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Discovery Research Department, Immunex Corporation, Seattle, WA 98101
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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-/- mice after immunization with Ag-pulsed DCs,
suggesting that the production of IFN- during the immune response
could interfere with the development of Th2 lymphocytes induced by DCs.
Coadministration of anti-Ox40 with DCs during Ag rechallenge
enhanced both Th1 and Th2 responses induced during a primary
immunization with DCs, and did not reverse an existing Th2 response.
This suggests that Ox40 costimulation amplifies an ongoing immune
response, regardless of Th differentiation potential. In an OVA-TCR
class II-restricted adoptive transfer system, anti-Ox40 treatment
greatly enhanced the level of cytokine secretion per Ag-specific
CD4+ T cell induced by immunization with DCs. In an OVA-TCR
class I-restricted adoptive transfer system, administration of
anti-Ox40 strongly enhanced expansion, IFN- secretion, and
cytotoxic activity of Ag-specific CD8+ T cells induced by
immunization with DCs. Thus, by enhancing immune responses induced by
DCs in vivo, the Ox40 pathway might be a target for immune intervention
in therapeutic settings that use DCs as Ag-delivery
vehicles. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, as well as IL-4
and IL-5 secretion, suggesting that Ox40/Ox40L interactions participate
in enhancing both Th1 and Th2 responses (13, 21).
Moreover, Ox40L-deficient mice demonstrated impairment in the induction
of alloantigen-specific CTLs (21), but showed normal CTL
responses to vesicular stomatitis virus in vivo (22). Among APCs, which in the mouse comprise dendritic cells (DCs), B cells, and macrophages, DCs appear to have the unique capacity to activate naive T cells in vitro and in vivo. DC pulsed extracorporeally can efficiently induce Th and CTL, as well as T cell-dependent humoral responses, in vivo (23). This property correlates with the capacity of DCs to express very high levels of antigenic peptide/MHC complexes and costimulatory molecules (24, 25, 26). Upon stimulation, they also show a high propensity to travel to lymphoid tissues such as the spleen and lymph nodes, where they interact with Ag-specific T cells and stimulate their activation (23, 27).
Recently, improved isolation techniques have led to the identification
of multiple DC subsets. Most investigators distinguish at least two
different subclasses of mouse DCs on the basis of their relative
expression of CD8
or CD11b (28, 29). In vivo
experiments have shown that either subset, pulsed in vitro with Ag, can
prime naive T cells, but CD8- DCs skew the T
cell response toward Th2 while CD8+ DCs skew
toward a Th1 response, in an IL-12-dependent manner (30, 31). In the study by Pulendran et al. (32), the
CD11c+CD11b- subset
(containing the CD8+ cells) induced high
levels of IFN- and IL-2, but little Th2 cytokines, whereas the
CD11c+ CD11b+ subset
induced large amounts of IL-4 and IL-10, in addition to IFN-
and IL-2.
Given the crucial role for DCs in the regulation of T cell responses, and the potentially beneficial role of Ox40 costimulation on the enhancement and the maintenance of T cell activation in vivo, we tested the effect of direct Ox40 costimulation on the outcome of T cell responses induced by DCs in vivo.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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All mice were used at 7–10 wk of age. Female BALB/c mice were
purchased from Taconic Farms (Germantown, NY). Female BALB/c IFN-
knockout (KO) mice were purchased from The Jackson Laboratory
(Bar Harbor, ME). DO11.10 TCR-transgenic mice specific for chicken OVA
peptide 323–339 (DO11p) in the context of I-Ad
(33) and OT-I CD45.1 TCR transgenic mice specific for
chicken OVA peptide 357–364 (OT-Ip) in the context of
H-2Kb (34) were bred at Immunex
(Seattle, WA). All mice were maintained at Immunex under specific
pathogen-free conditions according to federal guidelines.
Flow cytometry
Cells were analyzed by flow cytometry with a FACSCalibur
cytometer (BD Biosciences, Mountain View, CA). The cells were stained
in FACS buffer (PBS containing 2% FBS, 1% normal rat serum, 1%
normal hamster serum, 1% normal mouse serum, and 10 µg/ml 2.4G2 (a
rat anti-mouse FcR) mAb). All mAbs were from BD PharMingen (San
Diego, CA), except the anti-clonotypic KJ1-26 mAb specific for
DO11.10 T cells, which was produced and labeled at Immunex. The mAbs
used were as follows: FITC-anti-mouse CD11c (HL3),
FITC-anti-CD45.1 (A20), PE-anti-mouse CD8 (53-6.7),
PE-anti-mouse CD25 (PC61), PE-anti-mouse CD44 (IM7),
PE-anti-mouse CD62L (MEL-14), and APC-anti-mouse-CD4 (GK1.5).
