Rapid Clonal Expansion and Prolonged Maintenance of Memory CD8 + T Cells of the Effector (CD44 highCD62Llow) and Central (CD44 highCD62Lhigh) Phenotype by an This information is current as Archaeosome Adjuvant Independent of TLR2 of September 25, 2021. Lakshmi Krishnan, Komal Gurnani, Chantal J. Dicaire, Henk van Faassen, Ahmed Zafer, Carsten J. Kirschning, Subash Sad and G. Dennis Sprott

J Immunol 2007; 178:2396-2406; ; Downloaded from doi: 10.4049/jimmunol.178.4.2396 http://www.jimmunol.org/content/178/4/2396 http://www.jimmunol.org/ References This article cites 57 articles, 22 of which you can access for free at: http://www.jimmunol.org/content/178/4/2396.full#ref-list-1

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2007 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

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 Krishnan,2*† Komal Gurnani,* Chantal J. Dicaire,* Henk van Faassen,* Ahmed Zafer,* Carsten J. Kirschning,‡ Subash Sad,*† and G. Dennis Sprott*

Vaccines capable of eliciting long-term T 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 Downloaded from induced after immunization of mice with OVA-Methanobrevibacter smithii archaeosomes (MS-OVA). A single injection of MS- b ؉ OVA evoked a profound primary response but the numbers of H-2K OVA257–264-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 ϳ ؉ 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␣high)ofϳ5% included effector (CD44highCD62Llow) and high high central (CD44 CD62L ) phenotype cells. Recall response was observed even at >300 days. CFSE-labeled naive OT-1 http://www.jimmunol.org/

(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, ϳ25% 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؉ memory. The Journal of Immunology, 2007, 178: 2396–2406

he perception of a “danger signal” by APCs triggers the pathogen, the majority of Ag-specific T cells generated are innate immunity cascade which constitutes the first line of eliminated by and only a small population of memory by guest on September 25, 2021 T defense against infections. The danger signal context also cells survives for long-term maintenance (5). This stable mem- importantly directs subsequent adaptive immunity, as the APCs ory pool can provide life-long protection against reinfection, process and present pathogen-specific Ags on their MHC mole- although various factors such as homeostatic mechanisms and cules (1). T cells then embark on a programmed, intense, expan- heterologous infections can cause attrition of the T cell memory sion phase to counteract the rapid proliferation of pathogen. The pool over time (6, 7). ϩ ϩ two subsets of T cells, CD4 and CD8 , are contrastingly evoked Currently, effective vaccines are lacking for many diseases, in by Ag that is presented in the context of MHC class II or MHC ϩ particular chronic and intracellular infections, and cancer that re- class I, respectively (2). Classically, CD8 T cells are evoked quire a CD8ϩ T cell response for disease resolution. The paradigm against Ags generated in the cytosol, such as intracellular bac- ϩ ϩ of CD8 T cell induction, maintenance, and memory suggests an teria or viruses (3). CD8 T cells, with their unique ability to inherent difficulty in evoking CD8ϩ T cell immunity to subunit kill targets, are then able to eliminate the infected or vaccines, particularly in the absence of danger signal perception by (4). After the profound effector phase resulting in clearance of the host. Furthermore, CD8ϩ T cells segregate into effector and central memory subsets, the former with rapid effector function, *National Research Council-Institute for Biological Sciences, Ottawa, Ontario, and the latter with potent proliferative and lymph node homing Canada; †Department of Biochemistry, Microbiology, and Immunology, University properties (8). The manner in which an appropriate balance of ‡ of Ottawa, Ottawa, Ontario, Canada; and Institute for Microbiology, Immunology, these subsets can be evoked by vaccination for maximum benefit and Hygiene, Technical University, Munich, Germany to the host is unclear. Received for publication June 16, 2006. Accepted for publication November 28, 2006. Adjuvants are used in vaccines to provide immunostimulation The costs of publication of this article were defrayed in part by the payment of page and facilitate innate immunity and consequently can direct potent charges. This article must therefore be hereby marked advertisement in accordance adaptive immunity (9). Currently, only Alum and MF59 are ap- with 18 U.S.C. Section 1734 solely to indicate this fact. proved for widespread human use (10, 11) and both fail to evoke 1 This work was supported by funds from the Ontario Cancer Research Institute and a strong CD8ϩ T cell response. Thus far, the best choice for evok- the National Research Council (NRC) of Canada. This is NRC Publication Number ϩ 42512. ing CD8 T cell immunity to vaccines has been the use of live 2 Address correspondence and reprint requests to Dr. Lakshmi Krishnan, National recombinant bacterial and viral vectors as Ag delivery vehicles Research Council-Institute for Biological Sciences, 1200 Montreal Road, Building (12). Live vectors come with the risk of reconversion to virulence M-54, Ottawa, Ontario, Canada. E-mail address: [email protected] and may not be safe for use in aged and immunocompromised Copyright © 2007 by The American Association of Immunologists, Inc. 0022-1767/07/$2.00 individuals. Therefore, alternate strategies for evoking potent www.jimmunol.org The Journal of Immunology 2397

ϩ CD8 T cell immunity to vaccines are desired. Dendritic cells OVA (20 ␮g) in PBS (PBS-OVA), and OVA (25 ␮g) formulated in CFA (DCs)3 possess the unique ability to cross-present exogenous (CFA-OVA). The construction of LM-OVA has been described previously Ags on the MHC class I pathway and thus cross-prime CD8ϩ T (27). The immunization schedules were as described in figure legends. cells in vivo (13). Thus, novel immunostimulants that can har- Cell lines ness the cross-presentation properties of DCs hold promise for EL-4 (H-2Kb) was obtained from the American Type Culture Collection future vaccine development (14). (ATCC) and maintained in RPMI 1640 medium supplemented with 2-ME, Archaeal membranes are uniquely constituted of ether-linked 8% FBS, and 10 ␮g/ml gentamicin (R8 medium). EG.7 cells, a subclone of isoprenoid phytanyl core lipids (15, 16) that can be constituted into EL-4 stably transfected with the encoding OVA (28) were obtained stable Ag delivery vesicles, termed archaeosomes (17). The polar from the ATCC and cultured in R8 medium, additionally containing 400 ␮g/ml G418. B16OVA cells, expressing the gene for OVA were obtained lipid from the archaeon, Methanobrevibacter smithii (MS), is ad- from Dr. E. Lord (University of Rochester, Rochester, NY) and cultured in ditionally abundant in archaetidyl serine head groups (18), pro- R8 medium, additionally containing 400 ␮g/ml G418. All cells were main-

moting interaction with the phosphatidylserine (PS) on APCs tained at 37°C in 8% CO2. (19, 20). Thus, we have shown that MS archaeosomes, facilitate CTL assays cross-presentation of CD8ϩ T cell response (21) and activate DC ϫ 6 ϭ costimulation and production (19, 22). Furthermore, ar- Spleen cells (30 10 ) from pooled spleens (n 2–4) of immunized mice were cultured with 5 ϫ 105 irradiated (10,000 rad) EG.7 cells in 10 ml of chaeosomes evoke protective immunity against intracellular patho- R8 medium containing 0.1 ng/ml IL-2, in 25 cm2 tissue-culture flasks (Fal-

gens and cancer (23–25). In this study, we have characterized in detail con; BD Biosciences), kept upright. After 5 days (37°C, 8% CO2), the cells the CD8ϩ T cell induction, maintenance, and memory profile evoked were recovered from the flask and used as effectors in a standard 51Cr-

by MS archaeosomes. The T cell response has been followed kinet- release CTL assay (21). Supernatants were collected and radioactivity de- Downloaded from Ͼ tected by gamma counting. The percent-specific lysis was calculated using ically for 1 year in immunized mice and we have assessed the func- Ϫ Ϫ ϩ the formula: ((cpm experimental cpm spontaneous)/(cpm total cpm tionality of the CD8 T cells to kill targets in vivo. Furthermore, we spontaneous)) ϫ 100. One lytic unit is defined as the number of effector Ϫ Ϫ show that wild-type and TLR2 / mice evoke identical responses to cells per 106 spleen cells that yield 20% specific lysis of a population of ϩ 2.5 ϫ 104 targets. archaeosome Ag, suggesting archaeal lipids induce a potent CD8 T ϩ cell memory response in the absence of TLR2-mediated danger signal In vivo cytolytic activity of Ag-specific CD8 T cells was enumerated according to the protocol of Barber et al. (29). Donor spleen-cell suspen- perception. sions from syngenic mice were prepared and RBC lysed using Tris-buff- http://www.jimmunol.org/ ered ammonium chloride (RBC lysing solution). Cells were split in two

Materials and Methods aliquots. One aliquot was incubated for 30 min with OVA257–264 peptide Reagents (10 ␮g/ml) in R8 medium. Both cell aliquots were then stained with the dye PKH26 (4 ␮M). Then, the peptide-pulsed cell aliquot was stained with Sources for reagents were: OVA (grade VI), 2-ME, RBC lysing solution, 10ϫ CFSE (5 ␮M), whereas the other cell aliquot was stained with 1ϫ CFA, and the PKH26 Red Fluorescent Cell Linker , obtained from CFSE (0.5 ␮M) for 30 min in PBS. The two cell aliquots were mixed 1:1 Sigma-Aldrich; CFSE obtained from Molecular Probes; flow cytometric and injected (20 ϫ 106/mouse) into recipient mice that were immunized b Abs obtained from BD Biosciences; H-2K Ova257–264 tetramers ob- previously with MS-OVA. PBS-injected recipient mice served as controls. tained from Beckman Coulter; RPMI 1640 and gentamicin obtained At 24 h after the donor cell transfer, spleens were removed from recipients,

from Invitrogen Life Technologies; FBS obtained from HyClone; G418 single-cell suspensions were prepared, and cells were analyzed by flow by guest on September 25, 2021 obtained from Rose Scientific; and murine recombinant IL-2 obtained cytometry. The in vivo lysis percentage of peptide-pulsed targets was enu- from ID Labs. OVA257–264 (SIINFEKL) peptide was synthesized merated according to a previously published equation (29). in-house. Enumeration of IFN-␥-secreting cells Preparation and characterization of archaeosomes Enumeration of IFN-␥-secreting cells was done by ELISPOT assay. MS ALI (DSM 2375) was grown in fermenters, total lipids extracted from Briefly, spleen cells were incubated in anti-IFN-␥ Ab-coated ELISPOT frozen cell pastes, and the total polar lipids (TPL) were collected as the plates in various numbers (in a final cell density of 5 ϫ 105/well using acetone-insoluble fraction. Absence of detectable DNA in TPL was con- feeder cells) in the presence of IL-2 (1 ng/ml) and R8 medium or ␮ firmed by electrophoresis on agarose gels followed by ethidium bromide OVA257–264 (10 g/ml) for 48 h at 37°C, 8% CO2. The plates were staining. Archaeosomes were prepared by entrapping OVA (lacking frag- subsequently blocked, incubated with the biotinylated secondary Ab ments) in MS TPL by the dried-reconstituted vesicle method; the amount (4°C, overnight), followed by -peroxidase conjugate (room tem- of incorporated into the vesicle was estimated by modified SDS perature for 2 h). Spots were revealed using diamino benzidine. Lowry, as described in detail previously (17). These vesicles are referred to ϩ as MS-OVA. The ratio of protein to lipid was based on the salt-free dry Assessment of numbers and phenotype of Ag-specific CD8 weights of the vesicles and was 40–70 ␮g of OVA/1 mg of lipid. All T cells in vivo vesicles were unilamellar and diameters were in the range of 100–200 nm, ϩ The activation of Ag-specific CD8 T cells was tracked in vivo using the determined by number-weighted Gaussian size distributions using a tetramer assay. Briefly, spleen cells (10 ϫ 106) or PBLs (obtained from 100 Nicomp Particle sizer. All glassware used in the preparation of archaeo- ␮l of blood) were incubated in 200 ␮l of PBS plus 1% BSA (PBS-BSA) somes was prebaked (6 h at 180°C) to render them pyrogen-free and en- with anti-CD16/32 at 4°C. After 10 min., cells were stained with PE-H- dotoxin-free reagents were used throughout. b 2K OVA257–264 tetramer and various Abs (anti-CD8, anti-CD62L, anti- Mice and immunizations CD44, and anti-IL-7R␣) for 30 min at room temperature. Cells were washed with PBS and fixed in 0.5% formaldehyde and acquired on a BD Inbred, 6- to 8-wk-old female C57BL/6J, OT-1, and B6.PL mice were Biosciences FACS Canto Analyzer. Alternatively, to amplify the tetramer Ϫ Ϫ obtained from The Jackson Laboratory. Breeding pairs of TLR2 / mice signal, spleen cells (106) from OT-1 mice in HBSS, were injected i.v. into backcrossed onto a C57BL/6J background (26) were bred at the National recipient mice, at the same time that they received MS-OVA s.c. At various ϩ Research Council-Institute for Biological Sciences Animal Facility. Mice time points thereafter, the proportion of OVA257–264-specific donor CD8 were maintained in accordance with guidelines from the Canadian Council T cells that proliferated and differentiated in response to MS-OVA immu- on Animal Care. nization in the recipient mice was determined using the tetramer assay of Mice were immunized s.c. at the base of the tail with MS-OVA (15–25 spleen and/or blood . Analysis of IL-7R␣ϩ or CD44ϩCD62Lϩ ␮ ␮ b ϩ g of OVA in 0.2–0.5 mg of lipid/100 l of PBS), Listeria monocytogenes on H-2K OVA257–264-tetramer positive CD8 T cells was also done using expressing OVA (LM-OVA, 103 CFU/injection/100 ␮l of 0.9% saline), multiplex staining of the samples simultaneously. Assessment of in vivo cycling of Ag-specific CD8ϩ T cells 3 Abbreviations used in this paper: DC, dendritic cell; MS, Methanobrevibacter ϩ smithii; PS, phosphatidylserine; TPL, total polar lipid; LM, Listeria monocytogenes; Spleen cells from donor OT-1-transgenic mice (Thy1.2 ) were labeled PAMP, pathogen-associated molecular pattern; TE, effector T cell; TEM, effector with CFSE. Briefly, spleen cell suspensions were prepared and RBC re- ; TCM, central memory T cell; PRR, pathogen-recognition receptor. moved with RBC lysing solution. Spleen cells were resuspended in PBS 2398 MAINTENANCE OF CD8ϩ T CELL MEMORY BY ARCHAEOSOMES

(20 ϫ 106/ml) and an equal volume of CFSE (5 ␮M 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 ϫ 106) cells were injected i.v. into recipient B6.PL (Thy1.1ϩ) or C57BL/6J mice that had been immunized with MS-OVA. Four days later, recipient mice were euthanized and the numbers of donor ϩ b ϩ origin (Thy1.2 ) and H-2K OVA257–264-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 TLR2Ϫ/Ϫ 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 Induction of long-term CD8ϩ 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 Downloaded from potent CD8ϩ T cell immunity (21). We kinetically tracked the longevity of CTL for Ͼ1 year after initial immunization. Mice received two injections of MS-OVA 3 wk apart. At regular inter- vals we measured the in vitro CTL activity of splenocytes from representative mice (n ϭ 3/time point). In a standard chromium

release CTL assay, splenocyte effectors (generated after restimu- http://www.jimmunol.org/ lation 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 ϩ time, and is shown in Fig. 1b. A single immunization of MS-OVA 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) afforded ϳ10 lytic units/106 spleen cells on day 14 and a second with 15 ␮g of OVA entrapped in 0.285 mg of lipid. Spleen cells (n ϭ 3) immunization provided a booster effect. The CTL activity subse- were pooled and restimulated for 5 days in vitro with EG.7 cells and 0.1 ϳ by guest on September 25, 2021 quently underwent a gradual attrition, maintaining at 1 lytic unit/ ng/ml IL-2. a, Splenic effectors generated on day 327 or 449 from mice 6 10 spleen cells. A booster injection with Ag (in the absence of immunized twice on days 0 and 21 were assessed for ability to kill EL-4 adjuvant) at day 306 resulted in a dramatic increase in the CTL and EG.7 targets in a 51Cr-release assay. The percent-specific killing Ϯ SD intensity at day 327, in mice primed and then boosted at 3 wk with at the various E:T ratios is indicated. b, The percent-specific killing data MS-OVA. obtained from representative mice at various times were converted to lytic The number of Ag-specific IFN-␥ secreting CD8ϩ T cells was units. “I” indicates immunization on days 0 and 21, and “R” indicates a ␥ also determined for the same splenocyte samples (n ϭ 3/time recall injection with Ag on day 306. c, Frequency of IFN- -secreting cells point). The immunodominant CTL epitope of OVA for H-2Kb in the spleen of individual mice was determined by ELISPOT assay. Mean frequency Ϯ SD of IFN-␥-secreting cells per 106 spleen cells is indicated haplotype has been shown to be OVA257–264 (SIINFEKL) (30). ϭ ␥ for n 3 per time point. d, Mice were immunized with MS-OVA on days Therefore, IFN- production in response to stimulation with this 0 and 21 or days 0 and 77. At day 126 post first injection, mice were peptide was determined by ELISPOT assay. The frequency of Ag- euthanized, splenic effectors were generated, and the ability to kill EL-4 ϩ 6 specific IFN-␥-secreting CD8 T cells was ϳ200/10 splenocytes and EG.7 targets in a 51Cr-release assay was determined. The percent- at early time points after two injections of MS-OVA, gradually specific killing Ϯ SD at the various E:T ratios is indicated for effectors showing a decrease over time (Fig. 1c). The frequency then obtained from n ϭ 3 mice per group. showed an increase on recall with an Ag injection, nearly a year after immunization, demonstrating the potential functionality of the Ag-specific CD8ϩ T cells. Again, priming followed by a booster injection with MS-OVA was required to maintain a higher result of substantial in vitro expansion of the central subset cells. ϩ frequency of CD8 T cells in the long-term, as opposed to a single To ascertain the physiological relevance of the CD8ϩ CTL re- injection. sponse, we measured killing of specific targets in vivo after MS- Although these results illustrate the importance of a second in- OVA immunization (Fig. 2), using the recently developed assay jection, the timing for the booster was not critical. The magnitude (29). A single injection of MS-OVA resulted in the ability of the of the CTL response measured 18 wk after the first injection was host to eliminate Ͼ80% of the in vivo targets within 24 h, on day not significantly different for mice receiving the MS-OVA booster 7 after immunization (Fig. 2). However, this ability declined rap- after 3 or 11 wk (Fig. 1d). idly, with Ͻ15% of the targets being killed at day 21 and beyond (Fig. 2b). In contrast, two injections of MS-OVA on days 0 and 21 Induction of in vivo CTL activity by MS-OVA resulted in complete in vivo elimination of the targets within 24 h, In vitro CTL activity is measured after restimulation of the spleno- on day 30 (Fig. 2). Furthermore, the in vivo killing ability was cytes with Ag in vitro for 5 days, which could under- or overes- retained at Ͼ80% even 180 days after immunization, reflecting the timate the true functionality of CD8ϩ T cells in vivo. Thus, it was long-term functionality of the Ag-specific CD8ϩ T cell response possible that the dramatic CTL activity observed at Ͼ1 year was a evoked by MS-OVA (Fig. 2b). The Journal of Immunology 2399

