The CD1d-binding glycolipid α-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, Vα14⁺ 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 α-galactosylceramide (α-GC) enhances production of antibodies reactive with T-dependent antigen in vivo. α-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. α-GC also led to alterations in isotype switch, selectively increasing production of immunoglobulin G2b. Further analysis revealed that α-GC led to priming of class II-restricted Th cells in vivo. Additionally, we observed that α-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.

Introduction

Presentation of antigen-derived peptide by major histocompatibility complex (MHC) class II expressed on dendritic cells (DCs) activates T helper (Th) cells, which in turn provide help to stimulate the proliferation of antigen-specific B cells and their differentiation into memory B cells and/or antibody-producing plasma cells (reviewed in refs ^1–3). Many factors regulate B-cell functions leading to antibody production. These include presentation of class II/antigen complexes to class II-restricted T-cell receptors (TCR), interactions between CD154 on the Th cell and CD40 on the B cell, and the local cytokine environment created by activated Th cells.^4–6

CD1d is a non-polymorphic MHC class I-related protein expressed on antigen-presenting cells that activates CD1d-restricted natural killer-like T cells (NKT cells).^7–9 Type I invariant NKT cells are activated by antigen-presenting cells presenting the glycolipid antigen α-galactosylceramide (α-GC) bound to CD1d. α-GC-reactive murine NKT cells have a tightly restricted TCR repertoire expressing the Vα14/Jα18-arranged TCR gene.¹⁰ NKT cells can trigger both Th1 and Th2 responses in vivo.^11–14

In recent years, it has come to light that CD1d-restricted NKT cells influence antibody production.^15–20 For example, Galli and colleagues showed that ex vivo human peripheral blood NKT cells stimulated antibody production by autologous B cells in response to α-GC,¹⁶ suggesting that NKT cells provide help for antibody production. Campos and colleagues showed that contact allergen-induced immunoglobulin M (IgM) production by murine splenic marginal zone B cells was CD1d/NKT-dependent.¹⁵ Lisbonne and colleagues observed that production of allergen-specific IgE was deficient in Jα18^−/− mice and CD1d-blocking antibodies reduced IgE production in an experimental asthma model.²¹ Schofield and colleagues showed that the production of antibodies reactive to glycosyl-phosphatidylinositol-anchored circumsporozoite proteins from Plasmodium falciparum was abrogated in CD1d^−/− mice but not in class II^−/− mice.¹⁹ Conversely, in two other studies, antibody production appeared to be class II-dependent and CD1d-independent.^18,22 NKT cells may be important for antibody-mediated protection against pathogenic bacteria. CD1d^−/− mice infected with the spirochete Borrelia hermsii had impaired production of specific antibody and had a higher pathogen burden than CD1d-expressing controls.^23,24 Production of antibodies reactive with polysaccharide antigens from Streptococcus pneumoniae is also CD1d-dependent.²⁵ Importantly, this suggests that polysaccharide-derived antigen may bind CD1d and activate NKT cells. More recently, α-GC was shown to have an adjuvant effect following intranasal coadministration with protein antigen.²⁶ This could have consequences for inducing antibody-mediated immunity at mucosal surfaces.

Despite this accumulating information, it remains unknown whether α-GC-activated NKT cells provide B-cell help for in vivo antibody production in response to immunization with a T-dependent protein antigen or T-independent carbohydrate antigen. In this study, we tested the hypothesis that α-GC enhances or induces production of antibody reactive with T-dependent antigen in the presence or absence of class II/TCR cognate interactions. We show that α-GC leads to enhanced antibody production in a CD1d-dependent manner, and stimulates specific antibody isotype switch. Furthermore, we demonstrate that α-GC stimulates the priming of class II-restricted Th cells and has direct effects on B cells. Our data show that NKT-cell activation enhances humoral immunity and that there are several contributory mechanisms. Our findings support the notion that NKT cells can provide help for antibody production in vivo and suggest that adjuvant-like properties of α-GC may be exploited in situations where antibody-mediated immunity is known to be beneficial.

