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Departments of
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Microbiology and
Biochemistry, Cellular and Molecular Biology, University of Tennessee, Knoxville, TN 37996;
Department of Molecular Genetics and Biochemistry, University of Pittsburgh, Pittsburgh, PA 15261; and
§
Center for Cellular and Genetic Therapy, Department of Surgery, Duke University, Durham, NC 27710
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). Indeed, we have shown by in vitro studies with
DNA, that exposure of DC to plasmid DNA resulted in trivial
expression as compared with similarly treated M. DC do become
excellent APC upon successful transfection, although even transfection
by DNA can be difficult to accomplish (13). Although DC may act as the primary APC following DNA immunization, conceivably the DC take up exogenous proteins or peptides from other DNA-transfected cells. Such cross-priming has often been described for protein Ags, but it is unclear how immunogenic material is transferred, if the mechanism represents a common event during immune induction and the identity of cells involved in the process (14). Candidate mechanisms include the cell to cell transfer of MHC-binding peptides perhaps regurgitated from phagocytic cells (15) or alternatively molecular chaperones, such as hsp70 or gp96, may bind the peptides and mediate their transfer between cells (14). In addition, it has been suggested that apoptotic bodies released from cells may be taken up by the DC and the internalized material may somehow gain access to the endogenous Ag-processing machinery of the DC (16). Some evidence for each mechanism has been presented, although the topic has not been systematically investigated for DNA vaccines in which the bulk of material is delivered to cells not involved in Ag presentation.
In the present study, we have shown that in vitro interaction between
M and DC can result in CTL induction. This cell combination was
chosen because DC are the principal cell types involved in Ag
presentation after DNA immunization yet, unlike M, do not readily
express Ag when exposed in vitro to plasmid DNA. We demonstrate that
M, upon exposure to plasmid DNA, release factor(s) that, when added
to DC-T cell cultures, acts as APC to induce CTL or to activate an
Ag-specific T cell hybridoma to release IL-2. The immunogenic material
appeared to be a chaperone-bound peptide whose immunogenic activity was
dependent on the presence of ATP. We suggest that a similar mechanism
may occur in vivo involving cells such as myocytes and that DNA
vaccines may act to induce immunity in ways other than directly
transfecting APC.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Seven- to eight-week-old and retired breeder female C57BL/6 mice (H-2b) and BALB/c mice (H-2d) were obtained from Harlan Sprague-Dawley (Indianapolis, IN). In conducting the research described in this work, the investigators adhered to the Guide for the Care and Use of Laboratory Animals, as proposed by the committee on care of Laboratory Animal Resources Commission on Life Sciences, National Research Council. The facilities are fully accredited by the American Association for Accreditation of Laboratory Animal Care.
Cell line and cell culture
The tumor cell lines used were BALB/3T3 (BALB/c, H-2d fibroblast), EL4 (C57BL/6, H-2b lymphoma), EG7 OVA cells (EL4 cells transfected with the cDNA of chicken OVA) (17), EMT6 (BALB/c mammary adenocarcinoma cells, H-2d; kindly provided by Dr. Ed Cantin, City of Hope National Medical Center, Duarte, CA), CH.B2 (H-2b, B cell lymphoma kindly provided by Daniel Muller, University of North Carolina, Chapel Hill, NC), and YAC-1 (mouse lymphoma target for NK cells). All cell lines were cultured in DMEM (Life Technologies, Grand Island, NY) supplemented with 10% heat-inactivated FCS (Life Technologies). Target cells expressing HSV proteins, EMT6-gB, EL4-gB, BALB/3T3-gB, or ICP27 were generated by transfecting the cells overnight with DNA encoding the corresponding Ag (described under cytotoxicity assay). RF33.70 (T-T hybridoma) was obtained from Dr. Kenneth L. Rock (Dana-Farber Cancer Institute, Boston, MA) and grown in 10% RPMI 1640.
