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Promote Priming of Antigen-Specific CD8+ and CD4+ T Lymphocytes by Immunostimulatory DNA-Based Vaccines1
* Division of Hematology/Medical Oncology, Department of Medicine, New York Presbyterian Hospital and Cornell Medical Center, and Laboratory of Neuro-Oncology, Rockefeller University, New York, NY 10021; and
Department of Internal Medicine and The Sam and Rose Stein Institute for Research on Aging, University of California at San Diego, La Jolla, CA 92093
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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and -
, TNF-, and IL-6 and -12,
and to express costimulatory surface molecules such as CD40, B7-1, and
B7-2. Although ISS DNA has little direct effect on T cells by these
criteria, immunization of wild-type mice with ISS DNA and OVA results
in Ag-specific CTL and Th1-type T helper activity. This investigation
examines the mechanisms by which ISS DNA primes CD8+ and
CD4+ lymphocyte activities. In this report we demonstrate
that ISS DNA regulates the expression of costimulatory molecules and
TAP via a novel autocrine or paracrine IFN- pathway. Coordinated
regulation of B7 costimulation and TAP-dependent cross-presentation
results in priming of Ag-specific CD8+ CTL, whereas CD40,
B7, and IL-12 costimulation is required for priming of CD4+
Th cells by ISS-based vaccines. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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), IFN-,
TNF-, IL-6, and IL-12, and up-regulation of surface molecules such
as ICAM-1, CD40, B7-1 and -2, and MHC classes I and II
(5, 6, 7). By these criteria, ISS DNA have very little direct
effect on T cells. However, immunization with ISS-based vaccines
results in the priming of two T cell functions: Ag-specific CTL
activity and a Th1-biased immune response (6, 8).
Immunization of wild-type (wt), CD4-/-, or MHC
class II-/- mice with ISS oligodeoxynucleotide
(ODN)-based vaccines results in similar levels of CTL activation
(9), indicating that these vaccines efficiently prime CTL
against exogenous Ags (cross-priming) (10) independently
of Th cells. ISS-based vaccines appear to act upon APC to facilitate
cross-presentation and promote the expression of costimulatory
molecules, allowing them to directly prime naive
CD8+ lymphocytes.
It is likely that the priming interaction between ISS-stimulated APC
and CD4+ or CD8+
lymphocytes is mediated by the cytokines and surface molecules that are
induced by ISS DNA. However, it is unclear whether there are
differential costimulatory requirements for CTL vs Th1 priming, or
whether there is a temporal sequence to these signals. It is possible
that an early event is the lynchpin in the transition from innate to
Ag-specific immunity. One key class of mediators that may anchor the
adaptive response to ISS-based vaccines is IFN-, which have a
broad range of activities in both innate and adaptive immunities
(11). Like ISS DNA, IFN- promote maturation of APC
as measured by increased expression of costimulatory molecules
(12). In addition, proinflammatory cytokines, including
IFN- and TNF-, have been shown to modulate the expression of TAP
in many tissues, including macrophages (13). TAP plays a
critical role in the presentation of exogenous Ags (cross-presentation)
in the context of MHC class I (14). Although a role for
IFN- in regulating TAP in APC has not been reported to date,
IFN- stimulation has been shown to increase the expression of TAP in
human pulmonary epithelial cells (15). These findings
suggest a link between IFN- and the regulation of both
costimulation and cross-presentation by APC, two essential functions
for generating adaptive T cell activity against pathogens. ISS DNA
promotes the secretion of IFN- in both murine and human innate
immunity (16, 17, 18) and appears to facilitate both
costimulation and cross-presentation in priming T cell activity
(9). This suggests that ISS DNA promotes priming
interactions between APC and Ag-specific T cells via
IFN--dependent mechanisms.
In this report, we demonstrate that ISS-ODN stimulation of APC
regulates costimulatory molecules and TAP expression via IFN-
activity. The costimulation and cross-presentation mediated by this
mechanism result in the efficient priming of both CTL and Th cell
activity by ISS-based vaccines.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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wt C57BL/6 and 129 mice, and CD28-/-,
TAP1-/-, and
IL-12p40-/- mice were purchased from The
Jackson Laboratory (Bar Harbor, ME).
