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Dendritic Cell Function Can Be Modulated through Cooperative Actions of TLR Ligands and Invariant NKT Cells¹

Ian F. Hermans^2,3,*,, Jonathan D. Silk^2,*, Uzi Gileadi^2,*, S. Hajar Masri^*, Dawn Shepherd^*, Kathryn J. Farrand, Mariolina Salio^* and Vincenzo Cerundolo^4,*

^* Tumour Immunology Unit, Weatherall Institute of Molecular Medicine, John Radcliffe Hospital, Oxford, United Kingdom; and Malaghan Institute of Medical Research, Wellington, New Zealand

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
The quality of signals received by dendritic cells (DC) in response to pathogens influences the nature of the adaptive response. We show that pathogen-derived signals to DC mediated via TLRs can be modulated by activated invariant NKT (iNKT) cells. DC maturation induced in vivo with any one of a variety of TLR ligands was greatly improved through simultaneous administration of the iNKT cell ligand -galactosylceramide. DC isolated from animals treated simultaneously with TLR and iNKT cell ligands were potent stimulators of naive T cells in vitro compared with DC from animals treated with the ligands individually. Injection of protein Ags with both stimuli resulted in significantly improved T cell and Ab responses to coadministered protein Ags over TLR stimulation alone. Ag-specific CD8⁺ T cell responses induced in the presence of the TLR4 ligand monophosphoryl lipid A and -galactosylceramide showed faster proliferation kinetics, and increased effector function, than those induced with either ligand alone. Human DC exposed to TLR ligands and activated iNKT cells in vitro had enhanced expression of maturation markers, suggesting that a cooperative action of TLR ligands and iNKT cells on DC function is a generalizable phenomenon across species. These studies highlight the potential for manipulating the interactions between TLR ligands and iNKT cell activation in the design of effective vaccine adjuvants.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
Dendritic cells (DC)⁵ are the key mediators of adaptive immunity (1). In the absence of infection, DC in peripheral tissues are in a resting immature state with limited ability to stimulate naive T cells. Following infection, DC undergo maturation, a process characterized by phenotypic changes resulting in improved ability to promote T cell responses.

There is now increasing evidence suggesting that direct interactions between pathogens and DC help define the maturation process and the type of adaptive immune response initiated. DC interact with pathogens via pathogen-recognition receptors, which engage pathogen-associated molecular patterns common to microbial or viral products (2). Examples of pathogen-recognition receptors are the TLRs, which recognize structures such as LPS (TLR4), flagellin (TLR5), bacterial lipopeptides (TLR1/2/6), hypomethylated CpG DNA (TLR9), and viral dsRNA and ssRNA (TLR3, 7, and 8). These receptors can be expressed on the cell surface (TLR1, 2, 4, 5, and 6), or within endosomes, where they can interact with pathogen-derived products acquired through phagocytosis (TLR3, 7, 8, and 9). DC maturation, as defined by increased costimulatory capacity, can be induced by direct stimulation through DC-expressed TLRs, or indirectly by exposure to cytokines released by local immune or nonimmune cells stimulated via their own TLRs. A recent report has suggested that induction of full effector function in T cells is dependent on DC being directly stimulated via TLRs, thereby acquiring an activated phenotype characterized by the release of important proinflammatory cytokines, including IL-12 (3). Activated T cells play a considerable role in this process, providing key stimuli to DC, such as CD40/CD40L interactions (4), required for the enhanced release of proinflammatory cytokines once microbial signals have been received (5).

Monospecific T cell subpopulations, such as those restricted by CD1 molecules, can provide stimulatory signals for DC (6, 7, 8, 9), and by virtue of their significant frequency, may have a potent modulatory function. Significant among these populations are invariant NKT (iNKT) cells expressing an invariant TCR -chain encoded by V14-J18 gene segments in mice or V24-J18 in humans (10, 11). These cells are found at high frequency in the spleen, bone marrow, and liver and respond to both microbial (12, 13, 14, 15) and endogenous glycolipids (13, 16) in the context of CD1d.

iNKT cells can promote enhanced T cell responses when activated by a powerful stimulus such as the synthetic glycolipid, -galactosylceramide (-GalCer), implying that they can provide all of the signals required for DC activation (8, 9). Significantly, we have reported that microbial products can play a role in directing this immunity even in the presence of a robust iNKT cell response (17). When -GalCer treatment was combined with the TLR4 ligand monophosphoryl lipid A (MPL), a detoxified version of LPS, DC became even more potent promoters of adaptive responses.

