TLR9- and CD40-Targeting Vaccination Promotes Human B Cell Maturation and IgG Induction via pDC-Dependent Mechanisms in Humanized Mice

Abstract

Mice reconstituted with a human immune system (humanized mice) provide a robust model to study human immunology, vaccinology, and human infectious diseases. However, the development and function of B cells in humanized mice is impaired. B cells from humanized mice are immature and are impaired in IgM to IgG isotype switch in response to infection or vaccination. In the present study we report that Toll-like receptor 9 (TLR9) agonist CpG-B combined with CD40-targeting vaccination triggered human B cell immunoglobin class-switch from IgM⁺ to IgG⁺ B cells in humanized mice. Human B cells from mice vaccinated with CpG-B as adjuvant were more mature in phenotype and produced significant levels of both total IgG and antigen-specific IgG. We found that CpG-B treatment activated human pDCs (plasmacytoid dendritic cells) in vivo to induce interferon-alpha (IFN-α)expression in humanized mice. Pre-depletion of human pDC in vivo abrogated the adjuvant effect of CpG-B. Our results indicate that TLR9 and CD40-targeting vaccination triggers human B cell maturation and immunoglobulin class-switch in a pDC-dependent manner in humanized mice. The findings also shed light on induction of human IgG antibodies in humanized mouse models.

Keywords: B cell maturation; CD40-targeting vaccination; CpG-B; IFN-alpha; IgG induction; immunoglobin class-switch; plasmacytoid dendritic cell.

Figure 1

TLR9 agonist CpG-B promotes IgG responses in humanized mice vaccinated with CD40-targeting vaccine. Humanized mice were treated with PBS control (n=3) or vaccinated with αCD40-HIV5pep (n=3) alone or vaccinated with αCD40-HIV5pep plus CpG-B (n=4) at week0, week3 and week6. At week 7, mice were sacrificed. (A, B) The expression of IgM and IgG on B cells from PBMCs and spleens was detected by FACS. (C) The total level of IgM and IgG in the plasma was detected by ELISA. (D) Splenocyte from humanized mice were cultured ex vivo in the present of R848(1μg/ml) and IL-2(10 u/ml) for 48hours, the cells were used for total IgG detection by ELISpot. (E) Antigen specific IgG level in the plasma was detected by ELISA. (F) The expression of activation-induced cytidine deaminase (AID) in spleen cells was detected by RT-PCR. Each dot represents one individual mouse, bars indicate mean. *P < 0.05, **P < 0.01, by unpaired, two-tailed Student’s t-test comparing the two vaccinated groups.

Figure 2

CpG-B promotes maturation and activation of B cells in humanized mice. Humanized mice were vaccinated as in Figure 1 . The phenotype of human B cells (hCD45⁺CD19⁺) from spleens of mice was detected by FACS. Representative dot plot (A, C, E) and Summarized data (B, D, F) showing the expression of CD38and CD24, CD10 and CD86 on human B cells. Each dot represents one individual mouse with n=3 in PBS group, n=4 in Vac group and n=4 in CpG-B plus Vac group. Bars indicate mean. *P < 0.05, **P < 0.01, ***P < 0.001, by unpaired, two-tailed Student’s t-test comparing the two vaccinated groups.

Figure 3

CpG-induced IFN-a production in vivo is dependent on human pDCs in humanized mice. (A, B) Humanized mice were treated with PBS (n=3) or CpG-B (50ug/mouse, i.p., n=4). (A) IFN-α and IL-6 level in plasma at indicated timepoint post treatment were detected by ELISA. (B) The expression of CD40, CD86 and HLA-DR on pDC (CD4⁺CD303⁺) from spleens at 24 hours post-treatment was detected by FACS. (C) Humanized mice were pretreated with either isotype control (n=3) or pDC depletion monoclonal antibody (clone 15B, 200 μg/mouse, i.p., n=4) at day -3 and -1. The percentage of pDC in human CD45+ cells from blood were detected by FACS. (D) Humanized mice were pretreated with either isotype control (n=4) or pDC depletion monocolonal antibody (clone 15B, 200 μg/mouse, i.p., n=3) at day -3 and -1. At day0, mice were treated PBS (Mock, n=4) with CpG-B (n=4). IFN-a level in plasma was detected 24 hours after CpG-B treatment. Each dot represents one individual mouse, bars indicate mean. *P < 0.05, **P < 0.01, by unpaired, two-tailed Student’s t-test (A, C) or by one-way analysis of variance (ANOVA) and Tukey’s multiple comparisons test (D).

Figure 4

TLR9 and CD40-targeting vaccination depends on pDC to promote human B cell maturation and IgG induction in humanized mice. Humanized mice were vaccinated as in Figure1 except one group of the mice were treated with pDC depletion Ab before vaccination (A) IFN-α in the plasma 24 hours after vaccination. (B) Expression of CD10 on B cells from spleen at termination. (C) Total IgG level in serum was detected by ELISA. (D) Antigen specific IgG level in the plasma was detected by ELISA. Each dot represents one individual mouse, bars indicate mean. Shown are representative data (PBS, n=3; Vac, n=3; CpG-B+Vac, n=4; CpG+Vac+15B, n=3, for A and B) or combined data (PBS, n=6; Vac, n=3; CpG-B+Vac, n=9; CpG+Vac+15B, n=7, for C and D) of two independent experiments with mean values. *P < 0.05, by one-way analysis of variance (ANOVA) and Tukey’s multiple comparisons test. (E, F) Correlation analysis between the IFN-α levels in plasma and total IgG (E) and specific IgG (F) levels in plasma (Spearman rank correlation test). r, correlation coefficient.

Figure 5

CD40-targeting vaccination is required to induce IgG-response in humanized mice. Humanized mice were treated with PBS control (Vehicle, n=3) or vaccinated with recombinant hemagglutinin protein (HA, n=3, 10μg/mouse) alone or vaccinated with HA plus CpG-B (n=4) at week0, week3 and week6. (A) IFN-a levels in plasma in plasma at 24 hours post first vaccination was detected by ELISA. (B) The expression of CD86 on pDC (CD4⁺CD303⁺) from PBMC at 24 hours post-treatment was detected by FACS. (C) Antigen specific IgG level in the plasma was detected by ELISA. Each dot represents one individual mouse, bars indicate mean. *P < 0.05 by unpaired, two-tailed Student’s t-test comparing the two vaccinated groups.