Single-cell transcriptomics reveals monocytic glycolytic reprogramming in lymphoma-associated hemophagocytic lymphohistiocytosis Free

Background: Lymphoma-associated hemophagocytic lymphohistiocytosis (LAHS) is a life-threatening hyperinflammatory syndrome with poorly defined mechanisms distinguishing it from non-malignancy-associated HLH (NLAHS). Elucidating LAHS-specific immune signatures is crucial for improved diagnosis and therapy.

Methods: Single-cell RNA sequencing (scRNA-seq) was performed on peripheral blood mononuclear cells from 4 LAHS patients (2 diffuse large B-cell lymphoma, 2 peripheral T-cell lymphoma), 3 NLAHS patients (1 EBV-HLH, 1 macrophage activation syndrome, 1 familial HLH ), and 2 healthy controls (NC). Differential gene expression analysis identified LAHS signatures. Gene Set Variation Analysis (GSVA) and metabolic pathway scoring were applied. Key findings were validated by real-time quantitative PCR (qPCR) in an expanded cohort (LAHS n=13, NLAHS n=5, non-HLH lymphoma n=5, NC n=7) and targeted plasma metabolomics in an independent cohort (LAHS n=21, NLAHS n=11, non-HLH lymphoma n=5, NC=7).

Results: HLH patients (LAHS and NLAHS) exhibited significant expansion of monocytes, neutrophils, and proliferating T cells compared to NC, while monocyte proportions were comparable between LAHS and NLAHS. 79 unique LAHS specific differentially expressed genes (DEGs) were identified. These DEGs were highly enriched within monocytes, with significantly higher monocyte gene scores in LAHS vs. NLAHS or NC. GSVA of the 79 DEGs in monocytes showed top enrichment for acidification (lysosomal, Golgi ), pH regulation, and the glycolytic pathway.

Monocyte subsets included classical, non-classical, PTTG1hi classical, ZFP36L1hi classical , S100A8hi classical and dendritic cells (DCs). The PTTG1hi classical monocyte subset was predominantly enriched in LAHS and exhibited significantly elevated glycolytic pathway scores. Cytokine activity scores and cytokine receptor scores were comparable between LAHS and NLAHS monocyte.

scRNA-seq demonstrated high expression of glycolytic genes (e.g., SLC2A3, PKM, LDHA) in LAHS monocytes. qPCR validated significant upregulation of these key glycolytic genes in LAHS. Metabolomics demonstrated significantly elevated plasma lactate (P=0.011) and pyruvate (P=0.011) in LAHS.

Conclusion: Integrated scRNA-seq and metabolomic analysis identifies monocyte glycolytic reprogramming as a defining feature of LAHS. Enrichment of acidification/glycolysis pathways and the discovery of a highly glycolytic PTTG1hi monocyte subset in LAHS highlight aberrant monocyte metabolism as a critical pathogenic mechanism. These findings reveal novel diagnostic biomarkers and potential therapeutic targets (e.g., glycolytic inhibition) for LAHS.