Figure 3.

Activated APCS induced the differentiation and expansion of the PD-1⁺CXCR5⁻CD4⁺ Th-CXCL13 cell subset through TGF-β1. (A) Representative dot plots showing PD-1⁺CXCR5⁻CD4⁺ Th-CXCL13 cells in vitro (n=5). (B) Representative dot plots showing that TGF-β1 with or without other indicated cytokines gave rise to CXCL13-producing PD-1⁺CD4⁺ Th cells in vitro (n=5). (C) Secretion of CXCL13 by sorted PD-1⁺CXCR5⁻D4⁺ TILs (n=4) after stimulation for 8 days using anti-CD3/CD28, TGF-β1 (10 ng/mL) and/or TGF-β1 receptor inhibitor SB431542 (10 µM/mL). (D, E) The mRNA expression of the indicated cytokines was determined by RT-qPCR and the cytokine protein in supernatants was determined by ELISA (n=5). (F, G) MOs, DCs and Mφ were left untreated or stimulated with LPS for 5 hours and then cultured for 8 days with autologous T cells, naïve T cells and memory T cells from healthy donors (n=3). Proliferation (CFSE⁻) and expression of CXCL13⁺ cells were detected by FACS. (H) Representative dot plots showing that blocking of TGF-β1, IL-1β, IL-6, TNF-α and IL-12 changed the subset composition of Th-CXCL13 cells expanded by LPS-stimulated mos from healthy donors (n=3). (I) Representative dot plots and statistical analysis showing of the expression of the indicated markers on MOs from tumors and blood (n=5). (J) Representative dot plots showing that the addition of anti-TGF-β1 and/or IL-2 or TGF-β1 and/or anti-IL-2 changed the subset composition of Th-CXCL13 cells expanded by LPS-stimulated tumor-derived MOs (n=3). Data were expressed as the means±SD, and paired two-tailed Student t-test. *P<0.05, **P<0.001, ***P<0.0001. APC, antigen-presenting cell; DC, dendritic cell; FACS, fluorescence-activated cell sorting; IFN-γ, interferon gamma; Mφ, macrophage; MO, monocyte; RT-qPCR, quantitative reverse transcription PCR; TGF-β1, transforming growth factor beta 1; TIL, tumor-infiltrating lymphocyte; TLS, tertiary lymphoid structure; TNF-α, tumor necrosis factor alpha; MFI, mean fluorescence intensity.