Polyriboinosinic polyribocytidylic acid [poly(I:C)]/TLR3 signaling allows class I processing of exogenous protein and induction of HIV-specific CD8+ cytotoxic T lymphocytes
Chiaki Fujimoto1,2,
Yohko Nakagawa1,
Kunitoshi Ohara2 and
Hidemi Takahashi1
Departments of 1 Microbiology and Immunology and 2 Ophthalmology, Nippon Medical School, Tokyo 113-8602, Japan
Correspondence to: H. Takahashi; E-mail: htkuhkai{at}nms.ac.jp
Transmitting editor: K. Okumura
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Abstract
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In the case of viral infection, various viral proteins and genetic components are disseminated in the body. The former viral proteins may be captured by immature dendritic cells (DC) and the latter genetic components may stimulate the antigen-loading DC to maturate via specific Toll-like receptors (TLR), leading to the establishment of virus-specific cellular immunity; in particular, cytotoxic T lymphocytes (CTL) that control intracellular virions. Polyriboinosinic polyribocytidylic acid [poly(I:C)], which might reflect a natural genetic product from a variety of viruses during replication, has recently been identified as one of the critical stimuli for TLR3. Based on these observations, we speculated that stimulation of TLR3 with poly(I:C) might drive the direction of acquired/adaptive immunity to the cellular arm. Indeed, when BALB/c mice were immunized with purified recombinant HIV-1 envelope gp120 or influenza hemagglutinin (HA) protein together with poly(I:C), epitope-specific CD8+ class I MHC molecule-restricted CTL were primed from naive CD8+ T cells in vivo. In contrast, when the same proteins were immunized with lipopolysaccharide, a stimulant of TLR4, specific CTL were not primed at all. Moreover, we show here that immature DC could present processed antigen from captured purified protein in association with class I MHC molecules in the presence of poly(I:C), but not of LPS. These results indicate that we are able to manipulate the direction of acquired/adaptive effector immune responses using an appropriate stimuli and the findings presented in this paper will offer a new therapeutic strategy using poly(I:C) administration for priming antigen-specific CD8+ CTL with purified viral protein in vivo.
Keywords: cytotoxic T lymphocyte, dendritic cell, HIV-1, poly(I:C), Toll-like receptor
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Introduction
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Our internal defense equipment fights against various pathogens such as bacteria or viruses to protect our bodies and species integrity. Accumulating evidence indicates that the defense system is composed of two parts, innate immunity and acquired/adaptive immunity, which are closely linked in establishing machinery to conquer invading pathogens (1). The innate alert system is composed of a limited number of germline-encoded receptors without a requirement for genetic rearrangement and those receptors recognize conserved portions of microbial organisms to discriminate non-self from self (1). Indeed,
10 Toll-like receptors (TLR) (2) fixed on the sentinels of innate immunity respond to distinct pathogen-associated molecular patterns (PAMP) (3) found on the bacterial cell surface, in their genes or on virus components. For example, bacterial CpG DNA (4), lipopolysaccharide (LPS) of Gram-negative bacteria (5) and peptidoglycan of the bacterial cell wall (68) stimulate TLR9, TLR4 and TLR2 respectively. In addition to these bacteria-associated molecular patterns, double-stranded RNA (dsRNA), polyriboinosinic polyribocytidylic acid [poly(I:C)], which might reflect a natural product from a variety of viruses during replication, has recently been identified as a critical stimuli for TLR3 (9).
Viruses can only replicate inside mammalian cells using cellular machinery so that cellular immunity, particularly cytotoxic T lymphocytes (CTL) that recognize virus-derived peptide antigens in association with class I MHC molecules, seems to be the major effectors controlling intracellular virions. In contrast, most bacteria that can proliferate independently outside cells and which are covered with a thick cell wall might be regulated mainly by antibodies and neutrophils expressing Fc receptors for IgG that mediate bacterial opsonization. Thus, it is reasonable to elicit cellular acquired/adaptive immunity in the case of viral infection, whereas antibody-based humoral immunity is feasibly induced in the case of bacterial infection.
Dendritic cells (DC) are located at the interface of innate and acquired/adaptive immunity, and appear to function as connectors. When they are in an immature state like Langerhans cells in the skin, they might sample and uptake various antigenic molecules around them. Upon encountering a pathogen-derived component expressing PAMP, they differentiate into the matured phenotype that express both processed antigenic epitope in conjunction with MHC and co-stimulation molecules to activate acquired/adaptive immunity by priming naive antigen-specific T cells (10). Therefore, these matured DC seem to represent key commands for driving the direction of acquired/adaptive immune responses. Thus, some PAMP stimuli might drive the appropriate directions of acquired/adaptive effector immunity via TLR on DC.
In the present study, we examined whether PAMP stimuli via TLR3 by poly(I:C) turn the direction of acquired/adaptive immunity to the cellular arm when using soluble viral proteins that must be dispersed in the case of viral intrusion. We used purified recombinant HIV-1 envelope gp120 protein and influenza hemagglutinin (HA) protein as the known antigens. As expected, when BALB/c mice were immunized with these virus-derived proteins together with poly(I:C), epitope-specific CD8+ class I MHC molecule-restricted CTL were primed, whereas when the same proteins were immunized with LPS, a stimulant of TLR4, specific CTL were not primed. In addition, we show here that immature DC (iDC) could present processed antigen derived from purified protein in association with class I MHC molecules in the presence of poly(I:C), but not of LPS. These results indicate that the direction of acquired/adaptive effector immune responses could be manipulated using appropriate PAMP and suggest that poly(I:C) could be an attractive adjuvant for immunotherapy to drive cellular immune responses.
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Methods
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Mice
Female BALB/c (H-2d) mice were purchased from Charles River (Tokyo, Japan). Mice used in this study were at 612 weeks of age and maintained in a specific pathogen-free environment. All experiments were performed according to the guidelines of the NIH Guide for the Care and Use of Laboratory Animals.
Synthetic peptides
Epitope peptides for HIV-1 envelope-specific CTL, P18IIIB (315329, RIQRGPGRAFVTIGK) (11), for determining HIV-1 V3-specific antibodies of IIIB isolate by ELISA, Rp135 (NNTRKSIRIQRGPGRAFVTIGKIGC) (12), and for influenza HA-specific CTL, the HA of influenza A/PR/8/34 (H1N1) (518528, IYSTVASSLVL) (13), were synthesized using an Applied Biosystems model 430A peptide synthesizer (Foster City, CA). Synthetic peptides were further purified by gel filtration on Bio-gel P-4 and analyzed by HPLC on a C18 reversed-phase column. Peptide fractions containing >90% of the desired product were used for the experiments.
