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...

Exogenous CD1d-binding glycolipid (α-Galactosylceramide, α-GC) stimulates TCR signaling and activation of type-1 natural killer–like T (NKT) cells. Activated NKT cells play a central role in the regulation of adaptive and protective immune responses against pathogens and tumors. In the present study, we tested the effect of Bacillus anthracis lethal toxin (LT) on NKT cells both in vivo and in vitro. LT is a binary toxin known to suppress host immune responses during anthrax disease and intoxicates cells by protective antigen (PA)-mediated intracellular delivery of lethal factor (LF), a potent metalloprotease. We observed that NKT cells expressed anthrax toxin receptors (CMG-2 and TEM-8) and bound more PA than other immune cell types. A sub-lethal dose of LT administered in vivo in C57BL/6 mice decreased expression of the activation receptor NKG2D by NKT cells but not by NK cells. The in vivo administration of LT led to decreased TCR-induced cytokine secretion but did not affect TCR expression. Further analysis revealed LT-dependent inhibition of TCR-stimulated MAP kinase signaling in NKT cells attributable to LT cleavage of the MAP kinase kinase MEK-2. We propose that Bacillus anthracis–derived LT causes a novel form of functional anergy in NKT cells and therefore has potential for contributing to immune evasion by the pathogen.

Activation of Natural Killer-like T cells (NKT) with the CD1d ligand α-GC leads to enhanced production of anthrax toxin protective Ag (PA)-neutralizing Abs, yet the underlying mechanism for this adjuvant effect is not known. In the current study we examined the role of Th1 and Th2 type responses in NKT-mediated enhancement of antibody responses to PA. First, the contribution of IL-4 and IFNγ to the production of PA-specific toxin-neutralizing Abs was examined. By immunizing C57Bl/6 controls IL-4−/− mice and IFNγ−/− mice and performing passive serum transfer experiments, it was observed that sera containing PA-specific IgG1, IgG2b and IgG2c neutralized toxin in vitro and conferred protection in vivo. Sera containing IgG2b and IgG2c neutralized toxin in vitro but were not sufficient for protection in vivo. Sera containing IgG1 and IgG2b neutralized toxin in vitro and conferred protection in vivo. IgG1 therefore emerged as a good correlate of protection. Next, C57Bl/6 mice were immunized with PA alone or PA plus a Th2-skewing α-GC derivative known as OCH. Neutralizing PA-specific IgG1 responses were modestly enhanced by OCH in C57Bl/6 mice. Conversely, IgG2b and IgG2c were considerably enhanced in PA/OCH-immunized IL-4−/− mice but did not confer protection. Finally, bone marrow chimeras were generated such that NKT cells were unable to express IL-4 or IFNγ. NKT-derived IL-4 was required for OCH-enhanced primary IgG1 responses but not recall responses. NKT-derived IL-4 and IFNγ also influenced primary and recall IgG2b and IgG2c titers. These data suggest targeted skewing of the Th2 response by α-GC derivatives can be exploited to optimize anthrax vaccination.

T he role of immune cell memory in Clostridium difficile infection (CDI) remains poorly understood. CDI is complicated by a high frequency of recurrence, often after disease has apparently resolved, and can be associated with progressively worsening pathology and ultimately death (1). However, patients that develop antibodies (Ab) capable of neutralizing two toxins secreted by C. difficile (TcdA and TcdB) are less likely to experience recurrence (reviewed in reference 2). This suggests that memory B (Bmem) cells may contribute to resistance to reinfection by encoding toxin-neutralizing Ab. Bmem cells have typically undergone affinity maturation and Ab class switch in the germinal centers of secondary lymphoid organs (3). Bmem cells are therefore poised to respond rapidly to booster vaccinations or infection, by rapidly differentiating into plasma cells that secrete class-switched, highaffinity Ab (4). Such plasma cells may display a range of short-toextreme longevity, be associated with transient or sustained Ab titers, or secrete neutralizing or nonneutralizing Ab (5-7).In earlier studies, a correlation between bacterial load and advanced age was observed during CDI, with older individuals lacking toxin-neutralizing Ab (8). In more recent work, the probability of HIV-positive patients experiencing CDI increased as their CD4 ϩ T-cell counts declined (9), which could be partly attributable to altered CD4 ϩ T-cell-dependent B cell function (10). Indeed, there is growing concern about CDI in a variety of immunocompromised individuals, including organ transplant recipients (reviewed in reference 11). These observations highlight the wellrecognized importance of B cell responses and production of toxin-neutralizing antibodies in resisting CDI. However, the underlying characteristics of the Bmem cellular response and their contributions to production of toxin-neutralizing Ab have not been described.CDI is best known as a disease of the gastrointestinal tract, causing diarrhea, and this infection may progress to a severe pathological condition in which pseudomembranous colitis and toxic megacolon are evident (1, 2). However, CDI-associated mortality may be attributable to systemic sequelae of the disease (12). Although large-scale epidemiological studies are lacking, reported systemic complications include hepatic abscesses (13), ascites (14), pleural effusion with acute respiratory distress (15, 16), and severe sepsis and multiorgan dysfunction (17).TcdA and TcdB are large clostridial toxins that contribute sub-

