CD40L-null NKT cells provide B cell help for specific antibody responses

Abstract

CD1d-binding glycolipids exert potent adjuvant effects on T-dependent Ab responses. The mechanisms include cognate interaction between CD1d-expressing B cells and TCR-expressing Type I CD1d-restricted natural killer T cells (NKTs). However, the critical NKT-derived factors that stimulate B cells are poorly understood. We tested the hypothesis that CD1d-driven CD40L expression by NKT cells influences humoral immunity. Bone marrow chimeras with CD40L(+/+) or CD40L(-/-) NKT cells were immunized with Ag plus CD1d ligand before measuring Ab responses. CD40L(-/-) NKT cells stimulated higher endpoint Ab titers than controls expressing CD40L. In contrast, immunization of CD40L(-/-) mice revealed that CD40L(-/-) NKT cells could not provide B cell help when Th cells lacked CD40L. We report that CD40L(-/-) NKT cells can provide help for Ab production and do so cooperatively with CD40L(+/+) Th cells. We suggest that the manner in which NKT cells provide B cell help is distinct from that of Th cells.

Figure 1. NKT cells do not provide B cell help in CD40L^−/− mice

(A) C57Bl/6 mice were immunized with NP-KLH or NP-KLH plus α-GC. After 28 days, all mice received a booster vaccine (NP-KLH). Sera were collected on day 35 and endpoint IgM, IgG1, IgG2b, IgG2c and IgG3 titers determine by ELISA. (B) Thymocytes and splenocytes were obtained from CD40L^−/− and C57Bl/6 mice and then analyzed by flow cytometry. Dot plots (left) show CD1d tetramer⁺/TCRβ⁺ cells. Histograms (right) show expression of CD40L by gated CD1d tetramer⁺/TCRβ⁺ cells. Data show representative analyses from two CD40L^−/− mice and numerous (>50) C57Bl/6 mice. (C) C57Bl/6 and CD40L^−/− mice were immunized with NP-KLH plus α-GC on d 0 and boosted with NP-KLH on day 28 before bleeding on day 35. Endpoint IgM, IgG1, IgG2b, IgG2c and IgG3 titers in the sera collected on day 35 were then determined by ELISA. Each data point in (A) and (C) represents an individual mouse and line indicates geometric mean titer. Statistically significant differences between groups were determined using Mann Whitney U test.

Figure 2. NKT-derived CD40L is dispensable for Ab production

(A) Outlines the strategy used for generating mixed bone marrow chimeras. Jα18^−/− mice have a gene deletion in the TCR locus and do not rearrange the Vα14/Jα18 invariant TCR found on Type I NKT cells. (B) Spleens were obtained from immunized Jα18^−/−/C57Bl/6 and Jα18^−/−/CD40L^−/− chimeric mice and analyzed by flow cytometry for CD45.2^+/+ donor cells and residual CD45.1^+/+ recipient cells. (C) Graph shows expression of B cell, T cell and dendritic cell markers for each of the chimeras. (D) Dot-plots in upper panels show CD1d tetramer⁺/TCRβ⁺ NKT cells. Histograms in middle panels show CD1d expression. Anti-CD1d and isotype control mAb binding are represented by filled and empty histograms respectively. Dot-plots in lower panels show donor-derived (CD45.2^+/+) versus recipient-derived (CD45.2^−/−) tetramer-binding cells. Data in B-D are representative of three of each chimera. (E) CD45.2^+/+ (C57Bl/6) mice were lethally irradiated and engrafted with a 50/50 mixture of donor bone marrow cells from Jα18^−/− mice (CD45.2^+/+) and CD45.1^+/+ mice. After 12 weeks peripheral blood leukocytes were analyzed by flow cytometry for CD45.2 and CD45.1 expression. Graph shows relative percentages of CD45.1 and CD45.2 cells in the periphery of 5 re-constituted recipients. (F-H) Jα18^−/−/C57Bl/6 and Jα18^−/−/CD40L^−/− mixed chimeras were immunized as indicated. Sera were collected on d 28 (primary) and 35 (secondary) and ELISA assays were performed to analyze NP-specific (F) IgG1, (G) IgG2b and (H) IgG2c titers. Each data point represents an individual mouse and the lines indicate geometric mean titer. Two outliers were removed from the NP-KLH, primary sera group in (F). Data shown are representative of 2 independent experiments. Statistically significant differences between control and experimental mice were determined using Mann Whitney U test.