Single-Cell Analysis Reveals an Exhaustion of TEM and CTL CD8+ T Cells in Hemophagocytic Lymphohistiocytosis with EBV Infection

Background

Hemophagocytic lymphohistiocytosis (HLH) is a relatively rare and lethal syndrome characterized by uncontrolled hyperinflammatory reaction, often associated with Epstein-Barr virus (EBV) infection, which significantly complicates treatment approaches, posing challenges in studying T cell function across different stages of disease. T cell exhaustion is well-documented in chronic viral infections, yet its specific role remains unidentified in HLH with persistent EBV infection and inflammation.

Methods

We analyzed peripheral blood single-cell RNA sequencing (scRNA-seq) data from 15 individuals, including 3 healthy controls and 6 HLH patients with EBV infection, categorized into acute and remission phases. In total, 125, 737 cells including 30 cellular subtypes were analyzed. EBV-DNA level in different lymphocyte subsets was determined by flow cytometry and quantitative polymerase chain reaction (qPCR).

Results

We found that EBV infected various cellular subtypes in hematopoietic system including a minor population of hematopoietic stem cells (HSCs). Compared to individuals with EBV-infected B cells or CD4⁺ T cells, more cells expressing EBV-related genes were found in those with EBV-infected natural killer (NK)/NKT cells. Interestingly, there was a rapid increase in cells expressing EBV-related genes in individuals experiencing HLH relapse compared to those at initial diagnosis, particularly among those who progressed to lymphoblastic leukemia, where copy number variation (CNV) in lymphocyte cells often occurs. Specifically, CD8⁺ T effector memory (TEM) cells and cytotoxic T cells (CTL) in patients exhibited terminal exhaustion state characterized by increased expression of PDCD1, TIGIT, LAG3, HAVCR2, and TOX in HLH with EBV infection as compared to controls. Although improvement of exhaustion state was observed during disease remission in some cases, levels of exhaustion markers remained significantly higher than in healthy individuals. Furthermore, lymphocytopenia and a relative increase in myeloid cells persisted even after disease remission. Notably, myeloid cell subtypes, particularly monocytes and dendritic cells, exhibited suppressed antigen presentation and reduced cytokine secretion.

Conclusion

Through comprehensive analysis of PBMCs in HLH, we found that EBV may induce CD8+ TEM and CTL exhaustion and suppress the function of antigen presentation cell, contributing to HLH pathogenesis. Persistent or recurrent EBV elevation emerges as a pivotal factor in HLH relapse and progression. These insights offer crucial data for understanding and potentially targeting the mechanisms underlying this rare disease.