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The Journal of Immunology, 2003, 170: 2540-2548.
Copyright © 2003 by The American Association of Immunologists

NKT Cells Provide Help for Dendritic Cell-Dependent Priming of MHC Class I-Restricted CD8+ T Cells In Vivo 1

Detlef Stober, Ieva Jomantaite, Reinhold Schirmbeck and Jörg Reimann2

Institute of Medical Microbiology and Immunology, University of Ulm, Ulm, Germany


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Dendritic cells (DC) are potent APCs for naive T cells in vivo. This is evident by inducing T cell responses through adoptive DC transfer. Priming specific CTL responses in vivo often requires "help". We study alternative sources of help in DC-dependent priming of MHC class I-restricted CTL. Priming an anti-viral CTL response in naive B6 mice by adoptive transfer of antigenic peptide-pulsed DC required CD4+ T cell help. CTL priming was facilitated by providing MHC class II-dependent specific help. Furthermore, transfers of MHC class II-deficient pulsed DC into naive, normal hosts, or DC transfers into naive, CD4+ T cell-depleted hosts primed CTL inefficiently. Pretreatment of DC with immune-stimulating oligodeoxynucleotides rendered them more efficient for CD4+ T cell-independent priming of CTL. DC copresenting a Kb-binding antigenic peptide and the CD1d-binding glycolipid {alpha}-galactosyl-ceramide efficiently primed CTL in a class II-independent way. To obtain NKT cell-dependent help in CTL priming, the same DC had to present both the peptide and the glycolipid. CTL priming by adoptive DC transfer was largely NK cell-dependent. The requirement for NK cells was only partially overcome by recruiting NKT cell help into DC-dependent CTL priming. NKT cells thus are potent helper cells for DC-dependent CTL priming.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Dendritic cells (DC)3 are potent APCs for naive T cells. DC prime in vivo MHC class I-restricted CD8+ CTL responses (1, 2, 3, 4). Different human and murine DC subsets can efficiently prime in vitro and in vivo class I-restricted CTL responses with a wide range of specificities. Both CD8{alpha}+ and CD8{alpha}- DC subsets prime CD8+ T cells in vitro and induce protective antiviral CTL responses in vivo (4, 5, 6, 7). In many (but not all) studies on DC-mediated CTL priming in mouse and human, the establishment of a stable CTL immunity is facilitated by CD4+ T cell "help" (8, 9, 10, 11, 12). Highly immunogenic, minor histoincompatible DC fail to prime CTL in MHC class II-deficient mice indicating that even strong alloantigens cannot stimulate CD8+ T cell responses without help (10). CD4+ T cell help can 1) activate DC; 2) regulate the longevity of Ag presentation and of the activation status of the APC (on which the establishment of CTL immunity by DC critically depends) (13); 3) facilitate CTL infiltration into target tissues (14); and 4) facilitate in situ delivery of CTL effector functions (15). Help is not essential for establishing or maintaining CTL memory (16, 17, 18). Help operates at least partially at the level of the DC. The DC may either form a temporal bridge between CD4+ helper and CD8+ T cells thereby facilitating direct T-T interactions (19), or help may condition DC for more effective class I-restricted epitope presentation. Many studies point to CD40/CD40 ligand-dependent signals driving DC maturation (20, 21, 22). In addition to CD40-dependent CD4+ T cell help, CD40-independent DC sensitization events and direct cytokine-dependent CD4+/CD8+ T cell interaction may provide help (23, 24, 25). An activation signal for DC is provided by CpG-containing, immune-stimulating oligodeoxynucleotides (ODN) (11, 26, 27).

NKT cells are {alpha}{beta} T cells with an invariant TCR and intermediate level NK1 surface expression (in appropriate mouse strains) involved in anti-microbial immunity (28). The CD1d-binding glycolipid {alpha}-galactosyl-ceramide ({alpha}GalCer) stimulates most NKT cells, a specific recognition highly conserved through mammalian evolution (29). A key feature of NKT cells is their prompt cytokine secretion (30). {alpha}GalCer has been used as an adjuvant to facilitate priming of T cell immunity (31), an idea supported by the extensive bystander activation of T cells and NK cells after injection of this glycolipid (32, 33). Murine NKT cells activated in vivo by injecting {alpha}GalCer facilitate priming of either Th2- (34, 35) or Th1-biased immunity (36, 37, 38, 39, 40, 41). The interaction of NKT cells with {alpha}GalCer-pulsed DC enhances the capacity of the latter to prime NK cells and CTL (42, 43, 44, 45). We tested whether {alpha}GalCer-specific NKT cells can facilitate in vivo CD4+ T cell-independent priming of CTL responses by DC that present an epitope of the hepatitis B surface Ag (HBsAg) in the context of the class I molecule Kb.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mice

C57BL/6JBom (B6) mice (H-2b), B6 CD1d-/- (46), B6 A{alpha}-/- (47), and B6 A{beta}-/- (48, 49) mice were bred and maintained under standard pathogen-free conditions in the animal colony of Ulm University (Ulm, Germany). Male and female mice were used at 12–16 wk of age.

Peptides

The 8-mer Kb-binding S208–215 peptide ILSPFLPL of HBsAg (50), the 8-mer Kb-binding OVA257–264 peptide SIINFEKL, and the Ab-binding OVA323–339 peptide ISQAVHAAHAEINEAGR (51) of OVA were synthesized and purified by RP-HPLC by Jerini Biotools (Berlin, Germany). The peptides were stored at a concentration of 10 mg/ml and diluted before use with cell culture medium. OVA was purchased from Sigma-Aldrich (Munich, Germany; catalog no. A5503).

Cell culture

The in vitro generation of DC from murine bone marrow cells (BMC) has been described (52). Briefly, BMC were depleted of CD4+, CD8+, and B220+ lymphocytes and MHC class II+ cells (Ab-conjugated microbeads catalog nos. 130-049-201, 130-049-401, 130-049-501, 130-052-401 from Miltenyi Biotec, Bergisch Gladbach, Germany) by MACS following the manufacturer’s instructions. BMC depleted of T cells, B cells, and maturing myeloid cells were cultured at 0.75 x 106 cells/ml in 6 ml/well in UltraCulture medium (catalog no. 12-725F; BioWhittaker, Verviers, Belgium), supplemented with 10 ng/ml GM-CSF (catalog no. 315-03; PeproTech, Offenbach, Germany), 2 mM glutamine, antibiotics, and (if not indicated otherwise) 5% v/v FCS (catalog no. A15-649; PAA Laboratories, Linz, Austria). Cultures were incubated at 37°C in humidified air with 5% CO2. On day 3 and day 5, 50% of the cell culture medium was replaced by fresh, cytokine-supplemented medium. In some groups, DC were pretreated for 16 h with 2 µg/ml ODN (MWG Biotech, Ebersberg, Germany). The surface phenotype and the response to maturation-inducing stimuli of the DC used in this study have been previously described in detail (53, 54).

DC (5 x 106 cells/ml) suspended in GM-CSF-containing medium were pulsed for 2 h at 37°C with 40 µg/ml antigenic peptide. DC harvested from serum-free cultures were preincubated with 1 µg/ml human recombinant {beta}2 microglobulin ({beta}2 m) for 2 h before the peptide pulse, as this enhances class I-restricted presentation of pulsed DC (54). Thereafter, cells were washed before adoptive transfer. In some groups, DC were copulsed with 2 µg/ml {alpha}GalCer (kindly provided by Dr. Y. Koezuka; Kirin Brewery, Pharmaceutical Research Laboratory, Gunma, Japan). DC pulsed with 20–40 µg/ml peptide were found in preliminary experiments to optimally prime CTL responses.

CTL lines

CTL lines were generated by repeated in vitro stimulation of splenocytes from HBsAg-immune B6 mice with peptide-pulsed, irradiated RBL5 cells in the presence of 30 U/ml recombinant human IL-2 (54). Ag-pulsed or nonpulsed DC (5 x 104/well) from B6 or A{alpha}-/- mice were cocultured with CTL (5 x 104/well) for 24 h in 96-well U-bottom plates. Culture supernatants were harvested and analyzed for IFN-{gamma} by ELISA.