In this study, we have examined the combined influence of microbial stimulation and iNKT cell activation by -GalCer in more detail. We found that cooperation between TLR stimulation and iNKT cell activation on DC function is a generalized phenomenon exhibited by different TLR ligands, in both mouse and human systems. Significant quantitative and qualitative differences in the immune responses were generated depending on the stimuli provided.

	Materials and Methods

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Mice and cell lines

C57BL/6 (H-2^b) mice were obtained from breeding pairs originally from The Jackson Laboratory. Also used were mice lacking the J18 TCR gene segment (referred to as iNKT-deficient mice) (18) and CD1d^–/– mice (19), both of which are devoid of V14 iNKT cells. TLR4^–/– mice were provided by R. Grencis (University of Manchester, Manchester, U.K.) (20); F5-RAG^–/– mice transgenic (tg) for a TCR recognizing an H-2D^b-restricted epitope from influenza nucleoprotein (Flu-NP)_366–374, on a RAG^–/– background (referred to in text as Flu-NP-TCRtg), were provided by D. Kioussis (National Institute of Medical Research, London, U.K.) (21). All mice were maintained in the Biological Services Unit at John Radcliffe Hospital, and used according to established institutional guidelines.

In vitro culture medium and reagents

Cell lines were maintained in complete medium consisting of RPMI 1640 (Sigma-Aldrich), supplemented with 2 mM L-glutamine, 1% penicillin/streptomycin, 5 x 10^–5 M 2-ME (Invitrogen Life Technologies), and 10% heat-inactivated FCS (Globepharm). Chicken OVA grade VII was purchased from Sigma-Aldrich, or derived from germfree chickens, as described (22), and provided by T. Moran (Department of Microbiology, Mount Sinai School of Medicine, New York, NY). The iNKT cell ligand -GalCer (KRN7000) was supplied by Kirin Brewery and was solubilized in 150 mM NaCl, 0.5% Tween 20, referred to as vehicle. The following TLR ligands were used: MPL (Sigma-Aldrich), polyinosinic polycytidylic acid (poly(I:C)), flagellin (InvivoGen), and CpG DNA 1826 (Coley Pharmaceutical) (23). Peptides from Flu-NP (Flu-NP_366–374, binding to H-2 D^b) and OVA (OVA_257–264, binding to H-2 K^b) were synthesized in house. All Abs used for FACS analysis were obtained from BD Biosciences or eBioscience. Flow cytometry was performed on a FACSCalibur (BD Biosciences), and data analysis was conducted using CellQuest software (BD Biosciences).

Administration of Ags and adjuvants

All substances were diluted in PBS and administered i.v. The doses used were as follows (unless otherwise stated): 400 µg of OVA protein, 1 µg of -GalCer (or equivalent PBS-diluted vehicle solution), 25 µg of MPL, 50 µg of CpG, 2.5 µg of flagellin, and 2.5 µg of poly(I:C).

Restimulation of immune responses in vivo

OVA-specific T cell responses were boosted by i.v. injection of a recombinant vaccinia virus (5–10 x 10⁵ PFU/mouse) encoding full-length chicken OVA (vacc-OVA) (24).

Monitoring CD8⁺ T cell responses ex vivo with MHC class I/peptide tetramers

Mice were bled from the lateral tail vein and the PBL stained directly ex vivo with tetrameric H-2 K^b/OVA_257–264 peptide complexes, as described (25). In some experiments, the phenotype of the tetramer⁺ cells was established by four-color flow cytometry with Abs against CD127 and CD62L.

Phenotyping of APC

The maturation status of APC in response to different adjuvants was assessed 20 h after administration. Splenocyte preparations were obtained by teasing of splenic tissue through gauze in the presence of complete medium with 5 mM EDTA. RBC were lysed using RBC lysis fluid (Purescript). Expression of CD86 was assessed on CD11c⁺ cells (DC) and CD45R/B220⁺ cells (B cells) by flow cytometry. Anti-FcRII (BD Biosciences) was used to inhibit nonspecific staining.

T cell proliferation in vitro with isolated DC

Kits for CD11c⁺ DC isolation (Miltenyi Biotec) were used according to manufacturer’s instructions. DC were isolated from the spleens of animals treated with different adjuvant combinations and used to stimulate CFSE-labeled Flu-NP-TCRtg splenocytes in vitro. The DC were fixed with 1% paraformaldehyde, quenched with 0.1 mM glycine, and loaded with 1 µM Flu-NP_366–374 peptide. Responders were depleted of APC by adherence to tissue culture plates for 1 h, labeled with 67 nM CFSE (Molecular Probes), and cocultured with DC in a 96-well plate for 72 h (5 x 10⁴ responders/well against titrated doses of APC). Proliferation was determined by flow cytometry.