Materials and methods

Antibodies and fluorochromes

Allophyocyanin-conjugated CD1d tetramers loaded with ligand PBS57 were obtained from the National Institutes of Health NIAID Tetramer Facility at Emory University (Atlanta, GA). Anti-TCR-β, class II (IA/IE) and CD1d were purchased from BD Pharmingen (San Diego, CA). Horseradish peroxidase-conjugated anti-immunoglobulin antibodies were purchased from Southern Biotechnology (Birmingham, AL). Anti-CD40 monoclonal antibody (mAb)-producing hybridomas were a gift from Dr A. Rolink (University of Basel, Switzerland). The anti-CD40 mAb (FGK45) was purified by T-Gel chromatography (Pierce, Rockford, IL).

Antigens

Ovalbumin (OVA) was purchased from MP Biochemicals (Irvine, CA). OVA was treated with polymyxin B (15 μg/ml) to minimize endotoxin levels (Sigma, St Louis, MO). Nitro-iodophenol (NIP)-hapten-conjugated OVA, nitrophenol (NP)-hapten-conjugated keyhole limpet haemocyanin (KLH) and NP–Ficoll, hen egg lysozyme (HEL), NIP-bovine serum albumin (BSA) and NP-BSA were purchased from Biosearch Technologies Inc (Novato, CA). α-GC was obtained from Dr G.S. Besra (School of Biosciences, University of Birmingham, UK). The synthesis, purity, structural integrity and functionality have been described by us previously.^27,28

Mice

C57BL/6 mice were purchased from the National Cancer Institute (Bethesda, MD). The B6;129SH^dlAb1–Ea mouse strain was purchased from Jackson Laboratories (Bar Harbor, ME) and is referred to as class II^−/− mouse hereafter. Class II^−/− mice were purchased as breeding pairs and maintained in the Animal Resource Center at Dartmouth Medical School. Class II^−/− mice are homozygous null for all class II genes (Aα, Aβ, Eα, Eβ) as a result of the removal by homologous recombination.⁵ Class II^−/− mice have normal immunoglobulin levels, CD8⁺ cells, NK⁺ cells and NKT⁺ cells, but are deficient in CD4⁺ Th cells.⁵ CD1d^−/− mice on the C57BL/6 genetic background have been described previously²⁹ and were bred in the Animal Resource Center at Dartmouth Medical School for the experiments described herein. Male and female mice of 6–10 weeks of age were used for experiments described. All procedures were approved by the Institutional Animal Care and use Committee.

Staining and flow cytometry

Spleens were harvested into Hanks' balanced salt solution buffer and a single cell suspension of splenocytes was obtained by mechanical disruption. Contaminating erythrocytes were removed by ammonium chloride-mediated lysis according to the manufacturer's instructions (EBioscience, San Diego, CA). Cell viability was confirmed by trypan blue exclusion. Splenocytes were incubated at room temperature for 2 hr with a 1/250 dilution of allophycocyanin-conjugated CD1d/antigen tetramer and fluorescein isothiocyanante-conjugated anti-TCR-β antibody. The 2.4G2 mAb was added at a final concentration of 10 μg/ml to block FcR binding. Cells were then washed three times in phosphate-buffered saline (PBS) before fixation in 1% w/v paraformaldehyde in PBS. Cells were washed and re-suspended in PBS before analysis by flow cytometry using a Becton Dickinson FACScalibur flow cytometer (Franklin Lakes, NJ). Where indicated, whole blood leucocytes were analysed for CD1d and class II expression following erythrocyte lysis.

Retro-orbital eye bleed and serum collection

Mice were anaesthetized under 5% isofluorane/95% oxygen and 100 μl blood was collected by retro-orbital bleed with heparinized microcapillary tubes (Fisher, Hampton, NH). Samples were transferred immediately to polypropylene microcentrifuge tubes. Blood samples were incubated for 30 min at room temperature then allowed to clot overnight at 4°, before centrifugation at 13 000 g for 20 min at 4°. Sera were withdrawn with a pipette and stored in aliquots at −20°.

Immunization

Male and female mice between 6 and 10 weeks of age were used for all experiments described. In experiments with C57BL/6 and CD1d^−/− mice, a single subcutaneous (s.c.) immunization was administered over both flanks on day 0 immediately following collection of pre-bleed sera. Mice were then bled at days 14 and 28 post-immunization. Where appropriate mice were immunized with OVA and/or α-GC or challenged at day 14 with NIP-OVA. Four mice were immunized per group unless indicated otherwise. Following collection of pre-bleed sera, class II^−/− mice were immunized s.c. in both flanks with OVA and/or α-GC on days 0, 7 and 14. Mice were bled on days 14 and 28. Where appropriate, mice were injected s.c. with 100 μg agonistic anti-CD40 antibody (FGK45) or with a rat IgG isotype control antibody on days 1, 8 and 15. Fifty micrograms of antibody were administered to each flank. Between four and seven mice per group were used in experiments with class II^−/− mice. For experiments with T-cell-independent antigen, mice were pre-bled then immunized intraperitoneally with 5 μg NP–Ficoll alone or in combination with 4 μg α-GC. Mice were then bled on day 7 and sera were obtained.

