Effect of dendritic cells loaded with HBsAg on the differentiation of HBsAg-specific T helper 1 cells in patients with chronic hepatitis B

AIM: To investigate the influence of dendritic cells (DCs) loaded with HBsAg from peripheral blood monocytes of patients with chronic hepatitis B (CHB) on the differentiation of autologous T helper 1 (Th1). METHODS: Mononuclear cells were isolated from CHB patients using the methods of density gradient centrifugation (Ficoll-Hypaque) and adherence. The adherent monocytes were incubated with recombinant human GM-CSF and interleukin-4 (IL-4) to induce DC generation. The phenotypes on DCs including CD80, CD86, CD40 and HLA-DR were determined by flow cytometry (FCM). DCs' ability to stimulate allogenic mixed lymphocyte reaction (AMLR) was detected by CCK-8 assay. The concentration of IL-12 in DC supernate was tested by enzyme linked immunosorbent assay (ELISA). CD4⁺ Th cell subpopulation in peripheral blood of CHB patients was isolated and purified by immunomagnetic beads. Subsequently, purified CD4 ⁺ Th cells were co-cultured with autologous mature DCs. The differentiation of Th1/Th2 was detected by FCM using intracellular staining of interferon-γ (IFN-γ) and IL-4. ELISA assay was also used to quantify the IFN-γ and IL-4 in co-culture supernate. RESULTS: In comparison with those in the control group, the expression levels of CD80, CD86, CD40 and HLA-DR and the concentration of IL-12 in DC supernate of the IFN-γ group, HBsAg group and HBsAg + IFN-γ group were higher, and the capability of DCs to stimulate allogenic lymphocytes to proliferate in the three groups was also stronger. Meanwhile, the percentages of Th1 cells in CD4⁺ T cells and the level of IFN-γ in co-culture supernate of the IFN-γ group, HBsAg group and HBsAg + IFN-γ group were higher (Th1 cells: 10.76% ± 3.98%, 11.43% ± 4.32%, 15.28% ± 4.73% vs 7.84% ± 3.10%, P < 0.01; IFN-γ: 578 ± 47 mg/L, 496 ± 92 mg/L, 784 ± 97 mg/L vs 342 ± 34 mg/L, P < 0.05). However, the percentages of Th2 cells in CD4⁺ T cells and the level of IL-4 in the three groups were lower (Th2 cells: 1.43% ± 0.96%, 1.68% ± 0.16%, 0.92% ± 0.21% vs 2.61% ±1.27%, P < 0.01; IL-4:187 ± 52 mg/L, 169 ± 38 mg/L, 89 ± 37 mg/L vs 226 ± 48 mg/L, P < 0.05). The most significant effect was observed in the HBsAg + IFN-γ group. CONCLUSION: DCs loaded with HBsAg can improve the insufficient differentiation of Th1 cells resulted from dysfunction of DCs in vivo in CHB patients.