Characterization of Hemophagocytic Lymphohistiocytosis Associated with Myeloid Neoplasms at Single-Cell Resolution

Hemophagocytic lymphohistiocytosis (HLH) is a life-threatening hyperinflammatory syndrome, which is characterized by fever, hepatosplenomegaly and cytopenia. HLH include Familial HLH (FHL) that is triggered by genetic defects with impaired function of NK cells and cytotoxic T lymphocytes (CTLs) (Carvelli J, et al. Blood, 2020), while secondary HLH (sHLH) which is induced by infection, cancer or autoimmune disease (Rubin TS, et al. Blood, 2017; Noori T, et al. Blood, 2023). Interestingly, myeloid neoplasms (MN) patients generally have worse prognosis with sHLH. However, the definitive clinical outcome and associated pathophysiological mechanisms underlying sHLH with malignancy remain to be fully elucidated.

In this study, we firstly conducted a retrospective analysis of clinical and laboratory data from 16 MN patients with HLH and 48 age-, sex- and diagnosis- matched MN patients without HLH. We found that the overall survival (OS) was significantly lower in the MN-HLH group despite adjustments to the influence of infection factors. Additionally, in the enzyme-linked immunosorbent assay (ELISA) of supernatant from bone marrow (BM) aspirates, MN-HLH group showed higher levels of proinflammatory factors. These indicate that HLH associated with MN may lead to worse outcome through high cytokine levels.

To explore the molecular and cellular mechanisms of HLH in patients with MN, we performed single cell RNA sequencing (scRNAseq) of BM cells from 3 MN-HLH patients and 3 MN patients without HLH. We firstly identified cell types according to their expression of marker genes, including hematopoietic stem and progenitor cells (HSPCs), CD8 T cells, CD4 T cells, NK cells, monocytes and B cells. Notably, we found that the proportion of NK cells and T cells increased significantly, while other immune cells decreased in MN-HLH patients. Interestingly, our next-generation sequencing results showed that none of the FHL-related genes were identified in MN-HLH patients, while their lymphocyte cytotoxicity has reduced, in contrast to the reported FHL phenotypes. These data suggest that the function, degranulation, and cytotoxicity of NK cells in secondary HLH may not be reduced as in FHL. We next subdivided NK cells into 3 major groups, CD56^dimCD16^hi subset, CD56^brightCD16^lo subset and CD69⁺ subset, and found that CD56^dimCD16^hi subset had an increased proportion in MN-HLH group. Importantly, NK cells of MN-HLH group exhibited higher expression of cytotoxic effector genes including perforin (PRF1) and most granzyme (GZMB, GZMA, and GZMH), a potential mechanism for secondary HLH.

Moreover, differentially expressed genes (DEGs) and their functional enrichment between MN and MN-HLH groups of NK cells showed the main enriched pathway in MN-HLH group was inflammation-related pathways, such as interferon-gamma-mediated signaling pathway, positive regulation of tumor necrosis factor production and positive regulation of I-kappaB kinase/NF-kappaB (NF-κB) signaling. In addition, gene set enrichment analysis (GSEA) revealed the enrichment of retinoic acid inducible-gene I (RIG-I) related signaling pathway. RIG-I, as the key gene in RIG-I related signaling pathway, has been reported to contribute in phosphorylation of NF-κB (Zhu H, et al. J Cell Physiol, 2023 ), thus leading to the production of pro-inflammatory cytokines. Further analysis with scRNAseq data showed much higher cytokine gene expressions in MN-HLH group, and the expression of RIG-I showed a correlation with pro-inflammatory cytokines, such as CCL3 and CCL4, that are consistent with our clinical laboratory results. This suggests that the overexpression of RIG-I may regulate diverse inflammation pathways in NK cells tthat give rise to the increase of cytokines in sHLH.

In conclusion, our comprehensive analysis of clinical data, laboratory examination and scRNAseq data of HLH associated with MN, demonstrated the potential key role and mechanism of NK cells via RIG-I signaling pathway regulation unique in sHLH, which may be different from cytotoxicity reduction-related FHL. Further experiments are underway aiming to fully understand the underlying mechanisms and potential clinical treatments of this disease.