Download Enhancement of antigen-presenting cell surface molecules involved

International Immunology, Vol. 11, No. 7, pp. 1111–1118
© 1999 The Japanese Society for Immunology
Enhancement of antigen-presenting cell
surface molecules involved in cognate
interactions by immunostimulatory DNA
sequences
Elena Martin-Orozco, Hiroko Kobayashi, John Van Uden, Minh-Duc Nguyen,
Richard S. Kornbluth and Eyal Raz
Department of Medicine, University of California San Diego, 9500 Gilman Drive, La Jolla, CA 92093, USA
Keywords: activation markers, antigen-presenting cells, CpG motifs, immunostimulatory DNA sequences,
Th1 response
Abstract
Bacterial genomic DNA, plasmid DNA (pDNA) and synthetic oligodeoxynucleotides (ODN)
containing immunostimulatory DNA sequences (ISS) have been proposed to foster a Th1 response
via the release of type 1 cytokines from macrophages, dendritic cells, NK cells and B cells. In this
study, we show that ISS-enriched DNA up-regulates a distinct profile of cell surface molecules on
macrophages and B cells in vitro and in vivo. ISS-ODN and ISS-containing pDNA enhanced the
expression of antigen presentation molecules (MHC class I and II), co-stimulatory molecules (B7-1,
B7-2 and CD40), cytokine receptors (IFN-γ receptor and IL-2 receptor), an adhesion molecule
(ICAM-1) and an Fc receptor (Fcγ receptor) on murine B cells or bone marrow-derived
macrophages. The increased expression of these surface molecules is seen in purified cell
populations and is largely independent of the effects of type 1 cytokines. Splenic antigenpresenting cells stimulated with ISS-ODN in vivo efficiently activate naive T cells and bias their
differentiation toward a Th1 phenotype in vitro. Thus, the induction of both type 1 cytokines and a
distinct profile of cell surface molecules contributes to the potent immunostimulatory effects of
ISS-containing DNA on innate and adaptive immunity.
Introduction
The immunostimulatory properties of bacterial DNA were first
discovered when it was found that mycobacterial DNA (Bacille
Calmette-Guerin) elicited IFN (α, β and γ) production and
induced NK cell activation (1–3). Subsequent experiments
have shown that oligonucleotides containing immunostimulatory DNA sequences (ISS; also known as CpG motifs) induce
the release of IL-6, IL-12, tumor necrosis factor (TNF)-α and IL18, primarily from monocytes and macrophages (4–8). Indeed,
recent work has demonstrated the Th1-promoting activity of
synthetic oligodeoxynucleotides (ODN) containing ISS
sequences (ISS-ODN) on co-administered antigens (9–13).
Although the cytokine milieu induced by ISS is thought to play
an important role in amplifying the subsequent immune
response to the encountered antigen (6,10), cognate interactions between antigen-presenting cells (APC) and lympho-
cytes or among lymphocytes (e.g. B cell–T cell) also play a
crucial role in the primary activation of T and B cells. This study
was therefore designed to uncover the relationship between
ISS-induced cytokines and the activation of surface molecules
on different subsets of immunocytes, and to explore the subsequent functional properties induced by these ISS-driven
changes in the induction of the immune response in vitro and
in vivo. The results presented here indicate that (i) ISS-ODN
induces a distinctive profile of cell surface molecules on APC
[B cells and bone marrow-derived macrophages (BMDM)] but
not on T cells, (ii) this profile of cell surface markers is only
partially dependent on ISS-induced cytokine secretion, and (iii)
this distinctive activation profile plays an important role in vivo
in the generation of a potent antigen-specific T cell response
and its differentiation toward Th1 phenotype.
The first two authors contributed equally to this work
Correspondence to: E. Raz
Transmitting editor: K. Okumura
Received 9 September 199, accepted 25 March 1999
1112 Activation of APC by ISS
Methods
Animals
Female BALB/c mice, 6–10 weeks of age, were purchased from
The Jackson Laboratory (Bar Harbor, ME). TCR-OVA (DO11.10)
transgenic mice on the BALB/c background (14) were generously provided by Dr David Broide (UCSD) with permission
from Dr Dennis Loh. All experimental animal protocols were
approved by the UCSD Animal Subjects Committee.
