Journal: Cancer Biology & Medicine

Article Title: L1CAM overexpression promotes tumor progression through recruitment of regulatory T cells in esophageal carcinoma

doi: 10.20892/j.issn.2095-3941.2020.0182

Figure Lengend Snippet: L1CAM promotes the recruitment of Tregs by upregulating CCL22. (A) Volcano plot of differentially expressed genes (DEGs), on the basis of the median value of the L1CAM mRNA expression level. (B) The upregulated DEGs were used to perform GO enrichment analysis. (C) The secretion of cytokines and chemokines in the supernatants of shNC and shL1CAM-EC1 cells (up) or scramble and OE-L1CAM-KYSE450 cells (down) cultured for 48 h was assessed with a human multiplex bead-based kit. The expression of CCL22 was detected by qRT-PCR (D) and ELISA (E) between shNC and shL1CAM-EC1 cells, or scramble and OE-L1CAM-KYSE450 cells. (F) Expression of L1CAM and CCL22 mRNAs in the different groups tumor samples derived from Figure 2H . (G) IHC of tumor tissues in different groups derived from Figure 2H . Representative images are shown (200×). (H) The IHC scores of L1CAM and CCL22 in different groups. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: On days 23 and 26 after tumor inoculation, the nude mice were randomly selected for treatment via intraperitoneal injection with the IgG control (IgG2b: R&D Systems) or CCL22 neutralizing antibody (500 ng/mL; R&D, MAB336).

Techniques: Expressing, Cell Culture, Multiplex Assay, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Derivative Assay

Journal: Cancer Biology & Medicine

Article Title: L1CAM overexpression promotes tumor progression through recruitment of regulatory T cells in esophageal carcinoma

doi: 10.20892/j.issn.2095-3941.2020.0182

Figure Lengend Snippet: L1CAM promotes the recruitment of Tregs through CCL22 in vivo . (A) The correlation between FOXP3 and CCL22 in the ESCC TCGA database, n = 94. (B) The purity of CD4 + CD25 + CD127 - Tregs sorted from the peripheral blood of patients with ESCC, according to FACS. (C) The numbers of migrating Tregs obtained from ESCC patient blood samples was calculated for the shNC, shL1CAM, shL1CAM + recombinant protein CCL22 (100 ng/mL), shL1CAM + PBS, scramble, OE-L1CAM, OE-L1CAM + CCL22 antibody (500 ng/mL), and OE-L1CAM + IgG groups. (D) Flow chart of the in vivo experiments. (E) mRNA expression of FOXP3 and CCR4 in the indicated groups. (F) Percentage of CD4 + FOXP3 + Tregs and FOXP3 + CCR4 + Tregs recruited to tumor sites, as analyzed by flow cytometry. (G) Expression of FOXP3 in tumor tissues of different groups, determined by IHC (200×). (H) The expression of p-Akt, Akt, p-NF-κB, NF-κB, and CCL22 was detected by Western blot between shL1CAM and OE-L1CAM cells treated with or without p-Akt inhibitor (RF-04691502) and p-NF-κB inhibitor (QNZ). β-actin expression was used as a loading control. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: On days 23 and 26 after tumor inoculation, the nude mice were randomly selected for treatment via intraperitoneal injection with the IgG control (IgG2b: R&D Systems) or CCL22 neutralizing antibody (500 ng/mL; R&D, MAB336).

Techniques: In Vivo, Recombinant, Expressing, Flow Cytometry, Western Blot

Journal: Cancer Biology & Medicine

Article Title: L1CAM overexpression promotes tumor progression through recruitment of regulatory T cells in esophageal carcinoma

doi: 10.20892/j.issn.2095-3941.2020.0182

Figure Lengend Snippet: High expression of CCL22 predicts poor survival in patients with ESCC. (A–B) qRT-PCR analysis of CCL22 expression in paired fresh tissues from 47 patients with ESCC. GAPDH was used to normalize the data, which were analyzed with the 2 -ΔΔCt method. (C) Kaplan–Meier survival analysis of overall survival on the basis of high ( n = 24) and low ( n = 23) CCL22 expression by qRT-PCR. (D) The percentage of CD4 + FOXP3 + Tregs in tumor tissues obtained from patients with ESCC. Correlations between CD4 + FOXP3 + Tregs and L1CAM (E), CD4 + FOXP3 + Tregs, and CCL22 (F), L1CAM, and CCL22 in the tumor tissues of patients with ESCC (G) were evaluated with linear regression analysis. (H) Schematic diagram of the proposed molecular mechanism: L1CAM promotes the recruitment of Tregs into the tumor site through PI3K/Akt/NF-κB-CCL22-CCR4. Tregs produce TGF-β, which further induces L1CAM upregulation in ESCC tumor cells. ** P < 0.01.

Article Snippet: On days 23 and 26 after tumor inoculation, the nude mice were randomly selected for treatment via intraperitoneal injection with the IgG control (IgG2b: R&D Systems) or CCL22 neutralizing antibody (500 ng/mL; R&D, MAB336).

Techniques: Expressing, Quantitative RT-PCR

Journal: Stem Cell Research & Therapy

Article Title: Regulation of the inflammatory profile of stromal cells in human breast cancer: prominent roles for TNF-α and the NF-κB pathway

doi: 10.1186/s13287-015-0080-7

Figure Lengend Snippet: Impact of prolonged stimulation by Tumor CM on the release of inflammatory chemokines by the resulting CAFs. Human BM-derived MSCs were cultured with Tumor CM from MDA-MB-231 cells (MDA) (A) or MCF-7 cells (B) over a prolonged period of time (~30 days; MSCs + MDA CM or MSCs + MCF-7 CM, respectively). Twenty-four hours after medium exchange to fresh Tumor CM, cell supernatants were collected and the expression of CCL2 (A1, B1), CXCL8 (A2, B2) and CCL5 (A3, B3) was determined in comparison with supernatants of MSCs that were not supplemented with CM (MSCs) and with the original Tumor CM of MDA-MB-231 or MCF-7 cells alone (MDA CM or MCF-7 CM, respectively). Chemokine expression was determined by ELISA, in the linear range of absorbance. (A1), (A2), (B1) Representatives of n = 3 independent experiments that have shown similar results. (A3), (B2), (B3) Ratios between MSCs and MSCs + Tumor CM were not consistent in different experimental repeats. Therefore, in these panels, the findings are presented as mean ± standard deviation of normalized values (MSCs were given the value of 1) obtained in relevant experimental repeats (at least n = 3).