Reagents and antibodies
Poly(I:C) and LPS (from Escherichia coli) were purchased from Sigma (St Louis, MO). Recombinant HIV-1 gp120 protein of IIIB isolate was provided by the National Institute of Health (Bethesda, MD) or purchased from Immuno Diagnostics (Woburn, MA). Purified influenza HA protein was provided by Nissin Sei-yu (Yokohama, Japan). The purity of the proteins used in this study was >95%. The endotoxin content of all reagents was tested by the Limulus amebocyte lysate assay (Seikagaku, Tokyo, Japan) and the level was <20 pg/ml. The following mAb were used for depleting the T cell subset: anti-CD4 mAb (RL172.4; rat IgM) (14) and anti-CD8 mAb (3.115; rat IgM) (15).
Transfectants
BALB/c.3T3 (H-2d) fibroblast transfectants expressing HIV-1 gp160 of IIIB isolate (15-12) and control transfectants with only the selectable marker genes (Neo) were derived as described previously (11,16). Murine L cell (H-2k) clones stably transfected with H-2Dd (T4.8.3) (17), H-2Ld (T.1.1.1) (17) and H-2Kd (B4III2) (18) were used to determine class I MHC restriction of the generated CTL. All cells were maintained in complete T cell medium (CTM) (19), consisting of RPMI 1640 medium supplemented with 10% heat-inactivated FCS, 5 mM HEPES buffer solution (Gibco/BRL, Santa Clara, CA), 100 U/ml penicillin (Gibco/BRL), 100 µg/ml streptomycin (Gibco/BRL), 2 mM L-glutamine (Gibco/BRL), 2 mM sodium pyruvate (Gibco/BRL), 2 mM non-essential amino acid (Gibco/BRL), 2 mM modified vitamins (Dainippon Pharmaceutical, Tokyo, Japan) and 2 mM 2-mercaptoethanol (Sigma).
Generation of CTL lines
Spleen cells (5 x 106) from BALB/c mice previously immunized with 1 x 107 p.f.u. of vSC25 (recombinant vaccinia virus expressing the HIV-1 envelope glycoprotein gp160 of the IIIB isolate) (20) were re-stimulated in vitro with 1 x 105 mitomycin C (MMC)-treated 15-12 cells in 24-well plates containing 1.5 ml of CTM and 10% Rat T-STIM (Collaborative Biomedical Products, Bedford, MA). To establish CTL lines, the generated CTL were maintained by bi-weekly stimulation with MMC-treated 15-12 cells and named the LINE-IIIB cells.
Measurement of cytokine production
IL-12-p40, IL-10, IFN-
and IL-4 levels in mice serum samples were measured using specific ELISA kits (BioSource International, Camarillo, CA).
Immunization and CTL generation
Female BALB/c mice were immunized either i.p. or s.c. in the base of tail with 1 µg recombinant gp120 protein with or without either 100 µg poly(I:C) or 100 µg LPS. Four to 12 weeks later, 5 x 106 immune spleen cells were re-stimulated in vitro for 6 days with MMC-treated 15-12 cells in CTM and 10% Rat T-STIM. Similarly, 10 µg recombinant influenza HA (H1N1) protein was immunized with or without either 100 µg poly(I:C) or 100 µg LPS to prime HA-specific CTL.
CTL assay
After culture for 6 days, cytolytic activity of the re-stimulated spleen cells was measured as previously described (21) using a standard 51Cr-release assay with various 51Cr-labeled targets. In brief, the generated cells were harvested and mixed with various 51Cr-labeled target cells in 96-well, U-bottomed culture plates in RPMI 1640 containing 10% FCS at various E:T ratios. After incubation for 46 h at 37°C, the plates were centrifuged for 10 min at 330 g and cell-free supernatants were collected to measure radioactivity using a Packard Auto-Gamma 5650 counter (Hewlett-Packard Japan, Tokyo, Japan). Maximum release was determined from the supernatant of cells that had been lysed with 5% Triton X-100 and spontaneous release was determined from target cells incubated without added effector cells. The percent specific lysis was calculated as 100 x (experimental release spontaneous release)/(maximum release spontaneous release). SEM values of triplicate cultures were always <5% of the mean. Each experiment was performed at least 3 times.
Measurement of antigen-specific antibodies by ELISA
Serum samples were obtained 34 weeks after the immunization. The concentrations of V3 peptide-specific IgG1 and IgG2a antibodies in the sera was determined using sandwich ELISA assays. In brief, microtiter plates (cat. no. 439454; Nalge Nunc International, Tokyo, Japan) were coated overnight at 4°C with 100 µl/well of 10 µg/ml Rp135 peptide in 0.1 M sodium carbonate buffer (pH 9.6). After blocking with 1% BSA in PBS, serum samples were added to the plate and incubated for 1 h at 37°C. Thereafter, the plates were washed and horseradish peroxidase-conjugated rabbit anti-mouse IgG1 (A85-1) or IgG2a (R2-40) (2 µg/ml; Zymed, San Francisco, CA) was added to each well and incubated for 1 h at 37°C. Color was developed using tetramethylbenzidine and the optical density was measured at 450 nm using a microplate reader (model 3550; Bio-Rad, Hercules, CA).
Generation of DC and their sensitization with gp120 protein for CTL recognition
DC were generated from bone marrow precursors as described by Inaba et al. (22) with slight modifications. Briefly, bone marrow cells flushed out of the femurs from BALB/c mice were separated by centrifugation and red blood cells were lysed. After washing twice, cells were plated at a density of 5 x 105 cells/well in 1 ml of CTM containing 1000 U/ml of recombinant murine granulocyte macrophage colony stimulating factor (GM-CSF; Kirin Brewery, Gunma, Japan) in 24-well plates and cultured at 37°C in 5% CO2. On days 2 and 4, non-adherent cells were removed and replaced with fresh medium containing the same amount of GM-CSF. On day 5, non-adherent cells were harvested and used for CTL assays. Flow cytometry analysis revealed that >80% of the harvested cells were CD11c+ DC. These were further incubated at a density of 2 x 105 cells in 48-well plates for 24 h in the presence of 5 µg gp120 protein alone, or with either 100 µg/ml poly(I:C) or the same amount of LPS. After the incubation, these treated DC were harvested and used as targets for CTL assays or as stimulators for primed T cells.