Nucleic acid reagents and lipopolysaccharide (LPS)
Endotoxin-free (,1 ng/mg DNA) phosphorothioate singlestranded ODN (Trilink, San Diego, CA) were used in all the
experiments. The sequence of the ISS-ODN is 59-TGACTGTGAACGTTCGAGATGA-39 and the sequence of the mutant (M)ODN is 59-TGACTGTGAAGGTTAGAGATGA-39 (6). The underline indicates the ISS (CpG motif) and its corresponding 1 base
alteration (CG → GG). The plasmid pUC19 (bacterial plasmid
containing ISS) was purified with the Qiagen MaxiPrep kit
(Chastworth, CA). Plasmid DNA was phenol–chloroform
extracted and ethanol precipitated before use. The endotoxin
level was ,1 ng/mg DNA or ODN by the Pyrotel limulus amebocyte lysate (Associates Cape Cod, Woods Hole, MA). Escherichia coli LPS was purchased from Sigma (St Louis, MO).
Flow cytometry
Following incubation with Fc block (PharMingen, San Diego,
CA), sample cells were stained with conjugated antibodies
specific for cell populations and with antibodies specific for
surface molecules, as shown in Table 1. Isotype controls for the
specific surface markers are as follows: hamster IgG, hamster
IgM, rat IgG2a, rat IgG2b, rat IgM, mouse IgG2a and mouse
IgG2b (Caltag, San Francisco, CA or PharMingen). Propidium
iodide was included in the last wash at a concentration of 2 µg/
ml. Live cells (propidium iodide-negative) were analyzed on a
FACSCalibur flow cytometer (Becton Dickinson, San Jose, CA).
The data were analyzed with CellQuest (Becton Dickinson) and
FlowJo (Tree Star, San Carlos, CA) software. In most cases,
ISS-ODN and LPS treatment significantly increase non-specific
antibody binding or autoflorescence (seen as increases in isotype control antibody-stained mean florescence), so this is
controlled for with the mean florescence index ratio (MFIR).
MFIR 5 (mean florescence when stained for surface molecule)/
(mean florescence when stained with isotype control antibody).
MFIR represents the fold increase in surface marker expression
relative to background autoflorescence and non-specific antibody binding. We have found MFIR to be a conservative and
more accurate estimate of expression of surface molecules
when studying cells treated with ISS-containing DNA.
Spleen cell culture and B cell purification
Murine spleen cells (23106/ml) were cultured in RPMI supplemented with 10% FBS (Gemini Bioproducts, Calabasas, CA),
2 mM L-glutamine (BioWhitaker, Walkersville, MD), 2 mM penicillin/streptomycin (BioWhitaker) and 50 µM 2-mercaptoethanol (Sigma). Splenic B cells were purified by depletion of
non-B cells with antibodies and complement. Briefly, spleen
cells were incubated in supplemented RPMI for 1 h (adherent
step) and then incubated with anti-CD8 antibody (3.155), antiCD4 antibody (RL172) and anti-Thy-1 antibody (YTS 154) fol-
lowed by incubation with guinea pig complement (Pel-Freeze,
Rogers, AR). The cells were incubated for 48 h in the presence
of ISS-ODN (1 µg/ml), M-ODN (1 µg/ml), LPS (5 µg/ml), pUC19
(10 µg/ml) or media alone at 37°C with 5% CO2 and harvested
for FACS analysis as described above.
Cytokine ELISA and neutralization
The concentrations of cytokines in supernatants were analyzed
by commercial ELISA kits (PharMingen or Biosource, Camarillo, CA) according to the manufacturer’s instructions.
For neutralization, murine spleen cells (23106/ml) were cultured in the presence of ISS-ODN (1 µg/ml) plus commercial
neutralizing antibodies to IFN-α/β [polyclonal, cat. no.
