Journal: bioRxiv

Article Title: STING/RANTES Pathway in Airway Epithelium Stimulates Sensitization to Der p1 in an Asthma Model

doi: 10.1101/2023.07.30.550251

Figure Lengend Snippet: (A) HBEpC were cultured with cGAMP (14nM), H 2 O 2 , (100μM) or cGAMP+H 2 O 2 for 4 hours. (B-F) qPCR analysis of human IFNα (B) and IFNβ (C) and epithelial cytokines, IL-33 (D), TSLP (E), and RANTES (F). RANTES was stimulated by cGAMP and cGAMP+ H 2 O 2 . IFNβ was also stimulated by cGAMP+H 2 O 2 . Statistical analysis is performed by one-way ANOVA (means ± SEM, n=4). * p < 0.05, ** p < 0.01, **** p < 0.0001

Article Snippet: To elucidate the role of RANTES as an adjuvant during sensitization, the mice were sensitized with recombinant RANTES protein (R&D systems, Minneapolis, MN, USA, 478-MR) and 1 μg of HDM on Day 1.

Techniques: Cell Culture

Journal: bioRxiv

Article Title: STING/RANTES Pathway in Airway Epithelium Stimulates Sensitization to Der p1 in an Asthma Model

doi: 10.1101/2023.07.30.550251

Figure Lengend Snippet: (A) 8 weeks mice were treated with 20 ng of RANTES with 1 μg of HDM or 1 μg of HDM intra nasally on Day 1. Mice were challenged with 1 μg of HDM intranasally on Day 7, then lungs were extracted and analyzed on Day 8. (B) Pictures of lung sections of control and RANTES-adjuvanted, HDM-sensitized mice stained with H-E. Scale bar: 200 μm. Scale bar in picture of high magnification view: 40 μm. (C) Number of cells between bronchus and alveoli analyzed by ImageJ. Statistical analysis is performed by ordinary Mann Whitney’s U test (means ± SEM, 3 points/ section, n=5). (D) Pictures of lung sections in PBS and RANES-adjuvanted, HDM-sensitized mouse stained with PAS/Alcian blue. Scale bar: 80 μm. (E) Ratio of PAS/Alcianble area per epithelial cells (%). **** p < 0.0001

Article Snippet: To elucidate the role of RANTES as an adjuvant during sensitization, the mice were sensitized with recombinant RANTES protein (R&D systems, Minneapolis, MN, USA, 478-MR) and 1 μg of HDM on Day 1.

Techniques: Control, Staining

Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: CCR5 Inhibition Prevents Cardiac Dysfunction in the SIV/Macaque Model of HIV

doi: 10.1161/JAHA.114.000874

Figure Lengend Snippet: CCL5 decreases cardiomyocyte contractility via CCR5. Confocal microscopy showing expression of (A) CCR5 (red) on isolated adult rhesus macaque ventricular cardiomyocyte (RM VCM) merged with (B) sarcomeric actin (green). C, Representative twitch traces for VCM sequentially exposed to human recombinant CCL5 (100 nmol/L), then CCL5 (100 nmol/L) with maraviroc (MVC, 500 nmol/L). Compared to basal conditions (grey, left), CCL5 decreased contractility, shown by the reduced, flattened twitch amplitude (red, middle). The CCL5‐induced decline in contractility was reversed by subsequent addition of MVC with CCL5 (green, right). D, Representative twitch traces for reciprocal experiments showing VCM sequentially exposed to MVC (500 nmol/L) followed by a combination CCL5 (100 nmol/L) and MVC (500 nmol/L). Addition of MVC (blue, middle) did not alter contraction from basal (grey, left). Furthermore, addition of MVC prior to MVC+CCL5 (turquoise, left) prevented CCL5‐induced changes in contractility with twitch traces unchanged from basal. E, Summary data for RM VCM exposed to CCL5 (100 nmol/L) followed by MVC (500 nmol/L) with CCL5 (100 nmol/L). CCL5 significantly decreased fractional shortening compared to basal. Subsequent addition of MVC modulated the CCL5 effect towards basal shortening. F, CCL5 significantly increases the time from basal to 50% peak sarcomere length (t to pk) compared to basal conditions. Subsequent CCL5+MVC modulated t to pk shortening towards basal conditions. G, CCL5 did not significantly change the time from peak shortening to 50% baseline (t to bl) compared to basal conditions although t to bl in cells treated with CCL5+MVC was shorter than both basal and CCL5 treatment. Paired t ‐tests, mean value indicated by the top of bar with bars representing standard error.