Statistical analysis
Data are presented as mean values ± SD. Data were statistically analyzed by Students t-test. A value of P < 0.05 was considered as the level of significance.
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Results
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Kinetics of systemic and local cytokine production in mice after poly(I:C) or LPS injection
It has recently been demonstrated that poly(I:C) can activate innate immunity via TLR3 and stimulate IL-12 production in vivo using the C57BL/6 (H-2b) mouse strain when 50 µg poly(I:C) was inoculated i.p. (9). In order to confirm this phenomenon in different haplotype mice, we injected BALB/c (H-2d) mice with 0, 10, 30 or 100 µg poly(I:C), and pursued the amount of IL-12 in the sera by ELISA 2, 6 and 8 h after i.p. or s.c. injection. Figure 1(A, left panel) shows that the serum IL-12 level increased in a dose-dependent manner and at 100 µg poly(I:C) i.p., >10 ng/ml of IL-12 was detected in the sera 2 h later. This amount decreased thereafter over time. Because these observations showed that poly(I:C) affected systemic immune responses in vivo, we next examined the local effects of poly(I:C) when injected s.c. IL-12 elevation was also observed in sera from the tail vein of BALB/c mice inoculated with 100 µg poly(I:C) in the base of the tail (Fig. 1A, right panel). In contrast, IL-12 was undetectable in the sera of control mice injected both i.p. and s.c. with either PBS or the same dose of poly(A:U), which cannot activate the TLR3 pathway (9) (data not shown). We then injected BALB/c mice either i.p. or s.c. with the same amount of LPS that would stimulate innate sentinels via TLR4 as internal controls. Figure 1(B, left and right panel) shows that a little more IL-12 was detected in the sera of mice inoculated with 100 µg LPS both i.p. and s.c. The mice appeared unaffected even when injected with the highest dose (100 µg) of poly(I:C) and LPS. Thus, we decided to use 100 µg poly(I:C) and LPS for further experiments.

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Fig. 1. Cytokine levels in mice sera after poly(I:C) or LPS injection. (A) Female BALB/c mice were injected i.p. or s.c. with 0, 10, 30 or 100 µg poly(I:C) and the serum IL-12 levels was determined with ELISA kits 2, 6 and 8 h after the injection. (B) BALB/c mice were injected i.p. or s.c. with 0, 10, 30 or 100 µg LPS, and serum IL-12 level examined 2, 6 and 8 h after the injection. The amount of IL-10, (C) IFN- (D) and IL-4 (E) in the sera of mice injected i.p. or s.c. with 100 µg poly(I:C) (open columns) or the same amount of LPS (filled columns) was measured with by ELISA. Error bars show the SD. All results are representative of at least three independent experiments.
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Because IL-12 production by innate immunity reflects the activation of Th1-type cellular immune responses in acquired immunity (23,24), we also examined serum IL-10 production, which might correlate with Th2-type humoral immune responses by inhibiting IL-12 production in vivo (25,26). About 2000 pg/ml of IL-10 could be measured in the sera of LPS-inoculated mice immunized both i.p. and s.c., although very little IL-10 was detected in the sera of mice injected with poly(I:C) (Fig. 1C). Moreover, we detected IFN-
(Fig. 1D) in the sera of mice injected with 100 µg poly(I:C) and LPS. However, we could not observe any measurable amount of IL-4 in the sera of poly(I:C)-injected mice although a small amount of IL-4 was detected in sera from mice injected with LPS (Fig. 1E). Thus, in vivo i.p. and s.c. injections of poly(I:C) might help to dominantly generate Th1-type acquired immunity, whereas LPS might help to elicit both Th1- and Th2-type immunity.
Induction of HIV-1 gp120-specific killer cells by immunization with recombinant gp120 protein and poly(I:C)
The above findings suggest that the local presence of viral dsRNA such as poly(I:C) may help to induce virus-specific Th1-type cellular immune responses. Therefore, to generate killer cells, recombinant purified HIV-1 gp120 envelope protein was co-administered with 100 µg poly(I:C) into BALB/c mice via either the i.p. or s.c. route. After 4 weeks, immune spleen cells were re-stimulated in vitro with MMC-treated syngeneic BALB/c.3T3 fibroblasts expressing the HIV-1 gp160 envelope gene of IIIB isolate (15-12) and cytotoxicities were measured using a standard 51Cr -release assay. We found that gp120-specific killer cells were generated from mice immunized s.c. with 1 µg gp120 protein and 100 µg poly(I:C), whereas no cytotoxicity was observed in mice immunized i.p. with the same immunogens (Fig. 2A). These results indicate that s.c. inoculation was the optimal route for the induction of killer cells. Thus, we administered 100 µg poly(I:C) together with various amounts of gp120 protein s.c. to determine whether envelope-specific CTL were primed in vivo. As demonstrated in Fig. 2(B), HIV-1 envelope-specific cytotoxic activity was generated in a dose-dependent manner when immune spleen cells of BALB/c mice were re-stimulated in vitro with MMC-treated 15-12 cells. The generated killer cells obtained by s.c. immunization with 100 µg poly(I:C) together with 1 µg recombinant gp120 protein specifically killed fibroblast targets that either expressed whole envelope protein (15-12) or pulsed with a synthetic epitope peptide (P18IIIB: RIQRGPGRAFVTIGK) (Fig. 2C). In addition, when mice were immunized with 1 µg gp120 and various amounts of poly(I:C), specific cytotoxic activity was observed only in those injected with 100 µg poly(I:C) (Fig. 2D). Thus, 1 µg recombinant gp120 protein and 100 µg poly(I:C) were sufficient for priming virus-specific CTL in vivo. In contrast, we could not detect any specific cytotoxic activity when BALB/c mice were injected with 1 µg gp120 and either PBS (Fig. 2E) or 100 µg LPS (Fig. 2F), or a lower dose of LPS (data not shown). These results indicate that virus-specific CTL could be primed with poly(I:C) via TLR3 stimulation, but not with LPS via TLR4 using viral proteins for s.c. immunization, and thus suggest that the feasibility of vaccination to prime virus-specific CTL by immunization with recombinant viral proteins and poly(I:C).

