Single-cell transcriptomics reveals granzyme K–expressing cytotoxic Tfh cells in tertiary lymphoid structures in IgG4-RD

Background: Germinal center (GC) responses controlled by T follicular helper (Tfh) and T follicular regulatory (Tfr) cells are crucial for the generation of high-affinity antibodies. Acquired immune responses to tissue-released antigens might be mainly induced in tertiary lymphoid organs (TLOs) with GCs in affected tissues. IgG4-related disease (IgG4-RD) demonstrates polarized isotype switching and TLOs in affected tissues. We performed single-cell transcriptomics of tissue-infiltrating T cells from these TLOs to obtain a comprehensive, unbiased view of tissue-infiltrating GC-Tfh cells. Objective: To identify GC-Tfh-cell subsets in TLOs in patients with IgG4-RD using single-cell transcriptomics. Methods: Single-cell RNA sequencing of sorted CD3⁺ T cells and multicolor immunofluorescence analysis were used to investigate CD4⁺CXCR5⁺Bcl6⁺ GC-Tfh cells in affected lesions from patients with IgG4-RD. Results: Infiltrating CD4⁺CXCR5⁺Bcl6⁺ Tfh cells were divided into 5 main clusters. We detected HLA⁺ granzyme K⁺ (GZMK⁺) Tfh cells with cytotoxicity-associated features in patients with IgG4-RD. We also observed abundant infiltrating Tfr cells with suppressor-associated features in patients with IgG4-RD. These GZMK⁺ Tfh cells and Tfr cells clustered together in affected tissues from patients with IgG4-RD. Conclusions: This single-cell data set revealed a novel subset of HLA⁺GZMK⁺ cytotoxic Tfh cells infiltrating affected organs in patients with IgG4-RD, suggesting that infiltrating Tfr cells might suppress cytotoxic Tfh cells.