ϩ 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 CD8ϩ T cells and is a powerful measure of in vivo frequency. Mice were immunized with MS-OVA and, at various time points, the fre- b ϩ quency of H-2K OVA257–264-specific CD8 T cells in the pe- ripheral blood was assessed by tetramer staining. Fig. 3a shows representative tetramer staining on gated CD8ϩ T cells in the pe- ripheral blood. The kinetics of induction and maintenance of the Ag-specific CD8ϩ T cell frequencies are shown in Fig. 3b.Onday 7 after MS-OVA injection, 3–5% of CD8ϩ T cells were tetramer positive in the blood. This number dramatically expanded to ϳ25% of CD8ϩ T cells after the booster MS-OVA injection. A single injection of MS-OVA resulted in maintenance of the T cell frequency at Ͻ1%, whereas two injections of MS-OVA facilitated maintenance of the T cell frequency at ϳ5%. Furthermore, two injections of MS-OVA resulted in a gradual attrition of tetramer- positive cells over time. Priming of CD8ϩ T cell response with MS-OVA, a particulate immunogen, was similar in magnitude to a Downloaded from 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). CD8ϩ T cells have been classified into effector (CD44high CD62Llow) and central (CD44highCD62Lhigh) phenotype cells (8). http://www.jimmunol.org/ FIGURE 2. Archaeosomes evoke potent in vivo CTL response to en- Fig. 3c shows the kinetic distribution of effector and central phe- ␮ ϩ trapped Ag. C57BL/6J mice were immunized s.c. with 20 gofOVA notype OVA257–264-specific CD8 T cells. A single immunization in 0.5 mg of MS lipid once or twice (days 0 and 21). At various time of MS-OVA primed effectors but these underwent attrition rapidly, points thereafter, representative mice (n ϭ 4 per time point) were in- and primarily central phenotype cells were noted in the long-term. jected i.v. with equimolar proportions of nonspecific (labeled with 0.5 Priming followed by boosting with MS-OVA on day 21 facilitated ␮ ␮ M CFSE) and peptide-pulsed specific (labeled with 5 M CFSE) massive clonal expansion and the effectors generated underwent PKH26-labeled syngenic donor spleen cells. Twenty-four hours later, the proportion of the two CFSE-labeled populations of donor cells in gradual attrition. Furthermore, after the booster injection a stable

the spleen was determined by flow cytometry. a, Representative profile pool of both effector and central phenotype cells persisted. A sin- by guest on September 25, 2021 of PKH26-gated, CFSE-labeled populations in naive vs MS-OVA gle dose of LM-OVA also evoked profound effector response ini- immunized mice on day 7 after a single injection, or on day 30 (after tially, but later mainly central phenotype cells persisted (Fig. 3c). ϩ two injections of MS-OVA). b, The percent-specific killing Ϯ SEM To clearly monitor the phenotype of OVA-specific CD8 T deduced for the various time points after a single or two MS-OVA cells in the long-term, we used an alternate adoptive transfer injections. model, wherein 106 spleen cells (comprising ϳ105 CD8ϩ T cells) from OT-1 mice (with Ͼ90% of CD8ϩ T cells specific for

OVA257–264) were transferred i.v. into syngenic recipient mice.

FIGURE 3. Rapid induction of OVA257–264- tetramer-specific cells by MS-OVA. C57BL/6J mice (n ϭ 4) were immunized s.c. once or twice (days 0 and 21) with 20 ␮g of OVA in 0.3 mg of MS lipid, twice with 20 ␮g 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 tet- ramer staining, and expression of CD62L and CD44. a, Representative flow cytometric profile indicating the percentage of gated CD8ϩ T cells d positive for OVA257–264 H-2K tetramer. b, Mean percentage Ϯ SEM of CD8ϩ T cells that

were OVA257–264-specific is shown for the var- ious time points (n ϭ 4/group). c, The distribu- tion (mean percentage Ϯ SEM) of CD44high low high high CD62L , or CD44 CD62L , OVA257–264- specific cells for the various groups over time (n ϭ 4/time point), is shown. All data are de- duced from the analysis of 100,000 events ac- quired for each mouse per time point. 2400 MAINTENANCE OF CD8ϩ T CELL MEMORY BY ARCHAEOSOMES Downloaded from

ϩ high high FIGURE 4. A single injection of MS-OVA primes OVA257–264-specific CD8 T cells and results in the maintenance of CD44 CD62L 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 ␮g 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 CD8ϩ T cells positive for b ϩ OVA257–264 H-2K 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 Ϯ SEM of peripheral blood CD8ϩ T cells that were OVA -specific is shown for the various time points (n ϭ 3/group). The 257–264 http://www.jimmunol.org/ ϩ 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 CD8ϩ T cells that were CD44highCD62Lhigh (central phenotype) or CD44highCD62Llow (effector phenotype) on days 7, 14, 30, and 60. d, Mean percentage Ϯ SEM of peripheral blood Ag-specific CD8ϩ 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.

The recipient mice were then injected s.c. with MS-OVA or LM- 30, compared with preboost levels. Further, this resulted in main-

OVA and at various time points the percentage and phenotype of tenance of higher frequency of OVA257–264-tetramer-specific cells Ag-specific T cells were tracked. In this adoptive transfer model, beyond 50 days (ϳ8% in the spleen (Fig. 5a) and Ͼ20% in pe- by guest on September 25, 2021 parking naive OT-1 cells in the host before immunization in- ripheral circulation (Fig. 5b)). Interestingly, PBS-OVA failed to creased the initial frequency of responding Ag-specific cells, re- evoke any significant expansion of tetramer-specific cells even in sulting in amplification of the tetramer-positive response. There- this adoptive transfer model, wherein a higher frequency of naive fore, a single s.c. injection of MS-OVA to recipient mice results in OT.1 responders were made available. Fig. 5c shows the pheno- ϳ ϩ 20% OVA257–264-tetramer specific splenic CD8 T cells on day typic distribution of Ag-specific subsets in the peripheral blood 7 (Fig. 4a). This number subsequently dropped and was main- after two doses of MS-OVA and suggests a balanced maintenance tained at ϳ2% beyond 60 days. The magnitude of induction of of both effector and central phenotype memory cells. Ag-specific CD8ϩ T cells by MS-OVA was comparable to s.c. ␣ LM-OVA infection (Fig. 4a), a potent live vector. Fig. 4b shows Long-term IL-7R expression on Ag-specific cells evoked ϩ by MS-OVA correlates with a memory phenotype the frequency of OVA257–264-specific CD8 T cells in the periph- eral blood of mice administered a single injection of MS-OVA or CD44 and CD62L expression of CD8ϩ T cells is often not suffi- LM-OVA. Fig. 4c shows the splenic distribution of the effector and cient to discern early and late stage effectors. We therefore addi- central subset of tetramer-gated CD8ϩ T cells after s.c. MS-OVA tionally studied IL-7R␣ expression on Ag-specific cells in the or LM-OVA immunizations. At day 7 after immunization, the ma- peripheral blood evoked by MS-OVA. Fig. 6a shows the rep- ϩ ␣ ϩ jority (73%) of CD8 OVA257–264-specific cells evoked by MS- resentative profile of IL-7R on OVA257–264-specific CD8 T OVA were of the effector phenotype (CD44highCD62Llow). How- cells, on day 7 and day 28 after a single MS-OVA or LM-OVA ever, by day 30 the Ag-specific CD8ϩ T cells were predominantly injection. As expected in the early stages of the response, on day (83%) of the central phenotype (CD44highCD62Lhigh). Fig. 4d re- 7, a large proportion of the Ag-specific CD8ϩ T cells had down- iterates that a similar phenotypic distribution of cells occurred in regulated expression of IL-7R␣. However, by day 28, Ͼ75% of the the peripheral blood. Overall, a single injection of MS-OVA or cells had high IL-7R␣ expression. Fig. 6b shows that a second LM-OVA resulted in the maintenance of low numbers of Ag-spe- injection of MS-OVA (administered on day 21) did not evoke cific memory cells predominantly of the central (CD44high significant numbers of IL-7R␣low cells on day 28 (Fig. 6b). Thus, CD62Lhigh) phenotype. the second injection may have led to expansion of committed Fig. 5a shows the representative profiles of splenic tetramer memory (IL-7R␣high) cells that do not down-regulate IL-7R␣. The staining and phenotypic distribution of cells, after two injections kinetic distribution of Ag-specific CD8ϩ IL-7R␣high T cells over with MS-OVA on days 0 and 21. Fig. 5b shows the kinetics of the the long-term is shown in Fig. 6c, and suggests a similar functional frequency of OVA257–264-specific cells in the peripheral blood memory phenotype after single or two doses of MS-OVA. Simi- evoked by MS-OVA, in comparison to immunization with two larly, LM-OVA also evoked memory CD8ϩ T cells of the IL- doses of PBS-OVA. The second injection of MS-OVA resulted in 7Rhigh phenotype. CD27 expression on Ag-specific cells was also 10-fold expansion of numbers of Ag-specific cells at around day followed in the peripheral blood of MS-OVA-vaccinated mice and The Journal of Immunology 2401

ϩ

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 Downloaded from populations. Mice were first parked i.v. with 106 OT-1 spleen cells. They then received s.c. immunization of 20 ␮g of OVA in 0.3 mg of MS lipid, or 20 ␮ ϩ g 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 CD8ϩ T cells that were CD44highCD62Lhigh (central) or CD44highCD62Llow (effector) phenotype, at the various time points after MS-OVA Ϯ ϩ ϩ ϭ Ϯ 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 ϭ 5 mice per time point. All data are derived from 100,000 gated CD8ϩ events acquired for each sample. http://www.jimmunol.org/ indicated early down-regulation in response to activation on day 7 cells were cycling. In contrast, in OVA-archaeosome-immunized and subsequent up-regulation as the cells progressed to the mem- mice, when CFSE labeled OT-1 cells were transferred 3 days after ory phenotype (data not shown). immunization and tracked on day 7, profound cycling of trans- ferred cells (95%) based on CFSE reduction profile was noted OVA archaeosomes elicit profound in vivo Ag presentation indicating efficient Ag presentation and stimulation (Fig. 7). Trans- We next evaluated the in vivo Ag presentation by OVA archaeo- fer of CFSE labeled OT-1 cells at later time points resulted in somes in an adoptive transfer model, wherein naive OT-1-CFSE- lower, but significant, cycling of donor cells indicating the con- labeled spleen cells were injected into mice previously immunized tinued presentation of Ag by host APCs. For example, between by guest on September 25, 2021 with OVA archaeosomes and proliferation of Ag-specific CD8ϩ T day 9 and 13, 82% of donor cells cycled, and even on days 17–21 b cells was tracked 4 days later based on H-2K OVA257–264 tetramer and 48–52 25% of the donor cells cycled (Fig. 6). At all time staining and CFSE reduction. As hosts and donors differed in their points, mice injected with PBS showed little (2–5%) cycling (data expression of Thy1.2 allele, the proliferation of donor cells could not shown). We then compared this cycling profile to that induced be tracked specifically using an anti-Thy1.2 Ab. When OT-1 cells by s.c. infection of LM-OVA or OVA administered in CFA (Fig. were transferred into PBS injected mice, ϳ4% of donor CD8ϩ T 8). As with MS-OVA, at early time points after injection,

FIGURE 6. Profile of IL-7R␣ expression after MS- OVA immunization. IL-7R␣ (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 ␮g OVA per 0.89 mg of lipid/injection). a, Representative profile indicating the CD127 staining ϩ 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␣high cells. b, Repre- sentative profile indicating percentage of IL-7R␣high

OVA257–264-specific cells, 7 days after the second in- Ϯ ␣high jection. c, Mean SEM of IL-7R OVA257–264- specific cells at various time points (n ϭ 3/time point) after a single injection of LM-OVA or MS-OVA or two injections (days 0 and 21) of MS-OVA. 2402 MAINTENANCE OF CD8ϩ T CELL MEMORY BY ARCHAEOSOMES

FIGURE 7. Archaeosomes evoke prolonged Ag presentation. Thy1.1ϩ B6.PL mice were immunized s.c. with 25 ␮g of OVA entrapped in MS ar- chaeosomes (0.2 mg of lipid). On day 3, 9, 17, or 48, naive and MS-OVA-immunized Thy1.1ϩ recipients were injected i.v. with CFSE-labeled OT-1 (Thy1.2ϩ) cells. Four days later (on day 7, 13, 21, or 52 respectively), mice were euthanized, spleens removed, and the numbers of CD8ϩ T cells of donor origin (Thy1.2ϩ) was determined. Further analysis was conducted to deduce the reduction in CFSE of gated CD8ϩ 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 CD8ϩ T cells that were cycling based on CFSE reduction. b, The mean percentage Ϯ SEM of cycling (n ϭ 3–5 mice) for each of the time points mentioned above. Downloaded from

