The Journal of Experimental Medicine
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Published 21 July 2003. doi:10.1084/jem.20030324
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© Rockefeller University Press, 0022-1007/2003/7/267 $5.00
The Journal of Experimental Medicine, Volume 198, Number 2, 267-279

Activation of Natural Killer T Cells by {alpha}-Galactosylceramide Rapidly Induces the Full Maturation of Dendritic Cells In Vivo and Thereby Acts as an Adjuvant for Combined CD4 and CD8 T Cell Immunity to a Coadministered Protein

Shin-ichiro Fujii, Kanako Shimizu, Caroline Smith, Laura Bonifaz and Ralph M. Steinman

Laboratory of Cellular Physiology and Immunology and the Chris Browne Center for Immunology and Immune Diseases, The Rockefeller University, New York, NY 10021

Address correspondence to Ralph M. Steinman, Laboratory of Cellular Physiology and Immunology, The Rockefeller University, New York, NY 10021-6399. Phone: 212-327-8106; Fax: 212-327-8875; E-mail: steinma{at}mail.rockefeller.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The maturation of dendritic cells (DCs) allows these antigen-presenting cells to initiate immunity. We pursued this concept in situ by studying the adjuvant action of {alpha}-galactosylceramide ({alpha}GalCer) in mice. A single i.v. injection of glycolipid induced the full maturation of splenic DCs, beginning within 4 h. Maturation was manifest by marked increases in costimulator and major histocompatibility complex class II expression, interferon (IFN)-{gamma} production, and stimulation of the mixed leukocyte reaction. These changes were not induced directly by {alpha}GalCer but required natural killer T (NKT) cells acting independently of the MyD88 adaptor protein. To establish that DC maturation was responsible for the adjuvant role of {alpha}GalCer, mice were given {alpha}GalCer together with soluble or cell-associated ovalbumin antigen. Th1 type CD4+ and CD8+ T cell responses developed, and the mice became resistant to challenge with ovalbumin-expressing tumor. DCs from mice given ovalbumin plus adjuvant, but not the non-DCs, stimulated ovalbumin-specific proliferative responses and importantly, induced antigen-specific, IFN-{gamma} producing, CD4+ and CD8+ T cells upon transfer into naive animals. In the latter instance, immune priming did not require further exposure to ovalbumin, {alpha}GalCer, NKT, or NK cells. Therefore a single dose of {alpha}GalCer i.v. rapidly stimulates the full maturation of DCs in situ, and this accounts for the induction of combined Th1 CD4+ and CD8+ T cell immunity to a coadministered protein.

Key Words: {alpha}-galactosylceramide • dendritic cell maturation • dendritic cells • exogenous pathway • T cell–mediated immunity


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
It is important to identify new immune adjuvants to improve the efficacy of vaccines against human tumors and many infectious diseases such as tuberculosis, malaria, and AIDS (1, 2). Available adjuvants, like alum and CFA, are suboptimal in that alum can polarize the immune response toward a Th2 type (3), while CFA can induce suppressive macrophages (4) and cytokines (5).

Adjuvants potentially could act at many sites in the immune response, e.g., directly on T cells (6) or on DCs (7). DCs charged with antigens ex vivo are able to induce adaptive immunity upon injection into rodents and humans, both CD4+ and CD8+ T cells. To do so, the injected DCs need to be stimulated ex vivo to undergo an intricate differentiation process termed maturation (8–12). In this way, the injected antigen-bearing mature DCs serve as "nature's adjuvants."

It would be desirable to identify adjuvants that would harness the DC system directly in situ for the purpose of controlling immunity. For example DCs are specialized to present nonreplicating antigens on MHC class I products to CD8+ T cells (for reviews, see references 13 and 14). DC stimulation through toll-like receptors (15–17) and by CD4+ T cells (18, 19) induces some features of DC maturation in vivo. In recent experiments, antigen also has been targeted selectively to DCs in vivo together with an agonistic anti-CD40 antibody to stimulate differentiation of DCs and many other cell types. The combination of antigen targeting to DCs combined with anti-CD40 leads to strong Th1 CD4+ (20) and CD8+ (21, 22) T cell responses. The presentation of antigen leading to the induction of effector T cells and memory is consistent with the full maturation of DCs. While these approaches draw a correlation between DC maturation and the development of immunity, it remains to be shown directly that antigen-capturing maturing DCs in vivo are the mediators of immunization.

The synthetic glycolipid {alpha}-galactosylceramide ({alpha}GalCer)* enhances resistance to tumors (23–26) and several intracellular infections (27–29). The glycolipid is presented to NKT cells by CD1d molecules particularly on DCs (30). Presentation by DCs leads to a rapid innate response and then a more prolonged production of IFN-{gamma} by the NKT cells (31). It is also known that {alpha}GalCer acts as an adjuvant for CD8+ T cell–dependent protection against malaria infection, when the glycolipid is administered together with an irradiated sporozoite vaccine (32). These results are surprising because {alpha}GalCer has been used to reduce T cell–mediated autoimmune diseases (33–38). The mechanism of adjuvant action of {alpha}GalCer is therefore of interest to decipher, especially in the contexts of adjuvant action and DC maturation. Also this glycolipid appears to lack major toxicity in humans and may be useful in the design of vaccines and therapies (39). In this paper, we describe the capacity of {alpha}GalCer to act as a stimulus for the full maturation DCs in mice, and we show with an adoptive transfer approach that the mature DCs exclusively mediate the glycolipid's role as an adjuvant to help prime antigen-specific, Th1 CD4+ and CD8+ T cell–mediated immunity.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mice.
Pathogen-free C57BL/6 (B6), and TAP-/- female mice at 6–7 wk were purchased from The Jackson Laboratory. We were generously provided with OT-I and OT-II TCR transgenic mice by Dr. F. Carbone (University of Melbourne, Parkville, Victoria, Australia), MyD88-/- mice by Dr. S. Akira (Osaka University, Osaka, Japan), and J{alpha}281-/- mice by Dr. M. Taniguchi (Chiba University, Chiba, Japan). Mice were maintained under specific pathogen-free conditions in the animal facility of the Rockefeller University. All experiments were done in compliance with relevant laws and institutional guidelines.