Intracellular cytokine analysis

CFSE-labeled Flu-NP-TCRtg responder T cells from in vitro proliferation assays were also assayed for cytokine expression after 72 h. Cells were stimulated with 10^–6 M PMA (Sigma-Aldrich) and 1 µg/ml ionomycin (Sigma-Aldrich) or unstimulated. Brefeldin A (Sigma-Aldrich) was added after 1 h at 20 µg/ml, and cells were collected after a total of 6 h. Cells were fixed in 2% paraformaldehyde, treated with permeabilization solution (BD Biosciences), and stained with Abs to mouse IFN- and IL-2 for flow cytometry. When using ex vivo splenocytes, cells were washed, loaded with or without stimulating peptide (or control PMA and ionomycin, as above) for 1 h, followed by 5-h incubation with 5 µg/ml brefeldin A (Sigma- Aldrich). Cells were then fixed with 2% paraformaldehyde (Sigma- Aldrich) and washed. Cells were permeabilized using 0.5% saponin, 10 mM HEPES (Sigma-Aldrich), and 5% FCS, and stained with Abs to mouse IFN- and CD8 for flow cytometry.

In vivo cytotoxicity assay

The cytotoxic capacity of induced CD8⁺ T cell responses was measured by VITAL assay (26). Syngeneic splenocytes were loaded with 500, 50, or 5 nM OVA_257–264 peptide, and then labeled with CFSE at 1.65, 0.3, and 0.07 nM. A control population without Ag was labeled with 10 µM chloromethyl-benzoyl-aminotetramethyl-rhodamine (CMTMR). A mixture of all four populations was injected i.v into immune mice, and specific lysis of the peptide-loaded targets was monitored by FACS analysis of lymph node samples. Mean percentage of survival of peptide-pulsed targets was calculated relative to that of the control population, and cytotoxic activity was expressed as percent specific lysis (100 – mean percentage of survival of peptide-pulsed targets).

Cytokine ELISA

Concentrations of IL-12p40 and IL-12p70 in serum were determined using standard sandwich ELISAs using the C15.6 capture Ab (eBioscience) for IL-12p40, and the 9A5 capture Ab (BD Biosciences) for IL-12p70. Biotinylated C17.8 (BD Biosciences) was used as the detection Ab. Concentrations of IFN- were determined using R4-6A2 and biotinylated XMG1.2 Abs (eBioscience).

OVA-specific IgG ELISA

Microtiter plates were coated with OVA (Sigma-Aldrich) at 10 µg/ml for 16 h at 4°C. Serial dilutions of serum were added for 20 h at 4°C. OVA-specific IgGs were detected with HRP-conjugated sheep anti-mouse IgG (Amersham Biosciences).

Analysis of human DC maturation in vitro

Human DC were differentiated from peripheral blood monocytes, as described (27). The iNKT cell line SG was derived from PBL using -GalCer- loaded DC, as described, and purified by cell sorting (28). DC and iNKT cells were admixed at a ratio of 8:1 in the presence of combinations of vehicle, 150 ng/ml -GalCer, or 2.5 µg/ml MPL, and DC maturation was assessed after 40 h. The expression of CD83, CD80, CD86, and CD38 was assessed by flow cytometry.

	Results

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
In vivo cooperation between TLR4 stimulation and activation of iNKT cells improves stimulatory function of DC, leading to greater Ag-specific T cell and Ab responses.

To investigate whether immune responses to Ag can be modulated through the cooperative actions of iNKT cells and TLR ligands, chicken OVA protein was injected together with either the TLR4 ligand MPL, the iNKT cell ligand -GalCer, or a combination of both, into wild-type and TLR4-deficient (TLR4^–/–) mice. We have shown previously that injection of -GalCer with OVA induces DC maturation and enhances OVA-specific T cell responses, a process that was dependent upon the activation of iNKT cells (8, 17). The combination of -GalCer and MPL examined in this study had a synergistic effect on the induction of CD8⁺ T cell responses to OVA in wild-type animals, as measured in the blood on day 6 using fluorescent H-2K^b/OVA_257–264 tetramers (Fig. 1, A and B). In TLR4^–/– recipients, OVA-specific CD8⁺ T cell responses were no greater than those observed with injection of OVA with -GalCer alone, confirming a role for TLR4 signaling in the observed synergy between -GalCer and MPL (Fig. 1B) (17). The induction of CD4⁺ T cells to OVA was tested in these mice using a defined MHC class II-binding peptide to stimulate splenocytes in an IFN- ELISPOT assay. The OVA-specific CD4⁺ T cell responses were also strongest with the combination of -GalCer and MPL (data not shown). Total OVA-specific IgG responses were measured in serum 10 days after administration of OVA with -GalCer, MPL, or both. Although -GalCer or MPL alone served as effective adjuvants (Fig. 1C), the combination of -GalCer and MPL induced OVA-specific IgG responses that were significantly superior to either agent alone. Thus, both cell-mediated and Ab-mediated immune responses are improved by provision of iNKT cell-derived signals and TLR4 stimulation.

