Figure 5.

CD24⁺ CD38^hi B cells inhibit the anti-virus functions of T cells and NK cells. PBMCs from HBV patients at 12-36 weeks after the start of PEG-IFNα-2b therapy were cultured in 24-well flat-bottom plates at a density of 1–2 × 106 cells per well in a complete RPMI medium 1640 with 10% fetal bovine serum plus streptomycin and penicillin as well as IL-2 (100 U/ml). PBMC from peripheral blood mononuclear cells of CHB patients with or without CD19+CD24+CD38^hi B cells were sorted and the purity of depleting CD19+CD24+CD38^hi B cells was determined to be >95% by post-purification FACS analysis. PBMC with or without CD19+CD24+CD38^hi B cells were re-stimulated with 50 μg/ml of HBsAg (HyTest, 8HS7ay) for 72 h and cytokines were measured by intracellular staining. (A) The gating strategy for analyzing T- and NK cells. (B) Representative density plots of IFNγ, TNFα and CD107a expression on gated CD3⁺T cells or CD56⁺ CD3^-NK cells from HBV patients with or without CD24⁺ CD38^hiB cells. (C, D) Percentage analysis of IFNγ, TNFα and CD107a expression on gated CD3⁺T cells or CD56⁺NK cells from HBV patients with or without CD24⁺ CD38^hiB cells (Breg). n = 8 in each group. Paired t-tests. (E, F) Percentage analysis of IFNγ ⁺CD107a⁺, TNFα⁺CD107a⁺ cells in gated CD3⁺T cells or CD56⁺CD3^-NK cells from HBV patients with or without CD24⁺ CD38^hiB cells (Breg). Paired t test. (G) Analysis of IgG secretion from HBV patients with or without CD24⁺CD38^hiB cells. Paired t test. PBMC with or without CD19⁺CD24⁺CD38^hi B cells (Breg) were re-stimulated with 50 μg/ml of HBsAg for 72 h and cell-free supernatants of cultured PBMCs in each group were measured IgG by ELISA. (H) The IgG increase ratio (IgG level in PBMC after Breg depletion/IgG level in PBMC before Breg depletion) is positive correlated with the ratio between Breg cells and whole PBMC from HBV patients (Breg cell number/PBMC cell number×100). n = 9. Mean ± SEM, *p < 0·05, **P < 0·01, ***P < 0·005.