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Fig. 2. Induction of HIV-1 envelope protein-specific killer cells by immunization with gp120 protein and poly(I:C). (A) Female BALB/c mice were immunized with gp120 and poly(I:C) via distinct routes of inoculation. After 4 weeks, immune spleen cells were re-stimulated in vitro with MMC-treated 15-12 with Rat T-STIM (see Methods). After 6 days of culture, cytotoxic activities were tested against 51Cr-labeled 15-12 targets. The immune spleen cells from the syngeneic BALB/c mice inoculated s.c. with 1 µg gp120 protein together with 100 µg poly(I:C) (filled square) showed specific cytotoxicity against 15-12, whereas no cytotoxicity was observed in mice immunized i.p. with the same dose of gp120 protein and poly(I:C) (filled circle), s.c. with gp120 protein and PBS (open square) or i.p. with gp120 protein and PBS (open circle). (B) Female BALB/c mice were immunized s.c. with 100 µg poly(I:C) and various doses of purified recombinant gp120 protein [0 (open square), 1 (filled triangle) or 5 µg (filled square)]. Specific cytotoxic activity was detected in a dose-dependant manner. (C) To determine the specificity of generated killer cells, their cytotoxic activities were tested against the indicated 51Cr-labeled targets: 15-12 (filled circle), 1 µM P18IIIB (RIQRGPGRAFVTIGK)-pulsed BALB/c.3T3 fibroblasts (filled triangle) and control untreated BALB/c.3T3 fibroblasts, Neo (open circle). (D) To determine the optimal amount of poly(I:C) for immunization, 1 µg gp120 protein and several doses of poly(I:C) were examined: 0 (open circle), 10 (filled circle), 30 (filled triangle) and 100 µg (filled square). Only 100 µg poly(I:C) was able to prime specific killer cells. (E) As a negative control, 1 µg gp120 protein in PBS was immunized s.c. Re-stimulated effector cells were tested against 51Cr-labeled targets 15-12 (filled circle) or control fibroblasts, Neo (open circle). (F) To compare the effect of another TLR stimulant, 1 µg gp120 protein was administered s.c. with 100 µg LPS. Similarly, Re-stimulated effector cells were tested against 51Cr-labeled targets 15-12 (filled circle) or control fibroblasts, Neo (open circle). Results are representative of at least three independent experiments.
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Characterization of the induced killer cells
Because immunization with exogenous antigen generally primes MHC class II-restricted CD4+ T cells, we examined the phenotype and MHC restriction of the generated killer cells. When the killer cells were treated with anti-CD4 mAb plus complement or complement alone, their cytolytic activity was almost unchanged. However, treatment with anti-CD8 mAb plus complement led to a complete reduction in their cytotoxic activity (Fig. 3). To determine the class I MHC molecule restriction, we used the L cell-based transfectants, T4.8.3, T1.1.1 and B4III2, expressing Dd, Ld and Kd molecules respectively. The generated killer cells specifically recognized the immunodominant P18IIIB peptide presented by Dd, but not by Ld or Kd, MHC class I molecules (data not shown). Therefore, the killer cells induced by s.c. immunization with recombinant gp120 viral protein and poly(I:C) were conventional epitope peptide-specific CD8+ class I MHC molecule-restricted CTL.

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Fig. 3. Phenotype of the generated killer cells. Female BALB/c mice were immunized s.c. in the base of the tail with 1 µg gp120 protein and 100 µg poly(I:C). After 4 weeks, immune spleen cells were re-stimulated in vitro with MMC-treated 1 µM P18IIIB-pulsed syngeneic BALB/c.3T3 fibroblasts with Rat T-STIM. After 6 days of culture, cytotoxic activities were measured against 51Cr-labeled P18IIIB-pulsed BALB/c.3T3 fibroblasts. The effector cells were pre-treated with anti-CD4 mAb (RL172.4) plus complement (filled triangle), anti-CD8 mAb (3.155) plus complement (filled square) or complement alone (open circle). Results are representative of three independent experiments.
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Induction of influenza HA-specific CTL by immunization with recombinant HA protein and poly(I:C)
To generalize the observation that virus-specific CTL could be primed by immunization with viral protein and poly(I:C), we used purified influenza HA (H1N1) protein as the antigen. As demonstrated in Fig. 4(A), an immunodominant epitope of HA, HA518528 (IYSTVASSLVL), specific killer cells were generated when spleen cells of BALB/c mice immunized s.c. with 10 µg HA protein and 100 µg poly(I:C) were re-stimulated in vitro for 6 days with syngeneic fibroblasts pre-sensitized with HA518528 peptide. In contrast, specific cytolytic activity was undetectable when the mice were immunized with 10 µg HA protein with either PBS (Fig. 4B) or with 100 µg LPS (Fig. 4C). We confirmed that the generated killer cells were CD8+ Kd-restricted conventional CTL (data not shown). Therefore, both HIV-1 gp120-specific CTL and influenza HA-specific CTL could be primed by s.c. immunization with purified viral protein and poly(I:C).

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Fig. 4. Induction of HA (H1N1) protein-specific killer cells by immunization with HA protein and poly(I:C). (A) Female BALB/c mice were immunized s.c. in the base of tail with 10 µg HA protein and 100 µg poly(I:C). After 4 weeks, immune spleen cells were re-stimulated in vitro with MMC-treated 4 µM HA peptide (IYSTVASSLVL)-pulsed syngeneic BALB/c.3T3 fibroblasts with Rat T-STIM. After 6 days of culture, cytotoxic activities were tested against 51Cr-labeled targets: HA peptide-pulsed BALB/c.3T3 fibroblasts (Neo*HA) (filled circle) and control BALB/c.3T3 fibroblasts (Neo) (open circle). (B) As a negative control, 10 µg HA protein in 100 µl PBS was inoculated and immune spleen cells were tested for their ability to generate HA peptide-specific CTL. (C) To compare the effect of another TLR stimulant, 10 µg HA protein was administered s.c. with 100 µg LPS. Results are representative of three independent experiments.
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Analysis of antibodies in the sera of mice immunized with recombinant gp120 protein and adjuvant
We then examined the amount and type of gp120-specific antibodies in mice sera. As speculated from the pattern of serum cytokine production, predominant production of HIV-1 V3 (Rp135)-specific IgG2a (Th1-type) antibodies was measured in the sera of mice immunized s.c. with gp120 and poly(I:C) (Fig. 5A). In contrast, V3-specific IgG1 (Th2-type) antibodies were dominantly observed when mice were immunized s.c. with 1 µg gp120 and 100 µg LPS (Fig. 5B). Therefore, we confirmed that Th1-type systemic responses proceeded in the mice injected with poly(I:C), whereas Th2-type responses were induced by inoculation with LPS in vivo.