Reagents.
{alpha}GalCer (2S, 3S, 4R-1-O(a-galactopyranosyl)-2(N-hexacosanoylamino)-1,3,4-octadecanetriol) was provided by the Pharmaceutical Research Laboratory, Kirin Brewery (Gunma, Japan) and diluted in PBS. OVA protein was purchased from Seikagaku Corp. LPS was purchased from Sigma-Aldrich, and CpG-DNA from TriLink BioTechnologies. The following mAbs were purchased from BD Biosciences: FITC-conjugated anti-CD8{alpha} or PE-conjugated anti-CD11c, CD44, CD62L, biotinylated-isotype control, anti-CD40, CD80, CD86, I-Ab, and V{alpha}2. Biotinylated mAbs were detected with streptavidin-APC. The following mAbs were purified from hybridoma culture supernatants: anti-CD4, anti-CD8, anti-DEC205, and agonistic FGK45.5 anti-CD40 Ab. Rabbit polyclonal anti-asialo GM1, rabbit-IgG, and rat-IgG were purchased from Wako Pure Chemical USA Industries and Jackson ImmunoResearch Laboratories, respectively.

Flow Cytometry.
Cells were preincubated with 2.4G2 culture supernatant to block Fc{gamma} receptors, then washed and incubated with the indicated mAb conjugates for 30 min. Cells were washed and analyzed on a FACSCaliburTM flow cytometer (Becton Dickinson). For intracellular cytokine staining by FACS®, 5 x 106 spleen cells were cultured in 24-well plates for 6 or 8 h in the absence or presence of 1 µM OVA257–264 peptide (for CD8+ T cells), or 2 µM OVA323–339 peptide (for CD4+ T cells) with GolgiPlug (BD Biosciences) to accumulate cytokines intracellularly. The cells were incubated for 15 min at 4°C with the 2.4G2 anti-Fc{gamma}R mAb to block nonspecific staining. Then we stained cells with anti-CD4 or anti-CD8-FITC, or V{alpha}2-biotin and streptavidin-APC, for 20 min at room temperature. The cells were permeabilized using Cytofix/Cytoperm PlusTM (BD Biosciences) and stained with PE-conjugated anti-IFN-{gamma} (XMG1.2) or anti-IL-4 (11B11) mAb for 15 min at room temperature. Flow cytometry used a FACSCaliburTM instrument and CELLQuestTM software (BD Biosciences) or FlowJo (Tree Star).

DC Preparation from Spleen.
DCs were isolated from spleens using prior methods (40). In brief, splenocytes were released by homogenization followed by treatment with collagenase (collagenase D; Roche Diagnostics Corporation). A DC-enriched cell population was obtained as a low-density cell fraction using gradients. Collagenase-treated spleens were suspended in a dense BSA solution, overlaid with 1 ml of PBS, and centrifuged for 30 min. We separated low density CD11c+ and CD11c- fractions using anti-CD11c coated magnetic beads (Miltenyi Biotech). To isolate DC subsets on the basis of CD8{alpha} expression (41), we first depleted the low density spleen cells of CD5+ and CD19+ lymphocytes (T and B cells) with magnetic beads. From the lymphocyte-negative fraction, we selected CD8+ and then CD11c+ cells (CD8- DCs). No difference in viability was observed between CD8{alpha}+ and CD8{alpha}- DCs.

Cytokine Production by DCs.
After mice were given {alpha}GalCer, LPS, or PBS i.v., CD11c+ and CD11c- cells were isolated with anti-CD11c magnetic beads and cultured at 2 x 105 or 3 x 105 cells/well for 48–72 h in the absence or presence of FGK45.5 anti-CD40 mAb (10 µg/ml). The titers of IFN-{gamma}, IL-4, IL-10, IL-12p40, and IL-18 in the culture supernatants were determined by OptEIATM Kit (BD Biosciences), and IL-12p70 was determined by Quantikine ELISA kit (R&D Systems).

Stimulation of the Mixed Leukocyte Reaction by DCs.
Spleen DCs were isolated using anti-CD11c magnetic beads 8 h after administration of {alpha}GalCer, LPS, or PBS. Graded numbers of spleen DCs from C57BL/6 mice were irradiated and cultured with 2 x 105 allogeneic BALB/C or syngeneic T cells, isolated using T cell enrichment columns (R&D Systems), in a 96-well flat-bottom plate for 88 h. During the final 16 h, 3H-thymidine (1 µCi/well) was added. In some experiments, DCs were fixed with 0.75% paraformaldehyde (Electron Microscopy Science) for 30 min on ice.

