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International Immunology Advance Access originally published online on October 31, 2006
International Immunology 2006 18(12):1801-1813; doi:10.1093/intimm/dxl114
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© The Japanese Society for Immunology. 2006. All rights reserved. For permissions, please e-mail: journals.permissions@oxfordjournals.org

Lipid-containing mimetics of natural triggers of innate immunity as CTL-inducing influenza vaccines

Yuk Fai Lau1, Georgia Deliyannis1,2, Weiguang Zeng1, Ashley Mansell3, David C. Jackson1,2,* and Lorena E. Brown1,*

1 Cooperative Research Centre for Vaccine Technology, Department of Microbiology and Immunology, University of Melbourne, Victoria 3010, Australia
2 VacTX Pty. Ltd, Level 1, 123 Camberwell Road, Hawthorn East, Victoria 3123, Australia
3 Centre for Functional Genomics and Human Disease, Monash Institute of Reproduction and Development, Victoria 3165, Australia

Correspondence to: L. E. Brown; E-mail: lorena{at}unimelb.edu.au.


   Abstract
Top
 Abstract
Introduction
 Methods
 Results
 Discussion
 References
 
Anti-viral CD8+ T cell responses can be induced using synthetic lipopeptides and a range of different lipid moieties have been examined in a variety of model systems and in man for this purpose. Nevertheless, only limited data exist on comparative efficacy of different lipopeptides in a single model of protection so that the optimal composition for vaccination purposes remains unknown. In this study, we examined different lipid structures from bacterial or non-bacterial sources coupled to peptides representing influenza viral epitopes recognized by CD8+ and CD4+ T cells. These were assessed in the context of intra-nasal (i.n.) immunization in the absence of added adjuvant. The strongest immunogens were those containing bacterially derived lipids that induced dendritic cell (DC) maturation via Toll-like receptor 2 (TLR2) binding. The number of DCs induced to mature in vitro was directly associated with the strength of the CD8+ T cell-mediated viral clearing responses in primed mice. Mice immunized with the TLR2-binding lipopeptides showed greatly enhanced numbers of specific IFN-{gamma}-secreting CD8+ T cells at the site of infection after i.n. exposure to virus, which resulted in enhanced protection of the pneumonic lung. Importantly, lipopeptide-pulsed DCs were able to induce the appropriate T cells, indicating that the self-adjuvanting effects could occur in the absence of free lipopeptide interacting with additional TLR2-bearing cells in vivo. This study defines a hierarchy of lipopeptide constructs that can program DC to prime memory CD8+ T cells that on recall function to clear influenza virus from the infected lung.

Keywords: dendritic cells, lipids, lung, peptide, Toll-like receptors, virus


   Introduction
Top
 Abstract
 Introduction
Methods
 Results
 Discussion
 References
 
In the current context, a vaccine that will afford some degree of protection against the emergence of new subtypes of influenza is urgently required. Such a vaccine, if given prior to any knowledge of the emerging influenza strain, will need to target features in common with all influenza A viruses and most likely act to induce cross-reactive CD8+ T cells that recognize the internal conserved proteins of the virus. Influenza vaccines capable of inducing potent CD8+ T cell responses are yet to be developed. Inactivated influenza vaccines rely predominantly on the induction of neutralizing antibody against the surface glycoproteins of the virus subtypes currently circulating in the human population and have to be updated each year to cope with the antigenic variation occurring in these molecules; they are not expected to provide protection against a new subtype of influenza entering the human population. While live cold-adapted influenza vaccines (1, 2) have the potential to induce CD8+ T cells, the potency of these responses may be limited by the highly attenuated nature of these viruses. In a situation where more conventional experimental vaccines to induce strong CD8+ T cell responses have yet to show any potential in this area, we have explored the utility of lipopeptides for this purpose.

We have previously shown (3) that in order to generate long-lived memory CD8+ T cells for enhanced clearance of influenza, a peptide-based immunogen must have two features in addition to an epitope for CD8+ T cell recognition. One of these is a CD4+ T cell epitope for co-induction of Th, a finding supported by several other studies using infectious agents (4–6). This in itself, however, is not sufficient for memory cell development; although CD8+ T cells to a synthetic epitope from influenza virus were induced either in the presence of CD4+ T cells responding to a covalently linked CD4+ T cell epitope or when the CD8+ T cell epitope was delivered in CFA, the resulting CD8+ memory T cell response decayed at a rapid rate in both cases. The memory response was maintained, however, when elicited by immunogens that had, in addition to epitopes for CD4+ and CD8+ T cells, a covalently attached lipid moiety (3).

Attachment of lipid groups to synthetic immunogens has been used by us (3, 7–9) and others (reviewed in 10, 11) as a strategy to improve the immunogenicity of peptides, and the resulting lipopeptides have many features desirable for vaccination purposes. Foremost is the capacity to induce immunity in the absence of additional adjuvants, which not only increases their practicability for human use but also provides the added benefit of making them suitable for delivery by a variety of non-parenteral routes (7, 8, 12). Interaction of dendritic cells (DCs) with lipopeptides also confers on these cells the ability to provide prolonged presentation of the peptide epitopes to the immune system (13) which can be explained by the property of certain lipopeptides to drive DC maturation (8, 9, 14, 15).

The capacity of the DC to prime naive T cells is dependent on this maturation process, characterized by up-regulation of MHC and co-stimulatory molecules on the DC surface and migration of the DC, carrying its processed pathogen-derived antigens, to the regional lymph node where it can be accessed by a concentrated pool of T cells (16). A trigger for the maturation process is the interaction of microbial products with pattern-recognition receptors (PRRs) expressed by the DC (reviewed in 17). One such class of PRR is the Toll-like receptor (TLR) family of molecules, the engagement of which leads to nuclear factor {kappa}B (NF{kappa}B)-mediated signalling to the DC for the modulation of genes involved in the maturation process. Ligands specific for these receptors have been identified, and some are found to be lipids that act as anchoring structures of components of bacterial cell walls (17). For example, tripalmitoyl-S-glycerylcysteine (Pam3Cys, Pam3C or P3C), which is the synthetic version of a lipid component of Braun's lipoprotein located between the inner and outer membranes of Gram-negative bacteria, is capable of inducing the maturation of DC (18) and acts through interaction with TLR2 (19–21).

