Diagnostic accuracy and cellular origin of pleural fluid CXCR3 ligands for tuberculous pleural effusion.

BACKGROUND: Pleural biomarkers represent potential diagnostic tools for tuberculous pleural effusion (TPE) due to their advantages of low cost, short turnaround time, and less invasiveness. This study evaluated the diagnostic accuracy of two CXCR3 ligands, C-X-C motif chemokine ligand 9 (CXCL9) and CXCL11, for TPE. In addition, we investigated the cellular origins and biological roles of CXCL9 and CXCL11 in the development of TPE. METHODS: This double-blind study prospectively enrolled patients with undiagnosed pleural effusion from two centers (Hohhot and Changshu) in China. Pleural fluid on admission was obtained and levels of CXCL9 and CXCL11 were measured by an enzyme-linked immunosorbent assay (ELISA). The receiver operating characteristic (ROC) curve and the decision curve analysis (DCA) were used to evaluate their diagnostic accuracy and net benefit, respectively. THP-1 cell-derived macrophages were treated with Bacillus Calmette-Guérin (BCG), and quantitative real-time PCR (qRT-PCR) and ELISA were used to determine the mRNA and protein levels of CXCL9 and CXCL11. The chemoattractant activities of CXCL9 and CXCL11 for T helper (Th) cells were analyzed by a transwell assay. RESULTS: One hundred and fifty-three (20 TPEs and 133 non-TPEs) patients were enrolled in the Hohhot Center, and 58 (13 TPEs and 45 non-TPEs) were enrolled in the Changshu Center. In both centers, we observed increased CXCL9 and CXCL11 in TPE patients. The areas under the ROC curves (AUCs) of pleural CXCL9 and CXCL11 in the Hohhot Center were 0.70 (95 % CI: 0.55-0.85) and 0.68 (95 % CI: 0.52-0.84), respectively. In the Changshu Center, the AUCs of CXCL9 and CXCL11 were 0.96 (95 % CI: 0.92-1.00) and 0.97 (95 % CI: 0.94-1.00), respectively. The AUCs of CXCL9 and CXCL11 decreased with the advancement of age. The decision curves of CXCL9 and CXCL11 showed net benefits in both centers. CXCL9 and CXCL11 were upregulated in BCG-treated macrophages. Pleural fluid from TPE and conditioned medium from BCG-treated macrophages were chemotactic for Th cells. Anti-CXCL9 or CXCL11 neutralizing antibodies could partly block the chemotactic activity. CONCLUSIONS: Pleural CXCL9 and CXCL11 are potential diagnostic markers for TPE, but their diagnostic accuracy is compromised in elderly patients. CXCL9 and CXCL11 can promote the migration of peripheral Th cells, thus representing a therapeutic target for the treatment of TPE.

HIF-1α Mediates Immunosuppression and Chemoresistance in Colorectal Cancer by Inhibiting CXCL9, -10 and -11.

Uncertainty exists regarding the mechanisms by which hypoxia-inducible factors (HIFs) control CD8+T-cell migration into tumor microenvironments. Here, we found that HIF-1α knockdown or overexpression resulted in increased or decreased CXCL9, -10, and -11 expression in vitro, respectively. Gene Set Variation Analysis revealed that elevated HIF-1α levels correlated with a poor prognosis, severe pathological stage, and an absence of CD8+ T cells in the tumor microenvironment in colorectal cancer (CRC) patients. HIF-1α was inversely associated with pathways beneficial to anti-tumor immunotherapy and cytokine/chemokine function. In vivo, inhibiting HIF-1α or its upstream regulator BIRC2 significantly suppressed tumor growth and promoted CD8+ T-cell infiltration. CXCR3 neutralizing antibodies reversed these effects, implicating the involvement of CXCL9, -10, and -11/CXCR3 axis. The presence of HIF-1α weakened the upregulation of CXCL9, -10, and -11 by bleomycin and doxorubicin. Combining HIF-1α inhibition with bleomycin promoted CD8+ T-cell infiltration and tumor suppression in vivo. Moreover, doxorubicin could upregulate CXCL9, -10 and -11 by suppressing HIF-1α. Our findings highlight the potential of HIF-1α inhibition to improve CRC microenvironments and increase chemotherapy sensitivity.

