and MM1.S cells. Conclusions: CXCR3 might promote proliferation and invasion of MM cells. Sinomenine derivative YL064 could downregulate expressions of CXCR3 and its downstream signal regulators in MM cells, reduce abilities of clonal formation and migration, and induce apoptosis.

Background: Chemokines play a vital role in tumor progression, metastasis and prognosis; however, the profile and clinical significance of gamma interferon-inducible protein-10 (IP-10) and its receptor (CXCR3) in patients with hepatocellular carcinoma (HCC) have not been well evaluated. Methods: Liquid-phase chip technology was used to detect the serum IP-10 in 85 patients with HBV-related HCC, 50 patients with chronic hepatitis B (CHB) and 50 liver cirrhosis subjects (CS); simultaneously, the CXCR3 and Alpha fetoprotein (AFP) were determined. Additionally, their mRNA or protein expression levels in peripheral blood mononuclear cells (PBMC), liver tumor and paracancerous tissues were quantified using qRT-PCR or ELISA. Moreover, the IP-10 and CXCR3 expression was verified by the online data from Gene Expression Omnibus. Furthermore, the relationships of serum IP-10, CXCR3 and AFP levels with their overall survival rate were also analyzed. Results: The levels of IP-10 and CXCR3 in HCC group were significantly higher than those in CHB and CS groups, and their mRNA of PBMC is significantly positive correlation with those in their liver tissues or HBV DNA load (P < 0.0001), respectively. The serum IP-10 and CXCR3 in HCC were significantly correlated with tumor differentiation, metastases staging and distant metastasis (P < 0.05), but not related to gender, age and tumor size (P > 0.05, except IP-10 based on age). Conclusions: The serum IP-10 (142.6 pg/mL) and CXCR3 (241.2 pg/mL) could be differential diagnostic surrogates that distinguish HCC from CS, and the lower IP-10 level may be conducive to the postoperative survival of HCC patients. Moreover, the IP-10 and CXCR3 would be related to anti-tumor immunity in HCC patients and be a potential target for treatment of HCC. [ABSTRACT FROM AUTHOR]

Homeostasis of the host immune system is regulated by white blood cells with a variety of cell surface receptors for cytokines. Chemotactic cytokines (chemokines) activate their receptors to evoke the chemotaxis of immune cells in homeostatic migrations or inflammatory conditions towards inflamed tissue or pathogens. Dysregulation of the immune system leading to disorders such as allergies, autoimmune diseases, or cancer requires efficient, fast-acting drugs to minimize the long-term effects of chronic inflammation. Here, we performed structure-based virtual screening (SBVS) assisted by the Keras/TensorFlow neural network (NN) to find novel compound scaffolds acting on three chemokine receptors: CCR2, CCR3, and one CXC receptor, CXCR3. Keras/TensorFlow NN was used here not as a typically used binary classifier but as an efficient multi-class classifier that can discard not only inactive compounds but also low- or medium-activity compounds. Several compounds proposed by SBVS and NN were tested in 100 ns all-atom molecular dynamics simulations to confirm their binding affinity. To improve the basic binding affinity of the compounds, new chemical modifications were proposed. The modified compounds were compared with known antagonists of these three chemokine receptors. Known CXCR3 compounds were among the top predicted compounds; thus, the benefits of using Keras/TensorFlow in drug discovery have been shown in addition to structure-based approaches. Furthermore, we showed that Keras/TensorFlow NN can accurately predict the receptor subtype selectivity of compounds, for which SBVS often fails. We cross-tested chemokine receptor datasets retrieved from ChEMBL and curated datasets for cannabinoid receptors. The NN model trained on the cannabinoid receptor datasets retrieved from ChEMBL was the most accurate in the receptor subtype selectivity prediction. Among NN models trained on the chemokine receptor datasets, the CXCR3 model showed the highest accuracy in differentiating the receptor subtype for a given compound dataset. [ABSTRACT FROM AUTHOR]