Peptides and plasmid DNA-encoded Ags
The H-2b-specific HSV peptide (aa 498–505; SSIEFARL) and chicken OVA peptide (aa 257–264; SIINFEKL) were synthesized and deblocked to provide free amino and carboxyl ends (Research Genetics, Birmingham, AL). cDNA encoding the HSV-1 genes gB and ICP27 was inserted into the pcDNAI vector (Invitrogen, San Diego, CA), as described earlier (6). cDNA encoding chicken OVA was obtained from Dr. Michael Bevan (University of Washington, Seattle, WA) (17). The purity and concentration of plasmid DNA, which were produced LPS free using endo-free columns obtained from Qiagen (Chatsworth, CA), were analyzed by A260 and A280 and by agarose gel electrophoresis and ethidium bromide staining. Expression of the gB, ICP27, and OVA gene products was confirmed by Western blot analysis using commercially available Abs by transfection of splenic DC from BALB/c mice in the presence of the cationic lipid lipofectamine (Boehringer Mannheim Biochemicals, Indianapolis, IN). Plasmid pGREEN LANTERN-1 (catalog number 10642-015) was purchased from Life Technologies.
Biochemical reagents
ATP (Sigma, St. Louis MO; catalogue number A9187), adenosine 5'-O-(3-thiotrophosphate) (ATP-S; Sigma; catalog number A1388), CTP (Acros Organics, Pittsburgh, PA; catalog number 81012-87-5), GTP (Sigma; catalog number G8877), and UTP (Sigma; catalog number U6625). The reagents for the enzymatic treatment included a general protease-thermolysin (Boehringer Mannheim; catalog number 161586) (EDTA was used in parallel to negatively regulate the thermolysin activity as a control), apyrase (Sigma; catalog number A6410), which is an ATP and an adenosine 5'-diphosphatase, DNase I (Sigma; catalog number D4263), and RNase A (Sigma; catalog number R4875), ATP agarose (Sigma; catalog number 02065) for making columns.
Antibodies
Anti-hsp104 (StressGen, Victoria, BC, Canada; catalog number SPA 1040), anti-gp96 (gift from Dr. P. Srivastava, University of Connecticut), anti-hsp70 directed against a highly conserved 13-aa region near the N terminus of hsp70 (18), anti-hsp25 (StressGen; catalog number SPA 801), and normal rabbit serum (Zymed, San Francisco, CA; catalog number 01-6101). PE-labeled anti-CD11b (PharMingen, San Diego, CA; catalog number 01715B) for macrophages, PE-labeled anti-CD3 (PharMingen; catalog number 01085B) for T cells, FITC-labeled anti-B220 for B cells (PharMingen; catalog number 01125B), PE-labeled anti-CD11c (PharMingen; catalog number 09705B), PE-labeled anti-CD80 (PharMingen; catalog number 09605B), PE-labeled anti-CD86 (PharMingen; catalog number 09275B), and FITC-labeled anti-H-2b (PharMingen; catalog number 06044D) for dendritic cells were used.
Isolation and purity of APC and responder T cells
To isolate DCs, splenocytes were obtained as per the procedure mentioned elsewhere (19). Briefly, splenocytes were obtained from naive mice, and the cell concentration is adjusted to 2 x 107 cells in 3 ml of RPMI 1640 medium containing 10% FCS (RPMI-10% FCS). These cells were overlayed onto 2 ml of 14.5% metrizamide gradient column. After a low speed centrifugation (200 x g for 10 min), cells from the interface were collected and washed twice in RPMI-10% FCS. The pellet was resuspended in another 3 ml of the same medium and the above procedure was repeated. Cells from the interface were collected. The purity of the preparation was checked by surface staining with mAbs to 33D1 (kindly provided by Dr. Ralph Steinman, The Rockefeller University, New York, NY) and PE-labeled CD11c. The maturation status of the DCs, as analyzed by staining for MHC class II and costimulatory molecule (CD80 & CD86) expression, indicated it to be of heterogenous population.
