Figure 2. CD112R expression in immune cells and its effect on TCR signal. (A) Human CD112R transcript in human immune cells. RNAs were isolated from DCs, NK cells, and T cells stimulated by OKT3 plus CD28 mAb. The expression of CD112R was detected by PCR. G3PDH was used as a housekeeping gene. (B) HEK293T cells transduced with control or CD112R gene were stained with control (red) or CD112R mAb (clone 2H6; blue). (C) Cell lysate of HEK293T/CD112R transfectant was run in reducing (+DTT) and nonreducing (−) conditions and detected by CD112R mAb (clone 2H6). (D) Flow cytometry analysis of CD112R expression in human peripheral blood from healthy donors (n = 4 donors) stained with indicated cell surface markers. (E) CD112R expression on different NK cell subsets: CD16+ (CD56+CD16+) and CD16− (CD56+CD16−). The expression of CD112R (blue) in these two NK subsets is shown. (F) The CD112R expression on CD4+CD3+ and CD8+CD3+ T cell subsets. Graph (right) shows mean ± SD frequencies of CD112R-expressing cells in each subset. (G) CD8+ T cells were divided into two groups based on the expression of CD112R, and their expression of CD45RA and CCR7 was revealed. (H) Purified CD4+ T cells were left unstimulated (day 0) or activated by anti-CD3/CD28 Dynabeads for different times, and the CD112R expression on T cells was detected by biotinylated CD112R mAb. (I) HEK293T cells were transiently transfected with WT or tyrosine mutants of CD112R. Cells were treated with or without pervanadate before analysis for tyrosine phosphorylation on CD112R. (J) Molt4 cell lysates were immunoprecipitated with CD112R mAb or mouse IgG1 (control) and blotted with different phosphatase mAbs as indicated. The presence of CD112R and tyrosine phosphorylation was demonstrated by immunoblotting with CD112R and phosphorylated tyrosine (P-Tyr) mAbs, respectively. Whole cell lysate serves as a detective control. (K) Jurkat-NFAT-Luc cells transfected with different chimeras as indicated were stimulated with OKT3 in the presence or absence of a mouse CD28 agonistic mAb. Data shows mean ± SD of relative luciferase activity upon 4 h of stimulation. All data shown are representative of at least two independent experiments. IP, immunoprecipitation. F and K were analyzed by Student’s t test; *, P < 0.05; **, P < 0.01.
Figure 2.

CD112R expression in immune cells and its effect on TCR signal. (A) Human CD112R transcript in human immune cells. RNAs were isolated from DCs, NK cells, and T cells stimulated by OKT3 plus CD28 mAb. The expression of CD112R was detected by PCR. G3PDH was used as a housekeeping gene. (B) HEK293T cells transduced with control or CD112R gene were stained with control (red) or CD112R mAb (clone 2H6; blue). (C) Cell lysate of HEK293T/CD112R transfectant was run in reducing (+DTT) and nonreducing (−) conditions and detected by CD112R mAb (clone 2H6). (D) Flow cytometry analysis of CD112R expression in human peripheral blood from healthy donors (n = 4 donors) stained with indicated cell surface markers. (E) CD112R expression on different NK cell subsets: CD16+ (CD56+CD16+) and CD16− (CD56+CD16−). The expression of CD112R (blue) in these two NK subsets is shown. (F) The CD112R expression on CD4+CD3+ and CD8+CD3+ T cell subsets. Graph (right) shows mean ± SD frequencies of CD112R-expressing cells in each subset. (G) CD8+ T cells were divided into two groups based on the expression of CD112R, and their expression of CD45RA and CCR7 was revealed. (H) Purified CD4+ T cells were left unstimulated (day 0) or activated by anti-CD3/CD28 Dynabeads for different times, and the CD112R expression on T cells was detected by biotinylated CD112R mAb. (I) HEK293T cells were transiently transfected with WT or tyrosine mutants of CD112R. Cells were treated with or without pervanadate before analysis for tyrosine phosphorylation on CD112R. (J) Molt4 cell lysates were immunoprecipitated with CD112R mAb or mouse IgG1 (control) and blotted with different phosphatase mAbs as indicated. The presence of CD112R and tyrosine phosphorylation was demonstrated by immunoblotting with CD112R and phosphorylated tyrosine (P-Tyr) mAbs, respectively. Whole cell lysate serves as a detective control. (K) Jurkat-NFAT-Luc cells transfected with different chimeras as indicated were stimulated with OKT3 in the presence or absence of a mouse CD28 agonistic mAb. Data shows mean ± SD of relative luciferase activity upon 4 h of stimulation. All data shown are representative of at least two independent experiments. IP, immunoprecipitation. F and K were analyzed by Student’s t test; *, P < 0.05; **, P < 0.01.

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