Isolation of NKT cells

CD4+ NKT cells were isolated from spleens of A{beta}-/- mice by MACS using anti-mouse CD4-microbeads (catalog no. 130-049-01; Miltenyi Biotec) following the manufacturers’ instructions. The purity of the obtained NKT cell population assessed by flow cytometry (FCM) was >95%.

FCM analyses

Cells were suspended in PBS supplemented with 0.3% w/v BSA and 0.1% w/v sodium azide. Nonspecific binding of Abs to FcR was blocked by preincubating cells with 1 µg/106 cells of the anti-CD16/CD32 mAb 2.4G2 (BD Biosciences; catalog no. 01240D). Cells were incubated with 0.5 µg mAb per 106 cells for 30 min at 4°C and were washed twice. Cells stained with biotinylated Abs were incubated for 10 min at 4°C with a second-step reagent, washed twice, and analyzed using a FACScan (BD Biosciences, Heidelberg, Germany). Dead cells were excluded by propidium iodide staining. The following anti-murine mAbs (BD Biosciences) were used: FITC-conjugated anti-CD3 (catalog no. 01084D), PE-conjugated anti-CD11c (catalog no. 09705A), anti-Kb (catalog no. 06105A), anti-Ab (catalog no. 06045A), anti-CD1d (catalog no. 09905A), anti-CD80 (catalog no. 09605B), biotinylated anti-CD40 (catalog no. 09622D), and streptavidin-Red670 (catalog no. 19543-024; Life Technologies, Eggenstein, Germany).

Adoptive DC transfer

DC (4 x 105/mouse in 100 µl) were s.c. injected into the base of the tail. In some experiments, 30 µg ODN were coinjected.

In vivo suppression of CD4+ T cells

CD4+ T cells were suppressed in mice by repeated injections of anti-CD4 mAb (clone, YTS 191.1) as described (11). One day before, at the time of, and 2, 5, and 8 days after the DC transfer, mice were i.p. injected with 200 µl PBS containing 100 µg Ab. FCM analyses of PBMC and splenic mononuclear cell populations demonstrated that >99% of the CD4+ T cells were depleted 4 days after the last injection, and only 5–6% of the CD4+ T cell population reappeared within 2 wk after the last injection.

In vivo suppression of NK cells

NK cells were eliminated during CTL priming by injecting 30 µl {alpha}-asialoGM1 antiserum (catalog no. 986-10001; Wako Chemicals, Neuss, Germany) i.p. 3 days before and at the day of adoptive DC transfer. FCM analyses of spleen cell populations demonstrated that >92% of the DX5+ NK cells were deleted 48 h after the last injection, and >80% of the NK cells were depleted 4 days after the last injection, which confirmed our previously published data (45).

Cytokine detection by ELISA

Cytokines were detected in supernatants by double-sandwich ELISA. For capture and detection, the following mAbs (BD Biosciences) were used: mAb R4-6A2 (catalog no. 18181D) and biotinylated mAb XMG1.2 (catalog no. 18112D) were used for IFN-{gamma}; mAb C15.6 (catalog no. 18491D) or mAb RedT/G297-289 (catalog no. 20011D) were used for IL-12 p40 and IL-12 p70 capture, and biotinylated mAb C17.8 (catalog no. 18482D) was used for detection; mAb TN3-19.12 (catalog no. 23351D) and a biotinylated rabbit serum IgG (catalog no. 23442D) were used for TNF-{alpha} detection. The following recombinant mouse cytokines were used as standards: IFN-{gamma} (catalog no. 19301T) and IL-12 p70 (catalog no. 19361V), both from BD Biosciences, TNF-{alpha} (catalog no. 315-01A) from PeproTech (Rocky Hill, NJ), and IL-12 p40 (catalog no. 499-ML) from R&D Systems (Wiesbaden, Germany). Extinction was analyzed at 405/490 nm on a TECAN micro plate-ELISA reader (TECAN, Crailsheim, Germany) using the EasyWin software (TECAN). Detection limits of the cytokine ELISAs were 5 pg/ml for TNF-{alpha}, 10 pg/ml for IL-12 p40, and 20 pg/ml for IL-12 p70 and IFN-{gamma}.

Determination of specific, splenic CTL frequencies

Spleen cells were obtained 14 days postvaccination as specific CTL frequencies induced by s.c. injection of pulsed DC peak between day 12 and day 15 postimmunization. Spleen cells (7.5 x 106/ml) were stimulated with 2.5 µg/ml antigenic peptide for 4 h in RPMI 1640 medium in the presence of 5 µg/ml brefeldin A (catalog no. 350-019-M025; Alexis, Grünberg, Germany). Cells were harvested and surface-stained with PE-conjugated anti-CD8 mAb (catalog no. 01045B; BD Biosciences). Stained cells were fixed with 2% paraformaldehyde (in PBS), permeabilized with a saponin-containing buffer (HBSS, 0.5% BSA, 0.5% Saponin, 0.05% sodium azide), incubated for 30 min at room temperature with FITC-conjugated anti-IFN-{gamma} mAb (catalog no. 55441; BD Biosciences), and washed three times in permeabilization buffer. Stained cells were resuspended in PBS/0.3% w/v BSA supplemented with 0.1% w/v sodium azide, and analyzed by FCM. The frequencies of Ag-specific CTL were calculated as the number of IFN-{gamma}+ CD8+ T cells/105 CD8+ T cells. The mean frequency ± SD of three individual mice per group is shown. Differences in frequencies were evaluated using the unpaired t test (GraphPad Prism 3.0 software). Groups marked with an asterisk differ (p < 0.01).

Cytotoxic assay

In vitro expansion of CTL and detection of their specific cytotoxicity was performed as described (55). Briefly, 3 x 107 responder spleen cells and 1 x 106 peptide-pulsed, irradiated stimulator RBL5 cells were coincubated in 10 ml for 5 days at 37°C in humidified air with 5% CO2. Specific cytotoxicity was tested in a 51Cr-release assay. CTL were harvested and washed, and serial dilutions of effector cells were cocultured with 2 x 103 51Cr-labeled, peptide-pulsed targets in 200 µl round bottom wells. After a 4-h incubation at 37°C, 50 µl supernatant were collected for gamma-radiation counting. The percentage of specific lysis was calculated as [(experimental release - spontaneous release)/(total counts - spontaneous release)] x 100. Total counts were measured by resuspending target cells. Spontaneous release was always <15% of the total counts. Data shown are the mean of triplicate values.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
MHC class II-deficient and MHC class II-competent, bone marrow-derived DC

An established protocol was used to generate DC in serum-free or serum-supplemented cultures with GM-CSF from immature BMC precursors from either normal B6 mice or MHC class II-deficient A{beta}-/- (or A{alpha}-/-) B6 mice (52, 53). CD11c+ DC generated from either MHC class II-deficient or MHC class II-competent precursors expressed a similar surface marker profile. These DC expressed MHC class I (Kb) molecules, CD80, CD40, and CD1d molecules on the surface (Fig. 1A). As expected, DC from normal, but not MHC class II (Ab)-deficient mice expressed MHC class II molecules on the surface (Fig. 1A). We further tested "spontaneous" and inducible release of cytokines by DC generated from normal or MHC class II-deficient mice. Both DC populations released IL-12 p40 and TNF-{alpha} after CD40 ligation or stimulation with immune-stimulating ODN, but not spontaneously (Fig. 1B). DC from MHC class II-deficient and normal B6 mice presented the Kb/S208–215 peptide ILSPFLPL to specific CTL lines in vitro (Fig. 1C). Presentation of the antigenic peptide by DC generated in serum-free cultures was enhanced by preincubating the DC for 2 h before the peptide pulse with {beta}2 m (data not shown). DC from MHC class II-expressing (normal B6) or MHC class II-deficient (A{alpha}-/- or A{beta}-/- B6) mice thus have a similar surface phenotype, a similar inducible TNF-{alpha} or IL-12 response, and a similar capacity to present antigenic epitopes to CTL.