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Fig. 5. Type of HIV-1 V3-specific antibodies produced in the sera of mice immunized with recombinant gp120 protein and poly(I:C). Female BALB/c mice were immunized s.c. with 100 µl PBS, 100 µg poly(I:C), 1 µg gp120 protein, gp120 protein and poly(I:C) or gp120 protein and 100 µg LPS. Four weeks after the immunization, HIV-1 V3 (Rp135)-specific IgG1 and IgG2a levels were determined by ELISA. (A) Predominant production of V3-specific IgG2a (Th1-type) antibodies was observed in the sera of mice immunized s.c. with 1 µg gp120 and 100 µg poly(I:C). (B) V3-specific IgG1 (Th2-type) antibody production was dominantly observed when mice were immunized s.c. with 1 µg gp120 and 100 µg LPS. Data are representative of at least three independent experiments. Error bars indicate the SD. Statistical analysis was performed using paired Students t-test between immunization of recombinant gp120 protein with poly(I:C) and LPS; V3-specific IgG2a (*P = 0.0099) and V3-specific IgG1 (**P = 0.015).
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Sensitization of DC with recombinant gp120 protein in the presence of poly(I:C)
Finally, to analyze the mechanism of these phenomena, we examined whether immature bone marrow-derived DC (BM-DC) could uptake gp120 protein in the presence of poly(I:C) and present the processed peptide antigen to CTL in vitro. Based on the previous observations (22,27), immature BM-DC were incubated with 5 µg gp120 protein with or without 100 µg/ml of poly(I:C) or the same amount of LPS for 24 h at 37°C. After extensive washing to remove free antigenic protein and reagent, the cells were used as targets for 4-h CTL assay. As demonstrated in Fig. 6(A), BM-DC pulsed with gp120 protein and poly(I:C) were recognized and killed by a P18IIIB-specific CTL line (LINE-IIIB), whereas those pulsed with gp120 protein alone or with LPS were not lysed. In contrast, when BM-DC were pulsed with the same amount of gp120 protein and poly(A:U), they could not be lysed by LINE-IIIB (data not shown). These results indicate that the epitope peptide from gp120 protein was processed and presented to the MHC class I molecules in the presence of poly(I:C).

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Fig. 6. Sensitization of DC with recombinant gp120 protein in the presence of poly(I:C). (A) Five-day-cultured BM-DC were incubated with PBS, 100 µg/ml poly(I:C), 5 µg gp120 protein, gp120 protein and poly(I:C) or gp120 protein and 100 µg/ml LPS for 24 h in a 48-well flat-bottomed plate. As a positive control, BM-DC were pulsed with 1 µM P18IIIB. After being labeled with 51Cr and washed 3 times, the treated DC were co-cultured with P18IIIB-specific CTL (LINE-IIIB) to perform 4-h cytotoxic assays at various E:T ratios. (B) IL-12 and IL-10 production in the supernatant of 5-day-cultured BM-DC incubated with either 100 µg/ml poly(I:C) or 100 µg/ml LPS. The amount of cytokine production was measured with each specific ELISA kit. (C) Female BALB/c mice were immunized s.c. in the base of the tail with 1 µg gp120 protein and 100 µg poly(I:C). After 4 weeks, immune spleen cells were re-stimulated in vitro with the following MMC-treated DC and Rat T-STIM: DC alone (open circle); DC incubated with 5 µg gp120 protein (open square); DC with 5 µg gp120 protein and 100 µg/ml poly(I:C) (filled triangle); and DC with 5 µg gp120 protein and 100 µg/ml LPS (filled square). After 6 days of culture, cytotoxic activities were tested against 51Cr-labeled P18IIIB-pulsed BALB/c.3T3 fibroblasts (Neo) as targets. Data are representative of at least three independent experiments.
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We also examined the cytokine productions in the supernatant of those cultured BM-DC. Although both IL-12 and IL-10 production was detected in the supernatant after stimulation with LPS, IL-12, but not IL-10, could be measured in the poly(I:C)-stimulated supernatant (Fig. 6B). Moreover, the BM-DC sensitized with gp120 and poly(I:C) could specifically stimulate vSC25-primed splenic T cells, although BM-DC treated with gp120 and LPS or with gp120 alone did not stimulate the primed T cells (Fig. 6C). These results suggest that recombinant gp120 protein captured by iDC such as Langerhans cells in the skin was processed and presented in association with class I MHC molecules of these DC via maturation via poly(I:C), and that matured antigen-bearing DC might prime epitope-specific CD8+ CTL in vivo.
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Discussion
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The present study demonstrates that a single administration of viral protein such as HIV-1 gp120 or influenza HA could prime virus epitope-specific CD8+ class I MHC molecule-restricted conventional CTL when inoculated together with poly(I:C), an IFN inducer (28) that is sometimes used in models of viral infection, but not with LPS, reflecting the PAMP of the viral product recognized by TLR3 and the bacterial component by TLR4 respectively. It is reasonable to elicit cellular acquired/adaptive immunity, in particular virus-specific CD8+ CTL which might control intracellular virions, by disseminated viral proteins and genetic products after virus intrusion. The former viral proteins might be captured by iDC such as Langerhans cells and the latter virus-derived genetic products such as poly(I:C) might drive the direction of iDC with captured antigen towards the Th1 route for priming naive CD8+ T cells into viral protein-specific CTL. However, if LPS-bearing bacterial infection was overlapped at the same time, the direction of antigen-loaded DC might be shifted to Th2 and CTL generation would be disturbed. This would prolong the viral infection by inhibiting CTL induction via wrong manipulation of the DC with viral protein by LPS. However, it has recently been reported that bacteria-derived CpG DNA, PAMP for TLR9 recognition (4), might help to elicit antigen-specific CTL (29,30). Therefore, in contrast to LPS, some bacterial products may help to clear the virus-infected cells in vivo by turning the direction of T cell activation to Th1 route.
We have previously reported that DC are essential for priming CD8+ CTL and we could generate CTL by i.v. inoculation with epitope peptide-pulsed irradiated splenic DC (31). In that study, we noticed that the efficacy of CTL-priming by splenic DC was up to the conditions of peptide-loaded DC. In general, LPS is useful for the maturation of DC (32), which then secrete cytokines such as IL-12, tumor necrosis factor-
, IL-6 or IL-10. Actually it has been shown that i.v. immunization of epitope peptide-pulsed spleen cells activated in vitro by LPS could induce specific CTL (33). However, Kelleher and Beverley have recently reported that modulation of DC by LPS could not stimulate antigen-specific CTL from naive polyclonal CD8+ T cells in vitro (34). Our results similarly indicated that the maturation of antigen-captured DC by LPS would not help to prime specific CTL from naive splenic T cells in vivo. Moreover, as we will discuss below, LPS appeared to inhibit the MHC class I presentation pathway for captured intact viral protein via IL-10 production. Conversely, poly(I:C)-stimulated iDC seemed to efficiently proceed cross-presentation of captured viral protein probably by predominant secretion of IL-12 at the very early stage and low production of IL-10, as has been reported recently (35,36). Collectively, externally added IL-12 and anti-IL-10 may mimic the poly(I:C)-mediated environment where naive CD8+ T cells might be primed effectively.