The M were isolated as per method described previously
(19). Briefly, the splenic cells were allowed to adhere
onto a plastic tissue culture T-150 flask for 90 min at 37°C. The
nonadherent cells were processed for isolation of T cells described
elsewhere. The adherent cell population was scraped off and allowed to
readhere for another 1 h at 37°C. The readhered population was
dislodged and resuspended in RPMI 1640 with 5% FBS. Flow-cytometric
analysis was done after staining with mAb to CD11b to check for
purity.
T cell isolation was done by separating B cells from the nonadherent population from above by passing through a nylon wool column and subsequently panning on anti-Ig-coated plates. The separated cell population was analyzed for percentage of T lymphocytes by FACS analysis (anti-CD3 staining) and later used as responder naive T cells.
Flow-cytometric analysis
A portion of the isolated splenic populations was analyzed for
cell surface markers by flow cytometry to assess the purity of the
preparations. The cells were blocked with heat-inactivated FBS and
washed three times with FACS buffer (1x PBS with 1% BSA and 0.05%
NaN3). The cells were stained with mAb to 33D1,
and CD11c for DCs, PE-labeled anti-CD11b for M, PE-labeled
anti-CD3 for T cells, and FITC-labeled anti-B220 for B
cells.
In vitro CTL induction
T cells (5 x 106 cells/ml) and APC (5 x 105 cells/ml) were cultured in 100 µl of NCTC 109 and RPMI 1640 (1:1 v/v; Life Technologies), supplemented with 10% heat-inactivated FCS, 2 mM L-glutamine, 1 mM oxaloacetic acid, 0.2 U/ml bovine insulin, and 5 x 10-5 M 2-ME in 96-well U-bottom plates to give a responder:stimulator (R:S) ratio of 10:1. After 5 days, the cells were used as effectors in a standard 4-h 51Cr release assay.
M (5 x 105 cells/ml) were treated with
purified pcDNAgB or pcDNAICP27 (5–7 µg/ml) for 24 h in
96-well flat-bottom plates at 37 C. Following incubation for 24 h
at 37°C and 5% CO2, 100 µl of the
supernatant was harvested, passed through a 0.45-m filter, and added to
100 µl of DC-T cell microculture (R:S ratio of 10:1) in 96-well
U-bottom plates. Cultures were incubated at 37°C for 5 days,
following which the cells were pooled and used as effectors in a
standard 4-h 51Cr release assay.
Cytotoxicity assay
Target cells (5–10 x 106 cells) were incubated with pcDNAgB or pcDNAICP27 (100 µg) in the presence of 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) (50 µg) in Opti-MEM using standard transfection procedures for 12–15 h at 37°C to generate cells expressing gB or ICP27, followed by labeling with 100 µCi of 51Cr. For peptide-specific lysis, 2 x 106 target cells were labeled in 500 µl RPMI 1640 with 100 µCi of 51Cr for 90 min with appropriate peptides at a concentration of 7.5–10 µg/ml. For OVA-specific lysis, 2 x 106 EG7 were pulsed for 90 min with 100 µCi of 51Cr in 500 µl of RPMI 1640. After washing, 104 labeled targets and serial dilutions of effector cells were incubated in 200 µl of RPMI 1640 with 10% heat-inactivated FCS in 96-well V-bottom plates. The plates were centrifuged at 500 x g for 3 min and incubated at 37°C and 5% CO2 for 4 h. A total of 100 µl of the supernatant fluid was collected, and radioactivity was measured using a LKB gamma counter, and specific cytotoxic activity was determined using the formula: % specific release = [(experimental release - spontaneous)/(total release - spontaneous release)] x 100, where experimental release is the radioactivity present in test samples, while spontaneous release is the radioactivity of targets with the addition of media only and the total release is the measure of activity after the addition of 3% Triton-X. Each assay was performed in triplicate, and the spontaneous release was less than 22% of total release by detergent in all assays.