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  		FIGURE 1. DC generated from normal or MHC class II-deficient (A{alpha}-/-) C57BL/6 (B6) mice. A, Surface marker profile by DC. CD11c+ DC from normal (B6) or A{alpha}-/- mice were purified from day 7 cultures and stained with Abs to MHC class II (Ab), CD80, MHC class I (Kb), CD1d, or CD40. B, Cytokine release by DC from normal or A{alpha}-/- B6 mice. Purified CD11c+ DC from normal or A{alpha}-/- B6 mice were stimulated for 24 h with 4 µg/ml ODN, or CD40L-expressing J558/CD40L transfectants. TNF-{alpha}- and IL-12 p40-release into the supernatant was determined by ELISA. C, Stimulation IFN-{gamma} release of Kb/S208–215 (ILSPFLPL)-specific CTL lines by pulsed DC from normal or A{alpha}-/- B6 mice. Purified CD11c+ DC from normal or A{alpha}-/- B6 mice were pulsed with antigenic peptide for 2 h at 37°C, washed and cocultured with CTL for 24 h. IFN-{gamma} in the supernatants was determined by ELISA. Mean values ± SD of triplicates are shown.

 
CD4+ T cells facilitate CTL priming by adoptively transferred, immunogenic DC

CD11c+ DC generated from bone marrow progenitors of MHC class II-competent (B6) or MHC class II-deficient (A{alpha}-/- or A{beta}-/- B6) mice were pulsed with the Kb-binding S208–215 peptide and were washed. Pulsed or nonpulsed DC were injected s.c. into naive, syngeneic B6 hosts (4 x 105/mouse). Fourteen days after the single injection of DC, the number of specific, splenic CTL was determined directly ex vivo. Transfer of peptide-pulsed DC from normal B6 donor mice into naive, syngeneic B6 hosts primed a HBsAg-specific CTL response, whereas CTL priming by adoptive transfer of Ag-pulsed, MHC class II-deficient DC was severely deficient (Fig. 2A). Although the number of specific CTL found after adoptive transfer of Ag-pulsed, MHC class II-deficient DC was low, it was always above background (Fig. 2A). Transfer of nonpulsed or {beta}2 m-pulsed control DC did not induce CTL (Fig. 2A). High numbers of specific CTL were detected at day 12 to day 15 posttransfer (Fig. 2B). CTL priming by DC generated in vitro in serum-supplemented cultures was efficient while CTL priming by DC generated under serum-free conditions was deficient (Fig. 3A). Hence, peptides derived from heterologous serum supplements in the medium may provide help in this system. To provide specific, CD4+ T cell-dependent help, DC were pulsed with the Kb-binding, antigenic S208–215 peptide ILSPFLPL of HBsAg, and with either the Ab-binding, antigenic OVA323–339 peptide ISQAVHAAHAEINEAGR or purified OVA (Fig. 3B). DC that presented additional helper determinants of OVA primed OVA-specific CD4+ T cells (data not shown) and facilitated CTL priming to HBsAg (Fig. 3B).



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  		FIGURE 2. Priming CTL responses in vivo by adoptive transfer of pulsed DC. A, Purified CD11c+ DC from normal or MHC class II-deficient (A{alpha}-/-) B6 mice were pulsed for 2 h at 37°C with the Kb-binding S208–215 peptide ILSPFLPL of HBsAg and washed. Nonpulsed or pulsed DC were injected s.c. (4 x 105 cells per mouse) into naive, normal B6 mice. Groups marked with * differ, p < 0.01 (unpaired t test). B, Kinetics of appearance of specific CTL in the spleen after s.c. injection of peptide-pulsed DC. DC prepared as described in A were injected s.c., and the appearance of specific CTL in the spleen was determined at day 7, 9, 12, and 15 posttransfer. DC were pulsed either with peptide only or with peptide plus {alpha}GalCer (as described for Fig. 6). The data points represent two mice per time point (± SD).

 


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  		FIGURE 3. Priming CTL by adoptive DC transfer is help-dependent. A, DC were generated in 7-day cultures of BMC precursors in medium either supplemented with 5% v/v FCS, or serum-free. Cells were harvested, purified, pulsed, and transferred. DC generated under serum-free conditions were incubated with 1 µg/ml human recombinant {beta}2 m, washed, and subsequently pulsed with the antigenic peptide for 2 h to facilitate class I-restricted presentation. The preincubation with exogenous {beta}2 m. B, Specific CD4+ T cell help in the priming of CTL responses by adoptively transferred DC. Purified DC were pulsed with the Kb-binding antigenic peptide either alone, or together with either the Ab-binding OVA323–339 peptide, ISQAVHAAHAEINEAGR, or purified OVA. Cells were washed and transferred. On day 14 posttransfer, spleen cells were obtained and stimulated (in the presence of brefeldin A) for 6 h with the antigenic Kb-binding peptide. Specifically inducible IFN-{gamma}+ CD8+ T cells were detected by FCM. Mean numbers of splenic IFN-{gamma}+ CD8+ T cells per 105 CD8+ T cells ± SD of three mice per group are given. Groups marked with * differ, p < 0.01 (unpaired t test).

 
Depletion studies confirmed that specific priming of CD8+ CTL responses by adoptive transfer of immunogenic DC depends on CD4+ T cells. When peptide-pulsed DC were transferred into hosts depleted of CD4+ T cells (by repeated injections of anti-CD4 Ab), they did not prime a CTL response (Fig. 4). The specific activation of CD8+ CTL precursors by Ag-pulsed DC in vivo thus depends on help from CD4+ T cells supporting the interpretation that MHC class II-dependent help facilitates CTL priming by DC.



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  		FIGURE 4. Priming CTL responses by adoptive DC transfer depends on CD4+ T cells. Purified DC from normal B6 mice were either pulsed with the antigenic, Kb-binding peptide or nonpulsed. DC were transferred into naive B6 mice (4 x 105 cells per mouse) that were either untreated or depleted of CD4+ T cells by in vivo Ab treatment. On day 14 posttransfer, spleen cells were obtained and stimulated (in the presence of brefeldin A) for 6 h with the antigenic Kb-binding peptide. Specifically inducible IFN-{gamma}+ CD8+ T cells were detected by FCM. Mean numbers of splenic IFN-{gamma}+ CD8+ T cells per 105 CD8+ T cells ± SD of three mice per group are given. Groups marked with * differ, p < 0.01 (unpaired t test).

 
Activating DC before transfer renders CTL priming partially help-independent

Activation of DC with immune-stimulating ODN before adoptive transfer facilitates CTL priming and renders the induction of CTL responses helper-independent. Purified DC were treated in vitro with 2 µg/ml ODN for 16 h. This treatment stimulated release of IL-12 p40/p70 and TNF-{alpha}, and up-regulated surface expression of MHC class II and costimulator molecules (Fig. 1, data not shown) (56, 57, 58, 59, 60). Adoptive transfer of ODN-pretreated, Ag-pulsed DC from normal (MHC class II-competent) B6 mice primed CTL responses to the HBsAg-restricted epitope more efficiently than non-pretreated, Ag-pulsed DC (Fig. 5A). When ODN-pretreated, pulsed DC from MHC class II-deficient mice were transferred into normal B6 mice, they were ~10-fold more efficient in priming CTL than non-pretreated, pulsed DC (Fig. 5A). Thus, ODN-activated, pulsed DC efficiently primed a CTL response in the absence of CD4+ T cells. Pulsed, MHC class II-deficient DC were transferred into B6 host mice with either an intact CD4 T cell compartment, or a severely depleted CD4+ T cell compartment. CTL priming facilitated by pretreatment of DC with ODN was also detected in the absence of an intact CD4 T cell compartment (Fig. 5B). Pretreatment of DC with ODN thus delivers a helper signal that facilitates CD4+ T cell-independent priming of a CTL response.