Propidium iodide was added in the FACS buffer, and cells were
gated accordingly to eliminate dead cells and debris from analysis.
Reagents
Recombinant human Flt3 ligand (FL) (Chinese hamster ovary cell-derived) was produced at Immunex. Anti-Ox40 M5 Ab was generated as followed: Sprague Dawley rats were immunized with murine Ox40-Fc emulsified in CFA. After three boosts, rat serum was collected and tested for binding to Ox40-Fc and for blocking Ox40-Fc binding to Ox40L expressing cells. After the rat serum achieved a blocking titer, it was fused according to standard hybridoma protocols. The fusion was screened for reactivity to muOx40-Fc vs irrelevant Fc, and then screened for blocking binding of muOx40-Fc to Ox40L-expressing cells. M5 anti-Ox40 was derived after two subcloning procedures and blocked Ox40-Fc binding to Ox40L (data not shown).
Ribi and aluminum potassium sulfate (Alum) adjuvants were purchased from Corixa (Seattle, WA) and Sigma Aldrich (St. Louis, MI), respectively. Medium used to culture DCs was RPMI 1640 from Life Technologies (Rockville, MD), supplemented with 0.5% heat-inactivated normal mouse serum, penicillin, streptomycin, nonessential amino acids, sodium pyruvate, 2-ME, HEPES, and L-glutamine, or serum-free protein-free HyQ ADCF-mAb medium from HyClone Laboratories (Logan, UT).
Purification of DCs
Mice were injected i.p. with 10 µg of FL for 11 consecutive
days to induce DC expansion. Splenic DCs were purified from FL-treated
animals following a protocol described previously (30).
Briefly, spleens were digested with collagenase, further dissociated in
Ca2+-free medium in the presence of EDTA,
separated into low- and high-density fractions on a Nycodenz gradient,
and cultured overnight with 20 ng/ml recombinant murine GM-CSF. After
overnight culture, nonadherent cells contained at least 90% DCs, as
assessed by morphology and specific staining, using anti-CD11c mAb.
DCs were pulsed with 50 µg/ml KLH during overnight culture. For
peptide-pulsing, DCs were incubated after overnight culture in complete
culture medium containing 1 µM OVA peptide class II 323–339 (DO11p)
or 1 µM OVA peptide class I 357–364 (OT-Ip) for 1 h at 37°C,
and then washed twice with PBS. In some experiments, DCs were further
separated according to CD8 expression by FACS sorting. In brief,
cells were stained with FITC-anti-CD11c and PE-anti-CD8
mAbs, and sorted on a FACSVantage cytometer (BD Biosciences) into
CD11c+CD8- and
CD11c+CD8+ DCs.
Immunization protocol
Ag-pulsed DCs were washed in PBS and administered at a dose of 3 x 105 cells into the hind footpads of recipient mice. Alternatively, mice were immunized with 50 µg of KLH emulsified in adjuvant (Ribi or Alum). Some mice were injected at the time of immunization with 100 µg control rat Ig (Sigma Aldrich) or mAb anti-Ox40 M5 by the i.p. route. Draining popliteal lymph nodes were harvested 5 days after primary immunizations or 2 days after secondary immunizations.
In vitro assays
LN cells were restimulated in vitro in triplicate with graded
doses of Ag in Click’s medium supplemented with 0.5% heat-inactivated
normal mouse serum and additives (penicillin, streptomycin,
nonessential amino acids, sodium pyruvate, 2-ME, HEPES, and
L-glutamine). Triplicate culture supernatants were pooled
and assayed by two-site ELISA (BD PharMingen) for IL-2 after 24 h
of incubation, and for IL-4, IL-5, IL-10, and IFN- after 72 h
of incubation. An amplification system was used as described
(35). The detection limits were 50 pg/ml for IL-2, 50
pg/ml for IL-4, 50 pg/ml for IL-5, 50 pg/ml for IL-10, and 1 ng/ml for
IFN-.
Measurement of CD4+ T cell expansion in vivo and restimulation in vitro
Spleen and lymph node cell suspensions were prepared from
DO11.10 mice, and the percentage of T cells expressing transgenic TCR
was determined by flow cytometry, using anti-CD4 and
anti-clonotypic KJ1–26 mAb. The equivalent of 5 x
105 CD4+
KJ1-26+ T cells were injected i.v. into the
lateral tail vein of BALB/c recipients. One day later, recipients were
immunized with peptide-pulsed DCs in the hind footpads, popliteal LN
cells were harvested at different time points and counted, and T cell
expansion was measured by flow cytometry using anti-CD4 mAb and
anti-clonotypic mAb KJ1-26. The expression of CD25, CD44, and CD62L
on gated CD4+ KJ1-26+ cells
on LN cells from mice immunized with Ag-pulsed DCs 4 days earlier was
measured by FACS analysis. The LN cells were also restimulated in vitro
in triplicate with graded doses of the DO11p in Click’s medium
supplemented with 0.5% heat-inactivated mouse serum and additives.