Calculation of lytic units (LU)
A plot with percentage specific lysis value vs the log of the effector cell number for each effector cell preparation was done. A lysis value of 20% was selected through which most of the declining titration curves passed. One LU was the number of lymphocytes required to yield 20% lysis. If the titration curve of an effector cell preparation failed to reach the selected lysis value (20%), the activity was referred to as < x LU, where "x" is the calculated minimum level.
Induction of IL-2 in DC-RF33.70 cells
The RF33.70 generated by Rock (20) recognizes the
OVA epitope SIINFEKL presented on H-2b APC
and produces IL-2 in response. The macrophage-released factor (MRF)
collected from the M cultures treated with OVA DNA were serially
diluted in a 96-well plate; to this, DC and RF33.70 cells at a ratio of
1:2 (DC, 5 x 105 cells/ml; RF33.70, 1
x 106 cells/ml) were added and incubated for
36 h at 37°C and 5% CO2. The supernatants
from these cultures were analyzed for the presence of IL-2 using CTLL-2
cells obtained from Dr. Cynthia Watson (National Institutes of Health,
Bethesda, MD). The detail of the procedure for the CTLL-2 assay,
including the construction of a standard curve with rIL-2, was
described previously (21).
Ultrafiltration
The MRF were passed over a series of Centricon concentrators (Amicon, Beverly, MA) beginning with Centricon-100, in which molecules greater than 100 kDa were retained. The retentate was saved diluted to the original volume, and the flow-through was spun over a Centricon-30. Again the retentate, macromolecules larger than 30 kDa, was saved, diluted to the original volume, and the flow-through was spun over a Centricon-10. Finally, the flow-through from Centricon-10 was passed over Centricon-3 after diluting to the original volume. Each retentate and the flow-through from Centricon were tested by pulsing naive DC and tested for primary CTL induction in naive T cells.
Ultracentrifugation
The MRF were centrifuged to remove the MHC-containing vesicles or apoptotic bodies. The samples were loaded onto Beckman Quick-Seal centrifuge tubes and spun at high speed (117,000 x g) in a Beckman Optima LE 80 K ultracentrifuge with a Vti65.1 rotor for 1 h. The samples were later drawn with a sterile syringe and analyzed for activity.
Enzymatic treatment
The reagents for the enzymatic treatment included a general protease-thermolysin (10 U), EDTA (4 mM) was used in parallel to negatively regulate the thermolysin activity as a control; apyrase, which is an ATP and an adenosine 5'-diphosphatase; 4 µl of RNase-free DNase I (Sigma; catalog number D4263); and 1 µl of 10 mg/ml RNase A (Sigma; catalog number R4875). The MRF was treated with these enzymes and incubated for 30 min at 37°C in a water bath and later analyzed.
Nucleotides addition
The MRF were analyzed for the effect of addition of ATP, a
nonhydrolyzable ATP analogue ATP-
-S, a molar in excess, CTP, GTP,
and UTP. Because ATP had an enhancing effect on the induction of IL-2
by RF33.70 cells, an experiment for the dose response with different
concentration of ATP on the supernatant was done. Based on this, a
concentration of 2 mM was optimum concentration (data not shown), and
it was uniformly applied to other triphosphates as well so as to
compare their effect on the inducing factor. The MRF thus treated were
incubated at room temperature for 45–60 min. As a control for this
experiment, the DC and RF33.70 hybridoma were also treated with ATP (2
mM) for 30 min at 37oC, washed by passing
through a Histopaque (Sigma) column, and centrifuged twice and
resuspended in 10% RPMI 1640 to analyze the effect of ATP on DCs
directly, and a portion of it was mixed with RF33.70 cells and treated
with MRF.
Dialysis
The MRF was injected into Slide-A-Lyzer dialysis cassettes (Pierce, Rockford, IL; catalog number 66332CW), as per the manufacturer’s instruction, and dialyzed against PBS for 24 h at 4°C. The MRF were then analyzed for its potency to induce IL-2 either alone or with the addition of graded dose of ATP starting from 0.31 mM to 10 mM concentration.