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  		FIGURE 5. Pretreatment of DC by immune-stimulating ODN enhances their capacity to prime CTL responses in vivo. A, Pretreatment of DC with ODN in vitro makes them more potent APC for CTL precursors in vivo. DC from normal or MHC class II-deficient (A{alpha}-/-) B6 mice were pretreated with 2 µg/ml ODN for 16 h before the 2-h pulse at 37°C with antigenic peptide. Cells were washed and injected s.c. (4 x 105 DC per mouse). B, Pretreatment of DC with ODN facilitates CD4+ T cell help-independent priming of CTL. DC from A{alpha}-/- B6 mice were pretreated for 16 h with 2 µg/ml ODN, washed, pulsed for 2 h at 37°C with the Kb/S208–215-specific peptide ILSPFLPL, and washed. Nonpulsed or pulsed, pretreated DC were injected s.c. (4 x 105 DC per mouse) into CD4+ T cell-competent or -deficient B6 hosts. On day 14 posttransfer, spleen cells were obtained and stimulated (in the presence of brefeldin A) for 6 h with the antigenic Kb-binding peptide. Specifically inducible IFN-{gamma}+ CD8+ T cells were detected by FCM. Mean numbers of splenic IFN-{gamma}+ CD8+ T cells per 105 CD8+ T cells ± SD of three mice per group are given. Groups marked with * differ, p < 0.01 (unpaired t test).

 
NKT cells provide help for DC-mediated CTL priming in vivo

A T cell subset that activates DC are NKT cells (44). NKT cells rapidly deliver effector functions after recognizing the glycolipid {alpha}GalCer bound to CD1d. This MHC class I-like molecule is expressed by bone marrow-derived DC (Fig. 1A). The presentation of an antigenic peptide (by Kb) together with {alpha}GalCer (by CD1d) by DC enhanced their potency to prime CTL responses in vivo. The peptide- and {alpha}GalCer-pulsed DC showed more efficient, specific CTL priming in host B6 mice than did only peptide- but not {alpha}GalCer-pulsed DC (Fig. 6A) with a similar kinetics (Fig. 2B). B6 mice injected with peptide- and {alpha}GalCer-pulsed DC from MHC class II-deficient (A{alpha}-/- or A{beta}-/-) mice showed as efficient CTL priming to the HBsAg epitope as B6 mice injected with peptide- and {alpha}GalCer-pulsed DC from MHC class II-competent (normal) B6 mice (Fig. 6B). CTL specifically induced by adoptive transfer of peptide- and {alpha}GalCer-pulsed DC in vivo produced IFN-{gamma} (Fig. 6, A and B), and specifically lysed peptide-pulsed targets (Fig. 6D). These data were confirmed in another Ag system, i.e., OVA. DC from MHC class II-deficient (A{alpha}-/- or A{beta}-/-) B6 mice were pulsed with the Kb-binding peptide SIINFEKL from OVA. In some groups, DC were also pulsed with {alpha}GalCer. Following adoptive transfer into normal, naive B6 hosts, peptide-presenting DC also pulsed with {alpha}GalCer primed CTL responses more efficiently than peptide-pulsed DC not pulsed with {alpha}GalCer (Fig. 6C). NKT cells can thus provide potent help for specific CTL priming by DC in vivo, even when the conventional MHC class II-dependent CD4+ T cell help system is excluded.



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  		FIGURE 6. NKT cell recruitment by peptide-pulsed DC facilitates CTL priming in vivo. Purified DC from normal (A) or MHC class II-deficient A{alpha}-/- (B and D) B6 mice were pulsed with the antigenic Kb/S208–215 peptide plus (in some groups) 2 µg/ml of the CD1d-binding glycolipid {alpha}GalCer. DC were washed and transferred into naive, normal B6 mice. C, Purified DC from normal or A{alpha}-/- B6 mice were pulsed with the antigenic Kb/OVA323[infi]-329 peptide and (in some groups) {alpha}GalCer. DC were washed and transferred into naive, normal B6 mice (4 x 105 DC per mouse). On day 14 posttransfer, spleen cells were obtained and stimulated (in the presence of brefeldin A) for 6 h with either the antigenic Kb/S208–215 peptide ILSPFLPL (A and B), or the Kb/OVA257–264 peptide SIINFEKL (C). Specifically inducible IFN-{gamma}+ CD8+ T cells were detected by FCM. Mean numbers of splenic IFN-{gamma}+ CD8+ T cells per 105 CD8+ T cells ± SD of three mice per group are given. D, Spleen cells obtained from the same mice as in B were restimulated in vitro for 5 days with irradiated, peptide-pulsed RBL5 cells. Specific lysis at an E:T of 20:1 read out in a 4-h 51Cr-release assay was determined. Mean values ± SD of triplicates are shown. Groups marked with * differ, p < 0.01 (unpaired t test).

 
We tested whether {alpha}GalCer has to be presented by the DC that also presents the antigenic peptide to facilitate CTL priming. DC were either pulsed with the antigenic peptide ILSPFLPL and {alpha}GalCer, or one DC population was pulsed with the antigenic peptide ILSPFLPL and a second DC population was pulsed with {alpha}GalCer. The cells were washed, and the different populations were mixed at a 1:1 ratio (Fig. 7A). These DC populations were transferred into naive B6 hosts. Specific CTL priming was only facilitated by {alpha}GalCer when the same DC presented the antigenic peptide and {alpha}GalCer (Fig. 7A). Furthermore, neither the injection of a mixture of 5 µg peptide and 5 µg {alpha}GalCer nor an injection of peptide-pulsed DC together with 5 µg {alpha}GalCer into the same site facilitated CTL priming (data not shown). Hence, only presentation of {alpha}GalCer and antigenic peptide by the same DC facilitates CTL priming in vivo, while just mixing the Ag and {alpha}GalCer before injection is inefficient. Help mediated by ODN and by {alpha}GalCer had additive but no synergistic effects (Fig. 7B). Treating DC with ODN and pulsing them with {alpha}GalCer and antigenic peptide enhanced CTL priming after adoptive transfer into normal B6 hosts, when compared with only {alpha}GalCer-pulsed, or only ODN-pretreated pulsed DC. This suggests that ODN-mediated and NKT cell-mediated signals that activate DC are different.



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  		FIGURE 7. NKT cell help for peptide-pulsed DC in vivo for specific CTL priming. A, Purified DC from A{alpha}-/- B6 mice (DC 1) were pulsed for 2 h at 37°C with only Kb/S208–215 peptide or only {alpha}GalCer (2 µg/ml), or peptide plus {alpha}GalCer. In addition, purified DC from A{alpha}-/- B6 mice (DC 2) were either nonpulsed (-) or pulsed with {alpha}GalCer. DC were washed, and cells of the DC 1 and DC 2 groups were mixed at a ratio of 1:1. The DC 1/DC 2 mixture was injected into naive B6 mice (4 x 105 cells per mouse). B, Purified DC from MHC class II-deficient A{alpha}-/- B6 mice were not pretreated or pretreated for 16 h with ODN (2 µg/ml). DC were harvested, washed, and pulsed for 2 h at 37°C with the Kb/S208–215-specific peptide ILSPFLPL, in some groups together with {alpha}GalCer (2 µg/ml). On day 14 posttransfer, spleen cells were obtained and stimulated (in the presence of brefeldin A) for 6 h with the antigenic Kb-binding peptide. Specifically inducible IFN-{gamma}+ CD8+ T cells were detected by FCM. Mean numbers of splenic IFN-{gamma}+ CD8+ T cells per 105 CD8+ T cells ± SD of three mice per group are given. Groups marked with * differ, p < 0.01 (unpaired t test).