We have previously shown that peptides comprising a CD4+ T cell epitope and a CD8+ T cell epitope from influenza in a tandem linear arrangement, with two palmitic acids attached via an N-terminal lysine residue, were powerful inducers of CD8+ T cell-mediated influenza viral clearing responses when delivered by the subcutaneous route (3). However, preliminary studies showed these to be less effective by the intra-nasal route. Another study (9) showed that branched lipopeptides comprising these same epitopes but with a different lipid moiety, S-[2,3-bis(palmitoyloxy)propyl]cysteine or Pam2Cys, induced strong T cell immunity when delivered intra-nasally (i.n.), implying that the environment of the respiratory tract may have unique requirements for optimal T cell priming. As the intra-nasal route may be useful for mass delivery of vaccines, we decided to investigate the ability of different lipid-linked T cell epitope-based peptide immunogens to induce memory responses when delivered by this route for accelerated CD8+ T cell-mediated viral clearance from the influenza virus-infected lung. We have used lipid moieties that are known to stimulate DC through TLR2, as well as others whose DC stimulatory capacity is unknown. The aim is not only to define which lipopeptides function best at priming CD8+ T cell responses in the respiratory tract but also to understand what are the properties of immunogens that possess this function.


   Methods
Top
 Abstract
 Introduction
 Methods
Results
 Discussion
 References
 
Preparation of synthetic peptides and lipopeptides
A panel of immunogens were synthesized based on peptides representing a minimal epitope for CD8+ T cells and an epitope for CD4+ T cells, both from influenza virus. The peptide NP (147–155), referred to as CTL peptide, has the sequence TYQRTRALV from the nucleoprotein of A/Puerto Rico/8/34 virus (H1N1) and is the dominant CD8+ T cell epitope recognized by BALB/c mice in all type A influenza strains (22). The peptide HA2 (166–180), referred to as Th peptide, has the sequence ALNNRFQIKGVELKS and is an epitope present in the HA2 chain of A/Memphis/1/71 (H3N2) influenza haemagglutinin that elicits CD4+ T cells that are cross-reactive with all viruses of the H3 subtype (23). The immunogens used were based on a peptide comprising these two epitope sequences synthesized in tandem and were either used unmodified or modified by the addition of different lipid moieties.

The synthetic immunogens were assembled by conventional solid-phase methodology using Fmoc chemistry throughout (24). Lipopeptides were constructed as described (3, 8). A schematic diagram of each of the structures is shown in Fig. 1.


Figure 1
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  		Fig. 1 Representation of the peptide constructs used in this study. Peptide structures consisted of the I-Ed-restricted CD4+ helper T cell epitope (Th) representing residues 166–180 of the HA2 chain of influenza HA and an immunodominant H-2d-restricted CD8+ T cell epitope (CTL) derived from residues 147–155 of the viral nucleoprotein. These were either assembled as tandem linear sequences [Th]-K-[CTL] or as branched structures where a lipid moiety (PamXCys, with the structure shown) was attached through the {epsilon}-amino group of a lysine residue, K, situated between the two epitopes. In these constructs, two serine (Ser) residues (SS) were added between the peptide and lipid moiety. In one case, [Th]-K(Chol2KSS)-[CTL], two residues of cholesterol were attached to a terminal lysine coupled to the SS spacer. In another case, Pal2-KK[Th]-[CTL], two palmitic acid residues were attached to the {alpha}- and {epsilon}-amino groups of the N-terminal lysine residue and Th was attached to the {epsilon}-amino group of the penultimate lysine in the sequence.

 
Cell culture medium and buffers
T cell medium (TCM) consisted of RPMI-1640 without glutamine (JRH Biosciences), supplemented with 10% (v/v) heat-inactivated FCS (JRH Biosciences), penicillin (100 IU ml–1), streptomycin (180 µg ml–1), gentamicin (24 µg ml–1), glutamine (2 mM), sodium pyruvate (2 mM) and 0.1 mM 2-mercaptoethanol. DC medium consisted of Iscove's modified Dulbecco's medium containing 25 mM HEPES (JRH Biosciences), and additives as for TCM. It was further supplemented for DC culture with 30% conditioned supernatant from cultured NIH/3T3 cells and 5% supernatant from the plasmocytoma cell line X63-Ag8 transfected with the mouse gene for granulocyte macrophage colony-stimulating factor (25).

Mice and immunization protocol
The research complies with the University of Melbourne's Animal Experimentation Ethics guidelines and policies. Inbred 6- to 8-week old female BALB/c mice (H-2d) were bred in the Animal Facility of the Department of Microbiology and Immunology, University of Melbourne, under specific pathogen-free conditions. Mice were anaesthetized with penthrane and inoculated i.n. with synthetic immunogen in 50 µl of PBS.

Challenge protocol and assay of virus in lungs
Penthrane-anaesthetized mice were challenged i.n. with 104.5 plaque-forming units of infectious re-assortant influenza virus A/Memphis/1/71 (H3N2) x A/Bellamy/42 (H1N1), referred to as Mem71 (H3N1). After 5 days, supernatants of lung homogenates were prepared (3) and assayed for infectious virus by plaque formation (26).

Preparation of lymphocytes from broncheoalveolar lavage and lungs
The inflammatory cells in the airway were recovered following cervical dislocation of mice. The trachea was exposed and an incision was made near the larynx. A plastic cannula with a 1-ml syringe attached was inserted through the incision. The respiratory tract was then washed with HBSS containing 1% BSA using three separate 1-ml aliquots, each being infused and withdrawn three times. The cells were incubated in plastic petri dishes (Greiner-Bio One GmbH, Frickenhausen, Germany) for 60 min at 37°C to remove adherent cells.

The lungs were finely minced and treated with 4 mg collagenase A (from Clostridium histolyticum, Roche Diagnostics GmbH, Mannheim, Germany) in 2 ml RPMI per lung for 30 min at 37°C. The material was passed through wire mesh and pelleted by centrifugation. The pelleted cells were treated with 0.15 M NH4Cl in 17 mM Tris–HCl at pH 7.2 for 5 min at 37°C to lyse erythrocytes and incubated in plastic petri dishes as above.

Tetramer staining of peptide-specific CD8+ T cells
CD8+ T cells specific for the CTL peptide were identified using tetrameric complexes of the H-2Kd glycoprotein with bound CTL peptide. The class I/peptide in monomeric form was a gift from Stephen Turner and Peter Doherty, University of Melbourne. Tetramers were made by incubating the monomer with streptavidin–PE (Molecular Probes, Eugene, OR, USA) at a 4:1 molar ratio.

