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Secretogranin 2 binds LILRB4 resulting in immunosuppression

Nature Immunology volume 26, pages 1567–1580 (2025)Cite this article

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Abstract

Immunosuppressive myeloid cells are important in a variety of physiological and pathological contexts, including tumor development, but how hormones might regulate their activity is unclear. Secretogranins, a family of secretory proteins in endocrine and neuronal cells, are proposed to function as prohormones or hormones, but their specific receptors are unknown. Here we show that secretogranin 2 (SCG2), a granin family member, functionally interacts with leukocyte immunoglobulin-like receptor B4 (LILRB4) on monocytic cells. Tumor-derived SCG2 promotes tumor growth in myeloid-specific LILRB4 transgenic mice in a T cell-dependent manner, whereas SCG2 deficiency in host mice impairs tumor progression and reduces infiltration of immunosuppressive monocytic cells. Blockade of LILRB4 abrogates SCG2-induced signaling, immunosuppression and tumor growth. Mechanistically, this SCG2–LILRB4 interaction triggers SHP recruitment and SHP-independent STAT3 activation. These findings define a function for SCG2 in regulating monocytic immunosuppression and suggest that the SCG2–LILRB4 axis might be a therapeutic target.

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Fig. 1: SCG2 interacts specifically with LILRB4.
Fig. 2: SCG2 induces LILRB4 activation and function in cultured cells.
Fig. 3: SCG2 supports tumor development through LILRB4.
Fig. 4: SCG2 deficiency impairs tumor progression in LILRB4-Tg mice.
Fig. 5: LILRB4 enhances immunosuppressive activity of MDSCs.
Fig. 6: SCG2–LILRB4 interaction strengthens IL-6–STAT3 signaling.
Fig. 7: LILRB4 supports immunosuppressive function of human M-MDSCs.

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Data availability

The following publicly available datasets were accessed for this study. TCGA CESC and TCGA MESO survival data were accessed via the GEPIA2 online tool. Raw RNA-seq data for TCGA CESC and MESO were retrieved from TCGA. RNA-seq and survival data for GSE65858, GSE54236 and GSE1993 were obtained from the Tumor Immune Dysfunction and Exclusion (TIDE) database. scRNA-seq data from multiple human tumors were downloaded from GEO under accession code GSE154763. Bulk RNA-seq data generated in this study have been deposited in the GEO database under accession code GSE285725. All other data supporting the findings of this study are provided within the article and its Supplementary Information files. Additional raw data and unique/stable reagents generated during this study are available from the corresponding author upon reasonable request. Source data are provided with this paper.

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Acknowledgements

This work was supported by the National Cancer Institute (R01CA248736, R01CA263079 and Lung Cancer SPORE Development Research Program; C.C.Z.), the Cancer Prevention and Research Institute of Texas (RP220032, C.C.Z.; RP15150551 and RP190561, Z.A.), the Welch Foundation (AU-0042-20030616, Z.A.; I-1702, X.Z.), Immune-Onc Therapeutics (Sponsored Research Grant 111077, C.C.Z.) and National Institutes of Health (R35GM130289, X.Z.), the CPRIT training grant (RP210041, X.Y.; RP210041, L.C.). This work received support from University of Texas Southwestern Simmons Comprehensive Cancer Center’s Tissue Management Shared Resource and was supported by the National Cancer Institute of the National Institutes of Health (P30CA142543, C.L.).

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Authors and Affiliations

  1. Department of Physiology, University of Texas Southwestern Medical Center, Dallas, TX, USA

    Xing Yang, Ryan Huang, Meng Fang, Yubo He, Jingjing Xie, Xiaoye Liu, Chengcheng Zhang, Qi Lou, Mi Deng, Zade Sadek & Cheng Cheng Zhang

  2. Texas Therapeutics Institute, Brown Foundation Institute of Molecular Medicine, University of Texas Health Science Center, Houston, TX, USA

    Wei Xiong, Ningyan Zhang & Zhiqiang An

  3. Harold C. Simmons Comprehensive Cancer Center, University of Texas Southwestern Medical Center, Dallas, TX, USA

    Cheryl Lewis

  4. Center for Alzheimer’s and Neurodegenerative Diseases, University of Texas Southwestern Medical Center, Dallas, TX, USA