Blocking with anti-hsp Abs
The individual fractions of MRF were treated with polyclonal Abs to hsp104, gp96, hsp70, and hsp25 at a final concentration of 1 in 200 and incubated in a 37°C waterbath for 45 min to 1 h, and later the samples were added to the DC-RF33.70 culture to analyze the blocking of the activity of the MRF.
Western blot analysis
Different dilutions of the MRF were boiled in reducing SDS-PAGE sample buffer, run on 10–20% gradient SDS-PAGE. They were then electroblotted to polyvinyl difluoride membrane, and Western blots were performed with Abs to hsp70, hsp90, gp96 OVA, and hsp104. Secondary Abs were conjugated to alkaline phosphatase, and Luminolphos was used for detection of bands (enhanced chemiluminescent system of detection) by exposing the membrane to a hyper film (Amersham, Arlington Heights, IL) and later processed using Konica automatic film processor.
ATP agarose column
A 2-ml column was packed with 140 mg of ATP agarose and rinsed with 15 ml of PBS. The MRF was passed through the column at the rate of 1 ml every 5 min. The filtrate was collected and frozen, to be used later. The column was rinsed again with 15 ml PBS before elution. A solution containing 10 mM ATP in PBS was used to elute the bound hsp. The eluate was collected as 0.5-ml volume with fraction collector. Each fraction was analyzed for the presence of hsp70 by running it on a 10–20% gradient gel. The gel was fixed with ethanol:acetic acid:Milli Q water (40:10:50) and processed for silver staining, as per the procedure of Maniatis et al. (22)
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In previous studies, we demonstrated that transfection of DC with
plasmid DNA encoding gB or ICP27 of HSV caused a potent HSV-specific
CTL response by naive purified T cells (6). However,
without the transfecting agent, the addition of naked plasmid DNA to
DC-T cell cultures induced barely detectable CTL reactivity
(6). Table I
shows a similar
pattern of results obtained in DC-T cell cultures stimulated with
plasmid DNA encoding OVA. Thus, whereas transfection of DC with OVA DNA
induced specific cytotoxicity, when the plasmid was added to DC-T cell
cultures without transfection, responses were trivial unless the DC-T
cell cultures were exposed to high amounts of DNA (40 µg/ml or more).
With M as APC, primary CTL responses were induced upon exposure to 5
µg/ml of OVA DNA, but responses were less than observed when the APC
were transfected DC (data not shown). Additionally, it was shown using
DNA encoding the green fluorescent protein (GFP) that whereas M took
up and expressed the plasmid following exposure to it, GFP
expression only occurred in DC following transfection with the plasmid
DNA (Fig. 1).
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as APC supported indifferent in vitro primary CTL
responses, exposure of such cells for 24 h to naked DNA (5
µg/ml) encoding HSV or OVA proteins induced the production of
material (which is referred to as MRF), which, when added to naive DC-T
cell cultures, generated Ag-specific CTL. For example, the experiment
shown in Fig. 2 indicates that when
H-2d M were treated with either pcDNAgB,
pcDNAICP27, or control DNA-expressing luciferase (pcDNAlux), the
material generated CTL in DC-T cell cultures that were specific to the
protein encoded by the DNA. For example, MRF from pcDNAICP27-treated
M generated CTL, which lysed H-2d ICP27
targets, but not H-2d gB-expressing or
mock-transfected targets (Fig. 2 and data not shown). Similarly,
pcDNAgB induced MRF-generated gB-specific CTL. The pcDNAlux MRF failed
to generate any Ag-specific CTL. Once again, M-pcDNAgB MRF added to
syngeneic H-2b DC-T cell cultures generated CTL
that lysed H-2b gB targets, but not
H-2b ICP27 targets (see Fig. 3). The OVA DNA CTL induction data are
shown in Table II and discussed
subsequently.
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exposed
to pcDNA for 24 h. However, as shown in Fig. 3
, the activity was
first detected at about 6 h of M exposure to DNA.
