CXCL12/CXCR4 axis mediates CD8 ⁺ T cell overactivation in the progression of viral myocarditis

Abstract

Background

Myocarditis is a common inflammatory heart disease in children and young adults, with fulminant myocarditis (FM) being the most severe form due to its rapid onset and high mortality rate. However, the precise pathological immune subsets and molecular change in myocarditis, particularly FM, remain unknown.

Methods

We performed single-cell RNA sequencing of pediatric peripheral blood mononuclear cells during the acute and recovery phases of FM. A viral myocarditis (MC) mouse model was established using CVB3. Deletion and adoptive transfer of CD8⁺T cells, as well as blockade of CXCR4, were conducted in vivo. CD8⁺T cells were sorted and cultivated in vitro, then stimulated with CXCL12 and CXCR4 antagonists to investigate the mechanism of CD8⁺T cell overactivation.

Results

CD8⁺T cells show significant activation, amplification, enhanced cytotoxicity, and increased chemotactic ability in FM. Deletion of CD8⁺T cells alleviates myocardial injury and improves cardiac function in MC mice, while adoptive transfer of CD8⁺T cells from MC mice aggravates myocardial inflammation and injury. The transcriptomic analysis reveals elevated CXCR4 expression in CD8⁺T cells in acute FM. In vitro experiments demonstrate that the CXCL12/CXCR4 axis drives the overactivation and cytotoxicity of CD8⁺T cells. In vivo treatment with a CXCR4 antagonist effectively reduces CD8⁺T cell accumulation in the heart, alleviates myocardial inflammation, and improves cardiac function in MC mice.

Conclusions

These findings provide deeper insights into the immune landscape of pediatric FM, uncovering a novel role of the CXCL12/CXCR4 axis in driving CD8⁺T cell responses in myocarditis. Furthermore, they highlight the CXCL12/CXCR4 axis as a promising therapeutic target for myocarditis treatment.

Introduction

Myocarditis is a common inflammatory heart disease affecting children and young adults, often leading to heart dysfunction, sudden death, and secondary dilated cardiomyopathy [1, 2]. Fulminant myocarditis (FM), the most severe form of myocarditis, is characterized by sudden and severe diffuse cardiac inflammation, frequently resulting in death due to cardiogenic shock, ventricular arrhythmias, or multiorgan system failure [3, 4]. Despite its high mortality rate, the pathogenesis of myocarditis, particularly FM, remains poorly understood [5]. Current evidence suggests that the pathophysiology of myocarditis involves both direct myocardial injury caused by pathogens and indirect injury driven by autoimmune response and overwhelming inflammatory reactions [6, 7]. Early diagnosis of myocarditis is particularly challenging, and effective treatments are lacking. Therefore, a comprehensive understanding of its immunopathology is critical to facilitate early diagnosis, identify novel therapeutic targets, and improve clinical outcomes. Previous studies have highlighted key immune mechanisms in several myocarditis models. Xiumeng Hua et al. identified the association of macrophage cluster 2 and T-helper 17 cells with the inflammatory response in an experimental autoimmune myocarditis (EAM) model [8]. Han Zhu et al. reported that immune checkpoint inhibitor (ICI) associated myocarditis is linked to the expansion of specialized effector CD8⁺ T cells (Temra CD8⁺ cells) in the peripheral blood of affected patients [9]. Huihui Li et al. also demonstrated that self-recruited neutrophils trigger overactivation of the innate immune response and phenotypic change in cardiomyocytes in fulminant viral myocarditis model [10]. While immune profiling data in EAM, ICI myocarditis, and FM mouse models provide valuable insights, the precise pathological immune subsets and molecular changes in human fulminant myocarditis remain largely unexplored.

In this study, we present the comprehensive cellular and transcriptomic profiling of peripheral blood mononuclear cells (PBMCs) from children with acute FM and recovery phases, as well as from healthy control subjects, using single-cell RNA sequencing (scRNA-seq). Our findings reveal that a disordered proportion of immune cells and dysregulated immune functions are the most prominent features of PBMCs during the acute phase of FM, with most abnormalities being restored during the recovery phase, except for increased cytotoxicity of CD8⁺T cells. During the acute phase of FM, the proportion of CD8⁺T cells showed an upward trend. CD8⁺ Tn cells exhibited significant transcriptional change related to proliferation, differentiation, migration, and oxidation phosphorylation. CD8⁺ Tscm/cm cells and CD8⁺ Tem cells showed stronger cytotoxicity and chemotactic ability. Effector CD8⁺T cells upregulated cytotoxicity-related genes while downregulated inhibitory receptor genes. Transcriptomic analysis further revealed elevated expression levels of CXCR4 in CD8⁺T cells during the acute phase of FM. To verify the role and mechanism of CD8⁺T cells and the CXCR4 axis in myocarditis, a viral myocarditis (MC) mouse model was established. Deletion of CD8⁺T cells or CXCR4 blockade alleviated myocardial injury and improved cardiac function in MC mice. Furthermore, the adoptive transfer of CD8⁺T cells from MC mice aggravates myocardial inflammation and injury. In vitro experiments demonstrated that the CXCL12/CXCR4 axis promotes overactivation and enhances the cytotoxicity of CD8⁺T cells. These findings provide new insights to elucidate the pathogenesis and progression of myocarditis and suggest potential therapeutic targets for its treatment.

Results

Single-cell RNA profiling of PBMCs in children with fulminant myocarditis

We collected four acute-phase (P1-P4) and three recovery-phase peripheral blood samples (R1-R3) from four patients with fulminant myocarditis and three samples from age- and sex-matched healthy controls (C1-C3), following the inclusion and exclusion criteria. PBMCs were extracted and sequenced using the 10x Genomics platform (Fig. 1A). The Mean Reads per Cell for each sample exceeds 29 K/cell, and the Sequencing Saturation for each sample exceeds 67%. The specific data was shown in Supplementary Material 1. Rigorous quality control was applied to ensure that data were derived from single, viable cells with well-defined transcriptome phenotypes. Ultimately, a total of 79 542 cells passed quality control: 37 399 from the acute phase, 22 066 from the recovery phase, and 20,077 from healthy controls. We then used UMAP analysis to visualize the cells in two-dimensional space and found 21 cell clusters (Fig. 1B). Four major immune cell types were identified (Fig. 1C), T cells (CD3D, CD3E, CD3G, CD4, CD8A, 50.04%), natural killer (NK) cells (NCAM1, NKG7, FCGR3A, 15.96%), B cells (CD79A, CD19, MS4A1, JCHAIN, MZB1, 25.72%), and myeloid cells (CD14, FCN1, CST3, LYZ, S100A8, S100A9, 8.29%), based on known canonical markers (Fig. 1D). As expected, major immunological canonical markers were present in the immune subsets, as demonstrated by gene expression feature maps (Fig. 1F) and violin plots (Fig. 1G). We also tested the proportions of immune cell types in each group and found that the ratio of major immune cells varied significantly under the three conditions (Figure S1A). Furthermore, in the acute phase, we observed that T cells and NK cells showed a downward trend, while myeloid cells and B cells were upregulated. During the recovery phase, the proportion of T cells and myeloid cells tended to normalize, whereas NK cell percentages continued to decline (Fig. 1E). In order to verify the tendency of changing proportion of major immune cell types, we collected the cellular immunity data of 29 children with acute FM and 14 healthy controls from clinical laboratory tests, which were tested using flow cytometry. These findings were consistent with scRNA-seq data, showing a marked reduction in T cells and an increase in B cells during the FM acute phase (Fig. 1H).