Thus, the pattern of cytokine production by DC via TLR ligand such as LPS or poly(I:C) appears to be critical for determining the direction of acquired/adaptive immune responses. However, as has shown in Fig. 1, although the pattern of cytokine production observed after poly(I:C) administration in the base of the tail (s.c.) was similar to that systemically produced by i.p. inoculation, antigen-specific CTL could be primed in vivo only when mice were s.c. immunized with gp120 protein and poly(I:C). These results are consistent with those of others who reported that immunization with antigens at the base of the tail (s.c.) for CTL priming (3739). This notion of s.c. inoculation is also consistent with our previous observation using gp120 protein and immunostimulating complexes, consisting of materials derived from plants for the induction of gp120-specific CTL (40). Unfortunately, none of the reports have precisely analyzed and compared the routes of immunization for CTL induction. The iDC of the skin and mucosa, Langerhans cells, have been implicated as the first targets for HIV after sexual contact (41,42), and they constantly sample antigenic molecules from their environment until activation by a maturation signal. After maturation, such iDC located in non-lymphoid surface areas migrate to secondary lymphoid tissue, where they present processed antigen to T cells (43). Thus, our current results suggest that poly(I:C)/TLR3 signaling might activate such antigen-loaded iDC existing in the s.c. tissues, that allows them to cross-present exogenous antigens to prime CD8+ CTL. Taken together, s.c. immunization seems to be a better route for priming CD8+ CTL than the i.p. route. However, more precise analysis is required to prove those mechanisms.
Our current findings showed that antigenic peptides derived from purified HIV-1 gp120 protein could be cross-presented in association with class I MHC molecules by BM-DC in vitro in the presence of poly(I:C), but not of LPS. It is generally assumed that exogenously captured antigenic proteins are delivered to acidic endosomes, processed and efficiently presented on MHC class II molecules by professional antigen-presenting cells (44,45). Thus, there must be other unknown pathways that will be opened to cross-present those internally processed antigens on class I MHC molecules instead of class II MHC when protein-loaded iDC are stimulated by special substance like poly(I:C) via TLR. Indeed, consistent with our observation, very recently it has been shown that such cross-presentation of captured soluble proteins to class I MHC could be generated in BM-DC via maturation with a subset of TLR ligand such as immunostimulatory CpG DNA for TLR9 and poly(I:C) for TLR3 (46). Since the CpG DNA fail to develop CTL response in TAP-deficient BM-DC (47), the poly(I:C)-mediated TLR-dependent cross-presentation of captured cytosolic antigens also seems to require TAP and proteasomal processing, but not endosomal acidification. Moreover, the fact that TAP function could be inhibited by IL-10 (47,48) indicates that the low production of IL-10 maintained by both in vivo and in vitro treatment of iDC by poly(I:C) might activate the TAP-dependent pathway that enables it to cross-present processed antigen to class I MHC molecules. Further investigation will be required to examine the actual effect of poly(I:C)/TLR3 signaling on such cross-presentation and cross-priming.
In the case of certain viral infections, there must be a large number of iDC that have already captured disseminated viral protein fragments in vivo. The present study indicates the possibility that we could manipulate the direction of such iDC for priming neighboring CD8+ T cells to generate antigen-specific CTL effectors by poly(I:C). The findings presented here will offer a new therapeutic strategy using poly(I:C) administration to prime or activate antigen-specific CD8+ CTL both in vitro and in vivo.
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Acknowledgements
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We thank Dr Jay A. Berzofsky for critical reading of this manuscript and helpful suggestions, and Ms Masumi Shimizu for technical assistance. This work was supported in part by grants from the Ministry of Education, Science, Sport and Culture, from the Ministry of Health and Labor and Welfare, Japan, and from the Japanese Health Sciences Foundation.
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Abbreviations
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BM-DCbone marrow-derived dendritic cell
CTLcytotoxic T lymphocyte
CTMcomplete T cell medium
DCdendritic cell
dsRNAdouble-stranded RNA
GM-CSFgranulocyte macrophage colony stimulating factor
HAhemagglutinin
iDCimmature dendritic cell
LPSlipopolysaccharide
MMCmitomycin C
PAMPpathogen-associated molecular pattern
poly(I:C)polyriboinosinic polyribocytidylic acid
TLRToll-like receptor
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References
|
---|
- Fearon, D. T. and Locksley, R. M. 1996. The instructive role of innate immunity in the acquired immune response. Science 272:50.[Abstract]
- Kaisho, T. and Akira, S. 2002. Toll-like receptors as adjuvant receptors. Biochim. Biophys. Acta 1589:1.[ISI][Medline]
- Medzhitov, R. and Janeway, C., Jr. 2000. Innate immune recognition: mechanisms and pathways. Immunol. Rev. 173:89.[CrossRef][ISI][Medline]
- Hemmi, H., Takeuchi, O., Kawai, T., Kaisho, T., Sato, S., Sanjo, H., Matsumoto, M., Hoshino, K., Wagner, H., Takeda, K. and Akira, S. 2000. A Toll-like receptor recognizes bacterial DNA. Nature 408:740.[CrossRef][ISI][Medline]
- Poltorak, A., He, X., Smirnova, I., Liu, M. Y., Van Huffel, C., Du, X., Birdwell, D., Alejos, E., Silva, M., Galanos, C., Freudenberg, M., Ricciardi-Castagnoli, P., Layton, B. and Beutler, B. 1998. Defective LPS signaling in C3H/HeJ and C57BL/10ScCr mice: mutations in Tlr4 gene. Science 282:2085.[Abstract/Free Full Text]
- Takeuchi, O., Hoshino, K., Kawai, T., Sanjo, H., Takada, H., Ogawa, T., Takeda, K. and Akira, S. 1999. Differential roles of TLR2 and TLR4 in recognition of gram-negative and gram-positive bacterial cell wall components. Immunity 11:443.[ISI][Medline]
- Schwandner, R., Dziarski, R., Wesche, H., Rothe, M. and Kirschning, C. J. 1999. Peptidoglycan- and lipoteichoic acid-induced cell activation is mediated by toll-like receptor 2. J. Biol. Chem. 274:17406.[Abstract/Free Full Text]
- Takeuchi, J., Watari, E., Shinya, E., Norose, Y., Matsumoto, M., Seya, T., Sugita, M., Kawana, S. and Takahashi, H. 2003. Down-regulation of Toll-like receptor expression in monocyte-derived Langerhans cell-like cells: implications of low-responsiveness to bacterial components in the epidermal Langerhans cells. Biochem. Biophys. Res. Commun. 306:674.[CrossRef][ISI][Medline]
- Alexopoulou, L., Holt, A. C., Medzhitov, R. and Flavell, R. A. 2001. Recognition of double-stranded RNA and activation of NF-kappaB by Toll- like receptor 3. Nature 413:732.[CrossRef][ISI][Medline]
- Bendelac, A. and Medzhitov, R. 2002. Adjuvants of immunity: harnessing innate immunity to promote adaptive immunity. J. Exp. Med. 195:F19.