The current Bacillus anthracis vaccine consists largely of protective antigen (PA), the protein of anthrax toxin that mediates entry of edema factor (EF) or lethal factor (LF) into cells. PA induces protective antibody (Ab)-mediated immunity against Bacillus anthracis but has limited efficacy and duration. We previously demonstrated that activation of CD1d-restricted natural killer-like T cells (NKT) with a CD1d-binding glycolipid led to enhanced Ab titers specific for foreign antigen (Ag). We therefore tested the hypothesis that activation of NKT cells with the CD1d ligand (␣-galactosylceramide [␣-GC]) at the time of immunization improves PA-specific Ab responses. We observed that ␣-GC enhanced PA-specific Ab titers in C57BL/6 mice. In CD1d ؊/؊ mice deficient in type I and type II NKT cells the anti-PA Ab response was diminished. In J␣281 ؊/؊ mice expressing CD1d but lacking type I ␣-GC-reactive NKT cells, ␣-GC did not enhance the Ab response. In vitro neutralization assays were performed and showed that the Ab titers correlated with protection of macrophages against anthrax lethal toxin (LT). The neutralization capacity of the Ab was further tested in lethal challenge studies, which revealed that NKT activation leads to enhanced in vivo protection against LT. Anti-PA Ab titers, neutralization, and protection were then measured over a period of several months, and this revealed that NKT activation leads to a sustained protective Ab response. These results suggest that NKT-activating CD1d ligands could be exploited for the development of improved vaccines for Bacillus anthracis that increase not only neutralizing Ab titers but also the duration of the protection afforded by Ab.

Activation of iNKT cells with the CD1d-binding glycolipid adjuvant α-galactosylceramide (α-GC) enhances humoral immunity specific for coadministered T-dependent Ag. However, the relationship between the iNKT cell and the classic T helper (Th) or T follicular helper (Tfh) function following this immunization modality remains unclear. We show that immunization with the C-terminal domain (CTD) of Clostridium difficile toxin B (TcdB), accompanied by activation of iNKT cells with α-GC, led to enhanced production of CTD-specific IgG, which was CD1d- and iNKT cell-dependent and associated with increased neutralization of active TcdB. Immunization with CTD plus α-GC followed by NP hapten-linked CTD increased NP-specific IgG1 titers in an NKT-dependent manner, suggesting that iNKT activation could enhance Th or Tfh function or that iNKT and iNKTfh cells could provide supplemental, yet independent, B cell help. Th, Tfh, iNKT, and iNKTfh cells were, therefore, examined quantitatively, phenotypically, and functionally following immunization with CTD or with CTD plus α-GC. Our results demonstrated that α-GC-activated iNKT cells had no direct effect on the numbers, phenotype, or function of Th or Tfh cells. However, CD4 T cell-specific ablation of the Bcl6 transcription factor demonstrated that Tfh and iNKTfh cells both contributed to B cell help. This work extends our understanding of the immune response to vaccination and demonstrates an important contribution by NKTfh cells to humoral immunity.

CD1d-restricted invariant Natural Killer T (iNKT) cells boost humoral immunity to T-dependent Ags that are co-administered with the CD1d-binding glycolipid Ag α-galactosylceramide (α-GC). Observations that mice lacking iNKT cells have decaying Ab responses following vaccination has led to the hypothesis that iNKT cells express plasma cell (PC) survival factors that sustain specific Ab titers. Bone marrow (BM) chimeric mice in which the entire hematopoetic compartment or iNKT cells selectively lacked BAFF, APRIL, or both BAFF and APRIL were created and immunized with NP-KLH adsorbed to Alum or mixed with α-GC. In comparison to BAFF- or APRIL-sufficient BM chimeras, absence of hematopoetic compartment- and iNKT-derived BAFF and APRIL was associated with rapidly decaying Ab titers and reduced PC numbers. The iNKT cell-derived BAFF or APRIL assumed a greater role in PC survival when α-GC was used as the adjuvant for immunization. These results show that iNKT-derived BAFF and APRIL each contribute to survival of PCs induced by immunization. This study sheds new light on the mechanisms through which iNKT cells impact humoral immunity and may inform design of vaccines that incorporate glycolipid adjuvants.