Single-Cell RNA Sequencing Reveals Contradictory Immune Microenvironment in Hemophagocytic Syndrome
Authors: Qiu Guo , Xu Xi , Liu Qifa 
Published: December 2023

Hemophagocytic lymphohistiocytosis (HLH), a formidable and rare clinical syndrome impacting multiple organs, manifests as a perilous condition. However, the intricate pathophysiological mechanisms underlying HLH remain yet to be fully elucidated. In this study, we obtained peripheral blood samples from 9 HLH patients spanning the years 2018 to 2022. Employing single-cell RNA sequencing (scRNA-seq), we conducted a comprehensive analysis of the alterations in the immune microenvironment, delving into the role of EBV infection in HLH and its influence on pathogenesis.

By comparing with health donor peripheral blood , we identified a total of 11 significant immune cell populations among 72,779 single-cell transcriptomes, comprising 20,337 cells from healthy controls and 52,442 cells from HLH patients. Notably, we found that the proportion of T cells increased significantly, and other immune cells decreased in HLH patients. In terms of function, we found that the overall T cell killing ability was weakened, and the factor secretion ability was enhanced. Concerning myeloid cells, the impact of CD16+ monocytes on T cells exhibited a pronounced increase, along with an enhanced ability to secrete IL-18 factors, potentially serving as the main source of cells promoting T cell function.

Different subpopulations of T cells have significant changes in the pathogenesis of HLH. Remarkably, both CD4+ T cells and CD8+ T cells exhibited HLA-DR+GZMK+ cell subgroups with upregulated IFN-γ production and response, suggesting their association with the hyperinflammatory state in HLH. A correlation analysis between the proportion of HLA-DRA+ GZMK+ T cells and the severity score revealed a significant correlation (p&lt;0.05), with the ROC curve further affirming their predictability in assessing high inflammatory activity. Among CD4+ T cells, the proportion of Treg cells and the secretion of inhibitory factors such as TGFb1 decreased. Additionally, we noted the presence of a functionally impaired subset of CBLB+CD8+ T cells, indicating the considerable variability in T cell subset functionality in HLH.

Next, a comprehensive analysis of the alterations in myeloid cells, B cells, and NK cells in HLH patients was conducted. We observed a substantial reduction in the number of DC cells and immune-related functions within various myeloid cell subsets. Regarding B cells, an increase in anergy B cells was observed, whereas the population of plasma cells, an effective B cell type, exhibited a decrease. In the context of NK cells, there was a significant increase in the proportion of NK cell subsets associated with IFN-γ secretion.

Moreover, EBV-infected T cells in EBV-associated HLH were identified as the principal source of IFN-γ. Through single-cell EBV-CALL analysis, we found that IFN-γ secretion was primarily attributed to EBV-infected T cells, subsequently influencing CD16+ monocytes. Additionally, IL-16 production was predominantly observed in EBV-infected CD16+ monocytes, thereby impacting lymphocytes. Moreover, Monocytes were stimulated by EBV-infected CD4+ T cells and Atypical T cells, leading to the secretion of a pro-inflammatory factors such as MIF. Furthermore, our investigation unveiled that EBNA2 could promote MYC into the nucleus and enhance the proliferation and IFN-γ secretion of T cells.

In conclusion, our comprehensive analysis of the immune microenvironment changes following the onset of HLH has yielded valuable insights and novel perspectives for understanding the underlying mechanisms and potential clinical treatments of this condition.