recombinant ccl16  (R&D Systems)

Journal: International journal of molecular sciences

Article Title: Distinct Plasma Immune Profile in ALS Implicates sTNFR-II in pAMPK/Leptin Homeostasis.

doi: 10.3390/ijms24065065

Figure Lengend Snippet: Figure 3. Plasma levels of leptin, CCL16 and sTNF-RII measured by ELISA. (a) concentration (ng/mL) of leptin measured by ELISA in healthy controls (38) and sALS (51). (b,d) Concentration (ng/mL) of leptin (b), CCL16 (c) and sTNF-RII (d) measured by ELISA in slow (40) and fast (11) progressing ALS patients. (e,f) Concentration (ng/mL) of leptin in sALS and controls sub-grouped by sex (e) and by sex and rate of progression (f) (Data are mean ± SEM * p ≤0.05, ** p < 0.01, **** p < 0.0001, NS, not significant).

Article Snippet: Cells were also treated with different concentrations of recombinant CCL16 (R&D systems, Minneapolis, MN, USA, # 802-HC-025) or recombinant sTNF-RII (MyBioSource, MBS343136, London, UK).

Techniques: Clinical Proteomics, Enzyme-linked Immunosorbent Assay, Concentration Assay

Journal: Frontiers in Immunology

Article Title: Characterizing ligand-receptor interactions and unveiling the pro-tumorigenic role of CCL16-CCR1 axis in the microenvironment of hepatocellular carcinoma

doi: 10.3389/fimmu.2023.1299953

Figure Lengend Snippet: Reagent information used in this study.

Article Snippet: Recombinant flag-CCL16 , Novoprotein , C064.

Techniques: Enzyme-linked Immunosorbent Assay, Magnetic Beads, Transfection, Recombinant

Journal: Frontiers in Immunology

Article Title: Characterizing ligand-receptor interactions and unveiling the pro-tumorigenic role of CCL16-CCR1 axis in the microenvironment of hepatocellular carcinoma

doi: 10.3389/fimmu.2023.1299953

Figure Lengend Snippet: Reagent information used in this study.

Article Snippet: Recombinant flag-CCL16 , Novoprotein , C064.

Techniques: Enzyme-linked Immunosorbent Assay, Magnetic Beads, Transfection, Recombinant

Journal: Frontiers in Immunology

Article Title: Characterizing ligand-receptor interactions and unveiling the pro-tumorigenic role of CCL16-CCR1 axis in the microenvironment of hepatocellular carcinoma

doi: 10.3389/fimmu.2023.1299953

Figure Lengend Snippet: Primer sequences for qPCR.

Article Snippet: Recombinant flag-CCL16 , Novoprotein , C064.

Techniques:

Journal: Frontiers in Immunology

Article Title: Characterizing ligand-receptor interactions and unveiling the pro-tumorigenic role of CCL16-CCR1 axis in the microenvironment of hepatocellular carcinoma

doi: 10.3389/fimmu.2023.1299953

Figure Lengend Snippet: Tumor cell-derived CCL16 mediates the recruitment and M2 polarization of macrophages in the liver cancer microenvironment. (A) Expression of CCL16 and CCR1 in different cell types at the single-cell level. (B) Expression levels of CCL16 in different liver cancer cell lines from the CCLE database. (C) mRNA expression of CCL16 in different liver cancer cell lines detected by qPCR. (D) Validation of CCL16 knockdown in HEPG2 cell line. (E) Validation of CCL16 overexpression in SNU761 cell line. (F) Transwell assay to evaluate the recruitment of THP1 cells by control and CCL16 knockdown HEPG2 cells, and ELISA assay to measure CCL16 concentration in the culture supernatant of HEPG2 cells. (G) Transwell assay to evaluate the recruitment of THP1 cells by control and CCL16 overexpressing SNU761 cells, and ELISA assay to measure CCL16 concentration in the culture supernatant of SNU761 cells. (H) Schematic diagram illustrating the cell co-culture system and the macrophage migration assay. (I) Flow cytometry analysis to detect the proportion of M2-polarized cells after co-culture of THP1 cells with CCL16 knockdown or overexpressing tumor cells. Transwell Scale Bar = 100μm. Three independent replicates were conducted. Statistical data are presented as mean ± SD, and each data point represents an independent measurement. Unpaired Student’s t-test. ns, not significant; **: P < 0.01; ***: P < 0.001; ****: P < 0.0001.

Article Snippet: Recombinant flag-CCL16 , Novoprotein , C064.

