Figure 5. CD112 binds to CD112R to inhibit T cell response. (A) Human monocyte–derived DCs stimulated with LPS overnight were preincubated with mIgG1 or CD112 mAb (clone TX31) and then stained for CD112R protein binding. DCs stained with control FLAG protein are shown in red. Data shown are representative of three different experiments (n = 3 donors). (B) Human pancreatic cell line PANC198 was stained with isotype control (red) or CD112 mAb (blue) for CD112 expression (left). Cells were preincubated with control mIgG1 or CD112 mAb (clone TX31) before being stained by control (FLAG-Fc; red) or CD112R fusion protein (blue). Data shown are representative of two independent experiments. (C) Purified human T cells were CFSE labeled and stimulated with OKT3 together with plate-coated CD112-Fc or control protein (FLAG-Fc). Control (mouse IgG1) or CD112R mAb was added during cell culture. Cells were gated on CD8+ T cells, and their division was analyzed based on the dilution of CFSE. The CFSE-diluted cells indicated were counted as divided T cells. Data shown are representative of at least three independent experiments (n > 3 donors). (D and E) CFSE-labeled CD4+ T cells were cultured with mytomycin-treated CHO stimulators expressing CD112 or control CHO stimulator cells. Antibodies as indicated were added from the beginning of culture. After 5 d of culture, cell division was analyzed based on the dilution of CFSE (D). (E) IL-2 (day 2) and other cytokines (day 5) in the supernatant were measured by a human T helper cytokine panel. Data are representative of three independent experiments. (F) Purified human T cells were labeled with CFSE and were co-cultured with autologous DCs in the presence of TT. Control (mouse IgG1), CD112R mAb, or TIGIT mAb was included at the beginning of the culture. The proliferation of TT-specific CD4+ T cells was determined by CFSE dilution of the human CD3 and CD4 double-positive cells. Data are representative of three independent experiments (n = 3 donors). (G) In the same culture condition as in F, CD112R-Fc or control protein (FLAG-Fc) was included at the beginning to examine the effect on TT-specific T cell response. n = 5. *, P < 0.05 using two-way ANOVA. All bar graphs in C–F represent the mean ± SD results; *, P < 0.05; **, P < 0.01 (Student’s t test). The numbers in the histograms in C, D, F, and G refer to the percentages of divided T cells.
Figure 5.

CD112 binds to CD112R to inhibit T cell response. (A) Human monocyte–derived DCs stimulated with LPS overnight were preincubated with mIgG1 or CD112 mAb (clone TX31) and then stained for CD112R protein binding. DCs stained with control FLAG protein are shown in red. Data shown are representative of three different experiments (n = 3 donors). (B) Human pancreatic cell line PANC198 was stained with isotype control (red) or CD112 mAb (blue) for CD112 expression (left). Cells were preincubated with control mIgG1 or CD112 mAb (clone TX31) before being stained by control (FLAG-Fc; red) or CD112R fusion protein (blue). Data shown are representative of two independent experiments. (C) Purified human T cells were CFSE labeled and stimulated with OKT3 together with plate-coated CD112-Fc or control protein (FLAG-Fc). Control (mouse IgG1) or CD112R mAb was added during cell culture. Cells were gated on CD8+ T cells, and their division was analyzed based on the dilution of CFSE. The CFSE-diluted cells indicated were counted as divided T cells. Data shown are representative of at least three independent experiments (n > 3 donors). (D and E) CFSE-labeled CD4+ T cells were cultured with mytomycin-treated CHO stimulators expressing CD112 or control CHO stimulator cells. Antibodies as indicated were added from the beginning of culture. After 5 d of culture, cell division was analyzed based on the dilution of CFSE (D). (E) IL-2 (day 2) and other cytokines (day 5) in the supernatant were measured by a human T helper cytokine panel. Data are representative of three independent experiments. (F) Purified human T cells were labeled with CFSE and were co-cultured with autologous DCs in the presence of TT. Control (mouse IgG1), CD112R mAb, or TIGIT mAb was included at the beginning of the culture. The proliferation of TT-specific CD4+ T cells was determined by CFSE dilution of the human CD3 and CD4 double-positive cells. Data are representative of three independent experiments (n = 3 donors). (G) In the same culture condition as in F, CD112R-Fc or control protein (FLAG-Fc) was included at the beginning to examine the effect on TT-specific T cell response. n = 5. *, P < 0.05 using two-way ANOVA. All bar graphs in C–F represent the mean ± SD results; *, P < 0.05; **, P < 0.01 (Student’s t test). The numbers in the histograms in C, D, F, and G refer to the percentages of divided T cells.

This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal