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,
*
Department of Immunology and
Urological Institute, Cleveland Clinic Foundation, Cleveland, OH 44195;
Department of Biology, Case Western Reserve University, Cleveland, OH 44106; and
Department of Pathology, Case Western Reserve University School of Medicine, Cleveland, OH 44106
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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-producing cells, and CD4+ T
cells develop into IL-4/IL-5-producing cells. Administration of IL-12
during sensitization skews CD4+ T cell development to
IFN--producing cells, resulting in exaggerated CHS responses. In the
current report we tested the role of IL-12 on CD8+ T cell
development during sensitization and elicitation of CHS to
dinitrofluorobenzene. Administration of IL-12 during hapten
sensitization induced the expression of IL-12R
2 on both
CD4+ and CD8+ T cells, augmented IFN-
production by these T cell populations, and increased the magnitude and
duration of the CHS response to hapten challenge. CHS responses were
virtually identical in wild-type and IL-12 p40-/- mice.
Since engagement of CD40 on APC may stimulate IL-12 production, we also
tested the role of CD40-CD154 interactions on the development of
IFN--producing CD4+ and CD8+ T cells
following hapten sensitization. Development of IFN--producing
CD4+ T cells during hapten sensitization was absent in
wild-type mice treated with anti-CD154 mAb or in
CD154-/- mice. In contrast, the absence of CD40-CD154
signaling had little or no impact on the development of
IFN--producing CD8+ T cells. These results demonstrate
that the development of hapten-specific Th1 effector CD4+ T
cells in CHS requires both CD40-CD154 interactions and IL-12, whereas
the development of IFN--producing effector CD8+ T cells
can occur independently of these pathways. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-producing CD8+ T
cells and IL-4-, IL-5-, and IL-10-producing CD4+
T cells are observed in lymph nodes following hapten sensitization
(11).
Many factors, including the delivery of various costimulatory signals
and the cytokine environment during Ag priming, influence
CD4+ T cell development to a particular
cytokine-producing phenotype (12). The roles of such
factors during priming of hapten-specific CD4+
and CD8+ T cells for CHS remain unclear. Previous
studies from this laboratory indicated that administration of IL-12
during hapten sensitization skewed the development of
CD4+ T cells from an IL-4-producing to an
IFN--producing phenotype (13). The consequences of this
treatment were elimination of the regulatory component of the response,
resulting in CHS responses of increased magnitude and duration.
However, the role of IL-12 in CHS and its effect on hapten-specific
CD8+ T cell development remain undefined. In the
current report we have extended our previous studies and have
demonstrated that endogenous IL-12 is not required for CHS, but that
exogenous IL-12 amplifies IFN--producing CD8+
T cell development independently of the CD4+ T
cell compartment. Anti-CD154 mAb and CD154-/-
mice were used to show that the IL-12-driven development of
hapten-specific CD4+ T cells to IFN--producing
cells is dependent on CD40-CD154 interactions, whereas the development
of CD8+ T cells in CHS occurs independently of
this costimulatory signal. The results presented indicate for the first
time the role of endogenous IL-12 and the effect of exogenous IL-12 on
the development of hapten-specific CD8+ T cell
development.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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BALB/c and C57BL/6 mice were purchased through Dr. C. Reeder (National Cancer Institute, Frederick, MD). CD4-/-, CD8-/-, and CD154-/- mice on the C57BL/6 genetic background and IL-12 p40-/- mice on the BALB/c background were purchased from The Jackson Laboratory (Bar Harbor, ME). Adult female mice, 8–10 wk old, were used throughout these studies.
Abs and cytokines
mAb from the culture supernatant of the IgG-producing hybridomas
YTS191.1.2 and GK1.5 (anti-mouse CD4 mAb), 2.43 (anti-mouse CD8
mAb), 145.2C11 (anti-mouse CD3 mAb), KJ23a (anti-V17a mAb),
and MR1 (anti-mouse CD154 mAb) and 15.1.2 and 15.6.7
(anti-mouse IL-12 mAbs) were purified by protein G chromatography.
Capture and detection mAbs for IL-4- and IFN--specific ELISA and
rIFN- and IL-4 were purchased from PharMingen (San Diego, CA).
