Deep Profiling of Mouse Splenic Architecture with CODEX Multiplexed Imaging

TLDR

CODEX, a multiplexed cytometric imaging platform that uses DNA‑tagged antibodies, fluorescent dNTP analogs, and in situ polymerization indexing, was applied to normal and lupus murine spleens, and an algorithmic pipeline quantified single‑cell antigen expression and spatial neighborhoods to map lymphoid architecture. The study revealed that cellular neighborhoods profoundly influence immune receptor expression, uncovering extensive, previously uncharacterized splenic cell‑interaction dynamics between healthy and MRL/lpr lupus spleens, demonstrating the fidelity of multiplexed spatial cytometry for systemic tissue characterization.

Abstract

Highlights•Autoimmunity analyzed by multiplexed DNA-tagged antibody staining (CODEX)•CODEX data reveal pairwise interactions and niches changing with disease•First tier of neighbors significantly impacts marker expression in the index cells•Changes in splenic morphology correlate with shifts in cell frequenciesSummaryA highly multiplexed cytometric imaging approach, termed co-detection by indexing (CODEX), is used here to create multiplexed datasets of normal and lupus (MRL/lpr) murine spleens. CODEX iteratively visualizes antibody binding events using DNA barcodes, fluorescent dNTP analogs, and an in situ polymerization-based indexing procedure. An algorithmic pipeline for single-cell antigen quantification in tightly packed tissues was developed and used to overlay well-known morphological features with de novo characterization of lymphoid tissue architecture at a single-cell and cellular neighborhood levels. We observed an unexpected, profound impact of the cellular neighborhood on the expression of protein receptors on immune cells. By comparing normal murine spleen to spleens from animals with systemic autoimmune disease (MRL/lpr), extensive and previously uncharacterized splenic cell-interaction dynamics in the healthy versus diseased state was observed. The fidelity of multiplexed spatial cytometry demonstrated here allows for quantitative systemic characterization of tissue architecture in normal and clinically aberrant samples.Graphical abstract