Lymphocytes from the lungs were first treated with 20 µl of normal mouse serum (NMS) for 5 min at room temperature and then stained for 60 min with the tetrameric complexes. This was followed by staining with rat anti-mouse CD8{alpha} (53-6.7) conjugated with allophycocyanin (BD Biosciences PharMingen, San Diego, CA, USA) for 30 min on ice. Cells were washed twice and analyzed by flow cytometry. Data were collected on a FACSort flow cytometer using CellQuest software (BD Biosciences Immunocytometry Systems, San Jose, CA, USA). Further analysis was performed with FlowJo software (Tree Star Inc., CA, USA).

Intracellular cytokine production assay for IFN-{gamma}-secreting cells
Lymphocytes (106) from the lungs were cultured for 6 h in 96-well round-bottom plates (Nalge Nunc International, Rochester, NY, USA) in 200 µl of TCM containing 50 IU of human recombinant IL-2 (Roche Diagnostics GmbH) and 1 µl of Golgi plug, in the presence or absence of 1 µM CTL peptide. Peptide-specific IFN-{gamma}-secreting cells were detected by an intracellular cytokine production assay using the cytofix/cytoperm kit and antibodies from BD Biosciences PharMingen, first staining with PE-conjugated rat anti-mouse CD8{alpha} mAb (53-6.7), and after permeabilization, with FITC-conjugated rat anti-mouse IFN-{gamma} mAb (clone XMG 1.2). Cells were analyzed by flow cytometry.

Assay of D1 cell maturation
Immature DCs derived from BALB/c splenocytes (D1 cells) were prepared in our laboratory by the method of Winzler et al. (27). To assess maturation, cells were seeded into TC24 plates (Nalge Nunc International) at 2 x 105 cells per well in 900 µl complete DC medium. Dilutions of lipopeptide in PBS were added to the cultures in 100 µl. LPS purified from Escherichia coli serotype O111:B4 (Difco, Detroit, MI, USA) at a final concentration of 5 µg ml–1 was used as a positive control for maturation. After overnight incubation, the cells were harvested and washed once with 1% FCS in PBS. To prevent non-specific binding to Fc-RII/III, the cells were pre-incubated with 20 µl of NMS for 5 min at room temperature. Cells were then stained for 30 min on ice with 2.5 µg ml–1 FITC-conjugated mAb 14-4-4S [IgG2a, (28)], which is specific for I-Ek,d, or with mAb 36/1, specific for the haemagglutinin of influenza virus (29), as an isotype control. Samples were fixed with 2% PFA for 15 min on ice prior to flow cytometric analysis.

NF{kappa}B reporter gene assay
Cells of the human embryonic epithelial kidney cell line HEK293 were transiently transfected as described (30, 31) with 100 ng of an NF{kappa}B luciferase reporter gene (five NF{kappa}B sites upstream of luciferase), 70 ng of a ß-galactosidase-expressing plasmid and 5 ng of human TLR2-expressing plasmid using the FuGENE6 method (Roche Diagnostics, GmbH, Mannheim, Germany). The total amount of DNA (250 ng) was kept constant by supplementation with empty vector. Lipidated or non-lipidated peptides were added into the wells 24 h after transfection and lysates prepared 6 h after stimulation. Using enzyme assay kits (Promega Corporation, Madison, WI, USA), the luciferase and ß-galactosidase activities in the cell lysates were determined (31). The relative stimulation of NF{kappa}B activity was calculated by normalizing luciferase activity with ß-galactosidase activity.

Assay for IL-12
The amount of IL-12p40 released by D1 cells after overnight incubation with different lipopeptides was assayed by ELISA using anti-IL-12 mAbs (32) generously provided by Giorgio Trinchieri, Laboratory for Immunological Research, Schering-Plough Research Institute, Dardilly, France. IL-12 was captured from the supernatant on wells coated overnight with 50 µl of rat anti-mouse IL-12p40 mAb (clone C15.6) at 5 µg ml–1 of PBS. For detection of IL-12, 50 µl of biotin-conjugated rat anti-mouse IL-12p40 mAb (clone C17.8) at 2 µg ml–1 was used, followed, after washing, by 50 µl of HRP-conjugated streptavidin (Amrad Biotech, Victoria, Australia) diluted 1 in 400. The ELISA was carried out as previously described (33).

In vivo cytotoxicity assay
Analysis of CTL determinant-specific cytotoxicity in vivo was performed according to the method of Coles et al. (34). Targets were prepared from splenocytes of naive BALB/c mice that were pulsed with 9 µM CTL peptide at 37°C for 90 min, washed three times and labelled with a high concentration (3 µM) of 5-(and 6) carboxyfluorescein diacetate succinimidyl ester (CFSE; Molecular Probes). Antigen-specific lysis was controlled by co-injecting unpulsed syngeneic spleen cells that were labelled with a low concentration (0.5 µM) of CFSE. A mixture of 1.5 x 107 cells of each of these target cell populations was injected i.v. in 100 µl on day 4 after infection into pre-immunized mice and also into naive mice. The spleens were removed 16 h later and analyzed for the presence of CFSE-high and CFSE-low target cells by flow cytometry. A total of 1 x 106 lymphocytes were analyzed for each sample. The following formulae were used to calculate specific lysis: ratio = (percentage of CFSE low/percentage of CFSE high) and percentage of specific lysis = [1 – (ratio for unimmunized mice/ratio for immunized mice) x 100].

Statistical analysis
This was performed for the viral clearance studies using the non-parametric Mann–Whitney test, calculated using Prism version 4 (GraphPad Software Inc.).


   Results
Top
 Abstract
 Introduction
 Methods
 Results
Discussion
 References
 
Lipopeptides differ in their ability to mature DC and in the mechanism by which this occurs
A panel of lipopeptides were constructed, all of which had the same core structure comprising epitopes for CD4+ and CD8+ T cells derived from influenza virus, but different lipid moieties attached to the peptide backbone (Fig. 1). Their ability to induce DC maturation was assessed using spleen-derived D1 cells (Fig. 2). In unstimulated cultures, the majority of D1 cells displayed an immature phenotype, characterized by an intermediate surface expression of MHC class II molecules and low CD86 expression, while ~20% of the D1 cells expressed high levels of both markers, representing a population that has matured spontaneously. After overnight incubation with LPS, however, virtually all cells converted to the mature phenotype (Fig. 2A). When D1 cells were incubated with different concentrations of the lipopeptides, and the percentage of mature cells was determined (Fig. 2B), the data showed that, on an equimolar basis, not all lipopeptides had the same capacity to trigger maturation of the cells. The [Th]-K(Pam2CSS)-[CTL] lipopeptide was the most potent and showed 3- to 10-fold greater numbers of cells matured compared with the next most potent lipopeptide [Th]-K(Pam1CSS)-[CTL]. Both [Th]-K(Pam3CSS)-[CTL] and [Th]-K(Chol2KSS)-[CTL] had similar potencies and were ~10-fold less active again. Neither Pal2-KK[Th]-[CTL] nor the non-lipidated peptide could mature D1 cells at the concentrations tested.