Fig. 1

Analysis of peripheral blood mononuclear cell (PBMCs) populations in children with fulminant myocarditis. (A) Schematic of the experimental workflow. PBMCs were collected from healthy donors and children with fulminant myocarditis (FM) at the acute and recovery phases for single-cell RNA sequencing (scRNA-seq), single-cell T cell receptor sequencing (scTCR-seq), and single-cell B cell receptor sequencing (scBCR-seq). (B) Total PBMC clusters gained by the dimensional reduction using the uniform manifold approximation and projection (UMAP) across all samples. (C) Identification of PBMC clusters into four major immune cell types. (D) Dot plot showing the expression of marker genes of each cell type in each cluster. (E) Percentages of the four immune cell types among three groups. (F) Feature maps with major immunological canonical markers in UMAP. (G) Violin plots with major immunological canonical markers across all clusters. (H) Cellular immunity data from clinical laboratory tests by flow cytometry (FCM) showed the percentage of T cells (up), B cells (medium), and NK cells (down), in children with acute FM (n = 29) and control children (n = 14). Unpaired t-test was used for analysis. *p < 0.05; **p < 0.01; ***p < 0.001

CD8⁺T cells exhibit significant amplification, enhanced cytotoxicity, and chemotactic ability in FM

We combined NK and T cells and performed an unsupervised cluster analysis since these cells share similar growth patterns and gene expression profiles. Seventeen clusters were identified, including five T cell types (Fig. 2A) according to canonical markers (Figure S1B): CD4⁺T cells (CD4, IL7R, 28.90%), CD8⁺T cells (CD8A, GZMA, 39.55%), mucosal-associated invariant T cells (MAIT) (NCR3, KLRB1, SLC4A10, 1.68%), gamma-delta T cells (GDT) (TRDC, TRGC1, TRDV2, 5.47%), and proliferating T cells (MKI67, CD3D, 0.76%). Interestingly, the proportion of CD8⁺T cells showed an upward tendency in the acute phase (Fig. 2B). This finding was consistent with flow cytometry data from 29 FM patients, which also demonstrated a significant increase in the percentage of CD8⁺ T cells (Fig. 2C, D and E).

Fig. 2

Analysis of T cells, TCRs and subtypes of CD8⁺T cells. (A) Identification of peripheral blood NK cells and T cells across all samples into seven subtypes, including five T cell subtypes, and two NK cell subtypes. (B) Fraction of the five T cell subtypes and two NK cell subtypes among three groups. (C-E) Cellular immunity data from clinical laboratory tests by flow cytometry (FCM) showed the percentage of CD4⁺T cells (C), CD8⁺T cells (D), and the radio of CD4⁺T/CD8⁺T cells (E), in children with acute FM (n = 29) and control children (n = 14). Unpaired t-test was used for analysis. *p < 0.05; **p < 0.01; ***p < 0.001 (F) Identification of peripheral blood CD8⁺T cells across all samples into five subtypes. (G) Percentages of each CD8⁺ T cell subtype in total CD8⁺ T cells among three groups. (H) Dot plot showing the expression of marker genes of each CD8⁺T cell type in each cluster. (I-L) Dot plot of DEGs among three groups in CD8⁺ Tn cells(I), CD8⁺ Tscm/cm(J), CD8⁺ Tem(K), CD8⁺ Teff cells(L). (M) UMAP embedding of all CD8⁺ T cell subtypes, overlaid with the RNA velocity stream. (N) Subclustered NK cells and T cells colored by clonotype sizes (Left) and cell types (Right). (O) TCR clones detected among NK cell and T cell subsets, colored by their clonotype sizes. (P) Subclustered CD8⁺ T cells colored by clonotype sizes (Left) and cell types (Right). (Q) The clonal-size percentage across three conditions. Clonotypes whose clone size > 1 were identified as clonally expanded clones.

Next, we conducted an unsupervised clustering analysis on CD8⁺T cells, and based on the canonical cell markers, classified CD8⁺ naïve T cells (CD8⁺Tn, TCF7, CCR7, SELL, 39.54%), CD8⁺ stem cell-like memory/ central memory T cells (CD8⁺Tscm/cm, co-expression of TCF7, SELL, CXCR3, and LY6E, 11.02%), CD8⁺ T effector cells (CD8⁺Teff, NKG7, KLRD1, GZMA, 46.96%), CD8⁺ effector memory T cells (CD8⁺Tem, CX3CR1, NKG7 with low expression of CCR7, SELL, 1.82%) and CD8⁺ precursor exhausted T cells (CD8⁺Tpex, co-expression of TIGIT, HAVCR2, and TCF7 0.66%). The cluster and marker genes of CD8⁺T cells are shown in Fig. 2F and H. We found that the percentage of CD8⁺ Teff cells increased in the acute phase, along with the overall rise in CD8⁺T cells, while the proportions of CD8⁺ Tn and CD8⁺ Tscm/cm decreased (Fig. 2G). These changes in cell proportions tended to return to normal during the recovery phase (Fig. 2B and G).

CD8⁺ Naïve T cells (CD8⁺ Tn cells) exhibit transcriptional changes in proliferation, differentiation, migration, and oxidation phosphorylation in FM

We also analyzed the DEGs in CD8⁺ Tn cells between the FM acute phase and controls. Since naive T cells develop into effector T cells and migrate into certain sites after stimuli, we focused on genes related to T cell proliferation, differentiation, and migration. We found that in the FM acute phase, CD8⁺ Tn cells showed upregulation of IL7R, XBP1, JUNB, JUND, CCND3, TNFSF8, and CXCR4 (Fig. 2I), reflecting enhanced capabilities in these functions. Interestingly, a common set of DEGs was enriched in oxidative phosphorylation in CD8⁺ Tn cells. Most of these genes, including MT-ND1, MT-CO2, MT-ATP6, MT-CO3 MT-ND3, MT-ND4L, MT-ND4, and MT-CYB (Fig. 2I), were downregulated in the FM acute phase, suggesting reprogramed energy metabolism [11, 12].