Anti-serum immunoglobulin enzyme-linked immunosorbent assay

Immulon 4 enzyme-linked immunosorbent assay (ELISA) plates from Dynex Technologies Inc. (Chantilly, VA), were coated with OVA, NP-BSA or NIP-BSA as appropriate at 10 μg/ml in binding buffer (0·1 m Na₂HPO₄, pH 9·0), overnight at 4° before washing plates and blocking for 2 hr at room temperature with 1·0% w/v BSA in PBS/0·05% v/v Tween 20. Sera were diluted 100-fold in PBS/0·05% v/v Tween and subjected to two- or fourfold serial dilution before being added to OVA-coated, pre-blocked plates. Plates were incubated overnight at 4° with diluted sera, before washing four times in PBS/0·05% v/v Tween-20. Plates were washed and incubated for 2 hr at room temperature with horseradish peroxidase-conjugated anti-mouse IgM, IgG, IgG1, IgG2b, IgA, or IgE at a final concentration of 0·2–0·5 μg/ml. Plates were washed and developed using ABTS substrate (KPL, Gaithersburg, MD). Optical density of samples at an absorbance of 405 nm was then measured using a Dynex plate reader. End-point titres were determined as OD < 0·01 at 405 nm (equivalent to OD of 1/100 dilution of pre-bleed sera). Differences in antibody titre were assessed for statistical significance using Student's t-test or analysis of variance (anova).

Results

The CD1d antigen α-GC enhances antibody production in a CD1d-dependent manner

To confirm the NKT status of mice for our experiments, we obtained whole splenocytes from C57BL/6 and CD1d^−/− mice and analysed them for CD1d/antigen tetramer-reactive cells coexpressing TCR-β as well as CD1d- and class II-expressing cells. We observed a distinct population of TCR-β and tetramer double-positive cells in C57BL/6 mice (0·65–0·75% of total splenocytes) and a complete absence of this population in CD1d^−/− mice (Fig. 1a). C57BL/6 mice expressed CD1d which was absent in CD1d^−/− mice. Conversely, C57BL/6 mice and CD1d^−/− mice both expressed class II. As expected, C57BL/6 and CD1d^−/− produced similar anti-OVA IgG titres in response to s.c. immunization with high-dose OVA (400 μg) (Fig. 1b). C57BL/6 and CD1d^−/− mice were therefore used to assess the contribution of CD1d and NKT cells to antibody production in our experiments.

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.

To determine if α-GC influences production of antibodies reactive to class II/T-dependent antigen, we immunized C57BL/6 mice with a dose titration of OVA or OVA plus α-GC (Fig. 1c). We observed that, following immunization with low-dose OVA (5 μg), there was no increase in OVA-specific IgG1 titres relative to those in naive mice. In OVA/α-GC-immunized mice, there was a large increase in the OVA-specific IgG1 titre. At 50 μg OVA, there was a similar effect for α-GC, but less pronounced than for 5 μg OVA. At high-dose OVA (500 μg), α-GC had no effect on the total anti-OVA IgG1 titre. In the absence of OVA, α-GC did not induce any increase in the OVA-specific titre. In CD1d^−/− mice, immunized with OVA, there was no additional effect of administering α-GC (Fig. 1c), although antibody responses were intact in these mice (Fig. 1b). Antibody responses were also antigen-specific as there was no reactivity with HEL in sera from OVA-immunized mice (not shown). These data show that α-GC induces production of antigen-specific antibody at low but not high antigen concentrations in a CD1d-dependent manner.

We also observed that the data obtained with OVA could be reproduced using a highly immunogenic antigen KLH (Fig. 2). Immunization with KLH alone induced a significant anti-KLH titre that was greatly enhanced by α-GC co-immunization. In CD1d^−/− mice, α-GC did not induce enhanced anti-KLH responses. These results show that NKT-cell activation can enhance the production of antibodies reactive with different antigens and of different immunogenicities.

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.

