1598: Single-cell Spatial Proteomics Identifies Intraglomerular Myeloid Cells in Membranous Lupus Nephritis

Chen-Yu Lee¹, Caleb Marlin², Xiaoping Yang¹, Tayte Stephens², Alessandra Ida Celia³, Jeff Hodgin⁴, Peter Izmirly⁵, H Michael Belmont⁶, Jill Buyon⁷, Chaim Putterman⁸, Judith James², the Accelerating Medicines Partnership in RA/SLE⁹, Michelle Petri¹⁰, Joel Guthridge², Avi Rosenberg¹¹ and Andrea Fava¹, ¹Johns Hopkins University, Baltimore, MD, ²Oklahoma Medical Research Foundation, Oklahoma City, OK, ³John Hopkins University of Medicine, Rome, Italy, ⁴University of Michigan, Ann Arbor, MI, ⁵New York University School of Medicine, New York, NY, ⁶NYU School of Medicine, New York, NY, ⁷NYU Grossman School of Medicine, New York, NY, ⁸Albert Einstein College of Medicine, Bronx, NY, ⁹Multiple, Multiple, ¹⁰Department of Medicine, Division of Rheumatology, Johns Hopkins University School of Medicine, Timonium, MD, ¹¹Department of Pathology, The Johns Hopkins University School of Medicine, Baltimore, MD

Background/Purpose: Lupus nephritis (LN) leads to end-stage kidney disease (ESKD) in more than 20% of patients despite optimal treatment. Up to 30% of LN patients have membranous LN which is characterized by subepithelial immune deposits without immune infiltration in the glomeruli. Despite its association with ESKD, membranous LN is considered a milder type of LN with no consensus on the optimal use of immunosuppression. To develop mechanistic hypotheses of disease, we analyzed kidney samples from patients with LN using a whole slide spatially resolved proteomic approach as part of the Accelerating Medicines Partnership in RA/SLE. We report here the initial analysis.

Methods: We developed a serial immunohistochemistry (sIHC) staining workflow to stain for 18 antibodies, DNA, and PAS to be visualized on a single section via a cycle of staining, imaging, and destaining. This included incubation of FFPE slides with primary antibody, secondary HRP reagents, AEC-Red Chromogen, and Hematoxylin. Image processing was performed using HALO (Indica Labs) and included deconvolution of single-color channels, registration, fusion, cell segmentation, and automated tissue classification (glomeruli, tubulointerstitium, connective tissue, vasculature, background, and biopsy edges). To minimize batch effect, the analytical pipeline included within sample CLR-normalization and scaling, followed by harmonization (Harmony).

Results: In this initial analysis, we included 29 clinically indicated kidney biopsies classified as LN (13 pure proliferative, 10 pure membranous, 5 mixed, and 1 ISN class II). Patients were 79% female, 34% White, 31% Black, 10% Asian, and 24% identified with Other race/ethnicity. We detected 182,783 CD45+ cells out of 1,913,845 cell objects. Our analysis identified 10 immune cell clusters at low resolution (Figure 1A). Figure 1B displays the tissue distribution of each cell subset. B and T lymphocytes dominated the tubulointerstitium. The CD68+ myeloid subsets were the predominant cell type in the glomeruli (Figure 2). More than half of CD68+ cells expressed Ki67 indicating active proliferation. Surprisingly, we identified intraglomerular CD68+ cells (including endocapillary) also in patients with pure membranous LN, but at a lower tissue density than proliferative LN (Figure 2). Figure 3 demonstrates intraglomerular CD68+ cells in a biopsy classified as pure membranous LN by two experienced renal pathologists.

Conclusion: sIHC can be successfully employed to perform multiplexed whole slide analysis harnessing both the subcellular resolution (brightfield) and the reliability of IHC. Our analysis revealed intraglomerular CD68+ myeloid cells in pure membranous LN. By traditional clinical pathology, intraglomerular / endocapillary immune cells characterize proliferative LN and are not consistent with pure membranous LN. These findings implicate macrophages/monocytes in the glomerular disease in membranous LN with therapeutic implications. The analysis of 90 additional biopsies and a myeloid-focused panel is underway to validate and extend these findings.








C. Lee: None; C. Marlin: None; X. Yang: None; T. Stephens: None; A. Celia: None; J. Hodgin: AstraZeneca, 5, 6, Eli Lilly, 5, Gilead, 5, Janssen, 5, Moderna, 5, Novo Nordisk, 5, Regeneron, 5; P. Izmirly: None; H. Belmont: Alexion, 6, Aurinia, 6; J. Buyon: Bristol-Myers Squibb(BMS), 2, GlaxoSmithKlein(GSK), 2, Related Sciences, 1; C. Putterman: Equillium, 2, KidneyCure, 1, Progentec, 2; J. James: Bristol-Myers Squibb(BMS), 5, GlaxoSmithKlein(GSK), 2, Novartis, 2, Progentec Biosciences, 5; t. Accelerating Medicines Partnership in RA/SLE: None; M. Petri: Alexion, 1, Amgen, 1, AnaptysBio, 1, Annexon Bio, 1, Argenx, 1, Arhros-Focus Med/Ed, 6, AstraZeneca, 1, 5, Aurinia, 1, 5, 6, Axdev, 1, Biogen, 1, Boxer Capital, 2, Cabaletto Bio, 2, Caribou Biosciences, 2, CVS Health, 1, Eli Lilly, 1, 5, Emergent Biosolutions, 1, Exagen, 5, Exo Therapeutics, 2, Gilead Biosciences, 2, GlaxoSmithKlein(GSK), 1, 5, 6, Horizon Therapeutics, 2, Idorsia Pharmaceuticals, 2, IQVIA, 1, Janssen, 1, 5, Kira Pharmaceuticals, 2, MedShr, 6, Merck/EMD Serono, 1, Momenta Pharmaceuticals, 2, Nexstone Immunology, 2, Nimbus Lakshmi, 2, Proviant, 2, Sanofi, 2, Sinomab Biosciences, 2, Thermofisher, 5, UCB, 2; J. Guthridge: None; A. Rosenberg: None; A. Fava: Annexon Biosciences, 2, Sanofi, 1.