soluble anti-cd28 abs clone 37.51  (Bio X Cell)

Journal: Journal of leukocyte biology

Article Title: Ablation of RhoA impairs Th17 cell differentiation and alleviates house dust mite-triggered allergic airway inflammation

doi: 10.1002/JLB.3A0119-025RRR

Figure Lengend Snippet: (A and B) Purified CD4+ T cells were differentiated under Th17-polarizing conditions (TGF-β1 + IL-6) for 4 days and restimulated with PMA plus ionomycin for 5 h with BD GolgiPlug in the last 2 h. Cells were collected for IL-17 intracellular staining. Percentages of IL-17+ cells are shown in representative dot plots (A) and summarized in a bar graph (B). (C and D) Supernatants were collected from CD4+ T cells stimulated with or without anti-CD3/CD28 mAbs (C) or differentiated under Th17-polarizing conditions and restimulated with PMA/ionomycin (D)for ELISA assays of IL-17. (E) Total RNA was extracted from the cultured cells for real-time PCR analysis. The mRNA levels of Th17 effector cytokines as indicated are shown (arbitrary unit). The data are normalized to an 18S reference. Results (B-E) are expressed as mean + SD of triplicates, representative of 3 independent experiments. *P < 0.05, **P < 0.01 compared to WT cells

Article Snippet: Naïve T cells were activated with plate-bound anti-CD3 (clone 145-2C11, 10 μg/mL) plus soluble anti-CD28 (clone 37.51, 2 μg/mL) (BD Bioscience, San Jose, CA).

Techniques: Purification, Staining, Enzyme-linked Immunosorbent Assay, Cell Culture, Real-time Polymerase Chain Reaction

Journal: Journal of leukocyte biology

Article Title: Ablation of RhoA impairs Th17 cell differentiation and alleviates house dust mite-triggered allergic airway inflammation

doi: 10.1002/JLB.3A0119-025RRR

Figure Lengend Snippet: (A) Total RNA was extracted from splenic CD4+ T cells differentiated under Th17 conditions and restimulated with PMA/ionomycin for real-time PCR analysis. The mRNA levels of RORγt are shown. The data are normalized to an 18S reference. (B and C) Naïve splenic CD4+ T cells were stimulated with plate-bound anti-CD3 in the presence of free anti-CD28 for 3 days. Cells were washed and restimulated with recombinant mouse IL-6 (25 ng/mL) for the indicated time. Cells were harvested and processed for intracellular FACS staining for pStat3 (pY705) following BD Bioscience’s protocols. Representative histograms (B), the mean fluorescence intensity (MFI) and percentages of pStat3+ cells (C) are shown. Naïve CD4+ T cells were pooled from 5 to 6 mice. Results (means + SD of triplicates) are representative of 2 independent experiments. *p < 0.05, **P < 0.01 versus WT cells

Article Snippet: Naïve T cells were activated with plate-bound anti-CD3 (clone 145-2C11, 10 μg/mL) plus soluble anti-CD28 (clone 37.51, 2 μg/mL) (BD Bioscience, San Jose, CA).

Techniques: Real-time Polymerase Chain Reaction, Recombinant, Staining, Fluorescence

Journal: Journal of leukocyte biology

Article Title: Ablation of RhoA impairs Th17 cell differentiation and alleviates house dust mite-triggered allergic airway inflammation

doi: 10.1002/JLB.3A0119-025RRR

Figure Lengend Snippet: (A) Purified naïve CD4+ T cells (1 × 106/mL) pooled from 8 WT mice were stimulated with plate-bound anti-CD3 plus free anti-CD28 for 2 days without or with Y16 (0~50 μM). IL-17 and IL-21 in the culture supernatants were determined by ELISA. (B-D) Naïve CD4+ T cells were differentiated under Th17 conditions for 4 days and restimulated with PMA plus ionomycin for 5 h, in the presence of vehicle (Veh) or Y16 (30 μM) throughout the culture. Cells were collected for IL-17/IFN-γ intracellular staining. Percentages of IL-17+ or IFN-γ+ CD4+ T cells are shown in representative dot plots (B) and summarized in a bar graph (C). IL-17 and IL-21 in the culture supernatants were determined by ELISA (D). Data are representative of 2 independent experiments. **P < 0.01 compared to vehicle-treated groups

Article Snippet: Naïve T cells were activated with plate-bound anti-CD3 (clone 145-2C11, 10 μg/mL) plus soluble anti-CD28 (clone 37.51, 2 μg/mL) (BD Bioscience, San Jose, CA).

