fc tagged human cd28 protein  (Sino Biological)

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

Article Title: Cowpox virus encodes a protein that binds B7.1 and B7.2 and subverts T cell costimulation

doi: 10.1073/pnas.1909414116

Figure Lengend Snippet: M2 differentially blocks recognition of B7.1 and B7.2 by antibodies and the CD28 and CTLA4 receptors. (A, Left) MEFs transduced to express mouse B7.1 or B7.2 (MEF-mB7.1 or MEF-mB7.2) were preincubated with M2 at the indicated concentration before staining with 1 μg/mL anti-mouse B7.1 (16-10A1) or B7.2 (GL1) blocking antibody. (A, Right) Quantification of mean fluorescence intensity (MFI) of specific antibody staining in the presence of M2 relative to no M2 from 2 independent experiments is shown in the bar chart (mean ± SEM). ns, not significant. (B) Cells used in A were incubated with recombinant M2 or C8 (control PIE) and mouse CD28-Fc or CTLA4-Fc at the indicated concentration for 30 min before CD28-Fc/CTLA4-Fc binding was visualized by fluorescence-labeled anti-human IgG. (Upper) Representative flow cytometric plots of 3 independent analyses are shown. (Lower) Bar chart is the quantification of 3 analyses (mean ± SEM) showing MFI of CD28/CTLA4 in the presence of M2 relative to no M2. (C) Same experiment as in B was conducted with MEF-hB7.1 or MEF-hB7.2 and soluble human CD28-Fc or CTLA4-Fc. *P < 0.05, ***P < 0.001, ****P < 0001.

Article Snippet: The recombinant mouse and human CD28, CTLA4, and PD-L1, all fused with Fc of human IgG1 at the C terminus (except human PD-L1, fused with Fc of mouse IgG1) were from Sino Biological, Inc.

Techniques: Concentration Assay, Staining, Blocking Assay, Fluorescence, Incubation, Recombinant, Binding Assay, Labeling

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

Article Title: Cowpox virus encodes a protein that binds B7.1 and B7.2 and subverts T cell costimulation

doi: 10.1073/pnas.1909414116

Figure Lengend Snippet: ΔM2 CPXV elicits stronger primary T cell responses than WT CPXV. C57BL/6-CR mice were intraperitoneally infected with 50,000 platelet-forming units (PFU) of WT or ΔM2 CPXV. Spleens were analyzed 5 d postinfection. (A) B8R (peptide TSYKFESV) tetramer (TM) staining and total numbers (#) of tetramer-positive CD8 T cells. (B) Percentage of IFN-γ–producing CD8 T cells upon ex vivo stimulation with Δ12Δ203 CPXV-infected DC2.4 cells. (C) Percentage of IFN-γ–producing CD4 T cells upon ex vivo stimulation with peptide-pulsed DC2.4 cells. (D) CPXV titers in the spleen determined by DNA copy number. (E) C57BL/6-J WT and CD28−/− mice were intraperitoneally infected with 50,000 PFU of WT or ΔM2 CPXV and analyzed as in C. *P < 0.05, **P < 0.01, ***P < 0.001. ns. not significant.

Article Snippet: The recombinant mouse and human CD28, CTLA4, and PD-L1, all fused with Fc of human IgG1 at the C terminus (except human PD-L1, fused with Fc of mouse IgG1) were from Sino Biological, Inc.

Techniques: Infection, Staining, Ex Vivo