Immune profiling-based targeting of pathogenic T cells with ustekinumab in ANCA-associated glomerulonephritis

Abstract

Antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis is a life-threatening autoimmune disease that often results in kidney failure caused by crescentic glomerulonephritis (GN). To date, treatment of most patients with ANCA-GN relies on non-specific immunosuppressive agents, which may have serious adverse effects and be only partially effective. Here, using spatial and single-cell transcriptome analysis, we characterize inflammatory niches in kidney samples from 34 patients with ANCA-GN and identify proinflammatory, cytokine-producing CD4⁺ and CD8⁺ T cells as a pathogenic signature. We then utilize these transcriptomic profiles for digital pharmacology and identify ustekinumab, a monoclonal antibody targeting IL-12 and IL-23, as the strongest therapeutic drug to use. Moreover, four patients with relapsing ANCA-GN are treated with ustekinumab in combination with low-dose cyclophosphamide and steroids, with ustekinumab given subcutaneously (90 mg) at weeks 0, 4, 12, and 24. Patients are followed up for 26 weeks to find this treatment well-tolerated and inducing clinical responses, including improved kidney function and Birmingham Vasculitis Activity Score, in all ANCA-GN patients. Our findings thus suggest that targeting of pathogenic T cells in ANCA-GN patients with ustekinumab might represent a potential approach and warrants further investigation in clinical trials.

Fig. 1. Study overview.

34 patients from the Hamburg GN Registry underwent diagnostic kidney biopsy and multi-OMIC high dimensional immune profiling. Based on these results drug prediction revealed ustekinumab as the strongest candidate for treatment of ANCA-GN. Subsequently, 4 patients with severely relapsing ANCA-GN were treated with ustekinumab and followed up for 26 weeks.

Fig. 2. Spatial transcriptome analysis of the ANCA-GN exploratory group.

a Left, Representative section of an H&E-stained kidney biopsy. Right, Spatial distribution of renal compartments overlaid on the representative section. b UMAP embedding displaying annotated renal compartments across 10,763 spots from all ST slides. See Supplementary Fig. 1c for the expression of cell type markers in annotated compartments. c Barplots showing the composition of renal compartments in the control (21,420 spots) and ANCA-GN exploratory group. The significance of the difference in composition was assessed with differential population analysis and is presented in Supplementary Data 2. d Top 10 enriched gene ontology terms in inflamed compartments. Count: number of DE genes in the term. Gene ratios: ratio of the number of DE genes in the term to the number of all DE genes. The colors show adjusted p-values (p.adjust) computed using the enrichGO function from R package clusterProfiler with right-tailed Fisher’s exact test and Benjamini–Hochberg multiple test correction. e Scores of alpha-beta T cell-related gene ontology terms computed using GSVA in different renal compartments. f Graph showing the spatial proximity of renal compartments and the enrichment of T cell activation. The node sizes and edge widths are proportional to compartment size and spatial proximity, respectively. The nodes are colored by an increasing Th1 and Th17 cell differentiation term scores computed using GSVA. PT, proximal tubules. LOH, loop of Henle. DCT distal convoluted tubules, CNT connecting tubules, PC principal cells, IC intercalated cells. Source data are provided as a Source Data file.

Fig. 3. Single T cell transcriptome analysis and drug prediction.

a UMAP projection and cluster annotations of the combined human single cell atlas of renal and blood T cells. In total 72,416 cells are shown of which 22,187 stem from the kidney and 50,229 from the blood. See Supplementary Fig. 2b for the expression of cell type-specific marker genes. b Combined type 1-3 cytokine expression score (type 1: IFNG, TNF, IL2, IL18, LTA, CSF2; type 2: IL4, IL5, IL9, IL13; type 3: IL17A, IL17F, IL22, IL26) per cell overlayed on the UMAP projection. The positions of CD4⁺ and CD8⁺ T effector cell clusters are highlighted manually. The detailed expression per cytokine type and cell type is shown in Supplementary Fig. 3a. c Relative tissue composition per cell type. The frequencies are computed separately for blood and kidney cells, i.e., both sides add up to 100%. d CD4⁺ and CD8⁺ T effector cell subsets and their relative proportions. Both T effector cell subsets show some overlap due to the proximity of their respective expression profiles, e.g., the CD4⁺ Teff cluster contains a small proportion of CD8⁺ Tc1-like cells. The marker gene expression is detailed in Supplementary Fig. 3b, c. e Distribution of Teff cell subsets within the non-inflamed and inflamed compartments. For detailed proportions in individual non-inflamed compartments, see Supplementary Fig. 3e. Boxplots show the median (middle horizontal line), interquartile range (box), Tukey-style whiskers (lines beyond the box), outliers (data points beyond 1.5*interquartile or below—1.5*interquartile) for proportion of Teff subsets in 10,763 spots from all ANCA ST slides. f Ranking of drugs based on their interaction scores within Teff single cells, with colors representing their interaction scores specifically within inflamed renal compartments. Source data are provided as a Source Data file.

Fig. 4. Immune profiling of renal T cells.

a Representative flow cytometry plot showing the identification of chemokine receptor expression from cells isolated from biopsy samples of patients with ANCA-GN (exploratory cohort, n = 22). b Quantification of chemokine receptor expression CXCR3 (Th1/Tc1) and CCR6 (Th17/Tc17) from renal CD3⁺ T cells. Violin plots show mean, symbols represent individual data points. (n = 22). c Representative immunofluorescence staining of chemokine receptors CXCR3 and CCR6 on CD3⁺ T cells in human kidney tissue of ANCA-GN. Lower row zoomed in areas. Source data are provided as a Source Data file.

Fig. 5. Clinical outcome of the ustekinumab treatment cohort.

a Course of serum creatinine and albuminuria during ustekinumab treatment. Black arrowheads indicate cyclophosphamide and green arrowheads ustekinumab administration. Gray bands indicate low dose remission maintenance therapy with either AZA or MMF. b BVAS, ANCA levels measured via ELISA and CRP levels at baseline and 6 months after initiation ustekinumab treatment (n = 4). (CYC cyclophosphamide; AZA azathioprine; MMF mycophenolate mofetil; BVAS Birmingham Vasculitis Activity Score; CRP C-reactive protein; ACR albumin creatinine ratio). Source data are provided as a Source Data file.