View larger version (19K): [in this window] [in a new window]   FIGURE 1. Injection of -GalCer together with a TLR ligand enhances immune responses to coadministered protein Ag in vivo. OVA257–264-specific CD8+ T cell responses were assessed in the blood of recipient mice by FACS analysis using H-2Kb/OVA257–264 tetramers 6 days after immunization with OVA protein together with vehicle, 1 µg of -GalCer, 25 µg of MPL, or a combination of -GalCer and MPL. A, Representative FACS plots from each group, and B, mean proportions of tetramer+ cells as a percentage of CD8+ cells (±SEM) for each group (n = 5) are shown. Responses in C57BL/6 recipient mice were compared with those generated in TLR4–/– recipients. C, Serum was taken 10 days after Ag administration as in A (n = 5/group), and total OVA-specific IgG responses were measured by ELISA. Data are presented as the mean OD (±SEM).

To establish whether the enhanced immune responses observed with TLR4 and iNKT cell stimulation can be attributed to enhanced DC function, DC were isolated from treated animals and used to stimulate CD8⁺ T cells in vitro. Responder T cells bearing a TCR specific for the D^b-restricted epitope of Flu-NP (Flu-NP_366–374) were enriched from APC-depleted splenocytes. CFSE-labeled responders were combined with Flu-NP_366–374 peptide-loaded CD11c⁺ cells isolated from the spleens of mice previously injected with MPL and/or -GalCer. Proliferation was assessed 72 h later by FACS analysis, with cell division marked by a sequential loss of CFSE with each division. These analyses showed that DC isolated from animals treated with the combination of -GalCer and MPL provided the strongest stimulus to T cells (Fig. 2A). The same T cells showed enhanced production of IFN- and IL-2 (Fig. 2B and data not shown) with improved provision of cytokines apparent in each T cell division. In addition, analysis of the coculture supernatant by ELISA revealed that the most significant IFN- production was from T cells that had been stimulated with DC that had received both TLR4 and iNKT cell stimulation (Fig. 2C). These results indicate a fundamental difference in activated T cell phenotype with different DC stimuli.

View larger version (16K): [in this window] [in a new window]   FIGURE 2. DC from animals treated with a combination of -GalCer and MPL have increased capacity to induce TE in vitro. A, DC isolated from animals treated with the different ligands indicated were used to stimulate CFSE-labeled Flu-NP-TCRtg splenocytes in vitro. Proliferation was determined by FACS analysis after 72 h of culture. Representative FACS profiles from one of three animals per group are shown. B, Intracellular cytokine analysis was used to assess PMA/ionomycin-2 stimulated, T cell-derived cytokine release at 72 h after exposure to DC isolated from the treated animals. Representative FACS plots from one of three animals per group are shown. C, The culture supernatants were assessed by IFN- ELISA also after 72 h.

The previous in vitro and in vivo data suggested that the combination of iNKT cell and TLR4 stimulation could influence the phenotype of the responding T cells. To elaborate on this observation, a more detailed analysis of T cell kinetics in the blood of mice injected with -GalCer and MPL was conducted (Fig. 3A). The results of these experiments showed that clonal expansion induced with MPL in combination with -GalCer peaked at day 6, and was reduced to levels below -GalCer alone by day 13. In contrast, T cell responses induced in the presence of -GalCer alone peaked at day 22, and decayed more slowly than when combined with MPL.