DC Presentation of OVA.
To assess the role of {alpha}GalCer as an adjuvant for a T cell–mediated immune response, we used OVA as antigen in part because antigen presentation can be readily monitored using CD4+ and CD8+ OVA-specific TCR transgenic T cells. OVA was administered to mice i.v. either as a soluble protein (where 5 mg was required) or in association with dying osmotically shocked, syngeneic, TAP-/- splenocytes (where only 1 µg was associated with the injected cells; the dying cells efficiently target to DCs in spleen, accounting for the high efficiency of presentation as described [42]). Briefly, spleen cells were incubated with hypertonic medium at 37°C in the presence or absence of 10 mg/ml OVA for 10 min, and further incubated with hypotonic medium for 2 min to induce apoptosis, followed by washing with cold PBS (42). After injection of OVA in either soluble or cell-associated forms, the following tests for antigen presentation were done. (a) CD11c+ DC-enriched and CD11c- DC-depleted spleen cells were isolated 4 h after OVA injection and used to stimulate proliferation of OT-I or OT-II T cells in culture as described (42). (b) 7 d after OVA injection, mice were tested for T cell priming by quantifying OVA-specific, IFN-{gamma} and IL-4 producing T cells in the spleen as described above. (c) Mice were given 106 OT-I OVA-specific T cells i.v. and 1 d later the animals were primed with OVA and {alpha}GalCer; 3 d later, the OVA-specific T cells were monitored for expansion in cell numbers and intracellular IFN-{gamma} production (22). (d) CD11c+ DC-enriched and CD11c- DC-depleted spleen cells were isolated 4 h after OVA injection and used at a dose of 1 and 10 x 106 respectively to prime naive recipients, assessed as in approach (b) above.

Tumor Protection Experiments.
2 x 107 OVA pulsed apoptotic spleen cells were used to immunize mice, either mice given OT-I OVA-specific T cells i.v. 1 d earlier or naive animals, (42). 3 d later, 2 x 106 EG7, OVA-transduced tumor cells (American Type Culture Collection; CRL-2113) were inoculated s.c. The parental non-OVA transduced EL-4 thymoma was used as control tumor. To identify protective cells, we used blocking antibodies given i.p. 2 d before tumor inoculation and every 2 d after. The antibodies were control rat IgG, rat anti-CD4 (GK1.5) and CD8 (53–6.72), and rabbit anti-asialoGM1 Ab or control rabbit IgG in PBS. Mice were killed when tumor growth exceeded 400 mm2.

Statistical Analysis.
The statistical significance of differences between the experimental groups was determined by the Mann-Whitney exact rank sum test.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Maturation of the DC Surface After a Single I.V. Injection of {alpha}GalCer.
{alpha}GalCer enhances resistance to tumors and infections and is also presented by DCs to NKT cells (Introduction). We used several criteria to test if {alpha}GalCer leads to in vivo maturation of DCs, including their capacity to simultaneously capture and present antigen to diverse TCR{alpha}ß T cells and induce immunity. After i.v. administration of {alpha}GalCer, both CD8+ and CD8- DC subsets up-regulated several markers consistent with maturation. This included molecules involved in T cell costimulation (CD40, 80, 86), as well as antigen capture and presentation (MHC class II and the DEC-205 endocytic receptor; Fig. 1 A, compare the tracings for {alpha}GalCer with the black tracing for the vehicle control). These responses to glycolipid paralleled those seen with other known stimuli for DC maturation in vivo (Fig. 1 A), i.e., LPS (15), CpG deoxyoligonucleotides (16, 17), and agonistic anti-CD40 mAb (20–22).



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  		Figure 1. Maturation of DC surface markers with {alpha}GalCer and other stimuli in vivo. (A) 24 h after i.v. administration of LPS (25 µg), CpG ODN (10 µg), agonistic anti-CD40 mAb (50 µg), {alpha}GalCer (2 µg), or PBS (black tracing) to C57BL/6 mice, low density spleen cells were stained with PE-CD11c and FITC-CD8{alpha} (to identify DCs and their CD8+ or CD8- subsets) and with biotinylated isotype control, anti-CD40, CD80, CD86, I-Ab, and DEC205/CD205 followed by streptavidin APC. (B and C) As in (A), but the surface markers of CD8- or CD8+ DCs were followed at the indicated time points after i.v. administration of the maturation stimulus; data shown are for CD86 expression. (D) Minimal up-regulation of CD40 and CD86 on lymph node DCs (relative to splenic DCs) 24 h after {alpha}-GalCer i.v. Dotted line is the isotype control, gray shadow is antigen expression at time 0, and bold black line is CD40 and CD86 stain at 24 h. (E) Inability of {alpha}GalCer to induce the maturation of DCs from bone marrow progenitors, in contrast to LPS, as shown for up-regulation of CD86 on CD11c+ DCs from day 6–7 in culture.