 
CD1d expression in the host is required to facilitate CTL priming by {alpha}GalCer-pulsed DC

Development and activation of the {alpha}GalCer-reactive NKT cell population depends on CD1d molecules. This NKT cell subset (expressing CD4 and intermediate levels of CD3{epsilon} and NK1.1 on the surface) is severely depleted in CD1d-/- knockout mice (Fig. 8A). Peptide-pulsed DC from A{beta}-/- B6 mice transferred into naive, normal or CD1d-/- B6 hosts inefficiently primed CTL (Fig. 8B). DC from A{beta}-/- B6 mice pretreated with ODN and pulsed with the antigenic peptide efficiently primed CTL in naive, normal, or CD1d-/- B6 hosts (Fig. 8B), indicating that facilitated, specific CTL priming in vivo by ODN-stimulated, epitope-presenting DC is NKT cell-independent. In contrast, peptide- and {alpha}GalCer-pulsed DC from A{beta}-/- B6 mice transferred into naive, normal, or CD1d-/- B6 hosts efficiently primed CTL in normal but not CD1d-/- B6 hosts (Fig. 8B). Hence, NKT cell help is CD1d-dependent. These data furthermore support the notion that ODN-mediated and NKT cell-mediated signals facilitating DC-dependent CTL priming operate independently of each other.



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  		FIGURE 8. Recruitment of NKT cell help requires CD1d+. A, B6 mice deficient for CD1d (CD1d-/- B6) contain a reduced number of CD3int CD4+ NK1int T cells. B, DC from A{alpha}-/- B6 mice were purified and either not pretreated, or pretreated for 16 h with ODN. These DC were pulsed for 2 h at 37°C with the Kb/S208–215 peptide, either alone or together with {alpha}GalCer (2 µg/ml). The DC were washed and injected into naive, normal, or CD1d-/- B6 mice (4 x 105 cells per mouse). On day 14 posttransfer, spleen cells were obtained and stimulated (in the presence of brefeldin A) for 6 h with the antigenic Kb-binding peptide. Specifically inducible IFN-{gamma}+ CD8+ T cells were detected by FCM. Mean numbers of splenic IFN-{gamma}+ CD8+ T cells per 105 CD8+ T cells ± SD of three mice per group are given. Groups marked with * differ, p < 0.01 (unpaired t test).

 
The role of NK cells vs NKT cells in DC-dependent CTL priming

NKT cell-activated DC recruit NK cells and stimulate their IFN-{gamma} release (44, 45, 61). We tested whether NK cells are required to prime CTL by adoptive transfer of DC presenting an antigenic, Kb-binding peptide (Fig. 9). CTL priming was deficient, when pulsed DC were transferred into naive B6 mice depleted of NK cells before DC transfer (by repeated injections of {alpha}-asialoGM1 antiserum) (Fig. 9). Priming T cell help-dependent CTL responses by adoptively transferred, Ag-presenting DC thus requires NK cells.



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  		FIGURE 9. NK cell help and NKT cell help in DC-mediated CTL priming. Purified DC from normal or MHC class II-deficient A{alpha}-/- B6 mice were pulsed with the antigenic Kb/S208–215 peptide, plus (in some groups) the CD1d-binding glycolipid {alpha}GalCer. DC were washed and transferred (4 x 105 DC per mouse) into normal or NK cell-depleted, naive B6 mice. B6 mice were depleted of NK cells by repeated injections of the {alpha}-asialoGM1 Ab (as described in Materials and Methods). On day 14 posttransfer, spleen cells were obtained and stimulated (in the presence of brefeldin A) for 6 h with the antigenic Kb-binding peptide. Specifically inducible IFN-{gamma}+ CD8+ T cells were detected by FCM. Mean numbers of splenic IFN-{gamma}+ CD8+ T cells per 105 CD8+ T cells ± SD of three mice per group are given.

 
MHC class II-expressing or -deficient DC pulsed with antigenic peptide together with {alpha}GalCer were injected into normal or NK cell-depleted B6 hosts. In both systems, CTL priming by DC was less efficient in the absence of NK cells (Fig. 9). NK cells may help specific activation of CTL precursors either directly (through release of cytokines such as IL-15, type I IFNs, TNF, or IL-18) or indirectly (by enhancing the immunostimulatory phenotype of DC). Although recruitment of NKT cells help makes CTL priming by DC more efficient, it does not make it independent of NK cell help. Hence, NKT cell help can only partially replace NK cell help in specific CTL priming by adoptively transferred DC.


   Discussion
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
DC used in this study were generated from BMC precursors in 7 day GM-CSF-supplemented cultures. Similar to freshly isolated splenic or hepatic DC (44), these DC express high levels of CD1d, Kb, and Db but low/intermediate levels of the class II molecule Ab and the CD40, CD80, and CD86 costimulator molecules on the surface. No difference (except MHC class II expression) was found in the surface phenotype of bone marrow-derived DC from normal and MHC class II-deficient (A{alpha}-/- or A{beta}-/-) B6 mice. This phenotype, together with the lack of spontaneous release of IL-12 or TNF, characterizes these DC as immature. DC activated in vitro by either CpG-containing ODN or CD40 ligation up-regulated surface expression of MHC and costimulator molecules, and released IL-12 and TNF (Fig. 1). When pulsed with the Kb-binding peptide S208–215 of HBsAg, these DC specifically stimulated IFN-{gamma} release by cocultured CTL. When these DC were pulsed with {alpha}GalCer, they triggered IFN-{gamma}, IL-4, and IL-13 release by cocultured NKT cells (data not shown) (44). Hence, we have a source of large numbers of a fairly uniform, mostly immature DC available for in vivo studies that are inducible to maturation and specifically present ligands to class I-restricted CTL and CD1d-restricted NKT cells.

The adoptive transfer of DC pulsed with the Kb-binding antigenic HBsAg peptide into a syngeneic, naive recipient specifically primed a CD8+ CTL response (Fig. 2) confirming previous reports (2, 3, 4, 62). Transfer of pulsed DC into CD4+ T cell-depleted hosts primed CTL responses inefficiently (Fig. 4). This result indicates that a CD4+ cell subset facilitates CTL priming by DC in this system, which could be either a conventional, Ab-restricted CD4+ T cells or an {alpha}GalCer-reactive NKT cell. In these experiments, DC were pulsed only with the Kb-binding, immunogenic peptide. Transfer of peptide-pulsed, MHC class II-deficient DC into normal, naive B6 hosts was inefficient in priming CTL, whereas transfer of similarly pulsed MHC class II-expressing, congenic DC into the same hosts efficiently primed CTL (Fig. 2A). This suggests that antigenic peptides from the serum supplementing the culture medium are captured by Ab molecules on the surface of DC and mediate help in this system. This result was supported by the observation that pulsed, MHC class II-expressing DC generated in serum-free medium were inefficient in priming CTL responses in vivo (Fig. 3A). The data stress the point that helper-independence of CTL priming can be reliably assessed only with genetically engineered DC as culture or separation of DC cannot rule out noncontrolled, class II-restricted help. We confirmed the Ab-dependent, specific CD4+ T cell help by pulsing DC from MHC class II-expressing, normal B6 mice with the Kb-binding peptide and an Ab-binding helper peptide. The data in Fig. 3B indicate that specific CD4+ T cell help can facilitate CTL priming in vivo by adoptively transferred DC, as previously described (10, 11, 14). As specific help is in many vaccination situations either not well defined or specifically anergized or deleted, we asked: 1) can the requirement for class II-restricted CD4+ T cell help be bypassed by conditioning DC before transfer and 2) can the class II-restricted CD4+ T cell help be replaced or enhanced by an alternative helper cell.