IFN-R-/- mice were purchased from B & K
Universal (East Yorkshire, U.K.). CD40-/- mice
were the gift of N. Phillips (University of Massachusetts, Amherst, MA)
and H. Kikutani (Osaka University, Osaka, Japan). Animal protocols were
approved by the University of California-San Diego Office of Animal
Resources (San Diego, CA).
Oligonucleotides
Single-stranded phosphorothioate ISS-ODN (ISS motif underlined) (sequence 5'-TGA CTG TGA ACG TTC GAG ATG A-3') and mutated ODN (mODN; sequence 5'-TGA CTG TGA AGG TTG GAG ATG A-3') were purchased from Tri-Link Biotechnology (San Diego, CA).
Peptides
H-2b MHC class I-restricted peptides were purchased from PeptidoGenic (Fullerton, CA): OVA peptide, NH2-SIINFEKL-COOH; influenza virus nucleoprotein peptide (target control), NH2-ASNENMETM-COOH.
Cell lines and tissue culture
Murine thymoma EL4 cells were cultivated in RPMI (Irvine Scientific, Santa Ana, CA) supplemented with 10% (v/v) heat-inactivated FCS (Life Technologies, Gaithersburg, MD), 50 mM 2-ME (Sigma-Aldrich, St. Louis, MO), 2 mM L-glutamine (Cellgro, Herndon, VA), 100 U/ml penicillin, and 100 µg/ml streptomycin (Pen/Strep; Cellgro) (RP10). Cells were grown in 250-ml tissue culture flasks (Falcon, Franklin Lakes, NJ) at 37°C in 5% CO2.
Bone marrow-derived DC (BM-DC)
Mouse BM-DC were cultured as previously described
(19). Briefly, bone marrow from
IFN-R-/- or wt 129/SvEv mice was plated
in petri dishes (Fisher, Pittsburgh, PA) at 2 x
105 cells/ml in RP10, a tissue culture medium
preparation consisting of RPMI (Irvine Scientific) supplemented with
10% (v/v) FCS, supplemented with 5 ng/ml recombinant murine GM-CSF (BD
PharMingen, La Jolla, CA). On day 3 an equal volume of RP10 and GM-CSF
was added. On day 6 half the volume of RP10 and GM-CSF was replaced.
The nonadherent cells were harvested on day 7. The resultant population
consisted of 
70–80% CD11c+ DC.
FACS analysis
Day 7 BM-DC from wt or IFN-
R-/- mice were incubated with medium or ISS (1
µg/ml) for 48 h. All Abs were from BD PharMingen. The DC were
resuspended in FACS buffer (RPMI medium without phenol red,
supplemented with 3% FCS and 0.02% sodium azide) and Fc block (BD
PharMingen) for 10 min before staining for 30 min with FITC-labeled
anti-CD11c (clone HL3), PE-labeled anti-B7-1 (clone 16-10A1),
PE-labeled anti-B7-2 (clone GL1), PE-labeled anti-CD40 (clone
3/23), PE-labeled anti-H-2Kb (clone
AF6-88.5), PE-labeled anti-I-Ab (clone
M5114.15.2), or isotype control Abs. The cells were then washed and
analyzed on a FACSCalibur flow cytometer (BD Biosciences, Mountain
View, CA). Data analysis was performed using FlowJo 3.4 software
(Treestar, San Carlos, CA).
T cell subsets were quantified by the following method. Splenocytes
were recovered, and RBC were lysed by incubation in 1.0 ml ACK lysis
buffer (0.15 M NH4Cl, 1.0 mM
KHCO3, and 0.1 mM Na2EDTA
(pH 7.2)) for 5 min at room temperature. Splenocytes
(106 cells/sample) were washed and resuspended
in FACS buffer containing Fc block (BD PharMingen). After 10 min at
4°C anti-CD4-PE (clone RM25043; Caltag Laboratories, San
Francisco, CA), anti-CD8 FITC (clone RM2201; Caltag Laboratories),
or isotype control Abs were added. After a 30-min incubation at 4°C,
dead cells were stained by addition of propidium iodide at 5 µg/ml
for 5 min. The cells were then washed and analyzed on a FACSCalibur
flow cytometer (BD Biosciences).