 
When the kinetics of the splenic DC response to {alpha}GalCer was examined, as shown for CD86, maturation was evident within 4 h, and then began to subside at 48 h, returning to baseline at 72 h (Fig. 1, B and C). Axillary lymph node DCs from the same mice only showed minimal increases in CD86 and CD40 (Fig. 1 D). The kinetics of the splenic DC response to {alpha}GalCer was similar to that seen with LPS treatment (unpublished data). However, {alpha}GalCer was unable to directly stimulate DC maturation from bone marrow progenitors in culture, in contrast to LPS (Fig. 1 E). Therefore, {alpha}GalCer acts as a rapid and efficient inducer of splenic DC maturation in vivo, comparable in efficacy to other stimuli, but it does not have a direct effect on DCs, unlike other maturation stimuli.

NKT Cells Mediate DC Maturation in a MyD88-independent Manner.
NKT cells respond quickly to the presentation of {alpha}GalCer on CD1d molecules (43). To address the role of NKT cells in the rapid maturation of DCs, we tested mice lacking these T cells because of the deletion of essential TCR J{alpha}281 sequences (44). The DCs from J{alpha}281-/- mice did not mature in response to {alpha}GalCer in vivo and expressed comparable levels of CD86 and other maturation markers (CD40, CD80, MHC II) to wild-type mice given the PBS vehicle control (Fig. 2 A, left). As a positive control, DCs from J{alpha}281-/- mice were shown to mature in response to LPS and express high levels of CD86 (Fig. 2 A, right). We then tested DCs from MyD88-/- mice, where MyD88 is an essential adaptor protein for signaling cytokine production via Toll-like receptors (TLRs; references 45 and 46). The DCs from MyD88-/- mice did respond to {alpha}GalCer, maturing as effectively as DCs from wild type mice (Fig. 2 B). DCs from NK cell-depleted animals (using anti-asialoGM1 antibody treatment [31]) also matured in response to {alpha}GalCer (Fig. 2 C). Therefore, {alpha}GalCer in concert with NKT cells rapidly matures DCs in situ, as assessed by the surface markers of DCs in spleen, but the response does not require MyD88-based signaling or NK cells.



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  		Figure 2. DC maturation by {alpha}GalCer in vivo requires NKT cells. (A) Maturation, assessed by increased CD86 expression, did not occur in J{alpha}281-/- mice (lacking NKT cells) exposed to {alpha}GalCer (left), but did occur with LPS (right). (B) Maturation of DCs from MyD88 -/- mice in response to {alpha}GalCer and LPS, and (C) from mice depleted of NK cells by anti-asialoGM1.

 
Functional Maturation of {alpha}GalCer-stimulated DCs.
To demonstrate that DCs from {alpha}GalCer treated mice could stimulate resting T cells, we isolated DCs with anti-CD11c magnetic beads and tested them in some standard assays for DC maturation, e.g., as stimulators for allogeneic T cells in the primary mixed leukocyte reaction (MLR). DCs from all groups of mice (controls and mice treated with {alpha}GalCer or LPS) stimulated allogeneic T cell proliferation (Fig. 3 A) and to a much lesser extent, syngeneic T cell proliferation (Fig. 3 B). As splenic DCs are able to mature "spontaneously" in culture (47), we also tested DCs that were fixed in paraformaldehyde immediately upon isolation, to assess their MLR stimulating activity at the time of isolation. The fixed DCs from control mice no longer had stimulating activity for the MLR (Fig. 3 C), confirming prior work that most DCs in a spleen are functionally immature (20–22). In contrast, fixed DCs from {alpha}GalCer treated mice were potent stimulators of the allogeneic MLR (Fig. 3 C), suggesting the DCs had matured in the mice before isolation and fixation. With both live and fixed cells, CD11c- spleen cells lacked MLR stimulating activity at the doses tested (Fig. 3, A–D, open symbols). To prove that enhanced MLR stimulation by CD11c+ DCs was dependent upon NKT cells, we repeated the experiments on fixed DCs with J{alpha}281-/- mice. The increase in MLR stimulation was now ablated (Fig. 3 E).



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  		Figure 3. Functional maturation of splenic DCs from {alpha}GalCer treated mice. 8 h after i.v. administration of {alpha}GalCer, LPS or PBS, as in Fig. 1, spleen DCs were isolated using anti-CD11c–coated magnetic beads (purity >95 ± 2%). Graded numbers of spleen DCs from C57BL/6 mice were irradiated (30 Gy) and added to 2 x 105 allogeneic BALB/c (A) or syngeneic (B) T cells for 3 d in flat bottomed 96-well plates. In parallel, DCs were fixed with paraformaldehyde for 30 min, to block their maturation during the mixed leukocyte reaction, and also irradiated, followed by addition to allogeneic (C) and syngeneic (D) T cells. Proliferative responses were measured by [3H]-thymidine incorporation (Materials and Methods). The results are representative of four independent experiments. In E, the experiment in B was repeated comparing fixed DCs from wild-type and J{alpha}281-/- NKT deficient mice given {alpha}GalCer or vehicle. The results are averages of three independent experiments. (F and G) Mice were given PBS, {alpha}GalCer, or LPS i.v. and 8 h later, CD11c+ DC enriched and CD11c- DC-depleted, low density spleen cells were isolated with {alpha}-CD11c coated magnetic beads. In parallel, to test cytokine production by DC subsets, the low density spleen cells were first lymphocyte depleted with CD5+ and CD19+ magnetic beads, and then CD8+ and CD11c+ cells were selected successively with magnetic beads, providing enriched CD8+ and CD8- DC subsets respectively. IFN-{gamma} secretion (pg/ml) by each cell subset was measured by ELISA assays after culturing the cells for 48 h without further stimulation in 96 well plates at 2 x 105/well (F), while IL-12 production was measured in cells cultured 72 h in the presence of 10 µg/ml agonistic FGK45.5 anti-CD40 antibody at 3 x 105/well (G and H). All results required pools of three mice and represent averages of three independent experiments (*P < 0.05).