- Takahashi, H., Cohen, J., Hosmalin, A., Cease, K. B., Houghten, R., Cornette, J. L., DeLisi, C., Moss, B., Germain, R. N. and Berzofsky, J. A. 1988. An immunodominant epitope of the human immunodeficiency virus envelope glycoprotein gp160 recognized by class I major histocompatibility complex molecule-restricted murine cytotoxic T lymphocytes. Proc. Natl Acad. Sci. USA 85:3105.[Abstract]
- Javaherian, K., Langlois, A. J., McDanal, C., Ross, K. L., Eckler, L. I., Jellis, C. L., Profy, A. T., Rusche, J. R., Bolognesi, D. P., Putney, S. D., et al. 1989. Principal neutralizing domain of the human immunodeficiency virus type 1 envelope protein. Proc. Natl Acad. Sci. USA 86:6768.[Abstract]
- Tamura, M., Kuwano, K., Kurane, I. and Ennis, F. A. 1998. Definition of amino acid residues on the epitope responsible for recognition by influenza A virus H1-specific, H2-specific, and H1- and H2-cross-reactive murine cytotoxic T-lymphocyte clones. J. Virol. 72:9404.[Abstract/Free Full Text]
- Ceredig, R., Lowenthal, J. W., Nabholz, M. and MacDonald, H. R. 1985. Expression of interleukin-2 receptors as a differentiation marker on intrathymic stem cells. Nature 314:98.[ISI][Medline]
- Sarmiento, M., Glasebrook, A. L. and Fitch, F. W. 1980. IgG or IgM monoclonal antibodies reactive with different determinants on the molecular complex bearing Lyt 2 antigen block T cell-mediated cytolysis in the absence of complement. J. Immunol. 125:2665.[Abstract/Free Full Text]
- Takahashi, H., Houghten, R., Putney, S. D., Margulies, D. H., Moss, B., Germain, R. N. and Berzofsky, J. A. 1989. Structural requirements for class I MHC molecule-mediated antigen presentation and cytotoxic T cell recognition of an immunodominant determinant of the human immunodeficiency virus envelope protein. J. Exp. Med. 170:2023.[Abstract]
- Margulies, D. H., Evans, G. A., Ozato, K., Camerini-Otero, R. D., Tanaka, K., Appella, E. and Seidman, J. G. 1983. Expression of H-2Dd and H-2Ld mouse major histocompatibility antigen genes in L cells after DNA-mediated gene transfer. J. Immunol. 130:463.[Abstract/Free Full Text]
- Abastado, J. P., Jaulin, C., Schutze, M. P., Langlade-Demoyen, P., Plata, F., Ozato, K. and Kourilsky, P. 1987. Fine mapping of epitopes by intradomain Kd/Dd recombinants. J. Exp. Med. 166:327.[Abstract]
- Takahashi, H., Cease, K. B. and Berzofsky, J. A. 1989. Identification of proteases that process distinct epitopes on the same protein. J. Immunol. 142:2221.[Abstract/Free Full Text]
- Chakrabarti, S., Robert-Guroff, M., Wong-Staal, F., Gallo, R. C. and Moss, B. 1986. Expression of the HTLV-III envelope gene by a recombinant vaccinia virus. Nature 320:535.[ISI][Medline]
- Takahashi, M., Osono, E., Nakagawa, Y., Wang, J., Berzofsky, J. A., Margulies, D. H. and Takahashi, H. 2002. Rapid induction of apoptosis in CD8+ HIV-1 envelope-specific murine CTL by short exposure to antigenic peptide. J. Immunol. 169:6588.[Abstract/Free Full Text]
- Inaba, K., Inaba, M., Naito, M. and Steinman, R. M. 1993. Dendritic cell progenitors phagocytose particulates, including bacillus Calmette-Guerin organisms, and sensitize mice to mycobacterial antigens in vivo. J. Exp. Med. 178:479.[Abstract]
- Hsieh, C. S., Macatonia, S. E., Tripp, C. S., Wolf, S. F., OGarra, A. and Murphy, K. M. 1993. Development of TH1 CD4+ T cells through IL-12 produced by Listeria-induced macrophages. Science 260:547.[ISI][Medline]
- Manetti, R., Parronchi, P., Giudizi, M. G., Piccinni, M. P., Maggi, E., Trinchieri, G. and Romagnani, S. 1993. Natural killer cell stimulatory factor (interleukin 12 [IL-12]) induces T helper type 1 (Th1)-specific immune responses and inhibits the development of IL-4-producing Th cells. J. Exp. Med. 177:1199.[Abstract]
- De Smedt, T., Van Mechelen, M., De Becker, G., Urbain, J., Leo, O. and Moser, M. 1997. Effect of interleukin-10 on dendritic cell maturation and function. Eur. J. Immunol. 27:1229.[ISI][Medline]
- Huang, L. Y., Reis e Sousa, C., Itoh, Y., Inman, J. and Scott, D. E. 2001. IL-12 induction by a TH1-inducing adjuvant in vivo: dendritic cell subsets and regulation by IL-10. J. Immunol. 167:1423.[Abstract/Free Full Text]
- Bachmann, M. F., Lutz, M. B., Layton, G. T., Harris, S. J., Fehr, T., Rescigno, M. and Ricciardi-Castagnoli, P. 1996. Dendritic cells process exogenous viral proteins and virus-like particles for class I presentation to CD8+ cytotoxic T lymphocytes. Eur. J. Immunol. 26:2595.[ISI][Medline]
- Meegan, J. M. and Marcus, P. I. 1989. Double-stranded ribonuclease coinduced with interferon. Science 244:1089.[ISI][Medline]
- Horner, A. A., Datta, S. K., Takabayashi, K., Belyakov, I. M., Hayashi, T., Cinman, N., Nguyen, M. D., Van Uden, J. H., Berzofsky, J. A., Richman, D. D. and Raz, E. 2001. Immunostimulatory DNA-based vaccines elicit multifaceted immune responses against HIV at systemic and mucosal sites. J. Immunol. 167:1584.[Abstract/Free Full Text]