BM-derived macrophages
Bone marrow from wt C57BL/6 mice was washed and resuspended at 2 x 105/ml in DMEM high glucose (Irvine Scientific) with 10% (v/v) heat-inactivated FCS (Life Technologies), 2 mM L-glutamine (Cellgro), 100 U/ml penicillin, and 100 µg/ml streptomycin (Pen/Strep; Cellgro), and 30% (v/v) L cell-conditioned medium. These were cultivated for 6–8 days in petri dishes (Fisher) at 37°C in 5% CO2. BM-DM were harvested, replated at 5 x 105 cells/ml in six-well tissue culture plates (Costar, Cambridge, MA), and stimulated 10 µg/ml ISS-ODN or mODN. Stimulated BM-DM were harvested, and total RNA was recovered for FACS analysis or RT-PCR.
In vivo stimulation
Female 129 mice were injected i.v. with 20 µg ISS-ODN or mODN in sterile saline. Mice were sacrificed at the indicated time intervals, and total splenocytes were recovered for RNA isolation and RT-PCR.
RT-PCR
The following primers were used: TAP1: sense, 5'-CGG ACT CCA ACC ATG GAG GAA ATC ACA G-3'; antisense, 5'-TCA GTC TGC AGG AGC CGC AAG AGC C-3'; TAP2: sense, 5'-CAG GCG GCC TGT GCA GAC GAC TTC AT-3'; antisense, 5'-TCA TGC CTC CAG CCG CTG CTG TAC CAG GT-3'; and G3PDH: sense, 5'-ACC ACA GTC CAT GCC ATC AC-3'; antisense, 5'-TCC ACC ACC CTG TTG CTGTA-3' (Integrated DNA Technologies, Coralville, IA). Total RNA isolated from BM-DM or splenocytes was analyzed by RT-PCR using a Superscript preamplification system (Life Technologies) and Advantaq Plus DNA polymerase (Clontech Laboratories, Palo Alto, CA) according to the manufacturer’s instructions. Amplification was conducted at 94°C for 30 s and at 68°C for 1 min in a GeneAmp 9600 thermal cycler (PerkinElmer/Cetus, Norwalk, CT; 18 cycles for G3PDH and 24 cycles for TAP1 and -2).
Construction of BM chimeras
Total bone marrow was harvested from TAP1-/- and wt C57BL/6 donors and washed. BM cells were depleted of mature T cells by resuspension at 30 x 106/ml in RP2 containing anti-Thy1 mAb (clone YTS 154), anti-CD4 mAb (clone RL172), and anti-CD8 mAb (clone 3.155) on ice and then lysed with batch-tested guinea pig complement (Pel-Freez Biologicals, Rogers, AR) at a 1/10 dilution and 10 µg/ml DNase I (Sigma) at 37°C as previously described (8). Cells were washed and resuspended at 107/100 µl (108/ml) in RPMI, and 107 cells were injected i.v. into lethally irradiated recipient C57BL/6 mice. The mice were rested for 8 wk, then engraftment was verified by FACS analysis of peripheral blood. The chimeric mice were then vaccinated as described in Vaccination protocol.
Vaccination protocol
The test animals were vaccinated according to a previously described protocol (9). Briefly, wt animals received mAb blockade (no azide, low endotoxin formulation) i.p. 6 h before vaccination. Doses were 200 µg for anti-CD40 ligand (anti-CD40L; clone MR1, BD PharMingen) and 100 µg each for anti-CD80 (clone 16-10A1; BD PharMingen) and CD86 (clone PO3; BD PharMingen). Ab-treated and gene-deficient mice received 50 µg OVA (Sigma-Aldrich) and 50 µg ISS-ODN (Tri-Link) in sterile saline intradermally at the tail base on days 0 and 14. Control mice received 50 µg OVA alone intradermally or no treatment. Three days before sacrifice, mice received an i.v. boost of 50 µg OVA (Sigma-Aldrich) in sterile saline. Animals were sacrificed at 6 wk, and total splenocytes were recovered for FACS analysis, secondary CTL, and cytokine restimulation assays.