 
Maturation stimuli also can prime DCs in vivo to produce large amounts of immune enhancing cytokines such as IL-12 (48), IFN-{gamma} (49), and IFN-{alpha} (50). We studied cytokine production by cells from mice stimulated 8 h in vivo with {alpha}GalCer i.v., with LPS in comparison. We prepared CD11c+ DC-enriched and CD11c- DC-depleted cells, and further fractionated the DCs into CD8+ and CD8- subsets. The CD11c+ DCs contained few contaminating T and NK cells (0.5% CD3+ and <0.1% NK1.1+; unpublished data), while the DC subsets (CD8+ and CD8-) were obtained after depletion of T cells with anti-CD5 (Materials and Methods). CD11c+ DCs from {alpha}GalCer treated mice produced very high levels of IFN-{gamma} (Fig. 3 F). As prior work (48) showed that IL-12 production by DCs requires sequential stimuli from a microbe and then a T cell, e.g., through CD40 stimulation, we restimulated the DCs primed to {alpha}GalCer in vivo with anti-CD40 in culture. Now the DCs from {alpha}GalCer primed mice also produced IL-12 p70, in contrast to DCs from either PBS or LPS treated mice (Fig. 3, F and G). DCs from PBS, LPS, and {alpha}GalCer treated mice all produced IL-12 p40, possibly because of stimuli arising during their isolation and culture, but as in the case of IFN-{gamma}, production of IL-12 p40 and p70 were most active in the CD8- DC subset (Fig. 3, F and H). In contrast to these findings with DC stimulated in vivo with {alpha}GalCer, Ohteki et al. showed that the CD8+ DC subset from Listeria-infected mice was more active in IFN-{gamma} production after stimulation with IL-12 in vitro (49). We could not detect IL-4 and IL-10 (<10 pg/ml) in DCs from {alpha}GalCer primed mice (unpublished data). Interestingly, DCs stimulated by LPS in vivo responded to CD40 restimulation in culture by producing IL-18 rather than IL-12 (unpublished data). In sum, {alpha}GalCer matures DCs to produce enhancing cytokines and to more actively stimulate quiescent T cells in the mixed leukocyte reaction.

DCs Responding to OVA plus {alpha}GalCer Prime CD4+ and CD8+ T Cells in Culture.
To prove that DC maturation was associated with enhanced immunogenicity, we next injected mice with the protein antigen, OVA, either as a soluble protein or in association with dying TAP-/- splenocytes (the TAP-/- deletion ensured OVA presentation by MHC class I of recipient DCs rather than the injected splenocytes [21]), without or with {alpha}GalCer. Then we isolated the DCs and non-DC fractions from spleen and measured their capacity in tissue culture to stimulate CD8+ and CD4+, OVA-specific, naive OT-I and OT-II, TCR transgenic T cells respectively (Fig. 4 A). With both soluble (Fig. 4 B) and cell-associated OVA (Fig. 4 C) as antigen, the DCs selectively and actively stimulated proliferative responses by the naive CD4+ and CD8+ T cells in culture; CD11c- cells were inactive (Fig. 4, B and C). The T cells produced substantial IFN-{gamma}, >10 ng/ml culture, but no detectable IL-4 (unpublished data), indicating that antigen-capturing DCs were able to polarize the Th1 type of T cell differentiation.



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  		Figure 4. DCs capturing OVA protein and matured by {alpha}GalCer in vivo efficiently stimulate naive CD4+ and CD8+ T cells in culture. (A) Schematics for the experiments in Figs. 4, 6, and 7. Mice were given {alpha}GalCer or vehicle (PBS) together with OVA protein (5 mg/mouse) i.v. or osmotically shocked syngeneic splenocytes loaded with OVA protein as described (reference 21). 4 h later, CD11c+ cells or CD11c- cells were isolated and tested for their capacity to stimulate CD8+ and CD4+ TCR transgenic T cells from OT-I and OT-II mice, respectively. T cell proliferation was measured after [3H]-thymidine at 36–48 h. In some experiments (Fig. 5), OVA-specific T cell responses in spleen were evaluated 1 wk after immunization, while in others (Fig. 7), CD11c+ cells were transferred into naive mice to prime T cells in vivo. (B and C) The responses of CD4+ (OT-II) and CD8+ (OT-I) T cells to CD11c+ (top) and CD11c- (bottom) spleen cells from mice given soluble OVA (left panels) or cell-associated OVA (right panels) in the absence or presence of {alpha}GalCer; representative of three independent experiments. (D) As in B, but enriched populations of CD21high marginal zone B cells and CD21low follicular B cells were isolated by cell sorting and studied for CD1d and CD86 expression, as well as presentation of the injected soluble OVA to OT-I T cells. CD21high marginal zone B cells are the upper group of data on the right, while CD21low follicular B cells are the lower group (note much lower values on y-axis for D vs. B and C).