Techniques: Derivative Assay, Expressing, Over Expression, Transwell Assay, Enzyme-linked Immunosorbent Assay, Concentration Assay, Co-Culture Assay, Migration, Flow Cytometry

Journal: Frontiers in Immunology

Article Title: Characterizing ligand-receptor interactions and unveiling the pro-tumorigenic role of CCL16-CCR1 axis in the microenvironment of hepatocellular carcinoma

doi: 10.3389/fimmu.2023.1299953

Figure Lengend Snippet: The recruitment of tumor-associated macrophages mediated by CCL16 depends on the macrophage receptor CCR1. (A) Immunoprecipitation assay to detect the interaction between Flag-CCL16 and CCR1, CCR2, CCR5, CCR8 in THP1 cell culture medium. (B) Immunofluorescence assay to detect the co-localization of Flag-CCL16 and CCR1 in THP1 cells after the addition of Flag-CCL16. Scale bar = 20 μm. (C) qPCR validation of CCR1 knockdown in THP1 cells. (D) Transwell assay to evaluate the cell migration ability of CCR1 knockdown THP1 cells co-cultured with HEPG2 cells. (E) Recruitment of THP1 cells by control or 5μM BX471-treated CCL16 overexpressing SNU761 cells after 24 hours. (F) Recruitment of THP1 cells by CCL16 overexpressing SNU761 cells after CCR1 knockdown. Transwell Scale Bar = 100μm. Three independent replicates were conducted. Statistical data are presented as mean ± SD, and each data point represents an independent measurement. Unpaired Student’s t-test or two-way ANOVA was used for statistical analysis. ns, not significant; ***: P < 0.001; ****: P < 0.0001.

Article Snippet: Recombinant flag-CCL16 , Novoprotein , C064.

Techniques: Immunoprecipitation, Cell Culture, Immunofluorescence, Transwell Assay, Migration

Journal: Frontiers in Immunology

Article Title: Characterizing ligand-receptor interactions and unveiling the pro-tumorigenic role of CCL16-CCR1 axis in the microenvironment of hepatocellular carcinoma

doi: 10.3389/fimmu.2023.1299953

Figure Lengend Snippet: Clinical characteristics of patients with high and low expression of CCL16.

Article Snippet: Recombinant flag-CCL16 , Novoprotein , C064.

Techniques: Expressing

Journal: Frontiers in Immunology

Article Title: Characterizing ligand-receptor interactions and unveiling the pro-tumorigenic role of CCL16-CCR1 axis in the microenvironment of hepatocellular carcinoma

doi: 10.3389/fimmu.2023.1299953

Figure Lengend Snippet: High expression of CCL16 is positively correlated with the infiltration of M2 macrophages and the expression of CCR1 in clinical tissues. (A) Immunohistochemistry representative images and Pearson correlation analysis of CCL16 with CCR1, CD68, and CD206 in liver cancer patient samples, as well as the Mean Optical Density (MOD) values. Scale Bar = 100μm. (B) Immunofluorescence detection of CCR1+ macrophage infiltration. Scale Bar = 20μm. Statistical analysis of the difference in CD68+CCR1+ cell numbers between high and low CCL16 expression groups using unpaired Student’s t-test. **: P < 0.01. Pearson correlation analysis of CCL16 expression and CCR1+ macrophage infiltration, r = 0.5743, P < 0.001.

Article Snippet: Recombinant flag-CCL16 , Novoprotein , C064.

Techniques: Expressing, Immunohistochemistry, Immunofluorescence

Journal: Journal of Animal Science and Biotechnology

Article Title: Gene expression of CCL8 and CXCL10 in peripheral blood leukocytes during early pregnancy in cows

doi: 10.1186/s40104-018-0263-z

Figure Lengend Snippet: Effects of interferon-τ (IFNT; 100 ng/mL) and CCL16 (100 ng/mL) on the mRNA expression of ( a ) CCL8 , ( b ) CXCL10 , ( c ) ISG15 , ( d ) MX1 and ( e ) MX2 in cultured monocytes, granulocytes and lymphocytes. Data are shown as percentage of the control value ( n = 5). Different letters indicate significant differences ( P < 0.05)

Article Snippet: Cultured leukocytes were further incubated in this medium with recombinant proteins as follows: bovine IFNT (100 ng/mL: 1.1 × 10 5 units/mg, generated from HEK293 cells as described previously [ ]) or recombinant human CCL16 (100 ng/mL: #TP723266, OriGene Technologies, Inc., Rockville, MD, USA).

Techniques: Expressing, Cell Culture, Control

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: CCR8 + FOXp3 + T reg cells as master drivers of immune regulation

doi: 10.1073/pnas.1621280114

Figure Lengend Snippet: CCL1 selectively potentiates suppressive function in CD4+CD25+CD127low T cells. (A–C) CCL1 potentiates the suppressive function of human CD4+CD25+CD127low T cells. Freshly isolated human Treg cells were activated in the presence of each of the known CCR8 ligands used in suppression assay (SI Methods). Proliferation of Teff cells was detected by incorporation of [3H] thymidine incorporation (results in A are shown as mean of triplicates ± SE), or by CFSE staining of effector T cells (B and C). B shows the results of a representative experiment. C summarizes CFSE results of all 10 healthy donors as a scattered plot (P < 0.001 unpaired Student’s t test). (D) CCL1 directs its function via CCR8. Suppression assays were conducted as described for A above, with or without addition of an anti-CCR8 blocking mAb. Results of one of three independent experiments are shown as mean of triplicates ± SE. Significance was determined by two-tailed unpaired Student’s t test (*P < 0.01).

Article Snippet: Recombinant chemokines (CCL1, CCL8, CCL16, or CCL18) were added to CD4 + CD127 − CD25 + T reg cells (200 ng/mL, R&D Systems).