CTL assay
CTL activity was assayed as previously described (9). Briefly, 2 x 106 effector splenocytes were restimulated in culture for 5 days with 1.8 x 107 OVA peptide-pulsed stimulator splenocytes and 50 U/ml recombinant human IL-2 (BD PharMingen) in RP10. After restimulation, viable lymphocytes were recovered by centrifugation over Ficoll Lympholyte M (Cedarlane Laboratories, Hornby, Canada) and washed, then serially diluted to several E:T cell ratios in 96-well U-bottom culture plates (Costar) in colorless RPMI (Irvine Scientific) supplemented with 2% BSA (Sigma-Aldrich), 2 mM L-glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin. Target EL4 cells were pulsed with OVA or nucleoprotein peptide at 37°C for 1 h, washed, and added to effector cells. Plates were incubated for 4 h, and supernatants were recovered. Specific lysis was assayed with the CytoTox 96 kit (Promega, Madison, WI) according to the manufacturer’s instructions.
Cytokine ELISA
Cytokine restimulation was assayed as previously described
(9). Purified rat anti-mouse IFN- capture Ab and
purified, biotinylated rat anti-mouse IFN--detecting Ab were
purchased from BD PharMingen. Briefly, splenocytes were isolated as
described and incubated for 3 days with or without 50 µg/ml OVA
(Sigma-Aldrich) restimulation in 96-well plates (Costar). Aliquots of
tissue culture supernatant were removed for cytokine ELISA. Half-area
96-well plates (Costar) were coated with capture Ab diluted 1/1000 in
carbonate buffer (15 mM
Na2CO3 and 35 mM
NaHCO3 (pH 9.6)) overnight at 4°C. Plates were
washed with 1x BBS (160 mM NaCl, 40 mM NaOH, and 200 mM boric acid (pH
8.0)) and then blocked for 2 h at 37°C with blocking buffer (1%
BSA in BBS). Plates were washed and incubated with tissue culture
supernatants diluted 1/2 in blocking buffer overnight at 4°C. Plates
were washed and incubated with detecting Ab diluted 1/1000 in blocking
buffer at room temperature for 1 h. Plates were washed and
incubated with streptavidin-HRP conjugate (Zymed Laboratories, South
San Francisco, CA) diluted 1/2000 in blocking buffer at room
temperature for 1 h. Plates were washed and incubated with TMB
substrate (Kirkegaard & Perry Laboratories, Gaithersburg, MD). The
reaction was stopped with 1 M phosphoric acid (Sigma-Aldrich), and the
plates were read at 450 nm on a ThermoMax microplate reader (Molecular
Devices, Menlo Park, CA).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To examine the role of IFN- in regulating costimulatory
molecules, BM-DC from wt and IFN-R-/-
mice were stimulated with ISS-ODN in vitro and analyzed by FACS. wt
BM-DC stimulated with ISS-ODN increased surface expression of CD40,
B7-1 and -2, and MHC class I (Fig. 1
A, top row). In
contrast, only a subpopulation of BM-DC from
IFN-R-/- mice was capable of
up-regulating CD40 and B7 in response to ISS-ODN stimulation (Fig. 1A, bottom row).
IFN-R-/- BM-DC did not up-regulate MHC
class I under these conditions. This result indicated that ISS-ODN
signaling via IFN- promoted expression of these costimulatory
molecules on the majority of BM-DC.
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IFN- has been shown to modulate the expression of TAP
(15). To investigate the effect of ISS-ODN on TAP
expression in APC and the potential role of IFN- in this
activity, BM-DC from wt and IFN-R-/- mice
were treated in vitro with ISS-ODN or mODN that did not contain the CpG
motif, and total RNA was isolated at several time points. RT-PCR
demonstrated that ISS-ODN treatment increased the transcription of TAP1
mRNA 4 h after stimulation, whereas mODN treatment did not (Fig. 1
B, first column). Real-time semiquantitative
RT-PCR demonstrated a 1.5-fold increase in TAP1 transcription at 4
h in ISS-ODN-stimulated BM-DC, escalating to a 1.9-fold increase at
6 h (data not shown). TAP1 transcription did not increase in
response to ISS-ODN in BM-DC from
IFN-R-/- mice, suggesting that IFN-
mediated this activity as well. This result is consistent with the
absence of augmented MHC class I expression in
IFN-R-/- BM-DC following ISS-ODN
stimulation (Fig. 1A).
