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

Background: Germinal center (GC) responses controlled by follicular helper (Tfh) and T follicular regulatory (Tfr) cells 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. (J Allergy Clin Immunol 2024;153:513-20.)