We assessed the antibody classes and subclasses produced in responses to immunization with OVA or OVA plus α-GC. We observed that OVA- or OVA/α-GC-immunized (500 μg) C57BL/6 mice producing identical IgG1 titres also produced identical IgM titres and failed to stimulate IgA or IgE production (Table 1). However, the IgG2b titre was fivefold higher following OVA/α-GC immunization than OVA immunization. We also compared IgG2b production between C57BL/6 and CD1d^−/− mice immunized with OVA alone. We observed that CD1d^−/− mice produced less IgG2b than C57BL/6 mice even in the absence of NKT stimulation (Table 2). Our data suggest that NKT cells contribute to isotype switching and production of IgG2b in vivo.

Table 1.

Antibody classes and subclasses produced in the absence and presence of α-GC

	End-point anti-OVA titre
Class/subclass	500 μg OVA (n = 3)	500 μg OVA + 4 μg α-GC (n = 3)
IgM	7467 ± 2822	10 667 ± 2133
IgG1	> 25 600	> 25 600
IgG2b	6533 ± 3580	> 25 600
IgG3	< 100	< 100
IgA/IgE	< 100	< 100

Day 28 sera from C57BL/6 mice immunized s.c. with 500 μg OVA alone or OVA plus 4 μg α-GC were assessed by ELISA for OVA-specific antibody isotype and subclasses. Data show mean reciprocal anti-OVA titres ± SD for three mice per group.

Table 2.

Antibody classes and subclasses produced in the absence and presence of α-GC

Strain	Immunization	End-point anti-IgG2b titre
C57BL/6	500 μg OVA	8000 ± 2263 (n = 3)
CD1d−/−	500 μg OVA	1400 ± 320 (n = 4)

Day 28 sera were obtained from C57BL/6 and CD1d^−/− mice immunized s.c. with 500 μg OVA alone. Data show mean reciprocal anti-OVA IgG2b titres ± SD for three and four mice, respectively.

α-GC plus CD40 ligation induce anti-OVA antibody production in class II^−/− mice

Class II^−/− mice were assessed for NKT-cell development and CD1d expression. The abundance of CD1d tetramer-reactive cells was comparable with that observed for C57BL/6 mice and there was a similar amount of CD1d expression (Fig. 3a). Lack of class II expression was also confirmed by flow cytometry (Fig. 3a). These data are in accordance with previous reports^5,29 confirming that class II^−/− mice have normal levels of NKT cells while CD1d^−/− mice fail to develop this subset.

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.

We tested the hypothesis that α-GC-stimulated NKT cells were insufficient for inducing antibody production in the absence of class II-restricted Th cells. Class II^−/− mice were injected s.c. with 400 μg OVA or OVA plus 4 μg α-GC three times at 1-week intervals. We did not observe any increase in the end-point serum titre of OVA-specific IgG (Fig. 3b). This shows that in the absence of class II, α-GC is unable to independently stimulate help for production of IgG.

We tested the idea that antibody production could be induced by providing additional B-cell help. We there immunized class II^−/− mice s.c. with 400 μg OVA and 4 μg α-GC as described, but followed by s.c. injection of 100 μg of the agonistic anti-CD40 mAb 24 hr following each immunization (Fig. 3b). We observed an increase in the anti-OVA IgG titre in day 14 sera collected before the third immunization in four out of seven mice. The end-point titres in this group were variable, ranging from baseline to 2⁹ −2¹⁰. By the second bleed on day 28, the anti-OVA IgG end-point titre in the four responsive mice had increased further to 2¹⁰ −2¹¹. Importantly, these titres, while significant, were still two- to fourfold lower than in C57BL/6 or CD1d^−/− mice immunized with OVA. Immunization of class II^−/− mice with OVA and α-GC but followed 24 hr later by a rat IgG (isotype control for anti-CD40 mAb) did not result in significant increases in end-point titres above those of pre-bleed sera. Furthermore, injection of anti-CD40 alone or anti-CD40 24 hr following injection of OVA did not result in increased anti-OVA IgG titres.

We performed ELISAs on serum from the same mice to determine which antibody classes and subclasses were produced following stimulation with OVA, α-GC and anti-CD40. The IgG subclass produced was exclusively IgG2b and the results were identical to those of total IgG ELISAs.