AMC4014, 3 µg/ml 5 1000 neutralizing units (NU)/ml], IFN-γ
(polyclonal, cat. no. AMC4034, 10 µg/ml 5 1000 NU/ml), TNF-α
(polyclonal, cat. no. AMC3012, 1 µg/ml 5 1100 NU/ml), IL-6
(clone MP5-20F3, 1.25 µg/ml) or IL-12 (clone C15.6, 10 µg/
ml) (all from Biosource), or a cocktail of all the above. The
concentrations of the various antibodies were pre-determined
to neutralize cytokine levels induced by ISS-ODN. The cells
were harvested after 48 h incubation and activation markers on
B cells (gated on B2201 population) were analyzed by FACS.
As controls, sheep, rabbit (Irvine Scientific, Santa Ana, CA), rat
(PharMingen) and hamster (Caltag) sera were used.
Inhibition of B cell proliferation by irradiation or mitomycin C
(MMC) treatment
To evaluate the effect of the mitogenic properties of ISS (15,16)
on cell surface molecules, spleen cells were γ irradiated (1500
rad) or treated with MMC (50 µg/ml for 30 min at 37°C)
prior to stimulation. Cells were then incubated with ISS-ODN (1
µg/ml), M-ODN (1 µg/ml), LPS (5 µg/ml) or media alone for 48
h, then stained for expression of cell surface markers on B
cells (Table 1). BrdU staining (17) confirmed inhibition of B cell
proliferation by pre-treatment.
Preparation and activation of BMDM
Bone marrow cells were flushed from the femurs of 4- to-8week-old female BALB/c mice and cultured in 145 mm sterile
Petri dishes as previously described (18,19) Primary culture
media was DMEM (Irvine Scientific) supplemented with 10%
FBS (Gemini Bioproducts), 2 mM L-glutamine (BioWhitaker), 1
mM sodium pyruvate (BioWhitaker), 2 mM penicillin/streptomycin (BioWhitaker) with 30% L cell conditioned media (supernatant from L929 cells) (19). The cells had acquired macrophagelike morphology (BMDM), and FACS analysis at day 8 showed
the cell surface phenotype of the population to be: .90% Mac31, ,0.2% B2201, ,0.4% CD41 and CD81 cells. BMDM were
washed and re-cultured with supplemented DMEM media or
DMEM media containing ISS-ODN (1 µg/ml), M-ODN (1 µg/ml)
or LPS (5 µg/ml) for 48 h and then harvested for FACS analysis.
T cell proliferation assay and cytokine analysis
Splenic T cells and APC were prepared from TCR-OVA transgenic mice. To obtain APC, mice were injected i.d. with saline,
ISS-ODN (50 µg/mouse) or M-ODN (50 µg/mouse) on days 0
and 7, and sacrificed on day 14. To enrich APC, splenocytes
were treated with anti-CD8 antibody (3.155) and anti-CD4 antibody (RL172) followed by incubation with guinea pig complement as described (20), and treated with MMC (50 µg/ml, 30
Activation of APC by ISS 1113
Table 1. Antibodies for FACS Analysis in This Study
Antibody specificity (clone)
Common name
Category
Label
Source
H-2Kd (SF1-1.1)
I-Ad (AMS-32.1)
CD1d (1B1)
class I
class II
MHC analog
MHC molecules
FITC
FITC
FITC
PharMingen
PharMingen
PharMingen
co-stimulatory molecules
FITC
FITC
FITC
FITC
FITC
PEa
Caltag
PharMingen
PharMingen
PharMingen
PharMingen
PharMingen
CD28 (37.51.1)
CD80 (GL1)
CD86 (16-10A1)
CD40 (HM40-3)
CD40 ligand (MR1)
CD152 (UC10-4F10-11)
B7-1
B7-2
CTLA-4
CD16/32 (2.4G2)
CD23 (B3B4)
FcγIII/II receptor
FcεII receptor
Fc receptors
FITC
FITC
PharMingen
PharMingen
CD119 (GR20)
CD25 (7D4)
IL-1R (35F5)
IL-6 R (D7715A7)
IFN-receptor α chain
IL-2 receptor α chain
IL-1 receptor/p80
IL-6 receptor
cytokine receptors
FITC
PE
PE
PE
PharMingen
PharMingen
PharMingen
PharMingen
adhesion molecules
PharMingen
PharMingen
PharMingen
PharMingen
PharMingen
PharMingen
PharMingen
CD54/ICAM-1 (3E2)
CD102/ICAM-2 [3C4(MIC2/4)]
CD49a (Ha31/8)
CD49b (HMα2)
CD49d (R1-2)
CD49e [5H10-27 (MFR0)]
CD49f (GoH3)
integrin
integrin
integrin
integrin
integrin
chain
chain
chain
chain
chain
FITC
FITC
FITC
FITC
FITC
FITC
FITC
B220 (RA3-6B2)
integrin α6 chain
FITC
PharMingen
B220 (RA3-6B2)
CD4 (CT-CD4)
CD8 (Ly-2)
MAC-3 (M3/84)
B lymphocytes
helper/inducer T cells
cytotoxic/suppressor T cells
macrophages
PE,
PE,
PE,
PE,
Caltag
Caltag
Caltag
PharMingen
α1
α2
α4
α5
α6
cell population markers
FITC
FITC
FITC
FITC
aPhycoerythrin.
min at 37°C) prior to being used as accessory cells. Naive T
cells were purified from splenocytes obtained from naive TCROVA transgenic mice by depletion with antibody cocktail; J11D
(anti-HSA), CA4.12 (anti-Ia), RA36B2 (anti-B220), M5.114 (antiIa, isotype IgG2a), MAR.18 (mouse anti-rat IgG) and guinea
pig complement. The purity of resultant T cell preparations was
.95% by FACS staining. Naive T cells were incubated with the
same number of different accessory cells (APC) in the presence
of hen egg ovalbumin (OVA, 10–30 µg/ml, grade 5; Sigma).
After 4 days incubation, cells were pulsed with [3H]thymidine
(1 µCi/well; ICN Pharmaceuticals, Irvine, CA) for 18 h, then
harvested and [3H]thymidine incorporation was determined
with a 1450 Microbeta liquid scintillation counter (Wallac, Turku,
Finland). Supernatants were harvested for cytokine assay.
Cytokine levels (IFN-γ, IL-4 and IL-5) in supernatants were analyzed by commercial ELISA kits (PharMingen or Biosource).
Results
ISS-ODN induces in vitro up-regulation of a distinctive profile
of cell surface markers on B cells from spleen and blood
Incubation for 48 h with ISS-ODN produces a distinct profile
of surface molecules on purified splenic B cells (Fig. 1). The
expression of MHC class II, CD40 and CD16/32 was clearly
up-regulated, and the expression of MHC class I, IFN-γ
receptor and IL-2 receptor was slightly up-regulated in MFIR.
Interestingly, ISS-ODN suppressed CD23 (Fcε receptor)
expression. No differences in the expression of CD49b,
CD49a, CD49c–f, IL-1 receptor and IL-6 receptor (data not
shown) were observed in the ISS-ODN versus media or MODN treated cells. Stimulation with LPS resulted in a similar
pattern to that observed for ISS-ODN but with different intensity, except for CD23.
Splenic B cells are considered to have more of an activated/
memory phenotype than peripheral cells, so to evaluate
whether ISS-ODN could also activate resting naive B cells,
lymphocytes were Ficoll purified from mouse peripheral blood.
The lymphocytes were cultured in media alone, with ISS-ODN
or with M-ODN for 48 h, under the same conditions described
above. FACS analysis showed that ISS-ODN induces an
almost identical profile on blood B2201 cells (data not shown)
as on splenic B2201 cells.
While purified B cells alone readily respond to ISS-ODN
with surface marker expression, it may be that the complex
cellular interactions with other cell types (e.g. macrophages,
NK cells) have some enhancing or modulatory effects. To
address this, whole splenocytes (after red blood cell lysis)
were treated as above with ISS-ODN or controls, and B2201
cells were analyzed for the full set of surface marker expression profiles. The B cells in whole splenocyte mixtures showed
essentially the same profile of cell surface molecule expression as seen in Fig. 1 with purified B cells (data not shown).