CD8⁺ stem cell-like memory/ central memory T cells (CD8⁺ Tscm/cm cells) upregulate cytotoxicity genes in FM acute phase

CD8⁺ Tscm/cm cells, representing a transition stage from stem cell-like memory to central memory T cells, displayed increased expression of functional immune genes during the FM acute phase, specifically, cytotoxicity-related genes such as GZMA and SRGN were highly expressed. While in the recovery phase, S100A genes, rather than cytotoxicity-related genes, were observed to be increased in CD8⁺ Tscm/cm cells (Fig. 2J).

CD8⁺ effector memory T cells (CD8⁺ Tem cells) show enhanced cytotoxicity and chemotactic ability

In the FM acute phase, CD8⁺ Tem cells showed upregulated DEGs associated with chemokines/chemokine receptor (CXCR4, CX3CR1, CXCR3, CCL3, CCL4, and CCL5), cytotoxicity (GZMA, GZMH, NKG7, KLRD1), and S100A family. During the recovery phase, cytotoxicity genes (GZMA, GZMH, GZMB, and NKG7) and S100A genes remained persistently overexpressed (Fig. 2K). The elevated expression of cytotoxicity genes suggests that CD8⁺ Tem cells possess heightened cytotoxic potential, consistent with previous studies. Additionally, the upregulation of chemokines and chemokine receptor genes indicates a stronger capacity for migration to injured cardiac tissue. Notably, even after treatment and resolution of clinical symptoms, the cytotoxicity and chemotactic abilities of CD8⁺ Tem cells may persist.

CD8⁺ T effector cells (CD8⁺ Teff cells) show upregulated cytotoxicity genes and downregulated inhibitory receptor genes

As shown in Fig. 2L, we demonstrated that CD8⁺ Teff cells showed upregulated cytotoxicity genes GZMA and GZMH, and downregulated inhibitory receptor genes CD300A, TIGIT, KLRG1, and KLRC1 during both the acute and recovery phases of FM. However, the expression of activating receptor genes (NCR3, KLRF1, KLRD1, and CD160) varied between the two phases. NCR3 and KLRF1 were consistently downregulated, while KLRD1 was upregulated in the acute phase, and CD160 was upregulated in the recovery phase. Besides, chemokine receptor genes CXCR4 and CXCR3 maintained higher expression in the acute and recovery phases, respectively.

CD8⁺ precursor exhausted T cells (CD8⁺ Tpex cells) originate from CD8⁺ Tscm/cm cells and differentiate to CD8⁺ Teff cells

From the participants, we identified a small subset of exhausted CD8⁺ T cells, named CD8⁺ precursor exhausted T cells (CD8⁺ Tpex cells), which displayed stem-like properties characterized by the high expression of stem cell-associated genes like TCF7, SELL, IL7R, and CCR7. These cells exhibited self-renewal capacity, differentiation potential, and the ability to enhance immune responses in chronic viral infections and cancer. RNA velocity analysis indicated that CD8⁺ Tpex cells originated from CD8⁺ stem cell-like memory/ central memory cells T cells (CD8⁺ Tscm/cm cells) and had the potential to differentiate into effector cells (Fig. 2M). And the differentiation process CD8⁺ Tn→CD8⁺ Tscm/cm→CD8⁺ Teff or CD8⁺ Tpex was also observed, consistent with previous reports [13].

CD8⁺ T cells exhibit significant clonal amplification and respond to specific epitopes in FM

To explore the dynamics of TCR repertoires during FM, we analyzed scTCR-seq data. Clonotypes with a clone size > 1 were considered expanded. In our study, TCR exhibited significant amplification in both the acute and recovery phases of FM, with a more pronounced increase during the acute phase (Fig. 2Q). More specifically, the majority of clonally amplified T cells were CD8⁺T cells, with a substantial proportion of these clones having a clonotype size > 20, contributing to the enlarged CD8⁺ T cell clones (Fig. 2N and O). Furthermore, most of the clonotypes with a size > 20 were predominantly embedded within CD8 + Teff cells (Fig. 2P and Figure S1C). Using TCRMatch analysis [14], we identified the top five CDR3b patterns of CD8⁺T cells, recognizing putative epitopes derived from a variety of viruses, including Human cytomegalovirus, severe acute respiratory syndrome coronavirus 2, influenza A virus, and Epstein Barr virus (Supplementary Material 2). Interestingly, we found that adrenomedullin 2 and telomerase reverse transcriptase were also involved in putative epitopes, which haven’t been previously reported in the autoimmune genesis of myocarditis and may provide a new perspective for future research. All these results indicate that both virus-induced or injury-induced autoimmune responses could contribute to the pathogenesis of myocarditis.

CD8⁺T cells show significant amplification and activation in viral MC mouse models

Among all immune cell types, CD8⁺T cells caught our interest as they exhibited obvious amplification, activation, and stronger cytotoxicity in both two phases of FM. However, their specific role and mechanisms in myocarditis remain insufficiently understood. To address this, we established a viral MC mouse model using CVB3 to explore the function of CD8⁺T cells. Flow cytometry and immunofluorescence revealed a significant increase in the percentage of CD8⁺T cells in PBMCs (Fig. 3A and D), spleen (Fig. 3B and E), and heart of MC mice (Fig. 3C). CD8⁺T cells from mouse spleen were extracted by magnetic bead negative sorting. qRT-PCR analysis showed that cytotoxicity-related genes GZMA, GZMB, and Perforin, and Kill-associated cytokines IFN-γ and TNF-α were significantly upregulated in splenic CD8⁺T cells from MC mice compared with normal control (Fig. 3F-J). These results indicate that CD8⁺T cells undergo pronounced amplification and activation in MC.

Fig. 3

The accumulation and cytotoxicity of CD8⁺T cells in CVB3-induced viral myocarditis mice. (A) The representative flow cytometry images of CD8⁺T cells in PBMCs of myocarditis (MC) mice and control (Con) mice at day 7. (B) The representative flow cytometry images of CD8⁺T cells in spleens of MC mice and control mice at day 7. (C) Immunofluorescence revealed the enrichment of CD8⁺T cells (red) in the myocardium of MC mice at day 7. Scale bar, 275 μm. (D-E) The histogram of proportion of CD8⁺T cells in PBMCs (D) and spleens (E) of MC mice and control mice at day 7. (F-J) The expression of cytotoxic genes GZMA(F), GZMB(G), Perforin(H), IFN-γ(I) and TNF-α(J) was significantly upregulated in splenic CD8⁺T cells of MC mice compared with that in control mice tested by qRT-PCR. Unpaired t-test was used for analysis. Data are presented as mean ± SD (n = 3 biologically independent samples). *p < 0.05; **p < 0.01; ***p < 0.001

Deletion of CD8⁺T cells alleviates myocardial inflammation and myocardial injury in MC mice

To investigate the role of CD8⁺T cells in viral myocarditis, an anti-CD8 antibody was used to delete the CD8⁺T cells in mice (Fig. 4B), achieving a clearance efficiency of over 98%, as confirmed by flow cytometry (Fig. 4A). CVB3 infection was then induced in both CD8⁺T cell-depleted mice and control mice. We found that deletion of CD8⁺T cells improved the general health of the mice, as they showed slower body weight loss (Fig. 4C). The left ventricular ejection fraction (LVEF) and fraction shortening (FS) tested by echocardiography increased (Fig. 4D, E and F), indicating that the cardiac function improved significantly. In addition, the HE staining showed a marked reduction in cardiac immune cell infiltration (Fig. 4G), while the apoptosis rate of cardiomyocytes decreased (Fig. 4H). The expression levels of myocardial injury marker cTnT (Fig. 4I) and inflammatory cytokines TNF-α (Fig. 4J) were tested to be decreased significantly in serum, according to the ELISA. All the results indicate that deletion of CD8⁺T cells could alleviate myocardial inflammation and myocardial injury in MC mice.

