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The Journal of Immunology, 2007, 178: 2396-2406.
Copyright © 2007 by The American Association of Immunologists, Inc.

Rapid Clonal Expansion and Prolonged Maintenance of Memory CD8+ T Cells of the Effector (CD44highCD62Llow) and Central (CD44highCD62Lhigh) Phenotype by an Archaeosome Adjuvant Independent of TLR21

Lakshmi Krishnan2,*,{dagger}, Komal Gurnani*, Chantal J. Dicaire*, Henk van Faassen*, Ahmed Zafer*, Carsten J. Kirschning{ddagger}, Subash Sad*,{dagger} and G. Dennis Sprott*

* National Research Council-Institute for Biological Sciences, Ottawa, Ontario, Canada; {dagger} Department of Biochemistry, Microbiology, and Immunology, University of Ottawa, Ottawa, Ontario, Canada; and {ddagger} Institute for Microbiology, Immunology, and Hygiene, Technical University, Munich, Germany


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Vaccines capable of eliciting long-term T cell immunity are required for combating many diseases. Live vectors can be unsafe whereas subunit vaccines often lack potency. We previously reported induction of CD8+ T cells to Ag entrapped in archaeal glycerolipid vesicles (archaeosomes). In this study, we evaluated the priming, phenotype, and functionality of the CD8+ T cells induced after immunization of mice with OVA-Methanobrevibacter smithii archaeosomes (MS-OVA). A single injection of MS-OVA evoked a profound primary response but the numbers of H-2KbOVA257–264-specific CD8+ T cells declined by 14–21 days, and <1% of primarily central phenotype (CD44highCD62Lhigh) cells persisted. A booster injection of MS-OVA at 3–11 wk promoted massive clonal expansion and a peak effector response of ~20% splenic/blood OVA257–264-specific CD8+ T cells. Furthermore, contraction was protracted and the memory pool (IL-7R{alpha}high) of ~5% included effector (CD44highCD62Llow) and central (CD44highCD62Lhigh) phenotype cells. Recall response was observed even at >300 days. CFSE-labeled naive OT-1 (OVA257–264 TCR transgenic) cells transferred into MS-OVA-immunized recipients cycled profoundly (>90%) within the first week of immunization indicating potent Ag presentation. Moreover, ~25% cycling of Ag-specific cells was seen for >50 days, suggesting an Ag depot. In vivo, CD8+ T cells evoked by MS-OVA killed >80% of specific targets, even at day 180. MS-OVA induced responses similar in magnitude to Listeria monocytogenes-OVA, a potent live vector. Furthermore, protective CD8+ T cells were induced in TLR2-deficient mice, suggesting nonengagement of TLR2 by archaeal lipids. Thus, an archaeosome adjuvant vaccine represents an alternative to live vectors for inducing CD8+ T cell memory.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The perception of a "danger signal" by APCs triggers the innate immunity cascade which constitutes the first line of defense against infections. The danger signal context also importantly directs subsequent adaptive immunity, as the APCs process and present pathogen-specific Ags on their MHC molecules (1). T cells then embark on a programmed, intense, expansion phase to counteract the rapid proliferation of pathogen. The two subsets of T cells, CD4+ and CD8+, are contrastingly evoked by Ag that is presented in the context of MHC class II or MHC class I, respectively (2). Classically, CD8+ T cells are evoked against Ags generated in the cytosol, such as intracellular bacteria or viruses (3). CD8+ T cells, with their unique ability to kill targets, are then able to eliminate the infected or cancer cell (4). After the profound effector phase resulting in clearance of pathogen, the majority of Ag-specific T cells generated are eliminated by apoptosis and only a small population of memory cells survives for long-term maintenance (5). This stable memory pool can provide life-long protection against reinfection, although various factors such as homeostatic mechanisms and heterologous infections can cause attrition of the T cell memory pool over time (6, 7).

Currently, effective vaccines are lacking for many diseases, in particular chronic and intracellular infections, and cancer that require a CD8+ T cell response for disease resolution. The paradigm of CD8+ T cell induction, maintenance, and memory suggests an inherent difficulty in evoking CD8+ T cell immunity to subunit vaccines, particularly in the absence of danger signal perception by the host. Furthermore, CD8+ T cells segregate into effector and central memory subsets, the former with rapid effector function, and the latter with potent proliferative and lymph node homing properties (8). The manner in which an appropriate balance of these subsets can be evoked by vaccination for maximum benefit to the host is unclear.

Adjuvants are used in vaccines to provide immunostimulation and facilitate innate immunity and consequently can direct potent adaptive immunity (9). Currently, only Alum and MF59 are approved for widespread human use (10, 11) and both fail to evoke a strong CD8+ T cell response. Thus far, the best choice for evoking CD8+ T cell immunity to vaccines has been the use of live recombinant bacterial and viral vectors as Ag delivery vehicles (12). Live vectors come with the risk of reconversion to virulence and may not be safe for use in aged and immunocompromised individuals. Therefore, alternate strategies for evoking potent CD8+ T cell immunity to vaccines are desired. Dendritic cells (DCs)3 possess the unique ability to cross-present exogenous Ags on the MHC class I pathway and thus cross-prime CD8+ T cells in vivo (13). Thus, novel immunostimulants that can harness the cross-presentation properties of DCs hold promise for future vaccine development (14).

Archaeal membranes are uniquely constituted of ether-linked isoprenoid phytanyl core lipids (15, 16) that can be constituted into stable Ag delivery vesicles, termed archaeosomes (17). The polar lipid from the archaeon, Methanobrevibacter smithii (MS), is additionally abundant in archaetidyl serine head groups (18), promoting interaction with the phosphatidylserine (PS) receptor on APCs (19, 20). Thus, we have shown that MS archaeosomes, facilitate cross-presentation of CD8+ T cell response (21) and activate DC costimulation and cytokine production (19, 22). Furthermore, archaeosomes evoke protective immunity against intracellular pathogens and cancer (23, 24, 25). In this study, we have characterized in detail the CD8+ T cell induction, maintenance, and memory profile evoked by MS archaeosomes. The T cell response has been followed kinetically for >1 year in immunized mice and we have assessed the functionality of the CD8+ T cells to kill targets in vivo. Furthermore, we show that wild-type and TLR2–/– mice evoke identical responses to archaeosome Ag, suggesting archaeal lipids induce a potent CD8+ T cell memory response in the absence of TLR2-mediated danger signal perception.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Reagents

Sources for reagents were: OVA (grade VI), 2-ME, RBC lysing solution, CFA, and the PKH26 Red Fluorescent Cell Linker kit, obtained from Sigma-Aldrich; CFSE obtained from Molecular Probes; flow cytometric Abs obtained from BD Biosciences; H-2KbOva257–264 tetramers obtained from Beckman Coulter; RPMI 1640 and gentamicin obtained from Invitrogen Life Technologies; FBS obtained from HyClone; G418 obtained from Rose Scientific; and murine recombinant IL-2 obtained from ID Labs. OVA257–264 (SIINFEKL) peptide was synthesized in-house.