1114 Activation of APC by ISS
ISS-ODN induces in vivo up-regulation of cell surface markers
on splenic B cells
In order to examine the effect of ISS-ODN to the activation
profile in vivo, we injected BALB/c mice with ISS-ODN, MODN (100 µg in 200 µl saline per mouse) or saline i.p. Mice
were sacrificed at either day 2, 7 or 21 after the injection,
splenocytes were stained and surface markers on B cells
were analyzed by FACS. Figure 2 shows the activation profile
in vivo. The expressions of MHC class I, MHC class II, B7-1,
B7-2, CD40, ICAM-1, CD16/32 and IL-2 receptor were slightly
up-regulated, and most of them reached their peaks at
day 7 except for MHC class I, CD40 and CD16/32. Downregulation of CD23 expression was observed in vivo, as it
was in vitro (Fig. 1).
ISS-induced cytokines only partially mediate ISS-induced cell
surface marker up-regulation
In order to evaluate the effects of Th1-type cytokines on the
activation markers induced by ISS-ODN, various cytokines
(IL-12, IL-6, IFN-α/β, IFN-γ and TNF-α) were neutralized with
antibodies in vitro. The levels of these cytokines were initially
Fig. 1. In vitro induction of cell surface molecules on purified mouse
B cells. Purified B cells (23106/ml) obtained from spleens of naive
BALB/c mice were cultured with ISS-ODN (1 µg/ml), M-ODN (1 µg/
ml) or media alone for 48 h and then stained as described. The
x-axis represents a log scale of fluorescence intensity of each surface
marker and the y-axis represents the number of B2201 cells found
of that fluorescence. For each histogram, the surface moleculespecific antibody (black line) is compared to the appropriate antibody
isotype control (gray line). In most cases, ISS-ODN and LPS treatment
significantly increase non-specific antibody binding (seen with
increases in isotype control binding), so this is controlled for with the
MFIR, given in the top right corner of each histogram. Additionally,
stimulation of B cells with the ISS-containing plasmid pUC19 (at 10
µg/ml) gives the same profile of surface marker expression induction
as seen in this figure with ISS-ODN stimulation (data not shown). The
induction of surface molecules by ISS-ODN is not dependent upon
proliferation because ISS-ODN-stimulated B cells previously treated
with γ irradiation or MMC give essentially identical surface marker
expression changes as those seen in this figure (data not shown).
Data represents results from three independent experiments.
Fig. 2. In vivo induction of cell surface molecules on mouse splenic
B cells. BALB/c mice were injected i.p. with ISS-ODN (100 µg),
M-ODN (100 µg) or saline. Mice were sacrificed on day 2, 7 or 21
after injection, the spleens were harvested and the spleen cells were
stained for FACS analysis. The histograms show the expression of
each surface molecule within the B2201 cell population, in the same
format as that in Fig. 1. Data are representative of the results obtained
from three different mice per group.
Activation of APC by ISS 1115
assessed and the matching concentration of antibody for
each cytokine was determined by titration assay until complete
neutralization was observed (data not shown). Percent inhibition of activation markers was defined on splenocytes gated
on the B2201 cell population. As shown in Fig. 3, the enhanced
expression of MHC class II was not modified by neutralization
of cytokines described above. The neutralization of IFN-γ
inhibited CD40 and IL-2 receptor expression up to 40%, and
had a modest effect on ICAM-1. A cocktail of neutralizing
antibodies against all cytokines resulted in 30% inhibition of
CD40 and 70% inhibition of IL-2 receptor induction.
Induction of cell surface molecules on B cells is not dependent
upon B cell proliferation
To evaluate whether the effect of ISS-ODN on the expression
of surface molecules on B cells was related to its mitogenic
effect on B cells (15,16), splenocytes were γ irradiated or
treated with MMC and then incubated with ISS-ODN or MODN. The expression of surface markers was analyzed 48 h
later. The inhibition of proliferation was confirmed by the
lack of BrdU incorporation (data not shown). ISS-ODN upregulated the expression of cell surface molecules despite the
complete inhibition of B cell proliferation either by irradiation or
by MMC treatment (data not shown), indicating that the upregulation of surface molecules by ISS-ODN is not due to its
of mitogenic effect.