    Ankit Gupta

  5. Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas, TX, USA

    Lianqi Chen & Xuewu Zhang

  6. Quantitative Biomedical Research Center, Peter O’Donnell Jr. School of Public Health, University of Texas Southwestern Medical Center, Dallas, TX, USA

    Lei Guo & Lin Xu

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Contributions

C.C.Z. conceived the research, designed the study and wrote the paper. X.Y. designed the study, developed experimental protocols, performed experiments and wrote the paper. R.H., Y.H. and M.D. developed the experimental protocols. M.F. assisted with the analysis of published scRNA-seq data and paper editing. J.X., X.L., C.Z. and Q.L. performed experiments and assisted in paper editing. L.G. and L.X. assisted with The Cancer Genome Atlas (TCGA) data analysis and paper editing. L.C. and X.Z. assisted with the purification of SCG2 protein for key validation experiments. Z.S. performed experiments. C.L. provided human samples. A.G. performed biolayer interferometry assays. N.Z. and Z.A. directed and W.X. performed antibody engineering, production and evaluation of antibody properties.

Corresponding author

Correspondence to Cheng Cheng Zhang.

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Competing interests

Authors R.H., Y.H., M.D., W.X., N.Z., Z.A. and C.C.Z. are listed as inventors on relevant patent applications that were exclusively licensed to Immune-Onc Therapeutics by the Board of Regents of the University of Texas System. Authors M.D., Z.A., N.Z. and C.C.Z. hold equity in and had Sponsored Research Agreements with Immune-Onc Therapeutics. The University of Texas has a financial interest in Immune-Onc in the form of equity and licensing. The other authors declare no competing interests.

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Nature Immunology thanks Marco Colonna and Antonio Sica for their contribution to the peer review of this work. Peer reviewer reports are available. Primary Handling Editor: Nick Bernard, in collaboration with the Nature Immunology team.

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Extended data

Extended Data Fig. 1 SCG2 binds to LILRB4 specifically.

a, Summary of genome-wide screening performed by Retrogenix and our subsequent validation. b, Representative gating strategy for identifying T cells, B cells, NK cells, monocytes, and neutrophils from human peripheral blood cells analyzed by flow cytometry. c-d, LILRB4 reporter activation by coated or soluble SCG2, ApoE, or mouse SCG2. IgG or anti-LILRB4 antibody was added as indicated. GFP⁺ cells were analyzed by flow cytometry (n = 3 biological replicates). e, Co-IP of Flag-tagged SCG2 and Fc-tagged LILRB4 in HEK293T cells. f-g, Pull-down assays for LILRB4-ECD-Fc and ApoE–His or CNTFR–His in the presence or absence of SCG2-His. h, LILRB4 reporter cell activation induced by immobilized ApoE or SCG2 in combination with ApoE. GFP⁺ cells were analyzed by flow cytometry (n = 3 biological replicates). i, BioLayer Interferometry (Octet) measurement of binding kinetics between mouse SCG2-His and immobilized LILRB4-ECD, fit to a 1:1 model. j, Immunoblot analysis confirming the expression of SCG2 truncation mutants used in HEK293T reverse binding assays. k, Reverse binding assay assessing the specificity of SCG2 interaction with various members of LILRB family. HEK293T cells were transfected with plasmids encoding membrane-anchored SCG2 fusion proteins for 48 h. Cells were then harvested and incubated with purified recombinant Fc-tagged LILRB proteins for 1 hour at 4 °C. Binding was detected by flow cytometry. l, Co-IP of SCG2 with various Flag-tagged LILRB family receptors in HEK293T cells. m, Reporter cell activation assay for LILRAs and Pirb reporter cells incubated with SCG2 protein coated on plates overnight at 4 °C. GFP⁺ cells were analyzed by flow cytometry (n = 2 biological replicates). n, gp49b reporter cell activation assay. Reporter cells were stimulated for 24 hours with plate-coated SCG2, ApoE, or anti-gp49 antibody (positive control). GFP⁺ cells were analyzed by flow cytometry (n = 3 biological replicates). o, Activation assays using LILRB4 reporter cells incubated with different secretogranin family proteins. LILRB4 reporter cells were incubated for 24 h at 37 °C. GFP⁺ cells were analyzed by flow cytometry. All data are present as mean ± s.d.