Mycobacterium tuberculosis is a major human pathogen, and new vaccines are needed to prevent transmission. Mucosal vaccination may confer protection against M. tuberculosis by stimulating tissue-resident memory (TRM) CD4+ T cells in the lungs. The chemokine receptor CXCR3 promotes lung recruitment of T cells, but its role in TRM development is unknown. This study demonstrates the recombinant influenza A virus vaccine PR8.p25, expressing the immunodominant M. tuberculosis T cell epitope p25, induces CXCR3 expression on p25-specific CD4+ T cells in the lungs so that the majority of vaccine-induced CD4+ TRM expresses CXCR3 at 6 weeks. However, CXCR3−/− mice developed equivalent antigen-specific CD4+ T cell responses to wild-type (WT) mice following PR8.p25, and surprisingly retained more p25-specific CD4+ TRM in the lungs than WT mice at 6 weeks. The adoptive transfer of CXCR3−/− and WT P25 T cells into WT mice revealed that the initial recruitment of vaccine-induced CD4+ T cells into the lungs was independent of CXCR3, but by 6 weeks, CXCR3-deficient P25 T cells, and especially CXCR3−/− TRM, were significantly reduced compared to CXCR3-sufficient P25 T cells. Therefore, although CXCR3 was not essential for CD4+ TRM recruitment or retention, it provided a competitive advantage for the induction of M. tuberculosis-specific CD4+ TRM in the lungs following pulmonary immunization. [ABSTRACT FROM AUTHOR]

The transcription factor Fli-1, amember of the ETS family of transcription factors, is implicated in the pathogenesis of lupus disease. Reduced Fli-1 expression in lupus mice leads to decreased renal Cxcl10 mRNA levels and renal infiltrating CXCR3+ T cells that parallels reduced renal inflammatory cell infiltration and renal damage. Inflammatory chemokine CXCL10 is critical for attracting inflammatory cells expressing the chemokine receptor CXCR3. The CXCL10/ CXCR3 axis plays a role in the pathogenesis of various inflammatory diseases including lupus. Our data here demonstrate that renal CXCL10 protein levels are significantly lower in Fli-1 heterozygous MRL/lpr mice compared to wild-type MRL/lpr mice. Knockdown of Fli-1 significantly reduced CXCL10 secretion in mouse and human endothelial cells, and human mesangial cells, upon LPS or TNF stimulation. The Fli-1 inhibitor, Camptothecin, significantly reduced CXCL10 production in human monocyte cells upon interferon stimulation. Four putative Ets binding sites in the Cxcl10 promoter showed significant enrichment for FLI-1; however, FLI-1 did not directly drive transcription from the human or mouse promoters, suggesting FLI-1 may regulate CXCL10 expression indirectly. Our results also suggest that the DNA binding domain of FLI-1 is necessary for regulation of human hCXCR3 promotor activity in human T cells and interactions with co-activators. Together, these results support a role for FLI-1 in modulating the CXCL10-CXCR3 axis by directly or indirectly regulating the expression of both genes to impact lupus disease development. Signaling pathways or drugs that reduce FLI-1 expression may offer novel approaches to lupus treatment. [ABSTRACT FROM AUTHOR]

Background: In HTLV-1-associated malignant disease, adult T-cell leukaemia/lymphoma (ATLL), the interaction of virus and host was evaluated at the chemokines gene expression level. Also, IL-1β and Caspase-1 expressions were evaluated to investigate the importance of pyroptosis in disease development and progression. Methods and results: The expression of host CCR6 and CXCR-3 and the HTLV-1 proviral load (PVL), Tax, and HBZ were assessed in 17 HTLV-1 asymptomatic carriers (ACs) and 12 ATLL patients using the reverse transcription-quantitative polymerase chain reaction (RT-qPCR), TaqMan method. Moreover, RT-qPCR, SYBR Green assay were performed to measure Caspase-1 and IL-1β expression. HTLV-1-Tax did not express in 91.5% of the ATLLs, while HBZ was expressed in all ATLLs. The expression of CXCR3 dramatically decreased in ATLLs compared to ACs (p = 0.001). The expression of CCR6 was lower in ATLLs than ACs (p = 0.04). The mean of PVL in ATLL patients was statistically higher than ACs (p = 0.001). Furthermore, the expression of the IL-1β between ATLLs and ACs was not statistically significant (p = 0.4). In contrast, there was a meaningful difference between Caspase-1 in ATLLs and ACs (p = 0.02). Conclusions: The present study indicated that in the first stage of ATLL malignancy toward acute lymphomatous, CXCR3 and its progression phase may target the pyroptosis process. Mainly, HBZ expression could be a novel therapeutic target. [ABSTRACT FROM AUTHOR]

