Figure 1.
Flow cytometric analysis of HLA-A2 and HLA-DP4 expression of transgenic molecules.
(A) Splenocytes from HLA-A2/DP4 (bold histogram) and wild-type C57BL/B6 (shadowed histogram) mice were isolated and stained with PE-labeled anti-HLA-ABC mAb to observe the HLA-A2 expression. PE-labeled mouse IgG2a, k mAb was used as isotype control (thinned histogram) for staining HLA-A2/DP4 mouse. (B) Splenocytes from wild-type C57BL/B6 mice (Figure 1B) and HLA-A2/DP4 (Figure 1C) were isolated and stained with PE-labeled anti-CD19 and FITC-labeled anti-HLA-DP mAb to observe the HLA-DP4 expression.
Figure 2.
Flow cytometric analysis of peripheral CD8+ T lymphocytes.
Splenocytes from HLA-A2/DP4 (Figure 2A) and wild-type C57BL/B6 mice (Figure 2B) were isolated and CD3+ T cells were gated by staining APC-conjugated anti-CD3 mAb and CD8+ T cells were gated by staining PE-conjugated anti-mCD8 mAb. Frequencies of CD8+ T cells among total CD3+ T cells from 6 mice of each group were shown in Figure 2C.
Figure 3.
Flow cytometric analysis of peripheral mCD4+ and hCD4+ T lymphocytes.
Splenocytes from HLA-A2/DP4 (Figure 3A, 3B, 3D and 3E) and wild-type C57BL/B6 (Figure 3C and 3F) mice were isolated and CD3+ T cells were gated by staining with APC-conjugated anti-CD3 mAb. Meanwhile, FITC-conjugated anti-mCD4 and PECy7-conjugated anti-hCD4 antibodies were simultaneously used to observe the mCD4 and hCD4 expression in HLA-A2/DP4 and WT C57BL/B6 mice.
Figure 4.
HBs-specific antibody and cytotoxic responses.
(A) Sera were collected 10 days after the third immunization, and the titers of the antibody (IgG) against HBs particles in the immunized mice (solid bar) were determined and compared with the mean responses of six PBS-immunized mice (hollow bar) in an ELISA assay. (B) HBs epitope-specific IFN-γ production by CTLs was determined by measuring response to the HLA-A2-restricted epitopes HBsAg348–357 and HBsAg335–343 and to the H-2 Kb-restricted epitope HBsAg371–378 in immunized mice (solid bar) and PBS-immunized mice (hollow bar).
Figure 5.
HBs-specific antibody and proliferative response.
(A) The titers of antibody (IgG) from HLA-A2/DP4 transgenic mice (solid bar) and H-2 class II-deficient mice (hollow bar) against HBs particles and the preS2109–134 peptide were determined using an ELISA assay (Figure 5A). (B) The HLA-DP4-restricted proliferation of CD4+ T cells from HLA-A2/DP4 transgenic mice (solid bar) and H-2 class II-deficient mice (hollow bar) after stimulation with HBs particles, the previously described HBV DP4 restricted peptide, S181–S192, or the control HLA-DP4-restricted epitope, Mag-3243–258 (Figure 5B).
Table 1.
DP4 epitopes binding ability and proliferative response in HLA-A2/DP4 mice.
Table 2.
Proliferative response in DP4-positive donors.