Mature DC can prime CTL responses, whereas immature DC cannot (63). Immature DC acquire CTL priming capacity upon activation by Th-independent or -dependent stimuli (24). The maturation-inducing effect of CD4+ T cells operates to a large extent through CD40/CD40 ligand interactions (20, 64). As MHC class II-deficient DC do not prime CTL in vivo (22) (and shown in this study), the class II-restricted CD4+ T cell subset but not other CD40-expressing (e.g., NK, NKT, B) cells seem to be the main source of help under physiological conditions although CD8+ CTL themselves may also be helpers that induce DC maturation (65). Immune-stimulating DNA-based vaccines, CpG-containing oligonucleotides, or poly(I:C) induce DC maturation and CTL responses by a Th cell-independent mechanism (11, 23, 26, 27, 57, 58, 66, 67, 68). We confirm these data. CD4+ T cell-independent priming of CTL by DC in vivo is the target of the ODN adjuvant effect on CTL priming as 1) pulsed A{alpha}-/- or A{beta}-/- DC treated with ODN in vitro primed CTL as effectively as T help-supported DC in vivo, and 2) in vitro treatment of pulsed DC with ODN (followed by extensive washes), and the coinjection of pulsed DC with ODN into the mouse were equally effective in triggering the ODN adjuvant effect in vivo (suggesting that DC but not other cells recruited in vivo to the site of priming are the main target of the ODN adjuvant effect in this system).

The glycolipid {alpha}GalCer has been shown to promote adaptive immunity (reviewed in 31). NKT cells produce IFN-{gamma} (promoting Th1 immunity) and IL-4/IL-13 (promoting Th2 immunity), and facilitate in vivo priming of specific Th1 immunity (36, 69, 70) or Th2 immunity (34, 35). NKT cells have been shown to act as class II-independent helper cells in the generation of CD8+ effector function against intracellular infection (41, 71), and NKT cells can activate splenic and hepatic DC (31, 43, 44) and are expanded by {alpha}GalCer-pulsed DC (72). The question is are DC the target for the help of NKT cells? Our data indicate that {alpha}GalCer-pulsed, MHC class II-deficient DC presenting a class I-binding peptide efficiently prime CD8+ CTL responses in vivo. {alpha}GalCer-pulsed DC induce prolonged IFN-{gamma}-producing NKT responses (73), which may explain the exceptional adjuvant effect of this glycolipid. Under natural conditions, CD1d-binding ligands from either an exogenous source (e.g., pathogens) or an endogenous source (e.g., activated or damaged cells) may be available to facilitate CTL priming. Endogenous ligands may be either produced by DC themselves, or may be picked up by DC from the immediate vicinity. This adjuvant effect is certainly of interest for priming CTL responses in situations in which the CD4+ T cell system is either depleted (e.g., as in HIV infection), or specifically anergized (as in many chronic infections). The practical value of the approach in these clinical situations remains to be shown.


   Acknowledgments
 
We greatly appreciate the expert technical assistance of Tanja Güntert and Katrin Ölberger.


   Footnotes
 
1 This work was supported by a grant from the Federal Ministry of Research (DLR 01 GE9907) and the Deutsche Forschungsgemeinschaft Re549/10-1 (to J.R. and R.S.). Back

2 Address correspondence and reprint requests to Dr. Jörg Reimann, Department of Medical Microbiology and Immunology, University of Ulm, Helmholtzstrasse 8/1, D-89081 Ulm, Germany. E-mail address: joerg.reimann{at}medizin.uni-ulm.de Back

3 Abbreviations used in this paper: DC, dendritic cell; BMC, bone marrow cells; HBsAg, hepatitis B surface Ag; {alpha}GalCer, {alpha}-galactosyl-ceramide; {beta}2 m, {beta}2 microglobulin; NKT, {alpha}{beta} T cells expressing NK1.1; FCM, flow cytometry. Back

