In vivo inhibition of anti-hepatitis B virus core antigen (HBcAg) immunoglobulin G production by HBcAg-specific CD4(+) Th1-type T-cell clones in a hu-PBL-NOD/SCID mouse model

Abstract

Hepatitis B virus (HBV) core antigen (HBcAg)-specific CD4(+) T-cell responses are believed to play an important role in the control of human HBV infection. In the present study, HBcAg-specific, HLA-DR13*-restricted CD4(+) Th1-type T-cell clones were generated which secreted both gamma interferon and tumor necrosis factor alpha after in vitro antigen stimulation. These HBcAg-specific CD4(+) Th1-type T cells were able to lyse HBc peptide-loaded Epstein-Barr virus-transformed lymphoblastoid target cells in vitro. To examine whether these HLA-DR13*-restricted human CD4(+) Th1 T cells also display the same cytotoxic effects in vivo, we transferred peripheral blood leukocytes (PBL) derived from HBV-infected donors or an HBV-naïve donor sharing the DR13*, together with the HBcAg-specific CD4(+) Th1-type T cells and HBcAg, directly into the spleen of optimally conditioned Nod/LtSz-Prkdc(scid)/Prkdc(scid) (NOD/SCID) mice. The production of both secondary anti-HBc-immunoglobulin G (anti-HBc-IgG) and primary HBcAg-binding IgM in hu-PBL-NOD/SCID mice was drastically inhibited by HBcAg-specific CD4(+) Th1-type T cells. No inhibition was observed when CD4(+) Th1 cells and donor PBL did not share an HLA-DR13. These results suggest that HBcAg-specific CD4(+) Th1 T cells may be able to lyse HBcAg-binding, or -specific, B cells that have taken up and presented HBcAg in a class II-restricted manner. Thus, HBcAg-specific CD4(+) Th1-type T cells can modulate the function and exert a regulatory role in deleting HBcAg-binding, or -specific, human B cells in vivo, which may be of importance in controlling the infection.

FIG. 1

Phenotypic analysis of HBcAg-specific T-cell clone 121. HBcAg-specific T cells were double stained with different monoclonal antibodies (Becton Dickinson) and analyzed by flow cytometry. Clones 124 and 135 displayed the same pattern.

FIG. 2

HLA restriction of T-cell clones 124 and 135. HBcAg-specific T-cell clones were stimulated by HBcAg (0.1 μg/ml) and irradiated (2,500 rads) autologous PBL as APCs in the absence or presence of mouse anti-human HLA-DR, -DP, and -DQ antibodies and isotypic controls (1 μg/ml; Serotec). [³H]thymidine (0.5 μCi/well; Amersham International, Little Chalfont, United Kingdom) was added 18 h before harvesting on day 4. They were then assayed for [³H]thymidine incorporation by liquid scintillation counting in an LKB-Wallac 8100 counter (LKB, Bromma, Sweden). All cultures were performed in triplicate, and the data are expressed as the mean counts per minute. TT, tetanus toxoid.

FIG. 3

Intracellular cytokine staining of HBcAg-specific T-cell clones 121 and 124. Activated HBcAg-specific CD4 T cells were stained with both anti-CD4 (fluorescein isothiocyanate-labeled) and phycoerythrin-labeled anti-human cytokine (IFN-γ, TNF-α, and IL-4) antibodies as described. The two clones expressed both IFN-γ and TNF-α but no IL-4 and thus behaved like Th1-type CD4 cells.

FIG. 4

In vitro cytotoxic assays of HBcAg-specific CD4⁺ Th1 T-cell clones 124 and 135. JAM assays were performed as described in Materials and Methods. Peptide-loaded autologous EBV-transformed LCLs were used as target cells. Assays were set up at different effector/target ratios (90/1, 30/1, 10/1, 3.3/1, 1/1, and 0/1). All conditions were tested in triplicate. Specific lysis of target cells at different effector/target ratios is shown. Spontaneous lysis was always less than 20%.

FIG. 5

HLA class II restriction of the cytotoxicity of HBcAg-specific CD4⁺ Th1 T-cell clones 124 and 135. JAM assays were performed as described. Based on the results of experiments shown in Fig. 4, an effector/target ratio of 30/1 was selected in these experiments. Autologous and allogeneic EBV-transformed LCLs sharing different HLA class II molecules were loaded with the HBc peptide containing amino acids 141 to 160 and served as target cells. Specific lysis of the target cells is shown.