View larger version (23K): [in this window] [in a new window]   FIGURE 3. T cell responses induced in the presence of -GalCer adopt different kinetics and phenotypic profiles to those induced in the presence of -GalCer and MPL. Mice (n = 6) were injected with OVA and -GalCer, plus or minus indicated doses of MPL. A, The kinetics of OVA257–264-specific CD8+ T cell responses in the blood were monitored in animals injected with OVA and -GalCer ± 25 µg of MPL using H-2Kb/OVA257–264 tetramers. Mean proportions of tetramer+ cells as a percentage of CD8+ cells (±SEM) for each group are shown. B, Mice (n = 5) were injected with OVA and -GalCer ± 25 µg of MPL. After 6 days, expression of CD62L and CD127 was assessed on gated OVA257–264-specific CD8+ T cell responses detected in the blood. Mean percentage of tetramer+ CD8+ cells with effector (CD127low, CD62Llow), peripheral effector memory (CD127high, CD62Llow), or central memory (CD127high, CD62Lhigh) phenotype (±SEM) for each group is shown. C, Cytolytic activity of induced OVA257–264-specific responses was assessed in vivo on days 6 and 31 after administration of OVA and the indicated ligands. Cytotoxicity was assessed against CFSE-labeled splenocytes loaded with 5, 50, and 500 nM OVA257–264 peptide, as indicated, and a control population without peptide labeled with CMTMR. Analysis of Ag-specific lysis was calculated after 20 h using samples isolated from lymph nodes, with percent specific lysis calculated as the mean proportion of Ag-loaded cells depleted relative to control populations without Ag. D, Immune responses in mice primed with OVA and -GalCer ± 25 µg of MPL were restimulated by injection of vacc-OVA on day 15. OVA257–264-specific CD8+ T cell responses were measured in the blood 7 days later.

The cytotoxic capacity of T cells induced in the presence of TLR and iNKT cell stimulation was analyzed. Animals that had established OVA-specific CD8⁺ T cell responses resulting from injection of OVA in the presence of -GalCer, MPL, or both were injected with CFSE-labeled splenocytes loaded with OVA_257–264 peptide to serve as targets. A peptide-negative population labeled with CMTMR was included. Specific lysis of the peptide-loaded target populations was assessed relative to the control population 20 h after administration. Injection of OVA with the combination of -GalCer and MPL provided greater cytotoxic activity than either agent alone on day 8 (Fig. 3C), with cytolytic activity in this group saturating this assay at the highest peptide concentration. In contrast, analysis at day 31 showed less cytotoxicity in this group relative to animals treated with OVA and -GalCer alone. These kinetics of cytotoxic capacity reflect the observed kinetics of T cell accumulation in the blood shown in Fig. 3A.

We next investigated whether priming in the presence of an iNKT cell agonist, TLR ligands, or both had an impact on the capacity of T cells to be restimulated in vivo. Animals with established OVA-specific CD8⁺ T cell responses resulting from injection of OVA in the presence of -GalCer, MPL, or both were injected with vacc-OVA (Fig. 3D). This regime resulted in a significant boost to OVA-specific responses indicated by an increase in the percentage of OVA-specific CD8⁺ T cells in the blood. CD8⁺ T cell responses after boosting were comparable between animals primed with -GalCer and those primed with -GalCer/MPL. These data suggest that whereas the primary CD8⁺ T cell responses induced in the presence of -GalCer and MPL may be shorter lived than those induced with either ligand alone, the combination treatment does not impair the capacity of these responses to be restimulated.

Overall, the previous experiments demonstrate that the combination of iNKT cell stimulation and TLR-4-mediated signaling preferentially drives expansion of a population of short-lived T_E cells with cytotoxic function. This vaccination protocol also induces T cells with restimulatory capacity similar to those induced in the presence of -GalCer or MPL alone.

Cooperation between TLR stimulation and activation of iNKT cells is not restricted to TLR4

We investigated whether cooperation between iNKT cell-mediated signals and TLR4 stimulation can be extended to other TLRs. To this end, -GalCer was injected i.v. into mice with one of the following TLR ligands: poly(I:C) (TLR3), unmethylated CpG (TLR9), or flagellin (TLR5). DC maturation was examined in the spleen after 20 h (Fig. 4A). Administration of -GalCer or TLR ligand alone induced DC maturation, as defined by an increase in surface expression of the costimulatory molecule CD86 on CD11c⁺ cells. Increased expression of CD80, CD40, and MHC class I molecules was also observed (data not shown). When -GalCer was injected in combination with any one of the TLR ligands, the levels of CD86 induced were enhanced over those observed for animals treated with either agent alone. A hierarchy of responses was seen, with the most potent induction of CD86 on DC from animals treated with -GalCer and flagellin, whereas poly(I:C) was more potent than either CpG or MPL in combination with -GalCer. Similar maturation responses were observed with splenic B cells (data not shown).