 
The apparent lack of T cell stimulating activity in the CD11c- fractions was puzzling, because these cells should include marginal zone B cells that express high levels of CD1d (51, 52) and are activated by {alpha}GalCer in vivo (53). Therefore, in the OVA experiments of Fig. 4, we also enriched CD19+ CD21+ CD23- marginal zone B cells and CD19+ CD21- CD23+ follicular B cells. We confirmed that the former had higher levels of CD1d and that both types of B cell up-regulated CD86 in response to {alpha}GalCer (Fig. 4 D, left part of figure). Both B cell types failed to present OVA (Fig. 4 D, right part of figure), especially when compared with the CD11c+ DCs isolated from these same animals (Fig. 4 B). Thus, DCs are the major cell type that acquires stimulating activity for naive T cells in response to the combined administration of a foreign protein and {alpha}GalCer.

OVA Together with {alpha}GalCer as Adjuvant Primes CD8+ T Cells In Vivo and Increases Resistance to OVA-expressing Tumors.
To monitor the activation of OVA-specific CD8+ T cells more directly in vivo, mice were injected i.v. with 106 CFSE-labeled CD8+, OVA-specific, TCR transgenic T cells. 1 d later, OVA-loaded splenocytes were given without or with {alpha}GalCer. 3 d after injection of the OVA-loaded splenocytes, there was extensive expansion of the injected OT-I T cells and dilution of the CFSE label (proliferation). The injection of {alpha}GalCer together with antigen increased the frequency of cells in the spleen by threefold (Fig. 5 B) and induced them to express high levels of the activation antigen CD44 (unpublished data). The spleen cells were also cultured with or without OVA peptide to determine if the T cells, gated for expression of the V{alpha}2 OT-I TCR, had been primed to produce IFN-{gamma}. The use of the {alpha}GalCer adjuvant induced high level IFN-{gamma} production in most OT-I T cells (Fig. 5 B), but no detectable IL-4 (unpublished data). To verify that {alpha}GalCer-dependent priming was associated with enhanced resistance to OVA-expressing tumors, we gave mice 106 OT-I T cells i.v. and 1 d later, we administered {alpha}GalCer and OVA-loaded cells also i.v. 3 d later, we injected 2 x 106 EG7 cells into the skin, where EG7 is a murine thymoma transduced to express OVA in a secretory form. The mice primed with TAP-/- / OVA and {alpha}GalCer showed strong resistance to the tumor, whereas mice primed with TAP-/-/OVA splenocytes alone showed some resistance, but the EG7 tumors recurred (Fig. 5 C). We repeated the experiments in mice depleted of CD8+ or CD4+ T cells with monoclonal antibodies, or NK cells with anti-asialo GM1 (these depletions were begun 1 d after administering {alpha}GalCer to allow time for the NKT cells, which express CD4, to mature the DCs). Only CD8 depletion ablated the resistance induced by OVA-loaded splenocytes together with {alpha}GalCer (Fig. 5 D).



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  		Figure 5. {alpha}GalCer enhances immunity to OVA-bearing DCs in vivo. (A and B) 1 d after transferring 106 CFSE-labeled OT-I cells i.v. into C57BL/6 mice, four groups of mice were given either no treatment, osmotically shocked and OVA loaded TAP-/- spleen cells, {alpha}GalCer (2 µg/mouse), or both OVA loaded spleen cells and {alpha}GalCer. 3 d later, spleen cells were monitored for dilution of fluorescence in the CFSE labeled V{alpha}2+ OT-I cells in the spleen. IFN-{gamma} production was monitored in tandem after culturing the spleen cells without or with OVA257–264 (1 µM) for 6 h in the presence of GolgiPlug. Each panel shows the percentage of cytokine producing, CFSE-labeled, V{alpha}2+ cells in the spleen. (C and D) At day 3 after immunization as in A and B, the mice were injected s.c. with EG7 tumor cells, which stably express OVA as a secreted protein. Mean tumor sizes at 2 d intervals were plotted on the left, and the distribution of tumor sizes at day 18 in the individual animals shown on the right. The experiments in the bottom panels were similar to those in the top except that mice were immunized with OVA loaded spleen cells and {alpha}GalCer but depleted of individual lymphocyte subsets (from 2 d before tumor inoculation to the end of the experiments) with monoclonal antibodies as indicated.

 
To determine if {alpha}GalCer could serve as an adjuvant for generating specific CD4+ or CD8+ T cell responses to OVA in naive animals, we injected 2 x 107 dying cells loaded with OVA i.v., and half the animals also received {alpha}GalCer. 7 d later, we tested if splenic T cells would secrete IFN-{gamma} in response to rechallenge with OVA peptides presented on MHC class I or II products (Fig. 4 A for diagram of protocol). {alpha}GalCer did serve as an adjuvant to prime CD4+ and CD8+ T cells in vivo, as illustrated by intracellular cytokine staining for IFN-{gamma} in Fig. 6 A and summarized in Fig. 6 B. IL-4 production was not detectable (unpublished data). When we tried to prime J{alpha}281-/- mice lacking NKT cells; however, {alpha}GalCer no longer served as an adjuvant (Fig. 6 B, bottom). To establish that the observed T cell immunity in wild-type mice was associated with protective resistance, we verified that naive mice immunized with the combination of TAP-/-/OVA splenocytes and {alpha}GalCer showed resistance to OVA-transduced EG7 tumor cells, but no resistance to the parent EL-4 thymoma that was not transduced to express OVA (Fig. 6 C, compare left and right panels). In summary, a single intravenous dose of {alpha}GalCer acts as an adjuvant for combined Th1 type CD4+ and CD8+ T cell immunity in mice.