Preparation and characterization of archaeosomes

MS ALI (DSM 2375) was grown in fermenters, total lipids extracted from frozen cell pastes, and the total polar lipids (TPL) were collected as the acetone-insoluble fraction. Absence of detectable DNA in TPL was confirmed by electrophoresis on agarose gels followed by ethidium bromide staining. Archaeosomes were prepared by entrapping OVA (lacking fragments) in MS TPL by the dried-reconstituted vesicle method; the amount of protein incorporated into the vesicle was estimated by modified SDS Lowry, as described in detail previously (17). These vesicles are referred to as MS-OVA. The ratio of protein to lipid was based on the salt-free dry weights of the vesicles and was 40–70 µg of OVA/1 mg of lipid. All vesicles were unilamellar and diameters were in the range of 100–200 nm, determined by number-weighted Gaussian size distributions using a Nicomp Particle sizer. All glassware used in the preparation of archaeosomes was prebaked (6 h at 180°C) to render them pyrogen-free and endotoxin-free reagents were used throughout.

Mice and immunizations

Inbred, 6- to 8-wk-old female C57BL/6J, OT-1, and B6.PL mice were obtained from The Jackson Laboratory. Breeding pairs of TLR2–/– mice backcrossed onto a C57BL/6J background (26) were bred at the National Research Council-Institute for Biological Sciences Animal Facility. Mice were maintained in accordance with guidelines from the Canadian Council on Animal Care.

Mice were immunized s.c. at the base of the tail with MS-OVA (15–25 µg of OVA in 0.2–0.5 mg of lipid/100 µl of PBS), Listeria monocytogenes expressing OVA (LM-OVA, 103 CFU/injection/100 µl of 0.9% saline), OVA (20 µg) in PBS (PBS-OVA), and OVA (25 µg) formulated in CFA (CFA-OVA). The construction of LM-OVA has been described previously (27). The immunization schedules were as described in figure legends.

Cell lines

EL-4 (H-2Kb) was obtained from the American Type Culture Collection (ATCC) and maintained in RPMI 1640 medium supplemented with 2-ME, 8% FBS, and 10 µg/ml gentamicin (R8 medium). EG.7 cells, a subclone of EL-4 stably transfected with the gene encoding OVA (28) were obtained from the ATCC and cultured in R8 medium, additionally containing 400 µg/ml G418. B16OVA cells, expressing the gene for OVA were obtained from Dr. E. Lord (University of Rochester, Rochester, NY) and cultured in R8 medium, additionally containing 400 µg/ml G418. All cells were maintained at 37°C in 8% CO2.

CTL assays

Spleen cells (30 x 106) from pooled spleens (n = 2–4) of immunized mice were cultured with 5 x 105 irradiated (10,000 rad) EG.7 cells in 10 ml of R8 medium containing 0.1 ng/ml IL-2, in 25 cm2 tissue-culture flasks (Falcon; BD Biosciences), kept upright. After 5 days (37°C, 8% CO2), the cells were recovered from the flask and used as effectors in a standard 51Cr-release CTL assay (21). Supernatants were collected and radioactivity detected by gamma counting. The percent-specific lysis was calculated using the formula: ((cpm experimental – cpm spontaneous)/(cpm total – cpm spontaneous)) x 100. One lytic unit is defined as the number of effector cells per 106 spleen cells that yield 20% specific lysis of a population of 2.5 x 104 targets.

In vivo cytolytic activity of Ag-specific CD8+ T cells was enumerated according to the protocol of Barber et al. (29). Donor spleen-cell suspensions from syngenic mice were prepared and RBC lysed using Tris-buffered ammonium chloride (RBC lysing solution). Cells were split in two aliquots. One aliquot was incubated for 30 min with OVA257–264 peptide (10 µg/ml) in R8 medium. Both cell aliquots were then stained with the dye PKH26 (4 µM). Then, the peptide-pulsed cell aliquot was stained with 10x CFSE (5 µM), whereas the other cell aliquot was stained with 1x CFSE (0.5 µM) for 30 min in PBS. The two cell aliquots were mixed 1:1 and injected (20 x 106/mouse) into recipient mice that were immunized previously with MS-OVA. PBS-injected recipient mice served as controls. At 24 h after the donor cell transfer, spleens were removed from recipients, single-cell suspensions were prepared, and cells were analyzed by flow cytometry. The in vivo lysis percentage of peptide-pulsed targets was enumerated according to a previously published equation (29).

Enumeration of IFN-{gamma}-secreting cells

Enumeration of IFN-{gamma}-secreting cells was done by ELISPOT assay. Briefly, spleen cells were incubated in anti-IFN-{gamma} Ab-coated ELISPOT plates in various numbers (in a final cell density of 5 x 105/well using feeder cells) in the presence of IL-2 (1 ng/ml) and R8 medium or OVA257–264 (10 µg/ml) for 48 h at 37°C, 8% CO2. The plates were subsequently blocked, incubated with the biotinylated secondary Ab (4°C, overnight), followed by avidin-peroxidase conjugate (room temperature for 2 h). Spots were revealed using diamino benzidine.

Assessment of numbers and phenotype of Ag-specific CD8+ T cells in vivo

The activation of Ag-specific CD8+ T cells was tracked in vivo using the tetramer assay. Briefly, spleen cells (10 x 106) or PBLs (obtained from 100 µl of blood) were incubated in 200 µl of PBS plus 1% BSA (PBS-BSA) with anti-CD16/32 at 4°C. After 10 min., cells were stained with PE-H-2KbOVA257–264 tetramer and various Abs (anti-CD8, anti-CD62L, anti-CD44, and anti-IL-7R{alpha}) for 30 min at room temperature. Cells were washed with PBS and fixed in 0.5% formaldehyde and acquired on a BD Biosciences FACS Canto Analyzer. Alternatively, to amplify the tetramer signal, spleen cells (106) from OT-1 mice in HBSS, were injected i.v. into recipient mice, at the same time that they received MS-OVA s.c. At various time points thereafter, the proportion of OVA257–264-specific donor CD8+ T cells that proliferated and differentiated in response to MS-OVA immunization in the recipient mice was determined using the tetramer assay of spleen and/or blood lymphocytes. Analysis of IL-7R{alpha}+ or CD44+CD62L+ on H-2KbOVA257–264-tetramer positive CD8+ T cells was also done using multiplex staining of the samples simultaneously.

