# Immune checkpoint TIM-3 defines hyperactivated NK cells and predicts fatal outcome in severe fever with thrombocytopenia syndrome

## TL;DR

The study finds that immune checkpoint TIM-3 on NK cells is linked to severe outcomes in SFTS, suggesting potential biomarkers and therapies.

## Contribution

Identifies TIM-3⁺ NK cells and related biomarkers as novel indicators of fatal SFTS outcomes and therapeutic targets.

## Key findings

- TIM-3⁺ NK cells are significantly increased in SFTS patients, especially in fatal cases.
- Elevated sTIM-3 and sGalectin-9 levels correlate with fatal outcomes in SFTS.
- TIM-3 blockade reduces IFN-γ and TNF-α production, indicating a role in immune hyperactivation.

## Abstract

Severe fever with thrombocytopenia syndrome (SFTS) is a viral hemorrhagic fever with high mortality, primarily driven by immune dysregulation. This study aimed to characterize the expression profile of TIM-3 expression on peripheral NK cells in SFTS, explore the role of TIM-3 ⁺ NK cells in disease progression, and evaluate soluble TIM-3 (sTIM-3) and Galectin-9 (sGalectin-9) as prognostic biomarkers.

Public single-cell RNA sequencing (scRNA-seq) datasets were analyzed using weighted gene co-expression network analysis (WGCNA). Peripheral blood samples from 21 SFTS patients and 14 healthy donors collected at the First Affiliated Hospital, Zhejiang University School of Medicine, were analyzed by flow cytometry, functional assays, and serological assessments. NK cell cytotoxicity was assessed by granzyme B, perforin, IFN-γ, and TNF-α production. Serum sTIM-3 and sGalectin-9 were measured by Cytometric Bead Array.

TIM-3 ⁺ NK cells were significantly increased in SFTS patients, especially in fatal cases. WGCNA identified HAVCR2 (encoding TIM-3) as a mortality-associated hub gene. TIM-3 ⁺ NK cells exhibited enhanced granzyme B and perforin expression, while TIM-3 blockade significantly reduced IFN-γ and TNF-α production. Elevated sTIM-3 and sGalectin-9 levels were correlated with fatal outcomes.

Activated TIM-3 ⁺ NK cells were associated with fatal outcomes in SFTS. TIM-3 ⁺ NK cell proportion, sTIM-3, and sGalectin-9 may serve as novel prognostic biomarkers and therapeutic targets.

While previous studies have described immune dysregulation in SFTS (including cytokine storms, T cell exhaustion, and impaired B cell responses), the role of NK cells remained unclear. This study employs single-cell RNA sequencing, flow cytometry, and serological analysis to define a high-resolution landscape of circulating NK cell heterogeneity in SFTS. We identify significant upregulation of TIM-3 (HAVCR2) on NK cells, particularly in fatal cases, associated with hyperactivated cytotoxic function. Furthermore, we demonstrate that circulating TIM-3 ⁺ NK cells, soluble TIM-3 (sTIM-3), and Galectin-9 (sGal-9) serve as potential prognostic biomarkers for disease severity and outcome. Our findings indicate that TIM-3 ⁺ NK cells contribute to SFTS immunopathogenesis through hyperactivation. Serum Galectin-9 represent novel biomarkers for early risk stratification. These results also highlight the modulation of the TIM-3 immune checkpoint pathway as a promising therapeutic strategy to curb excessive immune activation in acute viral infections like SFTS.