Single-cell analysis reveals immune remodeling of monocytes, NK cells, T cell exhaustion, and Galectin-9–associated depletion of gamma delta and mucosal-associated invariant T cells in Long COVID with ME/CFS

Shima ShahbazNajmeh BozorgmehrAmirhossein RahmatiAmal AboudaHussain SyedMohammed OsmanShokrollah Elahi^*

• University of Alberta, Edmonton, Canada

The cellular mechanisms underlying Long COVID (LC) associated with myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) remain poorly understood. We performed single-cell RNA sequencing (scRNA-seq) on peripheral blood mononuclear cells collected 12 months after acute COVID-19 infection from female individuals with LC-ME/CFS and recovered (R) individuals. Comparative analysis was also performed using publicly available scRNA-seq datasets from idiopathic ME/CFS patients. Based on transcriptional signatures, LC-ME/CFS patients exhibited a marked reduction in naïve CD4⁺ and CD8⁺ T cells, regulatory T cells, MAIT cells, and γδ T cells, accompanied by an expansion of effector T cells. NK cells displayed reduced frequency and altered activation-associated transcriptional factors, consistent with impaired cytotoxic potentials. B cells in LC patients exhibited gene expression profiles indicative of heightened activation, while plasma cells revealed a distinct transcriptional subset expressing NK-associated genes. Platelets and low-density neutrophils were expanded and exhibited enrichment of activated-related transcripts. Monocyte subsets demonstrated transcriptional skewing characterized by reduced expression of phagocytosis-associated genes and increased expression of pro-inflammatory cytokine-related genes/pathways. In contrast, idiopathic ME/CFS patients exhibited less pronounced immune alterations at the transcriptional level: while T cell activation was evident, there was no reduction in MAIT or NK cells, nor signs of T cell exhaustion. Notably, FOXP3 expression was upregulated, and B cells and platelets demonstrated dysregulated signatures in idiopathic ME/CFS. Mechanistically, we identify Galectin-9–TIM-3 interaction as a potential pathway driving γδ and MAIT cell depletion in LC. Our results reveal extensive peripheral immune remodeling in LC-ME/CFS, distinct from idiopathic ME/CFS, and support a model of chronic immune activation and dysregulation. Our findings offer a cellular framework for understanding LC pathogenesis and point to potential biomarkers and therapeutic targets for intervention.