Assessment of in vivo cycling of Ag-specific CD8+ T cells

Spleen cells from donor OT-1-transgenic mice (Thy1.2+) were labeled with CFSE. Briefly, spleen cell suspensions were prepared and RBC removed with RBC lysing solution. Spleen cells were resuspended in PBS (20 x 106/ml) and an equal volume of CFSE (5 µM in PBS) was added. After 8 min at room temperature, an equal volume of FBS was added for quenching. After 1–2 min at 4°C, cells were washed with HBSS. CFSE-labeled OT-1 (20–25 x 106) cells were injected i.v. into recipient B6.PL (Thy1.1+) or C57BL/6J mice that had been immunized with MS-OVA. Four days later, recipient mice were euthanized and the numbers of donor origin (Thy1.2+) and H-2KbOVA257–264-tertramer-positive CD8+ T cells that were cycling (based on reduction in CFSE) were determined by flow cytometry.

Tumor model

C57BL/6J wild-type and TLR2–/– mice were injected with 106 B16OVA tumor cells (in PBS plus 0.5% normal mouse serum) in the shaved lower dorsal region. From day 5 onward, detectable solid tumors were measured using digital calipers. Tumor size, expressed in mm2, was obtained by multiplication of diametrically perpendicular measurements.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Induction of long-term CD8+ T cell response by MS-OVA

Entrapment of OVA in archaeosomes facilitates cross-presentation of Ag to the MHC class I pathway, resulting in activation of a potent CD8+ T cell immunity (21). We kinetically tracked the longevity of CTL for >1 year after initial immunization. Mice received two injections of MS-OVA 3 wk apart. At regular intervals we measured the in vitro CTL activity of splenocytes from representative mice (n = 3/time point). In a standard chromium release CTL assay, splenocyte effectors (generated after restimulation with Ag in vitro) from immunized mice, evoked a strong CTL response against specific EG.7 target cells on days 327 and 449, respectively (Fig. 1a). CTL data obtained from representative mice at various time points was converted to lytic units/106 spleen cells. This allowed stringent comparison of CTL activities over time, and is shown in Fig. 1b. A single immunization of MS-OVA afforded ~10 lytic units/106 spleen cells on day 14 and a second immunization provided a booster effect. The CTL activity subsequently underwent a gradual attrition, maintaining at ~1 lytic unit/106 spleen cells. A booster injection with Ag (in the absence of adjuvant) at day 306 resulted in a dramatic increase in the CTL intensity at day 327, in mice primed and then boosted at 3 wk with MS-OVA.


Figure 1
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  		FIGURE 1. Archaeosomes evoke long-term CD8+ T cell response. C57BL/6J mice were immunized s.c. either once or twice (days 0 and 21) with 15 µg of OVA entrapped in 0.285 mg of lipid. Spleen cells (n = 3) were pooled and restimulated for 5 days in vitro with EG.7 cells and 0.1 ng/ml IL-2. a, Splenic effectors generated on day 327 or 449 from mice immunized twice on days 0 and 21 were assessed for ability to kill EL-4 and EG.7 targets in a 51Cr-release assay. The percent-specific killing ± SD at the various E:T ratios is indicated. b, The percent-specific killing data obtained from representative mice at various times were converted to lytic units. "I" indicates immunization on days 0 and 21, and "R" indicates a recall injection with Ag on day 306. c, Frequency of IFN-{gamma}-secreting cells in the spleen of individual mice was determined by ELISPOT assay. Mean frequency ± SD of IFN-{gamma}-secreting cells per 106 spleen cells is indicated for n = 3 per time point. d, Mice were immunized with MS-OVA on days 0 and 21 or days 0 and 77. At day 126 post first injection, mice were euthanized, splenic effectors were generated, and the ability to kill EL-4 and EG.7 targets in a 51Cr-release assay was determined. The percent-specific killing ± SD at the various E:T ratios is indicated for effectors obtained from n = 3 mice per group.

 
The number of Ag-specific IFN-{gamma} secreting CD8+ T cells was also determined for the same splenocyte samples (n = 3/time point). The immunodominant CTL epitope of OVA for H-2Kb haplotype has been shown to be OVA257–264 (SIINFEKL) (30). Therefore, IFN-{gamma} production in response to stimulation with this peptide was determined by ELISPOT assay. The frequency of Ag-specific IFN-{gamma}-secreting CD8+ T cells was ~200/106 splenocytes at early time points after two injections of MS-OVA, gradually showing a decrease over time (Fig. 1c). The frequency then showed an increase on recall with an Ag injection, nearly a year after immunization, demonstrating the potential functionality of the Ag-specific CD8+ T cells. Again, priming followed by a booster injection with MS-OVA was required to maintain a higher frequency of CD8+ T cells in the long-term, as opposed to a single injection.

Although these results illustrate the importance of a second injection, the timing for the booster was not critical. The magnitude of the CTL response measured 18 wk after the first injection was not significantly different for mice receiving the MS-OVA booster after 3 or 11 wk (Fig. 1d).

Induction of in vivo CTL activity by MS-OVA

In vitro CTL activity is measured after restimulation of the splenocytes with Ag in vitro for 5 days, which could under- or overestimate the true functionality of CD8+ T cells in vivo. Thus, it was possible that the dramatic CTL activity observed at >1 year was a result of substantial in vitro expansion of the central subset cells. To ascertain the physiological relevance of the CD8+ CTL response, we measured killing of specific targets in vivo after MS-OVA immunization (Fig. 2), using the recently developed assay (29). A single injection of MS-OVA resulted in the ability of the host to eliminate >80% of the in vivo targets within 24 h, on day 7 after immunization (Fig. 2). However, this ability declined rapidly, with <15% of the targets being killed at day 21 and beyond (Fig. 2b). In contrast, two injections of MS-OVA on days 0 and 21 resulted in complete in vivo elimination of the targets within 24 h, on day 30 (Fig. 2). Furthermore, the in vivo killing ability was retained at >80% even 180 days after immunization, reflecting the long-term functionality of the Ag-specific CD8+ T cell response evoked by MS-OVA (Fig. 2b).