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  		Figure 6. Induction of strong immunity in naive mice with cell associated OVA together with {alpha}GalCer. (A) Priming of naive mice with the combination of 2 x 107 OVA-loaded, TAP-/- spleen cells and {alpha}GalCer (2 µg/mouse) i.v. 7 d later, spleen cells were cultured without or with OVA257–264 or OVA323–339 peptides to stimulate OVA specific CD8+ and CD4+ T cells respectively and T cell production of IFN-{gamma} was measured by intracellular cytokine staining (the frequency of T cells producing IL-4 was <0.04% for all groups). (B) Same as (A), but means are shown for four individual mice (*P < 0.05, **P < 0.01 for the wild-type mice primed with OVA-loaded, TAP-/- spleen cells and {alpha}GalCer vs. the other groups). CD4+ and CD8+ T cell responses were elicited by OVA323–339 and OVA257–264 peptides respectively. (C) 1 wk after immunization in each group, mice were challenged with either 2 x 106 EG7 or EL-4 tumor cells. The data are mean tumor sizes ± SEM of 6 (EG7) or 3–4 (EL4) individual mice in independent experiments. (TAP-/-/OVA + {alpha}GalCer vs. the other groups at day 18; P < 0.005).

 
The Adjuvant Action of {alpha}GalCer Is Due to Mature DCs.
Given the evidence for T cell responses to the coadministration of protein antigen and {alpha}GalCer, we wanted to prove that the mature DCs were responsible for the glycolipid's adjuvant action. To do so, we again gave mice OVA antigen as a soluble protein or in association with dying cells. Then we isolated CD11c+ DCs from spleen 4 h later and tested their capacity to generate CD4+ and CD8+ effector T cells in naive animals (Fig. 4 A for diagram of the protocol). We monitored T cell priming by looking at day 7 for IFN-{gamma} production by both T cell subsets using an intracellular cytokine secretion assay performed on lymph node and spleen cells cultured for 6 h with or without OVA peptides in the presence of brefeldin A (Materials and Methods).

We first obtained DCs from 4 groups of mice primed with vehicle, OVA only, {alpha}GalCer only, or both OVA and {alpha}GalCer. The latter DCs primed IFN-{gamma} producing CD4+ and CD8+ T cells in naive mice (Fig. 7, A and B ; top panels). The results were virtually identical in spleen (Fig. 7) and lymph node (unpublished data). Again, no IL-4 producing T cells could be detected (unpublished data). We were concerned that some of the primed IFN-{gamma} producing cells might be NKT cells, but very few of the CD4+ and CD8+ cytokine producers labeled for the NK1.1 marker (unpublished data), presumably because at day 6, the NKT response to {alpha}GalCer on DCs has largely subsided (31).



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  		Figure 7. DCs isolated from mice given OVA together with {alpha}GalCer are able to prime naive animals. Mice were given {alpha}GalCer or vehicle together with either OVA protein (5 mg/mouse) i.v. (A) or with osmotically shocked TAP-/- splenocytes without or with OVA loading (B). 4 h after injection, 106 CD11c+ DCs from each group were transferred to naive syngeneic mice. In the top panels, 7 d after immunization with DCs but no further antigen or {alpha}GalCer, spleen cells were cultured without (open bars) or with (closed bars) OVA peptides with GolgiPlug to allow accumulation of intracellular IFN-{gamma} as described in experimental procedures. Intracellular IFN-{gamma} staining was performed with double labeling for CD4 and CD8. In the bottom panels, the DCs were taken from mice given {alpha}GalCer together with either OVA protein (A) or OVA loaded splenocytes (B), but the DCs were injected into either rabbit Ig (control)-treated mice or mice lacking NKT and NK cells (see Results) to test priming of CD4+ and CD8+, IFN-{gamma} producing effector T cells. The data are mean ± SEM of three individual mice in independent experiments. *P < 0.05.

 
It remained possible that the transferred mature DCs in Fig. 7 were not priming naive T cells directly, but still required either NK or NKT cells in the recipient animals. We therefore repeated the experiments but injected the DCs (from mice primed with OVA splenocytes and {alpha}GalCer) into three groups of recipients: control mice treated with rabbit Ig, mice treated with rabbit anti-asialoGM1 to deplete NK cells (31), and mice depleted of NKT cells through deletion of the J{alpha} 281 genetic sequences (44). In all cases, the DCs from mice given OVA together with {alpha}GalCer were able to prime the recipients (Fig. 7, A and B; lower rows). Therefore the NKT cell dependent maturation of antigen capturing DCs by {alpha}GalCer in donor mice leads to the priming of Th1 CD4+ and CD8+ effector T cells in naive recipient animals.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Stimuli for the Maturation of DCs In Situ.
Most DCs in a mouse spleen are functionally immature in the steady-state, but they undergo rapid maturation in response to {alpha}GalCer. Several criteria have been used here to document the full maturation of DCs, which in prior studies has been monitored primarily at the level of increased expression of costimulatory molecules like CD86. Here we have emphasized the capacity of DCs in vivo to become potent stimulators of immunity, the classical criterion for maturation.