Source data

Extended Data Fig. 2 SCG2 does not bind LILRB4 F83A mutant.

a, Mapping SCG2-binding domain via LILRB4-D1 truncation constructs. HEK293T cells were co-transfected with Flag-tagged LILRB4-D1 truncation mutants and full-length SCG2. After 48 hours, cell lysates were subjected to IP with anti-Flag beads, IB with indicated antibodies (same below). b, Identification of SCG2-binding region with AA55-123 of LILRB4. HEK293T cells were co-transfected with SCG2 and FLAG-tagged LILRB4 mutants harboring internal deletions within amino acids 55–123 (AA55–123). Interactions were assessed by IP and immunoblotting. c, Single amino acid mapping within AA75-84. HEK293T cells were co-transfected with SCG2 and Flag-tagged LILRB4 single-point mutants containing single amino acid substitutions within the AA75–84 region. Interactions with SCG2 were analyzed by Co-IP. d, Activation assays comparing LILRB4 wild-type and F83A mutant reporter cells stimulated with plate-coated recombinant SCG2 protein or coated anti-LILRB4 antibody as a positive control. Reporter cells were incubated for 24 hours at 37 °C, and GFP⁺ cells were quantified by flow cytometry (n = 3 biological replicates). Data are present as mean ± s.d. e, Immunoblot analysis of LILRB4 phosphorylation and SHP2 recruitment in the presence of SCG2. HEK293T cells were co-transfected with Flag-tagged wild-type or mutant LILRB4 plasmids (2 μg), SCG2 (300 ng), SHP2 (300 ng), and Lyn (100 ng). Cell lysates were IP using anti-Flag beads and analyzed by IB with indicated antibodies.

Source data

Extended Data Fig. 3 Correlation of SCG2 with MDSCs, CTLs, or patient survival.

a, Kaplan–Meier survival curves comparing overall survival of patients stratified by high (red) versus low (blue) SCG2 expression in TCGA CESC, TCGA MESO, GSE65858, GSE54236, and GSE1993 datasets. Survival analyses for TCGA CESC and TCGA MESO datasets were performed using GEPIA2 online tool (http://gepia2.cancer-pku.cn/#index) with median SCG2 expression as the cutoff. Survival data and analyses for GSE65858, GSE54236, and GSE1993 were obtained from the Tumor Immune Dysfunction and Exclusion (TIDE) database (http://tide.dfci.harvard.edu/), using optimal cut-off values provided by TIDE. Statistical significance was determined using the log-rank (Mantel–Cox) test. b-c, Correlation analyses between SCG2 expression and infiltration of myeloid-derived suppressor cell (MDSC) or cytotoxic T lymphocyte (CTL) across multiple tumor datasets. RNA-seq data were obtained from GEO and TCGA. Gene expression values in each dataset were log2-transformed and normalized using the sample average for TIDE analysis. Cytotoxic T lymphocyte (CTL) infiltration scores were calculated based on the average expression of CD8A, CD8B, GZMA, GZMB, and PRF1 within each dataset. Pearson correlation analyses were conducted between SCG2 and MDSC infiltration, as well as between SCG2 and CTL infiltration. Scatter plots for these correlations were generated using the ggplot2 package in R.

Extended Data Fig. 4 LILRB4 transgene does not change immune profile.