Article Snippet: Supernatants from control and from stimulated cells were then collected and divided into groups as follows: supernatants from untreated control MSCs; supernatants from TNF-α-stimulated MSCs, treated by neutralizing antibodies against CCL2 (2 μg/ml, #MAB679; R&D Systems); and supernatants from TNF-α-stimulated MSCs, treated by isotype-matched nonrelevant antibody (2 μg/ml, #401201; BioLegend).

Techniques: Derivative Assay, Cell Culture, Expressing, Comparison, Enzyme-linked Immunosorbent Assay, Standard Deviation

Journal: Stem Cell Research & Therapy

Article Title: Regulation of the inflammatory profile of stromal cells in human breast cancer: prominent roles for TNF-α and the NF-κB pathway

doi: 10.1186/s13287-015-0080-7

Figure Lengend Snippet: TNF-α induces potent elevation in inflammatory traits in MSCs and Tumor-CM-generated CAFs. Human BM-derived MSCs were cultured with Tumor CM from MDA-MB-231 cells (MDA) (A) or from MCF-7 cells (B) over a prolonged period of time (~30 days). TNF-α (50 ng/ml) or its vehicle (in control cells) was added for the last 24 hours. Expression of CCL2, CXCL8 and CCL5 was then determined in supernatants of the cells. (A) Expression of the chemokines in the four experimental groups included in the study: MSCs grown in culture for ~30 days without any additional stimulus (MSCs); MSCs grown in culture for ~30 days in the presence of Tumor CM derived from MDA-MB-231 cells (MSCs + MDA CM); MSCs grown in culture for ~30 days and stimulated by TNF-α at the last 24 hours of culture (MSCs + TNF-α); and MSCs grown in culture for ~30 days in the presence of Tumor CM derived from MDA-MB-231 cells and stimulated by TNF-α at the last 24 hours of culture (MSCs + MDA CM + TNF-α). Expression of CCL2 (A1), CXCL8 (A2) and CCL5 (A3) was determined by ELISA, in the linear range of absorbance. (B) Experimental design as in (A), but with MCF-7-derived CM. In each panel, the findings are representatives of at least n = 3 experiments that have shown similar results. In comparisons between MSCs and all other groups: * P <0.05, ** P ≤0.01, *** P ≤0.001. NS, not significant. # Differences between the two indicated groups have shown variability in n ≥ 3 independent experiments and overall were not significant.

Article Snippet: Supernatants from control and from stimulated cells were then collected and divided into groups as follows: supernatants from untreated control MSCs; supernatants from TNF-α-stimulated MSCs, treated by neutralizing antibodies against CCL2 (2 μg/ml, #MAB679; R&D Systems); and supernatants from TNF-α-stimulated MSCs, treated by isotype-matched nonrelevant antibody (2 μg/ml, #401201; BioLegend).

Techniques: Generated, Derivative Assay, Cell Culture, Control, Expressing, Enzyme-linked Immunosorbent Assay

Journal: Stem Cell Research & Therapy

Article Title: Regulation of the inflammatory profile of stromal cells in human breast cancer: prominent roles for TNF-α and the NF-κB pathway

doi: 10.1186/s13287-015-0080-7

Figure Lengend Snippet: Basal and cytokine-induced release of inflammatory chemokines by patient CAFs. CAFs were isolated from lung metastasis of a breast cancer patient (CAFs #1) or from a primary tumor of a different breast cancer patient (CAFs #2). (A1, B1) Expression of the pro-malignancy chemokines CCL2, CXCL8 and CCL5 was determined by ELISA, in the linear range of absorbance. (A2, B2) Expression of CCL5 was determined following TNFa and IL-1β stimulation (TNF-α, 50 ng/ml; IL-1β, 500 pg/ml; 48 hours). Control cells were stimulated by vehicle. Expression of the chemokines was determined by ELISA, in the linear range of absorbance. In all panels, the findings are representatives of at least n = 3 independent experiments that have shown similar results.

Article Snippet: Supernatants from control and from stimulated cells were then collected and divided into groups as follows: supernatants from untreated control MSCs; supernatants from TNF-α-stimulated MSCs, treated by neutralizing antibodies against CCL2 (2 μg/ml, #MAB679; R&D Systems); and supernatants from TNF-α-stimulated MSCs, treated by isotype-matched nonrelevant antibody (2 μg/ml, #401201; BioLegend).

Techniques: Isolation, Expressing, Enzyme-linked Immunosorbent Assay, Control

Journal: Stem Cell Research & Therapy

Article Title: Regulation of the inflammatory profile of stromal cells in human breast cancer: prominent roles for TNF-α and the NF-κB pathway

doi: 10.1186/s13287-015-0080-7

Figure Lengend Snippet: TNF-α upregulates the inflammatory profile of MSCs via receptors TNF-RI and TNF-RII. (A) Relative induction of CCL2, CXCL8 and CCL5 in MSCs by TNF-α. Human BM-derived MSCs were stimulated by TNF-α (50 ng/ml) for 24 hours and the expression of the inflammatory chemokines CCL2, CXCL8 and CCL5 in supernatants of MSCs was determined by ELISA, in the linear range of absorbance. Results are from an experiment in which all three chemokines were analyzed in parallel, and the ratios between the three chemokines are representatives of the values obtained in many experimental repeats. (B) Human BM-derived MSCs were exposed to neutralizing antibodies against tumor necrosis factor receptors TNF-RI and TNF-RII or nonrelevant isotype-matched control antibodies (Control Ab) 1 hour prior to stimulation with TNF-α (50 ng/ml), and in the course of 24 hours of stimulation by the cytokine. Control, cells stimulated with vehicle. Expression of CCL2 was determined by ELISA, in the linear range of absorbance. The findings are representatives of at least n = 3 independent experiments that have shown similar results.