A useful functional marker for maturation is enhanced T cell stimulatory activity in the mixed leukocyte reaction. It seems that this is best assayed with chemically fixed DCs, as fixation should prevent DCs from undergoing "spontaneous" maturation in culture (47). When this is done, DCs from control mice are weak MLR stimulators, whereas formaldehyde-fixed DCs from {alpha}GalCer treated mice are potent. This maturation criterion is fully dependent upon NKT cells. It has also been reported that some features of DC maturation are induced by CD1d-restricted human NKT cell clones in vitro (54).

Maturation of DCs in mice also has been achieved by stimuli for toll like receptors, e.g., LPS (15) and CpG oligodeoxynucleotides (16, 17). Likewise TCR{alpha}ß T cells induce DC maturation in vivo, as assessed by the up-regulation of surface costimulatory molecules (18, 19). In this paper, {alpha}GalCer harnesses NKT cells to fully mature DCs as evidenced by the induction of immunity to antigens coadministered with the glycolipid. In contrast to T cell–mediated maturation, which is a part of an adaptive immune response, NKT cells are innate cells that mature most of the DCs in spleen very quickly, within 4 h of administering {alpha}GalCer by the intravenous route.

Mechanisms of Maturation via {alpha}GalCer.
The maturation of DCs after presentation of {alpha}GalCer to NKT cells seems to operate independently of TLR signaling. MyD88 is one of the adaptor proteins for signaling cytokine production through TLRs, but MyD88 is not required for the maturation of DCs by {alpha}GalCer. DCs have a MyD88-independent but TLR-dependent maturation pathway, especially through TLR4 (55, 56). However, this should lead to Th2 type responses (57), whereas DCs matured from {alpha}GalCer treated, MyD88 knockout mice induce Th1 type responses (unpublished data).

Transactivation of NK and B cells following administration of {alpha}GalCer is well known (58). As a high proportion of many cell types including DCs are activated in response to the glycolipid, a "cytokine storm" induced by {alpha}GalCer likely is playing a role. However, it is requiring a good deal of experimentation to identify the mechanisms of NKT-dependent DC maturation in vivo, and this will be the subject of a future report.

{alpha}GalCer as an Adjuvant for Th1 Type Cell-mediated Immunity.
It may be surprising that {alpha}GalCer matures splenic DCs and also acts as an adjuvant for strong Th1 type CD4+ and CD8+ T cell responses to antigens given i.v., as numerous reports have used this same compound to suppress experimental, Th1-dependent autoimmune diseases (33–38, 59). Naumov et al. have reported that {alpha}GalCer treatment of autoimmune prone animals induces a form of tolerogenic DCs in the lymph nodes that drain the target organ for the autoimmune disease (37). The basis for these ostensibly major differences in immunologic outcome is unclear, i.e., polarization in our study to Th1 responses versus polarization toward Th2 in other studies. However, the experimental designs are different. We have used a single intravenous dose of glycolipid to activate DCs to produce Th1 polarizing cytokines such as IFN-{gamma} and IL-12 at the same time that antigens are given i.v. for presentation by the maturing DCs. In contrast, repeated doses of {alpha}GalCer have been given to treat autoimmune prone mice. In other instances, a multiple dose regimen (60) or the s.c. administration of OVA together with CFA (61), have been associated with Th2 polarization to external antigens in response to {alpha}GalCer. A Th2 environment may originate from the finding that soluble {alpha}GalCer is able to anergize NKT cells after the initial cytokine response, such that the NKT cells can no longer respond to DCs and in particular to produce IFN-{gamma} (31).

To document the role of DC maturation for adjuvant function in vivo, we have isolated the CD11+ DC-enriched and CD11c- DC-depleted populations from the spleens after injection of either soluble or cell-associated OVA antigen in the absence or presence of {alpha}GalCer. Strong CD4+ and CD8+ T cell responses are induced by DCs from {alpha}GalCer-treated mice, whereas we do not detect immunogenicity with non-DCs from these animals. This adoptive transfer approach therefore establishes that mature DCs are responsible for the in vivo action of adjuvants, since the recipients of the DCs do not require additional exposure to OVA antigen, {alpha}GalCer adjuvant, or innate NKT and NK cells. In the case of cell-associated OVA, this antigen is known to induce peripheral tolerance in the absence of {alpha}GalCer (21), so that the protective tumor immunity we have observed overrides an otherwise tolerogenic situation. These data indicate that DCs are necessary and sufficient for the adjuvant role of {alpha}GalCer, including its capacity to enhance CD8+ T cell responses to nonreplicating forms of a protein antigen. To harness the adjuvant properties of this glycolipid, however, DCs need to capture the antigen or vaccine in question and also present the {alpha}GalCer to NKT cells.


   Acknowledgments
 
The authors are grateful to Judy Adams for help with graphics and to Drs. F. Carbone, S. Akira, and M. Taniguchi for valuable genetically deficient strains of mice.

S. Fujii was supported by the Uehara Memorial Foundation and R.M. Steinman by National Institutes of Health grants CA84512 and AI40874.

Submitted: March 3, 2003
Revised: April 30, 2003
Accepted: April 30, 2003


   Footnotes
 
* Abbreviations used in this paper: TLR, Toll-like receptor; MLR, mixed leukocyte reaction; {alpha}GalCer, {alpha}-galactosylceramide. Back


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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