ISS-ODN does not induce an activation of cell surface molecules on splenic T cells
Fig. 3. Neutralization of cytokines produced in response to ISS
partially inhibits the expression of some cell surface molecules.
Spleen cells (23106/ml) were cultured in the presence of ISS-ODN
(1 µg/ml) and neutralizing antibodies against IFN-α/β, IFN-γ, TNF-α,
IL-6 and/or IL-12. The neutralization effect of these cytokines was
confirmed by cytokine ELISA, then cells were stained to see the
inhibitory effect of cytokine neutralization on activation markers.
Percent inhibition of activation markers on the B2201 cell population
was defined as follows: 100%3(MFISS – MFISS 1 neutralizing antibody)/
(MFISS – MFmedia control), where MF is mean florescence. Data represent
results from one representative experiment out of three performed.
We also evaluated the differential expression of antigen
presentation and activation cell surface molecules on T cells
such as CD28, CTLA-4 and CD40 ligand in addition to the
markers described for B cells (Figs 1 and 2). Incubation of
splenocytes with ISS-ODN did not modify any of these cell
surface molecules on splenocytes gated on the CD41 or
CD81 cell population in vitro (data not shown). In addition,
injection with ISS-ODN in vivo also did not modify cell surface
molecules on splenocytes gated on the CD41 or CD81 cell
population (data not shown).
ISS-ODN induces up-regulation of a distinctive profile of cell
surface markers on BMDM in vitro
Recent work has demonstrated the role of ISS-ODN in activation of macrophages to release various cytokines such as
TNF-α, IFN-α/β and IL-12 (4–8). In this study we analyzed the
effect of ISS on the pattern of cell surface molecules expressed
on macrophages. As shown in Fig. 4, ISS-ODN clearly
Fig. 4. Induction of cell surface molecules on BMDM. BMDM (23105/ml) were incubated in vitro with ISS-ODN (1 µg/ml), M-ODN (1 µg/ml),
LPS (5 µg/ml) or media alone for 48 h, and then stained for Mac-3 (phycoerythrin) and activation markers (FITC) for FACS analysis. The
histograms represent the FITC staining of gated Mac-31 macrophages, in the same format as that in Fig. 1. Additionally, stimulation of BMDM
with the ISS-containing plasmid pUC19 (at 10 µg/ml) gives the same profile of surface marker expression induction as seen in this figure with
ISS-ODN stimulation (data not shown). Data represents results from one representative experiment out of three that were performed.
1116 Activation of APC by ISS
enhances the expression of MHC class I, B7-1, CD40, ICAM-1
and CD16/32 on BMDM.
pUC19 induces up-regulation of surface molecules on B cells
and BMDM
In previous work we and others have proposed that in gene
vaccination, the pDNA serves not only to encode the antigen,
but also as its intrinsic adjuvant (9,10,21). This effect is
mediated via the ISS motifs in the plasmid backbone (6,9,21).
To analyze the effect of pDNA on the cell surface profile,
we either incubated or transfected mouse splenocytes with
pUC19 or methylated pUC19 for 48 h prior to analysis by
FACS. Indeed, pUC19 but not methylated pUC19 induced a
similar cell surface expression profile on B splenocytes as well
as on BMDM under each of these conditions (data not shown)
as observed for ISS-ODN (see Figs 1 and 4 respectively).
ISS-ODN enhanced the functionality of splenic APC to activate
and to induce the differentiation of naive T helper cells
The data presented above indicate that ISS-ODN up-regulates
a distinct profile of cell surface molecules in addition to
promoting cytokine production (4–8). To evaluate the functionality of this expression profile, splenic APC were obtained
from TCR-OVA transgenic mice injected with ISS-ODN, MODN or saline and their ability to activate naive T cells in the
presence of OVA in vitro was examined. Indeed, splenic APC
obtained from mice injected with ISS-ODN induced 4-fold
more T cell proliferation (at 30 µg/ml of OVA) than splenic
APC obtained from mice injected with saline or M-ODN (Fig.
5A). This indicates that treatment with ISS-ODN enhanced
the capacity of the APC to stimulate naive T cells. Furthermore,
the subsequent activation of naive T cells resulted in OVAspecific IFN-γ production (Fig. 5B), indicating the differentiation of naive Th cells toward a Th1 phenotype by APC treated
with ISS-ODN.
