Lupus Susceptibility Loci Predispose Mice to Clonal Lymphocytic Responses and Myeloid Expansion

Lupus-prone mice have expanded splenic lymphocyte and myeloid populations. (A) Gene expression of cluster-defining genes projected onto UMAP of CD45 cells isolated from the spleens of B6 and SLE.yaa mice. (B) UMAP visualization of CD45 cells colored by unbiased cluster assignment. Mac, macrophages; PMN, polymorphonuclear cells; DC, dendritic cells; PC, plasma cells; NK, NK cells; pDC, plasmacytoid dendritic cells; Baso, basophils. (C) Stacked bar graph of percent of CD45 cells belonging to each cluster between B6 and SLE.yaa mice. (D) Flow cytometric quantification of B (CD19⁺B220⁺), germinal center (GC; CD19⁺B220⁺GL7⁺Fas⁺), plasma (PC; CD138⁺), T_CON (CD4⁺Foxp3⁻), T_REG (CD4⁺Foxp3⁺), T_FH (CD4⁺CXCR5⁺PD1⁺Foxp3⁻ICOS⁺), T_FR (CD4⁺CXCR5⁺PD1⁺Foxp3⁺ICOS⁺), CD8, macrophage (Mϕ; F4/80⁺CD68⁺), CD11c, and NK (CD49b⁺) cells from the spleens of B6 (black) or SLE.yaa (red) mice.

Macrophages from lupus-prone mice are less metabolically active. (A) Biplot of COMPASS principal-component scores of CD45 cells (dots) colored by scRNA-seq–based cluster assignment and top variable loadings (vectors). (B) COMPASS principal-component plot of CD45 cells colored according to genotype (B6, black; SLE.yaa, red) with marginal histogram of PC1 and PC2 scores. (C) Heatmap of spearman correlation of KEGG transcriptome signatures with top five COMPASS principal components. Only significant correlations (p < 0.05) are shown in color, and nonsignificant correlation coefficients are grayed out. (D) Volcano plots of differential COMPASS score activity between B cells, CD4 cells, CD8 cells, or macrophages from SLE.yaa and B6 mice. COMPASS metareactions are colored by their Recon2 pathways (outlined box). (E) Dot plots of differential COMPASS score activity of metabolic reactions in B cells, CD4 cells, CD8 cells, or macrophages from SLE.yaa and B6 mice. Reactions (dots) are partitioned by Recon2 pathways (rows), and significant reactions (adjusted p < 0.05) are colored by the sign of their Cohen’s d statistic. Nonsignificant reactions are grayed out.

Marginal zone B cells are decreased in lupus-prone mice. (A) UMAP visualization of B cells from B6 (left) or SLE.yaa (right) mice colored by unbiased cluster assignment. Fo, follicular; MZ, marginal zone; T2-B, transitional stage 2; PC, plasma cell; T1-B, transitional stage 1 B. (B) Stacked bar graph of percent of B cells belonging to each cluster between B6 and SLE.yaa mice. (C) Pseudotime scores of B cells calculated from gene expression data projected onto UMAP embeddings. (D) RNA velocity of B cells projected onto UMAP colored by cluster assignment. Velocity vector field is represented by streamlines that indicate speed and direction of cells. (E) Trajectory inference computed using partition-based graph abstraction of paths between B cell clusters. Topology data are represented by weighted edges whose thickness corresponds to the connectivity between two clusters. (F) Volcano plots of differentially expressed genes between SLE.yaa versus B6 mice within indicated B cell clusters. Adjusted p value <0.01 and absolute log₂FCs >0.2 are shown in red. (G) Dot plot of gene Ontology analysis of differentially expressed genes between follicular (left) or activated (right) B cells from SLE.yaa versus B6 mice. Size represents gene ratio, and color represents adjusted p. (H) Network plot of Gene Ontology analysis of differentially expressed genes between follicular B cells from SLE.yaa versus B6 mice. Tan circles represent gene sets, and colored dots represent genes colored by log₂FC in SLE.yaa compared with B6 mice. (I) Flow cytometry contour plots (left) and quantification (right) of marginal zone frequency among CD19⁺ cells from B6 (black) and SLE.yaa (red) mice. (J) Flow cytometric quantification of indicated proteins in B (CD19⁺B220⁺), germinal center (GC; CD19⁺B220⁺GL7⁺Fas⁺), plasma (PC; CD138⁺), or marginal zone (MZ; CD19⁺B220⁺CD21/35⁺CD1d⁺) cells from B6 (black) and SLE.yaa (red) mice.