Techniques: Isolation, Suppression Assay, Staining, Blocking Assay, Two Tailed Test

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: CCR8 + FOXp3 + T reg cells as master drivers of immune regulation

doi: 10.1073/pnas.1621280114

Figure Lengend Snippet: CCL1 induces Ca2+ flux in CHO-K1 cells overexpressing human CCR8. Each of the four known human CCR8 ligands was tested for its ability to induce intracellular Ca2+ flux in CHO-K1 cells overexpressing human CCR8 by fluorometric imaging plate reader (FLIPR) assay. Results of one of three independent experiments are shown as mean of triplicates ± SE. Significance was determined by two-tailed unpaired Student’s t test (*P < 0.001).

Article Snippet: Recombinant chemokines (CCL1, CCL8, CCL16, or CCL18) were added to CD4 + CD127 − CD25 + T reg cells (200 ng/mL, R&D Systems).

Techniques: Imaging, Two Tailed Test

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: CCR8 + FOXp3 + T reg cells as master drivers of immune regulation

doi: 10.1073/pnas.1621280114

Figure Lengend Snippet: CCL1 potentiates human Treg cells by inducing CCR8, FOXp3, CD39, granzyme B, and IL-10 expression. (A) CCL1 enhances the transcription of FOXp3, CCR8, CD39, granzyme B, and IL-10 in CD4+CD25+CD127low T cells. CCL1 was added to cultured CD4+CD25+CD127low T cells as described above, and 36 h later, the relative transcription of various genes was detected. Results are shown as mean of triplicates ± SE. Significance was determined by two-tailed unpaired Student’s t test (*P < 0.001). The results represent one of three different experiments with similar observations. (B) CC1 preferentially induces the expression of CCR8 on CD4+CD25+CD127low T-cells CCL1 was added to cultured CD4+CD25+CD127low and CD4+CD25−CD127low T cells as described above and expression of CCR8 was detected by flow cytometry. A representative plot is shown together with a scatterplot summarizing five different samples of healthy donors. Significance was determined by unpaired Student’s t test (C and D) CCL1 enhances the expression of FOXp3 (C), CD39, granzyme B, and IL-10 (D) in human CD4+CD25+CD127low T cells. CCL1 was added to cultured CD4+CD25+CD127low T cells as described above, and 36 h later the expression of various gene products was determined by flow cytometry. A representative plot is shown together with a scatterplot summarizing five different samples of healthy donors. Significance was determined by unpaired Student’s t test (B) P < 0.0001, (C) P < 0.01, and (D) P < 0.0001. (E) CCL1 does not convert FOXp3− T cells into Treg cells: CCL1 was added to cultured CD4+CD25− (FOXp3−) T cells undergoing anti–CD3-induced activation. TGF-β was used as a positive control for induction of iTreg cells.

Article Snippet: Recombinant chemokines (CCL1, CCL8, CCL16, or CCL18) were added to CD4 + CD127 − CD25 + T reg cells (200 ng/mL, R&D Systems).

Techniques: Expressing, Cell Culture, Two Tailed Test, Flow Cytometry, Activation Assay, Positive Control

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: CCR8 + FOXp3 + T reg cells as master drivers of immune regulation

doi: 10.1073/pnas.1621280114

Figure Lengend Snippet: CCL1 induces murine Treg cells via CCR8 in a STAT3-dependent manner. (A) CCL1 selectively induces the suppressive activity of murine FOXp3+ T cells: The three murine CCR8 ligands CCL1, CCL16, and CCL18 were examined for the induction of enhanced suppression of Teff proliferation by FOXp3+ Treg cells (isolated from FOXp3GFP mice as described above). Results of one of three independent experiments with similar data are presented as mean triplicates ± SE. Significance was determined by two-tailed unpaired Student’s t test (*P < 0.01). (B) The suppressive activity of CCL1 is CCR8 dependent. CCL1 was examined for the induction of enhanced suppression of FOXp3+ Treg cells that were obtained from wild-type or CCR8−/− mice. In this experiment, cells were separated based on CD4+CD25+. Results of one of three independent experiments with similar data are presented as mean triplicates ± SE. Significance was determined by two-tailed unpaired Student’s t test (*P < 0.001). (C) CCL1 enhances the transcription of FOXp3, CCR8, CD39, granzyme B, and IL-10 in CD4+FOXp3+ T cells. CCL1 was added to cultured CD4+FOXp3+ T cells (isolated from FOXp3–GFP reporter mice) as described in legend to Fig. 1, 16 h (Left) and 36 h (Right); later the relative transcription of various genes was detected. Results are shown as mean of triplicates ± SE. Significance was determined by two-tailed unpaired Student’s t test (*P < 0.001). The results represent one of three different experiments with similar observations. (D) A STAT3 inhibitor reverses the induction of IL-10 and granzyme B by CCL1. Cultured CD4+FOXp3+ T cells undergoing anti–CD3/anti–CD28-induced activation were supplemented with CCL1 with or without a STAT3 inhibitor. After 72 h, levels of granzyme B and IL-10 were recorded by ELISA. Results of one of three independent experiments with similar data are presented as mean triplicates ± SE. Significance was determined by two-tailed unpaired Student’s t test (*P < 0.001). (E) STAT3 inhibitor reverses the induction of CD39 by CCL1: CD39 expression by cultured mouse CD4+FOXp3+ T cells was detected by flow cytometry following anti–CD3/anti–CD28-induced activation in the presence of CCL1, with or without addition of a STAT3 inhibitor. A representative plot is shown together with scatterplot summarizing samples of five different experiments. Significance was determined by unpaired Student’s t test (P < 0.001). (F) CCL1 does not convert FOXp3− T cells into FOXp3+. FOXP3+ Treg cells were separated from FOXp3− cells from spleen cells of FOXp3GFP reporter mice and subjected to anti–CD3/anti–CD28-induced activation in the presence of CCL1 and analyzed for FOXp3 expression on CD4+ T cells. CCL1 failed in transforming FOXp3− T cells into FOXp3+.