Discussion
ISS-ODN has been shown to activate NK cells and macrophages to secrete a profile of cytokines including TNF-α, IFN
(α, β and γ), IL-12 and IL-18 (2–8). While these cytokines are
thought to play major roles in the amplification of the immune
response toward the encountered antigen (6,11–13), this
study indicates that the adjuvanticity of ISS-ODN is also
mediated via the induction of a distinctive profile of cell
surface molecules involved in cognate interaction.
ISS-ODN enhanced the expression of several categories
of cell surface molecules involved in antigen presentation on
splenic and peripheral B cells in vitro and in vivo. Antigen
presentation may be directly enhanced by the up-regulation
of MHC class I and II. Also, ISS-ODN slightly enhanced the
expression of co-stimulatory molecules, such as B7-1, B7-2
and CD40 in vivo. These molecules play a major role in T cell
priming, activation and differentiation as well as in T–B
cell collaboration. While the role of B7/CD28 interaction in
regulating Th1 and Th2 differentiation is complex and not fully
resolved (22–24), there is clear evidence for the role of
CD40–CD40 ligand co-stimulation in Th1 differentiation, B cell
activation and isotype switching (25–28). CD40–CD40 ligand
co-stimulation regulates these effects via the up-regulation of
Fig. 5. APC pre-primed with ISS in vivo efficiently activate naive T
cells in vitro. TCR-OVA transgenic mice were injected i.d. with ISSODN or M-ODN or saline. Naive T cells from TCR-OVA transgenic
mice (105/well) were mixed with the same number of accessory cells
(splenic APC) obtained from ISS-ODN, M-ODN or saline injected
mice, with different concentrations of OVA, for 4 days. (A) For T cell
proliferation assay, the cultures were incubated with [3H]thymidine
(1 µCi/well) for 18 h, and cells were harvested and [3H]thymidine
incorporation was determined. (B) Levels of IFN-γ in the culture
supernatants upon stimulation with different concentrations of OVA.
Levels of IL-5 and IL-4 were below detectable limits in this system.
Data represents the mean 6 SD of triplicates of one representative
experiment of three which were performed.
B7 expression on APC, and via the induction of IL-12 from
monocytes, macrophages and dendritic cells (29). Thus, the
induction of co-stimulatory molecules on APC by ISS-ODN
may provide the important signals for Th1 differentiation (30).
ISS-ODN increases the expression levels of IL-2 receptor
and IFN-γ receptor on B cells, but does not up-regulate the
expression of other cytokine receptors such as IL-1 receptor
or IL-6 receptor. The selective up-regulation of these cytokine
receptors can enhance the in vivo B cell proliferative response,
Ig synthesis and isotype switching to IgG2a upon exposure
to the corresponding cytokines (6,10).
The up-regulation of cell surface molecules on B cells was
observed for both splenic B cells (mainly memory cells) and
peripheral blood B cells (mainly naive cells). The levels of
expression in vivo were maximal 1 week after i.p. injection of
ISS-ODN and dropped back to the baseline levels after 21
days for most of the parameters evaluated, except for MHC
class I and CD40 which both displayed sustained expression.
Activation of APC by ISS 1117
Gamma irradiation or MMC treatment of B cells did not modify
the expression of the various cell surface molecules, ruling
out the mitogenic effect of ISS on B cells for this differential
expression. Cytokine neutralization resulted in a partial inhibition of the differential expression of some surface molecules
on B cells, indicating the limited role of type 1 cytokines in
the observed expression profile. In response to ISS-ODN
in vivo and in vitro, CD41 and CD81 cells did not significantly
increase their expression of a wide variety of surface molecules, although a recent paper has shown a modest indirect
induction of some markers in response to ISS-containing
Drosophila genomic DNA (31).
ISS-ODN enhanced the expression of surface molecules
on BMDM. The expression of MHC class I, B7-1, CD40, ICAM1 and CD16/32 was clearly up-regulated on BMDM, while
expression levels of MHC class II were not clearly modified.