Triplicate culture supernatants were pooled and assayed by ELISA for
IFN- after 72 h of incubation. ELISPOT plates were coated
overnight with 5 µg/ml anti-mouse IFN- mAb (BD PharMingen) in
PBS at 4°C, washed, and incubated 1–3 h at 37°C with PBS-10% FBS
to block nonspecific sites. Serial dilutions of LN cells from recipient
mice immunized 4 days earlier with Ag-pulsed DCs were restimulated with
2 µg/ml DO11p, or without Ag in Click’s medium, supplemented with
0.5% heat-inactivated normal mouse serum and additives for 24 h.
Plates were washed and incubated overnight at 4°C with biotinylated
anti-mouse IFN- (BD PharMingen) in PBS-10% FBS. Plates were
washed and incubated for 2 h at 37°C with a 1/1000 dilution of
HRP-streptavidin in PBS, washed, and the enzymatic activity was
revealed after incubation in 3,3'-diaminobenzidine (Sigma Fast Tablets;
Sigma Aldrich). The spots were counted under a binocular microscope by
two investigators independently. The data represent the mean values of
spots normalized for differences in transgenic T cell numbers at
culture inception. For intracellular IFN- staining, LN cells from
mice immunized with Ag-pulsed DCs 4 days earlier were stimulated with
50 ng/ml phorbol 12-myristate 13-acetate (PMA) and 500 ng/ml ionomycin
for 4 h in the presence of 5 µM monensin (Sigma Aldrich). After
harvest, cells were stained with anti-CD4 and anti-clonotypic
KJ1-26 mAb for 20 min, washed with PBS, and fixed for 20 min in
Cytofix/Cytoperm buffer (BD PharMingen). Cells were washed twice in
Cytoperm buffer and staining was performed for 30 min at 4°C with
PE-conjugated control Ig or anti-IFN- Ab (BD PharMingen) diluted
in Cytoperm buffer. After staining, cells were washed with Cytoperm
buffer, resuspended in PBS, and analyzed on a FACSCalibur (BD
Immunocytometry Systems, Mountain View, CA).
Measurement of CD8+ T cell expansion in vivo and restimulation in vitro
Spleen and lymph node cell suspensions were prepared from OT-I
CD45.1 mice, and the percentage of T cells expressing transgenic TCR
was determined by flow cytometry using anti-CD8, anti-CD45.1,
and anti-TCR V 2 mAbs. The equivalent of 1 x
106 CD8+ V
2+ T cells were injected i.v. into the lateral
tail vein of C57BL/6 recipients. One day later, recipients were
immunized with peptide pulsed DCs in the hind footpads and popliteal LN
cells were harvested at different time points, counted, and T cell
expansion was measured by flow cytometry using anti-CD8 mAb and
anti-CD45.1 mAb. Direct CTL activity was measured 4 days after DC
immunization in a standard 51Cr-release assay.
Briefly, C1498 targets cells were pulsed for 1 h at 37°C with or
without 1 µM OT-Ip in the presence of
51Cr in complete medium. Cells were washed three
times and 1 x 104 cells/well were added to
serial dilutions of LN cells isolated form DC-immunized animals in a
total volume of 200 µl. After 6 h of incubation at 37°C, 100
µl of supernatant was removed and radioactivity was counted in a
gamma-counter. Spontaneous and total release were determined by adding
culture medium or detergent to target cells, respectively. Percent
specific 51Cr release was calculated as 100
x (experimental release - spontaneous release)/(total
release - spontaneous release). For restimulation in vitro,
two-fold serial dilutions of LN cells were cultured in vitro with
1 x 105 C57BL/6 spleen cells pulsed with 1
µM OT-Ip in complete medium. The 3-day culture supernatants were
assessed for IFN- production by a two-site ELISA from BD
PharMingen.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We tested the effect of an exogenous source of Ox40 costimulation,
provided in the form of a rat anti-mouse Ox40 mAb (M5, described in
Materials and Methods), on the DC-induced immune response.
Mouse DCs, expanded after 11 days of treatment with FL, were isolated
from spleens and pulsed overnight with KLH in GM-CSF-containing medium.