ISS-ODN and IFN- also regulated TAP expression in vivo. wt and
IFN-R-/- mice were treated by i.v.
injection of ISS-ODN or mODN and were sacrificed at subsequent time
points. Splenocytes were isolated, and total RNA was analyzed by
RT-PCR. mRNA for TAP1 increased at 2 h in response to ISS-ODN, but
not mODN, and this effect was sustained up to 6 h (Fig. 1B, second column). In contrast, TAP1 was not
up-regulated in splenocytes from
IFN-R-/- mice.
IFN- is required for priming of Ag-specific CTL by ISS-ODN in
vivo
ISS-ODN appeared to regulate the expression of TAP and
costimulatory molecules via IFN-. To examine the role of
IFN- in priming of CTL by ISS-based vaccines, wt 129 and
IFN-R-/- mice were immunized with OVA and
ISS-ODN, and OVA-specific CTL activity was measured. wt mice
demonstrated efficient priming of OVA-specific CTL activity (Fig. 2A).
IFN-R-/- mice exhibited a 41–88%
reduction in CTL activity between 50:1 and 2:1 E:T cell ratios.
IFN-R-/- mice treated with mAb blockade
against B7-1 and -2 before immunization showed a similar reduction in
CTL priming, indicating that the observed reduction was due to the
absence of IFN- signaling. The
IFN-R-/- and wt mice had similar
CD4+ and CD8+ T cell
subsets, as determined by FACS analysis (Table I); therefore, the observed differences
were not an artifact due to reduction of the CD8+
population. These data demonstrated that IFN- played a critical
role and acted upstream of B7 costimulation in priming of CTL by
ISS-based vaccines.
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TAP activity is essential in certain animal models of
cross-priming against pathogen-derived Ags (14, 20). To
examine the role of TAP in cross-presentation by ISS vaccines,
TAP1-/- into wt
(TAP-/-
wt) and wtwt bone marrow chimeras
were generated. The chimeras were necessary because
TAP1-/- mice do not support the native
development of CD8+ CTL (21).
TAP1-/-wt chimeras did not prime CTL
activity in response to OVA and ISS-ODN vaccination, whereas wtwt
chimeras did, demonstrating that TAP was essential for ISS-induced
cross-presentation (Fig. 2B). The chimeric and wt mice also
had similar T cell subsets (Table I).
ISS-based vaccines bypass CD40 in priming naive CD8+ lymphocytes
Current models of cross-priming CTL activity against exogenous
protein Ags posit a "licensing" step between APC and
CD4+ Th cells, followed by an activation step
between licensed APC and naive CD8+ lymphocytes.
The licensing step between CD4+ T cells and APC
requires CD40/CD40L signaling (22, 23, 24), but the
costimulatory requirements for the activation interaction between
licensed APC and naive CD8+ lymphocytes are
unknown. As noted previously, ISS-based vaccines can bypass
CD4+ Th activity in priming CTL (9),
allowing evaluation of the activation interaction independent of the
licensing step. To examine the role of CD40, wt and
CD40-/- mice were immunized with OVA and
ISS-ODN. wt mice were also treated with blocking mAb to CD40L or
isotype control before immunization. wt mice immunized with OVA and
ISS-ODN demonstrated efficient priming of OVA-specific CTL activity
(Fig. 2C). Lytic activity from splenocytes of
CD40-/- mice and wt mice treated with
anti-CD40L mAb did not differ significantly from that of wt mice.
Treatment with an isotype control mAb did not affect CTL activation
(data not shown). The T cell subsets were also similar among these
groups (Table I). These data indicate that CD40/CD40L signaling did not
make a significant contribution to the priming interaction between APC
and CD8+ lymphocytes in response to ISS-based
vaccines.