Fig. 4

Depleting CD8⁺T cells in CVB3-induced viral myocarditis. (A) The clear efficiency of CD8⁺T cells confirmed by flow cytometry. (B) Schematic workflow of CD8⁺T cell deletion. (C) Weight changes of mice in different groups during the experimental process. (D) Representative echocardiographic image of different groups. (E-F) The ejection fraction (E) and fraction shortening (F) change of three groups. (G) Representative HE staining images in different groups. Scale bar, 20 μm. (H) Tunel staining revealed cell apoptosis in the myocardium in different groups. Broken DNA was labeled with CF488 (green). Nuclei were stained with DAPI (blue). Scale bar, 50 μm. (I-J) The plasma expression level of cardiac troponin T(cTnT) (I) and TNF-α (J) in different group. Unpaired t-test was used for analysis. Data are presented as mean ± SD (n = 5 biologically independent samples). *p < 0.05; **p < 0.01; ***p < 0.001

Adoptive transfer of CD8⁺T cells from MC mice aggravates myocardial inflammation and injury

To further confirm the pathogenic role of CD8⁺T cells in myocarditis, we extracted splenic CD8⁺T cells from MC mice (CD8⁺T^MC) and normal mice (CD8⁺T^Con) and adoptively transferred them into mice infected with a low dose of CVB3 (Fig. 5A and B). Mice receiving CD8⁺T^MC showed faster and more serious weight loss compared to those receiving CD8⁺T^Con (Fig. 5C). Correspondingly, transferring CD8⁺T^MC obviously damaged cardiac function (Fig. 5D and E), along with aggregated cardiac immune cell infiltration (Fig. 5F). Similarly, we also found that the apoptosis rate of cardiomyocytes (Fig. 5H) and the expression level of cTnT and TNF-α (Fig. 5G) increased markedly in the CD8⁺T^MC transferred group. These findings indicate that the adoptive transfer of CD8 + T^MC significantly aggravates myocardial inflammation and myocardial injury.

Fig. 5

Adoptive transfer of CD8⁺T cells in CVB3-induced viral myocarditis. (A-B) Schematic workflow of CD8⁺T cell transfer. (C)Weight changes of mice in different groups during the experimental process. (D) Representative echocardiographic image of different groups. (E)The ejection fraction (Left) and fraction shortening (Right) change of three groups. (F) Representative HE staining images in different groups. Scale bar, 20 μm. (G) The plasma expression level of cardiac troponin T(cTnT) (Left) and TNF-α (Right) in different group. (H)Tunel staining revealed the cell apoptosis in the myocardium of mice in different groups. Broken DNA was labeled with CF488 (green). Nuclei were stained with DAPI (blue). Scale bar, 50 μm. Unpaired t-test was used for analysis. Data are presented as mean ± SD (n = 5 biologically independent samples). *p < 0.05; **p < 0.01; ***p < 0.001

CXCL12/CXCR4 axis promotes the cytotoxicity of CD8⁺T cells

The above results demonstrated that CD8⁺T cells showed significant activation, amplification, and pathogenicity in FM patients and MC mice. To explore the molecular mechanism underlying CD8⁺ T cell activation, we focused on chemokines. Analysis of DEGs showed that the expression of chemokine receptor gene CXCR4 in CD8⁺T cells was highly upregulated in the FM acute phase, ranking among the top five DEGs (Fig. 6A and B). CXCR4 is a classic chemokine receptor, which may contribute to cardiac CD8⁺T cell infiltration and activation in MC. The qRT-PCR results confirmed the upregulation of CXCR4 expression in CD8⁺T cells (Fig. 6C) and the heart (Fig. 6D) of MC mice. Immunofluorescence further revealed the colocalization of CXCR4 and CD8⁺T cells in the heart of MC mice (Fig. 6H). Given the known ligand of CXCR4 is CXCL12, we investigated CXCL12 expression. Results showed significantly higher levels of CXCL12 in the hearts of MC mice compared to normal mice (Fig. 6E). As we know, cardiomyocytes, the predominant cell type in the heart, are the first to be affected during infection and secrete immunologically active substances. To determine whether cardiomyocytes are a source of CXCL12, we performed in vitro experiments using the HL-1 mouse cardiomyocyte line. Upon CVB3 infection, HL-1 cells showed significant upregulation of CXCL12 mRNA and secreted protein levels compared to PBS-treated cells (Fig. 6F and G).

Fig. 6

CXCL12/CXCR4 axis promoted the cytotoxicity of CD8⁺T cells. (A) Volcano plot exhibiting the top five upregulated and downregulated genes of CD8⁺T cells in FM acute phase compared with control. Red refers to upregulated genes, blue refers to downregulated genes. (B) Violin plots showing the expression of CXCR4 in three groups. (C-D) The expression of CXCR4 in splenic CD8⁺T cells (C) and heart (D) of mice in MC group and control group tested by qRT-PCR. (n = 5) (E) The expression of CXCL12 in the heart of mice in two groups tested by qRT-PCR. (F) The expression of CXCL12 in HL-1 cells stimulated by CVB3 or not, tested by qRT-PCR. (G) The level of CXCL12 in the supernatant of HL-1 cells stimulated by CVB3 or not, tested by ELISA. (H) Immunofluorescence revealed the colocalization of CXCR4 (green) and CD8⁺T cells (red) in the myocardium of MC mice at day 7. Nuclei were stained with DAPI (blue). Scale bar, 275 μm. (I-M) The expression of effective factors GZMA(I), GZMB(J), Perforin (K) and IFN-γ (L) in CD8⁺ T cells stimulated with three different conditions in vitro. Statistic analysis is shown in M. (N-O) The apoptosis rate of HL-1 cells co-cultured with CD8⁺ T cells under three different conditions, or with PBS in vitro. (P) The level of CXCL12 in serum of FM patients and healthy controls tested by ELISA. Unpaired t-test was used for analysis. Data are presented as mean ± SD (n = 3 biologically independent samples). *p < 0.05; **p < 0.01; ***p < 0.001 (Student’s t-test)

Based on these findings, we hypothesized that CXCL12 secreted by infected cardiomyocytes amplifies and activates CD8⁺T cells in MC through CXCL12/CXCR4 axis. To test this, CD8⁺ T cells were sorted, cultivated in vitro, and pre-activated by CD3 and IL2. They were then stimulated with recombinant CXCL12 in the presence or absence of a CXCR4 antagonist. Activation of CD8⁺ T cells was indicated by increased cytotoxicity, with CXCL12 treatment significantly raising the proportion of GZMA⁺ CD8⁺ T cells and the expression of GZMB, perforin, and IFN-γ, as determined by flow cytometry. Notably, the addition of a CXCR4 antagonist reversed the stimulatory effects of CXCL12 on CD8⁺T cell activation (Fig. 6I-M).