A total of 3 x 105 DCs were subsequently
injected into the hind footpads of syngeneic mice. Some mice were
injected with anti-Ox40 M5 (100 µg i.p.) at the time of
immunization with DCs. The popliteal lymph nodes were harvested 5 days
later, and immune responsiveness was analyzed by culturing LN cells
with graded doses of KLH for 3 days. Consistent with previously
published results (36, 37), lymph node cells from mice
immunized with KLH-pulsed splenic DCs produced IL-2, IL-4, IL-5, IL-10,
and IFN- when restimulated with the same Ag in vitro (Fig. 1
). By contrast, lymph node cells from
animals primed by injection of DCs together with anti-Ox40 mAb
produced higher levels of IL-2 and IFN-, but did not secrete
detectable amounts of IL-4, IL-5, and IL-10 under the same conditions
(Fig. 1). T cell priming was prevented by pretreatment with
anti-CD4 mAb in vivo, demonstrating that the response observed was
dependent upon the presence of CD4+ T lymphocytes
(data not shown). Anti-Ox40 mAb treatment alone without Ag-pulsed DCs
did not result in a detectable immune response (data not shown). This
result suggests that costimulation through Ox40 leads to a polarized
Th1 primary response induced by DCs.
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Two major subsets of splenic DCs have been characterized based on
the expression of CD8 or CD11b (28, 29), and recent
data suggest that these different subsets of Ag-pulsed DCs can
differentially regulate the development of Th1- or Th2-type responses
(30, 31, 32). The strong induction of a Th1-type response
induced by DCs in the presence of anti-Ox40 mAb prompted us to test
the effect of Ox40 costimulation on the outcome of the immune response
induced by subsets of DCs. DCs were isolated from spleens of FL-treated
animals, pulsed overnight with KLH in GM-CSF-containing medium, and
further separated according to CD8 expression by FACS sorting.
Re-analysis of the sorted cell populations showed a purity of >96%
(data not shown). Ag-pulsed DCs of either subset were subsequently
injected into the hind footpads of naive syngeneic recipients. Some
mice were also treated by i.p. administration of anti-Ox40 mAb at
the time of immunization with DCs. Consistent with previously published
data (30, 31), the subclasses of DCs demonstrated a
capacity to skew CD4+ T cell differentiation
toward Th2- or Th1-like profiles; i.e., the injection of
CD8- DCs resulted in the activation and
expansion of T cells secreting high levels of IL-4 and IL-5, low levels
of IFN-, and immunization with CD8+
DC-sensitized cells producing IFN-, but little IL-4 and IL-5 (Fig. 2). Coadministration of the anti-OX40
M5 mAb together with DCs (both subsets) induced the activation and the
development of cells secreting high levels of IFN-, and no IL-4 and
IL-5. This result suggests that both subsets of DCs induce a polarized
Th1-type immune response in the presence of Ox40 costimulation.
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To test whether Ox40 costimulation can directly favor the skewing
to a Th1 response and shift an established Th2 response to a Th1
response, we analyzed the effect of exogenous Ox40 costimulation on
secondary responses induced by DCs. To this end, a primary immune
response was induced by injection of unseparated KLH-pulsed DCs. Two
weeks later, when the primary response had declined to baseline levels,
mice were re-immunized with Ag-pulsed DCs with or without anti-Ox40
mAb. Draining LN cells were harvested 2 days after immunization, and
restimulated in vitro with graded doses of Ag, according to a protocol
described by Inaba et al. (38). Results in Fig. 3 indicate that mice immunized with DCs
and rechallenged with unseparated DCs developed a mixed secondary
cytokine secretion, consistent with previous reports (30).
The distinct kinetics of primary vs secondary responses provides
evidence that the T cell response of animals injected twice with DCs is
an anamnestic response (38, 39). Indeed, a single
injection of Ag-pulsed DCs 2 days before in vitro restimulation induced
no detectable cytokines compared with two injections of DCs, which
results in strong cytokine secretion by lymph node cells under the same
conditions (Fig. 3). The coadministration of anti-Ox40 mAb together
with DCs during the rechallenge enhanced both Th1 and Th2 responses
that were induced during the primary immunization by DCs (Fig. 3). This
suggests that Ox40 costimulation does not necessarily skew the immune
response to a Th1 pattern, but amplifies any ongoing immune
response.