Article Snippet: Cells were sequentially exposed to recombinant human CCL5 (278‐RN, R&D Systems) 100 nmol/L alone and with 500 nmol/L of MVC (NIH/AIDS Reagent).

Techniques: Confocal Microscopy, Expressing, Isolation, Recombinant

Journal: Oncotarget

Article Title: Lactate-activated macrophages induced aerobic glycolysis and epithelial-mesenchymal transition in breast cancer by regulation of CCL5-CCR5 axis: a positive metabolic feedback loop

doi: 10.18632/oncotarget.22786

Figure Lengend Snippet: (A) 3×10 5 THP-1 macrophages were treated with 15 mM lactate for 24 h, and the mRNA levels of chemokines were measured by quantitative PCR. The growth medium of control macrophages was titrated to pH6.1 using sterile HCl. (B) 3×10 5 THP-1 macrophages were incubated with different concentrations of lactate for 24 h, and CCL5 gene expression was determined with quantitative PCR. (C) 10 6 THP-1 macrophages were exposed to increasing concentrations of lactate for 48 h, and the secretion of CCL5 was measured by ELISA. (D) 10 6 human primary macrophages from breast cancer patients (n=9) were cultured with different concentrations of lactate for 48 h, and CCL5 production was detected. (E) 10 6 MDA-MB-231 cells were pre-treated with 15μM GSK 2837808A for 2 h, then the media were changed, and cells were cultured for another 24 h. The conditional media (MD-231 CM) were collected and applied to 10 6 THP-1 macrophages. CCL5 concentrations were detected with ELISA. (F) Immunohistochemical staining of CD68 and CCL5 in tumor adjacent tissues (control) and breast tumors (n=28). Scale bars represent 50 μm. * , P<0.05; ** , P<0.01.

Article Snippet: CCL5 (DRN00B), TGF-β1 (DB100B) ELISA kits and recombinant human CCL5 (278-RN/CF) and TGF-β1 (240-B) were bought from R&D Systems (Minneapolis, MN).

Techniques: Real-time Polymerase Chain Reaction, Control, Sterility, Incubation, Gene Expression, Enzyme-linked Immunosorbent Assay, Cell Culture, Immunohistochemical staining, Staining

Journal: Oncotarget

Article Title: Lactate-activated macrophages induced aerobic glycolysis and epithelial-mesenchymal transition in breast cancer by regulation of CCL5-CCR5 axis: a positive metabolic feedback loop

doi: 10.18632/oncotarget.22786

Figure Lengend Snippet: (A) 10 6 THP-1 macrophages were treated with 15 mM lactate for 48 h, and the expression of key regulators in Notch, NF-κB, STAT3 and HIF, were detected by western blot. (B) 3×10 5 THP-1 macrophages were cultured with 15 mM lactate for 24 h, and the mRNA levels of Notch ligands and receptors were measured by quantitative PCR. (C) Western blot for Notch ligands and receptors in THP-1 (10 6 ) macrophages after 48 h lactate treatment. (D) Lactate stimulated the expression of NICD in a time and dose-dependent manner. 10 6 THP-1 macrophages were treated with 15 mM lactic acid for 48 h. Data presented were representatives of at least three independent experiments. (E) 10 6 THP-1 macrophages were transfected with 50nM siNotch1, or pretreated with 50μM DAPT for 2 h, and then cultured with 15 mM lactate for 48 h. The secretion of CCL5 was measured by ELISA. * , P<0.05; ** , P<0.01.

Article Snippet: CCL5 (DRN00B), TGF-β1 (DB100B) ELISA kits and recombinant human CCL5 (278-RN/CF) and TGF-β1 (240-B) were bought from R&D Systems (Minneapolis, MN).

