Childhood-onset lupus nephritis is characterized by complex interactions between kidney stroma and infiltrating immune cells

Abstract

Children with systemic lupus erythematosus (SLE) are at increased risk of developing kidney disease, termed childhood-onset lupus nephritis (cLN). Single-cell transcriptomics of dissociated kidney tissue has advanced our understanding of LN pathogenesis, but loss of spatial resolution prevents interrogation of in situ cellular interactions. Using a technical advance in spatial transcriptomics, we generated a spatially resolved, single-cell resolution atlas of kidney tissue from eight patients with cLN and four control individuals. Annotated cells were assigned to 30 reference cell types, including major kidney subsets and infiltrating immune cells. Analysis of spatial distribution demonstrated that individual immune lineages localized to specific regions in cLN kidneys, including myeloid cells that trafficked to inflamed glomeruli and B cells that clustered within tubulointerstitial immune hotspots. Gene expression varied as a function of tissue location, demonstrating how incorporation of spatial data can provide new insights into the immunopathogenesis of SLE. Alterations in immune phenotypes were accompanied by parallel changes in gene expression by resident kidney stromal cells. However, there was little correlation between histologic scoring of cLN disease activity and glomerular cell transcriptional signatures at the level of individual glomeruli. Last, we identified modules of spatially correlated gene expression with predicted roles in induction of inflammation and the development of tubulointerstitial fibrosis. Single-cell spatial transcriptomics allowed insights into the molecular heterogeneity of cLN, paving the way toward more targeted and personalized treatment approaches.

Figure 1:. Single cell-resolution spatial transcriptomics in childhood-onset lupus nephritis (cLN).

(A) Dot plot of combined CosMX datasets showing cell type-specific marker gene expression. Dot intensity indicates scaled mean gene expression and dot diameter shows proportion expressing the indicated gene. Type A, Type B, and indistinct intercalated cells, and epithelial cell subtypes are shown as combined data in the dot plot. (B) Tissue sections from a patient with cLN (SLE6) showing Periodic acid–Schiff (PAS)-stained tissue sections (left) compared to sequential tissue sections annotated using spatial transcriptomics (right). Scale bars, 300μm. (C) Serial tissue sections (Control3) showing annotated cell types (left), WT1 immunohistochemistry (middle), and CD10 immunohistochemistry (right). Scale bars, 200μm. (D) Corresponding images from representative patient with cLN (SLE2) showing panCK⁺ immunofluorescent staining (left panel, green) overlapping with transcriptionally-defined connecting tubule cells (purple), principal cells (gray), type A intercalated cells (red), and type B intercalated cells (yellow). Scale bars, 50μm. (E) Mean fluorescence intensity (MFI) of panCK staining by transcriptionally-annotated cell types (Control2). (F) Podocyte gene expression in a cLN glomerulus (SLE6). PAS-stained histology is shown on the left, corresponding transcriptionally-defined glomerular cell types are shown in the middle, and VEGFA (red) and PLA2R1 (yellow) are shown on the right. Each dot indicates an individual gene transcript. Scale bars, 50μm. (G) Spatially-resolved expression of nephron segment genes (Control2). Immunofluorescence staining for panCK (green) and cell surface marker B2M/CD298 (red) is shown on the left. Transcriptionally-defined proximal convoluted tubule (PCT, red), thick ascending loop of Henle (LOH, gray), and connecting tubule (purple) cells is shown in the middle. Expression of PCT gene GPX3 (orange), thick ascending LOH gene EGF (gray), and connecting tubule genes AQP3 (red) and CALB1 (blue) is shown on the right. Each dot indicates an individual gene transcript. Scale bars, 50μm.