Intratumoral delivery of a highly active form of XCL1 enhances antitumor CTL responses through recruitment of CXCL9-expressing conventional type-1 dendritic cells.

Conventional type 1 dendritic cells (cDC1s) play a crucial role in antitumor immunity through the induction and activation of tumor-specific CD8+ cytotoxic T cells (CTLs). The chemokine XCL1 is a major chemotactic factor for cDC1s and its receptor XCR1 is selectively expressed on cDC1s. Here, we investigated the effect of intratumoral delivery of a highly active form of murine XCL1 (mXCL1-V21C/A59C) on cDC1-mediated antitumor immunity using a hydrophilic gel patch. The hydrophilic gel patch containing mXCL1-V21C/A59C increased cDC1 accumulation in the tumor masses and promoted their migration to the regional lymph nodes, resulting in enhanced induction of tumor-specific CTLs. Tumor-infiltrating cDC1s not only expressed XCR1 but also produced CXCL9, a ligand for CXCR3 which is highly expressed on CTLs and NK cells. Consequently, CTLs and NK cells were increased in the tumor masses of mice treated with mXCL1-V21C/A59C, while immunosuppressive cells such as monocyte-derived suppressive cells and regulatory T cells were decreased. We also confirmed that anti-CXCL9 treatment decreased the tumor infiltration of CTLs. The intratumoral delivery of mXCL1-V21C/A59C significantly decreased tumor growth and prolonged survival in E.G7-OVA and B16-F10 tumor-bearing mice. Furthermore, the antitumor effect of mXCL1-V21CA59C was enhanced in combination with anti-programmed cell death protein 1 treatment. Finally, using The Cancer Genome Atlas database, we found that XCL1 expression was positively correlated with tumor-infiltrating cDC1s and a better prognosis in melanoma patients. Collectively, our findings provide a novel therapeutic approach to enhance tumor-specific CTL responses through the selective recruitment of CXCL9-expressing cDC1s into the tumor masses.

Pharmacological Polarization of Tumor-Associated Macrophages Toward a CXCL9 Antitumor Phenotype.

Tumor-associated macrophages (TAM) are a diverse population of myeloid cells that are often abundant and immunosuppressive in human cancers. CXCL9Hi TAM has recently been described to have an antitumor phenotype and is linked to immune checkpoint response. Despite the emerging understanding of the unique antitumor TAM phenotype, there is a lack of TAM-specific therapeutics to exploit this new biological understanding. Here, the discovery and characterization of multiple small-molecule enhancers of chemokine ligand 9 (CXCL9) and their targeted delivery in a TAM-avid systemic nanoformulation is reported. With this strategy, it is efficient encapsulation and release of multiple drug loads that can efficiently induce CXCL9 expression in macrophages, both in vitro and in vivo in a mouse tumor model. These observations provide a window into the molecular features that define TAM-specific states, an insight a novel therapeutic anticancer approach is used to discover.

Quantifying dynamic pro-inflammatory gene expression and heterogeneity in single macrophage cells.