Developing strategies to activate tumor-cell-intrinsic immune response is critical for improving tumor immunotherapy by exploiting tumor vulnerability. KDM4A, as a histone H3 lysine 9 trimethylation (H3K9me3) demethylase, has been found to play a critical role in squamous cell carcinoma (SCC) growth and metastasis. Here we report that KDM4A inhibition promoted heterochromatin compaction and induced DNA replication stress, which elicited antitumor immunity in SCC. Mechanistically, KDM4A inhibition promoted the formation of liquid-like HP1γ puncta on heterochromatin and stall DNA replication, which activated tumor-cell-intrinsic cGAS-STING signaling through replication-stress-induced cytosolic DNA accumulation. Moreover, KDM4A inhibition collaborated with PD1 blockade to inhibit SCC growth and metastasis by recruiting and activating CD8+ T cells. In vivo lineage tracing demonstrated that KDM4A inhibition plus PD1 blockade efficiently eliminated cancer stem cells. Altogether, our results demonstrate that targeting KDM4A can activate anti-tumor immunity and enable PD1 blockade immunotherapy by aggravating replication stress in SCC cells.

Objective: To investigate the effects of cytokines IL-2 and GM-CSF on CXCR3 expression and chemotaxis of CAR-T cells. Background: 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. Materials and methods: 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. Results: 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. Conclusion: 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. [ABSTRACT FROM AUTHOR]

Chemokines are chemotactic-competent molecules composed of a family of small cytokines, playing a key role in regulating tumor progression. The roles of chemokines in antitumor immune responses are of great interest. CXCL9, CXCL10, and CXCL11 are important members of chemokines. It has been widely investigated that these three chemokines can bind to their common receptor CXCR3 and regulate the differentiation, migration, and tumor infiltration of immune cells, directly or indirectly affecting tumor growth and metastasis. Here, we summarize the mechanism of how the CXCL9/10/11–CXCR3 axis affects the tumor microenvironment, and list the latest researches to find out how this axis predicts the prognosis of different cancers. In addition, immunotherapy improves the survival of tumor patients, but some patients show drug resistance. Studies have found that the regulation of CXCL9/10/11–CXCR3 on the tumor microenvironment is involved in the process of changing immunotherapy resistance. Here we also describe new approaches to restoring sensitivity to immune checkpoint inhibitors through the CXCL9/10/11–CXCR3 axis. [ABSTRACT FROM AUTHOR]

Introduction: The pathogenesis of vitiligo is multifactorial; its diagnosis is mainly clinical and, in selected cases, is supported by histopathological studies that show the absence of melanocytes. The fundamental aspects of vitiligo are presented with emphasis on immunopathological events. Methodology: Narrative review. The PubMed® and Google Scholar search engines were used, with the following terms: "vitiligo", "vitiligo aetiology", "immunopathogenesis of vitiligo" and "vitiligo history". A total of 45 articles were selected, including Colombian literature. Results and discussion: Vitiligo is a disease as old as humanity. Until today, a clear causal phenomenon has not been established, but rather a set of events related to its origin and perpetuation. Thanks to decades of research, some genetic and environmental influences on melanocytes have been revealed, which lead to a greater susceptibility to oxidative damage and a decrease in their intercellular adhesion. In response to different noxa, innate and adaptive immunity are activated, leading to the destruction of the melanocyte mediated by cytotoxic CD8+ T cells. Participation of interferon-gamma (IFN-γ), the Janus kinase (JAK)/signal transducer and activator of transcription (STAT) pathway, especially JAK-1 and JAK-2, and the CXCR3B chemokine receptor are highlighted. Conclusions: Multiple biological phenomena converge in the etiopathogenesis of vitiligo; the result is the activation of CD8+ T lymphocytes, responsible for the destruction of melanocytes. The understanding of immunopathogenic pathways opens the door to the use of target therapies, such as JAK inhibitors and CXCR3B inhibitors. [ABSTRACT FROM AUTHOR]

Background: Vitiligo is melanocyte death that is affected by oxidative stress which elevated levels of reactive oxygen species can elicit an immune response, and ultimately cause melanocytic death. Objective: The current study aimed to investigate the impact of Narrowband Ultraviolet B (NBUVB) and excimer laser phototherapy on C-X-C motif chemokine receptor 3B expression in vitiligo patients and its impact on vitiligo improvement. Patients and methods: CXCR3B mRNA expression was measured in skin of 25 patients suffering from non-segmental vitiligo before and after generalized 'NBUVB' and targeted phototherapy 'Excimer laser'. All biopsies were kept in - 80C till RNA extraction that was performed utilizing QIAGEN RNeasy Little Pack, QuantiTect Switch Record Unit and QuantiNova SYBR Green PCR Unit. Results: After phototherapy patients with vitiligo had higher CXCR3B mRNA levels, in addition, this was higher in excimer bunch treatment than NBUVB. Conclusion: Such findings draw attention to CXCR3B as an inherited abnormality in vitiligo patients' skin and raise concerns about its potential contributions in maintaining vitiligo or triggering relapses & chronicity. [ABSTRACT FROM AUTHOR]

