Spatial Transcriptomics Reveals Compartment-Specific Immune Activation Signatures in Ileal and Lymph Node Tissue in Treated HIV Infection

People with HIV (PWH) on long-term antiretroviral therapy (ART) continue to experience elevated rates of morbidities and mortality driven by persistent immune activation despite viral suppression. Known contributors include low-level HIV provirus activity, microbial translocation in part from epithelial barrier dysfunction, microbiome dysfunction, and co-infections. However, how these interact and where they predominate across tissue compartments remains incompletely defined. Here, we applied spatial transcriptomics to characterize compartment-specific transcriptional programs in ileum (epithelium, Peyer's patches, lamina propria) and inguinal lymph nodes (B Cell follicles and T cell zone) from ten PWH on long-term ART, stratified by CD4/CD8 ratio into low-ratio and high-ratio groups, with low-ratio as a proxy for immune activation and increased risk for non-AIDS related serious event. Comparison of global expression found significant differences between groups in four of five compartments. Differential expression analysis identified 483 differentially expressed genes across four of five compartments, with the greatest burden in the T-cell zone and none in the lamina propria. Gene set enrichment analysis identified 116 enriched pathways predominantly in the low-ratio group, spanning immune activation, infection-associated, and metabolic programs, with Peyer's patches showing the broadest transcriptional divergence of any compartment. Cross-compartment signals included higher expression of ORMDL3 and ARL17B in the low-ratio group implicating mitochondrial stress and inflammasome activation, lower expression of CCL3L3 and FCMR in the low-ratio group suggesting impaired immune execution, and divergent ribosomal protein programs between B-cell follicles and the T-cell zone. Cell deconvolution identified compartment-specific differences in estimated immune cell proportions, and T-cell zone gene expression showed significant associations with HIV reservoir measures and plasma markers of microbial translocation and immune activation. Together these findings support spatially heterogeneous immune activation as a feature of persistent immune dysregulation in treated HIV infection and provide compartment-resolved, hypothesis-generating evidence for the tissue-specific mechanisms driving inflammation in this population.