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. 2022 Aug 25;13:941385. doi: 10.3389/fimmu.2022.941385

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Copyright © 2022 Murayama, Ikegami, Kamekura, Sakamoto, Yanagi, Kamiya, Sato, Sato, Shigehara, Yamamoto, Takahashi, Takano and Ichimiya

This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

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Phenotypic characteristics of GC-type Tfh cells in IgG4-RD lesions. (A) Flow cytometry profiles showing selected windows and gating strategy applied to identify GC-type Tfh cells (CD3⁺CD4⁺CXCR5^hiPD-1^hi) in lymphocytes of tonsils and SMG lesions of IgG4-RD. (B) Volcano plot identifying differentially expressed genes (p < 0.05) with more than two-fold expression in GC-type Tfh cells localized in SMG lesions of IgG4-RD versus GC-type Tfh cells in tonsils. The red and blue dots indicate upregulated and downregulated genes, respectively, in the Tfh cells of IgG4-RD. Data were obtained from microarray analysis of four specimens in each experiment group of IgG4-RD lesions or tonsils. (C) Heat map indicating relative abundances of transcripts identified in (A) for selected genes regulating helper CD4⁺ T cells and CTLs. Relative values of gene expression are indicated by color. (D) Relative expression levels of genes in GC-type Tfh cells in the SMG lesions of IgG4-RD and tonsils as indicated in (C) assessed by RT-qPCR analysis. GAPDH was used as a control (IgG4-RD, n = 4-6; tonsil, n = 4-6). (E) Representative flow cytometry profiles of the expression of CD8 and granzyme A (GZMA) in GC-type Tfh cells in SMG lesions of IgG4-RD and tonsils (upper panels). Graphs indicating the expression of CD8 (IgG4-RD, n = 31; tonsil, n = 71) and GZMA (IgG4-RD, n = 11; tonsil, n = 11) in GC-type Tfh cells as assessed by flow cytometry (lower panels). (F) Expression levels of CD8 on DP-Tfh cells and CD8⁺ CTLs in the lymphocytes of IgG4-RD lesions and tonsils (IgG4-RD, n = 8; tonsil, n = 8) assessed by flow cytometry. MFI, mean fluorescence intensity. (G) Transmission electron microscopy of FACS-sorted T cells, including GC-type Tfh cells from SMG lesions of IgG4-RD and tonsils and NKT cells (CD3⁺CD56⁺) from tonsils. Arrowheads indicate electron-dense granules in the cytosol. Scale bar: 1 μm. (H) DP-Tfh cells enriched in the CD3⁺CD8⁺CXCR5^hiPD-1^hi T cell population in tonsils. Representative flow cytometry profiles to detect DP-Tfh cells and CD3⁺CD4^-CD8⁺CXCR5^hiPD-1^hi T cells as indicated in the red square (left panels). A graph showing the CD3⁺CD8⁺CXCR5^hiPD-1^hi T cell population preferentially containing DP-Tfh cells (right). Data from the same tonsils are paired (n = 6). Data in (D–F, H) were analyzed by the Mann–Whitney U test. Data in (G) were obtained from three independent experiments.