Identity of immunogenic material generated by M
Several possibilities were considered as to the identity of the
immunogenic material released from the DNA-treated M. The idea that
the MRF might represent free peptide was made unlikely by the data
presented in Fig. 4, in which the MRF
were passed through ultrafiltration filters of various sizes before
assay for CTL induction. As is evident, the maximum CTL activity was
generated with fractions that were 30–100 kDa. Fractions that were
>100 kDa and 10–30 kDa generated some activity, but fractions less
than 10 kDa that would include peptides of 9–11 aa were without
activity.
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exposed to OVA DNA was the production system employed. As is shown
in Fig. 5A, the OVA
DNA-induced MRF that effectively generated primary OVA-specific CTL in
DC-T cell cultures also induced the production of IL-2 from DC-RF33.70
hybridoma cultures (Fig. 5B). The latter cultures also
responded to stimulation by the synthetic peptide SIINFEKL, although
the presence of DC to present the peptide was necessary for this to
occur. Similar concentrations (2–10 µg/ml) of peptide failed to
induce primary OVA-specific CTL responses in DC-T cell cultures. The
DC-hybridoma system failed to respond to MRF from vector DNA-treated
M and failed to react to OVA DNA unless exposed to high
concentrations (40 µg/ml or more). Because the active MRF were
generated at a DNA concentration of 5 µg/ml, most of which was taken
up or degraded, residual DNA (max levels of 0.7 µg/ml were detected)
would not appear to account for the IL-2-inducing effect.
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. Centrifugation had no
measurable effect on the activity of MRF (Fig. 6). In addition, the MRF appeared to be
neither RNA nor DNA because treatment with RNase and DNase failed to
diminish the activity (Fig. 6). However, treatment with the general
protease thermolysin (metalloendopeptidase) almost completely
eliminated the activity, an effect neutralized by chelating with EDTA
known to prevent the action of thermolysin (23) (Fig. 6).
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that treatment with ATP, but none of the other triphosphates, enhanced
the activity of MRF. This ATP-enhancing effect was largely counteracted
by pretreatment with a molar excess of the competitive inhibitor
ATP--S. Whereas 2 mM ATP enhanced the activity of the MRF, ATP
failed to enhance the IL-2 induction effect of any concentration of the
peptide SIINFEKL (Fig. 7). This further argues that the activity being
detected in the MRF was unlikely to be free peptide. In other
experiments, the effect of 2 mM ATP addition on the magnitude of the
OVA-specific CTL induction response was investigated. The presence of
ATP in supernatants enhanced the CTL induction response by about 25%
(Table II). In addition, treatment of DC with 2 mM ATP, followed by
cell washing before their exposure, failed to influence the response
significantly (Fig. 7). Thus, the effect of ATP may not be mediated by
nonspecific activation of DC or RF33.70.
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, dialysis reduced the activity,
although not to baseline, but the addition of 2 mM ATP enhanced the
activity to an even greater extent (up to 6-fold) than the ATP-mediated
enhancement observed with nondialyzed MRF (up to 1.9-fold).
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A). Western blot probing for
the presence of hsp and OVA protein was also additionally performed and
probed with Abs to OVA, hsp104, gp96, and hsp70. A highly sensitive
chemiluminescent detection system showed a prominent hsp70 band (Fig. 9B) as well as a very weak band for hsp104 and gp96
(however, our attempts to procure standard proteins were only
successful with hsp70 and OVA). The anti-OVA Ab failed to show a
45-kDa band that is indicative of intact OVA protein.
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, by
silver staining and Western blot analysis, hsp70 was readily
demonstrable in the MRF (Fig. 9A). Other chaperones such as
hsp90 (Fig. 9B), gp96, or hsp104 (data not shown) could not
be demonstrated. Soluble OVA protein was also not found in the MRF.
Purification of the MRF on ATP agarose column, followed by SDS-PAGE and
silver staining, revealed the presence of a 70-kDa but not 90-kDa
protein.