Figure 2. Spatial distribution of infiltrating immune cells in cLN

(A) Immune cell landscape in controls versus cLN. Immune cell infiltrates as % of total cells (top). Relative proportions of immune cell subsets (bottom). (B) Immune cell subsets are shown as % of total cells in controls (blue; n = 4) versus cLN (red; n = 10). Each dot indicates an individual sample and line indicates the median. * P<0.05, ** P<0.01, by two-tailed Mann-Whitney test. (C) Corresponding images showing major kidney cell types (top) and computationally-defined anatomical regions (bottom). Each dot indicates an individual cell colored by cell type (top) or spatial location (bottom). (D) Distribution of kidney stromal cells within the indicated kidney anatomical regions. Data generated using all healthy control (n=4) and cLN (n = 10) kidney samples. N per cell type is in Supplementary data file S1. (E) Proportion of infiltrating immune cells in cLN located within the 4 spatial kidney regions. N per cell type is in Supplementary data file S1. (F) Proportion of macrophages located inside (red) or bordering (grey) glomeruli versus tubulointerstitium (blue) in controls versus cLN. *P<10⁻¹⁶, by chi-squared test. (G) Total number of macrophages in/bordering glomeruli in individual control (n=4) and cLN (n-10) biopsy samples. Each dot equals macrophage number per glomerulus. Boxes show interquartile ranges over each tissue’s glomeruli. P values comparing each cLN sample with combined healthy control data are shown above the respective cLN box plots. Statistics calculated using a linear mixed model with tissue identification as a random effect. (H) Representative patterns of macrophage spatial distributions: i) macrophages (red) diffusely distributed in tubulointerstitium in control kidney; ii) cLN macrophages trafficking to glomeruli (dashed circle) in cLN; iii and iv) macrophages surrounding glomeruli (arrows) or clustered within tubulointerstitial immune hotspot (star) in cLN. (I) B cells (light blue) and plasma cells (dark blue) are shown within tubulointerstitial immune hotspots (arrows) in cLN. Macrophages (red) are also enriched within B cell/plasma cell foci. (J) Spatial distribution of CD4⁺ T cells (orange; arrows) and CD8⁺ T cells (pink; arrowheads) in cLN. (H-J) Scale bars, 200μm.

Figure 3:. Spatially resolved gene expression in cLN macrophages.

(A) Volcano plot showing DEG in cLN macrophages residing inside glomeruli versus within tubulointerstitium (n = 7843 macrophages). (B) Annotated cLN glomerulus showing expression of representative “glomerulus specific” genes by glomerular macrophages residing adjacent to mesangial cells and glomerular endothelial cells. Each colored dot represents a gene transcript. Blue, DAPI. Red, CD298/B2M segmentation marker. Scale bar, 10μm. (C) Representative images showing increased “glomerular gene score” expression by macrophages residing within cLN glomeruli (dashed borders) versus tubulointerstitial macrophages in controls (arrowheads). Each dot represents a macrophage annotated by metagene expression. Representative control sample (Control2) is shown on the left. Two representative cLN samples (SLE3, SLE6) are shown in the middle and on the right, respectively. Scale bar, 250 μm. (D) Reanalysis of AMP LN dataset (n = 466 myeloid cells) (5) showing “glomerular gene score” expression by myeloid subpopulations in adult LN. *** P<0.001, **** P<0.0001, by one-way ANOVA, followed by Tukey’s multiple comparison test. (E) Multiplexed ion beam imaging (MIBI) staining of representative control (Control2, left) and cLN (SLE3, right) glomeruli showing CD68 (green) and CD163 (red) (arrowheads) protein staining in cLN (dashed circle delineates a glomerulus). Blue, dsDNA. Scale bars, 100μm.

Figure 4:. Gene expression and cell interaction patterns within immune hotspots.

(A) UMAP of B cell lineage subclusters B.1 – B.4 (filtered for cells with >80 RNA counts, n = 2,040). (B) Gene expression of selected genes of interest in B cell subclusters. (C) Relative proportions of B cell subclusters in control and cLN. *P<0.05, by chi-squared test. (D) Histograms depicting number of the 50 closest cells to each B cell subset that are immune cells (color coded by B cell location and divided by disease status and B cell subcluster). (E) Annotated representative cLN kidney (SLE4) showing clusters (solid arrows) of B cells, plasma cells, macrophages, and rare CD4⁺ T cells located with tubulointerstitium. Dashed arrows indicate glomeruli. (F) Immunoglobulin gene transcripts (red=scaled gene expression) in a cLN plasma cell focus (SLE4). (G) UMAP of batch-corrected scRNA-seq data showing overlap of CosMx B cell subclusters (B.1 to B.4; n = 2897) and published AMP RA/SLE B cell subsets (CB0, CB1, CB2a, CB3, n = 418). Each dot represents a single cell, color coded by B cell subcluster. (H) Graphs of significantly enriched interactions between immune cell types, defined by cell centroids existing within 100 μm of one another. Edges indicate an interaction between two cell types occurring more frequently than expected given a hypergeometric distribution of randomly interacting cells (Benjamini-Hochberg (BH)-adjusted p < 0.05). Cells are colored by lineage and edge thickness is proportional to the ratio of observed:expected number of interactions across healthy controls (n interactions across all cells = 7,262) or patients with cLN (n = 234,055). (I) Volcano plots showing DEG in cLN macrophages residing inside versus outside “immune hotspots”. Colored dots represent genes upregulated (red) and downregulated (blue) in cLN “immune hotspots”. Threshold: Log₂(fold change) <−0.3 or >0.3, P<0.05. False Discovery Rate (FDR) < 0.05.

Figure 5:. Rituximab-resistant B cell foci in treatment-resistant cLN.