Techniques: Expressing, Western Blot, Cell Culture, Real-time Polymerase Chain Reaction, Transfection, Enzyme-linked Immunosorbent Assay

Journal: Oncotarget

Article Title: Lactate-activated macrophages induced aerobic glycolysis and epithelial-mesenchymal transition in breast cancer by regulation of CCL5-CCR5 axis: a positive metabolic feedback loop

doi: 10.18632/oncotarget.22786

Figure Lengend Snippet: (A) 10 6 THP-1 macrophages were treated with 15 mM lactate for 72 h, and then cells were washed twice and fresh media were added. Macrophages were cultured for another 24 h and the conditional media (lactate CM) was collected. The effect of CM on breast cancer cell migration was measured by double chamber transwell assay. 5μg/ml anti-CCL5 neutralizing antibody significantly decreased lactate CM-induced cell migration. (B) 10 6 MCF-7 cells were co-cultured with 15 mM lactate-activated macrophages in the presence of 5μg/ml anti-CCL5 antibody or not, and protein levels of EMT markers were tested by western blot. (C) 10 6 breast cancer cells were co-cultured with 10 6 lactate-activated THP-1 macrophages (or 10 6 lactate-activated primary macrophages) for different time points, and the expression of CCR5 was monitored by western blot. (D) MDA-MB-231 and MCF-7 cells were transfected with shCCR5 plasmids, or pre-treated with 5μM Maraviroc for 2 h, then cell migration induced by lactate CM was detected by double chamber transwell assay. Lactate CM was described in (A). (E) MCF-7 cells (10 6 ) were transfected with pcDNA3.1-CCR5, and then cultured with 10ng/ml CCL5 for 24 h. The expression of E-cadherin, N-cadherin and vimentin was investigated by western blot. (F) 10 6 Human primary macrophages (No. 4 and No. 9) were treated with 15 mM lactate for 72 h and CM was collected as described in (A). The migration of MDA-MB-231 cells was measured in the presence of primary macrophage CM. 5μg/ml anti-CCL5 neutralizing antibody, shRNAs designed against CCR5, or 5μM Maraviroc, significantly reduced primary macrophage CM-induced cell migration. * , P<0.05; ** , P<0.01.

Article Snippet: CCL5 (DRN00B), TGF-β1 (DB100B) ELISA kits and recombinant human CCL5 (278-RN/CF) and TGF-β1 (240-B) were bought from R&D Systems (Minneapolis, MN).

Techniques: Cell Culture, Migration, Transwell Assay, Western Blot, Expressing, Transfection

Journal: Oncotarget

Article Title: Lactate-activated macrophages induced aerobic glycolysis and epithelial-mesenchymal transition in breast cancer by regulation of CCL5-CCR5 axis: a positive metabolic feedback loop

doi: 10.18632/oncotarget.22786

Figure Lengend Snippet: (A) 3×10 5 MDA-MB-231 and MCF-7 cells were stimulated with 1-5ng/ml TGF-β1 for 24 h, and total RNA was isolated and tested for CCR5 mRNA by quantitative PCR. (B) Western blot for CCR5 protein in breast cancer cells (10 6 ) under TGF-β1 stimulation for 48 h. Data presented were representatives of at least three independent experiments. (C) MDA-MB-231 and MCF-7 cells (3×10 5 ) were co-transfected with pGL3-CCR5 and pRL-TK and exposed to different concentrations of TGF-β1 for 24 h, and luciferase activities were determined. (D) MDA-MB-231 and MCF-7 cells were pre-treated with 5μM SIS3 for 2 h, and cells were subjected to luciferase assay. (E) 10 6 MCF-7 cells were transfected with TGFβRI/ALK5 siRNA, and were then co-cultured with lactate-activated THP-1 macrophages (ratio 1:1) for 24 h. The protein levels of CCR5 were assayed by western blot. (F) The expression of TGF-β1, CCL5 and CCR5 in clinical samples obtained from breast cancer patients. The mRNA levels were measured by quantitative PCR, and the correlation between TGF-β1 and CCL5-CCR5 axis was shown. (G) Representative IHC staining for TGF-β1, CCL5 and CCR5 in breast cancer samples. The sample used was derived from 28 breast cancer cases. Scale bars represent 50 μm. * , P<0.05; ** , P<0.01.

Article Snippet: CCL5 (DRN00B), TGF-β1 (DB100B) ELISA kits and recombinant human CCL5 (278-RN/CF) and TGF-β1 (240-B) were bought from R&D Systems (Minneapolis, MN).

