Figure 3. Agonistic anti-DCIR mAb provides immunosuppressive function by increasing SHP2 binding to the ITIM.

(A) Human monocytes were treated with 5 μg/mL antibodies for 30 minutes. Antibody binding was quantitated by the 10 μg/mL PE anti-human IgG secondary antibody. Data are normalized to the isotype group. Representative data from 2 independent studies are shown. (B) Scheme of the immunosuppressive effect induced by the agonistic anti-DCIR mAb. (C) Human monocytes were treated with 5 μg/mL anti-DCIR mAbs or isotype for 30 minutes, followed by immunoprecipitation (IP) using anti-SHP2 antibody. SHP2-DCIR interaction was evaluated by the DCIR level analyzed by WB. SHP2 from IP lysate and GAPDH from whole cell lysate were probed as loading controls. (D and E) Human monocytes were pretreated with 5 μg/mL antibodies for 30 minutes, followed by 50 μg/mL anti-human IC: HSA stimulation for 30 minutes. SYK’s interactions with SHP2 and FcRγ chain were evaluated by immunoprecipitation using anti-SHP2 (D) and anti-FcRγ (E) antibodies. SHP2 and FcRγ chain from IP lysate and Actin from whole cell lysate were probed as loading controls. (A and C–E) Representative data from 2 studies are shown. (F and G) Human monocytes (n = 3) were pretreated with 10 μg/mL antibodies for 30 minutes, followed by 25 μg/mL ZymD stimulation overnight. Induction of TNF-α and IL-6 in supernatant were measured by ELISA and normalized to the isotype group. (H) Human neutrophils were pretreated with 10 μg/mL antibodies for 2 hours, and OCR were detected in real time after GM-CSF/PMA stimulation. AUC of the OCR was shown. Representative data from 3 independent studies are shown. (F–H) Means ± SEM are shown, and statistical significance is determined by 1-way ANOVA test with Dunnett’s correction compared with the isotype condition. *P < 0.05, **P < 0.01.