LM-OVA and CFA-OVA evoked profound cycling of donor at the injection site caused undesirable side effects. Mice were ϩ OVA257–264-specific CD8 T cells. However, with LM-OVA, the injected with various doses of Ag-free MS archaeosomes in one cycling declined to ϳ15% by day 10 and to Ͻ3% after day 15, footpad. The local edema was measured using calipers. Fig. 9a suggesting clearance of the Ag. CFA-OVA resulted in sustained indicates that any local reaction that was observed with a high dose cycling of donor cells at later time points (30 days) similar to of archaeosomes rapidly declined after the first 24 h, with no sig- http://www.jimmunol.org/ MS-OVA (Fig. 8), suggesting an Ag depot effect for these two nificant local edema or redness noted at later time points. Archaeo- adjuvants. Thus, archaeosomes were comparable to CFA and prob- somes, however, evoked an increase in the number of local pop- ably better than LM in terms of the extent and duration of Ag liteal lymph node cells (Fig. 9b) consistent with the facilitation of presentation. cell-mediated immunity and activation of DCs (22). However, this increase was not as profound as expected of adjuvants such as Lack of local edema after archaeosome injection CFA or for infections with associated inflammation. Because CFA CFA is known to provide a strong Ag depot effect but is a toxic is not approved to be used in such experiments due to exacer- adjuvant unsuitable for human applications. As MS-OVA also pro- bated footpad swelling and edema, our results suggest that ϩ moted sustained Ag presentation in vivo, suggesting an Ag depot archaeosomes induced potent CD8 T cell response without the by guest on September 25, 2021 effect, we tested whether the prolonged presence of archaeosomes undesirable inflammation. Induction of CD8ϩ T cell responses and protective immunity in TLR2Ϫ/Ϫ mice Pathogen-associated molecular patterns (PAMPs) interact with in- nate immune receptors, in particular TLRs on APCs to evoke a strong immune response. Some PAMPs thus constitute potent vaccine

FIGURE 8. Ag presentation evoked by MS-OVA is similar to CFA- FIGURE 9. MS-OVA injection does not cause local edema or inflam- OVA depot effect. Thy1.1ϩ B6.PL mice were immunized s.c. with 25 ␮g mation. Mice were immunized with varying dose of Ag-free MS archaeo- of OVA entrapped in MS (0.2 mg of lipid), or LM-OVA (103 CFU), or 25 somes in 50 ␮l of PBS in the right foot pad. The left foot pad received ␮g of OVA formulated in CFA. On day 3, 9, 17, or 24, naive and immu- equivalent amount of plain PBS. a, The footpad swelling was measured nized recipients (n ϭ 3 per time point) were injected i.v. with CFSE- using digital calipers, and the net footpad swelling was determined by labeled OT-1 (Thy 1.2ϩ cells). Four days later (on day 7, 13, 21, or 28), the subtracting the value for the left contralateral footpad receiving plain PBS. numbers of splenic CD8ϩ T cells of donor origin that were also cycling b, Eleven days after injection, the mice were euthanized, and the number based on CFSE reduction was deduced. In each panel, the boxed region of cells in the local popliteal lymph node on the side of the footpad that refers to the CFSE reduction and the numbers indicate percentage of donor- received the archaeosome injection was counted. Data represent mean Ϯ SD specific CD8ϩ T cells that were cycling. of n ϭ 5 mice per dose. The Journal of Immunology 2403

FIGURE 10. MS-OVA evokes Ag-specific CD8ϩ T cell response and tumor protection in TLR2Ϫ/Ϫ mice. Wild-type C57BL/6J (WT) and TLR2Ϫ/Ϫ mice were in- jected s.c. with 20 ␮g of OVA in 0.5 mg of MS lipid on days 0 and 21. At various time points, the blood was b collected, and stained with OVA257–264-H-2K tetramer and anti-CD8. a, Representative staining for CD8 and

OVA257–264 tetramer is indicated. b, Mean percent- age Ϯ SD of CD8ϩ T cells that were specific for ϭ OVA257–264 are plotted for the various times (n 6/time point). c, On day 70 after vaccination, vaccinated or naive, wild-type and TLR2Ϫ/Ϫ mice were challenged with 106 B16OVA tumor cells in the mid dorsal region. Downloaded from Mean tumor size Ϯ SEM, in n ϭ 4 mice/group is indicated. http://www.jimmunol.org/

adjuvants (14, 31). In the context of pathogen lipid recognition, munization with OVA entrapped in MS archaeosomes. We have TLR2 has often been implicated (32). To ascertain whether ar- shown that archaeosomes evoke profound in vivo Ag presentation chaeal lipid adjuvant effects were related to TLR2 activation, resulting in long-lasting in vivo CTL activity. Priming and boost- ϩ CD8 T cell immunity was evaluated in TLR2-deficient and wild- ing with MS-OVA evoked a CD8ϩ T cell response that is com- by guest on September 25, 2021 type mice after MS-OVA immunization. Fig. 10a shows the rep- parable in magnitude to a live replicating immunogen LM-OVA. resentative tetramer staining profile on days 7 and 28 in wild-type Furthermore, archaeosomes appear to provide a prolonged low- and TLR2Ϫ/Ϫ mice, whereas Fig. 10b shows the mean distribution level Ag presentation, without the undue side effects associated ϩ of CD8 OVA257–264-specific cells in the two strains of mice im- with depot adjuvants such as CFA. munized with MS-OVA. The first injection of MS-OVA yielded MS-OVA immunization is characterized by high initial burst of ϩ ␥ ϩ similar numbers of Ag-specific CD8 T cells in the peripheral OVA257–264-tetramer-positive and IFN- -secreting CD8 T cells, blood on day 7 for both groups. The second MS-OVA injection followed by a gradual attrition of the response thereafter. A single gave the expected booster effect. Thus, both priming and boosting injection of MS-OVA is less effective, whereas priming at day 0, occurred similarly in the presence or absence of TLR2 phenotype. followed by boosting at day 21 (or longer, up to at least 11 wk), We then tested tumor protection in wild-type and TLR2Ϫ/Ϫ provides a substantial increase in the Ag-specific CD8ϩ T cell mice. Mice were vaccinated twice with MS-OVA and, at 60 days response. At the peak of the response (a week after the second after the second immunization, mice were challenged s.c. with injection), endogenous tetramer frequencies of 20% of the total B16OVA tumor cells. The solid tumors progressed rapidly in naive CD8ϩ T cells in the peripheral blood are achieved. During the wild-type mice with tumor reaching ϳ400 mm2 in 2 wk (Fig. 10c). subsequent contraction phase, this response declines only 3- to Ϫ Ϫ In TLR2 / nonvaccinated mice, there was a slight delay in tumor 4-fold. The contraction phase in a secondary response to infection is progression (Fig. 10c). Nevertheless, all the mice had tumors of often less severe (3- to 5-fold) (34, 35), suggesting that the second Ͼ400 mm2 by day 30. As expected from the induction of similar injection with MS-OVA may have facilitated a response similar to a ϩ Ag-specific CD8 T cell numbers by MS-OVA in the two mice secondary challenge. strains, mice vaccinated prophylactically were profoundly pro- CD8ϩ T cells are heterogenous and in mice the subsets that have tected against B16OVA tumor cell challenge (Fig. 10c). Thus, in been characterized include: CD44highCD62LlowIL-7R␣low effec- high low ␣high the absence of TLR2, archaeosomes evoke a strong and functional tors (TE), CD44 CD62L IL-7R effector memory (TEM), ϩ high high CD8 T cell response to Ag. and CD44 CD62L central memory (TCM) (8, 36, 37). TEM traffic through nonlymphoid tissues and have strong cytolytic ac- Discussion tivity but relatively poor proliferative capability (38, 39). In con-