Techniques: Purification, Enzyme-linked Immunosorbent Assay, Staining

Journal: Mucosal immunology

Article Title: Protein Kinase 2 (CK2) Controls CD4 + T-cell Effector Function in the Pathogenesis of Colitis

doi: 10.1038/s41385-020-0258-x

Figure Lengend Snippet: (A) Naïve CD4 + T-cells from CK2α fl/fl or CK2α fl/fl dLck-Cre mice were polarized under Th1 conditions [anti-CD3 (1 μg/ml), anti-CD28 (1 μg/ml), IL-12 (10 ng/ml) and anti-IL-4 (10 μg/ml)] for 3 days. IFN-γ production by CD4 + T-cells was detected by intracellular staining. Representative flow cytometry profiles of IFN-γ production by CD4 + T-cells is shown (n = 5). (B) IFN-γ in the supernatant was measured by ELISA (n=3). (C) Naïve CD4 + T-cells from CK2α fl/fl mice or CK2α fl/fl dLck-Cre mice were activated with anti-CD3 (1 μg/ml) and anti-CD28 (1 μg/ml) for 24 h, stimulated with IL-12 (10 ng/ml) for the indicated time points, then phosphorylated P-STAT4 Y693 in CD4 + T-cells was detected by immunoblotting. (D) Relative ratio of phosphorylated P-STAT4 Y693 to total STAT4 at the indicated time points is shown (n=3). (E) Naïve CD4 + T-cells from CK2α fl/fl or CK2α fl/fl dLck-Cre mice were activated with anti-CD3 (1 μg/ml) and anti-CD28 (1 μg/ml) for 24 h, and expression levels of IL-12Rβ1 and IL-12Rβ2 detected by flow cytometry. Representative line graphs are shown. (F) Quantitation of MFI of IL-12Rβ1 and IL-12Rβ2 expression is shown. n=3 in each group. Bars represent the mean ± SD. * p<0.05, *** p< 0.001, **** p< 0.0001.

Article Snippet: For in vitro activation, naïve CD4 + T-cells were cultured in R10 medium and stimulated with plate-bound anti-CD3 (0.2 μg/ml, 0.5 μg/ml, 1.0 μg/ml) (Clone 145-2C11, BioX Cell, West Lebanon, NH) and soluble anti-CD28 Abs (1.0 μg/ml) (Clone 37.51, BioX Cell, West Lebanon, NH) for 24 and 48 h. For Th1 cell polarization, cells were stimulated with plate-bound anti-CD3 (1.0 μg/ml) and soluble anti-CD28 Abs (1.0 μg/ml) in the presence of IL-12 (10 ng/ml, Biolegend) and anti-IL-4 Ab (10 μg/ml) (Clone 11B11, BioX Cell, West Lebanon, NH) for 72 h. For phospho-STAT4 detection, cells were activated with plate-bound anti-CD3 (1.0 μg/ml) and soluble anti-CD28 (1.0 μg/ml) for 24 h, then stimulated with IL-12 (10 ng/ml) for the indicated time points.

Techniques: Staining, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Western Blot, Expressing, Quantitation Assay

Journal: Mucosal immunology

Article Title: Protein Kinase 2 (CK2) Controls CD4 + T-cell Effector Function in the Pathogenesis of Colitis

doi: 10.1038/s41385-020-0258-x

Figure Lengend Snippet: (A) CFSE labeled naïve CD4 + T-cells were activated with the indicated concentrations of anti-CD3 (0.2 μg/ml, 0.5 μg/ml, 1.0 μg/ml) and 1.0 μg/ml of anti-CD28 Abs for 72 h, and proliferation was assessed by CFSE dilution. Representative line graphs are shown. (B) Proliferation index of CD4 + T-cells is shown (n = 3). (C) 1 x 10 6 CFSE labeled naïve CD4 + T-cells from CK2α fl/fl or CK2α fl/fl dLck-Cre mice were transferred into Rag1 −/− mice by i.v. injection. Mice were sacrificed 5 days later, and proliferation of CD4 + T-cells from the spleen was assessed by CFSE dilution. Representative line graphs are shown. (D) Frequencies of cells undergoing proliferation is shown. Data represent pooled results from three independent experiments. CK2α fl/fl , n=6; CK2α fl/fl dLck-Cre, n=8. Bars represent the mean ± SD. ** p<0.01, *** p<0.001, **** p<0.0001.