B7-1 and -2/CD28 costimulation is required for priming of naive CD8+ lymphocytes by ISS-based vaccines
B7-1 and -2 have been shown to play a critical role in priming of
CTL activity in vivo (25). To examine the role of these
molecules in ISS-based vaccination, wt mice were treated with mAb
blockade to B7-1 and -2 before immunization with OVA and ISS-ODN. Both
B7 molecules were blocked, because previous studies demonstrated that
either signal alone can support the priming of CTL activity
(25). Mice deficient in CD28, the positive signaling
receptor for the B7 molecules (26), were also immunized.
Treatment with anti-B7 mAb or immunization of
CD28-/- mice resulted in a 52–80% reduction
(between 25:1 and 1:1 E:T ratios) in CTL activity compared with that in
wt mice, strongly suggesting that B7 signaling through CD28 provided
critical costimulation for CTL priming (Fig. 2D). Addition
of anti-CD40L mAb did not further reduce CTL activity, supporting
the findings in the previous section.
IL-12 makes a nonsynergistic contribution to priming of CD8+ lymphocytes by ISS-based vaccines
IL-12 is a proinflammatory cytokine that has been shown to promote
CTL proliferation (27). Both ISS DNA and IFN- have also
been shown to directly up-regulate IL-12 expression in APC
(6). Production of IL-12 by APC can also be stimulated by
CD40/CD40L and B7/CD28 interactions (28, 29). To determine
the role of IL-12 signaling in priming of CTL activity, wt and
IL-12p40-/- animals were immunized with OVA and
ISS-ODN, with one group of IL-12p40-/- mice
receiving anti-B7 and anti-CD40L mAb blockade.
IL-12p40-/- mice exhibited a 35% reduction in
CTL activation compared with wt mice at a 25:1 E:T cell ratio (Fig. 2E). The addition of blocking mAb against B7 and CD40L
resulted in an additional 46% reduction in CTL activation. This CTL
activity was comparable to mAb blockade of B7 alone (Fig. 2D), suggesting that B7 was the dominant costimulation in
priming of CTL by ISS-based vaccines. In this setting, IL-12 appeared
to make a nonsynergistic contribution to CTL priming in response to
ISS-based vaccines.
IFN- mediates priming of Th1-biased CD4+ activity
by ISS-ODN via CD40, B7, and IL-12
Priming of Th1-biased CD4+ helper activity
also exhibited dependence upon autocrine IFN-. Mice were
immunized as described in the previous sections, and splenocytes were
isolated and restimulated in culture with OVA. IFN- secretion was
quantified by ELISA as a measure of Th1-biased immune responses.
Splenocytes from immunized IFN-R-/- mice
secreted 62% less IFN- (Fig. 3, bar B) in restimulation assays compared with wt controls
(Fig. 3, 129 (+) control). IFN-R-/- mice
treated with mAb blockade against B7 molecules exhibited similarly
reduced levels of IFN- (Fig. 3, bar A), suggesting that
IFN- also acted upstream of B7 costimulation in
CD4+ priming. Thus, priming of Th1-biased
CD4+ lymphocyte activity by ISS-based vaccines
appeared to be partially dependent upon IFN-, consistent with its
regulation of CD40 and B7 expression (Fig. 1). Splenocytes from
TAPwt and wtwt bone marrow chimeras produced similar levels of
IFN- in restimulation assays (Fig. 3, bars C and D,
respectively), indicating that TAP did not make a significant
contribution to Th1 priming. It is likely that total body irradiation,
adoptive transfer of bone marrow, and older age of the chimeric animals
contributed to the lower overall IFN- secretion compared with wt
(Fig. 3, B6 (+) control).
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secretion (Fig. 3, bar E), and splenocytes from wt mice
treated with anti-CD40L mAb blockade demonstrated a 59% reduction
(Fig. 3, bar F) compared with wt controls (Fig. 3, B6 (+)
control). mAb blockade of B7 resulted in an 89% reduction in IFN-
secretion (Fig. 3, bar G), and
CD28-/- splenocytes had a 92% reduction (Fig. 3, bar I). Splenocytes from CD28-/- mice
treated with anti-CD40L mAb demonstrated complete inhibition of
IFN- secretion (Fig. 3, bar J). Splenocytes from
IL-12p40-/- mice secreted 2% IFN-
compared with wt (bar K), and this was not affected by mAb
blockade of B7 (Fig. 3, bar L).