To determine if the anti-OVA IgG/IgG2b response was antigen-specific, rather than reflective of a polyclonal B-cell expansion, we performed ELISAs to determine if an anti-HEL antibody response was triggered by stimulation with OVA/α-GC then anti-CD40 (Fig. 3c). No increase in the anti-HEL titre was detected, showing that the antibody response was OVA specific and therefore determined by the antigen chosen for immunization.

Our data show that CD1d-restricted NKT cells can induce limited antibody production in the absence of class II-restricted Th cells when additional B-cell help is provided via CD40 ligation.

α-GC enhances priming of class II-restricted Th cells

Having shown that α-GC affects antibody titre, isotype and affinity, we wished to determine whether the mechanism involved priming of class II-restricted Th cells. We immunized mice s.c. with 20 μg OVA or 20 μg OVA plus 4 μg α-GC. On day 14 post-immunization, we obtained primary bleed sera then immunized mice with NIP-OVA. On day 28 we obtained secondary bleed sera. We observed that initial immunization with OVA plus α-GC led to an enhanced anti-NIP IgG1 titre compared to OVA-immunized mice (Fig. 4a). We also observed that anti-OVA IgG1 titres were higher at day 14 following OVA/α-GC immunization, independently confirming the data presented in Fig. 1(c) (Fig. 4b). Since the anti-NIP antibody response was increased, even though α-GC was not administered during the NIP-OVA immunization, this indicates that α-GC led to enhanced priming of class II-restricted OVA-specific Th cells. These data indicate that NKT cells enhance the priming of Th cells, thus increasing antibody responses to T-dependent antigen.

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.

α-GC enhances production of antibody reactive with T-independent antigen

We considered the possibility that as well as priming Th cells, α-GC may lead to Th-independent effects on B cells. We therefore assessed the effect of α-GC on the production of antibodies reactive with the T-independent antigen Ficoll. Mice were immunized with NP-hapten-conjugated Ficoll alone or in combination with α-GC (Fig. 5). In C57BL/6 mice the α-GC treatment had no effect on the production of anti-NP IgM, IgG2b, IgG3, IgA, or IgE. However, α-GC selectively enhanced the production of anti-NP IgG1, with an approximate fivefold increase in antibody titre. Conversely, CD1^−/− mice responded equivalently to immunization with Ficoll plus α-GC as compared to Ficoll immunization of C57BL/6 mice, showing that the enhancement was indeed CD1d/NKT-dependent. These data show that NKT cells do not affect the initial production of IgM or switch to IgG3 in response to stimulation with T-independent antigen, but do stimulate IgG1 production.

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.

Discussion

We have demonstrated that the CD1d-binding glycolipid α-GC has adjuvant-like properties that enhance the production of antibodies reactive with T-cell-dependent antigen in vivo. The α-GC-enhanced antibody response was absent in CD1d-deficient mice, which failed to develop CD1d-restricted NKT cells. This indicates that NKT cells provide help for antibody production in vivo. The antibody responses were antigen-specific, suggesting that the same antigen-presenting cell (DC and/or B cell) has to capture both class II and CD1d antigens and co-present them to obtain appropriate Th-mediated and NKT-mediated help. Indeed one recent study showed that if α-GC was administered at the time of or within 24 hr of immunization with soluble antigen, then there was increased priming of antigen-specific CTL responses, but if administered several days apart, then there was no enhancement of function.³⁰ Consistent with this, s.c. immunization with OVA and α-GC into separate flanks failed to induce antibody production in class II^−/− mice (Lang, unpublished observation).

We have also shown that activation of NKT cells in class II-deficient mice induces limited production of antigen-specific antibody. Additional CD40-mediated help was required to induce antibody production, suggesting that NKT cells do not provide sufficient CD154-mediated help for antibody production in the absence of Th cells. CD154 expression was identical in NKT cells from C57BL/6 mice and class II^−/− mice, thus the requirement for anti-CD40 antibody was not attributable to lack of CD154 expression (Lang, unpublished observation). The inability of NKT cells alone to provide sufficient B-cell help in vivo is important because some non-protein antigens, such as Mycobacterium tuberculosis-derived phosphatidylinositol mannoside and lipoarabinomannan, which elicit an antibody response are also CD1-binding antigens.^31,32 It therefore seems that optimal production of antibodies reactive with such non-protein CD1-binding antigens will require association with and co-presentation of a class II-epitope to stimulate sufficient Th-mediated B-cell help (reviewed in ref. 33).