Received for publication September 23, 2002. Accepted for publication December 30, 2002.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Bender, A., L. K. Bui, M. A. Feldman, M. Larsson, N. Bhardwaj. 1995. Inactivated influenza virus, when presented on dendritic cells, elicits human CD8+ cytolytic T cell responses. J. Exp. Med. 182:1663.[Abstract/Free Full Text]
  2. Ludewig, B., S. Ehl, U. Karrer, B. Odermatt, H. Hengartner, R. M. Zinkernagel. 1998. Dendritic cells efficiently induce protective antiviral immunity. J. Virol. 72:3812.[Abstract/Free Full Text]
  3. Dhodapkar, M. V., R. M. Steinman, M. Sapp, H. Desai, C. Fossella, J. Krasovsky, S. M. Donahoe, P. R. Dunbar, V. Cerundolo, D. F. Nixon, N. Bhardwaj. 1999. Rapid generation of broad T-cell immunity in humans after a single injection of mature dendritic cells. J. Clin. Invest. 104:173.[Medline]
  4. Ruedl, C., M. F. Bachmann. 1999. CTL priming by CD8+ and CD8- dendritic cells in vivo. Eur. J. Immunol. 29:3762.[Medline]
  5. Thurner, B., I. Haendle, C. Roder, D. Dieckmann, P. Keikavoussi, H. Jonuleit, A. Bender, C. Maczek, D. Schreiner, P. von-den-Driesch, et al 1999. Vaccination with MAGE-3A1 peptide-pulsed mature, monocyte-derived dendritic cells expands specific cytotoxic T cells and induces regression of some metastases in advanced stage IV melanoma. J. Exp. Med. 190:1669.[Abstract/Free Full Text]
  6. Schlecht, G., C. Leclerc, G. Dadaglio. 2001. Induction of CTL and nonpolarized Th cell responses by CD8{alpha}+ and CD8{alpha}- dendritic cells. J. Immunol. 167:4215.[Abstract/Free Full Text]
  7. Norbury, C. C., D. Malide, J. S. Gibbs, J. R. Bennink, J. W. Yewdell. 2002. Visualizing priming of virus-specific CD8+ T cells by infected dendritic cells in vivo. Nat. Immun. 3:265.
  8. Fayolle, C., E. Deriaud, C. Leclerc. 1991. In vivo induction of cytotoxic T cell response by a free synthetic peptide requires CD4+ T cell help. J. Immunol. 147:4069.[Abstract]
  9. Bennett, S. R., F. R. Carbone, F. Karamalis, J. F. Miller, W. R. Heath. 1997. Induction of a CD8+ cytotoxic T lymphocyte response by cross-priming requires cognate CD4+ T cell help. J. Exp. Med. 186:65.[Abstract/Free Full Text]
  10. Livingstone, A. M., M. Kuhn. 1999. Dendritic cells need T cell help to prime cytotoxic T cell responses to strong antigens. Eur. J. Immunol. 29:2826.[Medline]
  11. Wild, J., M. J. Grusby, R. Schirmbeck, J. Reimann. 1999. Priming MHC-I-restricted, cytotoxic T lymphocyte responses to exogenous hepatitis B surface antigen is CD4+ T cell-dependent. J. Immunol. 163:1880.[Abstract/Free Full Text]
  12. de Veerman, M., C. Heirman, M. S. Van, S. Devos, J. Corthals, M. Moser, K. Thielemans. 1999. Retrovirally transduced bone marrow-derived dendritic cells require CD4+ T cell help to elicit protective and therapeutic antitumor immunity. J. Immunol. 162:144.[Abstract/Free Full Text]
  13. den Boer, A. T., L. Diehl, G. J. van Mierlo, E. I. van der Voort, M. F. Fransen, P. Krimpenfort, C. J. Melief, R. Offringa, R. E. Toes. 2001. Longevity of antigen presentation and activation status of APC are decisive factors in the balance between CTL immunity versus tolerance. J. Immunol. 167:2522.[Abstract/Free Full Text]
  14. Marzo, A. L., B. F. Kinnear, R. A. Lake, J. J. Frelinger, E. J. Collins, B. W. Robinson, B. Scott. 2000. Tumor-specific CD4+ T cells have a major "post-licensing" role in CTL mediated anti-tumor immunity. J. Immunol. 165:6047.[Abstract/Free Full Text]
  15. Stohlman, S. A., C. C. Bergmann, M. T. Lin, D. J. Cua, D. R. Hinton. 1998. CTL effector function within the central nervous system requires CD4+ T cells. J. Immunol. 160:2896.[Abstract/Free Full Text]
  16. Di, R. F., P. Matzinger. 1996. Long-lasting CD8 T cell memory in the absence of CD4 T cells or B cells. J. Exp. Med. 183:2153.[Abstract/Free Full Text]
  17. Stevenson, P. G., G. T. Belz, J. D. Altman, P. C. Doherty. 1998. Virus-specific CD8+ T cell numbers are maintained during {gamma}-herpesvirus reactivation in CD4-deficient mice. Proc. Natl. Acad. Sci. USA 95:15565.[Abstract/Free Full Text]
  18. Zhong, W., D. Marshall, C. Coleclough, D. L. Woodland. 2000. CD4+ T cell priming accelerates the clearance of Sendai virus in mice, but has a negative effect on CD8+ T cell memory. J. Immunol. 164:3274.[Abstract/Free Full Text]
  19. Ridge, J. P., R. F. Di, P. Matzinger. 1998. A conditioned dendritic cell can be a temporal bridge between a CD4+ T-helper and a T-killer cell. Nature 393:474.[Medline]
  20. Bennett, S. R., F. R. Carbone, F. Karamalis, R. A. Flavell, J. F. Miller, W. R. Heath. 1998. Help for cytotoxic-T-cell responses is mediated by CD40 signalling. Nature 393:478.[Medline]
  21. Ossendorp, F., E. Mengede, M. Camps, R. Filius, C. J. Melief. 1998. Specific T helper cell requirement for optimal induction of cytotoxic T lymphocytes against major histocompatibility complex class II negative tumors. J. Exp. Med. 187:693.[Abstract/Free Full Text]
  22. Hermans, I. F., D. S. Ritchie, A. Daish, J. Yang, M. R. Kehry, F. Ronchese. 1999. Impaired ability of MHC class II-/- dendritic cells to provide tumor protection is rescued by CD40 ligation. J. Immunol. 163:77.[Abstract/Free Full Text]
  23. Lu, Z., L. Yuan, X. Zhou, E. Sotomayor, H. I. Levitsky, D. M. Pardoll. 2000. CD40-independent pathways of T cell help for priming of CD8+ cytotoxic T lymphocytes. J. Exp. Med. 191:541.[Abstract/Free Full Text]
  24. Schuurhuis, D. H., S. Laban, R. E. Toes, C. P. Ricciardi-Castagnoli, M. J. Kleijmeer, E. I. van der Voort, D. Rea, R. Offringa, H. J. Geuze, C. J. Melief, F. Ossendorp. 2000. Immature dendritic cells acquire CD8+ cytotoxic T lymphocyte priming capacity upon activation by T helper cell-independent or -dependent stimuli. J. Exp. Med. 192:145.[Abstract/Free Full Text]
  25. Sato, M., K. Chamoto, T. Tsuji, Y. Iwakura, Y. Togashi, T. Koda, T. Nishimura. 2001. Th1 cytokine-conditioned bone marrow-derived dendritic cells can bypass the requirement for Th functions during the generation of CD8 CTL. J. Immunol. 167:3687.[Abstract/Free Full Text]
  26. Oxenius, A., M. M. Martinic, H. Hengartner, P. Klenerman. 1999. CpG-containing oligonucleotides are efficient adjuvants for induction of protective antiviral immune responses with T-cell peptide vaccines. J. Virol. 73:4120.[Abstract/Free Full Text]
  27. Cho, H. J., K. Takabayashi, P. M. Cheng, M. D. Nguyen, M. Corr, S. Tuck, E. Raz. 2000. Immunostimulatory DNA-based vaccines induce cytotoxic lymphocyte activity by a T-helper cell-independent mechanism. Nat. Biotechnol. 18:509.[Medline]
  28. Gumperz, J. E., M. B. Brenner. 2001. CD1-specific T cells in microbial immunity. Curr. Opin. Immunol. 13:471.[Medline]
  29. Brossay, L., M. Chioda, N. Burdin, Y. Koezuka, G. Casorati, P. Dellabona, M. Kronenberg. 1998. CD1d-mediated recognition of an {alpha}-galactosylceramide by natural killer T cells is highly conserved through mammalian evolution. J. Exp. Med. 188:1521.[Abstract/Free Full Text]
  30. Joyce, S.. 2000. Natural T cells: cranking up the immune system by prompt cytokine secretion. Proc. Natl. Acad. Sci. USA 97:6933.[Free Full Text]
  31. Bendelac, A., R. Medzhitov. 2002. Adjuvants of immunity: harnessing innate immunity to promote adaptive immunity. J. Exp. Med. 195:F19.
  32. Eberl, G., H. R. MacDonald. 2000. Selective induction of NK cell proliferation and cytotoxicity by activated NKT cells. Eur. J. Immunol. 30:985.[Medline]
  33. Eberl, G., P. Brawand, H. R. MacDonald. 2000. Selective bystander proliferation of memory CD4+ and CD8+ T cells upon NK T or T cell activation. J. Immunol. 165:4305.[Abstract/Free Full Text]
  34. Burdin, N., L. Brossay, M. Kronenberg. 1999. Immunization with {alpha}-galactosylceramide polarizes CD1-reactive NK T cells towards Th2 cytokine synthesis. Eur. J. Immunol. 29:2014.[Medline]
  35. Singh, N., S. Hong, D. C. Scherer, I. Serizawa, N. Burdin, M. Kronenberg, Y. Koezuka, L. Van-Kaer. 1999. Activation of NK T cells by CD1d and {alpha}-galactosylceramide directs conventional T cells to the acquisition of a Th2 phenotype. J. Immunol. 163:2373.[Abstract/Free Full Text]
