Spatially localized immune metaprograms reveal micro-niche organization in the human Dorsal Root Ganglion.

Dorsal root ganglion (DRG)-targeted local interventions are widely used in pain management; however, the cellular and spatial organization of immune transcriptional states within human DRG tissue remains poorly defined. In particular, it is unclear whether DRG immune activity reflects a single dominant inflammatory axis or a spatially heterogeneous microenvironment composed of parallel transcriptional programs. To address this question, we analyzed publicly available human DRG single-nucleus RNA sequencing data (GSE189501) to identify immune transcriptional metaprograms using rank-stable non-negative matrix factorization (NMF). A curated subset of 388 immune nuclei was analyzed to minimize lineage contamination, and rank selection was evaluated across k = 4-9, with k = 7 selected based on consensus stability metrics. Metaprograms were characterized using gene loading patterns, differential expression between high- and low-scoring cells, and Hallmark gene set enrichment analysis. Identified gene sets were then projected onto human DRG Xenium spatial transcriptomics data (GSE273557) by recalculating program scores from spatial transcript counts, and spatial aggregation of program-high cells was quantified using nearest-neighbor distance analysis with permutation-based significance testing. To evaluate robustness, the program gene signatures were further projected onto an independent human DRG snRNA-seq dataset (GSE168243). Unsupervised NMF identified seven reproducible immune metaprograms that did not converge on a single dominant inflammatory signature but instead represented parallel transcriptional states with distinct gene-loading patterns and functional annotations. One program exhibited receptor- and sensing-associated transcriptional features without strong classical inflammatory enrichment. Spatial projection revealed region-specific enrichment of metaprogram-high cells, and nearest-neighbor analysis demonstrated significant spatial aggregation in selected regions, supporting localized immune micro-niche organization rather than uniform tissue-wide activation. These findings indicate that human DRG immune organization is composed of multiple parallel transcriptional programs with spatially localized enrichment patterns, supporting a model of micro-niche-based immune architecture that may provide biological context for DRG-targeted local interventions.