Figure 2
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  		Fig. 2 Differential ability of lipopeptides to induce maturation of DCs and to signal through TLR2. (A) D1 cells were incubated overnight in the absence of stimulant (upper panels) or with LPS (+LPS; lower panels). Cells were examined for the level of surface expression of MHC class II (left panels) and CD86 (right panels) by flow cytometry to determine the numbers of mature DC that express high levels of these markers. (B) D1 cells were incubated overnight in different concentrations of the lipopeptides [Th]-K(Pam1CSS)-[CTL], closed squares; [Th]-K(Pam2CSS)-[CTL], closed triangles; [Th]-K(Pam3CSS)-[CTL], closed circles; Pal2-KK[Th]-[CTL], open circles; [Th]-K(Chol2KSS)-[CTL], open squares or the non-lipidated peptide [Th]-K-[CTL], open triangles, and then analyzed by flow cytometry as above. For each sample, 15 000 D1 cells were analyzed. Data are expressed as the percentage of D1 cells showing a mature phenotype. Each point represents the average of triplicate samples and the error bars are the SD. The broken lines indicate the level of spontaneous maturation of D1 cells incubated with medium alone. (C) The ability of various lipid moieties to signal via TLR2 was tested using HEK293 cells co-transfected with a TLR2-expressing plasmid and an NF{kappa}B luciferase reporter gene. Cells were seeded in 96-well cell culture plates and transfected 24 h later. Twenty-four hours after transfection, cells were exposed to 0.3 nmoles ml-1 of non-lipidated peptide or each of the different lipopeptides and 6 h later harvested into lysis buffer. Luciferase activity was then determined. Data are expressed as relative stimulation activity and were calculated from the mean and SD of triplicate samples. Data are representative of two separate experiments.

 
One mechanism by which lipid-containing moieties are known to trigger the maturation of DC is by their interaction with TLR2 or TLR4 (35). To determine whether the lipopeptides used in this study were capable of TLR4-dependent DC activation, D1 cells were prepared from C3H/HeJ mice. This strain bears a mutation in the TLR4 gene and consequently displays a phenotype of hyporesponsiveness to LPS, a bacterial cell wall component that signals DC via its lipid A portion through TLR4 (36). When the immunogens were titrated for their ability to induce C3H/HeJ D1 cell maturation, the profiles obtained were virtually indistinguishable from those obtained for BALB/c D1 cells examined in parallel (data not shown). This indicates that the DC maturation signal utilized by the immunogens is independent of TLR4.

To test for the ability to signal through TLR2, the lipopeptides, at a range of concentrations (0.03–1.4 nmoles ml–1), were incubated with HEK293 cells transfected with a TLR2-encoding plasmid. Signalling upon TLR2 engagement was measured by estimating luciferase produced by readout from an NF{kappa}B-dependent reporter gene construct. The results (Fig. 2C) for each of the lipopeptides (shown at 0.3 nmoles ml–1), indicate that [Th]-K(Pam1CSS)-[CTL], [Th]-K(Pam2CSS)-[CTL] and [Th]-K(Pam3CSS)-[CTL] could stimulate NF{kappa}B-dependent gene activation in TLR2 transfected cells. These lipopeptides did not stimulate cells lacking TLR2 (data not shown). The non-lipidated peptide and Pal2-KK[Th]-[CTL] failed to signal the cells, and the level of signalling mediated by [Th]-K(Chol2KSS)-[CTL] was very weak, even at the highest concentration tested (1.4 nmoles ml–1). These results indicate that DC maturation by the lipopeptides containing Pam1Cys, Pam2Cys or Pam3Cys was TLR2 dependent rather than TLR4 dependent and that Chol2, though able to induce DC maturation, did this by a mechanism distinct from the other lipopeptides and likely to be both TLR4 and TLR2 independent.

Lipopeptides differ in their ability to induce IL-12 production by D1 cells
In addition to the cell-to-cell interaction required for T cell priming, DC can also 'shape' the immune response through the release of soluble mediators. IL-12 is of particular interest because, apart from favouring the differentiation of precursor CD4+ T cells into Th1 effectors, it has also been implicated as playing a role in optimizing clonal expansion and differentiation of naive CD8+ T cells to effector CTL (37). The ability of the lipopeptides to induce the secretion of IL-12 by D1 cells was therefore tested. D1 cells were incubated overnight with the different lipopeptides, and the supernatants analyzed for the presence of IL-12p40 by ELISA. As shown in Fig. 3, supernatants from unstimulated D1 cells or from cells incubated with non-lipidated peptide contained low levels of IL-12; this was probably due to those spontaneously maturing D1 cells observed in the cultures. The lipopeptides containing Pam2Cys, Pam1Cys and Pam3Cys, despite displaying a range of DC maturing capacities (Fig. 2B), showed similar high levels of IL-12 production, comparable to those obtained following incubation with LPS. This indicates that the amount of IL-12 produced by D1 cells does not necessarily correlate with the number of mature DC in the cultures. Furthermore, the Chol2-containing lipopeptide, which showed the same level of DC maturation to that obtained with Pam3Cys-containing lipopeptide (Fig. 2B), produced 8-fold less IL-12, a level only slightly greater than the non-lipidated peptide. Our data therefore suggest that the physical properties of the lipid moieties dictate the biological activity of the DC and can determine the degree of maturation and, independently, the cytokine secretion. We next examined whether these observed differences had any impact on the immunity induced in response to the lipopeptides.


Figure 3
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  		Fig. 3 Lipopeptide-induced release of IL-12p40 by DC. D1 cells (1 x 106) were incubated overnight with 0.45 nmoles of different lipopeptides in 1 ml of DC medium. The supernatants were analyzed for the presence of IL-12p40 by ELISA. Cells incubated with medium alone were used to control for the release of IL-12p40 from spontaneously maturing D1 cells, and cells incubated with 5 µg ml-1 LPS were used as a positive control. DC culture medium (IMDM) alone was also included as a baseline for the assay.