Article Snippet: Recombinant chemokines (CCL1, CCL8, CCL16, or CCL18) were added to CD4 + CD127 − CD25 + T reg cells (200 ng/mL, R&D Systems).

Techniques: Activity Assay, Isolation, Two Tailed Test, Cell Culture, Activation Assay, Enzyme-linked Immunosorbent Assay, Expressing, Flow Cytometry

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: CCR8 + FOXp3 + T reg cells as master drivers of immune regulation

doi: 10.1073/pnas.1621280114

Figure Lengend Snippet: CCL1 potentiates human Treg cells in a STAT3-dependent manner. CCL1 induces STAT3 phosphorylation. (A) STAT phosphorylation was determined in human CD4+CD25+CD127low T cells by flow cytometry analyses. CCL1 was added (100 µg/mL) 24 h after CD3- and anti–CD28-induced activation. The data shown represent one of three independent experiments. (B) STAT3 inhibitor reverses the induction of CD39 by CCL1 in FOXp3+ Treg cells. Flow cytometry analyses of the expression of CD39 in cultured CD4+CD25+CD127low T cells undergoing anti–CD3- and anti–CD28-induced activation in the presence of CCL1 with or without addition of a STAT3 inhibitor (CP 690550, Santa Cruz Biotechnology, sc-202818, 20 µM). A representative plot is shown together with a scatterplot summarizing five different samples of healthy donors. Significance was determined by unpaired Student’s t test (P < 0.0001). (C and D) STAT3 inhibitor reverses the induction of granzyme B and IL-10 by CCL1. Cultured CD4+CD25+CD127low T cells undergoing anti–CD3/anti–CD28-induced activation were supplemented with CCL1 with or without a STAT3 inhibitor. After 72 h, granzyme B and IL-10 levels were recorded by ELISA. Results of one of three independent experiments with similar data are presented as mean triplicates ± SE. Significance was determined by two-tailed unpaired Student’s t test (*P < 0.001).

Article Snippet: Recombinant chemokines (CCL1, CCL8, CCL16, or CCL18) were added to CD4 + CD127 − CD25 + T reg cells (200 ng/mL, R&D Systems).

Techniques: Flow Cytometry, Activation Assay, Expressing, Cell Culture, Enzyme-linked Immunosorbent Assay, Two Tailed Test

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: CCR8 + FOXp3 + T reg cells as master drivers of immune regulation

doi: 10.1073/pnas.1621280114

Figure Lengend Snippet: An autocrine role for the CCL1–CCR8 axis in potentiating Treg cells at the autoimmune site. (A) The kinetics of CCL1 expression at the CNS with EAE disease course: C57BL/6 mice (FOXp3GFP transgenic) were subjected to active induction of disease. Representative mice (n = 3) were killed at different time points and the relative transcription of CCL1 at the lumbar spinal cord samples was quantitated and normalized to β2M by real-time PCR. (B) At the peak of disease in FOXp3–GFP reporter mice FOXp3+CD4+ and FOXp3−CD4+ populations were separated from the lumbar spinal cord. Each subtype was then analyzed by real-time PCR for the relative transcription of CCL1 (normalized to β2M), showing a 13.8-fold increase (P < 0.001) in its transcription by FOXp3+ Treg cells. The results of one of five experiments is shown (black bars) and a summary of all five experiments is shown at Right (P < 0.001 unpaired Student’s t test). (C) At the peak of disease in FOXp3–GFP reporter mice, FOXp3+CD4+ T cells and microglia cells were separated from the lumbar spinal cord and subjected to PCR analyses of CCL1 normalized by GAPDH. (D) Analyses of the differential expression of CD39, granzyme B, and IL-10 on CCR8+ and CCR8− Treg cells in CD4+ T cells isolated from the lumbar spinal cord of EAE mice at the peak of disease. Representative flow cytometry plot is accompanied by scatterplot of five different experiments (unpaired Student’s t test, P < 0.0001).

Article Snippet: Recombinant chemokines (CCL1, CCL8, CCL16, or CCL18) were added to CD4 + CD127 − CD25 + T reg cells (200 ng/mL, R&D Systems).