Article Snippet: Supernatants from control and from stimulated cells were then collected and divided into groups as follows: supernatants from untreated control MSCs; supernatants from TNF-α-stimulated MSCs, treated by neutralizing antibodies against CCL2 (2 μg/ml, #MAB679; R&D Systems); and supernatants from TNF-α-stimulated MSCs, treated by isotype-matched nonrelevant antibody (2 μg/ml, #401201; BioLegend).

Techniques: Derivative Assay, Expressing, Enzyme-linked Immunosorbent Assay, Control

Journal: Stem Cell Research & Therapy

Article Title: Regulation of the inflammatory profile of stromal cells in human breast cancer: prominent roles for TNF-α and the NF-κB pathway

doi: 10.1186/s13287-015-0080-7

Figure Lengend Snippet: CCL2 is expressed by CAFs localized in vicinity to breast cancer cells in tumors of invasive ductal carcinoma (IDC) patients. CCL2 expression was determined by immunohistochemistry in biopsy sections of patients diagnosed with IDC: (A) IDC #1, (B) IDC #2. CCL2 staining was detected in cancer cells and in adjacent fibroblasts. Some of the CCL2-expressing CAFs are indicated by arrows.

Article Snippet: Supernatants from control and from stimulated cells were then collected and divided into groups as follows: supernatants from untreated control MSCs; supernatants from TNF-α-stimulated MSCs, treated by neutralizing antibodies against CCL2 (2 μg/ml, #MAB679; R&D Systems); and supernatants from TNF-α-stimulated MSCs, treated by isotype-matched nonrelevant antibody (2 μg/ml, #401201; BioLegend).

Techniques: Expressing, Immunohistochemistry, Staining

Journal: Stem Cell Research & Therapy

Article Title: Regulation of the inflammatory profile of stromal cells in human breast cancer: prominent roles for TNF-α and the NF-κB pathway

doi: 10.1186/s13287-015-0080-7

Figure Lengend Snippet: Induction of CCL2 and CLXL8 in TNFα-stimulated MSCs is not mediated via the AP-1 pathway. (A) Human BM-derived MSCs were stimulated by TNF-α (50 ng/ml) for 5 and 10 minutes. Control cells were treated by the vehicle of TNF-α. c-Jun levels and phosphorylation were determined by western blot (WB) analyses. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as loading control. (B) Human BM-derived MSCs were transiently transfected by small interfering RNA (siRNA) to c-Jun or by control siRNA. (B1) c-Jun expression was determined by WB analyses. β-Tubulin was used as loading control. (B2) Following siRNA transfection, the cells were stimulated by TNF-α (25 ng/ml; in this part of the study we used a suboptimal concentration of TNF-α in order to facilitate detection of inhibitory effects) for 24 hours. Expression levels of CCL2 and CXCL8 in the supernatants of the cells were determined by ELISA, in the linear range of absorbance. # siRNA to c-Jun has yielded minor increases or reductions in CCL2 and CXCL8 secretion in different experiments (see Results and discussion), and thus overall there was no significant effect on CCL2 and CXCL8 secretion. In all panels, the findings are representatives of n = 3 independent experiments that have shown similar results.

Article Snippet: Supernatants from control and from stimulated cells were then collected and divided into groups as follows: supernatants from untreated control MSCs; supernatants from TNF-α-stimulated MSCs, treated by neutralizing antibodies against CCL2 (2 μg/ml, #MAB679; R&D Systems); and supernatants from TNF-α-stimulated MSCs, treated by isotype-matched nonrelevant antibody (2 μg/ml, #401201; BioLegend).

Techniques: Derivative Assay, Control, Phospho-proteomics, Western Blot, Transfection, Small Interfering RNA, Expressing, Concentration Assay, Enzyme-linked Immunosorbent Assay

Journal: Stem Cell Research & Therapy

Article Title: Regulation of the inflammatory profile of stromal cells in human breast cancer: prominent roles for TNF-α and the NF-κB pathway

doi: 10.1186/s13287-015-0080-7

Figure Lengend Snippet: NF-κB is essential in mediating TNF-α-induced release of chemokines by MSCs. (A) Human BM-derived MSCs were stimulated by TNF-α (50 ng/ml) for 15 minutes. The levels of IκBα (the negative regulator of the NF-κB pathway) were determined by WB analyses. GAPDH was used as a loading control throughout. (B) CAFs were generated by culturing MSCs with Tumor CM from MDA-MB-231 (MDA) or MCF-7 breast tumor cells over a prolonged period of time (~30 days). TNF-α (50 ng/ml) was added for the last 24 hours to MSCs + Tumor CM cells and IκBα levels were determined by WB analyses. (C) CAF #1 cells were stimulated for 48 hours by TNF-α (50 ng/ml). IκBα levels were determined by WB analyses. (D) Human BM-derived MSCs were stimulated with TNF-α (50 ng/ml) for 10 minutes. p65 phosphorylation was determined by WB analyses. (E) Human BM-derived MSCs were transiently transfected by siRNA to p65 or by control siRNA. (E1) p65 expression was determined by WB analyses. (E2) Following siRNA transfection, the cells were stimulated by TNF-α (25 ng/ml; a suboptimal concentration of TNF-α in order to facilitate detection of inhibitory effects) for 48 hours. Expression of CCL2 and CXCL8 in the supernatants of the cells was determined by ELISA, in the linear range of absorbance. In all panels, the findings are representatives of n = 3 independent experiments that have shown similar results.