Second, a role for hsp70 was analyzed by measuring the effects of
anti-hsp70 and other anti-chaperone proteins on the function of
MRF measured by the DC-hybridoma assay. As is evident in Fig. 10, the function of MRF was inhibited
by polyclonal anti-hsp70 Abs, but only marginally inhibited by
other anti-chaperone Abs. The anti-hsp70 serum appeared to have
no effect on either the DC or RF33.70 cells used in the assay
(Fig. 10).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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to plasmid DNA encoding viral proteins generates
Ag-specific material that, when presented in vitro by mature DC to
naive T cells, induces primary CTL responses. Administration of plasmid
DNA to the same system failed to induce CTL presumably because mature
DC take up and express the DNA poorly (6). The immunogenic
material released was proteinaceous in nature, free of apoptotic
bodies, and had an apparent m.w. much larger than a 9–11-aa
CTL-recognizable peptide. The cross-presentation process specifically
required hydrolyzable ATP and was inhibited by procedures that removed
or hydrolyzed ATP. These properties plus the ability of anti-hsp70
antiserum to partially abrogate the activity indicated the possible
involvement of a hsp70-linked peptide chaperone. This
cross-presentation mechanism of immune induction may represent a
principal method by which plasmid DNA delivered to cells such as
myocytes effectively shuttle Ag to DC or other APC to achieve CTL
induction in vivo.
Upon introduction as a vaccination strategy, i.m. injection was the
favored route for DNA immunization (8). Gene expression
was readily evident in myocytes, but such cells were not expected to
act as APC for immune induction. A more likely alternative is that rare
bone marrow-derived cells in muscles, or cells of bone marrow origin
transfected by plasmid DNA in the bloodstream, or lymphoid tissue act
as the APC for immune induction (5, 8). Recent reports
confirm the essential role of bone marrow-derived cells rather than
myocytes for CTL induction (3, 4), and transfected DC have
been observed in recipients of DNA vaccines (5, 26, 27, 28).
Consequently, immune induction following DNA vaccination could be
driven principally by APC, which themselves are transfected by plasmid
DNA. Although DC and other APC may contain plasmid DNA in vivo, DC,
especially in their mature Ag-presenting stage, take up plasmid DNA
ineffectively and in fact may sequester the DNA in endosomes that would
prevent expression (29). Accordingly, alternative means of
acquiring Ag for immune induction may operate. Our data provide
evidence that the cross-priming process initially described by Bevan
(17) represents one such mechanism. In our experiments,
the cross-presentation material was produced by M exposed in vitro
to plasmid DNA encoding either viral Ags or OVA. Whereas M readily
took up plasmid DNA, this was not the case for splenic DC. Only when
exposed to high concentrations of plasmid DNA (40 µg/ml) did DC-T
cell cultures respond. In contrast, M, although poor APC in the
primary antiviral system (6), rapidly produced the
cross-presentation material upon exposure to low levels of plasmid DNA
(5 µg/ml or less).
The types of events involved in cross-priming were succinctly reviewed by Bevan (14), although this review only dealt with responses to peptide/protein Ags. Our novel studies indicate that a cross-priming process can also occur during immune response to DNA vaccines, and that the process may involve an ATP-dependent chaperone protein such as hsp70 bound to the CD8+ T cell-recognized peptide. Thus, the MRF activity was protease sensitive, yet was resistant to both RNA and DNA nucleases. The MRF was unlikely to include apoptotic bodies as described by Bhardwaj and colleagues (16) as a means of Ag transfer between cells, nor could it simply be a MHC-binding small peptide. Accordingly, the activity was unaffected by high speed centrifugation and in being largely between 30 and 100 kDa in size, not a 9- to 11-aa peptide. The size profile could implicate peptides associated with either members of the hsp70 or hsp90 class of molecular chaperones (25). Furthermore, our data clearly showed that the addition of exogenous ATP enhanced the activity of MRF, as measured both in the CTL induction assay and an assay in which the material was presented by DC to an IL-2-producing T cell hybridoma. In fact, the stimulatory effect of exogenous ATP was even more marked when the MRF was dialyzed before assay to remove the endogenous ATP initially present. In this instance, the activity, at least as measured in the T cell hybridoma assay, was enhanced by ATP up to 6-fold. In contrast, the presence of exogenous ATP failed to influence the response of DC-hybridoma cultures to free peptide, and ATP treatment of DC in the absence of MRF failed to change their APC activity.