 
Increased numbers of antigen-specific CD8+ T cells are present at the site of infection following viral challenge of lipopeptide-primed mice
The CD8+ T cell responses induced by the lipopeptides were quantified by staining of lung lymphocytes with MHC class I tetramers complexed with the CTL peptide. Mice primed i.n. with [Th]-K(Pam2CSS)-[CTL], which was the most potent inducer of DC maturation, showed only very few CTL peptide-specific CD8+ T cells present in the lungs 9 days after inoculation (0.8% of CD8+ T cells stained tetramer positive above background; Fig. 4A). However, if challenged with virus at that time, ~8% of the CD8+ T cells present in the lungs 5 days later were specific for the CTL peptide. This increase was not due to a primary immune response to infection because none of these cells could be detected as early as 5 days after infection in the lungs of unprimed mice. This indicates that the lipopeptide induced an effective pool of cells that were rapidly expanded or recruited to the lungs on subsequent encounter with virus.


Figure 4
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  		Fig. 4 Presence of CTL determinant-specific CD8+ T cells in the lungs of vaccinated mice after viral challenge. (A) Lung lymphocytes from a mouse primed with 9 nmoles of [Th]-K(Pam2CSS)-[CTL] but not challenged with virus compared with those from a mouse primed with the lipopeptide and then challenged with virus 9 days later. Lungs were collected 5 days after infection and CTL peptide-specific CD8+ T cells in the lungs enumerated by co-staining with anti-CD8{alpha} antibody and tetrameric MHC class I complexes loaded with the CTL peptide. Lung lymphocytes from an unprimed mouse 5 days after infection were included to show the number of tetramer+ CD8+ T cells induced by the challenge virus at that time point. The percentage of lymphocytes in each quadrant is indicated. The background staining of lung lymphocytes from naive mice showed 0.9% of the CD8+ T cells were tetramer positive (data not shown). (B) The total number of tetramer+ CD8+ T cells present in the lungs of mice inoculated i.n. with 9 nmoles of the indicated lipopeptide or the non-lipidated peptide or with PBS. Lungs were collected 5 days after infection and lymphocytes analyzed as above.

 
Using this protocol, the number of CTL peptide-specific CD8+ T cells present at 5 days after infection in the lungs of mice primed i.n. with the immunogens was determined (Fig. 4B). Similar numbers were found in mice immunized with [Th]-K-[CTL] or PBS, highlighting the poor immunogenicity of the non-lipidated peptide in the absence of adjuvant. Mice inoculated with the lipopeptides had enhanced numbers of CTL peptide-specific CD8+ T cells in their lungs at this time. [Th]-K(Pam2CSS)-[CTL]-primed and [Th]-K(Pam1CSS)-[CTL]-primed mice had the highest numbers (corresponding to ~9% of the CD8+ T cells) and few were observed with [Th]-K(Pam3CSS)-[CTL] and [Th]-K(Chol2KSS)-[CTL]. The Pal2-KK[Th]-[CTL] construct was relatively poor at CD8+ T cell induction under these conditions. The results show that mice primed with lipopeptides not only have an accelerated CTL peptide-specific CD8+ T cell response at the site of infection but also that the particular lipid group in the immunogen can influence the magnitude of this response to an extent that closely mirrored their abilities to mature DC.

Lipopeptide-induced CD8+ T cells are functionally active on recall even several months after priming
As CTL peptide-specific CD8+ T cells can be detected by tetramer staining in the lungs of lipopeptide-primed mice 5 days after being challenged with influenza virus, it was of interest to examine the kinetics of appearance of these cells and whether they have any effector function. Mice were immunized i.n. with 9 nmoles of [Th]-K(Pam2CSS)-[CTL] in PBS and challenged with virus 9 days later. At different times after infection, the CTL peptide-specific CD8+ T cells present in the broncheoalveolar lavage (BAL) and lung cells were identified by intracellular IFN-{gamma} production assay. Consistent with the data from the tetramer study (Fig. 4A), few specific CD8+ T cells were detected in the BAL and lungs of those mice that were inoculated i.n. with [Th]-K(Pam2CSS)-[CTL] but not challenged with virus (Fig. 5A). When these [Th]-K(Pam2CSS)-[CTL]-primed mice were exposed to virus, a small rise in specific CD8+ T cells was seen as early as day 3 but there was a very large increase between days 4 and 5 after infection at both sites. At day 5 after infection, ~10% of the CD8+ T cells in the BAL and lungs were specific for the CTL peptide, consistent with the results of tetramer staining experiments.


Figure 5
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  		Fig. 5 Accelerated influx of CTL epitope-specific CD8+ T cells into the lungs of lipopeptide-primed mice after virus challenge. (A) Mice were inoculated i.n. with 9 nmoles of [Th]-K(Pam2CSS)-[CTL] or PBS and challenged with influenza virus 9 days later. Lymphocytes from the BAL and lungs were collected at the indicated times following viral challenge. (B) Mice were inoculated with [Th]-K(Pam2CSS)-[CTL] as in (A) and challenged with virus at the indicated times after inoculation. Lymphocytes from the lungs were collected 5 days later. (C) Mice were inoculated i.n. with 9 nmoles of the indicated lipopeptides or PBS and challenged with influenza virus 28 days later. Lymphocytes recalled to the lungs were collected 5 days later. In each case, the CTL determinant-specific CD8+ T cells were enumerated by an intracellular IFN-{gamma} production assay. The numbers on top of the bars in (A) represent the percentages of the total CD8+ T cells that were CTL determinant-specific and IFN-{gamma} secreting. The bars and error bars in (C) represent the mean and SD of three mice per group. Ten thousand CD8+ T cells were analyzed for each sample.

 
In addition to challenging [Th]-K(Pam2CSS)-[CTL]-primed mice 9 days after i.n. inoculation, an experiment was performed in which mice were challenged 1 or 3 months after immunization to determine whether specific CD8+ T cell memory had been established. Lungs were collected 5 days after infection and analyzed by intracellular IFN-{gamma} production. As shown in Fig. 5(B), a strong recall response was detected as much as 3 months after initial priming.