Article Snippet: Supernatants from control and from stimulated cells were then collected and divided into groups as follows: supernatants from untreated control MSCs; supernatants from TNF-α-stimulated MSCs, treated by neutralizing antibodies against CCL2 (2 μg/ml, #MAB679; R&D Systems); and supernatants from TNF-α-stimulated MSCs, treated by isotype-matched nonrelevant antibody (2 μg/ml, #401201; BioLegend).

Techniques: Derivative Assay, Control, Generated, Phospho-proteomics, Transfection, Expressing, Concentration Assay, Enzyme-linked Immunosorbent Assay

Journal: Stem Cell Research & Therapy

Article Title: Regulation of the inflammatory profile of stromal cells in human breast cancer: prominent roles for TNF-α and the NF-κB pathway

doi: 10.1186/s13287-015-0080-7

Figure Lengend Snippet: MSCs promote monocyte migration through TNF-α-induced secretion of CCL2. Supernatants (sups) were collected from human BM-derived MSCs that were stimulated by TNF-α (50 ng/ml) or by its vehicle for 24 hours. Thereafter, the sups were incubated with neutralizing antibodies for CCL2 or with nonrelevant isotype-matched control antibody (I.C.) for 30 minutes. The migration of monocytic cells in response to control medium (without chemokine), to recombinant human CCL2 (rhCCL2; 100 ng/ml) or to sups from TNF-α-stimulated or TNF-α nonstimulated MSCs was determined by Boyden chamber migration assays. Cells were counted under a high-power field (HPF). The findings are representatives of n = 3 independent experiments that have shown similar results.

Article Snippet: Supernatants from control and from stimulated cells were then collected and divided into groups as follows: supernatants from untreated control MSCs; supernatants from TNF-α-stimulated MSCs, treated by neutralizing antibodies against CCL2 (2 μg/ml, #MAB679; R&D Systems); and supernatants from TNF-α-stimulated MSCs, treated by isotype-matched nonrelevant antibody (2 μg/ml, #401201; BioLegend).

Techniques: Migration, Derivative Assay, Incubation, Control, Recombinant

Journal: Cancer research

Article Title: CCL22 DIVERTS T REGULATORY CELLS AND CONTROLS THE GROWTH OF MELANOMA

doi: 10.1158/0008-5472.CAN-16-0618

Figure Lengend Snippet: (A) Melanoma cells commonly metastasize to the lung, generating surface lesions detectable as pigmented spots. (B) In human melanoma, Treg are detectable by nuclear FoxP3 expression (red) and membrane CD3 staining (blue) resulting in double stained cells (purple arrows) that can be enumerated as a fraction of total T cells (C) Vaccination using plasmid DNA encoding Ccl22 is applied to ventral murine skin to provide proof of concept for (D) diverting Treg away from the circulation and towards skin.

Article Snippet: Primary antibodies include clones Ta99 to TRP-1 (Covance, Madison, WI), polyclonal rabbit antiserum to mouse and human FoxP3 (ThermoFisher, Rockford, IL) or MF-14 PE rat monoclonal to mouse FoxP3 (Biolegend, San Diego, CA), 145-2C11 bio, FITC or PE to mouse CD3ε (BD Biosciences, Franklin Lakes, NJ) or HIT3A FITC to human CD3 (BD Biosciences), PK138 Alexa Fluor 488 to mouse NK1.1 (BioLegend, San Diego, CA), 8P16-N4-L21 to human CCL1 (Enzo Life Sciences, Farmingdale, NY), 57203 to human CCL22 (R&D Systems, Minneapolis, MN) and MAB4391 to mouse Ccl22 (R&D Systems), TG6/CCR4 to human CCR4 (BioLegend, San Diego, CA) and 191704 to human CCR8 (R&D Systems).

Techniques: Expressing, Membrane, Staining, Plasmid Preparation

Journal: Cancer research

Article Title: CCL22 DIVERTS T REGULATORY CELLS AND CONTROLS THE GROWTH OF MELANOMA

doi: 10.1158/0008-5472.CAN-16-0618

Figure Lengend Snippet: (A) Expression of melanoma marker TRP-1 in a spinal metastasis is inversely related to (B) FoxP3 expression detected just outside a melanoma lesion whereas (C) such FoxP3 expression colocalizes with CCR4 chemokine receptor expression and (D) faint CCL22 expression detected in the same area. (E) CCR8 chemokine receptor expression and TRP-1 expression are likewise readily mutually exclusive whereas (F) CCL1 expression again colocalizes with its receptor CCR8 in areas surrounding the metastasis. In metastatic lesions of the (G) lung, (H) lymph node and (I) brain, similar distributions of CCL22-expressing cells are observed. Under (J) we quantified CCL22 expression in 3 melanoma in situ samples compared to 4 unaffected adult skin samples, detecting 5-fold upregulated chemokine expression whereas (K) an 8-fold upregulation of CCL1 expression was observed.

Article Snippet: Primary antibodies include clones Ta99 to TRP-1 (Covance, Madison, WI), polyclonal rabbit antiserum to mouse and human FoxP3 (ThermoFisher, Rockford, IL) or MF-14 PE rat monoclonal to mouse FoxP3 (Biolegend, San Diego, CA), 145-2C11 bio, FITC or PE to mouse CD3ε (BD Biosciences, Franklin Lakes, NJ) or HIT3A FITC to human CD3 (BD Biosciences), PK138 Alexa Fluor 488 to mouse NK1.1 (BioLegend, San Diego, CA), 8P16-N4-L21 to human CCL1 (Enzo Life Sciences, Farmingdale, NY), 57203 to human CCL22 (R&D Systems, Minneapolis, MN) and MAB4391 to mouse Ccl22 (R&D Systems), TG6/CCR4 to human CCR4 (BioLegend, San Diego, CA) and 191704 to human CCR8 (R&D Systems).

