B cell receptor-mediated uptake of CD1d-restricted antigen augments antibody responses by recruiting invariant NKT cell help in vivo

Abstract

Highly regulated activation of B cells is required for the production of specific antibodies necessary to provide protection from pathogen infection. This process is initiated by specific recognition of antigen through the B cell receptor (BCR), leading to early intracellular signaling followed by the late recruitment of T cell help. In this study we demonstrate that specific BCR uptake of CD1d-restricted antigens represents an effective means of enhancing invariant natural killer T (iNKT)-dependent B cell responses in vivo. This mechanism is effective over a wide range of antigen affinities but depends on exceeding a tightly regulated avidity threshold necessary for BCR-mediated internalization and CD1d-dependent presentation of particulate antigenic lipid. Subsequently, iNKT cells provide the help required for stimulating B cell proliferation and differentiation. iNKT-stimulated B cells develop within extrafollicular foci and mediate the production of high titers of specific IgM and early class-switched antibodies. Thus, we have demonstrated that in response to particulate antigenic lipids iNKT cells are recruited for the assistance of B cell activation, resulting in the enhancement of specific antibody responses. We propose that such a mechanism may operate to potentiate adaptive immune responses against pathogens in vivo.

Fig. 1.

B cells internalize particulate conjugates through the BCR and present αGalCer to iNKT cells. (A) (Upper) Silica beads were coated with αGalCer-containing liposomes (αGalCer), αGalCer-containing liposomes and HEL (αGalCer+HEL), or HEL alone (HEL). The binding of HEL to the beads was detected by Western blot with an anti-HEL antibody. (Lower) αGalCer-Alexa 488-coated beads were detected by FACS (black) compared with nonlabeled liposome-coated beads (gray). (B–H) B cell presentation of particulate αGalCer to iNKT cells. (B) MD4 B cells were stimulated with soluble αGalCer (15 ng/ml) or 0.1 μl of αGalCer-containing particles and cultured with iNKT cells. αGalCer presentation was measured by IL-2 production. (C) MD4 B cells were stimulated as in B with particles containing αGalCer (○), HEL (▴), or HEL-αGalCer (■). Control WT B cells were stimulated with HEL-αGalCer particles (▵). (D) MD4 marginal zone (MZ) and follicular (Fo) B cells were sorted by FACS and stimulated with particulate αGalCer-HEL before culture with iNKT cells. (E) MD4 B cells were primed with HEL-αGalCer particles and preincubated with CD1d-blocking antibody (αCD1d) or an isotype control (control Ig). (F) FACS analysis of αGalCer-containing particles coated with different HEL densities, expressed as mean fluorescence intensity (mfi). (G) MD4 B cells were stimulated with particles containing αGalCer and different densities of HEL, HEL^RDGN, or HEL^RKD (gray scale) before culture with iNKT cells. (H) Presentation of HEL-αGalCer conjugates by B cells expressing D1.3 IgM HEL-specific BCR (■) or a D1.3 IgM/H2 chimera (○). (I) MD4 B cells were stimulated with particles containing HEL-αGalCer (■), αGalCer (▴), HEL-Gal(α1→2)αGalCer (○) or Gal(α1→2)αGalCer (▵) before culture with iNKT cells.

Fig. 2.

iNKT cells help antigen-dependent B cell proliferation in vivo. MD4 B cells (or no cells, in mock) were transferred into WT mice challenged with particles containing αGalCer and/or HEL. (A) Proliferation of donor HEL-binding cells in the spleen was measured by FACS as CFSE dilution after 5 days. (B and D) Transfer experiments were also performed by using Jα18^−/− mice as recipients. Percentage of HEL⁺ (B) and CD138⁺HEL⁺ cells (D) recovered from recipient Jα18^−/− (gray bars) or WT (black bars) spleens are depicted. (C) Immunofluorescence microscopy was used to detect splenic follicular B cells (Fo, B220, red), HEL-binding cells (intracellular HEL, green), germinal centers (GC, PNA, blue), and EF PCs (EF) 5 days after MD4 cell transfer to recipient mice. (Magnification: ×10.) (E) ELISPOTS were used to detect HEL-specific IgMa secreting splenic cells in WT recipients. (F) Specific anti-HEL IgMa was measured in the sera of WT (filled symbols) and Jα18^−/− (unfilled symbols) recipient mice challenged with HEL-αGalCer particles (squares), αGalCer (triangles), or HEL (circles).

Fig. 3.

Antigen avidity modulates iNKT-dependent B cell proliferation and antibody production. MD4 transfer experiments were performed as in Fig. 2. WT recipient mice were challenged with particles containing αGalCer and HEL or HEL^RKD at different densities. (A) MD4 proliferation was detected as CFSE dilution in the HEL-binding population. (B) Percentage of HEL⁺ cells recovered from recipient spleens (high or low density). (C) Anti-HEL specific IgMa was detected on day 5 in the sera of recipient mice challenged with beads containing αGalCer and HEL high density (■), HEL low density (□), HEL^RKD high density (●), HEL^RKD low density (○). (D–F) WT mice received MD4 cells and particles containing HEL-αGalCer or HEL particles and soluble αGalCer. (D) Donor cell proliferation was detected as CFSE dilution after challenge with HEL-αGalCer conjugates (black line), HEL particles and soluble αGalCer (dotted line), or no beads (solid gray). (E) Percentage of HEL⁺ donor cells in mice receiving soluble (gray bar) or particulate (black bar) αGalCer-HEL. (F) Anti-HEL-specific IgMa in recipient mice that received beads containing HEL-αGalCer (■) or HEL beads plus soluble αGalCer (○).

Fig. 4.

Immunization with particulate antigen-αGalCer induces IgM and early class-switched specific antibodies. (A) C57BL/6 mice (four mice per group) were immunized with 10 μl of particles containing αGalCer (white bars), CGG (gray bars), or CGG-αGalCer (black bars). Specific CGG antibodies were detected in mice sera at 7 and 14 days after immunization. (B) WT (black bars) and Jα18^−/− (gray bars) mice (three mice per group) were immunized with particles containing CGG or CGG/αGalCer. (C) C57BL/6 mice (four mice per group) were immunized with 10 μl of particles containing αGalCer (white bars), HEL (gray bars), or HEL-αGalCer (black bars). Specific HEL antibodies were detected in mice sera at 7 and 14 days after immunization.

Fig. 5.

Cross-link of antigen and αGalCer enhances specific antibody responses. (A) C57BL/6 mice (four mice per group) were immunized with 1 μl of CGG-αGalCer-coated particles plus 1 μl of OVA-coated particles (gray bars) or 1 μl of OVA-αGalCer-coated particles plus 1 μl of CGG-coated particles (black bars). CGG- and OVA-specific antibodies were measured on day 7. (B) Specific CGG antibodies detected in C57BL/6 mice (three mice per group) immunized with 1 μl of particles containing CGG-αGalCer (black bars) or 1 μl of CGG-particles plus 150 ng of soluble αGalCer (gray bars). Specific CGG antibodies were detected on day 7.