Macrophages must respond appropriately to pathogens and other pro-inflammatory stimuli in order to perform their roles in fighting infection. One way in which inflammatory stimuli can vary is in their dynamics-that is, the amplitude and duration of stimulus experienced by the cell. In this study, we performed long-term live cell imaging in a microfluidic device to investigate how the pro-inflammatory genes IRF1, CXCL10, and CXCL9 respond to dynamic interferon-gamma (IFNγ) stimulation. We found that IRF1 responds to low concentration or short duration IFNγ stimulation, whereas CXCL10 and CXCL9 require longer or higherconcentration stimulation to be expressed. We also investigated the heterogeneity in the expression of each gene and found that CXCL10 and CXCL9 have substantial cell-to-cell variability. In particular, the expression of CXCL10 appears to be largely stochastic with a subpopulation of nonresponding cells across all the stimulation conditions tested. We developed both deterministic and stochastic models for the expression of each gene. Our modeling analysis revealed that the heterogeneity in CXCL10 can be attributed to a slow chromatin-opening step that is on a similar timescale to that of adaptation of the upstream signal. In this way, CXCL10 expression in individual cells can remain stochastic in response to each pulse of repeated stimulation, which we also validated by experiments. Together, we conclude that pro-inflammatory genes in the same signaling pathway can respond to dynamic IFNγ stimulus with very different response features and that upstream signal adaptation can contribute to shaping heterogeneous gene expression.

CXCL9:SPP1 macrophage polarity identifies a network of cellular programs that control human cancers.

Tumor microenvironments (TMEs) influence cancer progression but are complex and often differ between patients. Considering that microenvironment variations may reveal rules governing intratumoral cellular programs and disease outcome, we focused on tumor-to-tumor variation to examine 52 head and neck squamous cell carcinomas. We found that macrophage polarity-defined by CXCL9 and SPP1 (CS) expression but not by conventional M1 and M2 markers-had a noticeably strong prognostic association. CS macrophage polarity also identified a highly coordinated network of either pro- or antitumor variables, which involved each tumor-associated cell type and was spatially organized. We extended these findings to other cancer indications. Overall, these results suggest that, despite their complexity, TMEs coordinate coherent responses that control human cancers and for which CS macrophage polarity is a relevant yet simple variable.

Differential Anti-Tumor Effects of IFN-Inducible Chemokines CXCL9, CXCL10, and CXCL11 on a Mouse Squamous Cell Carcinoma Cell Line.

Chemokines are a group of cytokines involved in the mobilization of leukocytes, which play a role in host defense and a variety of pathological conditions, including cancer. Interferon (IFN)-inducible chemokines C-X-C motif ligand 9 (CXCL), CXCL10, and CXCL11 are anti-tumor chemokines; however, the differential anti-tumor effects of IFN-inducible chemokines are not completely understood. In this study, we investigated the anti-tumor effects of IFN-inducible chemokines by transferring chemokine expression vectors into a mouse squamous cell carcinoma cell line, SCCVII, to generate a cell line stably expressing chemokines and transplanted it into nude mice. The results showed that CXCL9- and CXCL11-expressing cells markedly inhibited tumor growth, whereas CXCL10-expressing cells did not inhibit growth. The NH2-terminal amino acid sequence of mouse CXCL10 contains a cleavage sequence by dipeptidyl peptidase 4 (DPP4), an enzyme that cleaves the peptide chain of chemokines. IHC staining indicated DPP4 expression in the stromal tissue, suggesting CXCL10 inactivation. These results suggest that the anti-tumor effects of IFN-inducible chemokines are affected by the expression of chemokine-cleaving enzymes in tumor tissues.

HDAC inhibitor HPTA initiates anti-tumor response by CXCL9/10-recruited CXCR3+CD4+T cells against PAHs carcinogenicity.

Polycyclic aromatic hydrocarbons (PAHs) exposure in food is closely associated with the occurrence and development of breast cancer, which may attribute to altered immunotoxicity and immune regulation. Currently, cancer immunotherapy aims to promote tumor-specific T cell responses, especially CD4+T helper cells (Th) for anti-tumor immunity. The histone deacetylase inhibitors (HDACis) are found to exert an anti-tumor effect by reshaping the tumor immune microenvironment, but the immune regulatory mechanism of HDACis in PAHs-induced breast tumor remains elusive. Here, using established breast cancer models induced by 7,12-dimethylbenz[a]anthracene (DMBA), a potent carcinogenic agent of PAH, the novel HDACi, 2-hexyl-4-pentylene acid (HPTA) exhibited anti-tumor effect by activating T lymphocytes immune function. HPTA recruited CXCR3+CD4+T cells into chemokines CXCL9/10-enriched tumor sites, and the increased secretion of CXCL9/10 was regulated by the NF-κB-mediated pathway. Furthermore, HPTA promoted Th1 differentiation and assisted cytotoxic CD8+T cells in the elimination of breast cancer cells. These findings support the proposition of HPTA as a potential therapeutic in the treatment of PAHs-induced carcinogenicity.