View larger version (23K): [in this window] [in a new window]   FIGURE 4. Coinjection of -GalCer and a range of TLR ligands enhances maturation of splenic DC and increases their immunostimulatory capacity in vitro. A, Animals were injected i.v. with 1 µg of -GalCer or vehicle solution, 25 µg of TLR4 ligand MPL, 50 µg of TLR9 ligand CpG, 2.5 µg of TLR5 ligand flagellin, or 2.5 µg of TLR3 ligand poly(I:C). Surface expression of CD86 was assessed on splenic CD11c+ cells 20 h later. Mean fluorescence index values are shown. B, Splenic DC isolated from animals treated with the different ligands indicated were loaded with Flu-NP366–374 peptide, and used to stimulate CFSE-labeled Flu-NP-TCRtg splenocytes in vitro. Proliferation was determined by FACS analysis after 72 h of culture. Representative FACS profiles from one of three animals per group are shown.

To extend this observation, -GalCer was combined with different TLR ligands to assess their impact upon T cell induction in vivo (Fig. 5). The concentration of TLR ligands used was calculated on the basis of titration experiments with the dose selected inducing one half-maximal CD86 expression on CD11c⁺ cells in the spleen (data not shown). Combinations of -GalCer and TLR ligand were injected i.v. with OVA, and OVA-specific CD8⁺ T cell responses were assessed in the blood 6 days later. When OVA was injected with -GalCer together with any of the TLR ligands, the response was greater than with -GalCer or the TLR ligand alone, regardless of the TLR ligand used. In the case of flagellin and poly(I:C), the combination with -GalCer on CD8⁺ T cell induction was additive at the concentrations tested. With CpG, as with MPL (see Fig. 1B), responses were greater than additive, implying a synergistic interaction of signals mediated via iNKT cells and TLR.

View larger version (26K): [in this window] [in a new window]   FIGURE 5. Injection of -GalCer together with a TLR ligand enhances immune responses to coadministered protein Ag in vivo. OVA257–264-specific CD8+ T cell responses in recipient animals were assessed as in Fig. 1B, after administration of OVA with the indicated ligands at the concentrations as used in Fig. 4.

Stimulation of DC with microbial signals increases expression of IL-12p40, whereas IL-12p70 is only released following engagement of CD40 on the DC (30). Because iNKT cells are a possible source of CD40L, we investigated whether IL-12 release was affected by the use of -GalCer with TLR stimulation (Fig. 6A). Injection of -GalCer or TLR ligands induced the release of IL-12p40 into the serum with the highest levels observed in mice treated with CpG. However, combining -GalCer with each TLR ligand did not further increase levels of IL-12p40 observed with injection of the TLR ligand alone (data not shown). In contrast, a quite different pattern emerged from analysis of IL-12p70. The majority of TLR ligands did not induce significant IL-12p70 release when administered alone, with the exception of CpG. However, when combined with -GalCer, each of the TLR ligands induced IL-12p70 release. The most dramatic was CpG, with the combination of -GalCer and CpG increasing the output of IL-12p70 by 9-fold. Interestingly, with the exception of CpG, the IL-12p70 released in the combination treatment was lower than with -GalCer alone. Analysis of the IL-12p70 produced with -GalCer and MPL at different times suggested that this was a reduction in the magnitude of the response, and not simply a change in kinetics (data not shown). Thus, the impact of the combination of iNKT cell and TLR stimulation upon IL-12 release was variable, but generally served to increase levels of bioactive IL-12 in the serum over TLR stimulation alone.

View larger version (16K): [in this window] [in a new window]   FIGURE 6. Coinjection of -GalCer with a TLR ligand increases the percentage of Ag-specific CD8+ T cells that can secrete IFN- upon stimulation. A, Serum IL-12p70 levels were assessed by ELISA 6 h after i.v. administration of -GalCer and/or the indicated TLR ligand at the concentrations as used in Fig. 4. B, IFN--producing, OVA257–264-specific CD8+ T cell responses in recipient animals were assessed in the spleen of recipient mice by intracellular cytokine staining following a 5-h incubation with OVA257–264 and brefeldin A. Splenocytes were analyzed 6 days after immunization with OVA with vehicle, 1 µg of -GalCer, 25 µg of MPL, 50 µg of CpG, or a combination of -GalCer and TLR ligand. Splenocytes from the same mice were analyzed by FACS using H-2Kb/OVA257–264 tetramer. Mean proportions of tetramer+ (sum of gray and black) or IFN-+ (black) cells are shown as a percentage of CD8+ cells (±SEM) for each group (n = 5). Numbers in parentheses indicate the average number of IFN--producing CD8+ lymphocytes as a percentage of the average number of H-2Kb/OVA257–264 tetramer+ lymphocytes.