Techniques: Expressing, Marker, In Situ

Journal: Cancer research

Article Title: CCL22 DIVERTS T REGULATORY CELLS AND CONTROLS THE GROWTH OF MELANOMA

doi: 10.1158/0008-5472.CAN-16-0618

Figure Lengend Snippet: To demonstrate that Ccl22 vaccination increases the fraction of skin Treg without converting T cells into Treg, we exposed 3 samples of T cells from a FoxP3 reporter mouse to Ccl22 or to TGF-β overnight. Splenocytes were gated for CD3+ T cells and exposed to CD4 and CD25 immunostaining. (A) FoxP3+CD25+ Treg in a representative untreated sample, (B) Treg abundance is increased in a representative sample of TGFβ treated cells whereas (C) exposure to Ccl22 does not induce a Treg phenotype in vitro. (D) Tregs are quantified, demonstrating a significant increase in response to 2 and 20 ng/mL TGF-β of up to 60%. (E) In a representative transwell migration assay among 3 performed, Treg abundance is increased among splenocytes migrating towards Ccl22, whereas (F) preferential Treg migration is not observed towards Ccl1.

Article Snippet: Primary antibodies include clones Ta99 to TRP-1 (Covance, Madison, WI), polyclonal rabbit antiserum to mouse and human FoxP3 (ThermoFisher, Rockford, IL) or MF-14 PE rat monoclonal to mouse FoxP3 (Biolegend, San Diego, CA), 145-2C11 bio, FITC or PE to mouse CD3ε (BD Biosciences, Franklin Lakes, NJ) or HIT3A FITC to human CD3 (BD Biosciences), PK138 Alexa Fluor 488 to mouse NK1.1 (BioLegend, San Diego, CA), 8P16-N4-L21 to human CCL1 (Enzo Life Sciences, Farmingdale, NY), 57203 to human CCL22 (R&D Systems, Minneapolis, MN) and MAB4391 to mouse Ccl22 (R&D Systems), TG6/CCR4 to human CCR4 (BioLegend, San Diego, CA) and 191704 to human CCR8 (R&D Systems).

Techniques: Immunostaining, In Vitro, Transwell Migration Assay, Migration

Journal: Cancer research

Article Title: CCL22 DIVERTS T REGULATORY CELLS AND CONTROLS THE GROWTH OF MELANOMA

doi: 10.1158/0008-5472.CAN-16-0618

Figure Lengend Snippet: (A) In a representative experiment of 2 performed, 5 mice/group were gene gun vaccinated with Ccl22-encoding or empty vector DNA as shown. (B) At euthanasia, lung tissues were collected to identify tumors (C) Significantly reduced pulmonary tumor counts were observed in Ccl22 vaccinated mice as quantified for 5 mice/group (P<0.05). (D) In therapeutic experiments, mice were tumor challenged 3 days before vaccination applied every 5 days as shown (E) Examples of pulmonary tissues from the Ccl22 (n=7) and empty vector control (n=8) groups. (F) Trimmed means (highest and lowest values removed from each group) were used to calculate that significantly reduced portions of the lung surface were covered by tumor in Ccl22 mice (representative experiment of 2 performed).

Article Snippet: Primary antibodies include clones Ta99 to TRP-1 (Covance, Madison, WI), polyclonal rabbit antiserum to mouse and human FoxP3 (ThermoFisher, Rockford, IL) or MF-14 PE rat monoclonal to mouse FoxP3 (Biolegend, San Diego, CA), 145-2C11 bio, FITC or PE to mouse CD3ε (BD Biosciences, Franklin Lakes, NJ) or HIT3A FITC to human CD3 (BD Biosciences), PK138 Alexa Fluor 488 to mouse NK1.1 (BioLegend, San Diego, CA), 8P16-N4-L21 to human CCL1 (Enzo Life Sciences, Farmingdale, NY), 57203 to human CCL22 (R&D Systems, Minneapolis, MN) and MAB4391 to mouse Ccl22 (R&D Systems), TG6/CCR4 to human CCR4 (BioLegend, San Diego, CA) and 191704 to human CCR8 (R&D Systems).

Techniques: Plasmid Preparation

Journal: Cancer research

Article Title: CCL22 DIVERTS T REGULATORY CELLS AND CONTROLS THE GROWTH OF MELANOMA

doi: 10.1158/0008-5472.CAN-16-0618

Figure Lengend Snippet: Treg in formalin-fixed cryosections by immunofluorescent double staining. CD3 shown in green and FoxP3 staining in red. DAPI counterstaining was used. Shown is a sequence representing (A) FoxP3 stained and (B) CD3 stained lung tissue as well as (C) an overlay, in an untreated mouse, including DAPI counterstaining and (D) the number of Treg was quantified as a ratio to total infiltrating T cells in pulmonary tissue, revealing a marked reduction in Ccl22 vaccinated mice (n=3, *P<0.05), representative experiment of 2 evaluated. The overall abundance of pulmonary (E) T cells and (F) NK cells was not affected. (G) Example TUNEL stainings of lung tissue from empty vector (top) and Ccl22 (bottom) treated mice in a prophylactic experiment. (H) The number of Treg is elevated in the skin of vaccinated mice. (I) Tregs in a sample of Ccl22 vaccinated skin.