Figure 2
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  		FIGURE 2. Archaeosomes evoke potent in vivo CTL response to entrapped Ag. C57BL/6J mice were immunized s.c. with 20 µg of OVA in 0.5 mg of MS lipid once or twice (days 0 and 21). At various time points thereafter, representative mice (n = 4 per time point) were injected i.v. with equimolar proportions of nonspecific (labeled with 0.5 µM CFSE) and peptide-pulsed specific (labeled with 5 µM CFSE) PKH26-labeled syngenic donor spleen cells. Twenty-four hours later, the proportion of the two CFSE-labeled populations of donor cells in the spleen was determined by flow cytometry. a, Representative profile of PKH26-gated, CFSE-labeled populations in naive vs MS-OVA immunized mice on day 7 after a single injection, or on day 30 (after two injections of MS-OVA). b, The percent-specific killing ± SEM deduced for the various time points after a single or two MS-OVA injections.

 
Induction of high frequency of OVA257–264-specific CD8+ T cells by MS-OVA and phenotype of the subsets evoked

The tetramer assay allows specific detection of Ag-specific CD8+ T cells and is a powerful measure of in vivo frequency. Mice were immunized with MS-OVA and, at various time points, the frequency of H-2Kb OVA257–264-specific CD8+ T cells in the peripheral blood was assessed by tetramer staining. Fig. 3a shows representative tetramer staining on gated CD8+ T cells in the peripheral blood. The kinetics of induction and maintenance of the Ag-specific CD8+ T cell frequencies are shown in Fig. 3b. On day 7 after MS-OVA injection, 3–5% of CD8+ T cells were tetramer positive in the blood. This number dramatically expanded to ~25% of CD8+ T cells after the booster MS-OVA injection. A single injection of MS-OVA resulted in maintenance of the T cell frequency at <1%, whereas two injections of MS-OVA facilitated maintenance of the T cell frequency at ~5%. Furthermore, two injections of MS-OVA resulted in a gradual attrition of tetramer-positive cells over time. Priming of CD8+ T cell response with MS-OVA, a particulate immunogen, was similar in magnitude to a live replicating immunogen, LM-OVA. In contrast, PBS-OVA (unencapsulated OVA administered in the absence of adjuvant) failed to evoke significant numbers of tetramer positive cells even after a booster injection (Fig. 3, a and b).


Figure 3
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  		FIGURE 3. Rapid induction of OVA257–264-tetramer-specific cells by MS-OVA. C57BL/6J mice (n = 4) were immunized s.c. once or twice (days 0 and 21) with 20 µg of OVA in 0.3 mg of MS lipid, twice with 20 µg of OVA in PBS, or once with 103 CFU of LM-OVA. At various time points, blood was obtained from the mice, and the PBLs processed for OVA257–264 tetramer staining, and expression of CD62L and CD44. a, Representative flow cytometric profile indicating the percentage of gated CD8+ T cells positive for OVA257–264 H-2Kd tetramer. b, Mean percentage ± SEM of CD8+ T cells that were OVA257–264-specific is shown for the various time points (n = 4/group). c, The distribution (mean percentage ± SEM) of CD44highCD62Llow, or CD44highCD62Lhigh, OVA257–264-specific cells for the various groups over time (n = 4/time point), is shown. All data are deduced from the analysis of 100,000 events acquired for each mouse per time point.

 
CD8+ T cells have been classified into effector (CD44highCD62Llow) and central (CD44highCD62Lhigh) phenotype cells (8). Fig. 3c shows the kinetic distribution of effector and central phenotype OVA257–264-specific CD8+ T cells. A single immunization of MS-OVA primed effectors but these underwent attrition rapidly, and primarily central phenotype cells were noted in the long-term. Priming followed by boosting with MS-OVA on day 21 facilitated massive clonal expansion and the effectors generated underwent gradual attrition. Furthermore, after the booster injection a stable pool of both effector and central phenotype cells persisted. A single dose of LM-OVA also evoked profound effector response initially, but later mainly central phenotype cells persisted (Fig. 3c).

To clearly monitor the phenotype of OVA-specific CD8+ T cells in the long-term, we used an alternate adoptive transfer model, wherein 106 spleen cells (comprising ~105 CD8+ T cells) from OT-1 mice (with >90% of CD8+ T cells specific for OVA257–264) were transferred i.v. into syngenic recipient mice. The recipient mice were then injected s.c. with MS-OVA or LM-OVA and at various time points the percentage and phenotype of Ag-specific T cells were tracked. In this adoptive transfer model, parking naive OT-1 cells in the host before immunization increased the initial frequency of responding Ag-specific cells, resulting in amplification of the tetramer-positive response. Therefore, a single s.c. injection of MS-OVA to recipient mice results in ~20% OVA257–264-tetramer specific splenic CD8+ T cells on day 7 (Fig. 4a). This number subsequently dropped and was maintained at ~2% beyond 60 days. The magnitude of induction of Ag-specific CD8+ T cells by MS-OVA was comparable to s.c. LM-OVA infection (Fig. 4a), a potent live vector. Fig. 4b shows the frequency of OVA257–264-specific CD8+ T cells in the peripheral blood of mice administered a single injection of MS-OVA or LM-OVA. Fig. 4c shows the splenic distribution of the effector and central subset of tetramer-gated CD8+ T cells after s.c. MS-OVA or LM-OVA immunizations. At day 7 after immunization, the majority (73%) of CD8+ OVA257–264-specific cells evoked by MS-OVA were of the effector phenotype (CD44highCD62Llow). However, by day 30 the Ag-specific CD8+ T cells were predominantly (83%) of the central phenotype (CD44highCD62Lhigh). Fig. 4d reiterates that a similar phenotypic distribution of cells occurred in the peripheral blood. Overall, a single injection of MS-OVA or LM-OVA resulted in the maintenance of low numbers of Ag-specific memory cells predominantly of the central (CD44highCD62Lhigh) phenotype.