Techniques: Isolation, Real-time Polymerase Chain Reaction, Western Blot, Transfection, Luciferase, Cell Culture, Expressing, Immunohistochemistry, Derivative Assay

Journal: Oncotarget

Article Title: Lactate-activated macrophages induced aerobic glycolysis and epithelial-mesenchymal transition in breast cancer by regulation of CCL5-CCR5 axis: a positive metabolic feedback loop

doi: 10.18632/oncotarget.22786

Figure Lengend Snippet: (A) Glucose uptake, lactic acid production and ATP levels in breast cancer cells co-cultured with lactate-activated THP-1 macrophages, with or without 5μg/ml anti-CCL5 neutralizing antibody. The co-culture system was described in Figure . (B) Western blots for glycolytic enzymes in breast cancer cells treated as in (A). (C) MDA-MB-231 cells were transfected with shRNAs designed against CCR5, or pre-treated with 5μM Maraviroc for 2 h, and then subjected to cell co-culture. Glucose uptake, lactic acid production and ATP levels were measured after co-culture. The co-culture system was described in Figure . (D) The protein levels of HK2, PKM2 and LDHA in MDA-MB-231 cells cultured as in (C). (E) Recombinant human CCL5 induced aerobic glycolysis in breast cancer cells. MDA-MB-231 and MCF-7/CCR5 cells were treated with increasing concentrations of CCL5 for 12 h, and glucose uptake, lactic acid production and ATP levels were detected. (F) Western blots for glycolytic enzymes in MDA-MB-231 and MCF-7/CCR5 cells after stimulation with CCL5. * , P<0.05; ** , P<0.01.

Article Snippet: CCL5 (DRN00B), TGF-β1 (DB100B) ELISA kits and recombinant human CCL5 (278-RN/CF) and TGF-β1 (240-B) were bought from R&D Systems (Minneapolis, MN).

Techniques: Cell Culture, Co-Culture Assay, Western Blot, Transfection, Recombinant

Journal: Oncotarget

Article Title: Lactate-activated macrophages induced aerobic glycolysis and epithelial-mesenchymal transition in breast cancer by regulation of CCL5-CCR5 axis: a positive metabolic feedback loop

doi: 10.18632/oncotarget.22786

Figure Lengend Snippet: (A) Western blot for AMPK, c-Myc, HIF-1α and Akt in breast cancer cells co-cultured with 15mM lactic acid-activated THP-1 macrophages (ratio 1:1) for 72 h. Results presented were representatives of at least three independent experiments. (B) The expression of AMPK downstream signaling target ACC in breast cancer cells co-cultured as in (A). (C) MDA-MB-231 and MCF-7 cells were transfected with 50 nM AMPKα1 siRNA, or pretreated with 10μM compound C for 4 h, and then incubated with 15mM lactic acid-activated THP-1 macrophages (ratio 1:1) for 48 h. The glucose uptake, lactic acid production and ATP levels were detected. (D) The inhibition of AMPK abrogated macrophage-induced EMT in MCF-7 cells. Cells were treated as described in (C). After co-culture, the expression of EMT markers, E-cadherin and vimentin, was measured by western blot. (E) Recombinant human CCL5 induced the phosphorylation of AMPK in MDA-MB-231 and MCF-7/CCR5 cells. 10 6 cells were treated with 50ng/ml CCL5 for defferent time points as indicated, and phosphorylated AMPK and total AMPK were investigated by western blot. (F) Inhibition of CCR5 in MDA-MB-231 cells significantly attenuated macrophage-induced AMPK phosphorylation. MDA-MB-231 cells were transfected with shRNAs designed against CCR5, or pre-treated with 5μM Maraviroc for 2 h, then co-cultured with 15 mM lactate-activated macrophages as described in (A). After co-culture, the phosphorylation of AMPK was detected by western blot. (G) Expressions of CCL5, CCR5 and p-AMPK in samples obtained from breast cancer patients (n =28). Scale bars represent 50 μm. * , P<0.05; ** , P<0.01.

Article Snippet: CCL5 (DRN00B), TGF-β1 (DB100B) ELISA kits and recombinant human CCL5 (278-RN/CF) and TGF-β1 (240-B) were bought from R&D Systems (Minneapolis, MN).