Enhanced maturation of human DC in vitro by TLR stimulation and activation of iNKT cells

We investigated whether cooperation between iNKT cell-mediated signals and TLR4 stimulation is a generalizable phenomenon observed across species. A human iNKT cell line was combined with DC cultured from healthy donors, and maturation of DC and cytokine release was assessed 40 h after addition of -GalCer and MPL. DC maturation was as assessed by analyzing expression of CD38, CD80, CD86, and CD83 (Fig. 7). When -GalCer was added, activation of iNKT cells was observed (as defined by down-regulation of TCR and secretion of IFN-; data not shown), and significant maturation of DC was induced. Notably, when - GalCer and MPL were added in combination, DC maturation was greater than that observed with -GalCer or MPL alone. This provides evidence that human DC phenotype can be modulated through cooperative actions of TLR4 ligand and iNKT cells.

View larger version (23K): [in this window] [in a new window]   FIGURE 7. Cooperation between TLR stimulation and activation of iNKT cells leads to maturation of human DC in vitro. A human iNKT cell line was combined with cultured human DC in the presence of the indicated stimuli. FACS profiles show the expression of indicated surface maturation markers after 40-h incubation: light gray profiles refer to DC alone plus or minus MPL; dark gray profiles are DC and NKT cells plus or minus MPL; black profiles are DC, NKT, and -GalCer plus or minus MPL.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

Enhanced DC maturation

For in vitro studies, DC isolated from mice treated with different combinations of ligands were fixed to prevent spontaneous maturation in culture. Therefore, the increased stimulatory capacity of DC from animals given -GalCer and TLR ligand must be attributed to differences in cell surface interactions, rather than soluble mediators. CD80 and CD86 were expressed at higher levels and are likely to contribute to the stronger proliferative responses and increased cytokine induction of the responder T cells. This does not rule out the possibility that soluble mediators are involved in the enhanced responses observed in vivo. In fact, analysis of IL-12 release in vivo indicated cooperation between iNKT cell stimulation and TLR stimulation at the level of IL-12p70 production, but not IL-12p40 production.

With the exception of CpG, TLR stimulation alone was insufficient to induce measurable levels of IL-12p70 in the serum. For these TLR ligands, combination with iNKT cell stimulation was required before measurable IL-12p70 release was observed. CpG-induced levels of IL-12p70, in contrast, were significant in the absence of iNKT cell stimulation, but then further enhanced when combined with -GalCer. This variability in IL-12 responses supports the proposition that DC possess significant plasticity in their cytokine responses that is influenced by pattern recognition (31). It has been reported that IL-12p70 requires a CD40 signal (30). In the system studied in this work, activated iNKT cells induced release of IL-12p70, whereas TLRs influenced the levels produced. CD40 signaling is critical to the release of IL-12p70 in this system, because no IL-12p70 was detected in -GalCer-treated CD40L-deficient animals (data not shown). Thus, one of the key signals provided by activated iNKT cells to DC is likely to be CD40L.

Enhanced T and B cell responses

Our data show that iNKT cell-dependent DC maturation is enhanced by the cooperative action of microbial stimuli mediated by TLR on the DC in both murine and human systems. The increased costimulatory capacity correlated with the induction of more potent T cell responses both in vitro and in vivo.

Following administration of OVA with -GalCer and MPL, the proportion of Ag-specific CD8⁺ T cells in the blood on days 6–7 was greater than with either ligand alone. The increased CD8⁺ T cell frequency was reflected in increased cytotoxicity at this time point. The proportions of tetramer-positive cells generated in the presence of -GalCer and MPL rapidly declined in the blood after day 7, whereas those generated in the presence of -GalCer peaked after day 20. The Ag-specific T cell populations detected in the blood had a larger proportion of cells with an effector phenotype (T_E) when induced in the presence of both iNKT cell and TLR4 stimulation. Such cells, which do not express the lymph node homing receptor CD62L, exhibit immediate effector function, but have limited in vivo survival (29). In contrast, the levels of T_CM induced in the presence of -GalCer were not affected by the combination treatment. It has been shown that T_CM have a greater capacity for in vivo restimulation, and it is argued that these cells are likely to be efficient in mediating long lasting protective immunity (32). Consequently, the ratio of subsets of memory T cells generated by vaccination may determine the efficacy of vaccine-driven immunity. Our results suggest that adjusting the balance of TLR to iNKT cell stimulation may be a means of increasing the pool of vaccine-induced immediate effector cells, while maintaining effective restimulatory capacity.