B cells are clonally expanded and mutated in lupus-prone mice. (A) Clone size topography (top) or mutational frequency (bottom) mapped onto UMAP visualization of B cells from B6 (left) or SLE.yaa (right) mice. Clonotypes defined by VDJC gene and size were calculated by the number of cells belonging to each clonotype. Mutational frequency was calculated by comparison with germline sequences. (B) Clonal overlap between B cell clusters represented by Morisita index of clones between two clusters. (C) Network interaction plot of clonotype sharing between clusters mapped onto UMAP visualization of B cells. Edge color represents proportion of clones shared between two clusters. Node sizes represents the number of unique clones in the underlying cluster. (D) Diversity curve of B cells from B6 (black) or SLE.yaa (red) mice representing the Hill diversity index (qD) over uniform resampling across diversity orders (q). Shading represents 95% confidence interval. (E) Hedgehog plot of somatic hypermutation mutability model for C (top) or A (bottom) nucleotides in B cells from B6 (left) or SLE.yaa (right) mice. Bar length represents the likelihood of a mutation in the given 5-mer. Bar colors represent known hot or cold spot motifs (WRC/GYW, red; WA/TW, green; SYC/GRS, blue; neutral, gray).

T cells from lupus-prone mice express markers of exhaustion. (A) UMAP visualization of T cells from B6 (left) or SLE.yaa (right) mice colored by unbiased cluster assignment. (B) Stacked bar graph of percent of T cells belonging to each cluster between B6 and SLE.yaa mice. (C) Volcano plots of differentially expressed genes between T cells from SLE.yaa versus B6 mice within indicated clusters. Adjusted p value <0.01 and absolute log₂FCs >0.2 are shown in red. (D) Gene set enrichment plot of indicated gene module against genes ranked by fold enrichment in SLE.yaa versus B6 cells within indicated T cell cluster. (E) Flow cytometry contour plots (left) and quantification (right) of indicated population among CD4 or CD8 cells from B6 (black) and SLE.yaa (red) mice. Flow cytometry plots are gated on total CD4 cells. (F) Flow cytometric quantification of indicated proteins in T_CON (CD4⁺Foxp3⁻), T_REG (CD4⁺Foxp3⁺), T_FH (CD4⁺CXCR5⁺PD1⁺Foxp3⁻ICOS⁺), T_FR (CD4⁺CXCR5⁺PD1⁺Foxp3⁺ICOS⁺), extrafollicular (EFO; CD4⁺CD62L⁻PSGL1⁻), or CD8 cells from B6 (black) and SLE.yaa (red) mice.