CD8+ T cells play a central role in antitumor immune responses. Epigenetic gene regulation is essential to acquire the effector function of CD8+ T cells. However, the role of Utx, a demethylase of histone H3K27, in antitumor immunity remains unclear. In this study, we examined the roles of Utx in effector CD8+ T‐cell differentiation and the antitumor immune response. In a murine tumor‐bearing model, an increased tumor size and decreased survival rate were observed in T‐cell‐specific Utx KO (Utx KO) mice compared with wild‐type (WT) mice. The number of CD8+ T cells in tumor‐infiltrating lymphocytes (TILs) was significantly decreased in Utx KO mice. We found that the acquisition of effector function was delayed and attenuated in Utx KO CD8+ T cells. RNA sequencing revealed that the expression of effector signature genes was decreased in Utx KO effector CD8+ T cells, while the expression of naïve or memory signature genes was increased. Furthermore, the expression of Cxcr3, which is required for the migration of effector CD8+ T cells to tumor sites, was substantially decreased in Utx KO CD8+ T cells. These findings suggest that Utx promotes CD8+ T‐cell‐dependent antitumor immune responses partially through epigenetic regulation of the effector function. [ABSTRACT FROM AUTHOR]

Background and purpose: The interaction of CXC motif chemokine receptor (CXCR) on the cell surface of multiple myeloma (MM) with chemokines in the bone marrow microenvironment is involved in proliferation, survival and extramedullary invasion of MM cells. Sinomenine derivative YL064 exerts its biological effects by targeting intracellular signaling regulators in MM cells. This study aimed to explore possible modulating and biological effects of sinomenine derivative YL064 on CXCR3 in MM cells. Methods: The MM cell lines H929 and MM1.S were used as in vitro models. H929-OE and MM1.S-OE cells with stable overexpression of CXCR3 were constructed with lentivirus vector. The effects of CXCR3 on clonal formation and migration of MM cells were detected by clone spot formation and transwell migration assay. Meanwhile, flow cytometry was used to analyze apoptotic rates of H929, H929-OE, MM1.S and MM1.S-OE cells treated with different concentrations of YL064. Furthermore, real-time fluorescence quantitative polymerase chain reaction (RTFQ-PCR) and Western blot assay were applied to detect expression levels of CXCR3 and its downstream signal regulators extracellular regulated protein kinase (ERK) and p-protein kinase B (p-AKT) in H929 and MM1.S cells before and after the treatment with YL064. Results: The clonal formation rates of H929-OE and MM1.S-OE cells with CXCR3 overexpression reached to 81.33%±5.79% and 73.00%±4.90%, which were significantly higher compared with H929 (58.33%±3.30%) and MM1.S cells (41.00%±3.14%). The migration proportion of H929-OE and MM1.S-OE cells were 7.90%±0.81% and 23.00%±1.63%, which were significantly higher compared with H929 (4.63%±0.37%) and MM1.S cells (14.63%±1.04%). After treatment with YL064, the apoptotic rates of H929-OE and MM1.S-OE cells were 29.80%±0.30% and 14.20%±0.26%, which were lower than those of H929 (33.40%±0.25%) and MM1.S cells (21.60%±0.21%). It was also shown that YL064 could reduce clonal formation and migration, inhibit CXCR3 gene transcription and downregulate expressions of CXCR3, ERK and AKT in H929 and MM1.S cells. Conclusions: CXCR3 might promote proliferation and invasion of MM cells. Sinomenine derivative YL064 could downregulate expressions of CXCR3 and its downstream signal regulators in MM cells, reduce abilities of clonal formation and migration, and induce apoptosis. [ABSTRACT FROM AUTHOR]