A similar experiment compared the responses with the different lipopeptides. Mice were inoculated i.n. with lipopeptides and challenged with virus 28 days later. After 5 days, the number of memory CTL peptide-specific IFN-{gamma}-secreting CD8+ T cells present in the lungs after infection was determined. As shown in Fig. 5(C), few specific CD8+ T cells were found in the lungs of mice primed with [Th]-K-[CTL] compared with those immunized with lipopeptides; the [Th]-K(Pam2CSS)-[CTL]-primed group had the largest population of CTL peptide-specific CD8+ T cells in the lungs, followed by the [Th]-K(Pam1CSS)-[CTL]-primed group. Similar levels of CTL peptide-specific CD8+ T cells were found in mice primed with [Th]-K(Pam3CSS)-[CTL], [Th]-K(Chol2KSS)-[CTL] and Pal2-KK[Th]-[CTL]. As before, mice receiving only PBS showed no specific IFN-{gamma}-producing CD8+ T cells in their lungs at this time point after infection, indicating that the enhanced levels of specific T cells were a result of lipopeptide priming.

An in vivo cytotoxicity assay showed that these lipopeptide-induced T cells also had cytolytic function upon recall with virus as demonstrated by disappearance of fluorescently labelled target cells transferred into the immunized mice 4 days after challenge (Fig. 6). This assay, which is very sensitive, could not distinguish between the cytotoxicity induced by [Th]-K(Pam1CSS)-[CTL], [Th]-K(Pam2CSS)-[CTL] and [Th]-K(Pam3CSS)-[CTL] whereas diminished activity was observed in mice immunized with Pal2-KK[Th]-[CTL] and [Th]-K(Chol2KSS)-[CTL].


Figure 6
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  		Fig. 6 Accelerated in vivo cytotoxic T cell activity in lipopeptide-primed mice. Groups of three mice were primed i.n. with 9 nmoles of the indicated lipopeptides in PBS and challenged with virus 28 days later. CTL determinant-specific cytotoxicity was assessed in vivo using CFSE-labelled CTL-pulsed splenocytes from naive BALB/c mice co-injected with unpulsed spleen cells labelled with a lower concentration of CFSE. A mixture of 15 x 106 cells of each of these target cell populations was injected i.v. in 100 µl on day 4 after infection into the primed mice and also into naive mice. The spleens were removed 16 h later and analyzed for the presence of CFSE-high and CFSE-low target cells by flow cytometry. A total of 1 x 106 lymphocytes were analyzed for each sample. Specific lysis was calculated as described in Methods. (A) The profiles are shown for a mouse immunized with [Th]-K(Pam2CSS)-[CTL] and subsequently challenged with virus, in which the peptide-pulsed target population is significantly reduced compared with a naive animal that has received similar CFSE-labelled target cells. (B) Specific in vivo lysis observed with mice primed with 9 nmoles of the indicated lipopeptide, the non-lipidated peptide or with PBS and then challenged with virus 28 days later, 4 days prior to injection of CFSE-labelled targets. Each closed circle represents an individual mouse and the bars represent the mean for each group.

 
Lipopeptide-pulsed DC can efficiently induce specific CD8+ T cells
Overall, it appeared that the lipids that were best at inducing the maturation of DC were also the most effective at T cell induction. This suggested that the DC may play a central role in mediating the enhanced immunity observed with the lipopeptides; however, it did not rule out the possibility that activation of DC was a correlate for other activation events taking place in vivo in response to the lipopeptides which were more directly responsible for the immune enhancement. To assess whether the lipopeptide–DC interaction was responsible for enhanced T cell immunity, D1 cells were pulsed overnight with the [Th]-K(Pam2CSS)-[CTL] lipopeptide in vitro and then pelleted onto a Ficoll cushion which retains the cells and not the free lipopeptide. The cells were injected into naive mice by the intravenous route and 28 days later the mice were challenged with influenza virus. On day 5 after infection, the CTL epitope-specific CD8+ T cells in the lungs were enumerated by intracellular IFN-{gamma} production (Fig. 7). The number of specific CD8+ T cells induced by the lipopeptide-pulsed DC increased as the number of cells delivered increased until a plateau of 7% of the total CD8+ T cells in the lungs was reached on injection of 105 DC. Similar levels of specific CD8+ T cells could be obtained with D1 cells pulsed with the non-lipidated peptide but it required 10-fold more DCs to achieve this. It is likely that the population of spontaneously maturing D1 cells that contaminate the immature population contribute to the T cell-inducing potential of the non-lipidated peptide-pulsed DC. The results indicate that DC matured and loaded with lipopeptide was sufficient to induce the specific T cell population present in the lungs 5 days after recall by viral infection.


Figure 7
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  		Fig. 7 Lipopeptide-pulsed DCs are potent inducers of antigen-specific CD8+ T cell responses. D1 cells (106) were incubated with 9 nmoles of [Th]-K(Pam2CSS)-[CTL] or [Th]-K-[CTL] in DC culture media overnight. The cells were then recovered, washed and separated from unbound lipopeptide by centrifugation on a Ficoll cushion. Cells positioned at the interface were recovered and washed three times. Naive mice received the indicated number of lipopeptide or non-lipidated peptide-pulsed DCs intravenously. On day 28 after inoculation, the mice were challenged with influenza virus. Lungs were collected on day 5 after infection and CTL determinant-specific CD8+ T cells were enumerated by an intracellular IFN-{gamma} production assay and expressed as a percentage of total lung CD8+ T cells. The error bars represent SD of three mice per group. Mice inoculated with material collected at the cushion interface onto which lipopeptide in the absence of DC had been pelleted failed to elicit a response (data not shown).

 
Lipopeptides constructed with different lipid groups induce a range of viral clearing responses
To determine whether the observed differences in the magnitude of the lipopeptide-induced CD8+ T cell response translate directly into differences in viral clearing capacity, the titres of virus remaining in the lungs of lipopeptide-primed mice 5 days after challenge with influenza virus were determined. As the core peptide used in the immunogens does not elicit antibody in BALB/c mice, either with or without adjuvant (data not shown), neutralization of the virus due to vaccine-induced antibody-dependent mechanisms will not contribute to any reduction seen in pulmonary virus titres.

Initially, clearance mediated by T cells present during the primary effector phase of the response to the lipopeptides was studied (Fig. 8A). Mice were immunized i.n. with 45 nmoles of different lipopeptides in PBS, and 9 days later, the mice were challenged with influenza virus. All lipopeptides elicited strong viral clearing responses with at least 95% reduction in viral titre compared with the PBS group (the range of P-values, P = 0.008–0.016, indicated a significant difference to the control group for all lipidated immunogens). The most potent immunogen was the Pam2Cys-containing lipopeptide, which induced responses capable of a 99% reduction in pulmonary virus load. Virus titres in the lungs of non-lipidated peptide-primed mice were not significantly reduced (only 19%; P = 1.000) relative to the PBS group.


