Extended Data Fig. 4: LILRB4 transgene does not change immune profile.
From: Secretogranin 2 binds LILRB4 resulting in immunosuppression
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.