The pronounced effect of treatments that alter the levels of ATP
suggests that the MRF requires ATP binding to function. In addition,
the ability of ATP--S to inhibit this activity indicates that ATP
hydrolysis is involved during MRF function. These properties are
consistent with the involvement of a class of molecular chaperone whose
function is influenced by ATP. The most likely candidates are
members of the hsp70 class of molecular chaperones that contain both an
ATP binding domain at the N terminus and a peptide domain at the C
terminus (30, 31). The two domains interact, and one
consequence of ATP binding is the release of peptide from its binding
domain (32, 33). The function of other chaperones such as
hsp90 may also be influenced by ATP, although in the case of hsp90, the
reported data are conflicting (34, 35, 36), and hsp90 appears
to lack known ATP-binding motifs (37).
The presence of a prominent hsp70 band and the absence of OVA
protein, as evidenced by Western blot analysis, strongly implicate
hsp70 chaperone in the process and negate a role for the soluble OVA
protein. An additional experiment also supported a likely role for an
hsp70 as principally involved in the cross-presentation activity of
MRF. Thus, the addition of antisera to hsp70 markedly reduced the
activity. The effect was almost unaffected by anti-gp96, perhaps
not surprising because this chaperone most likely functions
independently of ATP (38). Nevertheless, at least in some
tumor systems, in which peptide-bound chaperones can be isolated from
cells and shown to be immunogenic, gp96 as well as other chaperones
such as calreticulin can be involved as carriers of peptides
(39, 40, 41). The residual activity detected in our
experiments after anti-hsp70 treatment indicates the likely
involvement of other classes of molecular chaperones in the
cross-presentation process. Currently, we fail to understand the
mechanism by which the chaperone-bound peptides are generated within
M and released to the outside. This issue is under further
investigation.
It remains to be shown how the DNA-induced ATP-dependent chaperone-bound material transfers immunogenicity to DC, as well as to be established whether similar events also occur in vivo, perhaps involving myocytes and DC. With regard to the former issue, ATP activation is known to cause peptide release from chaperones that contain ATP binding domains (42). Conceivably, therefore, the increase in activity observed following the addition of ATP could represent a cell surface event with the released peptide exchanging with material bound to MHC class 1 on DC. However, due to the limited concentration of these peptides, a facilitated exchange/delivery system would most likely need to occur. Alternatively, the ATP-dependent chaperone could be internalized effectively following receptor binding to the surface of DC. This would need to be followed by a peptide exchange between the chaperone and newly formed MHC class I proteins. It is not clear if or where such exchanges occur in the cell. Clearly, additional experiments are required to define mechanisms by which ATP influences the cross-presentation process.
Finally, our results emphasize the importance of cross-priming as a mechanism by which DNA vaccines might achieve immunogenicity. If the process we describe is an important component of immune induction by DNA vaccines in vivo, it will be important to define the types of cells involved and devise optimal means of immunization that deliver material to the relevant cellular participants.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Barry T. Rouse, Department of Microbiology, University of Tennessee, Knoxville, TN 37996-0845.
3 Abbreviations used in this paper: DC, dendritic cell; ATP-S, adenosine 5'-O-(3-thiotrophosphate); GFP, green fluorescent protein; gp, glycoprotein; hsp, heat-shock protein; LU, lytic unit; M, macrophage; MRF, macrophage-released factor.
Received for publication September 15, 1999. Accepted for publication April 27, 2000.
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