Radiotherapy (RT) not only damages tumors but also induces interferon (IFN) expression in tumors. IFNs mediate PD-L1 to exhaust CD8+ T cells, but which also directly impact tumor cells and potentially activate anti-tumor immune surveillance. Little is known about the contradictory mechanism of IFNs in regulating CD8+ T-mediated anti-tumor activity in lung cancer. This study found that RT induced IFNs and CXCL9/10 expression in the RT-treated lung cancer cells. Specifically, RT- and IFNγ-pretreated A549 significantly activated CD8+ T cells, resulting in significant inhibition of A549 colony formation. RNAseq and consequent qPCR results revealed that IFNγ induced PD-L1, CXCL10, and ICAM-1, whereas PD-L1 knockdown activated CD8+ T cells, but ICAM-1 knockdown diminished CD8+ T cell activation. We further demonstrated that CXCR3 and CXCL10 decreased in the CD8+ T cells and nonCD8+ PBMCs, respectively, in the patients with lung cancer that expressed lower reactivation as co-cultured with A549 cells. In addition, inhibitors targeting CXCR3 and LFA-1 in CD8+ T cells significantly diminished CD8+ T cell activation and splenocytes-mediated anti-LL/2shPdl1. In conclusion, we validated that RT suppressed lung cancer and overexpress PD-L1, CXCL10, and ICAM-1, which exhibited different roles in regulating CD8+ T cell activity. We propose that CXCR3highCD8+ T cells stimulated by CXCL10 exhibit anti-tumor immunity, possibly by enhancing T cells-tumor cells adhesion through CXCL10/CXCR3-activated LFA-1-ICAM-1 interaction, but CXCR3lowCD8+ T cells with low CXCL10 in patients with lung cancer were exhausted by PD-L1 dominantly. Therefore, RT potentially activates CD8+ T cells by inducing IFNs-mediated CXCL10 and ICAM-1 expression in tumors to enhance CD8+ T-tumor adhesion and recognition. This study clarified the possible mechanisms of RT and IFNs in regulating CD8+ T cell activation in lung cancer. [ABSTRACT FROM AUTHOR]

Pancreatic ductal adenocarcinoma (PDA) is a lethal and metastatic malignancy resistant to therapy. Elucidating how pancreatic tumor-specific T cells differentiate and are maintained in vivo could inform novel therapeutic avenues to promote T cell antitumor activity. Here, we show that the spleen is a critical site harboring tumor-specific CD8 T cells that functionally segregate based on differential Cxcr3 and Klrg1 expression. Cxcr3+ Klrg1- T cells express the memory stem cell marker Tcf1, whereas Cxcr3-Klrg1 + T cells express GzmB consistent with terminal differentiation. We identify a Cxcr3+ Klrg1+ intermediate T cell subpopulation in the spleen that is highly enriched for tumor specificity. However, tumor-specific T cells infiltrating primary tumors progressively downregulate both Cxcr3 and Klrg1 while upregulating exhaustion markers PD-1 and Lag-3. We show that antigen-specific T cell infiltration into PDA is Cxcr3 independent. Further, Cxcr3-deficiency results in enhanced antigen-specific T cell IFNγ production in primary tumors, suggesting that Cxcr3 promotes loss of effector function. Ultimately, however, Cxcr3 was critical for mitigating cancer cell dissemination following immunotherapy with CD40 agonist + anti-PD-L1 or T cell receptor engineered T cell therapy targeting mesothelin. In the absence of Cxcr3, splenic Klrg1 + GzmB + antitumor T cells wain while pancreatic cancer disseminates suggesting a role for these cells in eliminating circulating metastatic tumor cells. Intratumoral myeloid cells are poised to produce Cxcl10, whereas splenic DC subsets produce Cxcl9 following immunotherapy supporting differential roles for these chemokines on T cell differentiation. Together, our study supports that Cxcr3 mitigates tumor cell dissemination by impacting peripheral T cell fate rather than intratumoral T cell trafficking. [ABSTRACT FROM AUTHOR]