  36. Nishimura, T., H. Kitamura, K. Iwakabe, T. Yahata, A. Ohta, M. Sato, K. Takeda, K. Okumura, L. Van Kaer, T. Kawano, et al 2000. The interface between innate and acquired immunity: glycolipid antigen presentation by CD1d-expressing dendritic cells to NKT cells induces the differentiation of antigen-specific cytotoxic T lymphocytes. Int. Immunol. 12:987.[Abstract/Free Full Text]
  37. Gonzalez, A., C. de Oliveira, M. Tomaska, S. Hong, R. O. Bruna, T. Nakayama, M. Taniguchi, A. Bendelac, L. Van-Kaer, Y. Koezuka, M. Tsuji. 2000. {alpha}-Galactosylceramide-activated V{alpha}14 natural killer T cells mediate protection against murine malaria. Proc. Natl. Acad. Sci. USA 97:8461.[Abstract/Free Full Text]
  38. Smyth, M. J., M. Taniguchi, S. E. Street. 2000. The anti-tumor activity of IL-12: mechanisms of innate immunity that are model and dose dependent. J. Immunol. 165:2665.[Abstract/Free Full Text]
  39. Hayakawa, Y., K. Takeda, H. Yagita, S. Kakuta, Y. Iwakura, L. Van Kaer, I. Saiki, K. Okumura. 2001. Critical contribution of IFN-{gamma} and NK cells, but not perforin-mediated cytotoxicity, to anti-metastatic effect of {alpha}-galactosylceramide. Eur. J. Immunol. 31:1720.[Medline]
  40. Gonzalez-Aseguinolaza, G., L. Van Kaer, C. C. Bergmann, J. M. Wilson, J. Schmieg, M. Kronenberg, T. Nakayama, M. Taniguchi, Y. Koezuka, M. Tsuji. 2002. Natural killer T cell ligand {alpha}-galactosylceramide enhances protective immunity induced by malaria vaccines. J. Exp. Med. 195:617.[Abstract/Free Full Text]
  41. Johnson, T. R., S. Hong, L. Van Kaer, Y. Koezuka, B. S. Graham. 2002. NK T cells contribute to expansion of CD8+ T cells and amplification of antiviral immune responses to respiratory syncytial virus. J. Virol. 76:4294.[Abstract/Free Full Text]
  42. Toura, I., T. Kawano, Y. Akutsu, T. Nakayama, T. Ochiai, M. Taniguchi. 1999. Inhibition of experimental tumor metastasis by dendritic cells pulsed with {alpha}-galactosylceramide. J. Immunol. 163:2387.[Abstract/Free Full Text]
  43. Takahashi, T., M. Nieda, Y. Koezuka, A. Nicol, S. A. Porcelli, Y. Ishikawa, K. Tadokoro, H. Hirai, T. Juji. 2000. Analysis of human Va24+ CD4+ NKT cells activated by {alpha}-glycosyl-ceramide-pulsed monocyte-derived dendritic cells. J. Immunol. 164:4458.[Abstract/Free Full Text]
  44. Trobonjaca, Z., F. Leithäuser, P. Moller, R. Schirmbeck, J. Reimann. 2001. Activating immunity in the liver. I. Liver dendritic cells (but not hepatocytes) are potent activators of IFN-{gamma} release by liver NKT cells. J. Immunol. 167:1413.[Abstract/Free Full Text]
  45. Trobonjaca, Z., A. Kroger, D. Stober, F. Leithäuser, P. Moller, H. Hauser, R. Schirmbeck, J. Reimann. 2002. Activating immunity in the liver. II. IFN-{beta} attenuates NK cell-dependent liver injury triggered by liver NKT cell activation. J. Immunol. 168:3763.[Abstract/Free Full Text]
  46. Smiley, S. T., M. H. Kaplan, M. J. Grusby. 1997. Immunoglobulin E production in the absence of interleukin-4-secreting CD1-dependent cells. Science 275:977.[Abstract/Free Full Text]
  47. Kontgen, F., G. Suss, C. Stewart, M. Steinmetz, H. Bluethmann. 1993. Targeted disruption of the MHC class II A{alpha} gene in C57BL/6 mice. Int. Immunol. 5:957.[Abstract/Free Full Text]
  48. Cosgrove, D., D. Gray, A. Dierich, J. Kaufman, M. Lemeur, C. Benoist, D. Mathis. 1991. Mice lacking MHC class II molecules. Cell 66:1051.[Medline]
  49. Cardell, S., S. Tangri, S. Chan, M. Kronenberg, C. Benoist, D. Mathis. 1995. CD1-restricted CD4+ T cells in major histocompatibility complex class II-deficient mice. J. Exp. Med. 182:993.[Abstract/Free Full Text]
  50. Schirmbeck, R., J. Wild, J. Reimann. 1998. Similar as well as distinct MHC-I-binding peptides are generated by exogenous and endogenous processing of hepatitis B virus surface antigen (HBsAg). Eur. J. Immunol. 28:4149.[Medline]
  51. Robertson, J. M., P. E. Jensen, B. D. Evavold. 2000. DO11.10 and OT-II T cells recognize a C-terminal ovalbumin 323–339 epitope. J. Immunol. 164:4706.[Abstract/Free Full Text]
  52. Inaba, K., M. Inaba, N. Romani, H. Aya, M. Deguchi, S. Ikehara, S. Muramatsu, R. M. Steinman. 1992. Generation of large numbers of dendritic cells from mouse bone marrow cultures supplemented with granulocyte/macrophage colony-stimulating factor. J. Exp. Med. 176:1693.[Abstract/Free Full Text]
  53. Stober, D., R. Schirmbeck, J. Reimann. 2001. IL-12/IL-18-dependent IFN-{gamma} release by murine dendritic cells. J. Immunol. 167:957.[Abstract/Free Full Text]
  54. Stober, D., Z. Trobonjaca, J. Reimann, R. Schirmbeck. 2002. Dendritic cells pulsed with exogenous hepatitis B surface antigen particles efficiently present epitopes to MHC class I-restricted cytotoxic T cells. Eur. J. Immunol. 32:1099.[Medline]
  55. Schirmbeck, R., J. Wild, D. Stober, H. E. Blum, F. V. Chisari, M. Geissler, J. Reimann. 2000. Ongoing murine T1 or T2 immune responses to the hepatitis B surface antigen are excluded from the liver that expresses transgene-encoded hepatitis B surface antigen. J. Immunol. 164:4235.[Abstract/Free Full Text]
  56. Jakob, T., P. S. Walker, A. M. Krieg, M. C. Udey, J. C. Vogel. 1998. Activation of cutaneous dendritic cells by CpG-containing oligodeoxynucleotides: a role for dendritic cells in the augmentation of Th1 responses by immunostimulatory DNA. J. Immunol. 161:3042.[Abstract/Free Full Text]
  57. Sparwasser, T., E. S. Koch, R. M. Vabulas, K. Heeg, G. B. Lipford, J. W. Ellwart, H. Wagner. 1998. Bacterial DNA and immunostimulatory CpG oligonucleotides trigger maturation and activation of murine dendritic cells. Eur. J. Immunol. 28:2045.[Medline]
  58. Hartmann, G., G. J. Weiner, A. M. Krieg. 1999. CpG DNA: a potent signal for growth, activation, and maturation of human dendritic cells. Proc. Natl. Acad. Sci. USA 96:9305.[Abstract/Free Full Text]
  59. Schirmbeck, R., K. Melber, J. Reimann. 1999. Adjuvants that enhance priming of cytotoxic T cells to a Kb-restricted epitope processed from exogenous but not endogenous hepatitis B surface antigen. Int. Immunol. 11:1093.[Abstract/Free Full Text]
  60. Askew, D., R. S. Chu, A. M. Krieg, C. V. Harding. 2000. CpG DNA induces maturation of dendritic cells with distinct effects on nascent and recycling MHC-II antigen-processing mechanisms. J. Immunol. 165:6889.[Abstract/Free Full Text]
  61. Carnaud, C., D. Lee, O. Donnars, S. H. Park, A. Beavis, Y. Koezuka, A. Bendelac. 1999. Cross-talk between cells of the innate immune system: NKT cells rapidly activate NK cells. J. Immunol. 163:4647.[Abstract/Free Full Text]
  62. Sandberg, J. K., P. Grufman, E. Z. Wolpert, L. Franksson, B. J. Chambers, K. Karre. 1998. Superdominance among immunodominant H-2Kb-restricted epitopes and reversal by dendritic cell-mediated antigen delivery. J. Immunol. 160:3163.[Abstract/Free Full Text]
  63. Dhodapkar, M. V., J. Krasovsky, R. M. Steinman, N. Bhardwaj. 2000. Mature dendritic cells boost functionally superior CD8+ T-cell in humans without foreign helper epitopes. J. Clin. Invest. 105:R9.
  64. Schoenberger, S. P., R. E. Toes, V. E. van-der, R. Offringa, C. J. Melief. 1998. T-cell help for cytotoxic T lymphocytes is mediated by CD40-CD40L interactions. Nature 393:480.[Medline]
  65. Mailliard, R. B., S. Egawa, Q. Cai, A. Kalinska, S. N. Bykovskaya, M. T. Lotze, M. L. Kapsenberg, W. J. Storkus, P. Kalinski. 2002. Complementary dendritic cell-activating function of CD8+ and CD4+ T cells: helper role of CD8+ T cells in the development of T helper type 1 responses. J. Exp. Med. 195:473.[Abstract/Free Full Text]
  66. Cella, M., M. Salio, Y. Sakakibara, H. Langen, I. Julkunen, A. Lanzavecchia. 1999. Maturation, activation, and protection of dendritic cells induced by double-stranded RNA. J. Exp. Med. 189:821.[Abstract/Free Full Text]
  67. Verdijk, R. M., T. Mutis, B. Esendam, J. Kamp, C. J. Melief, A. Brand, E. Goulmy. 1999. Polyriboinosinic polyribocytidylic acid (polyI:C) induces stable maturation of functionally active human dendritic cells. J. Immunol. 163:57.[Abstract/Free Full Text]
  68. Vabulas, R. M., H. Pircher, G. B. Lipford, H. Hacker, H. Wagner. 2000. CpG-DNA activates in vivo T cell epitope presenting dendritic cells to trigger protective antiviral cytotoxic T cell responses. J. Immunol. 164:2372.