CXCL9 influences the tumor immune microenvironment by stimulating JAK/STAT pathway in triple-negative breast cancer.

Triple-negative breast cancer (TNBC) is a subtype of breast cancer with a poor prognosis and limited effective treatment options. Notably, immunotherapy is a potential therapeutic approach for TNBC. This study performed single-cell RNA sequencing on TNBC and found highly expressed CXCL9 in M1 macrophages. An intercellular communication network was found between M1 macrophages and M2 macrophages, and M1 macrophages could differentiate into M2 macrophages over time. Meanwhile, CXCL9 expression started to decrease in association with cell differentiation from M1 macrophages to M2 macrophages. Additionally, the M1 macrophage had strong connections to the M2 macrophage in the MHC-II signaling network. Through GSVA analysis, the MHC-II pathway activity of the M1 macrophages was significantly stronger than that of the M2 macrophages. Furthermore, CXCL9 was enriched in the MHC-II signaling pathway. CXCL9 was significantly enriched in the JAK/STAT signaling pathway. Western blot revealed that CXCL9 overexpression promotes JAK1/STAT2 expression in MDA-MB-231 cells. These findings indicate that CXCL9 is a potential clinical biomarker of prognosis and immunotherapy efficacy for TNBC patients. Also, it stimulates JAK/STAT activity, which in turn modifies the tumor microenvironment.

Schistosoma mansoni infection decreases IL-33-mRNA expression and increases CXCL9 and CXCL10 production by peripheral blood cells.

Schistosoma mansoni infections, particularly egg antigens, induce Th2-dominant granulomatous responses accompanied by remarkable immunoregulatory mechanisms that avoid intense fibrosis. Interleukin (IL)-33 is a cytokine that stimulates the early activation of Th2 responses, and its soluble ST2 receptor (sST2) avoids granulomatous response, as well as CXCL9 and CXCL10 chemokines that have antifibrotic activity. However, in schistosomiasis, these molecules have not been suitably studied. Therefore, this study aimed to measure IL-33 and sST2 RNA, cytokines, and chemokines in peripheral blood cultures from individuals living in schistosomiasis-endemic areas. Peripheral blood cells from individuals with S. mansoni (n = 34) and non-infected individuals (n = 31) were cultured under mitogen stimulation. Supernatant chemokines and cytokines were evaluated using a cytometric bead array, and IL-33 and sST2 mRNA expression was measured using qPCR. Infected individuals showed higher levels of CXCL8, CXCL9, CXCL10, IFN-γ, TNF-α, IL-6, IL-2, IL-4, and IL-10; there was a lower expression of IL-33 mRNA and similar expression of sST2mRNA in infected than non-infected individuals. In conclusion, for the first time, we demonstrated lower IL-33mRNA expression and high levels of the antifibrotic chemokines CXCL9 and CXCL10 in schistosomiasis mansoni, which could control exacerbations of the disease in individuals from endemic areas.

Immunological Microenvironment Alterations in Follicles of Patients With Autoimmune Thyroiditis.