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-deficient animals
The strong bias toward a Th1 response that was induced by Ox40
costimulation in the primary immunization with both
CD8- and CD8+ DCs
may have been due to an effect of IFN-. To test this, KLH-pulsed,
wild-type (WT) DCs were injected, with or without anti-Ox40 M5 mAb,
into IFN--deficient, syngeneic-recipient animals. Five days later,
LN cells from these mice were restimulated in vitro with KLH, and
cytokine secretion was measured. Fig. 4
shows that KLH-pulsed DCs induced higher secretion of IL-4 and IL-5
when injected into IFN--deficient mice, compared with injection into
WT recipients, and Ox40 costimulation further enhanced the production
of IL-4 and IL-5. This result suggests that the production of IFN-
during the immune response could interfere with the development of
Th2-type lymphocytes induced by DCs.
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We next tested the effect of exogenous Ox40 costimulation on the
development of Th responses induced by Ag emulsified in adjuvants. Mice
were immunized subcutaneously in the hind footpads with KLH in Ribi or
Alum, with or without anti-Ox40 M5 mAb. The draining lymph node
cells were harvested 5 days after immunization, restimulated in vitro
with KLH, and cytokine production profiles were analyzed. The data in
Fig. 5 show that the adjuvants induced
distinct cytokine profiles, with Ribi and Alum preferentially driving
Th1- and Th2-like responses, respectively. Ox40 costimulation strongly
enhanced the cytokine response induced by each adjuvant. This supports
the notion that signaling through Ox40 enhances ongoing immune
responses without skewing toward a particular Th profile.
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To gain further insight into the mechanism by which Ox40
costimulation influences T cell responses, we used an adoptive transfer
system that allowed us to monitor the behavior of Ag-specific TCR
transgenic T cells in vivo after immunization with DCs with or without
direct Ox40 costimulation. To this end, 5 x
105 naive T cells from DO11.10 MHC class
II-restricted, OVA-specific transgenic mice were transferred into
naive, unirradiated BALB/c recipients. Their presence was detected with
the anti-clonotypic mAb KJ1.26. Mice were immunized s.c. in the
hind footpads with DCs pulsed in vitro with the
I-Ad-restricted OVA peptide, DO11p, with or
without anti-Ox40 M5 mAb. The ensuing T cell expansion in draining
lymph nodes was monitored by flow cytometry. The transferred DCs primed
T cell expansion in an Ag-specific manner, with similar kinetics, in
the presence or absence of anti-Ox40 M5 mAb (Fig. 6A). The magnitude of
expansion was significantly higher at day 4 (p
< 0.05), when mice received DCs and direct Ox40 costimulation. By day
14, the absolute numbers of transgenic T cells in the draining lymph
nodes returned to baseline levels in both groups. Furthemore, at day 4,
the percentage of T cells presenting an activated phenotype, as
assessed by increased expression of IL-2R (CD25), CD44, and
decreased expression of CD62L, was higher in the group treated with
anti-Ox40 M5 mAb, compared with control-Ig treated mice (Fig. 6B). At day 4 after priming, total popliteal LN cells were
restimulated in vitro with various concentrations of OVA peptide to
assess cytokine production by Ag-specific T cells primed in vivo with
DCs. Measurement of cytokine production in the cultures of LN revealed
that T cells primed in vivo by pulsed-DCs together with anti-Ox40
mAb exhibited a >100-fold increase in the Ag dose-dependent
sensitivity of IFN- production, compared with T cells primed by DCs
only (Fig. 6C). In addition, Ag-specific IFN--secreting
CD4+ T cells numbers at day 4 were quantified by
ELISPOT. The data are presented relative to the number of Ag-specific
cells per well. Fig. 6D shows that mice immunized with DCs
and anti-Ox40 mAb had 10-fold more IFN--secreting, Ag-specific
CD4+ T cells compared with mice immunized with
DCs only. Moreover, the size of the spots was bigger in the group of
mice immunized with DCs and anti-Ox40 mAb (Fig. 6E),
suggesting that the level of secretion per T cell was higher.
IFN- production on a per cell basis was directly assessed by
intracellular staining (Fig. 6F). Four days after primimg,
total popliteal LN cells were restimulated in vitro with PMA and
ionomycin for 4 h. Measurement of intracellular IFN- revealed
that a higher percentage of Ag-specific T cells primed in vivo by
pulsed-DCs together with anti-Ox40 mAb, secreted IFN-, and the
level of cytokine secretion was higher on a per cell basis, compared
with T cells primed by DCs only. Taken together, these data suggest
that both the number of activated T cells and the level of cytokine
secretion per T cell were higher when mice were immunized with DCs and
anti-Ox40, compared with mice immunized with DCs only.
