Single-cell and spatial transcriptomics reveal shared and divergent features of fulminant lymphocytic and giant cell myocarditis

Introduction

Fulminant myocarditis (FM) is the most severe form of acute myocarditis. Fulminant lymphocytic myocarditis (FLM) represents the most common subtype, whereas fulminant giant cell myocarditis (FGCM) is rare and often autoimmune-related. However, their shared and subtype-specific peripheral immune signatures remain poorly defined.

Methods

To address this, we enrolled 43 patients with histologically proven FM. In the multi-omics subgroup, scRNA-seq and scTCR-seq were performed on PBMCs from five FM patients, including three with FLM and two with FGCM, and validated these findings in the overall FM cohort.

Results

We obtained 98,146 high-quality PBMCs from scRNA-seq. Cellular fraction revealed a dramatic expansion of myeloid cells in the acute phase of FM. Differential gene expression analysis showed that myeloid cells underwent the most pronounced transcriptional changes. Among them, Monocytes_S100A8 were the only subset significantly expanded in FM and exhibited the strongest S100A signature. DIA proteomic profiling of the validation cohort revealed elevated levels of S100A8 and S100A9 in FM. Quantification of CD8⁺ T cells showed a relative increase in CD8⁺ effector T cells in FM. Compared with other subsets, CD8⁺ effector subsets displayed large clonal expansions. Trajectory analysis revealed two distinct differentiation paths originating from naive T cells: one toward FM-associated effector T cells (FM state) and the other toward control state. Spatial transcriptomics of cardiac tissues from FM mice further revealed that S100a8⁺Cd14⁺ monocytes showed strong spatial colocalization with Cd8⁺ effector T cells.

Cell fraction analysis further revealed a marked expansion of T cells in FGCM compared with FLM, particularly within the CD8⁺ effector T-cell subset. Relative to FLM, CD8⁺ effector T cells in FGCM displayed an upregulated activator protein-1 (AP-1) transcriptional program. NicheNet further identified myeloid cells as the predominant source of ligands with strong regulatory potential for inducing AP-1 activation in FGCM CD8⁺ effector T cells, with interleukin-6 (IL-6) among the top-ranked candidates. Plasma profiling in an independent validation cohort demonstrated elevated IL-6 levels in FGCM (n = 10) compared with FLM (n = 20) (P < 0.001). Receiver operating characteristic analysis revealed excellent diagnostic performance for IL-6 in discriminating FGCM from FLM (AUC = 0.915). Moreover, higher IL-6 levels (IL-6 > 10 pg/ml) were associated with worse outcomes.

Conclusions

This study identifies Monocytes_S100A8 as a principal source of S100A-family alarmins, which may promote CD8⁺ effector T-cell activation, contributing to myocardial inflammation in FM. Furthermore, elevated circulating IL-6 levels in FGCM patients are associated with AP-1 activation in CD8⁺ effector T cells and also serve as a clinically relevant marker that helps differentiate FGCM from FLM and correlates with adverse outcomes.Shared peripheral immune signatures in FMSubtype-specific signatures in FLM and FGCM