Interestingly, mAb blockade of CD40L on CD4+
lymphocytes was not equivalent to the absence of CD40 on APC (Fig. 3, bars E and F). The addition of anti-CD40L mAb blockade to
either anti-B7 mAb blockade (Fig. 3, bar H) or
IL-12p40-/- mice (Fig. 3, bar L) resulted
in higher levels of IFN- secretion than either condition alone, but
the addition of anti-CD40L to CD28-/- mice
(Fig. 3, bar J) reduced IFN- secretion from 8% of the control
value to zero. These data suggest that engagement of the blocking mAb
to CD40L may transduce partial positive signaling to the
CD4+ cell, and this stimulation is dependent upon
the presence of intact CD28.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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mechanism to regulate the expression of
costimulatory molecules and TAP. These molecules then mediate the
priming interactions between APC and naive CD4+
and CD8+ lymphocytes via cell-to-cell
interactions and cross-presentation of Ag in the context of MHC class
I. Priming of CD8+ CTL by ISS-based vaccines
requires TAP activity and B7, and costimulation is partially dependent
upon IL-12 and is independent of CD40. In contrast, priming of
CD4+ Th1 lymphocytes requires CD40, B7, and IL-12
costimulation and is independent of TAP activity.
Priming of Ag-specific CTL by ISS-based vaccines is independent of
CD4+ T helper activity (9). The data
presented in this study suggest the following sequence of events in
this activity (depicted in the right half of Fig. 4). First, ISS-ODN and Ag are acquired by
the APC. ISS-ODN promotes autocrine signaling via IFN- (Fig. 4, arrow 1), which up-regulates TAP (Fig. 4, arrow 2) and B7
expression (Fig. 4, arrow 3). TAP-dependent cross-presentation of
exogenous Ag in the context of MHC class I (Fig. 4, arrow 4) in
conjunction with B7 costimulation results in priming of naive CTL.
Although IL-12 appears to make a contribution to CTL priming (Fig. 4, arrow 5), it is unclear whether this is a direct effect on CTL.
Because the experiments reported in this study were conducted in
animals with an intact CD4+ compartment, it is
possible that IL-12 acts via priming of Th cells to promote B7
up-regulation rather than by directly affecting CTL. The enhanced
inhibition of CTL priming observed in
IL-12p40-/- mice treated with anti-B7 mAb
blockade supports this hypothesis. Further investigation will be
required to discern among these possibilities.
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(Fig. 4, arrow 1), which, in turn,
up-regulates CD40 and B7 costimulatory molecules (Fig. 4, arrow
6). ISS-ODN also up-regulates IL-12 expression (Fig. 4, arrow
7), and this may be a direct effect of ISS-ODN or may also be
mediated by autocrine IFN- (6). Priming of Th1-type
responses (depicted in the left half of Fig. 4) is achieved
by cognate interaction between the TCR of naive
CD4+ Th cells and Ag in the context of MHC class
II and receptor-ligand interactions between CD40/CD40L, B7/CD28, and
IL-12/IL-12R. CD40- and B7-dependent signaling is known to enhance
IL-12 production, which may provide amplification of the stimulatory
signal (29, 30). These costimulatory paths act upon
CD4+ lymphocytes to promote Th1-type priming. As
expected, CD4+ lymphocyte priming is not
dependent upon TAP.
TAP activity is required for cross-presentation and CTL priming by
ISS-based vaccines. The data presented in this study appear to be the
first report of regulation of TAP expression in APC by IFN-,
suggesting a potential mechanism for facilitation of cross-presentation
of exogenous Ags by ISS-based vaccines. Cytokine-induced increases in
TAP expression have been shown to promote Ag presentation and
stabilization of MHC class I complexes (31, 32). These
findings suggest that ISS-ODN facilitates cross-presentation at least
in part by regulation of TAP expression via IFN--dependent
signals.