Article Snippet: For in vitro activation, naïve CD4 + T-cells were cultured in R10 medium and stimulated with plate-bound anti-CD3 (0.2 μg/ml, 0.5 μg/ml, 1.0 μg/ml) (Clone 145-2C11, BioX Cell, West Lebanon, NH) and soluble anti-CD28 Abs (1.0 μg/ml) (Clone 37.51, BioX Cell, West Lebanon, NH) for 24 and 48 h. For Th1 cell polarization, cells were stimulated with plate-bound anti-CD3 (1.0 μg/ml) and soluble anti-CD28 Abs (1.0 μg/ml) in the presence of IL-12 (10 ng/ml, Biolegend) and anti-IL-4 Ab (10 μg/ml) (Clone 11B11, BioX Cell, West Lebanon, NH) for 72 h. For phospho-STAT4 detection, cells were activated with plate-bound anti-CD3 (1.0 μg/ml) and soluble anti-CD28 (1.0 μg/ml) for 24 h, then stimulated with IL-12 (10 ng/ml) for the indicated time points.

Techniques: Labeling, Injection

Journal: Mucosal immunology

Article Title: Protein Kinase 2 (CK2) Controls CD4 + T-cell Effector Function in the Pathogenesis of Colitis

doi: 10.1038/s41385-020-0258-x

Figure Lengend Snippet: RNA sequencing of CK2α fl/fl CD4 + T-cells (WT) and CK2α fl/fl dLck-Cre CD4 + T-cells (KO) from the colon at 6 weeks after colitis induction was performed (three to five mice were combined per experiment, two experiments in each group). (A) Summary of genes differentially regulated by CK2α using the following cutoffs are shown: p < 0.05, fold change >1.5. (B) GSEA plot shows enrichment of RNA sequencing data compared with KEGG pathway dataset. (C) Heat map shows TCR signaling relative gene expression. (D) Selected gene expression was validated in CD4 + T-cells from colitis Rag1 −/− mice by qRT-PCR. CK2α fl/fl , n=5; CK2α fl/fl dLck-Cre, n=4. (E) Naive CD4 + T-cells from CK2α fl/fl and CK2α fl/fl dLck-Cre mice were activated with 0.5 μg/ml of anti-CD3 and 1.0 μg/ml of anti-CD28 Abs for 24 h, and expression of NFAT2 was detected by flow cytometry. Representative line graphs and quantitation are shown. n=3 in each group. (F) Naïve CD4 + T-cells from CK2α fl/fl and CK2α fl/fl dLck-Cre mice were activated and transduced with caNFAT2 or empty control vector. GFP + transduced cells were sorted and stimulated with 0.1 μg/ml of anti-CD3 and 1.0 μg/ml of anti-CD28 Abs for 48 h, and expression of ICOS, CD40L and BATF was detected by flow cytometry. Representative line graphs are shown. (G) Quantitation of relative MFI of ICOS, CD40L and BATF is shown. n=3 in each group. (H) GFP + cells were sorted and stimulated with 0.1 μg/ml of anti-CD3 and 1.0 μg/ml of anti-CD28 Abs for 48 h, and proliferating cells detected by Ki-67 staining. Representative line graphs and quantitation are shown. (I) Quantitation of absolute number of Ki-67 + CD4 + T-cells is shown. n=3 in each group. Bars represent the mean ± SD. * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001.

Article Snippet: For in vitro activation, naïve CD4 + T-cells were cultured in R10 medium and stimulated with plate-bound anti-CD3 (0.2 μg/ml, 0.5 μg/ml, 1.0 μg/ml) (Clone 145-2C11, BioX Cell, West Lebanon, NH) and soluble anti-CD28 Abs (1.0 μg/ml) (Clone 37.51, BioX Cell, West Lebanon, NH) for 24 and 48 h. For Th1 cell polarization, cells were stimulated with plate-bound anti-CD3 (1.0 μg/ml) and soluble anti-CD28 Abs (1.0 μg/ml) in the presence of IL-12 (10 ng/ml, Biolegend) and anti-IL-4 Ab (10 μg/ml) (Clone 11B11, BioX Cell, West Lebanon, NH) for 72 h. For phospho-STAT4 detection, cells were activated with plate-bound anti-CD3 (1.0 μg/ml) and soluble anti-CD28 (1.0 μg/ml) for 24 h, then stimulated with IL-12 (10 ng/ml) for the indicated time points.

