Extended Data Fig. 7: SCG2-LILRB4 axis strengthens MDSC suppression.
From: Secretogranin 2 binds LILRB4 resulting in immunosuppression
a, T cell proliferation assay in BM-MDSC-T cell co-culture assay. CFSE-labeled CD3+ T cells were co-cultured with LILRB4+ MDSCs at indicated MDSC-to-T cell ratios. T cell proliferation was analyzed by flow cytometry (n = 3 biological replicates). b, ELISA quantification of IFN-γ in culture supernatants from co-cultures described in Fig. 5a (n = 3 biological replicates). c, Nitric oxide (NO) concentrations in culture supernatants from co-cultures described in Fig. 5a, using Griess reagent assay (n = 3 biological replicates). d, ROS production in BM-MDSCs from WT or LILRB4-Tg mice, cultured for 24 h on plates coated with BSA or SCG2 (10 µg/ml). ROS levels were quantified by flow cytometry using DCFH-DA staining (n = 3 biological replicates). e, T cell proliferation of CFSE-labeled CD3+ T cells co-cultured with BM-MDSCs (1:1 ratio) from LILRB4-Tg mice. Co-cultures were stimulated with anti-CD3/CD28 beads in plates coated with BSA or SCG2, and treated with the NOS inhibitor L-NMMA (500 µM, MCE) or vehicle (DMSO). T cell proliferation was analyzed by flow cytometry (n = 3 biological replicates). f, Transwell migration assay of purified human CD14+ monocytes or T cells toward SCG2 (10 µg/ml) or BSA (control). Cells were placed in the upper chamber, and after 16 h incubation, migrated cells in the lower chamber were quantified by flow cytometry (n = 3 biological replicates). g, Flow cytometry analysis of CD80, CD86 and CD163 expression in BMDMs from WT and LILRB4-Tg mice. Cells were cultured for 24 h with LPS (100 ng/ml) in 96-well plates coated with BSA or SCG2, and treated with the anti-LILRB4 or IgG1 antibodies (n = 3 biological replicates). h-i, KEGG pathway (h) and Gene Ontology (GO) (i) enrichment analyses of genes differentially expressed in M-MDSCs isolated from LILRB4-Tg mice compared to LILRB4-Tg SCG2−/- mice. Significantly enriched pathways are indicated. Statistical significance was calculated by hypergeometric test with Benjamini-Hochberg correction for multiple testing. j, Gene set enrichment analysis (GSEA) demonstrating enrichment of JAK-STAT signaling pathway genes in M-MDSCs from LILRB4-Tg mice compared with those from LILRB4-Tg SCG2−/- mice. Data are present as mean ± s.d. p values were determined by one-way ANOVA with Holm-Sidak’s multiple comparisons test (b-g).
