Journal: Nature immunology

Article Title: Secretogranin 2 binds LILRB4 resulting in immunosuppression

doi: 10.1038/s41590-025-02233-4

Figure Lengend Snippet: a – c , Tumor growth of SCG2-expressing or vector tumor cells in LILRB4 -Tg mice subcutaneously injected with MC38 ( a ; n = 7 or 9), B16F10 ( b ; n = 6 or 8) or LLC ( c ; n = 7) cells. Tumor size was measured every 2 days (same as below). d , MC38 tumor growth in LILRB4 -Tg mice intratumorally injected with recombinant SCG2 protein (10 μg in 50 μl of PBS) or PBS ( n = 7). e , f , B16F10 lung metastasis model in WT and LILRB4 -Tg mice. Representative lung images at the experimental endpoint ( e ; n = 7) and survival analysis ( f ; n = 6 or 7) are shown; i.v., intravenous. g , MC38-Vec and MC38-SCG2 tumor growth in LILRB4 -Tg mice treated with anti-LILRB4 and isotype control ( n = 8). h , CD4 + /CD8 + T cell depletion in MC38-bearing LILRB4 -Tg mice. Tumor-bearing mice were treated with isotype control or anti-CD4/CD8 ( n = 6 or 8). i , Myeloid subset cell depletion in MC38-bearing LILRB4 -Tg mice. For G-MDSC depletion, mice were treated with anti-Ly6G ( n = 5). For M-MDSC depletion, mice were treated with anti-CSF1R ( n = 5). j,k , Immune profiling of B16F10 tumors collected from WT or LILRB4 -Tg mice on day 15. Percentages of MDSCs ( j ) and CD4 + and CD8 + T cells and regulatory T (T reg ) cells ( k ) were measured by flow cytometry ( n = 6 or 7). Data on tumor size are presented as mean ± s.e.m.; all other data are shown as mean ± s.d. P values were determined by two-way analysis of variance (ANOVA) with a Tukey’s multiple comparisons test ( a – c and g – i ) or a Bonferroni’s multiple comparisons test ( d ), one-way ANOVA with a Holm–Sidak’s multiple comparisons test ( e , j and k ) or log-rank (Mantel–Cox) test for survival curves ( f ).

Article Snippet: Ly6G + cells were removed using Ly6G beads (Miltenyi, 130–120-337).

Techniques: Expressing, Plasmid Preparation, Injection, Recombinant, Control, Flow Cytometry

Journal: Nature immunology

Article Title: Secretogranin 2 binds LILRB4 resulting in immunosuppression

doi: 10.1038/s41590-025-02233-4

Figure Lengend Snippet: 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 + Ly6C hi ), 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 + Ly6 hi Ly6G − ), 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.

Article Snippet: Ly6G + cells were removed using Ly6G beads (Miltenyi, 130–120-337).

Techniques: Expressing, Control, Western Blot, Flow Cytometry, Derivative Assay, Isolation, Single Cell, Marker, Two Tailed Test

Journal: Nature immunology

Article Title: Secretogranin 2 binds LILRB4 resulting in immunosuppression

doi: 10.1038/s41590-025-02233-4

Figure Lengend Snippet: 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 antiLy6G 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). I , 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’st-test ( d , i , and I ), 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).

Article Snippet: Ly6G + cells were removed using Ly6G beads (Miltenyi, 130–120-337).

Techniques: Western Blot, Expressing, Infection, Plasmid Preparation, Injection, Control, Recombinant, Two Tailed Test