Techniques: Expressing, Transgenic Assay, Real-time Polymerase Chain Reaction, Isolation, Flow Cytometry

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: CCR8 + FOXp3 + T reg cells as master drivers of immune regulation

doi: 10.1073/pnas.1621280114

Figure Lengend Snippet: Generation of CCL1–Ig and its in vivo pharmacokinetics. (A) Schematic view of the CCL1–Ig construct. (B) Western blot analysis of CCL1–Ig fusion protein. (C) CCL1–Ig maintains its ability to attract CCR8-expressing cells. Chemotaxis assay using Transwell containing CCL1 and CCL1–Ig showing that CCL1–Ig preserves the chemotactic properties of CCL1. (D) CCL1–Ig phosphorylates ERK1/2 in bw5147 thymoma cells. The 5 × 106 bw5147 cells were incubated in DMEM 5% with 250 ng CCL1–Ig for different durations and tested for the phosphorylation of erk1/2 in Western blot. (E) PK of CCL1–Ig: C57BL/6 mice were injected with mouse CCL1–Ig (i.p. 100 µg). Blood was drawn at the indicated time points. Plasma extracted and the presence of CCL1–Ig was detected by Western blot using anti-His HRP-conjugated antibodies. IgG light chain served as a loading control.

Article Snippet: Recombinant chemokines (CCL1, CCL8, CCL16, or CCL18) were added to CD4 + CD127 − CD25 + T reg cells (200 ng/mL, R&D Systems).

Techniques: In Vivo, Construct, Western Blot, Expressing, Chemotaxis Assay, Incubation, Injection

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: CCR8 + FOXp3 + T reg cells as master drivers of immune regulation

doi: 10.1073/pnas.1621280114

Figure Lengend Snippet: Pharmacodynamics of CCL1–Ig. At the onset of MOG35–55 induced EAE in FOXp3–GFP C57BL/6 mice that were administered at the onset of disease with CCL1–Ig (i.p. 100 µg/mouse) or control IgG. Spleen and lumbar spinal cord CD4+ T cells were subjected to FACS analysis for CD39 and FOXp3 at different time points. Results of five mice per group are shown.

Article Snippet: Recombinant chemokines (CCL1, CCL8, CCL16, or CCL18) were added to CD4 + CD127 − CD25 + T reg cells (200 ng/mL, R&D Systems).

Techniques:

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: CCR8 + FOXp3 + T reg cells as master drivers of immune regulation

doi: 10.1073/pnas.1621280114

Figure Lengend Snippet: CCL1–Ig suppress ongoing EAE. (A) C57BL/6 female mice were injected with MOGp35-55 to induce active EAE and at the onset of disease (day 12) were separated into groups with comparable disease scores (n = 9 mice per group, from which three were killed at the peak of disease). On days 13, 15, 17, and 19 after the induction of disease mice were injected (i.p.) with either PBS, 300 µg per mouse of mCCL1–Ig or IgG isotype control. An observer blind to the experimental protocol monitored the development and progression of disease. The results (n = 9 mice per each group until day 17 and n = 6 from day 17 onward) are shown as the mean maximal score ± SE. The results show one of three independent experiments with similar data. One-way ANOVA for paired data was used to determine the significance of the time–response curves (*P < 0.01). The arrows indicate the days of mCCL1–Ig or IgG administration. (B) Histopathological evaluation: At the peak of disease (day 17), three representative mice per group were killed and lumbar spinal cord was subjected to histological analysis (18 sections per spinal cord) using a score of 0–3 as described in ref. 63 (see also SI Methods). The mean histological score ± SE was calculated for each treatment group. Representative histological sections are shown, and a statistical analysis of all sections is also given. Significance was determined by two-tailed unpaired Student’s’s t test (*P < 0.001).

Article Snippet: Recombinant chemokines (CCL1, CCL8, CCL16, or CCL18) were added to CD4 + CD127 − CD25 + T reg cells (200 ng/mL, R&D Systems).

Techniques: Injection, Two Tailed Test

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: CCR8 + FOXp3 + T reg cells as master drivers of immune regulation

doi: 10.1073/pnas.1621280114

Figure Lengend Snippet: Therapy with CCL1–Ig increases the relative level of Treg cells expressing high levels of FOXp3, CD39, IL-10, and granzyme B and also their proliferative rate (BrdU uptake). (A) Comparative flow cytometry analyses of T cells isolated from spleen and spinal cords of control and CCL1–Ig-treated mice (day 17). (Left) Staining for FOXp3 vs. CD4. (Right) Gating on CD4+ analysis of CCR8 expression on FOXp3+ CD4+ T cells. In each experiment a representative plot is shown as well as analyses of three independent experiments (unpaired Student’s t test, P < 0.001). (B) BrdU uptake of FOXp3+ T cells. T cells isolated from the spinal cords of control and CCL1–Ig-treated mice by flow cytometry (gated on CD4−). In each experiment a representative plot is shown as well as analyses of three independent experiments (unpaired Student’s t test, P < 0.001). (C) Analyses of the expression of CD39, granzyme B, and IL-10 by T cells isolated from the spinal cords of control and CCL1–Ig-treated mice by flow cytometry (gated on CD4+CCR8+). In each experiment a representative plot is shown as well as analyses of three independent experiments (unpaired Student’s t test, P < 0.001).

Article Snippet: Recombinant chemokines (CCL1, CCL8, CCL16, or CCL18) were added to CD4 + CD127 − CD25 + T reg cells (200 ng/mL, R&D Systems).

