The CD1d-binding glycolipid alpha-galactosylceramide enhances humoral immunity to T-dependent and T-independent antigen in a CD1d-dependent manner

Abstract

Specific interaction of class II/peptide with the T-cell receptor (TCR) expressed by class II-restricted CD4+ T helper (Th) cells is essential for in vivo production of antibodies reactive with T-dependent antigen. In response to stimulation with CD1d-binding glycolipid, Valpha14+ TCR-expressing, CD1d-restricted natural killer T (NKT) cells may provide additional help for antibody production. We tested the hypothesis that the CD1d-binding glycolipid alpha-galactosylceramide (alpha-GC) enhances production of antibodies reactive with T-dependent antigen in vivo. alpha-GC enhanced antibody production in vivo in a CD1d-dependent manner in the presence of class II-restricted Th cells and induced a limited antibody response in Th-deficient mice. alpha-GC also led to alterations in isotype switch, selectively increasing production of immunoglobulin G2b. Further analysis revealed that alpha-GC led to priming of class II-restricted Th cells in vivo. Additionally, we observed that alpha-GC enhanced production of antibodies reactive with T-independent antigen, showing the effects of NKT cells on B cells independently of Th cells. Our data show that NKT cells have multiple effects on the induction of a humoral immune response. We propose that NKT cells could be exploited for the development of novel vaccines where protective antibody is required.

Figure 1

α-GC enhances T-dependent antibody production in a CD1d-dependent manner. (a) Splenocytes were harvested from C57BL/6 and CD1d^−/− mice and analysed for binding of CD1d tetramer versus TCR-β (top row). Whole blood cells were analysed for CD1d (middle row) and class II expression (bottom row). (b) Mice were pre-bled (naive) and immunized s.c. with 400 μg OVA before obtaining immune primary and secondary bleed sera on days 14 and 28, respectively. Data show the end-point anti-OVA IgG1 titre in C57BL/6 and CD1^−/− mice. Each data point represents an individual mouse for four mice per group. (c) Pre-bleed sera (naive) were obtained from C57BL/6 mice followed by s.c. immunization with α-GC alone (4 μg), OVA alone (5, 50, or 500 μg) or OVA mixed with α-GC. Immune sera were then obtained at day 14 post-immunization. CD1d^−/− mice were treated, bled, and then immunized with 5 µg OVA either alone or mixed with α-GC, before obtaining immune sera at day 14. Data show mean ±SD end-point anti-OVA IgG1 titres for four mice per group. Statistically significant differences, as determined by Student's t-test, are indicated. Flow cytometry data in (a) are representative of at least six experiments. Data in (b) and (c) are representative of four similar experiments.

Figure 2

α-GC enhances anti-KLH titres in a CD1d/NKT-dependent manner. C57BL/6 and CD1d^−/− mice were immunized s.c. with 10 μg KLH, 4 μg α-GC or KLH plus α-GC. On day 14, sera were obtained and anti-KLH IgG1 titres were assessed by ELISA. Data show the mean end-point titre ±SD for four mice per group and are representative of three similar experiments.

Figure 3

CD40-ligation following α-GC stimulation induces specific T-dependent antibody production in class II^−/− mice. (a) Shows: CD1d tetramer binding (left panel); CD1d expression (middle); and class II expression (right) in class II^−/− mice. (b) Mice were immunized s.c. on days 0, 7 and 14 with 400 μg OVA + 4 μg α-GC, OVA + α-GC then 100 μg rat IgG, OVA + α-GC + 100 μg anti-CD40, anti-CD40 alone, or OVA then anti-CD40. Anti-IgG ELISAS were performed on sera collected on days 0, 14 and 28. Data show the mean ± SD end-point titre for seven mice per group. Four out of seven mice were responsive in the OVA/α-GC/anti-CD40 group (the data from the three non-responsive mice are not shown). (c) Sera from the four responding OVA/α-GC/anti-CD40 immunized mice were tested for the presence of anti-HEL-specific IgG. Results from positive control sera (C57BL/6 mouse injected with HEL) are shown.

Figure 4

α-GC enhances the priming of Th cells. Pre-bleed sera (naive) were obtained from C57BL/6 mice followed by s.c. immunization with OVA alone (20 μg) or OVA plus α-GC (4 μg). Mice were bled (immune primary) then challenged s.c. with 20 μg NIP-conjugated OVA on day 14. On day 28, mice were bled (immune). (a) Anti-NIP-ELISAs were performed on day 28 sera. (b) Anti-OVA ELISAs were performed on day 14 sera. Data shows mean end-point antibody titre ± SD for four mice per group. Statistically significant differences are indicated.

Figure 5

α-GC enhances antibody responses to T-independent antigen. C57BL/6 and CD1d^−/− mice were bled (naive) then immunized i.p. with 5 μg NP–Ficoll or NP–Ficoll plus 4 μg α-GC. On day 7, mice were bled (immune). ELISAs were then performed to assess anti-NP IgM, IgG1, IgG2b, IgG3, IgA and IgE titres in sera. Data show the mean end-point anti-NP titre ± SD for four mice per group. Statistically significant differences are indicated.