Three parameters that may be expected to influence the success of trast, TCM home to lymph nodes and possess high proliferative T cell vaccines are the frequency, phenotype, and persistence of potential (40) allowing rapid expansion into effectors on encounter memory cells. In previous studies, we have shown that archaeo- with Ag. However, it still unclear as to which subset is most ben- some adjuvants evoke a potent Ab, CD4ϩ and CD8ϩ T cell im- eficial for conferring long-term protective memory (5). Tracking munity to subunit vaccines (21, 21, 33). In this study, we profiled the subsets of CD44 and CD62L coexpressing Ag-specific cells in detail the CD8ϩ T cell response for Ͼ1 year in mice after im- after a single MS-OVA vaccination, we observed that during the 2404 MAINTENANCE OF CD8ϩ T CELL MEMORY BY ARCHAEOSOMES

first week, the effector phase included predominantly CD44high tion schedule of priming and boosting with Ag adjuvant. Indicators CD62Llow phenotype cells. Furthermore, at this time the majority of CD8ϩ T cell fitness including IL-7R␣ and CD27 expression of Ag-specific CD8ϩ T cells had low IL-7R␣ expression sugges- suggest no significant difference in the functionality of CD8ϩ T tive of their highly activated state. However, by 3 wk a large pro- memory cells evoked after single or two injections of MS-OVA. portion of Ag-experienced cells were CD44highCD62Lhigh However tetramer analysis indicates a lower frequency of memory ␣high IL-7R indicating the generation of TCM cells. The persistence T cells after a single injection in comparison to two injections of of a high proportion of central phenotype cells before the booster MS-OVA. Thus, it may essentially be the magnitude of the re- explains the dramatic expansion of Ag-specific CD8ϩ T cells in sponding memory T cell pool that dictated a stronger recall re- response to the second injection of MS-OVA. However, after the sponse after two injections of MS-OVA. second injection, there was no significant increase in cells with Adjuvants have long been essential components of vaccines, down-regulated IL-7R␣ expression. During the activation of naive influencing the quantity and quality of immune responses. Never- CD8ϩ T cells, it has been suggested that the cells that down- theless, the mechanism of action of many adjuvants has not been regulate IL-7R␣ die, and only the cells that maintain high levels of fully elucidated. Further, it is only recently that a direct link be- IL-7R␣ expression during the peak response phase become mem- tween recognition of PAMPs by host pathogen-recognition recep- ory cells (37). Thus, the down-regulation of IL-7R␣ appears to be tors (PRRs) and its consequence in directing subsequent adaptive coupled with apoptosis. Reduced down-regulation of IL-7R␣ ex- immunity has come to (47). PRRs constitute a limited number pression on memory cells in response to a booster MS-OVA dose of germline-encoded recognition receptors for the host, of which may therefore have facilitated their long-term survival. Neverthe- the TLR family constitute the most broad spectrum receptors that less, beyond 60 days, while a single injection of MS-OVA facil- recognize molecular patterns shared by bacteria, viruses, fungi, Downloaded from itated maintenance of primarily OVA257–264-specific TCM, two in- and parasites (32). Although several TLRs are known, those that jections of MS-OVA resulted in a balanced proportion of both TEM consistently recognize lipids include the subfamily of TLR1, and TCM cells. TLR2, and TLR6 (32). Of these, TLR2 has been implicated in a It has been suggested that the Ag load influences the proportions large number of lipid/lipoprotein interactions, including recogni- of the T cell subsets. TCM persist after rapid clearance of acute tion of lipoarabinomannan from mycobacteria (48), lipoteichoic infections, and are more effective in controlling secondary infec- acid from group B Streptococcus (49), and porins from Neisseria http://www.jimmunol.org/ tion with pathogens such as lymphocytic choriomeningitis virus species (50). MS-OVA evoked similar quantitative (Fig. 10) and (8). In contrast, chronic viral infections are reported to promote phenotypic (data not shown) responses in TLR2Ϫ/Ϫ mice, suggest- survival of TEM (41, 42). The persistence of primarily TCM cells at ing noninteraction of MS lipids with TLR2. We have previously later times after a single MS-OVA immunization suggests a clas- shown the ability of MS lipids to augment cytokine production sical acute Ag exposure. Indeed profound cycling of Ͼ90% of from DCs. Consistent with the CD8ϩ T cell response in TLR2Ϫ/Ϫ Ag-specific cells was seen in the first few days after MS-OVA mice, MS lipids evoked cytokine production from TLR2Ϫ/Ϫ DCs vaccination. These levels are similar to that seen with acute LM- as well (data not shown). Another TLR that is unusual in its ability OVA infection or CFA-OVA vaccination. However, after acute to recognize diverse structurally unrelated ligands is TLR4. For LM-OVA infection, cycling of Ag-specific cells declined rapidly. example, TLR4 recognizes, LPS, heat shock , the plant by guest on September 25, 2021 In contrast, MS-OVA promoted low-level cycling of Ag-specific diterpene paclitaxel and fibronectin that have different structures cells for Ͼ50 days. This suggests continued low-level presentation (32). We have however shown that archaeosomes serve as adju- of Ag to the CD8ϩ T cells by APCs at later time points and pre- vants in TLR4-deficient C3H/HeJ mice (33). However, we cannot dicts an Ag-depot effect for archaeosomes. MS lipids are rich in fully exclude the possible involvement of TLR2 and TLR4 by the bipolar, membrane-spanning isoprenoids (16, 18) that confer in- lipids in the mixture of polar lipids, where engagement of either creased stability to the vesicles (43), and thus may be speculated to receptor alone may be sufficient for adjuvant activity. Archaea are survive in the host for prolonged periods. Indeed CFA, known to nonpathogenic, MS is a normal habitant of human colon, and to provide an Ag-depot effect also allows prolonged cycling of Ag- date there is only one rather speculative, suggestive, disease asso- specific cells. However, we noted that the intensity of cycling does ciation with archaeal methanogens (51). Thus, it is understandable decline from Ͼ90% at initial times to ϳ20% later. Thus, Ag burst if archaeal cell components do not interact with PRRs. Neverthe- appears to occur classically within the first week of MS-OVA vac- less, archaeosomes evoke DC maturation and cytokine production cination, whereas, low-level Ag persistence may proceed at later (22), although not massive inflammation (19) as seen with many times. However, there is a clear absence of overt inflammation at TLR ligands. We have shown that MS archaeosomes are recog- the injection site, as seen with toxic