Resolving the renal microenvironment: a 5-plex immunofluorescence workflow to quantify B-lineage cells in FFPE lupus...

Accurate identification and spatial enumeration of B lineage cells in formalin-fixed, paraffin-embedded (FFPE) lupus nephritis (LN) kidney tissue is critical for understanding disease pathogenesis and CD20-directed therapeutic responses. We developed a targeted 5-plex immunofluorescence and digital image analysis workflow for simultaneous enumeration of B cells, plasma cells (PCs), and plasmablasts (PBs) in FFPE human tissues that is amenable to deployment in LN clinical trials. We validated this workflow on two tonsils and eight LN biopsy tissue blocks. Comparison of B lineage markers confirmed that CD79a provides far superior sensitivity for interstitial B cells compared to CD19, establishing it as the requisite anchor for B cell burden assessment in FFPE lupus nephritis tissue. Accordingly, B cells were defined as CD79a⁺/CD138^-; PCs as CD138⁺/CD38⁺/Ki-67^-; and PBs as CD138⁺/CD38⁺/Ki-67⁺. These definitions ensured unambiguous cell classifications, overcoming the challenge of variable CD138 expression in renal tubular epithelium. Whole slide analysis of LN tissues revealed comparable average frequencies of B cells versus PCs (~250 cells/mm²), with far fewer PBs (~14 cells/mm²). Comparison with an exploratory "permissive" gating strategy (CD38⁺/Ki-67⁺) confirmed the absence of CD138^- B-lineage plasmablasts, validating the sufficiency of CD138 for tissue ASC enumeration. Most PCs and PBs were CD79a⁺, indicating the retention of a functional B-cell program that may contribute to disease pathogenesis. This robust, validated, fit-for-purpose methodology is poised for deployment in larger clinical LN cohorts to evaluate local tissue impacts of B-cell depletion therapies, deepening our understanding of disease pathogenesis and treatment responses.