CD4 clonal expansion is associated with differential gene expression in lupus-prone mice. (A) UMAP visualization of T cells from B6 and SLE.yaa mice colored by cluster assignment. (B) Clone size mapped onto UMAP visualization of transcriptomic data of individual T cells from B6 (left) or SLE.yaa (right) mice. Clonotypes are defined by paired full-length TCRα and TCRβ sequences, and clone sizes are number of individual cells within a given clonotype. (C) Bar plot of number of individual cells belonging to each clonotype in B6 (black) or SLE.yaa (red) mice. (D) Pie charts of clonal expansion of T cell clusters identified by scRNA-seq (columns) in B6 (top) or SLE.yaa (bottom) mice. Number of cells with both TCRα and TCRβ successfully identified is shown below each pie chart. For clonotypes expressed by two or more cells, the number of cells expressing that clone is shown by a distinct color. (E) Unweighted network analysis of expanded clonotypes (more than one individual cell) from B6 (black) and SLE.yaa (red) mice. Clonotypes are defined by paired full-length TCRα and TCRβ sequences. Individual samples (m232, m233, m234, m235) are depicted as colored circles, and clonotypes are depicted as gray circles and sized according to the number of cells belonging to a given clonotype. Edges represent clonotype membership to individual samples. (F) Volcano plot of differentially expressed genes between expanded CD4 clonotypes from SLE.yaa versus B6 mice. Adjusted p value <0.01 and absolute log₂FCs >0.2 are shown in red. (G) Scatter plot comparing log fold change of gene expression between expanded versus unexpanded CD4 clones in B6 and SLE.yaa mice. Log₂FCs >0.1 are indicated in black (correlated in both conditions), red (correlated in SLE.yaa mice only), or red (correlated in B6 mice only). (H) Scatter plot comparing GLIPH2 specificity group size between B6 and SLE.yaa mice and colored according to disease class of predicted Ag. Size of specificity groups represents total number of samples in which the given specificity group is observed. (I) Mapping of predicted disease class onto UMAP visualization of T cell transcriptomic data from B6 (left) or SLE.yaa (right) mice. Cells for which disease class prediction was not possible are left gray.

Myeloid subset distribution is altered in lupus-prone mice. (A) UMAP visualization of myeloid cells from B6 (left) or SLE.yaa (right) mice colored by unbiased cluster assignment. (B) Dot plot of averaged log-normalized expression of top six differentially expressed genes (columns) for each myeloid subcluster (row). Size represents percentage of cells in cluster expressing gene and color represents expression level. (C) Stacked bar graph of percent of myeloid cells belonging to each cluster between B6 and SLE.yaa mice. (D) Volcano plots of differentially expressed genes between myeloid cells from SLE.yaa versus B6 mice within indicated clusters. Adjusted p value <0.01 and absolute log₂FCs >0.2 are shown in red. (E) Dot plot of gene Ontology analysis of differentially expressed genes between myeloid cells from SLE.yaa versus B6 mice within indicated clusters. Size represents gene count, and color represents adjusted p value.

Myeloid cell differential gene expression is validated by flow cytometry and histology. (A) Flow cytometry contour plots of gating strategy to identify DP1 (CD11c^hiCD11b⁺), DP2 (CD11c^medCD11b⁺), and CD11c (CD11c⁺CD11b⁻) in B6 (left) and SLE.yaa (right) mice. (B) Flow cytometric quantification of indicated proteins in macrophages (Mϕ; F4/80⁺CD68⁺) and DP1, DP2, or CD11c cells from B6 (black) and SLE.yaa (red) mice. (C) Confocal microscopy of spleens from B6 (top) or SLE.yaa (bottom) mice stained for indicated markers. Insets (below) show higher magnification. White arrows indicate myeloid cells that are positive for CD5L, CD74, CD16/32, or CD16.2. Hollow arrowhead indicates CD5L expression by follicular dendritic cells (FDCs).

Myeloid cells from lupus-prone mice are less capable of presenting Ag to CD4 cells. (A) Flow cytometry histograms (left) and quantification (right) of I-A/I-E expression in macrophages (Mϕ; F4/80⁺CD68⁺) and DP1 (CD11c^hiCD11b⁺), DP2 (CD11c^medCD11b⁺), and CD11c (CD11c⁺CD11b⁻) cells from B6 (black) or SLE.yaa (red) mice. (B) Flow cytometry histograms (left) and quantification (right) of CellTrace Violet (CTV), CD44, CD69, ICOS, or PD-1 expression in OT-II CD45.1⁺ CD4 cells following coculture with splenocytes isolated from B6 (blue) or SLE.yaa (red) mice with or without OVA. Data are representative of four independent experiments.