Autoimmune thyroiditis (AIT) is the most prevalent autoimmune endocrine disease, with a higher incidence in women than in men. Immunological abnormalities may lead to the impairment of ovarian folliculogenesis; however, whether the presence of AIT affects immunological microenvironment in follicles remains controversial. We performed a cross-sectional study including 122 patients, aged 20-40 years, who underwent IVF/ICSI treatment owing to isolated male or tube factor infertility. Patients were divided into AIT and control groups according to clinical presentation, thyroid function, and thyroid autoantibody measurements. Follicular fluid was collected and the distribution of cytokines/chemokines in follicular fluid was measured by flow cytometry using multiplex bead assays between the two groups. Based on differences in levels of intrafollicular chemokines and cytokines between the AIT and control groups, the relevant inflammatory cascade was further demonstrated. Among the 12 chemokines analyzed, three (CXCL9, CXCL10, and CXCL11) showed significantly elevated levels in the follicular fluid of patients with AIT. Among the 11 cytokines detected, compared with those in the control group, significantly higher levels of IFNγ were observed in patients with AIT. IFNγ dose-dependently stimulated the expression and secretion of CXCL9/10/11 in cultured primary granulosa cells. The percentage of CXCR3+ T lymphocytes was significantly elevated in the follicular microenvironment of patients with AIT. We concluded that the IFNγ-CXCL9/10/11-CXCR3+ T lymphocyte inflammatory cascade is activated in the follicular microenvironment of patients with AIT. These findings indicate that a considerable immune imbalance occurred in the follicular microenvironment of patients with AIT.

C-X-C Motif Chemokine Ligand 9 and Its CXCR3 Receptor Are the Salt and Pepper for T Cells Trafficking in a Mouse Model of Gaucher Disease.

Gaucher disease is a lysosomal storage disease, which happens due to mutations in GBA1/Gba1 that encodes the enzyme termed as lysosomal acid ß-glucosidase. The major function of this enzyme is to catalyze glucosylceramide (GC) into glucose and ceramide. The deficiency of this enzyme and resultant abnormal accumulation of GC cause altered function of several of the innate and adaptive immune cells. For example, augmented infiltration of T cells contributes to the increased production of pro-inflammatory cytokines, (e.g., IFNγ, TNFα, IL6, IL12p40, IL12p70, IL23, and IL17A/F). This leads to tissue damage in a genetic mouse model (Gba19V/-) of Gaucher disease. The cellular mechanism(s) by which increased tissue infiltration of T cells occurs in this disease is not fully understood. Here, we delineate role of the CXCR3 receptor and its exogenous C-X-C motif chemokine ligand 9 (CXCL9) in induction of increased tissue recruitment of CD4+ T and CD8+ T cells in Gaucher disease. Intracellular FACS staining of macrophages (Mϕs) and dendritic cells (DCs) from Gba19V/- mice showed elevated production of CXCL9. Purified CD4+ T cells and the CD8+ T cells from Gba19V/- mice showed increased expression of CXCR3. Ex vivo and in vivo chemotaxis experiments showed CXCL9 involvement in the recruitment of Gba19V/- T cells. Furthermore, antibody blockade of the CXCL9 receptor (CXCR3) on T cells caused marked reduction in CXCL9- mediated chemotaxis of T cells in Gba19V/- mice. These data implicate abnormalities of the CXCL9-CXCR3 axis leading to enhanced tissue recruitment of T cells in Gaucher disease. Such results provide a rationale for blockade of the CXCL9/CXCR3 axis as potential new therapeutic targets for the treatment of inflammation in Gaucher disease.

Therapeutic effects of CXCL9-overexpressing human umbilical cord mesenchymal stem cells on liver fibrosis in rats.

