This study aims to integrate flow cytometry of peripheral blood with single-cell RNA-sequencing of aortic tissue to characterize NKT-cell subsets and their functional states in TAK, and to identify mechanistic biomarkers and therapeutic targets.

We performed flow cytometric analysis of peripheral blood from 98 TAK patients and 50 healthy controls to phenotype NKT cells and their CD8⁺ subsets using CD3, CD56 and CD8, and to assess functional potential via granzyme B, perforin and IFN-γ expression. In parallel, This study utilized single-cell transcriptome analysis on aortic specimens from three TAK patients, with control data sourced from a publicly available database (GSE155468). Differentially expressed genes (DEGs) were defined at false-discovery rate < 0.05. Functional enrichment used Gene Ontology and KEGG pathways, and CellChat inferred intercellular ligand–receptor communication.

Flow cytometry reveals reduced peripheral blood NKT and CD8⁺ NKT cells with increased IL‑4⁺ NKT cells in patients with Takayasu arteritis

Peripheral blood samples from 98 patients with Takayasu arteritis (TAK) and 50 healthy controls were analyzed, with human NKT cells defined as CD3⁺CD56⁺ lymphocytes (Fig. 1a). Both the frequencies and absolute counts of NKT and CD8⁺ NKT cells among total gated lymphocytes were significantly lower in TAK patients than in healthy controls (P < 0.0001) (Fig. 1b). Concurrently, expression levels of granzyme B and interferon‑γ were decreased, while interleukin‑4 was increased (Fig. 1c), indicating a functional shift of NKT cells from cytotoxicity toward immunoregulation. No significant differences in NKT cell proportions were observed between active and inactive disease stages (Fig. 1d), suggesting that this phenotype represents an inherent disease feature rather than a marker of disease activity.

Single‑cell RNA‑sequencing identifies expanded FCGR3A⁺ NKT cells in aortic lesions of patients with Takayasu arteritis

Single‑cell RNA‑sequencing was performed on aortic specimens from 3 TAK patients and 3 healthy controls, identifying nine transcriptionally distinct cell subsets (Fig. 2a). Further clustering of T cell subsets resolved six subpopulations (Fig. 2b). Compared with controls, FCGR3A⁺ NKT cells were significantly expanded in aortic tissue from TAK patients, whereas KLRC1⁺ NKT cells were virtually absent (Fig. 2c). Expression levels of granzyme B, perforin, and interferon‑γ were markedly higher in FCGR3A⁺ NKT cells from the TAK group than in KLRC1⁺ NKT cells from controls, indicating enhanced cytotoxic function of NKT cells in TAK lesions (Fig. 2d). Subsequent Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses identified the top 20 GO terms and KEGG pathways. FCGR3A⁺ NKT cells in the TAK group were enriched in autoimmune and pro‑inflammatory pathways (Fig. 3a), whereas KLRC1⁺ NKT cells in controls were enriched in NK cell‑mediated cytotoxicity and immune regulatory pathways (Fig. 3b), with convergence in core immune functions. Based on these results, we further performed cell‑cell interaction analyses focusing on FCGR3A⁺ NKT cells in TAK and KLRC1⁺ NKT cells in controls. Interaction network analysis demonstrated enhanced crosstalk between FCGR3A⁺ NKT cells and CD8⁺ effector T cells, driving inflammation, whereas KLRC1⁺ NKT cells interacted actively with regulatory T cells (Tregs) to maintain immune homeostasis (Fig. 2e). Pro‑inflammatory transcription factors were strongly upregulated in FCGR3A⁺ NKT cells, establishing these cells as a central hub in TAK‑associated inflammatory networks.

By integrating flow cytometric analysis of peripheral blood and single-cell transcriptomic profiling of aortic tissues from TAK patients and healthy controls, we identified compartmentalized alterations of NKT cells in TAK. Specifically, circulating NKT and CD8⁺ NKT cells were decreased in conjunction with an expanded IL‑4⁺ NKT cell population, while FCGR3A⁺ NKT cells were markedly enriched in aortic lesions. Interaction analysis further demonstrated that FCGR3A⁺ NKT cells preferentially engaged with CD8⁺ effector T cells to promote inflammatory responses, whereas KLRC1⁺ NKT cells in controls interacted actively with regulatory T cells to preserve immune homeostasis. These findings highlight divergent NKT cell profiles between the peripheral circulation and vascular lesions in TAK. We hypothesize that cytotoxic FCGR3A⁺ NKT cells may undergo polarization and migrate from peripheral blood to aortic lesions, where they exert pro‑inflammatory and cytotoxic effects that contribute to vascular inflammation and remodeling.