a, Schematic diagram illustrating the generation of myeloid-specific LILRB4-Tg mice. The human LILRB4 gene was introduced downstream of a loxP-flanked STOP cassette containing a puromycin resistance gene (Pur/Stop cassette), driven by the constitutive CAG promoter. These mice were crossed with LysM-Cre mice expressing Cre recombinase under the control of the myeloid-specific LysM promoter. Upon Cre-mediated recombination, the STOP cassette was excised specifically in myeloid lineage cells, resulting in cell-specific expression of the human LILRB4 transgene. b-c, Immunoblot analysis (b) and Flow cytometry analysis (c) validating LILRB4 expression on bone marrow-derived macrophages (BMDMs) isolated from wild-type WT and LILRB4-Tg mice (n = 3 biological replicates). d, Representative flow cytometry gating strategy for dendritic cells (DCs, CD11c+MHC-II+), macrophages (CD11b+F4/80+), monocytes (CD11b+Ly6Chi), and neutrophils (CD11b+Ly6G+) in spleen single-cell suspensions from LILRB4-Tg mice. e, Quantification of LILRB4 expression levels (MFI) and percentages of LILRB4-positive cells among DCs, macrophages, monocytes, and neutrophils in spleens from LILRB4-Tg mice (n = 5 mice per group). f, Comparative analysis of cell surface marker expression profiles (CD40, CD80, CD86, and CD206) on monocytes, macrophages, and DCs isolated from spleens of WT and LILRB4-Tg mice (n = 5 mice per group), assessed by flow cytometry. g-j, Detailed gating strategies employed for the comprehensive immunophenotyping of spleen immune cell populations from WT and LILRB4-Tg mice, including analysis of monocytes (CD11b+Ly6ChiLy6G−), neutrophils (CD11b+Ly6C−Ly6G+), cDC1 (CD11c+MCHII+XCR1+), cDC2 (CD11c+MCHII+CD172a+), T cells (CD3+CD4+ or CD3+CD8+), NK cells (CD3−NK1.1+), and Treg cells (CD3+CD4+CD25+CD127−). Quantification of percentages of these immune cell subsets in the spleen is shown (n = 3 biological replicates). All data are present as mean ± s.d. p values were determined by two-tailed unpaired Student’s t-test.

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Extended Data Fig. 5 SCG2-LILRB4 interaction promotes tumor growth.

a, Immunoblot of SCG2 expression in MC38, B16F10 and LLC cells infected with lentiviruses encoding pLVX-SCG2-puro or empty vector (pLVX-puro). b, Tumor weights from WT or LILRB4-Tg mice subcutaneously injected with indicated SCG2-expressing or vector control MC38 (n = 7 or 9), B16F10 (n = 6 or 8), or LLC (n = 7) cells. Tumors were harvested and weighed at the endpoint. c, Tumor growth curve and tumor weights in LILRB4-Tg mice and WT mice injected subcutaneously with WT MC38 cells or mouse SCG2-expressing MC38 cells (n = 7 or 9). d, Tumor weights of MC38 tumor-bearing LILRB4-Tg mice intratumorally injected with recombinant SCG2 or PBS (n = 7). e, Tumor weights and representative images from MC38-Vec and MC38-SCG2 tumors in LILRB4-Tg mice intraperitoneally injected with anti-LILRB4 antibody or control IgG (n = 8). f, Flow cytometric analysis of LILRB4 expression on tumor-infiltrating M-MDSCs and G-MDSCs from tumor-bearing mice treated with anti-LILRB4 antibody (n = 8). g, Frequency of CD4+ and CD8+ T cells in peripheral blood of mice treated intraperitoneally with anti-CD4/CD8 antibodies or control IgG. h, Tumor weights and representative images from MC38-Vec and MC38-SCG2 tumor-bearing mice treated with anti-CD4/CD8 antibodies or control IgG (n = 6 or 8). i, Percentages of peripheral blood M-MDSCs, G-MDSCs, and macrophages after intraperitoneal treatment with anti-Ly6G or anti-CSF1R antibody (n = 5). j, Tumor weights from MC38 tumor-bearing mice treated with anti-Ly6G or anti-CSF1R antibodies (n = 5). k, Immune cell infiltration analysis of B16F10-Vec and B16F10-SCG2 tumors from WT and LILRB4-Tg mice (n = 7). l, Frequency of GZMB+ cells among tumor-infiltrating CD8+ T cells in MC38-Vec and MC38-SCG2 tumors (n = 9). m-n, Analysis of tumor-infiltrating immune cell populations in MC38-Vec and MC38-SCG2 tumors from mice treated with anti-LILRB4 antibody or control IgG (n = 7). Tumor growth curves are present as mean ± s.e.m.; other data are present as mean ± s.d. p values were determined by two-tailed unpaired Student’s t-test (d, i, and l), one-way ANOVA with Holm-Sidak’s multiple comparisons test (b, c (right), e, f, h, j, k, m, n), or two-way ANOVA with Tukey’s multiple comparisons test (c, left).