Article Snippet: Primary antibodies include clones Ta99 to TRP-1 (Covance, Madison, WI), polyclonal rabbit antiserum to mouse and human FoxP3 (ThermoFisher, Rockford, IL) or MF-14 PE rat monoclonal to mouse FoxP3 (Biolegend, San Diego, CA), 145-2C11 bio, FITC or PE to mouse CD3ε (BD Biosciences, Franklin Lakes, NJ) or HIT3A FITC to human CD3 (BD Biosciences), PK138 Alexa Fluor 488 to mouse NK1.1 (BioLegend, San Diego, CA), 8P16-N4-L21 to human CCL1 (Enzo Life Sciences, Farmingdale, NY), 57203 to human CCL22 (R&D Systems, Minneapolis, MN) and MAB4391 to mouse Ccl22 (R&D Systems), TG6/CCR4 to human CCR4 (BioLegend, San Diego, CA) and 191704 to human CCR8 (R&D Systems).

Techniques: Double Staining, Staining, Sequencing, TUNEL Assay, Plasmid Preparation

Journal: Frontiers in Endocrinology

Article Title: CCL17 and CCL22 chemokines are upregulated in human obesity and play a role in vascular dysfunction

doi: 10.3389/fendo.2023.1154158

Figure Lengend Snippet: Circulating CCL17 and CCL22 chemokine levels in morbidly obese patients and age-matched controls. (A) CCL17 and (B) CCL22 levels were measured in plasma samples from obese patients (n = 60) and controls (n = 20). Scatter dot plots showing median with interquartile range. Comparison between groups were made by Mann Whitney test. Spearman test shows a positive correlation between CCL17 and CCL22 with HOMA-IR Index (C, D) and BMI (E, F) (n = 20 control subjects and n = 60 morbidly obese patients).

Article Snippet: Antigen was unmasked with proteinase K (cat#S3020, Dako, Santa Clara, CA) and blocked with 15% horse serum for 1 h. Samples were incubated with the following primary antibodies overnight at 4°C: mouse anti-human CCL17 (1:50, cat#DY364-05, R&D Systems), mouse anti-human CCL22 (1:50, cat#DY336, R&D Systems), goat anti-human CCR4 (1:100, ab1669; Abcam, Cambridge, UK), rabbit anti-human CD3 (1:100, cat#C7930, Sigma-Aldrich, St. Louis, MO), rabbit polyclonal anti-human CD31 (1:50, cat#ab32457, Abcam) and rat anti-human Mac-3 (1:100, cat#sc19991, Santa Cruz Biotechnology, Dallas, TX).

Techniques: Clinical Proteomics, Comparison, MANN-WHITNEY, Control

Journal: Frontiers in Endocrinology

Article Title: CCL17 and CCL22 chemokines are upregulated in human obesity and play a role in vascular dysfunction

doi: 10.3389/fendo.2023.1154158

Figure Lengend Snippet: Expression of CCL17 and CCL22 is increased in VCAT from morbidly obese patients. Relative quantification of mRNA levels for (A) CCL17 and (B) CCL22. Comparisons between groups were made by Wilcoxon matched-pair signed-rank test. Values are expressed as mean ± SEM (n = 33). (C) CCL17 and (D) CCL22 chemokine release into conditioned media was determined after 48 h of SCAT and VCAT explant culture. Chemokine secretion is expressed as pg/ml in the supernatant. Values are expressed as mean ± SEM (n = 22). Comparison between groups were made by Mann Whitney test. (E) Immunofluorescence representative images showing colocalization of CCL17 with CD3 (lymphocytes), CD31 (endothelial cells) and Mac-3 (macrophages); or CCL22 with CD3, CD31, Mac-3 in VCAT. Immunoreactivity was visualized using Alexa Fluor 594 (CCL17 and CCL22, red ) and Alexa Fluor 488 (CD31, CD3, Mac-3, green ) secondary antibodies. Nuclei were stained with Hoechst ( blue ). Scale bar, 20 μm. Nuclei were stained with Hoechst ( blue ).

Article Snippet: Antigen was unmasked with proteinase K (cat#S3020, Dako, Santa Clara, CA) and blocked with 15% horse serum for 1 h. Samples were incubated with the following primary antibodies overnight at 4°C: mouse anti-human CCL17 (1:50, cat#DY364-05, R&D Systems), mouse anti-human CCL22 (1:50, cat#DY336, R&D Systems), goat anti-human CCR4 (1:100, ab1669; Abcam, Cambridge, UK), rabbit anti-human CD3 (1:100, cat#C7930, Sigma-Aldrich, St. Louis, MO), rabbit polyclonal anti-human CD31 (1:50, cat#ab32457, Abcam) and rat anti-human Mac-3 (1:100, cat#sc19991, Santa Cruz Biotechnology, Dallas, TX).

Techniques: Expressing, Quantitative Proteomics, Comparison, MANN-WHITNEY, Immunofluorescence, Staining

Journal: Journal of Leukocyte Biology

Article Title: CCL22 regulates experimental autoimmune encephalomyelitis by controlling inflammatory macrophage accumulation and effector function

doi: 10.1189/jlb.0810442

Figure Lengend Snippet: Mice were immunized with PLP139–151 in CFA, and CNS tissue was harvested at various time-points corresponding to milestones in disease development. (A) Gene expression analysis was performed by Agilent whole mouse genome array on laser capture microdissected tissues. The data are expressed as fold induction in immunized mice compared with naïve controls. The results indicate high CCL22 expression in CNS lesion and peri-lesion areas. (B) Real-time RT-PCR analysis of CCL22 expression was conducted on spinal cord homogenates corresponding to the indicated times after and associated with disease development using SYBR Green chemistry. The data are expressed as mean (±sd) fold induction from individual mice (n=3) calculated by the Δ comparative threshold method relative to the housekeeping gene G3PDH. (C) CCL22 protein expression in the spinal cord was determined by assessing CNS homogenates from PLP139–151-immunized mice at the indicated time-points after disease induction by ELISA. The data are expressed as mean (±sd) from individual mice (n=3) at each time-point. *P < 0.05 for all experiments where statistical analysis was performed. These results are representative of multiple experimental replicates with similar results.