Figure 4
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  		FIGURE 4. A single injection of MS-OVA primes OVA257–264-specific CD8+ T cells and results in the maintenance of CD44highCD62Lhigh central memory subset. C57BL/6J mice were first parked i.v. with 106 OT-1 spleen cells. Subsequently, they were injected s.c. with 20 µg of OVA in 0.3 mg of MS lipid, or 103 CFU of LM-OVA. a, Representative flow cytometric profile indicating the percentage of gated splenic CD8+ T cells positive for OVA257–264 H-2Kb tetramer on days 7, 14, 30, and 60. The percentage of CD8+ T cells that were OVA257–264-specific is indicated within each panel. b, Mean percentage ± SEM of peripheral blood CD8+ T cells that were OVA257–264-specific is shown for the various time points (n = 3/group). The CD8+OVA257–264-specific T cells were then further analyzed for the expression of CD62L and CD44. c, Representative profile of spleen indicating Ag-specific CD8+ T cells that were CD44highCD62Lhigh (central phenotype) or CD44highCD62Llow (effector phenotype) on days 7, 14, 30, and 60. d, Mean percentage ± SEM of peripheral blood Ag-specific CD8+ T cells that are of the central or effector phenotype after a single injection of MS-OVA or LM-OVA. Data are derived from 100,000 events acquired for each individual spleen and/or blood sample. Spleen profiles are representative of percentages seen among four to five mice for each time point.

 
Fig. 5a shows the representative profiles of splenic tetramer staining and phenotypic distribution of cells, after two injections with MS-OVA on days 0 and 21. Fig. 5b shows the kinetics of the frequency of OVA257–264-specific cells in the peripheral blood evoked by MS-OVA, in comparison to immunization with two doses of PBS-OVA. The second injection of MS-OVA resulted in 10-fold expansion of numbers of Ag-specific cells at around day 30, compared with preboost levels. Further, this resulted in maintenance of higher frequency of OVA257–264-tetramer-specific cells beyond 50 days (~8% in the spleen (Fig. 5a) and >20% in peripheral circulation (Fig. 5b)). Interestingly, PBS-OVA failed to evoke any significant expansion of tetramer-specific cells even in this adoptive transfer model, wherein a higher frequency of naive OT.1 responders were made available. Fig. 5c shows the phenotypic distribution of Ag-specific subsets in the peripheral blood after two doses of MS-OVA and suggests a balanced maintenance of both effector and central phenotype memory cells.


Figure 5
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  		FIGURE 5. MS-OVA boost causes potent expansion of Ag-specific CD8+ T cells and results in the balanced maintenance of central and effector memory populations. Mice were first parked i.v. with 106 OT-1 spleen cells. They then received s.c. immunization of 20 µg of OVA in 0.3 mg of MS lipid, or 20 µg of OVA in PBS on days 0 and 21. a, A representative profile indicating the percentage of splenic OVA257–264-specific CD8+ T cells and the percentage of Ag-specific CD8+ T cells that were CD44highCD62Lhigh (central) or CD44highCD62Llow (effector) phenotype, at the various time points after MS-OVA immunization. b, Mean ± SEM of CD8+ OVA257–264 tetramer+ cells in the peripheral blood (n = 3). c, Mean ± SEM of Ag-specific central and effector cell subsets. Splenic data are representative of percentages and trends seen for n = 5 mice per time point. All data are derived from 100,000 gated CD8+ events acquired for each sample.

 
Long-term IL-7R{alpha} expression on Ag-specific cells evoked by MS-OVA correlates with a memory phenotype

CD44 and CD62L expression of CD8+ T cells is often not sufficient to discern early and late stage effectors. We therefore additionally studied IL-7R{alpha} expression on Ag-specific cells in the peripheral blood evoked by MS-OVA. Fig. 6a shows the representative profile of IL-7R{alpha} on OVA257–264-specific CD8+ T cells, on day 7 and day 28 after a single MS-OVA or LM-OVA injection. As expected in the early stages of the response, on day 7, a large proportion of the Ag-specific CD8+ T cells had down-regulated expression of IL-7R{alpha}. However, by day 28, >75% of the cells had high IL-7R{alpha} expression. Fig. 6b shows that a second injection of MS-OVA (administered on day 21) did not evoke significant numbers of IL-7R{alpha}low cells on day 28 (Fig. 6b). Thus, the second injection may have led to expansion of committed memory (IL-7R{alpha}high) cells that do not down-regulate IL-7R{alpha}. The kinetic distribution of Ag-specific CD8+ IL-7R{alpha}high T cells over the long-term is shown in Fig. 6c, and suggests a similar functional memory phenotype after single or two doses of MS-OVA. Similarly, LM-OVA also evoked memory CD8+ T cells of the IL-7Rhigh phenotype. CD27 expression on Ag-specific cells was also followed in the peripheral blood of MS-OVA-vaccinated mice and indicated early down-regulation in response to activation on day 7 and subsequent up-regulation as the cells progressed to the memory phenotype (data not shown).


Figure 6
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  		FIGURE 6. Profile of IL-7R{alpha} expression after MS-OVA immunization. IL-7R{alpha} (CD127) expression was monitored in peripheral blood of recipient C57BL/6J mice that were adoptively transferred with 106 OT.1 spleen cells and then vaccinated with LM-OVA (103) or MS-OVA (20 µg OVA per 0.89 mg of lipid/injection). a, Representative profile indicating the CD127 staining of gated CD8+ T cells that were OVA257–264-tetramer specific, on days 7 and 28 after a single injection of LM-OVA or MS-OVA. The number within each panel refers to the percentage of IL-7R{alpha}high cells. b, Representative profile indicating percentage of IL-7R{alpha}high OVA257–264-specific cells, 7 days after the second injection. c, Mean ± SEM of IL-7R{alpha}high OVA257–264-specific cells at various time points (n = 3/time point) after a single injection of LM-OVA or MS-OVA or two injections (days 0 and 21) of MS-OVA.