To validate the cytotoxicity of CD8⁺ T cells on cardiomyocytes, we conducted a T cell-mediated cell-killing assay by co-culturing CD8⁺T cells and HL-1 cells under the same conditions. We found that when CD8⁺ T cells were activated by CD3 and IL2, the damage to cardiomyocytes was significantly increased. In addition, CXCL12 treatment markedly increased the cardiomyocyte apoptosis rate, while the addition of a CXCR4 antagonist significantly reduced apoptosis (Fig. 6N). In summary, CXCL12 secreted by infected cardiomyocytes contributes to the activation and cytotoxicity of CD8⁺ T cells via the CXCL12/CXCR4 axis, ultimately leading to myocardial injury in MC.

In addition, the serum level of CXCL12 in 12 FM patients and 7 controls have been tested by ELISA. The results showed that CXCL12 was higher in FM patients’ serum compared with healthy controls (Fig. 6P), which suggested that CXCL12 might be an important inflammatory indicator in FM. In the following study, the expression level and clinical value of CXCL12 in FM patients’ serum will be further investigated in more clinical samples.

CXCR4 Blockade reduces CD8⁺ T cell accumulation in the heart, alleviates myocardial inflammation, and improves cardiac function in MC mice

Considering that CXCR4 blockade reduced the activation of CD8⁺T cells and myocardial apoptosis in vitro, we evaluated its therapeutic effects in vivo using MC mice. Treatment with a CXCR4 antagonist significantly decreased CXCR4 expression and CD8⁺T cell infiltration in the hearts of MC mice, as verified by immunofluorescence and WB (Fig. 7A, B and Figure S1E). This was accompanied by reduced cytotoxicity of CD8⁺T cells (Fig. 7C to G). Consistently, blocking CXCR4 improved the general health of MC mice, as indicated by slower body weight loss (Fig. 8B). Echocardiography revealed significantly enhanced cardiac function, with increased LVEF and FS (Fig. 8C, E and F). Moreover, cardiac immune cell infiltration (Fig. 8D) and cardiomyocyte apoptosis, as determined by the TUNEL assay (Fig. 8G), were markedly reduced. In addition, the expression levels of cTnT and TNF-α tested by ELISA in serum decreased significantly when blocking CXCR4 in MC mice (Fig. 8H and I). These results indicated that targeting the CXCL12/CXCR4 axis by a CXCR4 antagonist could reduce the accumulation and lethality of CD8⁺T cells and improve cardiac function in MC by alleviating myocardial inflammation.

Fig. 7

CXCR4 blockade reduced CD8⁺T cell accumulation in the heart. (A) Immunofluorescence revealed the infiltration of CD8⁺T cells (red) in the myocardium among control group, MC group and CXCR4 blockade group. (B) The expression of CXCR4 in the heart of three groups tested by qRT-PCR. (C-G) The expression of cytotoxic genes GZMA(C), GZMB(D), Perforin(E), IFN-γ(F) and TNF-α(G) were significantly decreased in splenic CD8⁺T cells in CXCR4 blockade mice compared with that in MC mice tested by qRT-PCR. Unpaired t-test was used for analysis. Data are presented as mean ± SD (n = 3–5 biologically independent samples). *p < 0.05; **p < 0.01; ***p < 0.001

Fig. 8

Blocking CXCR4 could alleviate myocardial inflammation and improve cardiac function. (A) Schematic workflow of blocking CXCR4. (B) Weight changes of mice in different groups during the experimental process. (C) Representative echocardiographic image of different groups. (D) Representative HE staining images in different groups. Scale bar, 20 μm. (E-F) The ejection fraction (E) and fraction shortening (F) change of three groups. (G) Tunel staining revealed the cell apoptosis in the myocardium of mice in different groups. Broken DNA were labeled with CF488 (green). Nuclei were stained with DAPI (blue). Scale bar, 50 μm. (H-I) The plasma expression level of cardiac troponin T(cTnT) (H) and TNF-α (I) in different group mice. Data are presented as mean ± SD (n = 5 biologically independent samples). *p < 0.05; **p < 0.01; ***p < 0.001 (Student’s t-test)

Discussion

The pathogenesis of myocarditis remains poorly understood. It is generally believed that the disease’s pathophysiology involves a direct myocardial injury caused by pathogens and subsequent autoimmune reactions. FM, characterized by its rapid onset and accelerated progression, is one of the most severe diseases in children. It can lead to devastating outcomes, including heart failure, severe hemodynamic changes, fatal arrhythmia, and cardiogenic shock. Despite its clinical significance, the mechanisms underlying FM pathogenesis and potential targeted therapeutic strategies remain inadequately understood. Although immune profiling studies have been conducted in autoimmune myocarditis mouse models [8], checkpoint inhibitor-induced myocarditis [9], and FM mouse model [10], a comprehensive exploration of the immune phenotyping in pediatric FM is lacking. Peripheral blood has been shown to be a rich resource of immunological information that may reflect immune changes occurring at the tissue level. In this study, we utilized peripheral blood to investigate the immune landscape in pediatric FM, with a particular focus on CD8⁺T cell profiles. Moreover, using scTCR-seq technology, we provided the first detailed analysis of peripheral TCR repertoires in exploring specific adaptive immune responses in pediatric FM.

Our results showed that T cells constituted the largest proportion of PBMCs, accounting for nearly 50%, followed by B cells, NK cells, and myeloid cells. T cells are considered a critical driver of cardiac damage in myocarditis. Upon pathogen stimulation, naïve T cells are activated into effector T cells, which clonally expand and differentiate to perform effector functions such as killing infected cells, producing cytokines, and modulating immune responses. The study revealed a notable decrease in the proportion of total T cells in the peripheral blood of pediatric FM patients in acute phase. Further subset analysis demonstrated that this decline was predominantly driven by a reduction in CD4 ⁺ T cells, accompanied by an increase in CD8 ⁺ T cells, resulting in a significantly altered CD4/CD8 ratio. These findings align with clinical observations of diminished CD4 ⁺ T cell counts, elevated CD8 ⁺ T cell proportions, and a disrupted CD4/CD8 balance. In healthy children, CD4 ⁺ T cells typically outnumber CD8 ⁺ T cells, with a physiological CD4/CD8 ratio ranging from 2:1 to 3:1 [15]. Thus, the observed reduction in total T cell proportion likely stems from the selective depletion of CD4 ⁺ T cells rather than global T cell loss. Similar patterns of CD4 ⁺ T cell depletion with preserved or increased CD8 ⁺ counts have been documented in other acute viral infections, including influenza [16] and COVID-19 [17].