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Extended Data Fig. 6 SCG2 loss restricts tumor progression in LILRB4-Tg mice.

a, Immunoblot analysis of SCG2 expression levels in brain tissues and serum from LILRB4-Tg and LILRB4-Tg SCG2−/- mice (n = 5). b, Percentages of immune cell populations in spleens from WT, SCG2−/-, LILRB4-Tg, and LILRB4-Tg SCG2−/-, mice (n = 6). c, Representative images of excised tumors (MC38, EO771, LLC, and CT2A models) from indicated mouse groups at study endpoint. d-e, Tumor growth curves and final tumor weights showing no significant difference between SCG2−/- mice and WT mice bearing MC38 (n = 6) or LLC (n = 6) tumors. Tumor volumes were measured every two days; tumors were excised and weighed at endpoint. f, MC38 tumor growth in WT, LILRB4-Tg, mice and LILRB4-Tg SCG2−/- mice. Tumor size was measured every two days, and tumor weights were measured on the final day (n = 9 or 10). g, Tumor growth in WT or ApoE−/- mice. Mice were subcutaneously implanted with LLC cells (5×105) on the right flank. Tumor size was measured every two days, and tumor weights were measured on the final day (n = 7 or 8). h, Representative immunofluorescence staining and quantification of CD8+ T cells and M-MDSCs in MC38 tumors from LILRB4-Tg mice and LILRB4-Tg SCG2−/- mice (3 independent experiments, n = 10). Scale bars, 100 µm. i, Quantification of immune cell infiltration (M-MDSCs, G-MDSCs, macrophages, cDC1, cDC2, CD4+ and CD8+ T cells) in tumor tissues (B16F10 (n = 8), LLC (n = 9), EO771 (n = 8) and CT2A (n = 8)) isolated from LILRB4-Tg mice and LILRB4-Tg SCG2−/- mice at endpoint. Tumor growth data are present as mean ± s.e.m.; all other data are present as mean ± s.d. p values were determined by two-tailed unpaired Student’s t-test or two-way ANOVA with Bonferroni’s multiple comparisons test (tumor volume (d-g)).

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Extended Data Fig. 7 SCG2-LILRB4 axis strengthens MDSC suppression.

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).

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Extended Data Fig. 8 SCG2-LILRB4 interaction upregulates IL-6-STAT3 activation.

a, Quantification and statistical analysis of Western blot results shown in Fig. 6a (n = 3 biological replicates). b-c, Immunoblot (b) and quantification (c) of p-STAT3 levels in BM-MDSCs from LILRB4-Tg mice stimulated with or without IL-6 for 10, 20, 30 min in plates coated with BSA, SCG2 or ApoE (n = 3 biological replicates). d-e, Immunoblot (d) and quantification (e) of p-STAT3 levels in BM-MDSCs from WT, LILRB4-Tg, or LILRB4-Tg ApoE−/- mice. Cells were stimulated with or without IL-6 for 10 min in plates coated with BSA, SCG2 or ApoE (n = 3 biological replicates). f, Quantification and statistical analysis of Western blot results shown in Fig. 6b (n = 3 biological replicates). g, mRNA level of Arg1 in BM-MDSCs stimulated with IL-6 under different conditions. Cells were incubated with varying concentration of IL-6 for 24 h or with IL-6 for indicated times in plates coated with BSA or SCG2 (n = 3 biological replicates). h, IP Flag-tagged LILRB4 and endogenous STAT3 in THP-1 cells. THP-1 LILRB4-Flag stable cells were subjected to IP with anti-Flag beads followed by IB with anti-STAT3 antibody. i,Co-IP of Flag-tagged STAT3 domain deletion and HA-tagged LILRB4 in HEK293T cells. j, SCG2 induced phosphorylation of STAT3 in a recombinant HEK293T system. HEK293T cells were co-transfected with LILRB4-Flag, ITIM-mutant LILRB4-Flag (2 μg), STAT3 (1 μg), Lyn (100 ng), and SCG2 (300 ng) as indicated. At 48 h post-transfection, cell lysates were collected and subjected to IP/IB analysis. k, Quantification and statistical analysis of Western blot results shown in Fig. 6j (n = 3 biological replicates). l, Immunoblot of p-STAT1 and p-STAT6 in BM-MDSCs from LILRB4-Tg mice stimulated with or without IFN-γ or IL-4 (20 ng/mL) for 10 min in plates coated with BSA or SCG2. m, Quantification and statistical analysis of Western blot results shown in Fig. 6l (n = 3 biological replicates). Data are presented as mean ± s.d. p values were determined by one-way ANOVA with Tukey’s multiple comparisons test (a, f, k, and m), two-way ANOVA with Tukey’s multiple comparisons test (c and e) or two-way ANOVA with Bonferroni’s multiple comparisons test (g).