adjuvants such as CFA. The nized by a PS receptor on APCs through interaction with archaeti- overall weaker stimulation at later times may have supported the dyl-PS (19). It is possible that PS-receptor interaction also facili- presence of a constant population of TCM as well as the slower tates DC activation to some extent. Alternatively, other PRRs such attrition of the TEM cells. For example, chronic Mycobacterium as C-type lectins and NOD proteins (47) may be involved in ar- bovis, bacillus Calmette-Gue´rin infection, evokes an attenuated in- chaeal lipid recognition, as glycol-head groups are abundant on ϩ fection, and predominantly TCM phenotype of CD8 T cells (44). archaeal lipid structures (16). In chronic viral infections, Ag persistence induced the exhaus- The types of vaccines considered to be suited for evoking CD8ϩ tion of CD8ϩ T cells (45) or persistence of nonfunctional CD8ϩ T T cell immunity include attenuated viruses or bacteria (live vac- cells (46). However, CD8ϩ T cells evoked after MS-OVA vacci- cines), replication-deficient recombinant viruses or bacteria (live nation were highly functional as evident by the memory recall vectored vaccines), DNA vaccines, and vaccines that use the CTL and IFN-␥ response Ͼ1 year after vaccination. Further, in prime-boost strategy with heterologous live vector adjuvants (52). vivo killing of specific targets was retained at Ͼ80% until 180 These approaches combine the ability to provide sufficient immu- days. Thus the low-level, long-term Ag presentation evoked by nostimulation, as well as targeting of CD8ϩ T cell immunity. Of MS-OVA did not lead to T cell exhaustion and the CD8ϩ T mem- these approaches, live vectors come with the risk of reconversion ory cells were capable of rapidly responding to an antigenic chal- to virulence (53) and DNA vaccines often require either high doses lenge. It is intriguing however, that a single injection of MS-OVA or special delivery approaches for sustained immunity (54). Lipo- appeared to evoke weaker memory in comparison to a two injec- somes composed of conventional ester lipids have been considered The Journal of Immunology 2405 for T cell vaccine development, but often require a codelivered from the human archaea Methanobrevibacter smithii and Methanosphaera stadt- immunostimulant for sufficient augmentation of innate immunity manae and its relevance to the adjuvant activities of their liposomes. Biochim. Biophys. Acta 1440: 275–288. (55, 56). A heterologous prime-boost approach that combines 19. Gurnani, K., J. Kennedy, S. Sad, G. D. Sprott, and L. Krishnan. 2004. Phospha- DNA vaccines with a live-vectored boost is promising (57), but tidylserine receptor-mediated recognition of archaeosome adjuvant promotes en- docytosis and MHC class I cross-presentation of the entrapped antigen by pha- pre-existing immunity to live vectors in many humans (58) can gosome-to-cytosol transport and classical processing. J. Immunol. 173: 566–578. compromise efficacy. In this study, we have demonstrated that ar- 20. Sprott, G. D., S. Sad, L. P. Fleming, C. Dicaire, G. B. Patel, and L. Krishnan. chaeosomes are well-suited for evoking the full spectrum of CD8ϩ 2003. Archaeosomes varying in lipid composition differ in receptor-mediated endocytosis and differentially adjuvant immune responses to entrapped antigen. T cell response, and thus may be an attractive choice for T cell Archaea 1: 151–164. vaccine development. Furthermore, we have shown that priming 21. Krishnan, L., S. Sad, G. B. Patel, and G. D. Sprott. 2000. Archaeosomes induce ϩ and boosting with archaeosomes provides substantial enhancement long-term CD8 cytotoxic T cell response to entrapped soluble protein by the exogenous cytosolic pathway in the absence of CD4ϩ T cell help. J. Immunol. of the initial T cell burst, similar to heterologous prime-boost 165: 5177–5185. strategies. 22. Krishnan, L., S. Sad, G. B. Patel, and G. D. Sprott. 2001. The potent adjuvant A number of challenges remain for T cell vaccine development, activity of archaeosomes correlates to the recruitment and activation of macro- phages and dendritic cells in vivo. J. Immunol. 166: 1885–1893. but the recent renaissance in innate immunity and the explosion of 23. Krishnan, L., S. Sad, G. B. Patel, and G. D. Sprott. 2003. Archaeosomes induce immunological assays for determining efficacy of vaccines holds enhanced cytotoxic T responses to entrapped soluble protein in the absence of interleukin 12 and protect against tumor challenge. Cancer Res. 63: great promise for the future. Although, the ultimate test for the 2526–2534. efficacy of many adjuvants will come from human trials, studies 24. Krishnan, L., and S. G. Dennis. 2003. Archaeosomes as self-adjuvanting delivery such as these provide a necessary first step for the validation of systems for cancer vaccines. J. Drug Target 11: 515–524. 25. Conlan, J. W., L. Krishnan, G. B. Patel, and G. D. Sprott. 2001. Immunization of novel adjuvants and shed light on the immune correlates for suc- mice with lipopeptide antigens encapsulated in specialized liposomes prepared Downloaded from cessful development of T cell vaccines in general. What our results from the total polar lipids of various archaeobacteria elicits rapid prolonged pro- have shown here is that archaeosomes are a good alternative to live tective immunity against the facultative intracellular pathogen Listeria monocy- togenes. Vaccine 19: 3509–3517. vaccines as they induce responses similar in magnitude to LM, an 26. Wooten, R. M., Y. Ma, R. A. Yoder, J. P. Brown, J. H. Weis, J. F. Zachary, intracellular pathogen that is considered to be a potent inducer of C. J. Kirschning, and J. J. Weis. 2002. Toll-like receptor 2 is required for innate, ϩ but not acquired, host defense to Borrelia burgdorferi. J. Immunol. 168: CD8 T cell memory. 348–355.

27. Dudani, R., Y. Chapdelaine, H. H. Faassen, D. K. Smith, H. Shen, L. Krishnan, http://www.jimmunol.org/ Acknowledgments and S. Sad. 2002. Multiple mechanisms compensate to enhance tumor-protective ϩ ϩ We are thankful to Dr. Gordon Willick for peptide synthesis support. We CD8 T cell response in the long-term despite poor CD8 T cell priming ini- tially: comparison between an acute versus a chronic intracellular bacterium ex- acknowledge the technical assistance provided by Perry Fleming and Lise pressing a model antigen. J. Immunol. 168: 5737–5745. Deschatelets for archaeal growth and lipid extraction. 28. Moore, M. W., F. R. Carbone, and M. J. Bevan. 1988. Introduction of soluble protein into the class I pathway of antigen processing and presentation. Cell 54: Disclosures 777–785. 29. Barber, D. L., E. J. Wherry, and R. Ahmed. 2003. Cutting edge: rapid in vivo The authors have no financial conflict of interest. killing by memory CD8 T cells. J. Immunol. 171: 27–31. 30. 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