Techniques: Western Blot, Cell Culture, Expressing, Transfection, Incubation, Inhibition, Co-Culture Assay, Recombinant, Phospho-proteomics

Journal: Oncotarget

Article Title: Lactate-activated macrophages induced aerobic glycolysis and epithelial-mesenchymal transition in breast cancer by regulation of CCL5-CCR5 axis: a positive metabolic feedback loop

doi: 10.18632/oncotarget.22786

Figure Lengend Snippet: (A) MDA-MB-231 cells were co-cultured with 15 mM lactate-activated THP-1 macrophages for 7 days, in the presence of 5μg/ml anti-CCL5 neutralizing antibody or not. MDA-MB-231 cells were then collected and injected into the tail vein of nude mice. After two weeks, animals were sacrificed and metastatic nodules on lung surfaces were counted. (B) CCR5, HK2 and p-AMPK were immunostained in MDA-MB-231 metastases. Scale bars represent 50 μm. * , P<0.05; ** , P<0.01.

Article Snippet: CCL5 (DRN00B), TGF-β1 (DB100B) ELISA kits and recombinant human CCL5 (278-RN/CF) and TGF-β1 (240-B) were bought from R&D Systems (Minneapolis, MN).

Techniques: Cell Culture, Injection

Journal: Journal of Oncology

Article Title: Palbociclib Enhances Migration and Invasion of Cancer Cells via Senescence-Associated Secretory Phenotype-Related CCL5 in Non-Small-Cell Lung Cancer

doi: 10.1155/2022/2260625

Figure Lengend Snippet: Palbociclib induces SASP-related CCL5 in NSCLC cells. (a) The β -galactosidase staining in NSCLC cells induced by palbociclib. Left: images of β -Gal-stained H1650 and H226 cells with/without palbociclib. Scale bars represent 100 μ m. Right: the bar charts display the calculated numbers of β -Gal positive cells. (b) Protein expression levels of the genes involved in cell senescence after treatment with 2 μ M palbociclib. Left: gel images of western blot for genes' expression, with GAPDH as a loading control. Right: the bar charts display the relative expression of target genes on protein level. P16, P21, p-RB, and corresponding GAPDH were tested in the same experiment. And β -Gal, STING, and corresponding GAPDH were tested in another experiment. Western blot result of every target protein cropped from the same gel in the same experiment and the proteins with similar size came from different gels in the same experiment. (c) CCL5 mRNA expression levels in NSCLC cells treated with palbociclib of gradient concentration (left) and secreted CCL5 in the supernatants of NSCLC cell with 2 μ M palbociclib (right). ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001, and ∗∗∗∗ P < 0.0001.

Article Snippet: Thus, the secreted CCL5 levels in the media of palbociclib-treated cells and the control cells were assessed by enzyme-linked immunosorbent assay (ELISA) using CCL5 ELISA kit (EK0494, Boster Biological Technology, CA, USA) according to the manual instruction.

Techniques: Staining, Expressing, Western Blot, Control, Concentration Assay

Journal: Journal of Oncology

Article Title: Palbociclib Enhances Migration and Invasion of Cancer Cells via Senescence-Associated Secretory Phenotype-Related CCL5 in Non-Small-Cell Lung Cancer

doi: 10.1155/2022/2260625

Figure Lengend Snippet: Blocking SASP-related CCL5 inhibits the migration ability induced by Palbociclib. (a) The effect of antagonists of CCR5 TAK-779 on the migration ability of NSCLC cells treated with 2 μ M Palbociclib. Upper: Images of migrated cell with Palbociclib or TAK-779, bar: 100 μ m. Lower: The results were analyzed using unpaired t -test. (b) Diagram showing how Palbociclib contributes to the migration and invasion of NSCLC via SASP-related CCL5.

Article Snippet: Thus, the secreted CCL5 levels in the media of palbociclib-treated cells and the control cells were assessed by enzyme-linked immunosorbent assay (ELISA) using CCL5 ELISA kit (EK0494, Boster Biological Technology, CA, USA) according to the manual instruction.

Techniques: Blocking Assay, Migration

Journal: Life Metabolism

Article Title: Blockade of Arf1-mediated lipid metabolism in cancers promotes tumor infiltration of cytotoxic T cells via the LPE-PPARγ-NF-κB-CCL5 pathway