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To determine the effect of anti-Ox40 treatment on the
activation of CD8+ T cells induced by DCs in
vivo, we used an adoptive transfer system in which 1 x
106 naive T cells from OT-I CD45.1 MHC class
I-restricted, OVA-specific transgenic mice were transferred into naive,
unirradiated congenic C57BL/6 CD45.2 recipients. Mice were immunized
s.c. in the hind footpads with DCs pulsed in vitro with the
H-2Kb-restricted OVA peptide, OT-Ip, with or
without anti-Ox40 M5 mAb. Accumulation of transgenic
CD8+ T cells in the draining lymph nodes was
monitored by flow cytometry. The data in Fig. 7A show that Ag-pulsed DCs
were able to drive the expansion of CD8+ T cells
in vivo, which peaked at day 4 and returned to baseline levels at day
14. The magnitude of expansion was significantly higher at the peak of
the response (p < 0.05) when mice received DCs
and direct Ox40 costimulation (Fig. 7A). The development of
effector functions by CD8+ T cells was also
measured at the peak of the response. Lymph-node cells from adoptively
transferred recipients were recovered 4 days after immunization with
Ag-pulsed DCs, and cytotoxic activity was assayed directly on
peptide-coated target cells. As seen in Fig. 7B, DCs alone
were able to prime for a low level of cytotoxicity. In contrast, in
vivo treatment with anti-Ox40 M5 resulted in a strong increase in
specific cytotoxicity. Lymph node cells from mice immunized with DCs
were also restimulated in vitro with syngeneic spleen cells pulsed with
the OT-I peptide and the production of IFN- was assessed from the
3-day culture supernatants (Fig. 7C). In vivo priming with
Ag-pulsed DCs induced IFN- secretion by the LN cells upon in vitro
restimulation. Anti-Ox40 M5 administration at the time of
priming strongly increased this production.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Previously, it had been proposed that the Ox40-Ox40L pair of
costimulatory molecules plays an important role in the development of
Th2 responses. For instance, results from in vitro studies suggested
that Ox40 stimulation is necessary to induce IL-4, but not IFN-,
secretion by DC-activated human T cells, and promotes the activation of
Th2-type immune responses (7). Strong expression of Ox40
on T cells was also found in a Th2-type cytokine environment in a model
of Th2-mediated, systemic autoimmunity (40). However, Chen
et al. (22) demonstrated that DCs from Ox40L KO mice show
a reduced capacity to induce IL-2, IL-4, and IFN- production by
allogeneic CD4+ T cells. Similarly, Ox40L KO mice
exhibited impaired T cell priming and Th cytokine production when
immunized with KLH in adjuvant (21). In another study,
Ox40-deficient animals exhibited reduced primary Th1 and Th2 responses
to KLH (13). Blocking of Ox40L-Ox40 interactions in
several models of autoimmunity involving Th1-type cells ameliorates the
diseases (18, 19, 20), and reduces the levels of Th1-related
cytokines such as IFN-, IL-12, and TNF-, suggesting that Ox40
costimulation may also participate in the enhancement of Th1-type
immune responses. Our results show that the activation of naive T cells
induced by DCs in vivo can be dramatically enhanced through Ox40
ligation. Administration of anti-Ox40 mAb during primary
immunization with either CD8- or
CD8+ DCs induced a bias toward a Th1-type
immune response. By contrast, administration of anti-OX40 mAb only
during antigenic rechallenge enhanced both Th1 and Th2 responses that
were established following priming with DCs (Fig. 3
). Thus, Ox40
costimulation does not appear to redirect an ongoing Th2 response
toward a Th1 response; rather, it will amplify the cytokine profile
established during a primary response. We also show that Ox40
costimulation strongly enhances KLH-specific Th1- and Th2-type
responses preferentially induced by Ribi and Alum, respectively.
Together, these results do not support a role for Ox40-signaling in the
differential polarization of CD4+ T cells, but do
support the notion that costimulation through Ox40 is required for
optimal activation or maintenance of CD4+ T
cells, and may serve as an amplification system for an ongoing immune
response.