Of note, the NKT-cell population(s) responsible for antibody production in class II^−/− mice may not be equivalently represented in C57BL/6 mice. This is because class II^−/− mice have a higher proportion of atypical NK1.1^−/− CD1d-restricted NKT cells than C57BL/6 mice.³⁴ NK1^−/− NKT cells are potent IL-4 producers and could be contributing to the antibody production we have observed.³⁴

Interestingly, in class II-deficient mice, antibody isotype was switched to the production of IgG2b in response to α-GC and anti-CD40 mAb. On examination of antibody subclasses in C57BL/6 mice, we also observed that α-GC led to an increase in IgG2b production. Furthermore, CD1d^−/− mice had lower anti-OVA IgG2b titres than C57BL/6 mice even in the absence of α-GC, suggesting a steady-state effect of NKT cells on antibody production. The reasons for NKT-dependent IgG2b production are unclear at present, particularly as little is known about switching to IgG2b. LPS stimulation or T-cell help in the presence of transforming growth factor-β promotes IgG2b production.^35,36 The large IgG1 response in the presence of Th activation is consistent with IL-4 production by Th cells and NKT cells.³⁷ Interferon-γ is a major cytokine produced by NKT cells³⁸ and suppresses IgG1 production in vivo.³⁹ This suggests that NKT cells providing help for antibody production might be major producers of interleukin-4 rather than interferon-γ. It therefore appears that NKT cells in the context of Th cells can alter the cytokine environment to affect isotype switching. Further examination of these events is warranted.

In the present study, we wished to determine how NKT cells enhance antibody production. We observed that immunization with OVA/α-GC led to enhanced anti-NIP antibody production following challenge with NIP-OVA, demonstrating that OVA-specific Th cells had been primed in response to α-GC. Hermanns and colleagues demonstrated that OVA/α-GC immunization led to enhanced CD4⁺ and CD8⁺ T-cell and cytotoxic T-cell responses against OVA-expressing tumours,³⁰ suggesting that NKT cells enhance the functions of class II- and class I-restricted T cells. The authors also showed that OVA/α-GC-treated DCs enhanced T-cell function, suggesting that NKT cells could condition DCs to maximize T-cell priming. Support for this idea was provided by the Steinman laboratory demonstrating that α-GC can mature DCs in vivo.⁴⁰ An alternative, but as yet untested, hypothesis is that NKT cells and Th cells could interact directly with each other via receptor pairing and/or cytokine signals in the context of priming by DCs. A recent study by our laboratory also showed that B cells expressing CD1d could stimulate enhanced presentation of B-cell antigen receptor-targeted CD1d antigen to NKT cells,²⁸ suggesting a third possibility, that a B-cell co-presenting T-dependent antigen and α-GC could further activate DC-primed Th cells to maximize B-cell help.

NKT cells may also have direct effects on B cells to enhance antibody production as well as ‘indirect effects’ via Th priming. We observed that α-GC enhanced antibody responses to the T-cell-independent antigen, NP–Ficoll. While T-independent antigens do not require Th help for antibody production, we have raised the possibility that NKT cells could directly interact with CD1d-expressing B cells to enhance antibody production. Our data indicate that this mechanism is probably restricted to T-independent antigens as NKT cells did not provide sufficient help for the production of anti-OVA antibodies in the absence of Th cells. Therefore, NKT cells may differentially regulate the functions of B cells depending on whether the cell is specific for a T-dependent or T-independent antigen.

In summary, CD1d-restricted NKT cells enhance the production of antibodies reactive with T-dependent and T-independent antigens in vivo. NKT cells may have numerous effects including isotype switch, Th priming and Th-independent effects on B cells. Studies are now underway in our laboratory to determine the mechanisms by which NKT cells enhance humoral immunity. This is important for determining how to harness NKT cells for the development of vaccines where good humoral immunity is required.

Abbreviations

α-GC

α-galactosylceramide

BSA

bovine serum albumin

DC

dendritic cell

ELISA

enzyme-linked immunosorbent assay

HEL

hen egg lysozyme

IGM

immunoglobulin M

KLH

keyhole limpet haemocyanin

mAb

monoclonal antibody

MHC

major histocompatibility complex

NIP

nitro-iodophenol

NKT

natural killer-like T cell

NP

nitrophenol

OVA

ovalbumin

PBS

phosphate-buffered saline

s.c.

subcutaneous

TCR

T-cell receptor

Th

T helper