Figure 8
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  		Fig. 8 Induction and recall of pulmonary viral clearing responses in mice inoculated with synthetic immunogens. (A) Groups of five mice were inoculated i.n. with 45 nmoles of the specified lipopeptide immunogens or PBS and challenged 9 days later with virus. In addition, groups of five mice were immunized i.n. with 45 nmoles (B) or 9 nmoles (C) of the immunogens and challenged with virus 28 days later. Titres of infectious virus in lung homogenates sampled 5 days after challenge were determined by plaque formation. Each closed circle represents the lung virus titre of an individual mouse and the bars represent the geometric mean titre of the group of mice. The percentage reduction in mean viral titre relative to the PBS control group is shown beside each column of data.

 
To investigate the effectiveness of the memory responses, mice were inoculated with 45 nmoles (Fig. 8B) or 9 nmoles (Fig. 8C) of lipopeptide i.n. and challenged with virus 28 days later. The results for the higher dose (Fig. 8B) showed that lipopeptide-primed mice all had some long-term benefit of vaccination in contrast to those immunized with the non-lipidated peptide where the pulmonary viral titres were indistinguishable from those of the control group. Pam2Cys-, Pam1Cys- and Pam3Cys-containing lipopeptides induced memory T cell responses capable of at least 90% reduction of the challenge virus (P = 0.008 in each case). Mice inoculated with Chol2- and Pal2-containing lipopeptides showed weaker responses, with 84% (P = 0.008) and 78% (P = 0.159) reduction, respectively. At the lower dose (Fig. 8C), the viral clearing responses were reduced, except in the group immunized with [Th]-K(Pam2CSS)-[CTL], which still maintained a 93% reduction in pulmonary viral load (P = 0.008), indicating that Pam2Cys is the most potent of the lipid groups. The hierarchy for the effectiveness of the lipid moieties in conferring self-adjuvanting properties on a peptide immunogen for the induction of CD8+ T cell-mediated viral clearance is therefore Pam2Cys > Pam1Cys, Pam3Cys, Chol2 > Pal2. This broadly reflects the relative efficacy of the lipids in measures of memory CD8+ T cell induction and in their relative ability to mature DC.


   Discussion
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
References
 
The data presented here demonstrate that effective priming of CD8+ T cell-mediated viral clearing responses can be achieved with a single dose of lipopeptide immunogen delivered by the intra-nasal route. We also show that not all lipid components of these immunogens are equally effective in this regard. Alterations to the number or orientation of the palmitic acid groups or use of an alternate lipid, cholesterol, led to subtle differences in the very early interactions between the immunogen and the innate immune system, in particular the priming DC. The capacity to mature DC appeared to be closely linked to the magnitude of the resulting adaptive immune response, which was in turn, translated into highly significant differences in the potency of the immune response induced. The core immunogen we have used in this study is a very simple 25 amino acid residue peptide, based on a single epitope for CD8+ T cells and another for CD4+ T cells from influenza virus, to which different lipid structures have been coupled. Our results confirm our earlier studies (3, 9) in which immunogens with similar components are sufficient to induce potent CD8+ T cell and viral clearing responses in the absence of additional adjuvant.

Previous studies have shown that Pam3Cys, when coupled to appropriate peptides, can induce CTL responses in the absence of any other external adjuvants (38, 39). This lipid moiety is known to interact with TLR2 (18, 40) and we have shown, using TLR2 transfected cells, that the synthetic immunogen containing this lipid structure can likewise signal through TLR2 for NF{kappa}B-dependent gene readout and for the induction of DC maturation. We also confirm our earlier observation (9) that the related Pam2Cys, which is found naturally as a component of macrophage-activating lipopeptide-2 from mycoplasma (41), can likewise signal through TLR2 when present as a component of a synthetic immunogen and extend these findings to include Pam1Cys. Pam1Cys-, Pam2Cys- and Pam3Cys-containing lipopeptides were also potent inducers of IL-12p40 production by cultured DC. Nevertheless, the interaction of these three lipopeptides with DC did not result in an equivalent degree of maturation. Although almost all the D1 population could undergo maturation when incubated with each of these lipopeptides at 0.5 nmoles ml–1, at lower doses it was evident that the lipopeptide containing Pam2Cys was ~3- to 10-fold more potent than that containing Pam1Cys which was again ~3- to 10-fold more potent than that containing Pam3Cys.

The superiority of the Pam2Cys-containing lipopeptide was reflected in the level of T cell responses induced and the subsequent viral clearance seen upon challenge. As Pam2Cys is recognized by a heterodimeric structure that consists of TLR2 and TLR6 (42, 43) while Pam3Cys is recognized by TLR1/TLR2 heterodimers (44), it is possible that Pam2Cys is a more potent DC activator because it has a higher affinity for its receptor than does Pam3Cys for its receptor. Alternatively, signalling through these different heterodimers may lead to a different spectrum of gene products being produced (45) that may influence the quality of the T cell priming capacity of the DC.

The ability of lipopeptides functioning as TRL2 ligands to prime for strong CTL responses can be explained by their DC targeting function a well as their DC maturing capacity. However, a study comparing ligands for different TLR molecules to enhance CD8+ T cell induction by a co-administered virus-like particle vaccine showed that the TLR2 agonist was relatively poor in this regard (46). This is compatible with our earlier data which show that, even in 5-fold molar excess, an admix of Pam3Cys with a peptide hormone-based immunogen was insufficient to provide an immune response, whereas a 1:1 conjugate with the same peptide was highly immunogenic (8). Therefore, it would seem that a critical determinant for the success of TLR2 ligands in promoting the induction of T cell immunity is that they are physically linked to the antigen. This notion is compatible with the finding, using an anti-TLR2 antibody as a surrogate ligand, that TLR2 acts as an endocytic receptor (47). This provides a mechanism whereby the peptide cargo in the vaccines described here can efficiently enter the cell and access the class II processing and presentation pathway. It will be of interest to determine whether, once internalized, the lipid moiety is responsible for endosomal escape to allow access to the cytosol and the class I processing pathway.

