Fig 2: S100a9 expression is elevated within Gr1highCD11b+ cells of male lupus-prone mice and regulates B cell differentiation in vitro and antibody production in vivo. Gr1highCD11b+ and Gr1lowCD11b+ cells were isolated from 9 week old male and female NZBWF1 lupus-prone mice by flow cytometry and levels of S100a9 (A), S100a8 (B), and S100a4 (C) mRNA were determined by RT-PCR analysis (n = 4). Graphs show mean +/- SEM. (D) Supernatants from overnight cultured Gr1highCD11b+ cells from 4 wk old NZBWF1 male mice suppress IFNα/CD40L-driven B cell differentiation. Shown is the average (± SEM) of 4 independent experiments. (E) S100A8/A9 heterodimer levels in serum, spleen and bone marrow from 9 wk old NZBWF1 mice. Each symbol represents one mouse. Male vs. female: p = 0.07; two-way ANOVA. (F) Gr1highCD11b+ cells from 9 wk old S100a9-deficienct male NZBWF1 mice fail to suppress cytokine-driven B cell differentiation in vitro. IgM/IgG-secreting cells were enumerated by ELISPOT and are presented as number of cells per 105 plated B cells. Data shown represent the mean ± SEM of 4 independent assays. *P < 0.05; **P < 0.01, Student’s unpaired t test.

Fig 3: The aggressive niche harbors an expanded and spatially restricted myeloid population.a–c UMAP was used for dimension reduction of single-cell data. Immune fractions from indolent and aggressive lesions were computationally merged (a) for cluster analysis and identification (b). Proportion of clusters per group shown in c. NK natural killer, pDC plasmacytoid dendritic cell, cDC conventional dendritic cell. d Heatmap of top differentially expressed (DE) genes for each cluster, ordered in decreasing cluster size. Clusters indicated by top horizontal bar, with inferred immune identity noted. Cells derived from indolent or aggressive tumor sample indicated by second horizontal bar. DE genes are listed on the left. e Immunohistochemical staining for major lymphoid (T cells—CD3; B cells—CD19) and myeloid (macrophages—F4/80; neutrophils—MPO) populations associated with indolent and aggressive lesions. Arrows point to selected positively stained cells. Representative images shown from n = 4, 3, 3, 4 animals for CD3, CD19, F4/80, and MPO, respectively. f Immunofluorescent staining for neutrophils using an alternative marker S100A8. Left panel shows low power magnification view of adjacent indolent and aggressive lesions, with S100A8+ infiltration tightly localized with the aggressive niche. Boxes correspond by color to high magnification images shown middle (blue, indolent) and right (yellow, aggressive) panels. Representative images shown from n = 7 animals. g Immune clusters were analyzed by lesion type to calculate a change in KEGG pathway enrichment score (GSVA delta) in aggressive vs. indolent lesions. Positive score indicates increased enrichment score in aggressive lesions. Top 5 pathways most commonly increased or decreased across populations were identified to ascertain potential niche-wide changes associated with aggressive lesions. See also Supplementary Figs. 5–7. Source data are provided as a Source Data file.

Fig 4: H2R deficiency promoted DNA synthesis in bone marrow cells. (A) Immunofluorescence staining (magnification: 200× and 1000×) of S100A8 (green), CXCR2 (white), and TLR4 (red) in WT and H2R-deficient bone marrow cells. (B) The mean numbers in each field of TLR4-negative neutrophils at 400× power (n = 10). (C) DNA content of different phases of bone marrow cells determined by flow cytometry (n = 5). (D) The expression levels of Myc in HDNs and LDNs of WT and Hdc KO mice by qRT-PCR (n = 3). In all panels, means ± SD are shown. The P value is shown in each panel. CXCR2, C-X-C Motif Chemokine Receptor 2; DAPI, 4',6-diamidino-2-phenylindole; mRNA, messenger RNA.

Fig 5: Confirmation of the distinct gene signatures of LDNs and HDNs. (A–C) Expression of representative genes in MYC target, NF-?B pathways, and surface markers of LDNs and HDNs by qRT-PCR (n = 4). Means ± SD are shown. The P value is shown under each panel. (D) Immunofluorescence staining (magnification: 200× and 1000×) of S100A8 (green), CXCR2 (pink), and TLR4 (red) in LDNs and HDNs. (E) The expression levels of S100a8 and S100a9 in LDNs and HDNs by qRT-PCR (n = 4). Means ± SD are shown. P values are shown on the panel. CXCR2, C-X-C Motif Chemokine Receptor 2; DAPI, 4',6-diamidino-2-phenylindole.