While earlier studies primarily focused on CD4 ⁺ T cells, recent attention has shifted to CD8⁺T cells. Known as cytotoxic T cells, CD8⁺ T cells destroy infected cells via granzymes, perforins, and FAS-induced apoptosis. They also contribute to viral clearance by producing TNF-αand IFN-γ [18, 19]. In our study, the proportion of CD8⁺T cells was found to increase during the acute phase of FM, a finding further corroborated by clinical cellular immunity data. To gain deeper insights into CD8⁺T cells in FM, we analyzed each identified subset and observed characteristic transcriptional changes across all cell types. For CD8⁺ Tn cells, the reduced level of oxidative phosphorylation during the acute phase suggested specific reprogramming of energy metabolism adapted to FM. While naïve T cells typically depend on oxidative phosphorylation of glucose, antigen stimulation drives their differentiation into effector cells, shifting their energy metabolism to aerobic glycolysis to meet increased energy demands [11, 12]. Notably, all CD8⁺ Tscm/cm cells, CD8⁺Tem cells, and CD8⁺Teff cells showed heightened cytotoxicity in the FM acute phase, while the elevated cytotoxicity of CD8⁺Tem cells and CD8⁺Teff cells persisted into the recovery phase. A unique cell type we found was CD8⁺Tpex cells with very small amounts. These cells have self-renewal capacity, differentiation potential, and the ability to enhance immune response in chronic viral infections and cancer [20]. Consistent with previous reports [13], we found CD8⁺ Tpex cells originated from naïve and memory T cells and had the potential to differentiate into effector cells upon stimulation.

CD8⁺T cells are closely related to autoimmunity, as dysfunction of autoreactive CD8⁺ T cells can undermine self-tolerance mechanisms, leading to autoimmune diseases mediated by CD8⁺T cells [21]. With the increasing application of immune checkpoint inhibitors (ICI) in tumor therapy, cases of ICI-associated myocarditis have gradually risen. Studies have shown that in both patients with ICI myocarditis [9] and corresponding mouse models [22], CD8⁺T cells, especially CD8⁺Teff cells, are significantly expanded and highly expressed cytotoxic markers and cardiophilic chemokines. These activated CD8⁺T cells target myocardial-specific antigens via TCR, mistakenly causing injury to cardiomyocytes. In viral myocarditis, while over-reactive CD8⁺T cells contribute to viral clearance [23], they also enhance heart damage [24]. In addition, the “bystander T cell” hypothesis suggests that CD8⁺ T cells are attracted to inflammatory sites but are not necessarily specific to the disease-causing antigens. Instead, they may recognize autoantigens, leading to collateral damage of normal tissues [25]. In our study, CD8⁺T cells constituted the majority of extended TCR clonotypes and exhibited clonal amplification in FM. Several viral putative epitopes and two self-antigen epitopes, including adrenomedullin 2 and telomerase reverse transcriptase antigens, were identified in CD8⁺T cells of FM patients. Adrenomedullin 2 belongs to the wider calcitonin gene-related peptide/calcitonin family of peptides and is widely distributed throughout the body, including the heart [26]. Telomerase reverse transcriptase, the core subunit of telomerase [27], has been shown to prevent cellular aging by maintaining telomere length and exerting anti-inflammatory, antioxidant, and cardioprotective properties in cardiovascular diseases [28, 29]. Our study provides a theoretical basis for the role of expanded and highly cytotoxic autoreactive CD8⁺T cells in the autoimmune processes underlying viral myocarditis. Furthermore, we explored the pathogenic role of CD8⁺T cells in a CVB3-induced viral MC mouse model. The percentage of CD8⁺T cells in PBMCs, spleen, and heart of CVB3-induced MC mice was significantly higher than in control mice. In CD8⁺T cell-deleted MC mice, weight loss was slower and less severe compared to general MC mice. These mice also exhibited reduced myocardial inflammatory cell infiltration, a marked decrease in cardiomyocyte apoptosis, significant improvement in cardiac ejection function, and lower levels of cardiac troponin T and TNF-α in peripheral blood plasma. In contrast, mice receiving adoptive transfer of CD8⁺T cells from MC mice showed more severe weight loss and myocardial injury than mice transferred with CD8⁺T cells from healthy mice. Collectively, these findings suggest that prolonged and excessive activation of CD8⁺T cells play a significant role in the pathophysiology of viral myocarditis.

The factors promoting the overactivation of CD8⁺T cells in myocarditis remain poorly understood. In this study, we identified the CXCL12/CXCR4 axis as a potential regulator of CD8⁺T cell overactivation in myocarditis through analysis of DEGs. Previous studies have shown that the expression of CXCL12 increases in the heart of MC mice [30] and CXCR4 facilitates leukocyte recruitment during the inflammation by binding to CXCL12 [31]. However, the role of CXCL12/CXCR4 axis in viral myocarditis has not been previously explored. In our study, we observed increased CXCR4 expression in CD8⁺T cells and the hearts of MC mice, alongside elevated CXCL12 levels in the heart. In vitro examinations confirmed that cardiomyocytes serve as a source of CXCL12. CD8⁺T cells exhibited higher cytotoxicity and lethality after CXCL12 stimulation, a process effectively interrupted by CXCR4 antagonists. Furthermore, CXCR4 blockade reduced CD8⁺T cell accumulation in the hearts of MC mice. Finally, we demonstrated the therapeutic efficacy of a CXCR4 antagonist in the CVB3-induced viral myocarditis model. Several studies have highlighted the importance of CXCR4 in the immunological function of CD8⁺T cells. Marieke Goedhart et al. demonstrated that CXCR4, highly expressed on both naive and memory CD8⁺ T cells in bone marrow, is crucial for the homing of CD8⁺T cell subsets to the bone marrow in mice [32]. Another study found that the CXCL12-CXCR4 signaling pathway regulates the migration of CD8⁺T cells, and blocking CXCR4 limits lymphatic discharge, promotes the retention of CD8⁺T cells in the tumor, and enhances the effectiveness of immunotherapy [33]. Our results indicate that CXCR4 is a critical regulator of CD8⁺T cell accumulation, clonal expansion, and activation in the heart, playing a key role in modulating CD8⁺T cell immunoregulatory functions in viral myocarditis.