Umbilical cord mesenchymal stem cells (UC-MSCs) transplantation has become a promising treatment for liver fibrosis. However, UC-MSCs have limited anti-fibrosis ability, and their homing ability of UC-MSCs to the injured liver seems to be poor. In our study, we aimed to determine if the CXCL9-overexpressing UC-MSCs could have synergistic anti-fibrosis effects and whether it can promote the homing ability of UC-MSCs. Overexpression of CXCL9 in UC-MSCs (CXCL9-UC-MSCs) was attained by transfecting the lenti-CXCL9-mCherry to naive UC-MSCs. The therapeutic effect of transducted CXCL9-UC-MSCs on both repairing of hepatic fibrosis and target homing were evaluated by comparing with the control of UC-MSCs transfected with empty lenti-mCherry vector. The results revealed that the liver function of CXCL9-UC-MSCs treated group was significantly improved when compared with that of control UC-MSCs (P < 0.05), and the histopathology indicated an obvious decrease of the collagen fiber content and significant disappearing of pseudo-lobules with basically normal morphology of hepatic lobules. Furthermore, liver frozen sections confirmed that CXCL9-UC-MSCs have significantly stronger chemotaxis and stable persistence in the injured liver tissues. In summary, overexpression of CXCL9 could improve the efficacy of UC-MSCs therapy for liver fibrosis repairing on account of an enhanced ability of UC-MSCs in homing to and staying in the injured sites of liver fibrosis in rat models.

Genomic biomarkers in chronic beryllium disease and sarcoidosis.

Background Previous gene expression studies have identified genes IFNγ, TNFα, RNase 3, CXCL9, and CD55 as potential biomarkers for sarcoidosis and/or chronic beryllium disease (CBD). We hypothesized that differential expression of these genes could function as diagnostic biomarkers for sarcoidosis and CBD, and prognostic biomarkers for sarcoidosis. Study Design/Methods We performed RT-qPCR on whole blood samples from CBD (n = 132), beryllium sensitized (BeS) (n = 109), and sarcoidosis (n = 99) cases and non-diseased controls (n = 97) to determine differential expression of target genes. We then performed logistic regression modeling and generated ROC curves to determine which genes could most accurately differentiate: 1) CBD versus sarcoidosis 2) CBD versus BeS 3) sarcoidosis versus controls 4) non-progressive versus progressive sarcoidosis. Results CD55 and TNFα were significantly upregulated, while CXCL9 was significantly downregulated in CBD compared to sarcoidosis (p < 0.05). The ROC curve from the logistic regression model demonstrated high discriminatory ability of the combination of CD55, TNFα, and CXCL9 to distinguish between CBD and sarcoidosis with an AUC of 0.98. CD55 and TNFα were significantly downregulated in sarcoidosis compared to controls (p < 0.05). The ROC curve from the model showed a reasonable discriminatory ability of CD55 and TNFα to distinguish between sarcoidosis and controls with an AUC of 0.86. There was no combination of genes that could accurately differentiate between CBD and BeS or sarcoidosis phenotypes. Interpretation CD55, TNFα and CXCL9 expression levels can accurately differentiate between CBD and sarcoidosis, while CD55 and TNFα expression levels can accurately differentiate sarcoidosis and controls.

MIG/CXCL9 exacerbates the progression of metabolic-associated fatty liver disease by disrupting Treg/Th17 balance.

CD4+CD25+ regulatory T (Treg) cells and Th17 cells play important roles in the progression of metabolic-associated fatty liver disease (MAFLD). However, the contribution of monokine induced by interferon-gamma (MIG)/CXCL9 to the Treg/Th17 imbalance in MAFLD is only partially understood. In the present study, we detected increased levels of MIG/CXCL9 and a Treg/Th17 imbalance in the setting of metabolic-associated steatohepatitis (MASH). Recombinant adeno-associated virus-mediated gene transfer and silencing of MIG/CXCL9 expression in mice alleviated MASH and increased the Treg/Th17 ratio. Furthermore, the percentage of Th17 cells, but not Treg cells, differentiated from splenic CD4+ T cells was significantly increased by administration of MIG/CXCL9. MIG/CXCL9 also promoted Th17 cell proliferation, and its effects were dose dependent. Levels of phosphorylated c-Jun N-terminal kinase (JNK) decreased dramatically when MIG/CXCL9 was inhibited in a murine MASH model. In cultured Treg cells, phosphorylated JNK levels decreased dose-dependently in response to MIG/CXCL9 inhibition, but increased in cultured Th17 cells. This effect was blocked in the presence of a JNK inhibitor. These findings underline the fundamental importance of MIG/CXCL9 in maintaining the Treg/Th17 balance in MAFLD and provide the foundations for a novel approach to preventing and treating MAFLD.