Techniques: Expressing, Flow Cytometry, Isolation, Staining

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: CCR8 + FOXp3 + T reg cells as master drivers of immune regulation

doi: 10.1073/pnas.1621280114

Figure Lengend Snippet: The in vivo suppressive activity of CCL1 on Treg cells is CCR8 dependent. C57BL/6 CCR8−/− female mice (n = 54) were subjected to active induction of MOGp35-55-induced EAE. One day postinduction, EAE-induced mice were separated into three groups, with or without injection with 5 × 105 CD4+CD25+ cells from a wild-type donor. At the onset of symptomatic disease (day 11) the mice of each group were separated into three groups based on disease score (n = 6 mice per group). On days 12, 14, 16, and 18 (arrows) after the induction of disease, mice were injected i.p. with either PBS control (squares), 200 μg per mouse of mCCL1–Ig (circles), or IgG isotype control (rectangles). An observer blind to the experimental protocol monitored the development and progression of disease. The results are shown as the mean maximal score ± SE. (A) CCR8−/− mice, (B) WT mice, (C) CCR8−/− mice reconstituted with Treg cells from CCR8+/+ mice, and (D) CCR8−/− mice reconstituted with Treg cells from CCR8−/− mice. The results show one of three independent experiments with similar data. One-way ANOVA for paired data was used to determine the significance of the time–response curves (*P < 0.01).

Article Snippet: Recombinant chemokines (CCL1, CCL8, CCL16, or CCL18) were added to CD4 + CD127 − CD25 + T reg cells (200 ng/mL, R&D Systems).

Techniques: In Vivo, Activity Assay, Injection

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: CCR8 + FOXp3 + T reg cells as master drivers of immune regulation

doi: 10.1073/pnas.1621280114

Figure Lengend Snippet: CCL1 potentiates Treg cells from IL-10 deficient mice. (A) CCL1 enhance the in vitro suppressive activity of Treg cells from IL-10 KO mice. In vitro suppression assay was established as described in the legend to Fig. 4 A and B. CD25+ Treg cells were isolated from either WT or IL-10 KO mice. We show that CCL1 enhances the suppressive activity of Treg cells from both WT and IL-10 KO mice. (B) CCL1–Ig enhances the in vivo suppressive activity of Treg cells from IL-10 KO mice. CCR8−/− mice were administered with 5 × 105 CD4+CD25+ cells from WT or IL-10−/− donors. After the onset of disease (days 13, 15, 17, and 18) the mice were injected i.p. with 200 µg of mCCL1–Ig and monitored for the development and progression of disease. Results are shown as mean EAE score ± SE.

Article Snippet: Recombinant chemokines (CCL1, CCL8, CCL16, or CCL18) were added to CD4 + CD127 − CD25 + T reg cells (200 ng/mL, R&D Systems).

Techniques: In Vitro, Activity Assay, Suppression Assay, Isolation, In Vivo, Injection

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: CCR8 + FOXp3 + T reg cells as master drivers of immune regulation

doi: 10.1073/pnas.1621280114

Figure Lengend Snippet: CCL1 does not enhance IL-4 expression by CD4+ Th2 T cells. Primary CD4+ T cells (CD44loCD62Lhi) were purified from spleen of BALB/C mice. T cells were stimulated for 2 d by anti-CD3 (10 µg/mL; BioLegend) and anti-CD28 (1 µg/mL; BioLegend) in the presence of IL-2 (2.5 ng/mL), IL-4 (10 ng/mL; PeproTech), and mAb to IFN-γ (5 µg/mL; R4-6A2, BioLegend). Cells were cultured for an additional 3 d without stimulation of the TCR with or without CCL1 (200 ng/mL). Intracellular staining was done for IFN-γ and IL-4 (BioLegend). We show that CCL1 has no effect on IL-4 production by Th2 cells.

Article Snippet: Recombinant chemokines (CCL1, CCL8, CCL16, or CCL18) were added to CD4 + CD127 − CD25 + T reg cells (200 ng/mL, R&D Systems).

Techniques: Expressing, Purification, Cell Culture, Staining

Journal: International Journal of Molecular Sciences

Article Title: Possible Roles of CC- and CXC-Chemokines in Regulating Bovine Endometrial Function during Early Pregnancy

doi: 10.3390/ijms18040742

Figure Lengend Snippet: Comparison of mRNA levels for selected chemokines in the endometrium of pregnant vs. non-pregnant cows as determined by microarray analysis (Fold > 2.0; p < 0.05).

Article Snippet: Cultured endometrial tissues were further incubated in the medium with recombinant proteins as follows: bovine CCL2 (RP0027B, Kingfisher Biotech., Inc. St. Paul, MN, USA), human CCL8 (281-CP, R&D Systems, Inc. Minneapolis, MN, USA), bovine CCL11 (RP0071B, Kingfisher Biotech.), human CCL14 (1578-HC, R&D Systems), human CCL16 (TP723266, OriGene Technologies, Inc., Rockville, MD, USA), bovine CXCL10 (RP0079B, Kingfisher Biotech.), bovine tumor necrosis factor-α (TNF; 2279-BT, R&D Systems), human leukemia inhibitory factor (LIF; TP723270, OriGene Technologies), bovine IFNT (1.1 × 10 5 U/mg, generated from HEK293 cells as described previously; Takahashi et al., 2017 [ ]) or supernatant derived from homogenized fetal trophoblast on day 18 of pregnancy (FMP).

