Fig 2: The pro-fibrogenic features of circulating CD161+CD4+ T cells in chronic HBV settings. (a–c) Human pHSCs were co-cultured with circulating CD4+ T subsets from LC cohort (n = 6) at a cell-to-cell ratio of 1:1 for 72 h. After co-cultures, adherent pHSCs were collected for qRT-PCR analysis of genes involved in liver fibrogenesis including a-SMA, collagen I, TGF-ß1 and TIMP1. (a) Co-cultures with circulating CD161+CD4+ T cells or homologous CD161-CD4+ T cells in the contact or transwell manner. (b, c) Co-cultures with CD161+CD4+ T cells in the transwell (b) or contact (c) manner in the presence or absence of neutralising anti-IL-17, anti-IL-23 or anti-IFN-? Ab. (d) pHSCs were stimulated with IFN-? or co-cultured with circulating CD161+CD4+ T cells from LC cohort (n = 4) in the presence or absence of the neutralising anti-IFN-? Ab. After 72 h, pHSCs were collected for qRT-PCR analysis of the APCs phenotypic markers of CD80, CD86, MHC-II, CD1d, CD1b and IL-23. (e) Homologous CD161+CD4+ T cells and CD161-CD4+ T cells from the circulation of LC cohort (n = 6) were, respectively, treated with recombinant IL-23 at different concentrations for 72 h. Supernatant samples were collected to determine IL-17 concentration by ELISA. (f) pHSCs were treated with recombinant IL-17 at different concentrations for 72 h to be determined by qRT-PCR analysis of fibrosis markers (*P < 0.05; **P < 0.01; ***P < 0.001).

Fig 3: Dectin-1 directs TH17 polarization via release of IL-1ß and IL-23.a-e, Flow cytometry analyses of TH polarization by staining for intracellular IL-17 and IFN-? expression (FI, fluorescence intensity) in restimulated T cells, outgrown in vitro by co-culture of either naive (Tn, a,b,) or memory (Tm, c-e,) CD4+ T cells with immature DCs (iDC) or DCs primed for 48 h with curdlan or C. albicans, in the presence of blocking IL-1ß, IL-23 or IL-12 antibodies (Ab) or isotype IgG control antibodies, at day 5 (a,b, n = 3; c-e, n = 6) or day 11–14 (c-e, n = 6). In a,c,d, representative dot plots for independent donors are shown, with the percentage positive cells indicated in each quadrant. In b,e, the percentage IL-17+ cells per total amount of T cells are shown. Data in b,e represent mean ± s.d. of independent donors. **P < 0.01 (Student’s t-test, paired, two-tailed), calculated between untreated and treated samples that were likewise stimulated. Source data

Fig 4: Dectin-1 does not instruct IFN-ß-mediated non-pathogenic TH17 polarization via IL-1ß, IL-6, IL-23, IL-12 or IL-27.a,b, ELISA for quantification of IL-1ß, IL-6, IL-23, IL-12 and IL-27 in the supernatant of unstimulated DCs or after stimulation of DCs with curdlan or C. albicans, in the presence of blocking IFN-a/ßR antibodies (a,b) or rhIFN-ß (b) at 28 h (a, n = 5; b, IFN-a/ßR antibody n = 5, rhIFN-ß n = 3). Data in a,b represent mean ± s.d. of independent donors. **P < 0.01, *P < 0.05 (paired, two-tailed Student’s t-test), calculated between untreated and treated samples that were likewise stimulated. c–e, Flow cytometry analyses of TH polarization by staining for intracellular IL-17 and IFN-? expression (FI) in restimulated T cells, outgrown in vitro by co-culture of memory CD4+ T cells with iDC or DCs primed for 48 h with curdlan or C. albicans, in the presence of neutralizing IL-27 antibodies or rhIL-27 (both during DC-T co-culture), at day 11–14 (IL-27 antibody n = 7, rhIL-27 n = 4). In c, representative dot plots for independent donors are shown, with percentage positive cells indicated in each quadrant. In d, the percentage IL-17+ cells per total amount of T cells are shown and in e, the percentage IFN-?- and percentage IFN-?+ cells per IL-17+ T cells are shown. Data in d,e represent mean ± s.d. of independent donors.Source data

Fig 5: Effect of the affinity for topo I in topo I-reactive B cells on the development of fibrosis in the topo I-induced systemic sclerosis (SSc) model mice.Topo I-reactive B cells (104 cells) from mice immunized once or four times with topo I were adoptively transferred into wild-type (WT) mice, and 14 days later, these WT mice were immunized with topo I three times every 2 weeks (A). 56 days after the adoptive transfer, the skin (original magnification, ×40) and lung tissues (×100) were obtained, and the dermal thickness and lung fibrosis score were examined histologically (B). Similarly, after 14 days of the adoptive transfer of topo I-reactive B cells (104 cells) from mice immunized once or four times with topo I into non-immunized WT mice (C), the dermal thickness and lung fibrosis score were measured (D). Serum anti-topo I antibody levels were elevated in both first and fourth models compared with non-immunized WT mice. Furthermore, topo I-reactive B cells were adoptively transferred with either anti-IL-6 (1 mg/week, administrated subcutaneously) or anti-IL-23 (100 µg/week, administrated subcutaneously) antibodies (Abs), and dermal thickness and lung fibrosis score were examined 14 days after the adoptive transfer (E). WT mice, IL-6-deficient (IL-6KO), and IL-23-deficient (IL-23KO) mice were immunized four times with topo I. Then, 104 cells of topo I-reactive B cells obtained were transferred to non-immunized WT mice, and the dermal thickness and lung fibrosis score were measured 14 days later. Serum anti-topo I antibody levels were elevated in both IL-6 KO mice and IL-23 KO mice compared with non-immunized WT mice (F). These results represented six experiments. The bar graphs show the mean + SD. *p<0.05. Figure 6—source data 1.Source file for the effect of the affinity for topo I in topo I-reactive B cells on the development of fibrosis in the topo I-induced systemic sclerosis (SSc) model mice.This archive contains all data used for the quantitative analysis shown in Figure 6.