NK T cells provide lipid antigen-specific cognate help for B cells

Abstract

The mechanisms of T cell help for production of antilipid antibodies are largely unknown. This study shows that invariant NK T cells (iNK T cells) and B cells cooperate in a model of antilipid antigen-specific antibody responses. We use a model haptenated lipid molecule, 4-hydroxy-3-nitrophenyl-alphaGalactosylCeramide (NP-alphaGalCer), to demonstrate that iNK T cells provide cognate help to lipid-antigen-presenting B cells. B cells proliferate and IgG anti-NP is produced from in vivo-immunized mice and in vitro cocultures of B and NK T cells after exposure to NP-alphaGalCer, but not closely related control glycolipids. This B cell response is absent in CD1d(-/-) and Jalpha18(-/-) mice but not CD4(-/-) mice. The antibody response to NP-alphaGalCer is dominated by the IgM, IgG3, and IgG2c isotypes, and marginal zone B cells stimulate better in vitro lipid antigen-driven proliferation than follicular B cells, suggesting an important role for this B cell subset. iNK T cell help for B cells is shown to involve cognate help from CD1d-instructed lipid-specific iNK T cells, with help provided via CD40L, B7-1/B7-2, and IFN-gamma, but not IL-4. This model provides evidence of iNK T cell help for antilipid antibody production, an important aspect of infections, autoimmune diseases, and vaccine development. Our findings also now allow prediction of those microbial antigens that would be expected to elicit cognate iNKT cell help for antibody production, namely those that can stimulate iNKT cells and at the same time have a polar moiety that can be recognized by antibodies.

Fig. 1.

Characterization of NP-aGalCer and NP-BGalCer. (A) NP-αGalCer and NP-βGalCer. (B) Haptenated NP-αGalCer, but not NP-βGalCer, stimulates iNK T cells in vitro and in vivo. CD1d-restricted hybridoma (DN32D3) is stimulated with 1.25 nM αGalCer, NP-αGalCer, GD1a, C95 dolichol, or media alone. IL-2 is measured by ELISA and is expressed as mean/SE of duplicate wells. Representative of two experiments is shown (*, significantly different from media). (C) iNK T TcR Tg splenic T cells were labeled ex vivo with αGalCer-loaded mouse CD1d tetramer, αCD3, and anti-CD69 2 h after PBS/0.05% BSA, 0.5 μg αGalCer, and 0.5 μg NP-αGalCer or 30 min after 0.5 μg αCD3 immunization. Data are expressed as MFI of CD69 expression on CD3, tetramer double positive NKT cells.

Fig. 2.

NP-αGalCer immunization stimulates IgG anti-NIP. (A) Intraperitoneal administration of NP-αGalCer (0.5 μg per mouse) but not NP-KLH (2.2 μg per mouse), NP-βGalCer (0.5 μg per mouse), or αGalCer (0.5 μg per mouse) stimulates early anti-NIP IgG in vivo. (B) IgG anti-NIP from mixed splenic B1–8^hi B and iNK T TcR Tg T cell culture supernatants 7 days after media, 15 μg/ml αIgM, 1 μg/ml LPS, 0.2 μg/ml αCD3, 8.6 μg/ml NP-KLH, 1 ng/ml αGalCer, 1 ng/ml NP-αGalCer, or 1 ng/ml NP-βGalCer stimulation (*, significantly different from PBS/media group).

Fig. 3.

CD1d and Jα18 iNK T cells, but not CD4, are required for in vivo IgG and IgM anti-NIP response to NP-αGalCer. IgG (A) and IgM (B) anti-NIP from B6 WT, B6 CD1d^−/−, or B6 Jα18^−/− mice immunized with 0.5 μg per mouse NP-αGalCer, 0.5 μg per mouse αGalCer, or 200 μl of PBS/0.05% BSA, detected by ELISA between days 3 and 21 (n = 4–5 mice per group) (*, significantly different from WT).

Fig. 4.

iNK T cells provide in vivo and in vitro cognate help for antihapten antibody production. (A) B6 WT mice were immunized with two separate injections of 2.2 μg of NP-KLH (i.p.) and 0.5 μg of αGalCer (s.c.) or one injection of 2.2 μg of NP-KLH mixed with 0.5 μg of αGalCer (i.p. only). (B) Comparable mice were immunized with two separate injections of 0.5 μg of NP-βGalCer (i.p.) and αGalCer (s.c) or one injection of 0.5 μg of NP-βGalCer mixed with αGalCer (i.p. only) (n = 5 per group). (C) Three-day culture of CFSE-labeled 1 × 10⁵ Igh^a B6 CD1d^+/+ splenic B cells, 1 × 10⁵ Igh^b B6 CD1d^−/− splenic B cells, and 1 × 10⁵ iNK T TcR Tg splenic T cells plus media, 50 ng/ml NP-αGalCer, 50 ng/ml αGalCer, 50 ng/ml βGalCer, or 1 μg/ml LPS. IgM^a+ CD1d^−/− B cells, IgM^b+ CD1d^+/+ B cells, and Thy1.2+ T cells were identified by FACS. Proliferation represents percentage of cells that have divided from a pool of triplicate wells. Representative of two experiments is shown.

Fig. 5.

MZ B cells stimulate stronger proliferation than FO B cells when mixed with iNK T TcR Tg T cells plus NP-αGalCer. A total of 1 × 10⁵ splenic MZ B cells or 1 × 10⁵ splenic FO B cells were mixed with 1 × 10⁵ iNK T TcR Tg splenic T cells and incubated for 3 days with 10 ng/ml NP-αGalCer or 0.1 ng/ml NP-αGalCer. Media were <200 cpm for all cell types. Individual populations proliferate <4,000 CPM in response to NP-αGalCer, and αCD3 and LPS controls confirmed active T and B cell populations but are not shown for simplicity. Proliferation was measured by ³[H]thymidine incorporation. Pool of three experiments is shown. *, significantly different from FO B cells + 10 ng/ml Ag; **, significantly different from FO B cells + 0.1 ng/ml Ag.

Fig. 6.

NP-αGalCer preferentially induces IgM, IgG2c, and IgG3 anti-NIP. Serum collected 7–10 days after immunization of C57BL/6 WT mice with 0.5 μg NP-αGalCer in PBS/0.05% BSA (A) or 50 μg NP-KLH in alum (B) was tested by ELISA for NIP-specific antibodies. Serum titer represents the first dilution with OD >3× background. ELISA limit of detection is 1:100. Shown is pool of two experiments. Each point = one mouse (n = 5–9 mice per condition).

Fig. 7.

B7–1/2, CD40L, and IFN-γ are important for the in vivo antibody response to NP-αGalCer. (A) B6 WT and B7–1/2^−/− mice immunized with 0.5 μg NP-αGalCer, 50 μg NP-KLH/alum, or PBS/0.05% BSA. (B and C) B6 WT, IFNγ^−/−, IL-4^−/−, and CD40L^−/− mice immunized with 0.5 μg NP-αGalCer/PBS/0.05% BSA (B) or 50 μg NP-KLH/alum (C). Shown is mean/SE of two to three mice per group and one representative of two experiments.

Fig. 8.

iNK T cells provide cognate help for lipid-specific B cells. Recognition of CD1d by the TcR on the iNK T cell, plus engagement of costimulatory molecules CD40 and B7–1/2, are important for cognate iNK T cell help for B cell antibody and proliferation responses to lipid antigens. IFN-γ also contributes to isotype class switch during the B cell antibody response.