The effects of T-DXd on the expression of HLA class I and chemokines CXCL9/10/11 in HER2-overexpressing gastric cancer cells.

Trastuzumab deruxtecan (T-DXd), a HER2-targeting antibody-drug conjugate with a topoisomerase I inhibitor deruxtecan (DXd), exhibits an excellent anti-tumor effect in previously treated HER2-positive tumors. A recent study demonstrated that T-DXd not only suppressed tumor growth but also enhanced anti-tumor immunity through increasing the number of tumor-infiltrating CD8+ T cells and enhancement of major-histocompatibility-complex class I expression on tumor cells in a mouse model. However, the effect of T-DXd on anti-tumor immune responses in human cancers is largely unknown. We investigated the effect of T-DXd on the expression of HLA class I and CXCL9/10/11, T-cell chemoattractants, in HER2-positive human gastric cancer (GC) cells. We found that T-DXd significantly inhibited GC cell proliferation in a HER2-dependent manner, while it slightly increased the expression of HLA class I in HER2-positive GC cells. Moreover, we revealed that T-DXd significantly induced mRNA expression of CXCL9/10/11 in HER2-positive GC cells. T-DXd-triggered up-regulation of these chemokines was mediated through the activation of DNA damage signaling pathways. These results suggest that T-DXd triggers anti-tumor immune responses at least in part through induction of the expression of HLA class I and CXCL9/10/11 on HER2-positive GC cells, resulting in the enhancement of anti-tumor immunity in human GC.

Immune infiltration profiling in gastric cancer and their clinical implications.

The abundance and type of immune cells in the tumor microenvironment (TME) significantly influence immunotherapy and tumor progression. However, the role of immune cells in the TME of gastric cancer (GC) is poorly understood. We studied the correlations, proportion, and infiltration of immune and stromal cells in GC tumors. Data analyses showed a significant association of infiltration levels of specific immune cells with the pathological characteristics and clinical outcomes of GC. Furthermore, based on the difference in infiltration levels of immune and stromal cells, GC patients were divided into two categories, those with "immunologically hot" (hot) tumors and those with "immunologically cold" (cold) tumors. The assay for transposase-accessible chromatin using sequencing and RNA sequencing analyses revealed that the hot and cold tumors had altered epigenomic and transcriptional profiles. Claudin-3 (CLDN3) was found to have high expression in the cold tumors and negatively correlated with CD8+ T cells in GC. Overexpression of CLDN3 in GC cells inhibited the expression of MHC-I and CXCL9. Finally, the differentially expressed genes between hot and cold tumors were utilized to generate a prognostic model, which predicted the overall survival of GC as well as patients with immunotherapy. Overall, we undertook a comprehensive analysis of the immune cell infiltration pattern in GC and provided an accurate model for predicting the prognosis of GC patients.

Exercise Training Improves Tumor Control by Increasing CD8+ T-cell Infiltration via CXCR3 Signaling and Sensitizes Breast Cancer to Immune Checkpoint Blockade.