Techniques: Microarray

Journal: International Journal of Molecular Sciences

Article Title: Possible Roles of CC- and CXC-Chemokines in Regulating Bovine Endometrial Function during Early Pregnancy

doi: 10.3390/ijms18040742

Figure Lengend Snippet: Changes in relative amounts of mRNA for ( a ) CCL2, ( b ) CCL8, ( c ) CCL11, ( d ) CCL14, ( e ) CCL16, and ( f ) CXCL10 in the endometrium at days 15 and 18 of non-pregnant cows (NP) and pregnant cows (P). Data are means ± SEM of four cows per stage and are expressed as relative ratios of the mRNAs to SUZ12 polycomb repressive complex 2 subunit (SUZ12). p -Values show significant differences between NP and P.

Article Snippet: Cultured endometrial tissues were further incubated in the medium with recombinant proteins as follows: bovine CCL2 (RP0027B, Kingfisher Biotech., Inc. St. Paul, MN, USA), human CCL8 (281-CP, R&D Systems, Inc. Minneapolis, MN, USA), bovine CCL11 (RP0071B, Kingfisher Biotech.), human CCL14 (1578-HC, R&D Systems), human CCL16 (TP723266, OriGene Technologies, Inc., Rockville, MD, USA), bovine CXCL10 (RP0079B, Kingfisher Biotech.), bovine tumor necrosis factor-α (TNF; 2279-BT, R&D Systems), human leukemia inhibitory factor (LIF; TP723270, OriGene Technologies), bovine IFNT (1.1 × 10 5 U/mg, generated from HEK293 cells as described previously; Takahashi et al., 2017 [ ]) or supernatant derived from homogenized fetal trophoblast on day 18 of pregnancy (FMP).

Techniques:

Journal: International Journal of Molecular Sciences

Article Title: Possible Roles of CC- and CXC-Chemokines in Regulating Bovine Endometrial Function during Early Pregnancy

doi: 10.3390/ijms18040742

Figure Lengend Snippet: Localization of CCR1 (binds to CCL8, CCL14, and CCL16), CCR2 (binds to CCL2, CCL8, and CCL16), CCR3 (binds to CCL11), and CXCR3 (binds to CXCL10) in the bovine endometrium and fetal trophoblast obtained from cows in their 18th day of pregnancy. Intensive immunoreactivity was observed in endometrial epithelial cells, glandular epithelial cells, or fetal trophoblast. No positive immunoreactivity was observed in the negative control (Control). Scale bar = 50 µm.

Article Snippet: Cultured endometrial tissues were further incubated in the medium with recombinant proteins as follows: bovine CCL2 (RP0027B, Kingfisher Biotech., Inc. St. Paul, MN, USA), human CCL8 (281-CP, R&D Systems, Inc. Minneapolis, MN, USA), bovine CCL11 (RP0071B, Kingfisher Biotech.), human CCL14 (1578-HC, R&D Systems), human CCL16 (TP723266, OriGene Technologies, Inc., Rockville, MD, USA), bovine CXCL10 (RP0079B, Kingfisher Biotech.), bovine tumor necrosis factor-α (TNF; 2279-BT, R&D Systems), human leukemia inhibitory factor (LIF; TP723270, OriGene Technologies), bovine IFNT (1.1 × 10 5 U/mg, generated from HEK293 cells as described previously; Takahashi et al., 2017 [ ]) or supernatant derived from homogenized fetal trophoblast on day 18 of pregnancy (FMP).

Techniques: Negative Control

Journal: International Journal of Molecular Sciences

Article Title: Possible Roles of CC- and CXC-Chemokines in Regulating Bovine Endometrial Function during Early Pregnancy

doi: 10.3390/ijms18040742

Figure Lengend Snippet: Effects of the supernatant derived from homogenized fetal trophoblast (FMP; 200 ng/mL) and interferon-τ (IFNT; 100 ng/mL) on the mRNA expression of ( a ) CCL2, ( b ) CCL8, ( c ) CCL11, ( d ) CCL14, ( e ) CCL16, and ( f ) CXCL10 in cultured bovine endometrial tissues. Homogenization buffer was added at the control group. Data are means ± SEM of five cows and are expressed as relative ratios of the mRNAs to SUZ12. p -Values show significant differences between treated group and control group.

Article Snippet: Cultured endometrial tissues were further incubated in the medium with recombinant proteins as follows: bovine CCL2 (RP0027B, Kingfisher Biotech., Inc. St. Paul, MN, USA), human CCL8 (281-CP, R&D Systems, Inc. Minneapolis, MN, USA), bovine CCL11 (RP0071B, Kingfisher Biotech.), human CCL14 (1578-HC, R&D Systems), human CCL16 (TP723266, OriGene Technologies, Inc., Rockville, MD, USA), bovine CXCL10 (RP0079B, Kingfisher Biotech.), bovine tumor necrosis factor-α (TNF; 2279-BT, R&D Systems), human leukemia inhibitory factor (LIF; TP723270, OriGene Technologies), bovine IFNT (1.1 × 10 5 U/mg, generated from HEK293 cells as described previously; Takahashi et al., 2017 [ ]) or supernatant derived from homogenized fetal trophoblast on day 18 of pregnancy (FMP).

