HBVsvp-Pulsed Dendritic Cell Immunotherapy Induces Th1 Polarization and Hepatitis B Virus-Specific Cytotoxic T Lymphocytes Production

Abstract

Background: In chronic hepatitis B virus (CHB) patients, both dendritic cells (DCs) and T cells are functionally impaired and consequently the HBV-specific cellular immune responses are downregulated. The present study aims to investigate whether monocyte-derived DC (MoDCs)-pulsed-HBV subviral particles (HBVsvp) can polarize Th1 cells to induce HBV-specific cytotoxic T-lymphocytes (CTL) responses in CHB patients.

Methods and materials: To this end, the human hepatoma HepG2.2.15 cell line was used to produce HBVsvp as a culturing system, and HBVsvp were concentrated for highly virus titer using the polyethylene glycol protocol. Peripheral blood mononuclear cells (PBMCs), collected from CHB patients and healthy donors, were differentiated into MoDCs and T cells. PBMCs-derived MoDCs were first pulsed with HBVsvp and then cultured with PBMCs-derived T cells. MoDCs and/or T subsets cells were identified for phenotypic activation by FACS analysis. The cytokine secretion of IL-4, IL-12, and IFN-γ in the culture supernatants was detected.

Results: The MoDCs were restored for their activation upon pulsing with HBVsvp in vitro, as identified by significantly overexpression of both CD86 and HLA-DR, and overproduction of IL-4 and IL-12. Furthermore, MoDCs-pulsed-HBVsvp induced Th1 frequencies and activated HBV-specific CTL to produce significantly highest amount of IFN-γ. Enhanced HBV-specific CTL led to strong cytolytic capacity against HepG2.2.15.

Conclusion: Overall, our data suggest that in vitro activation of MoDCs with HBVsvp overcomes the functionally impaired DCs and T cells in CHB patients offering a promising tool for therapeutic or vaccine-based approaches against HBV.

Keywords: CHB; HBV-specific CTL; MoDCs-pulsed-HBVsvp; immunotherapy.

Figure 1

Showed FACS analysis of MoDCs, identified by expression of CD11c after 7 days of maturation, approximately 79.5% of the cultured cells were positive for CD11c, indicating the differentiation of monocytes towards MoDCs.

Figure 2

Showed CD86-PE activating marker of MoDCs detected in healthy donors and chronic patient groups at different treatment. Small letters represent the significance between normal control and chronic patient within each treatment. Capital letters represent the significance of normal control or chronic patient at different treatments. Error bars represent standard error (SE).

Figure 3

Showed HLA DR-PE activating marker of MoDCs detected in healthy donors and chronic patient groups at different treatment. Small letters represent the significance between normal control and chronic patient within each treatment. Capital letters represent the significance of normal control or chronic patient at different treatments. Error bars represent standard error (SE).

Figure 4

Showed MoDCs from healthy donors and chronic patients were secreted significantly larger amounts of the effector cytokine IL-4 into the culture medium when co-cultured with HBVsvp+ LPS. Capital letters represent the statistical significance difference between normal control and chronic patient at different treatments and/or within each treatment. Error bars represent standard error (SE).

Figure 5

Showed MoDCs from healthy donors and chronic patients were secreted significantly larger amounts of the effector cytokine IL-12 into the culture medium when co-cultured with HBVsvp+ LPS. Capital letters represent the statistical significance difference between normal control and chronic patient at different treatments and/or within each treatment. Error bars represent standard error (SE).

Figure 6

Showed ability of MoDCs-pulsed-HBVsvp to induce an autologous Th1/Tc polarization response in vitro for both healthy donors and chronic patients.

Figure 7

Showed HBV-specific-CTL from healthy donors and chronic patients were secreted significantly larger amounts of the effector cytokine IFN-γ into the culture medium when co-cultured with MoDCs-pulsed-HBVsvp. Capital letters represent the statistical significance difference between normal control and chronic patient at different treatments and/or within each treatment. Error bars represent standard error (SE).

Figure 8

Showed the cytotoxicity assay for HBV-specific-CTL against HepG2.2.15 cell line in different ratio for different chronic patients and different healthy donors (**p < 0.001, *p < 0.05).