Article Snippet: Biotinylated goat anti-mouse CCL22 (5 ng/well; R&D Systems) was added, and plates were incubated for 1 h at 37°C.

Techniques: Gene Expression, Expressing, Quantitative RT-PCR, SYBR Green Assay, Enzyme-linked Immunosorbent Assay

Journal: Journal of Leukocyte Biology

Article Title: CCL22 regulates experimental autoimmune encephalomyelitis by controlling inflammatory macrophage accumulation and effector function

doi: 10.1189/jlb.0810442

Figure Lengend Snippet: Mice were immunized with PLP139–151 in CFA at Day 0 and treated with normal goat Ig or polyclonal anti-murine CCL22 at Days –2 and 0 relative to immunization. (A) Anti-CCL22 treatment significantly (*P<0.05) inhibited acute and relapsing clinical disease. The data represent the mean clinical disease score at each specific day postimmunization. (B) In the same experiment, mean cumulative clinical score (±sd) was calculated, and there was a significant (*P<0.05) reduction in the anti-CCL22-treated group. (C) The mean number of relapses (±sd) was calculated for each group, and there was a significant (*P<0.05) reduction in the anti-CCL22-treated mice. The clinical results are representative of three independent experiments. (D) Anti-CCL22 treatment at Day +10, relative to disease induction, did not significantly inhibit acute clinical disease. The data represent the mean clinical disease score at each specific day postimmunization.

Article Snippet: Biotinylated goat anti-mouse CCL22 (5 ng/well; R&D Systems) was added, and plates were incubated for 1 h at 37°C.

Techniques:

Journal: Journal of Leukocyte Biology

Article Title: CCL22 regulates experimental autoimmune encephalomyelitis by controlling inflammatory macrophage accumulation and effector function

doi: 10.1189/jlb.0810442

Figure Lengend Snippet: (A) Representative photomicrograph (original magnification, 100×) of the H&E-stained lumbar spinal cord section from a control Ig-treated mouse. The data indicate extensive mononuclear cell infiltration (arrows). (B) Representative photomicrograph (original magnification, 100×) of a H&E-stained lumbar spinal cord section from an anti-CCL22-treated mouse. The data indicate a lack of mononuclear cell infiltration. (C) Representative flow cytometric analysis of CNS-infiltrating mononuclear cells from a control Ig-treated mouse. The dot plots are derived from a combined forward- and side-scatter and CD45+ gate. The percentages of cells in each quadrant are indicated: 38% lymphocytes, 40% macrophages/DCs, and 11% microglia. (D) Representative flow cytometric analysis of CNS-infiltrating mononuclear cells from an anti-CCL22-treated mouse. The dot plots are derived from a combined forward- and side-scatter and CD45+ gate. The percentages of cells in each quadrant are indicated: 10% lymphocytes, 2% macrophages/DCs, and 83% microglia. (E) Representative flow cytometric analysis of CNS-infiltrating CD4 and CD8 T cells from a control Ig-treated mouse. The dot plots are derived from a combined forward- and side-scatter and CD45+ gate. The percentages of cells in each quadrant are indicated: 40% CD4 T cells and 4% CD8 T cells. (F) Representative flow cytometric analysis of CNS-infiltrating CD4 and CD8 T cells from an anti-CCL22-treated mouse. The dot plots are derived from a combined forward- and side-scatter and CD45+ gate. The percentages of cells in each quadrant are indicated: 8% CD4 T cells and 3% CD8 T cells. (G) Quantification of the total number of CNS-infiltrating CD4 and CD8 T cells and macrophages. Individual mice (n=5) in control- and anti-CCL22-treated groups were assessed for the presence of mononuclear cells in the spinal cord at the time of peak clinical disease in the control-treated group by flow cytometry. The data are expressed as the mean cell number (±sd) and demonstrate significantly (*P<0.05) fewer CD4 and CD8 T cells and macrophages in the anti-CCL22-treated mice. The results are representative of three independent histologic experiments and two independent flow cytometric analyses.

Article Snippet: Biotinylated goat anti-mouse CCL22 (5 ng/well; R&D Systems) was added, and plates were incubated for 1 h at 37°C.

Techniques: Staining, Control, Derivative Assay, Flow Cytometry

Journal: Journal of Leukocyte Biology

Article Title: CCL22 regulates experimental autoimmune encephalomyelitis by controlling inflammatory macrophage accumulation and effector function

doi: 10.1189/jlb.0810442

Figure Lengend Snippet: (A) CNS-infiltrating CD11b+Ly6Chi macrophages from acute EAE express CCR4. Mice were immunized to develop EAE, and at the peak of acute clinical disease, the CNS mononuclear cells were harvested and assessed for CCR4 expression by flow cytometry. The results are shown for a representative animal (out of a total of five), the data are derived from a CD45hiCD11b+ gate, and the results show CCR4 expression as a function of Ly6Chi expression. (B) CNS-infiltrating CD11b+Ly6Chi macrophages from relapsing EAE express CCR4. Mice were immunized to develop EAE, and at the peak of relapsing clinical disease, the CNS mononuclear cells were harvested and assessed for CCR4 expression by flow cytometry. The results are shown for a representative animal (out of the remaining five), the data are derived from a CD45hiCD11b+ gate, and the results show CCR4 expression as a function of Ly6Chi expression. (C) The presence of Ly6ChiCD11b+ macrophages was assessed in the CNS of control Ig-treated mice at peak clinical disease using flow cytometry. The results are displayed from a representative animal (out of a total of three) as the percentage of total CD11b+ macrophages out of the CD45+ gate (R1=37%) and the percentage of only Ly6Chi cells from the CD45+ gate (R2=18%). (D) The presence of Ly6ChiCD11b+ macrophages was assessed in the CNS of anti-CCL22-treated mice at the time controls were showing peak clinical disease using flow cytometry. The results are displayed from a representative animal (out of a total of three) as the percentage of total CD11b+ macrophages out of the CD45+ gate (R3=22%) and the percentage of only Ly6Chi cells from the CD45+ gate (R4=5%). The results are representative of two similar experimental replicates.