Polyclonal hamster IgG was purchased from Jackson ImmunoResearch
Laboratories (West Grove, PA). Recombinant mouse IL-12 was a gift from
Genetics Institute (Cambridge, MA).
Hapten sensitization and elicitation of CHS
For sensitization to DNFB, mice were painted on days 0 and 1 with 25 µl 0.25% DNFB (Sigma, St. Louis, MO) on the shaved abdomen and 5 µl on each footpad. On day 5 mice were challenged with 10 µl 0.2% DNFB on both sides of each ear. The increase in ear swelling was measured at 24-h intervals after challenge using an engineer’s micrometer (Mitutoyo, Elk Grove Village, IL) and was expressed in units of 10-4 in. as previously described (8). The magnitude of the ear swelling responses is presented as the mean increase in each group of four sensitized or nonsensitized mice (i.e., eight ears) ± SEM.
Cell culture
Lymph node cells (LNC) were obtained from nonsensitized mice and
from hapten-sensitized mice on day 4. For in vitro depletion of
CD4+ or CD8+ T cells, LNC
were incubated with specific Ab-coated magnetic beads (Dynabeads;
Dynal, Oslo, Norway). Ab staining and flow cytometric analyses
performed in each experiment indicated that the efficiency of this
depletion was >95% for the target T cell population (data not shown).
Enriched CD4+ and CD8+ T
cell populations were stimulated to produce cytokines by culture on
anti-CD3 mAb-coated wells. The 96-well U-bottom tissue culture
plates were coated with 30 µl/well anti-CD3 mAb 145.2C11 (25
µg/ml) for 90 min at 37°C. As a negative control, wells were coated
with an Ab to a V region not expressed by BALB/c or C57BL/6 mice,
anti-V17a mAb KJ 23a. The wells were washed, and 2 x
105 cells were delivered to each well in 200 µl
complete RPMI medium and cultured for 48 h in a 7%
CO2 humidified incubator at 37°C. The culture
supernatants were harvested and assayed for cytokine production by
ELISA. Previous results have demonstrated that lymph node T cells from
hapten-sensitized, but not from naive, mice produce cytokines during
anti-CD3 Ab-mediated stimulation similar to cytokine production
during culture with hapten-presenting Langerhans cells
(11).
Cytokine-specific ELISA
Polyvinylchloride ELISA plates were coated with capture
anti-IFN- or anti-IL-4 mAb in 0.1 M bicarbonate buffer, pH
8.6, overnight at 4°C and then blocked with 5% FCS/0.5% gelatin in
PBS. Duplicate aliquots of all supernatant dilutions were tested
undiluted and in at least two dilutions. Each plate also included
titrated recombinant cytokine as a positive control and to obtain a
standard curve for quantitation. Following incubation overnight at
4°C, each plate was washed, and the biotin-labeled anti-cytokine
Abs were added. The plate was incubated for 2 h at 37°C and
washed, and alkaline phosphatase-conjugated streptavidin (Fisher
Scientific, Pittsburgh, PA) was added. Following a final incubation for
1 h at 37°C the plate was washed, and the assay was developed by
addition of the substrate p-dinitrophenylphosphate (Sigma).
Results were read at 405 nm, and mean values were calculated. The
amount of cytokine in each test supernatant was calculated using the
standard curve on each plate.
ELISPOT assay
Hapten-specific IFN--producing T cells were enumerated using
ELISPOT assays as previously described (14). Briefly,
ELISA spot plates (Unifilter 350, Polyfiltronics, Rockland, MA) were
coated with 4 µg/ml IFN--specific mAb and incubated overnight at
4°C. The plates were blocked with 1% BSA in PBS and then washed four
times with PBS. LNC from unsensitized or DNFB-sensitized mice were
prepared on day 5 after hapten sensitization and used as responder
cells. Syngeneic spleen cells from naive mice were treated with
mitomycin C, then labeled with 100 µg/ml DNBS and used as stimulator
cells as previously described (15). Responder and
stimulator cells were cultured in serum-free HL-1 medium (BioWhittaker,
Walkersville, MD) supplemented with 1 mM L-glutamine.