(A) Diagram depicting immunosuppressive therapies and timing of serial kidney biopsies in individual SLE8. Stars indicate individual doses of rituximab (1000 mg, red), intravenous (IV) methylprednisolone (500 mg, yellow), and cyclophosphamide (900–1200 mg, blue). Bars indicate duration of oral medication use. (B) Trajectory of clinical biomarkers of cLN disease activity and (C) circulating CD19⁺ B cell numbers in SLE8. Each dot indicates an individual laboratory value. Stars indicate values above/below the limit of detection. Dashed lines indicate normal range for clinical assay. (D) Intrarenal B cells (black circle) and plasma cells (open circle), as percentage of total kidney cells, as determined by CosMx spatial transcriptomics, in 3 serial biopsies from individual SLE8. Grey bars indicate %B cells and %plasma cells in control kidney. (E) Representative CosMx images from individuals SLE8.1 and SLE8.3 showing persistent foci of tubulointerstitial B cells (orange) and plasma cells (blue). SLE8.1 was obtained prior to rituximab treatment and SLE8.3 post-rituximab in the setting of circulating B cell depletion. B2M/CD298 (red); PanCK (green). Scale bars, 200 μm. (F) PAX5 immunohistochemistry staining for post-rituximab tubulointerstitial B cells in SLE8.3. Arrowheads indicate PAX5⁺ B cells. Scale bar, 100 μm.

Figure 6:. Lupus nephritis is characterized by altered gene expression in resident glomerular cells.

(A) Volcano plots showing DEG in glomerular endothelial cells (left), mesangial cells (middle), and podocytes (right). Red dots represent genes upregulated in cLN and blue dots represent genes upregulated in controls. Threshold: Log₂(fold change) <−0.5 or >0.5; P<0.05. (B) Gene ontology (GO) analysis showing enriched Reactome pathways in resident glomerular cells. The color scale for each dot represents the P value adjusted for multiple hypotheses as standard for pathway analysis, and dot size indicates the number of DEG represented in each pathway. (C) Volcano plot showing number of DEGs in glomerular endothelial cells (upper), mesangial cells (middle), and podocytes (lower) as a function of whether cell populations reside within control glomeruli (Control), cLN glomeruli exhibiting no pathologic changes (No path), or cLN glomeruli exhibiting any lupus-associated morphologic changes (Path). Red dots represent genes upregulated in cLN and blue dots genes upregulated in controls. Threshold: Log₂(fold change) <−0.5 or > 0.5; P<0.05. (D) UMAP of individual glomeruli colored by patient ID (left), slide number and CosMx batch number (middle), and presence or absence of lupus-defining histopathology (right). Each dot indicates an individual glomerulus.

Figure 7:. Spatially correlated gene modules in cLN.

(A) InSituCor connection graph showing gene pairs with spatial correlations > 0.1. Each point represents a gene. Node color shows clusters of mutually correlated genes. (B) Spatial correlation network for interferon stimulated gene (ISG) module. (C) ISG module expression in cellular neighborhoods. For each tissue, the FOVs with the maximum (top) module scores are shown. Glomeruli are outlined in cyan. Scale bars, 250μm. (D) Spatial correlation network for two macrophage-associated modules (“Complement cluster”, red; “Class II HLA cluster”, blue). (E) Class II HLA and complement module expression in cellular neighborhoods. Regions of increased complement module expression are shown in blue, class II HLA module in red, and high expression of both modules in pink. For each tissue, the FOVs with the maximum (top) sum of these two module scores are shown. Glomeruli are circled in yellow. Scale bars, 250μm.

Figure 8:. cLN is characterized by spatially correlated expression of fibrosis-associated genes.

(A) Spatial correlation network for fibrosis-associated gene module, calculated across all cells from control (n=4) and cLN (n=10) tissue samples. (B) Heatmap showing expression of individual fibrosis module genes by kidney cell type. (C) Fibroblasts (as % of total cells) in controls (blue) versus cLN (red). Each dot indicates an individual sample and line indicates the median. ** P<0.01, by two-tailed Mann-Whitney test. (D) Fibrosis module expression in cellular neighborhoods. For each tissue, the FOVs with the maximum (top) module scores are shown. Glomeruli are circled in cyan. Scale bars, 250μm. (E) Mean fibrosis module score per cell in control (n=4) and cLN (n=10) tissue samples. **** P<0.0001, by two-tailed t-tests comparing fibrosis module scores in single cells from each cLN sample vs. single cells from all controls. (F) Masson trichrome stain of representative fields from the three cLN samples with the highest “fibrosis gene module” expression (SLE4, SLE6, SLE7). Scale bars, 300μm.