Techniques: Derivative Assay, Expressing, Cell Culture, Homogenization

Journal: International Journal of Molecular Sciences

Article Title: Possible Roles of CC- and CXC-Chemokines in Regulating Bovine Endometrial Function during Early Pregnancy

doi: 10.3390/ijms18040742

Figure Lengend Snippet: Effects of CCL2, CCL8, CCL11, CCL14, CCL16, and CXCL10 (50 ng/mL each) on the mRNA expression of ( a ) interferon-stimulated gene 15 (ISG15), ( b ) myxovirus-resistance gene 1 (MX1), ( c ) cyclooxygenase 2 (COX2), ( d ) oxytocin receptor (OTR), and ( e ) estrogen receptor α (ESR1) in cultured bovine endometrial tissues. Data are means ± SEM of five cows and are expressed as relative ratios of the mRNAs to SUZ12. p -Values show significant differences between treated group and control group.

Article Snippet: Cultured endometrial tissues were further incubated in the medium with recombinant proteins as follows: bovine CCL2 (RP0027B, Kingfisher Biotech., Inc. St. Paul, MN, USA), human CCL8 (281-CP, R&D Systems, Inc. Minneapolis, MN, USA), bovine CCL11 (RP0071B, Kingfisher Biotech.), human CCL14 (1578-HC, R&D Systems), human CCL16 (TP723266, OriGene Technologies, Inc., Rockville, MD, USA), bovine CXCL10 (RP0079B, Kingfisher Biotech.), bovine tumor necrosis factor-α (TNF; 2279-BT, R&D Systems), human leukemia inhibitory factor (LIF; TP723270, OriGene Technologies), bovine IFNT (1.1 × 10 5 U/mg, generated from HEK293 cells as described previously; Takahashi et al., 2017 [ ]) or supernatant derived from homogenized fetal trophoblast on day 18 of pregnancy (FMP).

Techniques: Expressing, Cell Culture

Journal: International Journal of Molecular Sciences

Article Title: Possible Roles of CC- and CXC-Chemokines in Regulating Bovine Endometrial Function during Early Pregnancy

doi: 10.3390/ijms18040742

Figure Lengend Snippet: Primers used in real-time PCR.

Article Snippet: Cultured endometrial tissues were further incubated in the medium with recombinant proteins as follows: bovine CCL2 (RP0027B, Kingfisher Biotech., Inc. St. Paul, MN, USA), human CCL8 (281-CP, R&D Systems, Inc. Minneapolis, MN, USA), bovine CCL11 (RP0071B, Kingfisher Biotech.), human CCL14 (1578-HC, R&D Systems), human CCL16 (TP723266, OriGene Technologies, Inc., Rockville, MD, USA), bovine CXCL10 (RP0079B, Kingfisher Biotech.), bovine tumor necrosis factor-α (TNF; 2279-BT, R&D Systems), human leukemia inhibitory factor (LIF; TP723270, OriGene Technologies), bovine IFNT (1.1 × 10 5 U/mg, generated from HEK293 cells as described previously; Takahashi et al., 2017 [ ]) or supernatant derived from homogenized fetal trophoblast on day 18 of pregnancy (FMP).

Techniques: Sequencing

Journal: International Journal of Molecular Sciences

Article Title: Possible Roles of CC- and CXC-Chemokines in Regulating Bovine Endometrial Function during Early Pregnancy

doi: 10.3390/ijms18040742

Figure Lengend Snippet: Hypothetical model for inhibition of luteolysis by IFNT and chemokines. Although this model is not concerned with the effects of steroids or growth factors, IFNT, CCL2, CCL8, CCL16, CXCL10, and LIF may block TNF-stimulated-COX2 expression in bovine endometrial cells, leading to the reduction of TNF-induced PGF2α output from the cells. Furthermore, IFNT and CCL16 may stimulate anti-viral activity by up-regulating ISG15 and MX1 expression at the time of maternal recognition in cows. Red and blue arrows show stimulatory and inhibitory actions of each substance, respectively. IFNT may stimulate both CCL8 and CXCL10 production and inhibit CCL14 production from bovine endometrium. Effects of CCL11 on bovine endometrial function are still unclear, although its receptor (CCR3) is expressed in the endometrial epithelial cells.

Article Snippet: Cultured endometrial tissues were further incubated in the medium with recombinant proteins as follows: bovine CCL2 (RP0027B, Kingfisher Biotech., Inc. St. Paul, MN, USA), human CCL8 (281-CP, R&D Systems, Inc. Minneapolis, MN, USA), bovine CCL11 (RP0071B, Kingfisher Biotech.), human CCL14 (1578-HC, R&D Systems), human CCL16 (TP723266, OriGene Technologies, Inc., Rockville, MD, USA), bovine CXCL10 (RP0079B, Kingfisher Biotech.), bovine tumor necrosis factor-α (TNF; 2279-BT, R&D Systems), human leukemia inhibitory factor (LIF; TP723270, OriGene Technologies), bovine IFNT (1.1 × 10 5 U/mg, generated from HEK293 cells as described previously; Takahashi et al., 2017 [ ]) or supernatant derived from homogenized fetal trophoblast on day 18 of pregnancy (FMP).

Techniques: Inhibition, Blocking Assay, Expressing, Activity Assay