doi: 10.1093/lifemeta/load036

Figure Lengend Snippet: The Arf1 inhibitors promote T-cell infiltration by upregulating CCL5 chemokine. (a) Chord diagram of gene correlation in the Arf1-ablated MYC-ON liver tumors ( n = 4 in each group). (b) qRT-PCR for chemokines in CT26 cells treated with DMSO, DU101, or DU102. (c) Serum CCL5 levels in the CT26 allografts with the indicated treatments ( n = 6 in each group). (d) CCL5 mRNA levels in the liver tumors of MYC-ON mice that were treated with DMSO, DU101, or DU102 ( n = 9 in each group). (e) Experimental design for the co-culture system. (f) FACS analysis of the migration of CD3 + T cells in CT26 cells that were treated with DMSO, DU101, or DU102. (g) The mRNA levels of CCL5 in CT26 cells with the indicated knockdowns and treatments. (h) FACS analysis of CD3 + T-cell migration in CT26 cells with the indicated knockdowns and treatments. (i and j) Tumor volumes (i) and tumor weights (j) of CT26 allografts with the indicated knockdowns and treatments ( n = 10 in each group). (k) Percentages of infiltrated CD3 + T cells in CT26 allografts with the indicated knockdowns and treatments ( n = 3 in each group). Data are shown as mean ± SEM. Student’s t test. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. n.s., no significance.

Article Snippet: The level of chemokine CCL5 in cultured medium and mouse serum was detected by Mouse RANTES ELISA Kit (BOSTER, Cat# EK0495) according to the manufacturer’s instructions.

Techniques: Quantitative RT-PCR, Co-Culture Assay, Migration

Journal: Life Metabolism

Article Title: Blockade of Arf1-mediated lipid metabolism in cancers promotes tumor infiltration of cytotoxic T cells via the LPE-PPARγ-NF-κB-CCL5 pathway

doi: 10.1093/lifemeta/load036

Figure Lengend Snippet: The Arf1 inhibitors activate the PPARγ pathway by inducing unsaturated fatty acid (LPE). (a) Heatmap of different lipid fractions in the Arf1-deficient CT26 cells. (b) The mRNA levels of downstream genes of PPARγ were detected in CT26 cells treated with different concentrations of LPE (0, 100, 200, 250, and 300 μmol/L for 24 h) by qRT-PCR. (c and d) The mRNA levels of PPARγ in CT26 cells (c) and MYC-ON liver tumors (d) treated with DMSO or Arf1 inhibitors were detected by qRT-PCR. (e) The CCL5 mRNA levels in CT26 cells treated with different concentrations of LPE were detected by qRT-PCR. (f) The CCL5 mRNA levels in CT26 cells with vesicle or GW9662 treatment were detected by qRT-PCR. (g) The CCL5 levels in cell medium collected from CT26 cells with the indicated treatments were determined by ELISA. (h) The percentages of the infiltrated CD3 + T cells in CT26 cells with the indicated treatments. Data are shown as mean ± SEM. Student’s t test. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. n.s., no significance.

Article Snippet: The level of chemokine CCL5 in cultured medium and mouse serum was detected by Mouse RANTES ELISA Kit (BOSTER, Cat# EK0495) according to the manufacturer’s instructions.

Techniques: Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay

Journal: Life Metabolism

Article Title: Blockade of Arf1-mediated lipid metabolism in cancers promotes tumor infiltration of cytotoxic T cells via the LPE-PPARγ-NF-κB-CCL5 pathway

doi: 10.1093/lifemeta/load036

Figure Lengend Snippet: The Arf1 inhibitors promote T-cell tumor infiltration by activating the NF-κB-CCL5 pathway. (a) The phosphorylated p65 in CT26 cells treated with vesicle, DU101, or DU102 was detected by western blot analysis. (b) The phosphorylated p65 in MYC-ON liver tumors after DMSO, DU101, or DU102 treatment was detected by IHC staining. The right was the statistical analysis of p-p65 + cells per field. (c) The relative CCL5 enrichment in CT26 cells with the indicated treatments was analyzed by ChIP-PCR assay. (d) The CCL5 mRNA levels in CT26 cells with the indicated treatments were detected by qRT-PCR. (e) The CCL5 levels in the cell medium collected from CT26 cells with the indicated treatments were detected by the mouse CCL5 ELISA kit. (f) FACS analysis of the infiltrated CD3 + T cells in CT26 cells with the indicated treatments. (g and h) The proportions of the infiltrated CD3 + T cells (g) and CD3 + CCR5 + T (h) cells in Hepa1-6 cells with the indicated treatments were detected by FACS analysis. Data are shown as mean ± SEM. Student’s t test. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. n.s., no significance.

Article Snippet: The level of chemokine CCL5 in cultured medium and mouse serum was detected by Mouse RANTES ELISA Kit (BOSTER, Cat# EK0495) according to the manufacturer’s instructions.