Another lipopeptide tested in this study contained cholesterol, instead of palmitic acid, in a simple linkage through lysine rather than cysteine. The Chol2-containing lipopeptide was able to induce the maturation of DC to a similar extent as the Pam3Cys-containing lipopeptide but the interaction lead to ~8-fold less induction of IL-12p40, a level that was only slightly above that found in cultures of unstimulated cells. This result is in accord with that of Zhu et al. (15) who showed that an anti-herpes simplex virus antibody-inducing lipopeptide, with a single cholesterol at the N-terminus of the peptide, was relatively weak in inducing the production of IL-12 from DC and tended to favour a Th2-type response. The Chol2-containing lipopeptide in the present study could not stimulate D1 cells from mice deficient in the TLR adapter protein MyD88 (data not shown), nor was its ability to stimulate the maturation of D1 cells derived from the TLR4 mutant C3H/HeJ mice compromised (data not shown), together suggesting that maturation of DC by this lipopeptide is dependent on interaction with a TLR other than TLR4. It is possible that this lipopeptide could function as a very weak TLR2 ligand as it stimulated NFkB-dependent gene readout in TLR2 transfected cells to a level slightly but significantly above the non-lipidated peptide at each of the concentrations tested. However, it cannot be ruled out that it delivers its maturation signal optimally via a heterodimer containing TRL2 paired with a different TLR chain that is not represented in the TLR2 transfected cell type used in this study, or by a TLR2-independent mechanism. Despite its inability to induce IL-12p40, the Chol2-containing lipopeptide, which showed comparable DC maturation to the Pam3Cys-containing lipopeptide, also induced comparable levels of CTL peptide-specific CD8+ T cells as shown by tetramer staining and by intracellular staining for IFN-{gamma} in the lungs following virus challenge. Likewise, the Chol2-containing lipopeptide and the Pam3Cys-containing lipopeptide induced similar levels of viral clearance when the mice were challenged in the primary effector phase of the anti-peptide response and also when the memory cells were recalled by viral challenge 28 days after priming with either a high or low dose of the lipopeptides. This suggests that the ability of a lipopeptide to induce the production of high levels of IL-12p40 by DC is not a prerequisite for CD8+ T cell induction.

The remaining lipopeptide examined in this study was the Pal2-containing immunogen, which failed to mature D1 cells and did not appear to signal through TLR2. Despite being able to induce cells with lytic capacity, the tetramer-positive cells were under-represented in the lungs following challenge with the virus 28 days after immunization and this translated into levels of viral clearance that were the least potent of all the lipopeptides at this time point. This finding was somewhat unexpected as the Pal2-containing lipopeptide has previously been shown by us to be very effective at inducing viral clearing responses when delivered by the subcutaneous route (3). In the present study, all lipopeptides were delivered by the intra-nasal route and therefore potentially come into contact with a different population of DC than those available to subcutaneously delivered lipopeptide. This highlights how the route of inoculation is critical to the success of these immunogens. In this regard, other components of the innate immune system may come into play to enhance the priming of T cells at particular sites. For example, it has been shown that HEK293 cells transfected with TLR2, and a human lung epithelial cell line, which constitutively expresses TLR2 (48), as well as primary human tracheobronchial epithelial cells (49), secrete human ß-defensin 2 in response to bacterial lipoprotein. In addition to having a role in direct killing of bacteria, human ß-defensin 2 has been shown to function as a chemoattractant for immature DC (50). A similar peptide (Defb2) is also found in the mouse (51). The i.n. administered lipopeptide therefore has the potential to invoke defensin production in response to TLR2 ligation, recruiting additional immature DC to the site of infection for antigen loading and thereby further increasing the magnitude of the adaptive immune response generated. Another example is the bronchus-associated and alveolar macrophages, which may be triggered by the lipid to secrete cytokines to increase activation of DC and indirectly enhance peptide presentation. Clearly, an understanding of the influence of the microenvironment of different inoculation sites, including the subsets of DC that are present and the expression of TLR on different tissues, on the success of these immunogens will be an important area for future study.

Despite the potential for interactions between lipopeptide and TLR on epithelial and other tissues to enhance the overall immunity, we have shown that DC pulsed with the Pam2Cys-containing lipopeptide is sufficient to induce the presence of a substantial (7% of total CD8+ T cells) specific CD8+ T cell population in the lungs early (5 days) after infection, at a time prior to when significant numbers of infection-induced T cells are present in unprimed animals. It is not necessarily the case that the transferred cells themselves actually present the peptide; perhaps these activated cells can in turn cross-present their peptide cargo to other antigen-presenting cells for T cell priming. Nevertheless, a specific T cell population is induced in vivo that has the capacity for rapid expansion within or infiltration into the infected lung. In fact, we showed that the ability of the lipopeptides to induce D1 cell maturation in vitro was an excellent predictor of how efficacious they were as immunogens in vivo. This not only reinforces the link between lipopeptide-induced DC maturation and T cell priming, providing the mechanism for self-adjuvanticity, but also indicates that the D1 cells, which are derived from precursors in the spleen, may share many important characteristics with those cells encountered by the lipopeptides in the MLN after priming. Overall, we show that immunogens incorporating bacterial triggers of innate immunity can function very efficiently to induce strong anti-viral cellular responses, independently of potentially poorly tolerated exogenous adjuvants. We also identify the TLR2-targeting Pam2Cys-containing lipopeptides as highly potent in this regard.

This study, which links the early events of immunogen recognition to the functional outcome of protective efficacy of the immune response to pathogen challenge, provides insight into the mechanism that may explain the success of lipopeptides as self-adjuvanting immunogens and forms a basis for the construction of highly efficacious CD8+ T cell-inducing vaccines. The ability of this type of vaccine to speed the clearance of influenza from the infected lung may provide a pathway for the design of pandemic influenza vaccines that could offer a reduction in morbidity and mortality in infected individuals in that time before sufficient specific antibody-inducing vaccines can be produced.


   Acknowledgements
 
This work was supported by grants from the National Health and Medical Research Council of Australia and the Australian Government's Cooperative Research Centres Program.


   Abbreviations
 
BAL, bronchioalveolar lavage
CFSE, 5-(and 6) carboxyfluorescein diacetate succinimidyl ester
Chol, cholesterol
DC, dendritic cell
i.n., intra-nasally
K, lysine
NF{kappa}B, nuclear factor {kappa}B
NMS, normal mouse serum
Pal, palmitic acid
Pam2Cys, Pam2C, S-[2,3-bis(palmitoyloxy)propyl]cysteine
Pam3Cys, Pam3C, tripalmitoyl-S-glycerylcysteine
PRR, pattern-recognition receptor
S or Ser, serine
TCM, T cell medium
TLR, Toll-like receptor

   Notes
 
* These authors are the co-senior authors. Back

Transmitting editor: D. Tarlinton Back

Received 16 December 2005, accepted 27 September 2006.


   References
Top
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 Introduction
 Methods
 Results
 Discussion
 References
 

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