It has been reported that IFN- secreted by ISS-stimulated APC can
act directly upon T cells to induce partial activation
(33). However, our data show that costimulatory molecules
and TAP-dependent cross-presentation act downstream of IFN-,
because interruption of these activities in animals with intact
IFN- signaling prevents CTL and Th1 priming. The addition of
anti-B7 mAb blockade does not enhance the reduction of CTL priming
in IFN-R-/- mice, also indicating that
costimulation acts downstream of IFN-. Interestingly, IFN-
have been shown to act directly upon activated T cells to promote their
survival (34). Thus, ISS-based vaccines may prime T cells
via indirect IFN--dependent mechanisms and maintain T cell
activity by a direct mechanism. The in vitro model presented
demonstrates that autocrine IFN- signaling can support the
regulation of costimulatory molecules and TAP. However, ISS-ODN also
stimulate the secretion of IFN- by other cellular components of
innate immunity. The more rapid induction of TAP in vivo strongly
suggests that other sources of IFN- may contribute to this
activity. Further investigation is needed to determine whether
autocrine or paracrine signaling predominates in vivo.
ISS-ODN is an ideal molecular probe for examining the cross-priming
interaction between APC and naive CD8+
lymphocytes, because it achieves this activity independently of
CD4+ T cell help. The results reported in this
study provide further refinement of the licensing model of
cross-priming, which invokes a three-cell interaction between Th cells,
APC, and naive CD8+ lymphocytes
(22, 23, 24). By directly up-regulating B7 and TAP, ISS-based
vaccines bypass CD40-dependent T cell help. This model implies that a
major function of Th-dependent licensing in cross-priming is the
up-regulation of B7 expression on APC via CD40/CD40L interaction
(35). Although this model explains many aspects of
cross-priming, there are still unresolved issues that require further
investigation. First, it is apparent that a subpopulation of
IFN-R-/- BM-DC is capable of
up-regulating costimulatory molecules in response to ISS-ODN
stimulation. What are the signaling pathways that mediate this
redundant activity? Second, do novel members of the CD40/CD40L
(36, 37) and B7/CD28 (38) families of
signaling molecules play roles in this system? Third, what are the
interactions between IFN- and the pathways critical to ISS DNA
signaling, Toll-like receptor 9 (39), and DNA-PK
(40)? Finally, do ISS-ODN have different activities on
different subsets of DC, and how does this affect T cell priming?
ISS-based vaccines are uniquely suited to efficiently prime CTL
activity due to their coordinated regulation of cross-presentation and
costimulation. CTL priming by ISS-based immunization has been
demonstrated for experimental Ags OVA and -galactosidase
(41) and for viral Ags HIV gp120 (41, 42) and
hepatitis B virus surface Ag (43). This immunization
strategy has already demonstrated efficacy in an animal model of cancer
(9). Although the efficient priming of Ag-specific tumor
immunity has been an elusive goal, ISS-based vaccines represent a
promising new approach to this problem. Further investigation of the
formulation of vaccines with naturally occurring tumor-associated Ags
could result in effective cancer immunotherapy.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Hearn Jay Cho, Division of Hematology/Medical Oncology, New York Presbyterian Hospital, 525 East 68th Street, New York, NY 10021. E-mail address: hjc2001{at}med.cornell.edu
3 Abbreviations used in this paper: ISS, immunostimulatory sequence; DC, dendritic cell; BM-DC, bone marrow-derived DC; ODN, oligodeoxynucleotide; CD40L, CD40 ligand; mODN, mutated ODN; wt, wild type.
Received for publication November 19, 2001. Accepted for publication March 8, 2002.
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). This decrease was not affected by
addition of anti-B7-1 and -2 mAb blockade (
). Untreated wt 129
mice did not exhibit OVA-specific CTL activity (
). Target cells
loaded with an irrelevant H-2b-restricted peptide derived
from influenza nucleoprotein were not lysed, indicating that CTL
activity was Ag specific (data not shown). B, C57BL6
wt
) did not exhibit OVA-specific CTL
activity, nor did untreated negative control mice (
and 
) did not further decrease CTL activity,
indicating that B7/CD28 mediated the dominant costimulatory interaction
between APC and naive CTL. E, IL-12p40-/-
mice (•) exhibited an intermediate reduction of OVA-specific CTL
activity compared with wt controls (