 
OVA archaeosomes elicit profound in vivo Ag presentation

We next evaluated the in vivo Ag presentation by OVA archaeosomes in an adoptive transfer model, wherein naive OT-1-CFSE-labeled spleen cells were injected into mice previously immunized with OVA archaeosomes and proliferation of Ag-specific CD8+ T cells was tracked 4 days later based on H-2KbOVA257–264 tetramer staining and CFSE reduction. As hosts and donors differed in their expression of Thy1.2 allele, the proliferation of donor cells could be tracked specifically using an anti-Thy1.2 Ab. When OT-1 cells were transferred into PBS injected mice, ~4% of donor CD8+ T cells were cycling. In contrast, in OVA-archaeosome-immunized mice, when CFSE labeled OT-1 cells were transferred 3 days after immunization and tracked on day 7, profound cycling of transferred cells (95%) based on CFSE reduction profile was noted indicating efficient Ag presentation and stimulation (Fig. 7). Transfer of CFSE labeled OT-1 cells at later time points resulted in lower, but significant, cycling of donor cells indicating the continued presentation of Ag by host APCs. For example, between day 9 and 13, 82% of donor cells cycled, and even on days 17–21 and 48–52 25% of the donor cells cycled (Fig. 6). At all time points, mice injected with PBS showed little (2–5%) cycling (data not shown). We then compared this cycling profile to that induced by s.c. infection of LM-OVA or OVA administered in CFA (Fig. 8). As with MS-OVA, at early time points after injection, LM-OVA and CFA-OVA evoked profound cycling of donor OVA257–264-specific CD8+ T cells. However, with LM-OVA, the cycling declined to ~15% by day 10 and to <3% after day 15, suggesting clearance of the Ag. CFA-OVA resulted in sustained cycling of donor cells at later time points (30 days) similar to MS-OVA (Fig. 8), suggesting an Ag depot effect for these two adjuvants. Thus, archaeosomes were comparable to CFA and probably better than LM in terms of the extent and duration of Ag presentation.


Figure 7
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  		FIGURE 7. Archaeosomes evoke prolonged Ag presentation. Thy1.1+ B6.PL mice were immunized s.c. with 25 µg of OVA entrapped in MS archaeosomes (0.2 mg of lipid). On day 3, 9, 17, or 48, naive and MS-OVA-immunized Thy1.1+ recipients were injected i.v. with CFSE-labeled OT-1 (Thy1.2+) cells. Four days later (on day 7, 13, 21, or 52 respectively), mice were euthanized, spleens removed, and the numbers of CD8+ T cells of donor origin (Thy1.2+) was determined. Further analysis was conducted to deduce the reduction in CFSE of gated CD8+ T cells of donor origin. a, Representative flow cytometric profile for mice injected with PBS or MS-OVA and adoptively transferred with donor-CFSE cells. Numbers within each panel indicate the percentage of donor CD8+ T cells that were cycling based on CFSE reduction. b, The mean percentage ± SEM of cycling (n = 3–5 mice) for each of the time points mentioned above.

 

Figure 8
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  		FIGURE 8. Ag presentation evoked by MS-OVA is similar to CFA-OVA depot effect. Thy1.1+ B6.PL mice were immunized s.c. with 25 µg of OVA entrapped in MS (0.2 mg of lipid), or LM-OVA (103 CFU), or 25 µg of OVA formulated in CFA. On day 3, 9, 17, or 24, naive and immunized recipients (n = 3 per time point) were injected i.v. with CFSE-labeled OT-1 (Thy 1.2+ cells). Four days later (on day 7, 13, 21, or 28), the numbers of splenic CD8+ T cells of donor origin that were also cycling based on CFSE reduction was deduced. In each panel, the boxed region refers to the CFSE reduction and the numbers indicate percentage of donor-specific CD8+ T cells that were cycling.

 
Lack of local edema after archaeosome injection

CFA is known to provide a strong Ag depot effect but is a toxic adjuvant unsuitable for human applications. As MS-OVA also promoted sustained Ag presentation in vivo, suggesting an Ag depot effect, we tested whether the prolonged presence of archaeosomes at the injection site caused undesirable side effects. Mice were injected with various doses of Ag-free MS archaeosomes in one footpad. The local edema was measured using calipers. Fig. 9a indicates that any local reaction that was observed with a high dose of archaeosomes rapidly declined after the first 24 h, with no significant local edema or redness noted at later time points. Archaeosomes, however, evoked an increase in the number of local popliteal lymph node cells (Fig. 9b) consistent with the facilitation of cell-mediated immunity and activation of DCs (22). However, this increase was not as profound as expected of adjuvants such as CFA or for infections with associated inflammation. Because CFA is not approved to be used in such experiments due to exacerbated footpad swelling and edema, our results suggest that archaeosomes induced potent CD8+ T cell response without the undesirable inflammation.


Figure 9
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  		FIGURE 9. MS-OVA injection does not cause local edema or inflammation. Mice were immunized with varying dose of Ag-free MS archaeosomes in 50 µl of PBS in the right foot pad. The left foot pad received equivalent amount of plain PBS. a, The footpad swelling was measured using digital calipers, and the net footpad swelling was determined by subtracting the value for the left contralateral footpad receiving plain PBS. b, Eleven days after injection, the mice were euthanized, and the number of cells in the local popliteal lymph node on the side of the footpad that received the archaeosome injection was counted. Data represent mean ± SD of n = 5 mice per dose.

 
Induction of CD8+ T cell responses and protective immunity in TLR2–/– mice

Pathogen-associated molecular patterns (PAMPs) interact with innate immune receptors, in particular TLRs on APCs to evoke a strong immune response. Some PAMPs thus constitute potent vaccine adjuvants (14, 31). In the context of pathogen lipid recognition, TLR2 has often been implicated (32). To ascertain whether archaeal lipid adjuvant effects were related to TLR2 activation, CD8+ T cell immunity was evaluated in TLR2-deficient and wild-type mice after MS-OVA immunization. Fig. 10a shows the representative tetramer staining profile on days 7 and 28 in wild-type and TLR2–/– mice, whereas Fig. 10b shows the mean distribution of CD8+OVA257–264-specific cells in the two strains of mice immunized with MS-OVA. The first injection of MS-OVA yielded similar numbers of Ag-specific CD8+ T cells in the peripheral blood on day 7 for both groups. The second MS-OVA injection gave the expected booster effect. Thus, both priming and boosting occurred similarly in the presence or absence of TLR2 phenotype.


Figure 10
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  		FIGURE 10. MS-OVA evokes Ag-specific CD8+ T cell response and tumor protection in TLR2–/– mice. Wild-type C57BL/6J (WT) and TLR2–/– mice were injected s.c. with 20 µg of OVA in 0.5 mg of MS lipid on days 0 and 21. At various time points, the blood was collected, and stained with OVA257–264-H-2Kb tetramer and anti-CD8. a, Representative staining for CD8 and OVA257–264 tetramer is indicated. b, Mean percentage ± SD of CD8+ T cells that were specific for OVA257–264 are plotted for the various times (n = 6/time point). c, On day 70 after vaccination, vaccinated or naive, wild-type and TLR2–/– mice were challenged with 106 B16OVA tumor cells in the mid dorsal region. Mean tumor size ± SEM, in n = 4 mice/group is indicated.

