Journal: Signal Transduction and Targeted Therapy
Article Title: Oligomeric cystatin C supports the immunosuppressive activity of myeloid cells through interaction with inhibitory receptors
doi: 10.1038/s41392-025-02462-x
Figure Lengend Snippet: a Percentages of LILRB2 reporter cells activated on plates coated with or exposed to soluble commercial cystatin C Sino (20 μg/mL) or cystatin C EX (20 μg/mL) in the presence or absence of an anti-LILRB2 (20 μg/mL) blocking antibody. n = 3 biological replicates. The threshold of activation is defined as twice that of the negative control treatment. b Co-IP analysis of LILRB2-specific phosphotyrosine (P-Tyr) and SHP-1/2 recruitment in HEK293T cells cotransfected with LILRB2-Flag, Lyn, SHP-1, and SHP-2 plasmids, with or without anti-LILRB2 antibody treatment, followed by incubation with cystatin C EX-CELL-conditioned medium. Band intensities were quantified relative to the input and are presented as the means ± SD. n = 3 biological replicates. c Co-IP analysis of LILRB2-specific P-Tyr in stable THP-1-LILRB2 cells after a 10-min incubation on plates coated with cystatin C (20 μg/mL). β-actin served as the internal control. Band intensities were quantified relative to the input and are presented as the means ± SD. n = 3 biological replicates. d M1 macrophages were differentiated in vitro from CD14 + monocytes isolated from fresh human PBMCs. The cells were treated with cystatin C EX and anti-LILRB2 antibodies from day 0, when differentiation started. Top: Cell morphology on day 6, Scale bar = 100 µm. Bottom: MFIs of CD86, CD14, and CD163 were measured via flow cytometry on day 6. n = 3 biological replicates. e Dendritic cells were differentiated in vitro from CD14 + monocytes isolated from fresh human PBMCs. Cystatin C EX and anti-LILRB2 antibodies were included in the differentiation from day 0. The MFIs of CD14, CD40, and CD86 were measured on day 8 via flow cytometry, which was 2 days after stimulation. n = 3 biological replicates. f , g Primary human neutrophils were incubated for 24 h in the presence of an N1 polarization cocktail, including the pan-caspase inhibitor Q-VD-Oph. The cells were treated with cystatin C EX and anti-LILRB2 antibodies beginning on day 0, when polarization started. The percentage of CD62L low cells and the MFIs of CD16, CD182 and CD66b were measured via flow cytometry. Neutrophils that were treated with only Q-VD-Oph served as controls. n = 3 biological replicates. h Primary human neutrophils were cultured in serum-free RPMI 1640 medium under the indicated treatment conditions for 12 h in the presence of the pan-caspase inhibitor Q-VD-Oph. The cells were then incubated with 0.1 mg/mL FITC-conjugated dextran at 37 °C for 45 min to allow endocytosis. The MFI of FITC was measured via flow cytometry. A parallel group incubated at 4 °C served as a control for nonspecific binding and passive uptake. n = 3 biological replicates. i Primary human neutrophils were pretreated with either an isotype control or anti-LILRB2 blocking antibody in serum-free RPMI 1640 medium for 4 h at 37 °C in the presence of the pan-caspase inhibitor Q-VD-Oph. Neutrophil suspensions were added to the upper chambers of transwell inserts, and the indicated serum-free RPMI 1640 medium was added to the lower chambers. The cells were incubated at 37 °C for 17 h. Neutrophils that migrated to the lower chambers were collected and quantified by flow cytometry. n = 3 biological replicates. The data are presented as the means ± SD. P values were determined by one-way ANOVA with Dunnett’s multiple comparisons test ( a ) or Holm‒Sidak’s multiple comparisons test ( d – i ). See also Supplementary Fig.
Article Snippet: Normal human CD14 + monocytes were isolated from the mononuclear cell fraction of normal peripheral blood via the AutoMACS Pro Separation System (Miltenyi Biotec).
Techniques: Blocking Assay, Activation Assay, Negative Control, Co-Immunoprecipitation Assay, Incubation, Control, In Vitro, Isolation, Flow Cytometry, Cell Culture, Binding Assay
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