Expanded circulating follicular dendritic cells facilitate immune responses in chronic HBV infection

Abstract

Background: The restoration of host hepatitis B virus (HBV)-specific antiviral immunity is an effective strategy for hepatitis B recovery. Follicular dendritic cells (FDCs) play a crucial role in immune regulation. The goal of the present study was to investigate the characteristics and functions of FDCs in chronic HBV infection.

Methods: The frequencies of FDCs in peripheral blood, liver, and spleen were measured in patients with chronic HBV infection. Isolated FDCs from splenic tissues of HBV-related liver cirrhosis-induced hypersplenism patients were cultured with autologous intrasplenic CD4⁺ T cells and CD19⁺ B cells.

Results: We observed that patients with chronic HBV infection had a significantly increased frequency of circulating FDCs compared to that of healthy controls. Additionally, the frequency of circulating FDCs was positively correlated with that of intrahepatic and intrasplenic counterparts. Moreover, positive correlations were observed between the frequencies of circulating FDCs and plasmablast and memory B cells, as well as C-X-C motif chemokine receptor type 5 (CXCR5)⁺CD4⁺ T cells and CXCR5⁺CD8⁺ T cells. Notably, in vitro experimental results demonstrated that FDCs derived from splenic tissues of chronic HBV patients facilitated interferon-γ and interleukin-21 production from autologous intrasplenic CD4⁺ T cells and promoted the proliferation of autologous intrasplenic CD19⁺ B cells.

Conclusions: Expanded FDCs in patients with chronic HBV infection may favor host immune responses against HBV. The identification of this unique population of cell may contribute to a better understanding of the immune regulatory mechanisms associated with chronic HBV infection and provide a potential immunotherapeutic target for this disease.

Keywords: B cells; Follicular dendritic cells; Hepatitis B virus; T cells.

Fig. 1

Circulating FDCs and intrahepatic and intrasplenic FDCs frequencies are positively correlated in chronically HBV-infected patients. a Gating strategy for the identification of FDCs (CD14⁺CD21^highFDC⁺) using flow cytometry. FDCs population was calculated as a percentage of CD14⁺ cells. b Comparison of the frequencies of circulating (n = 55), intrahepatic (n = 22) and intrasplenic (n = 17) FDCs in patients with chronic HBV infection. c The frequency of circulating FDCs was correlated with that of intrasplenic FDCs (left panel, n = 11) and intrahepatic FDCs (right panel, n = 11). ***P < 0.001. FSC-A, forward scatter; SSC-A, side scatter; Live/Dead, fixable dead cell stain

Fig. 2

Circulating FDCs are expanded in patients with chronic HBV infection. a Comparison of the frequencies of FDCs between healthy controls (HCs, n = 10) and patients with chronic HBV infection (HBV, n = 31). b Comparison of the frequencies of FDCs between immune tolerant carrier (IT, n = 13), HBeAg-positive CHB (CHB, n = 9), and inactive carrier (IC, n = 9) patients. c–e Analyses of the correlations between the frequency of circulating FDCs and serum levels of ALT, AST, HBsAg, HBeAg, and HBV DNA in chronic HBV patients (n = 31). *P < 0.05, **P < 0.005

Fig. 3

Correlations between the frequencies of FDCs and immune cell subsets in chronic HBV-infected patients. a Correlations between the frequencies of FDCs and plasmablast (CD19⁺CD38⁺CD27⁺), memory B cells (CD19⁺CD27⁺CD38⁻), naïve B cells (CD19⁺CD10⁻CD27⁻), and immature B cells (CD19⁺CD10⁺CD27⁻) in peripheral blood in chronic HBV patients (n = 16). b Correlations between the frequencies of FDCs and CXCR5⁺CD4⁺ T cell and CXCR5⁺CD8⁺ T cell subsets in peripheral blood in chronic HBV patients (n = 5). c Correlations between the frequency of circulating FDCs and plasma levels of IL-6, IL-7, IL-15, BAFF, and CXCL13 in chronic HBV patients (n = 31)

Fig. 4

FDCs facilitate CD4⁺ T cell cytokine production and B cell proliferation in vitro. a Cultured FDCs were observed by optical microscopy at days 1, 7, 14, and 25. b Cultured FDCs were stained for FDC expression and analyzed by confocal microscopy, where red and blue indicate FDCs and DAPI, respectively. Images at ×40 magnification. Cultured FDCs were stimulated with the indicated reagents for 3 days and the supernatants were collected for IL-6 (c) and CXCL13 (d) detection by ELISA. Cultured FDCs were co-cultured with purified autologous CD4⁺ T cells in the presence of IL-2 (10 ng/mL) and anti-CD3/CD28 (10 μg/mL) for 3 days and the supernatant was collected for IL-21 (e) and IFN-γ (f) detection by ELISA. g Purified autologous CD19⁺ B cells were labeled with CFSE and then co-cultured with cultured FDCs in the presence of CPG (10 μg/mL) for 7 days and the proliferation of B cells was measured by flow cytometry