- Maurer, T., Heit, A., Hochrein, H., Ampenberger, F., OKeeffe, M., Bauer, S., Lipford, G. B., Vabulas, R. M. and Wagner, H. 2002. CpG-DNA aided cross-presentation of soluble antigens by dendritic cells. Eur. J. Immunol. 32:2356.[CrossRef][ISI][Medline]
- Takahashi, H., Nakagawa, Y., Yokomuro, K. and Berzofsky, J. A. 1993. Induction of CD8+ cytotoxic T lymphocytes by immunization with syngeneic irradiated HIV-1 envelope derived peptide-pulsed dendritic cells. Int. Immunol. 5:849.[Abstract]
- Verhasselt, V., Buelens, C., Willems, F., De Groote, D., Haeffner-Cavaillon, N. and Goldman, M. 1997. Bacterial lipopolysaccharide stimulates the production of cytokines and the expression of costimulatory molecules by human peripheral blood dendritic cells: evidence for a soluble CD14-dependent pathway. J. Immunol. 158:2919.[Abstract]
- Kakugawa, K., Udaka, K., Nakashima, K., Inaba, K., Oka, Y., Sugiyama, H., Tamamura, H. and Yamagishi, H. 2000. Efficient induction of peptide-specific cytotoxic T lymphocytes by LPS-activated spleen cells. Microbiol. Immunol. 44:123.[ISI][Medline]
- Kelleher, M. and Beverley, P. C. 2001. Lipopolysaccharide modulation of dendritic cells is insufficient to mature dendritic cells to generate CTL from naive polyclonal CD8+ T cells in vitro, whereas CD40 ligation is essential. J. Immunol. 167:6247.[Abstract/Free Full Text]
- Verdijk, R. M., Mutis, T., Esendam, B., Kamp, J., Melief, C. J., Brand, A. and Goulmy, E. 1999. Polyriboinosinic polyribocytidylic acid (poly(I:C)) induces stable maturation of functionally active human dendritic cells. J. Immunol. 163:57.[Abstract/Free Full Text]
- Cella, M., Salio, M., Sakakibara, Y., Langen, H., Julkunen, I. and Lanzavecchia, A. 1999. Maturation, activation, and protection of dendritic cells induced by double-stranded RNA. J. Exp. Med. 189:821.[Abstract/Free Full Text]
- Corradin, G., Etlinger, H. M. and Chiller, J. M. 1977. Lymphocyte specificity to protein antigens. I. Characterization of the antigen-induced in vitro T cell-dependent proliferative response with lymph node cells from primed mice. J. Immunol. 119:1048.[Abstract]
- Ahlers, J. D., Dunlop, N., Alling, D. W., Nara, P. L. and Berzofsky, J. A. 1997. Cytokine-in-adjuvant steering of the immune response phenotype to HIV-1 vaccine constructs: granulocyte-macrophage colony-stimulating factor and TNF-alpha synergize with IL-12 to enhance induction of cytotoxic T lymphocytes. J. Immunol. 158:3947.[Abstract]
- Maitre, N., Brown, J. M., Demcheva, M., Kelley, J. R., Lockett, M. A., Vournakis, J. and Cole, D. J. 1999. Primary T-cell and activated macrophage response associated with tumor protection using peptide/poly-N-acetyl glucosamine vaccination. Clin. Cancer. Res. 5:1173.[Abstract/Free Full Text]
- Takahashi, H., Takeshita, T., Morein, B., Putney, S., Germain, R. N. and Berzofsky, J. A. 1990. Induction of CD8+ cytotoxic T cells by immunization with purified HIV-1 envelope protein in ISCOMs. Nature 344:873.[CrossRef][ISI][Medline]
- Hussain, L. A. and Lehner, T. 1995. Comparative investigation of Langerhans cells and potential receptors for HIV in oral, genitourinary and rectal epithelia. Immunology 85:475.[ISI][Medline]
- Lin, C. L., Sewell, A. K., Gao, G. F., Whelan, K. T., Phillips, R. E. and Austyn, J. M. 2000. Macrophage-tropic HIV induces and exploits dendritic cell chemotaxis. J. Exp. Med. 192:587.[Abstract/Free Full Text]
- Banchereau, J. and Steinman, R. M. 1998. Dendritic cells and the control of immunity. Nature 392:245.[CrossRef][ISI][Medline]
- Takahashi, H. 1993. Antigen processing and presentation. Microbiol. Immunol. 37:1.[ISI][Medline]
- Takahashi, H. 2003. Antigen presentation in vaccine development. Comp. Immunol. Microbiol. Infect. Dis. 26:309.[CrossRef][ISI][Medline]
- Datta, S. K., Redecke, V., Prilliman, K. R., Takabayashi, K., Corr, M., Tallant, T., DiDonato, J., Dziarski, R., Akira, S., Schoenberger, S. P. and Raz, E. 2003. A subset of Toll-like receptor ligands induces cross-presentation by bone marrow-derived dendritic cells. J. Immunol. 170:4102.[Abstract/Free Full Text]
- Salazar-Onfray, F., Charo, J., Petersson, M., Freland, S., Noffz, G., Qin, Z., Blankenstein, T., Ljunggren, H. G. and Kiessling, R. 1997. Down-regulation of the expression and function of the transporter associated with antigen processing in murine tumor cell lines expressing IL-10. J. Immunol. 159:3195.[Abstract]
- Petersson, M., Charo, J., Salazar-Onfray, F., Noffz, G., Mohaupt, M., Qin, Z., Klein, G., Blankenstein, T. and Kiessling, R. 1998. Constitutive IL-10 production accounts for the high NK sensitivity, low MHC class I expression, and poor transporter associated with antigen processing (TAP)-1/2 function in the prototype NK target YAC-1. J. Immunol. 161:2099.[Abstract/Free Full Text]