Summary 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.

NKT cell activation with CD1d-binding glycolipid a-galactosylceramide (a-GC) enhances antibody responses to co-administered T-dependent antigen. The efficacy of a-GC relative to other CD1d-binding glycolipids and adjuvants is not known. There is little information on how NKT cells affect antibody production beyond initial booster-stimulated recall responses. We therefore tested the hypothesis that a-GC stimulates induction of plasma cells and antibody responses as effectively as Th1-and Th2-skewing variants of a-GC and several other adjuvants. C57BL/6 and CD1d -/-mice were immunized with nitrophenolconjugated keyhole limpet hemocyanin (NP-KLH) plus a-GC or NP-KLH plus adjuvants before administration of an NP-KLH booster and assessing antibody responses and plasma cell frequency. a-GC boosted long-term antibody responses as efficiently as all other agents tested and induced plasma cells that were detected in bone marrow 13 weeks after immunization. We then determined whether NKT cells were required in the presence of other adjuvants. CD1d -/-mice had a reduced induction of plasma cells in response to NP-KLH/Alum as compared to C57BL/6 mice. However, NKT cells were not required for the continued presence of those cells that were induced. Although NKT cells are capable of inducing persistent plasma cell responses, they may not play a major role in supporting longevity post-induction.

IntroductionCD1d is expressed by antigen-presenting cells (APCs) and presents glycolipids, including ␣-galactosylceramide (␣-GC) to CD1d-restricted natural killer-like T (NKT) cells. 1-3 NKT cells stimulate Th1 and Th2 responses in vivo and are thus implicated in asthma, infectious disease, cancer, and autoimmunity. [4][5][6][7][8] A newly appreciated aspect of NKT function is their potential to regulate, enhance, and sustain humoral immune responses against foreign Ags, including those expressed by parasites, bacteria, and viruses. [9][10][11][12][13][14][15][16][17][18][19] Despite clear demonstrations that NKT cells impact humoral immunity, the mechanisms are not defined. We therefore tested the hypothesis that CD1d glycolipid presentation by B cells in vivo was required for NKT-enhanced humoral immunity. After reconstitution of B cell-deficient MT mice with CD1d ϩ/ϩ or CD1d Ϫ/Ϫ B cells and subsequent immunization, we observed that NKTenhanced antibody (Ab) responses were deficient without CD1d expression by B cells. Our data introduce an important concept in humoral immunity: the direct interaction of B cells and NKT cells to facilitate enhanced Ab responses. Methods MiceC57Bl/6 mice were from the National Cancer Institute (Bethesda, MD) and MT mice from the Jackson Laboratory (Bar Harbor, ME). CD1d Ϫ/Ϫ mice have been described previously. 20 All procedures were approved by the Institutional Animal Care and Use Committee at University of Oklahoma Health Sciences Center and performed on female mice between 6 and 10 weeks of age. Antibodies and fluorochromesAnti-CD1d, -TCR␤, and isotype control mAbs were from BD Biosciences (San Jose, CA), anti-B220 and -CD19 mAbs from Biolegend (San Diego, CA), anti-CD4, -Thy1.2, and 2.4G2 mAbs from BioXpres (West Lebanon, NH), CD1d/␣-GC tetramers from the National Institute of Allergy and Infectious Diseases (NIAID) Tetramer Facility (Emory University, Atlanta, GA), and carboxyfluorescein diacetate succinimidyl ester (CFSE) from Invitrogen (Carlsbad, CA). Cells and adoptive transfersSpleens and thymi were subjected to mechanical disruption. Splenic erythrocytes were removed by hypotonic lysis. NKT and T cells were depleted by Lo-Tox complement-mediated lysis of anti-CD4/Thy1.2-labeled splenocytes (Cedarlane, Burlington, NC). Magnetic sorting was performed according to manufacturer's instructions using anti-fluorescein isothiocyanate microbeads in conjunction with a fluorescein isothiocyanateanti-CD19 mAb (Miltenyi Biotech, Auburn, CA). A BD FACScalibur was used to confirm cell purity. Where indicated, cells were labeled with CFSE (9 M in phosphate-buffered saline [PBS]/0.1% w/v bovine serum albumin) for 10 minutes at 37°C. This was followed by incubation for 5 minutes at 4°C and washing with PBS. Para-orbital injection was used to adoptively transfer 2 ϫ 10 7 cells into recipient mice. Immunization and sera collectionRecipient mice (MT) were immunized intraperitoneally 24 hours after adoptive transfer with either 20 g NP-KLH (Biosearch Technologies, Novato, CA) or 20 g NP-KL...

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