Our study has several limitations. First, the small sample size used for scRNA-seq might have reduced the statistical power in differential proportion and differential expression analysis. This could explain why some cell type proportions changed obviously but without statistical significance. Second, although the PBMCs provide valuable insights into systemic immune responses during FM, they may not fully reflect the immune infiltration status in myocardial tissue. Furthermore, the lack of myocardial biopsy prevented analysis of clonotype overlaps between blood and heart tissue. Third, the precise mechanisms by which the CXCL12/CXCR4 axis influences intracellular downstream signaling pathways and its effects on CD8⁺ T cell phenotypes require further in-depth investigation. Additionally, the impact of CXCR4 antagonists on peripheral and myocardial immune cells in MC mice, apart from CD8⁺ T cells, was not assessed. Another limitation is the absence of confirmation regarding the activation and heightened toxicity of CD8 ⁺ T cells, validation of the key genes in various T cell types in the scRNA data, as well as the evaluation of CXCR4 expression in CD8 ⁺ T cells within the peripheral blood of FM patients. These aspects will be subjected to further exploration in subsequent study.

In conclusion, this study provides the first comprehensive cellular and transcriptomic profiling of PBMCs, particularly CD8⁺T cells, in children in FM at a single-cell level. CD8⁺T cells in the FM acute phase showed significant amplification and overactivation, suggesting an important pathogenic role in myocarditis. We also elucidated a novel mechanism of CD8⁺T cell activation in myocarditis, where infected cardiomyocytes secreted CXCL12, recruiting and activatingCD8⁺T cells via CXCR4, ultimately causing myocardial injury. Taken together, our findings enhance the understanding of the immune landscape of pediatric FM, reveal a novel role of the CXCL12/CXCR4 axis in driving CD8⁺T cell responses in myocarditis, and present a promising therapeutic target for the disease.

Methods and materials

Ethics statement

The study was carried out in compliance with the Helsinki Declaration and was authorized by the Institutional Ethics Committee of Shandong Provincial Hospital. Informed consent was obtained from all donors or their guardians. The animal study was approved by the Institutional Animal Research Committee of Shandong Provincial Hospital. All procedures complied with Directive 2010/63/EU of the European Parliament on the protection of animals used for scientific purposes.

Patients

At Shandong Provincial Hospital, peripheral blood samples were collected from four children with FM during the acute phase (P1-P4) and three of them during the recovery phase (R1-R3). Additionally, samples from three healthy children (C1-C3) were obtained as controls. These samples were utilized for single-cell RNA sequencing. We also collected serum samples from 12 FM patients and 7 controls. The demographics and clinical features of the participants were detailed in Supplementary Material 3. In addition, clinical laboratory data on cellular immunity were collected from 29 FM patients in the acute phase and 14 healthy controls. All FM patients were diagnosed based on the criteria outlined in the 2013 Position Statement of the European Society [34], the 2019 Chinese Society of Cardiology Expert Consensus [3], and the 2021 American Heart Association scientific statement on the diagnosis and treatment of childhood myocarditis [2]. All the patients or their guardians signed the informed consent.

PBMC collection, single-cell RNA sequencing (scRNA-seq), and data processing

Venous blood (2 mL) from each donor was collected in EDTA anticoagulant tubes. PBMCs were isolated using Ficoll-Paque density gradient centrifugation. Single-cell suspensions were prepared and processed on the 10x Chromium platform to capture 5,000–12,000 cells per sample, following the manufacturer’s instructions. Subsequent cDNA amplification and library construction were performed using standard protocols. The scRNA-seq, scBCR-seq, and scTCR-seq libraries were sequenced on an Illumina NovaSeq 6000 platform (150 bp paired-end) at a minimum depth of 20,000 reads per cell by LC-Bio Technology Co., Ltd. (Hangzhou, China). Sequencing data were demultiplexed and converted to FASTQ format using Illumina bcl2fastq software. Sample demultiplexing, barcode processing, and single-cell 5’ gene counting were performed with the Cell Ranger pipeline (version 3.1.0), aligning the scRNA-seq data to the Ensembl GRCh38 reference genome. A total of 88,801 single cells were processed using the 10X Genomics Chromium Single Cell 5’ Solution.

The output from Cell Ranger was analyzed using Seurat (version 3.1.1) for dimensional reduction, clustering, and downstream analysis. After quality control, 79,542 cells passed the thresholds: genes expressed in more than three cells, 500–5,000 genes expressed per cell, UMI counts > 500, and mitochondrial gene content < 25%.

Dimension reduction and clustering

Dimensional reduction of all 79 542 cells into a 2D space was performed using Seurat and Uniform Manifold Approximation and Projection (UMAP). Gene expression values were normalized using the LogNormalize method within the Seurat “Normalization” function. Principal component analysis (PCA) was then conducted, and the top 20 principal components were used for clustering and UMAP analysis.

Clusters were identified using a weighted Shared Nearest Neighbor (SNN) graph-based clustering method. Marker genes for each cluster were determined using the “FindAllMarkers” function in Seurat with the “bimod” test (default parameters). Representative genes were selected if they were expressed in more than 10% of cells within a cluster and had an average log2 Fold Change (log2FC) ≥ 0.26.

Differential expression and functional enrichment analysis

Differential expression analysis for each cell type among the three groups was performed using the “bimod” test via the “FindAllMarkers” tool. Differentially expressed genes (DEGs) were identified based on the criteria: p < 0.01, expression in more than 10% of cells, and|log2FC| ≥ 0.26. Functional enrichment analysis, including Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) pathway analysis, was conducted using the “clusterProfiler” package (version 4.0) in R.

RNA velocity analysis

RNA velocity was calculated using the “scvelo” Python package to predict future cellular states. Spliced and unspliced reads were annotated, and RNA velocity for each cell was computed with default settings. Velocity fields and pseudotime values were then projected onto the UMAP plot.

TCR data analysis

scTCR-seq data were processed using the Cell Ranger VDJ Software Suite (version 4.0.0, 10x Genomics) and mapped to the GRCh38 VDJ reference. Only full-length, productive contigs with assigned raw clonotype IDs were retained. Clonotypes were characterized by V gene usage, and expanded clones (clone size > 1) were identified. Analysis of clonal expansion in CD8 ⁺ T cells from FM patients revealed the top five CDR3b patterns, which were analyzed using the TCRMatch tool with a medium filtration threshold (> 0.90).