 
We then tested tumor protection in wild-type and TLR2–/– mice. Mice were vaccinated twice with MS-OVA and, at 60 days after the second immunization, mice were challenged s.c. with B16OVA tumor cells. The solid tumors progressed rapidly in naive wild-type mice with tumor reaching ~400 mm2 in 2 wk (Fig. 10c). In TLR2–/– nonvaccinated mice, there was a slight delay in tumor progression (Fig. 10c). Nevertheless, all the mice had tumors of >400 mm2 by day 30. As expected from the induction of similar Ag-specific CD8+ T cell numbers by MS-OVA in the two mice strains, mice vaccinated prophylactically were profoundly protected against B16OVA tumor cell challenge (Fig. 10c). Thus, in the absence of TLR2, archaeosomes evoke a strong and functional CD8+ T cell response to Ag.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Three parameters that may be expected to influence the success of T cell vaccines are the frequency, phenotype, and persistence of memory cells. In previous studies, we have shown that archaeosome adjuvants evoke a potent Ab, CD4+ and CD8+ T cell immunity to subunit vaccines (21, 21, 33). In this study, we profiled in detail the CD8+ T cell response for >1 year in mice after immunization with OVA entrapped in MS archaeosomes. We have shown that archaeosomes evoke profound in vivo Ag presentation resulting in long-lasting in vivo CTL activity. Priming and boosting with MS-OVA evoked a CD8+ T cell response that is comparable in magnitude to a live replicating immunogen LM-OVA. Furthermore, archaeosomes appear to provide a prolonged low-level Ag presentation, without the undue side effects associated with depot adjuvants such as CFA.

MS-OVA immunization is characterized by high initial burst of OVA257–264-tetramer-positive and IFN-{gamma}-secreting CD8+ T cells, followed by a gradual attrition of the response thereafter. A single injection of MS-OVA is less effective, whereas priming at day 0, followed by boosting at day 21 (or longer, up to at least 11 wk), provides a substantial increase in the Ag-specific CD8+ T cell response. At the peak of the response (a week after the second injection), endogenous tetramer frequencies of 20% of the total CD8+ T cells in the peripheral blood are achieved. During the subsequent contraction phase, this response declines only 3- to 4-fold. The contraction phase in a secondary response to infection is often less severe (3- to 5-fold) (34, 35), suggesting that the second injection with MS-OVA may have facilitated a response similar to a secondary challenge.

CD8+ T cells are heterogenous and in mice the subsets that have been characterized include: CD44highCD62LlowIL-7R{alpha}low effectors (TE), CD44highCD62LlowIL-7R{alpha}high effector memory (TEM), and CD44highCD62Lhigh central memory (TCM) (8, 36, 37). TEM traffic through nonlymphoid tissues and have strong cytolytic activity but relatively poor proliferative capability (38, 39). In contrast, TCM home to lymph nodes and possess high proliferative potential (40) allowing rapid expansion into effectors on encounter with Ag. However, it still unclear as to which subset is most beneficial for conferring long-term protective memory (5). Tracking the subsets of CD44 and CD62L coexpressing Ag-specific cells after a single MS-OVA vaccination, we observed that during the first week, the effector phase included predominantly CD44highCD62Llow phenotype cells. Furthermore, at this time the majority of Ag-specific CD8+ T cells had low IL-7R{alpha} expression suggestive of their highly activated state. However, by 3 wk a large proportion of Ag-experienced cells were CD44highCD62LhighIL-7R{alpha}high indicating the generation of TCM cells. The persistence of a high proportion of central phenotype cells before the booster explains the dramatic expansion of Ag-specific CD8+ T cells in response to the second injection of MS-OVA. However, after the second injection, there was no significant increase in cells with down-regulated IL-7R{alpha} expression. During the activation of naive CD8+ T cells, it has been suggested that the cells that down-regulate IL-7R{alpha} die, and only the cells that maintain high levels of IL-7R{alpha} expression during the peak response phase become memory cells (37). Thus, the down-regulation of IL-7R{alpha} appears to be coupled with apoptosis. Reduced down-regulation of IL-7R{alpha} expression on memory cells in response to a booster MS-OVA dose may therefore have facilitated their long-term survival. Nevertheless, beyond 60 days, while a single injection of MS-OVA facilitated maintenance of primarily OVA257–264-specific TCM, two injections of MS-OVA resulted in a balanced proportion of both TEM and TCM cells.

It has been suggested that the Ag load influences the proportions of the T cell subsets. TCM persist after rapid clearance of acute infections, and are more effective in controlling secondary infection with pathogens such as lymphocytic choriomeningitis virus (8). In contrast, chronic viral infections are reported to promote survival of TEM (41, 42). The persistence of primarily TCM cells at later times after a single MS-OVA immunization suggests a classical acute Ag exposure. Indeed profound cycling of >90% of Ag-specific cells was seen in the first few days after MS-OVA vaccination. These levels are similar to that seen with acute LM-OVA infection or CFA-OVA vaccination. However, after acute LM-OVA infection, cycling of Ag-specific cells declined rapidly. In contrast, MS-OVA promoted low-level cycling of Ag-specific cells for >50 days. This suggests continued low-level presentation of Ag to the CD8+ T cells by APCs at later time points and predicts an Ag-depot effect for archaeosomes. MS lipids are rich in bipolar, membrane-spanning isoprenoids (16, 18) that confer increased stability to the vesicles (43), and thus may be speculated to survive in the host for prolonged periods. Indeed CFA, known to provide an Ag-depot effect also allows prolonged cycling of Ag-specific cells. However, we noted that the intensity of cycling does decline from >90% at initial times to ~20% later. Thus, Ag burst appears to occur classically within the first week of MS-OVA vaccination, whereas, low-level Ag persistence may proceed at later times. However, there is a clear absence of overt inflammation at the injection site, as seen with toxic adjuvants such as CFA. The overall weaker stimulation at later times may have supported the presence of a constant population of TCM as well as the slower attrition of the TEM cells. For example, chronic Mycobacterium bovis, bacillus Calmette-Guérin infection, evokes an attenuated infection, and predominantly TCM phenotype of CD8+ T cells (44).

