Reduction of CD1d expression in vivo minimally affects NKT-enhanced antibody production but boosts B-cell memory

Abstract

The CD1d-binding glycolipid α-galactosylceramide exerts potent adjuvant effects on T-dependent humoral immunity. The mechanism is driven by cognate interaction between CD1d-expressing B cells and TCR-expressing type I CD1d-restricted NKT cells. Thus, far positive effects of alpha-galactosylceramide have been observed on initial and sustained antibody titers as well as B-cell memory. Following vaccination, each of these features is desirable, but good B-cell memory is of paramount importance for long-lived immunity. We therefore tested the hypothesis that CD1d expression in vivo differentially affects initial antibody titers versus B-cell memory responses. CD1d(+/+) and CD1d(+/-) mice were generated and immunized with antigen plus CD1d ligand before analysis of cytokine expression, CD40L expression, initial and longer term antibody responses and B-cell memory. As compared with CD1d(+/+) controls, CD1d(+/-) mice had equivalent numbers of total NKT cells, lower cytokine production, fewer CD40L-expressing NKT cells, lower initial antibody responses, similar long-term antibody responses and higher B-cell memory. Our data indicate that weak CD1d antigen presentation may facilitate good B-cell memory without compromising antibody responses. This work may impact vaccine design since over-stimulation of NKT cells at the time of vaccination may not lead to optimal B-cell memory.

Fig. 1.

Comparison of NKT cells from CD1d^+/+ and CD1d^+/− mice. Female CD1d^+/+ C57Bl/6 mice were bred to male CD1d^−/− C57Bl/6 mice to generate CD1d^+/− C57Bl/6 mice. (A) Shows sample PCR results to amplify Neo cassette and CD1d gene sequences in parents and offspring. (B–F) Splenocytes from age-matched female CD1d^+/+ and CD1d^+/− C57Bl/6 mice were analyzed by flow cytometry as detailed in Methods. (B) Shows CD1d expression in splenocytes. (C) Shows staining and gating strategy to detect and enumerate type I α-GC-specific splenic NKT cells. (D) Shows expression of CD4 and CD8 by NKT cells (TCRβ/CD1d-tetramer gate applied). (E) Shows TCR Vβ expression by NKT cells. (F) Shows percent of NKT cells expressing of markers of activation (CD69, NKG2D and CD62L). Mean fluorescence intensity of these markers was comparable (not depicted). (G) Shows IL-4 and IFNγ secretion following i.p. administration of α-GC (4 μg per mouse in 100 μl of PBS/0.05% polysorbate-20). IL-4 and IFNγ were measured at 6 and 22 h, respectively. Data in (A–F) are representative of four independent experiments. Graph in (C) shows pooled data from 10 mice per substrain. Data in (G) are representative of two independent experiments.

Fig. 2.

CD1d antigen presentation regulates IFNγ and IL-4 secretion by NKT cells. (A, B) Splenocytes from CD1d^+/+, CD1d^+/− and CD1d^−/− C57Bl/6 mice were cultured with media (vehicle) or with α-GC-containing media for 48 h. (C, D) NKT cells were enriched using magnetic beads and cultured for 48 h with anti-CD3 and anti-CD28 mAbs. Culture supernatants were harvested and cytokine concentrations measured. Data show the mean ±SD IFNγ and IL-4 concentrations in the cell supernatants. Data are representative of four independent experiments.

Fig. 3.

CD1d antigen presentation regulates CD40L expression by NKT cells. Splenocytes from CD1d^+/+ and CD1d^+/− C57Bl/6 mice were cultured with media (vehicle) or with α-GC-containing media for 18 h. Cells were then harvested and stained with anti-TCRβ mAb, α-GC-loaded CD1d tetramer and anti-CD40L mAb and analyzed by flow cytometry. Plot on left demarcates NKT cells (red gate) and T cells (black gate). Data on right show TCRβ versus CD40L expression with and without α-GC treatment. Data are representative of three independent experiments.

Fig. 4.

Similar antibody titers in CD1d^+/+ and CD1d^+/− mice. CD1d^+/+ and CD1d^+/− C57Bl/6 mice were bled and then immunized s.c. with 10 μg NP-KLH or NP-KLH plus 4 μg α-GC. After 28 days, mice were bled (A, primary) and given a booster vaccine (10 μg NP-KLH s.c.) before collecting sera on day 35 (B, secondary) and day 140 (C, tertiary). Each data point represents the end-point anti-NP IgG1 titer for a single mouse. Data are representative of three independent experiments.

Fig. 5.

Comparison of transferable B-cell memory in immunized CD1d^+/+ and CD1d^+/− mice. CD1d^+/+ and CD1d^+/− mice were immunized with NP-KLH plus α-GC and after 60 days, splenocytes were harvested. (A) Total splenocytes were adoptively transferred to CD45.1 C57Bl/6 recipients before administration of NP-KLH and collection of sera after 7 and 21 days. ELISAS were performed to determine end-point anti-NP IgG1 titers. (B) B cells were adoptively transferred to IgH^a C57Bl/6 congenic mice before administration of NP-KLH and collection of sera on day 21. ELISAS were performed to determine NP-specific IgH^b antibody responses. NP-specific IgH^a was not detected (not depicted). Data show (A) mean ± SEM end-point titer or (B) absorbance at a one in 200 dilution of sera for three mice per group.