Techniques: Western Blot, Immunohistochemistry, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay

Journal: Journal for immunotherapy of cancer

Article Title: EBV promotes TCR-T-cell therapy resistance by inducing CD163+M2 macrophage polarization and MMP9 secretion.

doi: 10.1136/jitc-2023-008375

Figure Lengend Snippet: Figure 2 EBV-infected tumors secrete CCL5 to recruit T cells and monocytes. (A, B) Representative images (left) and quantification (right) of CD8+T cells (A) or CD14+monocytes (B) recruited to the lower chamber by EBV-infected or EBV- uninfected tumor supernatants. Magnification: ×400. Mean±SD, n=3, two-tailed t-test. (C, D) Left: Representative images of EBV-uninfected or EBV-infected nasopharyngeal and gastric cancer pathology sections stained with antibodies targeting CCL5 (C) or CXCL10 (D). Magnification: ×200. Right: Measurement of CCL5 (C) and CXCL10 (D) levels in the supernatant of EBV-uninfected or EBV-infected nasopharyngeal and gastric cancer cell lines by ELISA. Mean±SD, n=4, two-tailed t-test. (E, F) Quantification of CD8+T cells (E) or CD14+ monocytes (F) recruited to the lower chamber by 200 nM CCL5, 200 nM CXCL10 or serum-free RPMI 1640. CD8+T cells and CD14+monocytes were pretreated overnight with AGS-EBV or HK1-EBV cell supernatants. Mean±SD, n=3, one-way ANOVA. (G, I) Quantification of CD8+T cells (G) and CD14+monocytes (I) recruited to the lower chamber. CD8+T cells and CD14+monocytes were pretreated overnight with AGS-EBV or HK1-EBV cell supernatant, and anti-CCR5 or CCL5+anti-CCR5 groups were simultaneously treated with 200 nM CCR5 antibody overnight to block CCR5. CD8+T cells or CD14+ monocytes were then recruited with 200 nM CCL5 or serum-free RPMI 1640. Mean±SD, n=3, one-way ANOVA. (H, J) CD8+T cells or CD14+ monocytes were treated with or without CCR5 antibody overnight to block CCR5. The above cells were recruited with EBV-infected or EBV-uninfected tumor supernatants. Quantification of CD8+T cells (H) and CD14+ monocytes (J) recruited to the lower chamber. Mean±SD, n=3, one-way ANOVA. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. ANOVA, analysis of variance; EBV, Epstein-Barr virus.

Article Snippet: We used a CCL5 ELISA kit (Proteintech, KE00093), CXCL10 ELISA kit (Neobioscience, EHC157.96), CSF1 ELISA kit (Neobioscience, EHC028.96), IL10 ELISA kit (Neobioscience, EHC157.96) and MMP9 ELISA kit (Neobioscience, EHC115.96) to measure the levels of CCL5, CXCL10, CSF1, IL10 and MMP9 in the cell supernatant according to the manufacturer’s protocol.

Techniques: Infection, Two Tailed Test, Staining, Enzyme-linked Immunosorbent Assay, Blocking Assay, Virus

Journal: Journal for immunotherapy of cancer

Article Title: EBV promotes TCR-T-cell therapy resistance by inducing CD163+M2 macrophage polarization and MMP9 secretion.

doi: 10.1136/jitc-2023-008375

Figure Lengend Snippet: Figure 3 EBV-infected tumors promote CD163+M2 macrophages polarization. (A) Flow cytometry analysis of the CD163+M2 phenotype in THP-1 or CD14+ monocytes treated with EBV-infected or EBV-uninfected tumor supernatants. Mean±SD, n=4 (HK1), n=3 (AGS), two-tailed t-test. (B) Level of CSF1 in EBV-infected and EBV-uninfected tumor supernatants as measured by ELISA. Mean±SD, n=4, two-tailed t-test. (C) Flow cytometry analysis of the CD163+M2 phenotype of CD14+monocytes treated with 50 ng/mL CSF1, 80 ng/mL IL10, or both together. Mean±SD, n=3, one-way ANOVA. (D) Level of IL10 in EBV- infected and EBV-uninfected tumor supernatants as measured by ELISA. Mean±SD, n=4, two-tailed t-test. (E) Levels of IL10 in supernatants of mononuclear macrophages treated with EBV-infected and EBV-uninfected tumor supernatants for 3 days as measured by ELISA. Mean±SD, n=3, two-tailed t-test. (F) Levels of IL10 in mononuclear macrophage supernatants treated for 3 days with EBV-infected tumor supernatant, EBV-infected tumor supernatant and CSF1R inhibitor, 50 ng/mL CSF1, or no treatment as measured by ELISA. Mean±SD, n=4, two-tailed t-test. (G) Flow cytometry analysis of the CD163+M2 phenotype of CD14+monocytes treated with EBV-infected tumor supernatant (control), EBV-infected tumor supernatant and CSF1R inhibitor, EBV-infected tumor supernatant and anti-IL10R antibody, or EBV-infected tumor supernatant and CSF1R inhibitor and anti- IL10R antibody. Mean±SD, n=3, one-way ANOVA. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. ANOVA, analysis of variance; EBV, Epstein-Barr virus.