Animals

4-6-week-old male BALB/c mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd and randomly divided into two or three groups. A viral myocarditis mouse model was established using coxsackie virus type B3 (CVB3). Mice in the CVB3 + group received an intraperitoneal injection of 200 µl 10⁶ tissue culture infectious dose 50 (TCID50) /100µl of CVB3. An equivalent phosphate buffer solution (PBS) was used as a control. In the CVB3 + CD8⁺T-deletion group, mice were treated with anti-mouse CD8a antibody (100746, BioLegend, San Diego, USA) intravenously at a dose of 200 ug per mouse one day before CVB3 infection, with additional injections given every other day (Fig. 4B). CD8⁺T depletion was confirmed on day 7 using FACS. For adoptive transfer, 1 × 10⁶ CD8⁺T cells extracted from MC mice (CD8⁺T^MC) and normal mice (CD8⁺T^Con) were first injected intravenously, followed by the injection of a low dose of CVB3 (100 µl of the previously described CVB3 solution) one day later (Fig. 5A and B). Mice in the CVB3 + CXCR4-block group were treated daily with a CXCR4 antagonist (Motixafortide, MCE, Shanghai, China) subcutaneously at 200 ug per mouse from day 0 to day 4 (Fig. 8A). Weight was recorded daily until day 7. For anesthesia, mice were placed in an induction chamber with 4% isoflurane, and maintained with 2% isoflurane at a flow rate of 0.6–1 L/min via facemask. Echocardiography was performed using KOLO^® SiliconWave 60 with a 30 MHz L38-22K3 scan head, recording left ventricular ejection fraction and fractional shortening. All animals were sacrificed on day 7 by spinal cord dislocation following anesthesia, and blood, spleens, and hearts were collected for analysis.

Histological staining

Mouse cardiac tissues were fixed in 4% paraformaldehyde and embedded in paraffin. Tissue slices with 4 μm thickness were prepared and subjected to hematoxylin-eosin staining, apoptosis detection, and immunofluorescence analysis (details provided below).

Immunofluorescence

For paraffin-embedded mouse myocardial tissue sections, the samples were dewaxed, permeabilizated, blocked, and stained overnight at 4° with CXCR4 Monoclonal antibody (1:200; 60042-1-Ig, Proteintech, Wuhan, China) or Recombinant Anti-CD8 alpha Rabbit mAb (1:50; GB15068-100, Servicebio, Wuhan, China) after PBS washing, the sections were incubated with CoraLite488-conjugated goat anti-mouse (1:200; SA00013-1, Proteintech, Wuhan, China) or Alexa Fluor594-conjugated goat anti-rabbit antibody (1:200; GB28301, Servicebio, Wuhan, China) for 1 h at room temperature. Nuclei were also stained with DAPI for 10 min. Images were collected using an OLYMPUS BX63 fluorescence microscope and an EVOS M7000 microscope.

Tissue and cell apoptosis detection

Cardiac tissue apoptosis was assessed using the CF488 Tunel Cell Apoptosis Detection Kit (Servicebio, Wuhan, China) following the manufacturer’s instructions. Images were collected by OLYMPUS BX63 fluorescence microscope. Apoptosis in HL-1 cardiomyocytes was measured using Annexin V-phycoerythrin (PE)/7-aminoactinomycin D (7AAD) kit (BD Biosciences, NJ, USA), following the manufacturer’s protocol.

Mouse Splenic CD8⁺T cells isolation

Mouse splenic CD8⁺T cells were isolated using the mouse CD8⁺T cell isolation kit (Vazyme, Nanjing, China) per the manufacturer’s instructions. The purity of isolated CD8⁺T cells was verified by flow cytometry (Figure S1D).

In vitro stimulation of CD8⁺T cells

Extracted mouse splenic CD8⁺Tcells were stimulated for 24 h using Ultra-LEAF™ Purified anti-mouse CD3ε antibody (3 µg/ml, BioLegend, San Diego, USA) and IL-2 (100ng/ml, MCE, Shanghai, China). Additional stimulation with CXCL12 (100ng/ml, MCE, Shanghai, China) was performed both in the presence and absence of a CXCR4 antagonist (100 ng/mL, MCE, Shanghai, China) for 24 h. Intracellular cyttoxicity proteins and cytokines were then analyzed.

Cell lines

The murine cardiac muscle cell line HL-1, purchased from Shanghai Zhong Qiao Xin Zhou Biotechnology Co., Ltd., was incubated at 37 °C with 5% CO₂ in Minimum Essential Medium (MEM) supplemented with 10% fetal bovine serum (Gibco, CA, USA) and 1% penicillin-streptomycin (HyClone, UT, USA). Cells were seeded in 6-well plates and treated with 200 µl 10⁶ TCID50/100µl of CVB3 at 70–80% confluence. Before stimulation, cells were starved in a serum-free medium for 12 h and harvested 24 h after stimulation.

Quantitative Real-Time PCR (RT-qPCR)

RNA was extracted from isolated splenic CD8 ⁺ T cells using SparkZol Reagent (Shandong SparkJade Biotechnology Co., Ltd., Shandong, China). cDNA synthesis and RT-qPCR were performed using Evo M-MLV RT Premix for qPCR and the SYBR Green Premix Pro Taq HS qPCR Kit (ACCURATE BIOTECHNOLOGY, Hunan, China), following the manufacturer’s protocols. Primers for Gzma, Gzmb, Ifnγ, Tnfa, Prf1, and Actin were synthesized by BioSune Co., Ltd. (Shanghai, China) listed in Supplementary Material 4. Gene expression levels were normalized to Actin, and relative quantification was calculated using the 2^-∆∆CT method.

Enzyme-linked immunosorbent assay

The mouse plasma levels of Troponin-T, and TNF-αwere measured using a standard enzyme-linked immunosorbent assay (ELISA) kits (Cat.E-EL-M1801, Cat.E-EL-M3063, Elabscience, Wuhan, China), following the manufacturer’s instructions. The serum CXCL12 levels in FM patients and controls and CXCL12 levels in the supernatant of HL-1 cells were measured using the ELISA kit (RE2857H, RE2857M, ReedBiotech, Wuhan, China).

Flow cytometry

Mouse spleens were isolated and prepared into single-cell suspensions after red blood cell lysis. CD8⁺ T-cell subsets were identified using PerCP anti-mouse CD8a Antibody (Cat.100731, BioLegend, San Diego, USA) and FITC anti-mouse CD3 Antibody (Cat. 100203, BioLegend, San Diego, USA). The cytotoxic protein and effective factors of CD8⁺T cells were analyzed with PE anti-mouse Granzyme A Antibody, APC anti-human/mouse Granzyme B Recombinant Antibody, PE anti-mouse Perforin Antibody, and PE anti-mouse IFN-γ Antibody (Cat.149703, Cat.372203, Cat.154405, Cat.163503, BioLegend, San Diego, USA) respectively.

Statistical analysis

SPSS 25 software was used for statistical analysis, and GraphPad Prism version 9.0 software was used for graphing. Differences between the two groups were evaluated using unpaired two-sided student’s t-test, and differences among multiple groups were assessed using analysis of variance (ANOVA), followed by Dunnett’s multiple comparisons tests. A p < 0.05 was considered statistically significant.