The mechanisms behind the antitumor effects of exercise training (ExTr) are not fully understood. Using mouse models of established breast cancer, we examined here the causal role of CD8+ T cells in the benefit acquired from ExTr in tumor control, as well as the ability of ExTr to improve immunotherapy responses. We implanted E0771, EMT6, MMTV-PyMT, and MCa-M3C breast cancer cells orthotopically in wild-type or Cxcr3-/- female mice and initiated intensity-controlled ExTr sessions when tumors reached approximately 100 mm3 We characterized the tumor microenvironment (TME) using flow cytometry, transcriptome analysis, proteome array, ELISA, and immunohistochemistry. We used antibodies against CD8+ T cells for cell depletion. Treatment with immune checkpoint blockade (ICB) consisted of anti-PD-1 alone or in combination with anti-CTLA-4. ExTr delayed tumor growth and induced vessel normalization, demonstrated by increased pericyte coverage and perfusion and by decreased hypoxia. ExTr boosted CD8+ T-cell infiltration, with enhanced effector function. CD8+ T-cell depletion prevented the antitumor effect of ExTr. The recruitment of CD8+ T cells and the antitumor effects of ExTr were abrogated in Cxcr3-/- mice, supporting the causal role of the CXCL9/CXCL11-CXCR3 pathway. ExTr also sensitized ICB-refractory breast cancers to treatment. Our results indicate that ExTr can normalize the tumor vasculature, reprogram the immune TME, and enhance the antitumor activity mediated by CD8+ T cells via CXCR3, boosting ICB responses. Our findings and mechanistic insights provide a rationale for the clinical translation of ExTr to improve immunotherapy of breast cancer.

LINC00152 mediates CD8+ T-cell infiltration in gastric cancer through binding to EZH2 and regulating the CXCL9, 10/CXCR3 axis.

This study aimed to annotate the role of long intergenic non-coding RNA 152 (LINC00152) in CD8+ T cells mediated immune responses in gastric cancer (GC) and the underlying mechanism. LINC00152 expression levels were detected through RT-PCR. For tumor engraftment, HGC-27 cells that received LINC00152 shRNA, LINC00152 overexpression vectors, enhancer of zeste homolog 2 (EZH2) shRNA or combination transfection were injected into mice. Chromatin immunoprecipitation (ChIP) assay was used to explore the interaction between LINC00152, Cys-X-cys ligand 9 (CXCL9) and Cys-X-cys ligand 10 (CXCL10). Flow cytometry was adopted to measure the CD8+ T-cell infiltration in tumor issue. In this study, we found increased LINC00152 expression levels are positively associated with the poor prognosis of GC patients and negatively associated with the CD8 levels. ChIP assay verified that LINC00152 recruits EZH2 to the promoters of CXCL9 and CXCL10, thus the silencing of LINC00152 promoted the production of CXCL9 and CXCL10. Knockdown of LINC00152 suppressed tumor cells growth in vivo and in vitro, increased tumor-infiltrating CD8+ T cells numbers and promoted the expression of CXCL9, CXCL10 and C-X-C Motif Chemokine Receptor 3 (CXCR3) in xenograft tumors. While CD8+ T cell depletion reversed the tumor suppression effect of LINC00152 silence. Besides, the silencing of EZH2 partly inhibited the promotion effect LINC00152 on tumor growth. Our study indicated that LINC00152 inhibition suppressed the tumor progress may through promoting CD8+ T-cell infiltration.

CXCL9-expressing tumor-associated macrophages: new players in the fight against cancer.

Tumor-associated macrophages (TAMs) are among the main contributors to immune suppression in the tumor microenvironment, however, TAM depletion strategies have yielded little clinical benefit. Here, we discuss the concept that TAMs are also key regulators of anti-PD(L)-1-mediated CD8 T cell-dependent immunity. Emerging data suggest that expression of the chemokine CXCL9 by TAMs regulates the recruitment and positioning of CXCR3-expressing stem-like CD8 T (Tstem) cells that underlie clinical responses to anti-PD(L)-1 treatment. We evaluate clinical and mechanistic studies that establish relationships between CXCL9-expressing TAMs, Tstem and antitumor immunity. Therapies that enhance anti-PD(L)-1 response rates must consider TAM CXCL9 expression. In this perspective, we discuss opportunities to enhance the frequency and function of CXCL9 expressing TAMs and draw on comparative analyzes from infectious disease models to highlight potential functions of these cells beyond Tstem recruitment.