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Extended Data Fig. 9 LILRB4 promotes human M-MDSC suppressive function.

a, T cell proliferation assay of CFSE-labeled CD3⁺ T cells co-cultured with CD14⁺ MDSCs isolated from cancer patient peripheral blood. Cells were cultured in plates coated with BSA or SCG2 in the presence of anti-CD3/CD28 activation beads. Anti-LILRB4 blocking antibodies were added as indicated. T cell proliferation was assessed by flow cytometry (n = 3 biological replicates). b, Quantification and statistical analysis of Western blot results shown in Fig. 7c (n = 3 biological replicates). c, Immunoblot of CD14+ cells from cancer patient blood stimulated with or without IL-6 for 10 min in 96-well plates coated with BSA or SCG2. Lysates were analyzed using the indicated antibodies. d-e, Immunoblot (d) and quantification (e) of IL-6-STAT3 signaling activity in CD14+ cells from health blood stimulated with or without IL-6 for 5, 10 min in plates coated with BSA or SCG2 (n = 3 biological replicates). f, Percentage of human CD45+ cells in peripheral blood of humanized mice 8 weeks after CD34+ cells injection into NSG mice. Human cell engraftment was evaluated by flow cytometry. g, Immunoblot analysis confirming SCG2 ectopic expression in A375 melanoma cells transduced with pLVX-SCG2 lentivirus. h, Representative tumor images of the A375 tumor model in humanized mice intraperitoneally injected with anti-LILRB4 antibody or control IgG (n = 9 or 10). Data are presented as mean ± s.d. p values were determined by one-way ANOVA with Holm-Sidak’s multiple comparisons test (a), one-way ANOVA with Tukey’s multiple comparisons test (b), or two-way ANOVA with Bonferroni’s multiple comparisons test (e).

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Extended Data Fig. 10 Conserved function of LILRBs in M-MDSC activation.

a, Proportions of myeloid cell subsets (monocytes, macrophages, DCs, and mast cells) across eight tumor types, analyzed from publicly available single-cell RNA-seq datasets. b, Bubble heatmap showing expression of selected signature genes across myeloid subsets. Dot size represents the percentage of cells expressing each gene; colored intensity indicates normalized average expression levels. c, Co-IP of Flag-tagged LILRB1-5 and STAT3 in the presence or absence of Lyn in HEK293T cells. HEK293T cells were co-transfected with Flag- LILRB1-5 (2 μg), Lyn (500 ng), and STAT3 (2 μg) plasmids. After 48 h, lysates were subjected to IP with anti-Flag beads and immunoblotting. d, Co-IP analysis of STAT3 binding to Flag-tagged LILRB1, LILRB4, or achimeric receptor combining the ECD of LILRB1 and ICD of LILRB4 (ECDLILRB1-ICDLILRB4), co-transfected in HEK293T cells. After 48 h, lysates were subjected to IP with anti-Flag beads and immunoblotting. e-f, Immunoblot (e) and quantification (f) of p-STAT3, p-SHP1, and p-SHP2 in human CD14+ monocytes from healthy donors stimulated with or without IL-6 (20 ng/mL) for 10 min in 96-well plates coated with BSA or indicated antibodies (n = 3 biological replicates). Data are presented as mean ± s.d. p values were determined by one-way ANOVA with Tukey’s multiple comparisons test.

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Supplementary information

Reporting Summary

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Supplementary Table 1

Primer sequences used for quantitative PCR experiments.

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Yang, X., Huang, R., Fang, M. et al. Secretogranin 2 binds LILRB4 resulting in immunosuppression. Nat Immunol 26, 1567–1580 (2025). https://doi.org/10.1038/s41590-025-02233-4

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