Article Snippet: Biotinylated goat anti-mouse CCL22 (5 ng/well; R&D Systems) was added, and plates were incubated for 1 h at 37°C.

Techniques: Expressing, Flow Cytometry, Derivative Assay, Control

Journal: Journal of Leukocyte Biology

Article Title: CCL22 regulates experimental autoimmune encephalomyelitis by controlling inflammatory macrophage accumulation and effector function

doi: 10.1189/jlb.0810442

Figure Lengend Snippet: Mice were immunized with PLP139–151 in CFA and treated with control Ig or anti-CCL22 at Days –2 and 0 relative to antigen priming. At the indicated time-points, three mice/group/time were assessed for proliferative and cytokine responses. (A) Splenic PLP139–151-specific T cell-proliferative responses were determined from control- or anti-CCL22-treated mice at Day 10 postimmunization and at times corresponding to peak clinical disease, remission, and peak-relapsing disease. The data are displayed as mean cpm (±sd), and there are no significant differences at any time-point measured. (B) Splenic PLP139–151-specific IFN-γ T cell responses were determined using ELISPOT analysis of cells from each group of mice at 10 days postimmunization. The data are displayed as mean number of cytokine-secreting cells (±sd) at each dose of peptide (0, 5, 50, and 100 nM). There are no significant differences between the two groups at any peptide dose measured. (C) Splenic, PLP139–151-specific IL-17 T cell responses were determined using ELISPOT analysis of cells from each group of mice at 10 days postimmunization. The data are displayed as mean number of cytokine-secreting cells (±sd) at each dose of peptide (0, 5, 50, and 100 μM). There are no significant differences between the two groups at any peptide dose measured. (D) The presence of Tregs in the LN and spleen was assessed by flow cytometric analysis of Foxp3 expression by CD4+CD25+-gated lymphocytes from control- or anti-CCL22-treated mice at the time when control mice showed clinical disease. The data are expressed as mean percentage of Foxp3+ cells from the CD4+ T cell population (±sd) in the LN and spleen of control (n=3)- and anti-CCL22-treated mice (n=3). There are no significant differences between the two groups in either organ analyzed. These results are representative of at least two independent experimental replicates.

Article Snippet: Biotinylated goat anti-mouse CCL22 (5 ng/well; R&D Systems) was added, and plates were incubated for 1 h at 37°C.

Techniques: Control, Enzyme-linked Immunospot, Expressing

Journal: Journal of Leukocyte Biology

Article Title: CCL22 regulates experimental autoimmune encephalomyelitis by controlling inflammatory macrophage accumulation and effector function

doi: 10.1189/jlb.0810442

Figure Lengend Snippet: (A) Two groups of recipient mice (n=10) received i.v. injections of 5 × 106 PLP139–151-primed and reactivated lymphocyte blasts and were treated with control or polyclonal anti-murine CCL22 at Days 4 and 6 relative to T cell transfer. The data represent the mean clinical disease score at each specific day post-cell transfer and significantly demonstrate (*P<0.05) disease development in the anti-CCL22-treated group. (B) The CNS of control- or anti-CCL22-treated mice was evaluated by flow cytometry for leukocyte infiltration. The results show a decreased percentage of lymphocytes (CD45hiCD11b–) and macrophages (CD45hiCD11b+) in a representative anti-CCL22-treated animal compared with control. (C) The CNS of control- or anti-CCL22-treated mice was evaluated by flow cytometry for inflammatory macrophage infiltration. The results show a decreased percentage of inflammatory macrophages (CD45hiF4/80+Ly6Chi) in a representative anti-CCL22-treated animal compared with control. (D) The number of CNS-infiltrating leukocytes was determined in control- and anti-CCL22-treated mice (n=5, each group). The results show a significantly (*P<0.05) decreased number of total leukocytes, macrophages, and Ly6Chi inflammatory macrophages in the CNS of anti-CCL22-treated mice. (E) CNS-infiltrating Th1 and Th17 cells from mice with EAE do not express CCR4. The results shown are representative of two similar experimental replicates.

Article Snippet: Biotinylated goat anti-mouse CCL22 (5 ng/well; R&D Systems) was added, and plates were incubated for 1 h at 37°C.

Techniques: Control, Flow Cytometry

Journal: Journal of Leukocyte Biology

Article Title: CCL22 regulates experimental autoimmune encephalomyelitis by controlling inflammatory macrophage accumulation and effector function

doi: 10.1189/jlb.0810442

Figure Lengend Snippet: (A) Cytokine expression patterns from CD11b+Ly6Chi macrophages from control- or anti-CCL22-treated mice were determined by ELISA from cells sorted from control- or anti-CCL22-treated mice. The data are displayed as mean cytokine production (±sd) and indicate a significantly (*P<0.05) decreased TNF level and a significantly (P<0.05) increased IL-10 level by CD11b+Ly6Chi macrophages from anti-CCL22- compared with control-treated mice. (B) Stimulation of Ly6Chi macrophages with CCL22 and LPS together results in significantly increased TNF and significantly (*P<0.05) decreased IL-10 compared with macrophages. These results are representative of two similar experimental replicates.

Article Snippet: Biotinylated goat anti-mouse CCL22 (5 ng/well; R&D Systems) was added, and plates were incubated for 1 h at 37°C.

Techniques: Expressing, Control, Enzyme-linked Immunosorbent Assay