Notably, the up-regulation of ICAM-1 was more pronounced
on BMDM than on B cells. ICAM-1 stabilizes interactions
between APC and lymphocytes by binding to its ligand LFA1, thus reducing the threshold number of cognate interactions
required for activation (32). Recently, it has been demonstrated that dendritic cells also can be stimulated by ISScontaining DNA to up-regulate a similar profile of surface
molecules (33,34).
In recent studies we have proposed that ISS (CpG motifs)
in the pDNA backbone induce the necessary initial cytokine
milieu (IFN and IL-12) which fosters the characteristic Th1
response seen in gene vaccination (6,9). In light of this
finding, the pDNA provides a source for both antigen and
adjuvant (10). Indeed, pUC19, but not methylated pUC19,
up-regulated the expression levels of cell surface molecules
on B cells or BMDM in vitro, with a similar profile to that
observed with ISS-ODN stimulation. This suggests that the
role of ISS as an adjuvant in the plasmid backbone of DNA
vaccines is not limited to cytokine stimulation, but also includes
increases in important cell surface molecules.
It has previously been shown that the local cytokine milieu
(35,36) and the expression of surface molecules on APC (37)
enhance the subsequent immune response. In our study
splenic APC stimulated with ISS in vivo enhanced the T cell
proliferative response to antigen stimulation and induced
OVA-specific IFN-γ production in vitro. This indicates that ISSactivated splenic APC can instruct and bias the subsequent Th
response toward a Th1 phenotype, emphasizing the instructive
role of innate immunity (i.e. APC) in the development of the
adaptive immune response (38).
In summary, this study demonstrates that ISS-ODN or pDNA
including ISS up-regulates cell surface receptors involved
in cognate interactions. These include antigen presentation
(MHC class I and II), co-stimulation (B7 and CD40), antigen
uptake (Fcγ receptor), cell–cell adhesion (ICAM-1) and cytokine receptors. Combined with cytokine production, i.e. TNFα, IFN, IL-12 and IL-18, ISS-induced increases in these surface
molecules appear to provide the basis for the observed strong
Th1-inducing adjuvant properties. ISS-ODN were initially discovered in the mycobacterial genome as DNA sequences
that enhanced NK cell activity (2). Indeed, a recent study
demonstrated that the infection of human dendritic cells with
mycobacteria resulted in up-regulation of MHC class I, B7,
CD40 and ICAM-1, and the release of IL-12 and TNF-α (39),
an activation profile which is very similar to the pattern induced
by ISS in this study. Thus, microbial ISS plays a major role in
priming and activation of APC while synthetic ISS-ODN mediate these effects without the risks posed by infection. These
immunostimulatory properties of synthetic ISS-ODN could be
applied in vaccine design against infectious and malignant
diseases as well as for allergen immunotherapy.
Acknowledgements
We wish to thank Arash Ronaghy, Pei-Ming Chen and Patricia Charos
for their excellent technical assistance; Dr Hans Spiegelberg, Dr
Dennis A. Carson, Dr Maripat Corr and Dr Malini Sen for helpful
discussions and the review of the manuscript; and Nancy Noon and
Jane Uhle for editorial assistance. This work was supported in part
by NIH grant AI-40682 and by Dynavax Technologies Corp. E. M.-O.
is a recipient of a fellowship from the Spanish Ministry of Education
and Culture (MEC); H. K. is a recipient of a scholarship from the
Fukushima Medical Foundation. J. V. U. is a Medical Scientist Training
Program Trainee (NIGMS grant GM 07198) and a Lucille P. Markey
Charitable Trust Fellow.
Abbreviations
APC
BMDM
ISS
ISS-pDNA
ISS-ODN
LPS
MFIR
MMC
M-ODN
NU
ODN
OVA
pDNA
TNF
antigen-presenting cell
bone marrow-derived-macrophages
immunostimulatory DNA sequences
pDNA containing ISS
ODN containing ISS sequences
lipopolysaccharide
mean florescence index ratio
mitomycin C
oligodeoxynucleotides containing the mutant
sequence
neutralizing units
oligodeoxynucleotide
ovalbumin
plasmid DNA
tumor necrosis factor
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