It is not clear how the combined adjuvant effect of MPL and -GalCer treatment results in more T_E than iNKT stimulation alone, nor why the expansion in numbers of T cells with this treatment is short-lived. It is possible that the increased costimulatory capacity of DC drives an increased proliferative burst of T cells with limited longevity. Provision of IL-12 at priming is closely associated with improved intrinsic survival properties of memory CD8⁺ T cells (33), although this finding was not reflected by vaccination of IL-12-sufficient vs IL-12-deficient animals (34). Our experimental system also showed no correlation between levels of IL-12 produced and longevity of the CTL response. The kinetics of CTL response following treatment with the combination of iNKT cell agonist and CpG were similar to the kinetics of CTL response seen with -GalCer and MPL, despite significant differences in IL-12p70 resulting from these different treatments (data not shown). It should be noted that other members of the IL-12 family of cytokines, including IL-23 and IL-27, whose release may also be differentially influenced by TLR and iNKT cell stimulation, may affect the phenotype of the induced T cells (35).

We extended these results to B cell responses by demonstrating that injection of -GalCer in combination with MPL results in high OVA-specific IgG responses. These results were further extended by showing an enhanced expression of CD86 on B cells, as compared with injection of -GalCer or MPL alone (data not shown). Although it remains to be established whether iNKT cell-dependent activation of B cells requires a direct interaction between iNKT and B cells, these results underscore important clinical implications of combining TLR stimulation with iNKT cell activation to elicit elevated Ab titers specific to subunit vaccines.

Cross-talk between human DC and human iNKT cells

We have combined human DC and human iNKT cells in vitro and shown that the levels of costimulatory molecules are dramatically enhanced in the presence of both -GalCer and MPL compared with either stimulus alone (Fig. 7). The levels of IL-12p70 detected were also significantly elevated (M. Salio and V. Cerundolo, manuscript in preparation). These data suggest that a cooperative action of TLR ligands and iNKT cells on DC function is a generalizable phenomenon that applies across species. It may be possible to exploit this interaction in the design of vaccines for human disorders. In this context, the ability to specifically target iNKT cells with -GalCer has already been shown in patients (36, 37, 38). Recent developments in biotechnology have made possible the large-scale production of recombinant proteins and peptides encoding clinically relevant Ags for preventative and therapeutic vaccines. Preclinical data suggest that -GalCer may be a useful adjuvant to improve potency of such subunit vaccines (8, 9, 17, 39). It remains to be shown whether the adjuvant properties ascribed to this ligand can be replicated in patients. On the basis of data presented in this study, we suggest that administration of a TLR ligand together with -GalCer may represent a potentially flexible adjuvant combination that, when used together with subunit vaccines, may improve T cell and Ab responses. It may also be possible to tailor the phenotype of responses induced through selection of an appropriate TLR ligand or by using analogues of -GalCer that invoke qualitatively different responses from iNKT cells (40 ). Structural analyses of CD1d molecules loaded with iNKT cell ligands (41, 42) may facilitate the process of rational optimization of -GalCer analogues for such fine-tuning of iNKT cell activation in vivo (43, 44).

	Acknowledgments

 
We thank the personnel of the Biomedical Services Unit of John Radcliffe Hospital for animal husbandry, and Kirin Breweries for providing -GalCer. We are very grateful to Thomas Moran and Bruno Moltedo for the supply of endotoxin-free OVA.

	Disclosures

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
The authors have no financial conflict of interest.

	Footnotes

 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ This work was funded by Cancer Research U.K. (Programme Grant C399-A2291), Harry Mahon Cancer Research Trust, Medical Research Council and FP6 project DC-VACC (LSHB-CT-2003-503037) (to V.C.), and a New Zealand Health Research Council Sir Charles Hercus Fellowship (to I.F.H.).

² I.F.H., J.D.S., and U.G. contributed equally to this work.

³ Current address: Malaghan Institute of Medical Research, P.O. Box 7060, Wellington, New Zealand.

⁴ Address correspondence and reprint requests to Dr. Vincenzo Cerundolo, Tumour Immunology Unit, Weatherall Institute of Molecular Medicine, John Radcliffe Hospital, Oxford OX3 9DS, U.K. E-mail address: vincenzo.cerundolo{at}imm.ox.ac.uk

⁵ Abbreviations used in this paper: DC, dendritic cell; -GalCer, -galactosylceramide; CMTMR, chloromethyl-benzoyl-aminotetramethyl-rhodamine; Flu-NP, influenza nucleoprotein; iNKT, invariant NKT; MPL, monophosphoryl lipid A; poly(I:C), polyinosinic polycytidylic acid; T_CM, central memory T cell; T_E, effector T cell; tg, transgenic; vacc-OVA, recombinant vaccinia virus encoding full-length chicken OVA.

Received for publication June 16, 2006. Accepted for publication December 13, 2006.

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