BackgroundIdentifying where human immunodeficiency virus (HIV) persists in people living with HIV and receiving antiretroviral therapy is critical to develop cure strategies. We assessed the relationship of HIV persistence to expression of chemokine receptors and their chemokines in blood (n = 48) and in rectal (n = 20) and lymph node (LN; n = 8) tissue collected from people living with HIV who were receiving suppressive antiretroviral therapy.MethodsCell-associated integrated HIV DNA, unspliced HIV RNA, and chemokine messenger RNA were quantified by quantitative polymerase chain reaction. Chemokine receptor expression on CD4+ T cells was determined using flow cytometry.ResultsIntegrated HIV DNA levels in CD4+ T cells, CCR6+CXCR3+ memory CD4+ T-cell frequency, and CCL20 expression (ligand for CCR6) were highest in rectal tissue, where HIV-infected CCR6+ T cells accounted for nearly all infected cells (median, 89.7%). Conversely in LN tissue, CCR6+ T cells were infrequent, and there was a statistically significant association of cell-associated HIV DNA and RNA with CCL19, CCL21, and CXCL13 chemokines.ConclusionsHIV-infected CCR6+ CD4+ T cells accounted for the majority of infected cells in rectal tissue. The different relationships between HIV persistence and T-cell subsets and chemokines in rectal and LN tissue suggest that different tissue-specific strategies may be required to eliminate HIV persistence and that assessment of biomarkers for HIV persistence may not be generalizable between blood and other tissues.

Introduction: Basal cell carcinoma (BCC) is the most common type of skin cancer worldwide. The pathogenesis of BCC involves interplay between various environmental and genetic factors. It is believed that chemokines play a significant role in the modulation of cancer growth by generating autocrine and paracrine signaling effects. The present study was conducted to elucidate the expression of chemokine, CXCL11, and its receptor CXCR3, and their interaction with tumor cells and peri-tumoral stroma in various subtypes of BCC. Aim and Objectives: The aim of this study was to evaluate the immunohistochemical expression of chemokine CXCL11 and its receptor CXCR3 in various subtypes of BCC. Materials and Methods: The study included 40 cases of histopathologically confirmed BCC. Clinical and histopathological features of various tumor subtypes were noted. Immunohistochemistry was performed using antibodies against CXCL11 and CXCR3, and these were assigned scores 0, 1, and 2 on the basis of immunohistochemical expression. Results: The median age of study participants was 65.0 ± 12.2 years with a male-to-female ratio of 1.5:1. The most common site was face, followed by neck, scalp, and back. The tumor subtypes included in the study were nodular (n = 20), pigmented (n = 8), infiltrating (n = 5), superficial (n = 4), and adenoid (n = 3). On immunohistochemistry, CXCR3 expression was seen in 34 (85%) cases with stromal inflammatory cells immunopositivity in 29 (72.5%) cases and tumor cells immunopositivity in 5 (12.5%) cases. CXCL11 expression was seen in 36 (90%) cases with weak expression in stroma and tumor in 18 cases and strong expression in the rest 18 cases. In individual subtypes, higher immunopositivity for CXCR3 and CXCL11 in tumor cells and peri-tumoral stroma was seen for nodular, infiltrating, and pigmented subtypes, compared to adenoid and superficial subtypes. Conclusion: Our study shows the enhanced expression of chemokine CXCL11 and its receptor CXCR3 in tumor cells and peri-tumoral stroma of BCC. This expression is greater in tumor cells of aggressive subtypes, i.e. nodular, infiltrating, and pigmented types. This suggests that receptor ligand pathway involving CXCR3 and CXCL11 plays a key role in pathogenesis of BCC, and blocking this pathway may result in inhibition of tumor growth. Thus, these chemokines may serve as future potential targets in developing novel therapeutic regimens against BCC.

Abstract Background The chemokines, CXCL12 and CXCL11, are upregulated in tumors from many organs and control their progression. CXCL12 and CXCL11 affect tumor cell functions by either binding their prime receptors, CXCR4 and CXCR3, respectively, and/or CXCR7 as a common second chemokine receptor. In humans, CXCR3 exists in the functional splice variants, CXCR3A and CXCR3B, which either have pro- or anti-tumor activity, respectively. Despite the intimate crosstalk between the CXCL12- and CXCL11-system, the impact of a combination of CXCL12 and CXCL11 on tumor progression remains vague. Methods In the present work, we have analyzed CXCL12 and CXCL11 for combined effects on migration, invasion, proliferation, and cytostatic-induced apoptosis of the human tumor cells, A549, A767, A772, DLD-1, and MDA-MB-231. Results We demonstrate that the mode of interaction differs with respect to cell type and function and allows for either potentiation, attenuation or no changes of cellular responses. The divergent responses are not the result of the distinct use of different CXCL12- and CXCL11-receptors by the respective tumor cells, but in case of cell migration seem to be associated with the activation of p38 signaling pathways. Conclusions Our findings point to therapeutic limitations of ongoing efforts to selectively target CXCR3, CXCR4, or CXCR7 in cancer patients, and rather favor individualized targeting strategies.