Abstract Introduction Sarcoidosis is an inflammatory disorder characterized by granuloma formation resulting from persistent activation of macrophages and dendritic cells. In cardiac sarcoidosis, granulomas cause structural damage and disrupt electrical conductivity. While T-cell immune checkpoints such as cytotoxic T-lymphocyte–associated protein 4 (CTLA-4) have been implicated in peripheral sarcoidosis, their role in cardiac tissue remains unknown. This study investigates CTLA-4 pathway activation within the transcriptomic landscape of cardiac sarcoidosis and validates CTLA-4 protein expression in affected human heart tissue. Methods Online single-nucleus RNA-sequencing (snRNA-seq) datasets from cardiac sarcoidosis patients and healthy controls were integrated and analyzed after stringent quality control and normalization. Clustering and annotation were based on canonical marker genes. Differential gene expression between groups was assessed using MAST, adjusted for dataset source, patient, and unique molecular identifiers. Gene Ontology (GO) enrichment analysis was performed to assess associated processes. Formalin-fixed paraffin-embedded (FFPE) cardiac tissue samples (n = 4) from sarcoidosis patients were stained for CTLA-4, CD28 (activating counterpart of CTLA-4), and CD68 (pan-macrophage marker) by immunofluorescence and visualized using confocal microscopy. Results Analysis of 45,442 nuclei (20,532 healthy, 24,910 sarcoidosis) identified macrophages as the predominant immune population. CTLA-4 and CD28 expression were significantly upregulated in sarcoidosis macrophages (CTLA-4 log2FC 3.60, p 0.05; CD28 log2FC 0.12, p 0.01). Among four macrophage clusters, inflammatory HLADR+ and SYTL3+ macrophages specifically expressed CTLA-4, whereas resident CD163+ macrophages expressed CD28. GO analysis revealed CTLA-4+ macrophages were enriched in activation and proliferation pathways, consistent with an M1-like phenotype, while CTLA-4− macrophages showed M2-like homeostatic and fibrotic signatures. In patient cardiac tissue, immunofluorescence confirmed CTLA-4 protein expression in CD68+ macrophages forming compact granulomas, with broader and stronger CTLA-4 staining compared to CD28. Conclusion This study identifies CTLA-4 as a marker of highly inflammatory macrophages in cardiac sarcoidosis, linking this immune checkpoint to active or expanding granulomas. CTLA-4+ macrophages display M1-associated activation, whereas CD28+ macrophages exhibit M2-like fibrotic characteristics. Beyond its known T-cell function, CTLA-4 may serve as a macrophage-specific marker for active cardiac sarcoidosis and a potential indicator of granuloma phase progression. These findings support exploration of CTLA-4–targeted modulation as both a diagnostic and therapeutic strategy in cardiac sarcoidosis.
Yousif et al. (Fri,) studied this question.