In chronic viral infections, Ag persistence induced the exhaustion of CD8+ T cells (45) or persistence of nonfunctional CD8+ T cells (46). However, CD8+ T cells evoked after MS-OVA vaccination were highly functional as evident by the memory recall CTL and IFN-{gamma} response >1 year after vaccination. Further, in vivo killing of specific targets was retained at >80% until 180 days. Thus the low-level, long-term Ag presentation evoked by MS-OVA did not lead to T cell exhaustion and the CD8+ T memory cells were capable of rapidly responding to an antigenic challenge. It is intriguing however, that a single injection of MS-OVA appeared to evoke weaker memory in comparison to a two injection schedule of priming and boosting with Ag adjuvant. Indicators of CD8+ T cell fitness including IL-7R{alpha} and CD27 expression suggest no significant difference in the functionality of CD8+ T memory cells evoked after single or two injections of MS-OVA. However tetramer analysis indicates a lower frequency of memory T cells after a single injection in comparison to two injections of MS-OVA. Thus, it may essentially be the magnitude of the responding memory T cell pool that dictated a stronger recall response after two injections of MS-OVA.

Adjuvants have long been essential components of vaccines, influencing the quantity and quality of immune responses. Nevertheless, the mechanism of action of many adjuvants has not been fully elucidated. Further, it is only recently that a direct link between recognition of PAMPs by host pathogen-recognition receptors (PRRs) and its consequence in directing subsequent adaptive immunity has come to light (47). PRRs constitute a limited number of germline-encoded recognition receptors for the host, of which the TLR family constitute the most broad spectrum receptors that recognize molecular patterns shared by bacteria, viruses, fungi, and parasites (32). Although several TLRs are known, those that consistently recognize lipids include the subfamily of TLR1, TLR2, and TLR6 (32). Of these, TLR2 has been implicated in a large number of lipid/lipoprotein interactions, including recognition of lipoarabinomannan from mycobacteria (48), lipoteichoic acid from group B Streptococcus (49), and porins from Neisseria species (50). MS-OVA evoked similar quantitative (Fig. 10) and phenotypic (data not shown) responses in TLR2–/– mice, suggesting noninteraction of MS lipids with TLR2. We have previously shown the ability of MS lipids to augment cytokine production from DCs. Consistent with the CD8+ T cell response in TLR2–/– mice, MS lipids evoked cytokine production from TLR2–/– DCs as well (data not shown). Another TLR that is unusual in its ability to recognize diverse structurally unrelated ligands is TLR4. For example, TLR4 recognizes, LPS, heat shock proteins, the plant diterpene paclitaxel and fibronectin that have different structures (32). We have however shown that archaeosomes serve as adjuvants in TLR4-deficient C3H/HeJ mice (33). However, we cannot fully exclude the possible involvement of TLR2 and TLR4 by the lipids in the mixture of polar lipids, where engagement of either receptor alone may be sufficient for adjuvant activity. Archaea are nonpathogenic, MS is a normal habitant of human colon, and to date there is only one rather speculative, suggestive, disease association with archaeal methanogens (51). Thus, it is understandable if archaeal cell components do not interact with PRRs. Nevertheless, archaeosomes evoke DC maturation and cytokine production (22), although not massive inflammation (19) as seen with many TLR ligands. We have shown that MS archaeosomes are recognized by a PS receptor on APCs through interaction with archaetidyl-PS (19). It is possible that PS-receptor interaction also facilitates DC activation to some extent. Alternatively, other PRRs such as C-type lectins and NOD proteins (47) may be involved in archaeal lipid recognition, as glycol-head groups are abundant on archaeal lipid structures (16).

The types of vaccines considered to be suited for evoking CD8+ T cell immunity include attenuated viruses or bacteria (live vaccines), replication-deficient recombinant viruses or bacteria (live vectored vaccines), DNA vaccines, and vaccines that use the prime-boost strategy with heterologous live vector adjuvants (52). These approaches combine the ability to provide sufficient immunostimulation, as well as targeting of CD8+ T cell immunity. Of these approaches, live vectors come with the risk of reconversion to virulence (53) and DNA vaccines often require either high doses or special delivery approaches for sustained immunity (54). Liposomes composed of conventional ester lipids have been considered for T cell vaccine development, but often require a codelivered immunostimulant for sufficient augmentation of innate immunity (55, 56). A heterologous prime-boost approach that combines DNA vaccines with a live-vectored boost is promising (57), but pre-existing immunity to live vectors in many humans (58) can compromise efficacy. In this study, we have demonstrated that archaeosomes are well-suited for evoking the full spectrum of CD8+ T cell response, and thus may be an attractive choice for T cell vaccine development. Furthermore, we have shown that priming and boosting with archaeosomes provides substantial enhancement of the initial T cell burst, similar to heterologous prime-boost strategies.

A number of challenges remain for T cell vaccine development, but the recent renaissance in innate immunity and the explosion of immunological assays for determining efficacy of vaccines holds great promise for the future. Although, the ultimate test for the efficacy of many adjuvants will come from human trials, studies such as these provide a necessary first step for the validation of novel adjuvants and shed light on the immune correlates for successful development of T cell vaccines in general. What our results have shown here is that archaeosomes are a good alternative to live vaccines as they induce responses similar in magnitude to LM, an intracellular pathogen that is considered to be a potent inducer of CD8+ T cell memory.


   Acknowledgments
 
We are thankful to Dr. Gordon Willick for peptide synthesis support. We acknowledge the technical assistance provided by Perry Fleming and Lise Deschatelets for archaeal growth and lipid extraction.


   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 supported by funds from the Ontario Cancer Research Institute and the National Research Council (NRC) of Canada. This is NRC Publication Number 42512. Back

2 Address correspondence and reprint requests to Dr. Lakshmi Krishnan, National Research Council-Institute for Biological Sciences, 1200 Montreal Road, Building M-54, Ottawa, Ontario, Canada. E-mail address: Lakshmi.Krishnan{at}nrc-cnrc.gc.ca Back

3 Abbreviations used in this paper: DC, dendritic cell; MS, Methanobrevibacter smithii; PS, phosphatidylserine; TPL, total polar lipid; LM, Listeria monocytogenes; PAMP, pathogen-associated molecular pattern; TE, effector T cell; TEM, effector memory T cell; TCM, central memory T cell; PRR, pathogen-recognition receptor. Back

Received for publication June 16, 2006. Accepted for publication November 28, 2006.


   References
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 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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