Techniques: RNA Sequencing, Gene Expression, Quantitative RT-PCR, Expressing, Flow Cytometry, Quantitation Assay, Transduction, Control, Plasmid Preparation, Staining

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Conserved region C functions to regulate PD-1 expression and subsequent CD8 T cell memory 1

doi: 10.4049/jimmunol.1601464

Figure Lengend Snippet: A) Representative enrichment by MACS of CD8 T cells from total splenocytes used for ex vivo experiments. B–D) Enriched CD8 T cells were stimulated with CD3/CD28 antibodies. B) mRNA was harvested at 0 and 24 hours, and analyzed by real-time PCR. Values for Pdcd1 and IL2 mRNA are graphed as a percentage of 18s rRNA. C and D) Cells were harvested at 0, 24, 48, and 96 hours after stimulation as above and stained for flow cytometry. Representative plots (left) and MFI (right) at each time point are shown for PD-1 (C) and CD69 (D). Data are graphed as mean plus standard deviation, and represent six independent experiments. E) Naïve CD8 T cells were enriched by MACS and stimulated with the following doses of anti-CD3/CD28 antibodies: 0.04/0.08 μg/ml (1), 0.2/0.4 μg/ml (2), 1/2 μg/ml (3), 2.5/5 μg/ml (4), 5/10 μg/ml (5), and 10/20 μg/ml (6). Cells were harvested at 24 h after stimulation and stained for flow cytometry. Representative flow plots for both WT and CRC− cells are shown on the left, and average plus standard deviation from three independent replicates is graphed to the right. Panel B used a Student’s T test and Panels C, D, and E used a two-way ANOVA to determine statistical significance. * P< 0.05; *** P<0.001

Article Snippet: On the next day, the plate was washed twice with 500μl sterile PBS, and 0.5x10 6 primary CD8 T cells were incubated in each well with 10 μg/ml soluble anti-CD28 antibody (clone 37.51, Fisher Scientific) at 37 °C for the time points indicated.

Techniques: Ex Vivo, Real-time Polymerase Chain Reaction, Staining, Flow Cytometry, Standard Deviation

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Conserved region C functions to regulate PD-1 expression and subsequent CD8 T cell memory 1

doi: 10.4049/jimmunol.1601464

Figure Lengend Snippet: A) Representative enrichment by MACS of CD8 T cells from total splenocytes used for ex vivo experiments. B–D) Enriched CD8 T cells were stimulated with CD3/CD28 antibodies. B) mRNA was harvested at 0 and 24 hours, and analyzed by real-time PCR. Values for Pdcd1 and IL2 mRNA are graphed as a percentage of 18s rRNA. C and D) Cells were harvested at 0, 24, 48, and 96 hours after stimulation as above and stained for flow cytometry. Representative plots (left) and MFI (right) at each time point are shown for PD-1 (C) and CD69 (D). Data are graphed as mean plus standard deviation, and represent six independent experiments. E) Naïve CD8 T cells were enriched by MACS and stimulated with the following doses of anti-CD3/CD28 antibodies: 0.04/0.08 μg/ml (1), 0.2/0.4 μg/ml (2), 1/2 μg/ml (3), 2.5/5 μg/ml (4), 5/10 μg/ml (5), and 10/20 μg/ml (6). Cells were harvested at 24 h after stimulation and stained for flow cytometry. Representative flow plots for both WT and CRC− cells are shown on the left, and average plus standard deviation from three independent replicates is graphed to the right. Panel B used a Student’s T test and Panels C, D, and E used a two-way ANOVA to determine statistical significance. * P< 0.05; *** P<0.001

Article Snippet: On the next day, the plate was washed twice with 500μl sterile PBS, and 0.5x10 6 primary CD8 T cells were incubated in each well with 10 μg/ml soluble anti-CD28 antibody (clone 37.51, Fisher Scientific) at 37 °C for the time points indicated.

Techniques: Ex Vivo, Real-time Polymerase Chain Reaction, Staining, Flow Cytometry, Standard Deviation