Article Snippet: We used a CCL5 ELISA kit (Proteintech, KE00093), CXCL10 ELISA kit (Neobioscience, EHC157.96), CSF1 ELISA kit (Neobioscience, EHC028.96), IL10 ELISA kit (Neobioscience, EHC157.96) and MMP9 ELISA kit (Neobioscience, EHC115.96) to measure the levels of CCL5, CXCL10, CSF1, IL10 and MMP9 in the cell supernatant according to the manufacturer’s protocol.

Techniques: Infection, Flow Cytometry, Two Tailed Test, Enzyme-linked Immunosorbent Assay, Control, Virus

Journal: Journal for immunotherapy of cancer

Article Title: EBV promotes TCR-T-cell therapy resistance by inducing CD163+M2 macrophage polarization and MMP9 secretion.

doi: 10.1136/jitc-2023-008375

Figure Lengend Snippet: Figure 6 MMP9 secreted by EBV-induced CD163+M2 macrophages suppresses TCR-T-cell function in vitro. (A) Levels of MMP9 in the supernatants of mononuclear macrophages treated with different conditions (left) as well as in EBV-infected or EBV-uninfected tumor supernatants (right) measured by ELISA. Mean±SD, n=4, two-tailed t-test. (B) Left: Representative images of EBV-positive or EBV-negative nasopharyngeal and gastric cancer tissues stained with MMP9 antibody. Magnification: ×200. Right: IHC scores of MMP9. Mean±SD, n=5 (GC and NPC+), n=4 (NPC−), two-tailed t-test. (C) Flow cytometry analysis of functional molecules in TCR-T cells after 3 days of coculture with M2 macrophages induced by CSF1+IL10, M2 macrophages and MMP9 inhibitors, untreated monocytes (control), or untreated monocytes and MMP9 inhibitors (without C666-1-A11- LMP2A). Mean±SD, n=4, one-way ANOVA. (D) Images of C666-1-A11-LMP2A cells killed by TCR-T cells at 0 hour and 16 hours which were cocultured with M2 macrophages, M2 macrophages+MMP9 inhibitor, untreated monocytes or untreated monocytes+MMP9 inhibitor for 3 days and then isolated via MACS. Magnification: ×100. (E) Statistical analysis of LDH released from C666-1-A11-LMP2A cells after 16 hours of killing by TCR-T cells with different treatment. Mean±SD, n=5, two-tailed t-test. (F) Apoptosis analysis of C666-1-A11-LMP2A cells killed by TCR-T cells for 16 hours which were cocultured with M2 macrophages, M2 macrophages+MMP9 inhibitor, untreated monocytes or untreated monocytes+MMP9 inhibitor for 3 days and then isolated via MACS Mean±SD, n=4, two-tailed t-test. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. ANOVA, analysis of variance; EBV, Epstein-Barr virus; IHC, immunohistochemistry.

Article Snippet: We used a CCL5 ELISA kit (Proteintech, KE00093), CXCL10 ELISA kit (Neobioscience, EHC157.96), CSF1 ELISA kit (Neobioscience, EHC028.96), IL10 ELISA kit (Neobioscience, EHC157.96) and MMP9 ELISA kit (Neobioscience, EHC115.96) to measure the levels of CCL5, CXCL10, CSF1, IL10 and MMP9 in the cell supernatant according to the manufacturer’s protocol.

Techniques: Cell Function Assay, In Vitro, Infection, Enzyme-linked Immunosorbent Assay, Two Tailed Test, Staining, Flow Cytometry, Functional Assay, Control, Isolation, Virus, Immunohistochemistry