Two recent reports have highlighted a role for different subsets of DCs
in the differentiation of distinct T helper populations (30, 32). In one study, CD11c+
CD11bdull DCs (containing a majority of the
CD8+ DCs) loaded in vitro with an OVA peptide,
and injected into the footpads of syngeneic mice transferred with T
cells from OVA-specific DO11.10 TCR transgenic mice, induced high
levels of IFN- and IL-2, but only low levels of Th2 cytokines. The
CD11c+ CD11b+ subset (which
is CD8- DCs) induced large amounts of IL-4
and IL-10, in addition to IFN- and IL-2 (32). In
another study, CD8- and
CD8+ DCs, when pulsed ex vivo with KLH and
injected into syngeneic mice, induced Th2- or Th1-type immune
responses, respectively (30). Cytokines present in the
microenvironment at the time of priming, as well as the relative
expression of costimulatory molecules, might be the decisive elements
driving the CD4+ T cell response toward a
particular Th profile. Indeed, functional blockade of CD86 at the time
of DC injection does not affect the priming capacity of DCs, but
abrogates IL-4 secretion and leads to enhanced secretion of IFN- by
T cells, suggesting that CD86 might play a role in the development of
Th2 responses (37). Neutralization of CD80 in this system
had little effect on cytokine production. In contrast, IL-12 appears to
be the key factor biasing toward Th1 immune responses in vivo, as
evidenced in part by the observation that injection of exogenous IL-12,
along with either subset of Ag-pulsed DCs led to a polarized Th1
response (30). Alternatively, DCs from IL-12 p40 KO mice
are incapable of inducing a Th1-type response after injection into a WT
host (30). Here, we show that the presence of IFN-
during priming with Ag-loaded DCs may have a negative effect on the
development of IL-4 producing cells. Injection of Ag-pulsed DCs into an
IFN--/- host led to increased production of
IL-4 compared with injection into a WT recipient. This effect was
further enhanced by administration of anti-Ox40 mAb, suggesting
that the strong Th1 bias induced by Ox40 costimulation during primary
immunization with both CD8- and
CD8+ DCs might be dependent upon the early
production of IFN- during the immune response. Of note, when
anti-Ox40 was administered on day 3 rather than at the time of
immunization, high levels of IFN- and moderate levels of IL-4 were
induced upon in vitro restimulation (data not shown). In the same line
of evidence, it is interesting to note that
CD8- DCs, when pulsed ex vivo with KLH plus
IFN- and injected into syngeneic animals, induce a Th1-type immune
response as opposed to the Th2-type immune response normally observed
with this DC subset (R. Maldonado-Lopez, unpublished
observation). Whether or not this effect is dependent on an
increased production of IL-12 by DCs after IFN- incubation is still
a matter of speculation. Of note, several reports have indicated that
IL-12 production by DCs requires prior activation with microbial agents
or T cell derived factors (41, 42, 43, 44, 45). For instance, IFN-
was shown to enhance production of both (p40)2 homodimeric and p75
heterodimeric forms of IL-12 (41). Thus, these results
suggest that DCs drive Th2 development in a neutral environment and
this may occur as the default response in the absence of IL-12 or other
proinflammatory cytokines such as IFN-. Our results also suggest
that the capacity to skew to particular Th responses by different
subsets of DCs is not fixed, and may depend upon microenvironmental
factors to which DCs are exposed around the time of interaction with
Ag-specific T cells.
Expression of Ox40L has been reported on cells of the immune system including B and dendritic cells (21, 46, 47, 48). Stimulation of DCs via CD40 may be a critical signal for expression of Ox40L (21). Because the expression of CD40L is induced on T cells shortly after activation (49), one might speculate that Ox40L expression in vivo is closely regulated in time, and that Ox40L only appears on the surface of DCs upon interaction with Ag-specific T cells. CD40L-/- mice present the same impairment of in vivo T cell priming in response to protein Ags as Ox40L-deficient mice (50). Other costimulatory molecules such as CD80 and CD86 are highly expressed on mature DCs, and their blockade completely abrogates the priming capacity of DCs in vivo. As the DCs used in this study expressed high levels of CD80, CD86, and CD40 costimulatory molecules but no Ox40L (data not shown) at the time of injection, our results suggest that Ox40 costimulation could further enhance CD80/CD86-CD28 costimulation, but also could bypass the need for a further activation of DCs in vivo by Ag-specific T cells through CD40. In conclusion, the close relationship between costimulatory systems such as CD80/CD86-CD28 and CD40-CD40L suggests that they may work cooperatively, but the plausibility of this scheme in the development of T cell responses will require further investigation.
The exact mechanism of action of Ox40 costimulation is still a matter
of speculation. An agonistic mAb to Ox40 was shown to enhance clonal
expansion and survival of Ag-specific CD4+ T
cells (13), suggesting that the effect of Ox40
costimulation regulates the extent of T cell expansion and
differentiation, rather than T cell activation and effector functions.
