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 Western blot analysis of cystatin C in DMEM- or EX-CELL-conditioned medium from HEK293T cells expressing cystatin C via an anti-cystatin C monoclonal antibody under nonreducing conditions. b Representative flow cytometry histograms showing the binding of cystatin C-Flag in DMEM- or EX-CELL-conditioned medium to various primary immune cell populations from the peripheral blood of healthy donors. FMO (fluorescence minus one) and isotype antibodies served as negative controls. The mean fluorescence intensities (MFIs) are indicated. c Flow cytometry analysis of cystatin C-HA in DMEM or EX-CELL-conditioned medium binding to HEK293T cells expressing LILRBs. d Co-IP assay results showing that the cystatin C oligomers bind to LILRB2 in cotransfected HEK293T cells. Band intensities were quantified relative to those of the input and are presented as the means ± SD. n = 3 biological replicates. e Co-IP assays revealed that cystatin C oligomers bind to LILRB2 on the surface of HEK293T cells expressing LILRB2 following incubation with cystatin C-containing EX-CELL supernatant. f Co-IP assays revealed that cystatin C binds to the ECD of LILRB2 in EX-CELL-conditioned medium (CM) from cotransfected HEK293T cells. g Pull-down assays revealed that cystatin C EX directly binds to the LILRB2 ECD. h Titration curves of cystatin C EX binding to LILRB2 ECD-Fc measured by ELISA. The data are presented as the means ± SD. i Left: Western blot analysis of WT and mutated cystatin C in EX-CELL-conditioned medium from HEK293T cells expressing cystatin C via an anti-cystatin C monoclonal antibody under nonreducing conditions. Right: Flow cytometry analysis of WT and mutated cystatin C-HA in EX-CELL-conditioned medium bound to HEK293T cells expressing LILRB2 or LILRB5. j Top: Schematic diagram showing the design of the LILRB2 domain-swapping mutants. The B2D1D2 and B2D3D4 mutants were generated by replacing the D3D4 and D1D2 domains of LILRB2 with the corresponding domains from LILRB1, respectively. Bottom: Flow cytometry analysis of cystatin C-Flag in EX-CELL-conditioned medium bound to HEK293T cells expressing chimeric LILRB2. See also Supplementary Figs. ,

Article Snippet: Circulating levels of human Cystatin C were quantified via a Quantikine ELISA Kit (R&D Systems, cat. no. DSCTC0) according to the manufacturer’s instructions.

Techniques: Western Blot, Expressing, Flow Cytometry, Binding Assay, Fluorescence, Co-Immunoprecipitation Assay, Incubation, Titration, Enzyme-linked Immunosorbent Assay, Generated

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 Representative flow cytometry histograms showing that cystatin C oligomers enhance the T-cell suppressive activity of monocytes in a CSFE assay. CD14 + monocytes from healthy donors were cocultured with CFSE-stained autologous T cells for 5 days. Cystatin C EX and anti-LILRB2 antibodies were included in the coculture system from day 0. b Percentages of proliferative CD4 + and CD8 + T cells in the monocyte:T coculture system under the indicated treatment conditions. c Representative flow cytometry histograms showing that cystatin C oligomers enhance the T-cell suppressive activity of MDSCs in a CSFE assay. MDSCs from cancer patients were cocultured with CFSE-stained autologous T cells for 5 days. Cystatin C EX and anti-LILRB2 antibodies were included in the coculture system from day 0. d Percentage of proliferative CD4 + and CD8 + T cells in the MDSC:T coculture system under the indicated treatment conditions. e ELISA analysis of IFN-γ secretion from the coculture system under the indicated treatment conditions. n = 3 biological replicates. f , g Enriched MDSCs from cancer patients were cultured for 2 days. Cystatin C EX and anti-LILRB2 antibodies were included from day 0. The percentage of live cells ( f ) and the MFIs of surface CD14 and CD163 ( g ) were measured via flow cytometry. n = 3 biological replicates. The data are presented as the means ± SD. P values were determined via one-way ANOVA with Holm‒Sidak’s multiple comparisons test. See also Supplementary Fig.

Article Snippet: Circulating levels of human Cystatin C were quantified via a Quantikine ELISA Kit (R&D Systems, cat. no. DSCTC0) according to the manufacturer’s instructions.

Techniques: Flow Cytometry, Activity Assay, Staining, Enzyme-linked Immunosorbent Assay, Cell Culture

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: Fifty microliters (20 μg/ml) of commercial cystatin C Sino (Sino Biological, cat. no. 10439-H08H) or purified cystatin C EX were pre-coated onto 96-well plates at 37 °C for 3 h. After two washes with PBS, 1 × 10 5 LILRB reporter cells were seeded in each well.

Techniques: Blocking Assay, Activation Assay, Negative Control, Co-Immunoprecipitation Assay, Incubation, Control, In Vitro, Isolation, Flow Cytometry, Cell Culture, Binding Assay

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 Kaplan–Meier survival analysis of the correlations between CST3 expression and overall survival in patients with esophageal cancer (ESCA), glioblastoma (GBM), kidney clear cell carcinoma (KIRC), lower grade glioma (LGG), liver cancer (LIHC), lung squamous cell carcinoma (LUSC), stomach cancer (STAD), and ocular melanoma (UVM). Patients were stratified by low levels of CST3 (blue) or high levels of CST3 (red) from the TCGA database. Statistical significance was calculated via the log-rank test. b Bioinformatic analysis of the correlation between CST3 and LILRB2 expression across the indicated cancer types was performed via TIMER 2.0. For each tumor type, RNA-seq expression values (TPM, transcripts per million) from TCGA were log2-transformed, and Spearman’s rank correlation coefficients (Rho) were calculated. Statistical significance was assessed by P values provided by TIMER 2.0, with multiple-testing correction across cancer types via the Benjamini–Hochberg method. Correlations were defined as positive when Rho > 0 with P < 0.05 and negative when Rho < 0 with P < 0.05. c SK-MEL-5-vector or SK-MEL-5-hcystatin C melanoma cells were inoculated subcutaneously into humanized mice. Tumor growth curves and tumor weights in each group ( n = 7) are shown. d Humanized mice were inoculated subcutaneously with SK-MEL-5-hcystatin C melanoma cells on day 0 and treated with isotype or anti-LILRB2 monoclonal antibody every 7 days from day 28 to day 70. Tumor growth curves and tumor weights in each group ( n = 7) are shown. The data are presented as the means ± SEM. P values were determined via two-tailed Student’s t test ( c , d ). See also Supplementary Fig.

Article Snippet: For the CST3 -overexpressing cell lines, CST3 cDNA was cloned from commercial plasmids (Sino Biological, cat. no. HG10439-G) with a C-terminal FLAG tag and inserted into the pLVX lentiviral expression vector under the control of the Ef1α promoter.

Techniques: Expressing, RNA Sequencing, Transformation Assay, Plasmid Preparation, Two Tailed Test

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: The biosensors were subsequently dipped into varying concentrations of monomeric cystatin C Sino (Sino Biological, cat. no. 10439-H08H; 156, 313, 625, 1250, 2500, 5000 nM) or oligomeric cystatin C EX (156, 313, 625, 1250, 2500, 5000 nM) proteins.

Techniques: Blocking Assay, Activation Assay, Negative Control, Co-Immunoprecipitation Assay, Incubation, Control, In Vitro, Isolation, Flow Cytometry, Cell Culture, Binding Assay