This effect of Ox40 costimulation on T cell expansion was particularly
striking at low doses of Ag, which produced a weak T cell response
alone (13). Our results are somewhat in accordance with
these data. Indeed, in an adoptive transfer system of transgenic
CD4+ T cells specific for OVA, we have shown that
Ox40 costimulation, together with pulsed DCs, induced a significant
increase in the number of Ag-specific T cells 4 days after immunization
and a 5- to 10-fold increase in the number of cells producing IFN-
upon restimulation, compared to immunization with DC alone. Moreover,
at the peak of the response, Ag-specific T cells from mice immunized
with DCs and anti-Ox40 had a phenotype of activated cells (high
expression of CD25 and CD44, low expression of CD62L) and secreted
higher levels of IFN- on a per cell basis, compared to Ag-specific T
cells from animals immunized with DCs alone. These results suggest that
not only the number of T cells, but also the level of activation are
increased through Ox40 costimulation.
In this report, we show that CD8+ T cell
expansion and the development of specific cytotoxic activity and
secretion of IFN- induced after immunization with Ag-pulsed DCs are
strongly increased by treatment with anti-Ox40 mAb. The prevalent
theory for the development of CTL responses induced by DCs postulates
that specific helper CD4+ T cells are essential
(51). These cells were originally thought to provide
cytokines, such as IL-2, that facilitate activation and survival of
CD8+ T cells. Recent reports have suggested an
alternative model wherein CD4+ T helper cells
must interact with and activate DCs, which are then capable of
stimulating pre-CTLs. The pretreatment of DCs with agonistic
anti-CD40 can replace CD4+ T cell help,
suggesting that interactions between CD40 and CD40L are responsible for
the "conditioning" of the DCs by CD4+ T cells
(51, 52, 53). However, recent work by Lu et al.
(54) has suggested that in addition to CD40-dependent
activation of DCs, important components of CD4+
help CD8+ CTLs to comprise CD40-independent DC
sensitization and direct
CD4+-CD8+ T cell
communication through the release of cytokines. The source of
CD40-independent activation of CD8+ T cells is
still a matter of debate, but the Ox40-Ox40L pair may be promising
candidates. Franco et al. (55) have also recently shown
that priming and memory generation of Ag-specific
CD8+ CTL does not require help when the immunogen
binds MHC class I molecules with high affinity. The OVA peptide
used in this study (SIINFEKL) is a high affinity peptide
(34), and CD4+ cells are dispensable
for the DC-induced CD8+ T cell activation in vivo
in this system (T. De Smedt, J. Smith, and C. Maliszewski, manuscript
in preparation). This suggests that the enhancement of CTL
activity induced by DCs observed with anti-Ox40 treatment is a
direct effect on CD8+ T cells.
Due to their powerful activity in the generation of immune responses,
several approaches are being taken to use DCs to modulate immune
responses (23, 56). However, the unique capacity of DCs to
sensitize naive T cells is not constitutive, and develops through an
activation process termed maturation, which is characterized by an
increase in the expression of MHC molecules and costimulatory molecules
such as CD80, CD86, and CD40 (23). In general, strategies
that use DCs as vaccine adjuvants aim at increasing the maturation
and/or activation of DCs through the use of stimuli, such as TNF-,
CD40L, and PGE2 (57). An alternative
or complementary approach would be to increase expansion and effector
functions of DC-activated T cells by targeting appropriate
costimulatory molecules on T cells. The data that we present in this
report are relevant to the enhancement of anti-tumor responses
induced by treatment with anti-Ox40 mAb observed in different
murine tumor models (58, 59). Given the strong effect of
Ox40 costimulation that we observed, the combination of DCs with
activating agents such as anti-Ox40 mAbs may prove to be more
effective than either regimen alone in attempts to generate effective
immune responses against tumors. This approach would also have the
advantage of maintaining the specificity of the response because only
recently activated Ag-specific T cells express the Ox40R. Conversely,
altering the interactions between DCs and T cells by blocking the
Ox40-Ox40L pathway might prove to be useful in deleterious immune
responses such as autoimmunity.
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Acknowledgments |
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Footnotes |
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2 Abbreviations used in this paper: Ox40L, Ox40 ligand; DC, dendritic cells; OT-Ip, OVA peptide class I 357–364; KO, knockout; FL, Flt3 ligand; WT, wild type.
Received for publication July 3, 2001. Accepted for publication November 12, 2001.
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) or with control
Ig (thick lines and 
) 5 days earlier were cultured with graded doses
of KLH. Cytokine production was measured as described in
Materials and Methods. 
, Untreated animals.
, DCs + anti-Ox40 mAb. Statistical analysis was conducted using
a paired t test, and the results were considered
significant at p < 0.05. B, FACS
analysis for CD25, CD44, and CD62L expression on gated CD4+
KJ1-26+ cells in LN cells from mice immunized with
Ag-pulsed DCs, together with (thick line) or without anti-Ox40 mAb
(thin line) 4 days earlier. Dotted line, isotype controls.
C, IFN-