DNP-labeled stimulator or, as a control, unlabeled cells were plated at
5 x 105 cells/well with 2 x
105 responder cells/well. After 24 h of cell
culture at 37°C in 5% CO2, cells were removed
from the plate by extensive washing with PBS. Biotinylated
anti-IFN- mAb (2 µg/ml) was added, and the plate was incubated
overnight at 4°C. The following day the plate was washed three times
with PBS/0.05% Tween 20, and conjugated streptavidin-alkaline
phosphatase for IFN- was added to each well. After 2 h at room
temperature the plates were washed with PBS, and nitro blue
tetrazolium-5-bromo-4-cloro-3-indolyl substrate (Kirkegaard & Perry,
Gaithersburg, MD) was added for the detection of IFN--producing
cells. The resulting spots were counted with an ELISA spot image
analyzer (developed at Case Western Reserve University, Cleveland, OH)
using Optimas software (Optimas, Botheled, WA) that was designed to
detect ELISA spots with predetermined criteria of spot size, shape, and
colorimetric density.
RNase protection assay
LNC from naive or sensitized mice were obtained on day 5 following hapten sensitization. CD4+ and CD8+ T cells cell populations were positively selected using Dynabeads and lysed in TRIzol reagent (Life Technologies, Grand Island, NY). Total RNA was isolated by phenol-chloroform extraction, followed by precipitation with isopropyl alcohol. IL-12R mRNA was detected by RNase protection assay, using the MCR-3 template from PharMingen.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-producing
CD4+ and CD8+ T cells in CHS
The effect of rIL-12 administration during hapten sensitization on
the development of hapten-specific CD4+ and
CD8+ T cells was tested. To initiate these
studies, CHS responses in wild-type, CD4-/-,
and CD8-/- cells given IL-12 during DNFB
sensitization were compared. Consistent with our previous observations
(13), IL-12 treatment during sensitization of wild-type
mice increased the magnitude of ear swelling responses following
challenge (Fig. 1
A). IL-12
also increased CHS responses in CD4-/- mice
(Fig. 1B). Whereas CHS was absent in sensitized and
challenged CD8-/- mice, IL-12 treatment
restored the responses in these mice (Fig. 1C).
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-producing CD4+ and
CD8+ T cells in response to DNFB sensitization
with or without IL-12 treatment was examined in each group by ELISPOT
assay. Although sensitization of wild-type mice did not induce many
CD4+ T cells producing IFN- to DNP-labeled
stimulator cells, this was reversed by IL-12 treatment (Fig. 2A). IL-12 treatment also
increased the number of IFN--producing CD8+ T
cells >3-fold in sensitized wild-type mice. Similarly, IL-12 increased
the number of hapten-specific IFN--producing
CD8+ T cells in DNFB-sensitized
CD4-/- mice, although not to the same degree as
observed in wild-type mice (Fig. 2B). Few IFN--producing
CD4+ T cells were observed in DNFB-sensitized
CD8-/- mice correlating with the absent CHS
responses to hapten challenge (Fig. 2C). Again, this was
reversed by IL-12 treatment during DNFB sensitization of
CD8-/- mice. These data indicated that
exogenous IL-12 enhanced the development of IFN--producing
CD4+ and CD8+ T cells in
response to DNFB sensitization.
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The ability to alter hapten-specific T cell development and the
CHS response by administration of IL-12 during sensitization suggested
that the induction of CHS was not dependent on IL-12. To test this
directly, groups of BALB/c mice were first depleted of
CD4+ T cells by treatment with anti-CD4 mAb
on days -4, -3, and -2, and these mice and control mice were treated
with control rat IgG or with two different anti-IL-12 mAb on each
day of hapten sensitization (i.e., days 0 and 1). Treatment with
anti-IL-12 Ab did not alter the magnitude of ear swelling observed
in sensitized mice with or without CD4+ T cells
following hapten challenge (Fig. 3A). In addition, wild-type
BALB/c and IL-12-deficient p40-/- mice were
sensitized with DNFB, and 5 days later the ear swelling responses to
hapten challenge were compared. As shown in Fig. 3B,
responses were virtually identical in the two groups. These results
indicated that endogenous IL-12 was not required for CHS responses.
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2 on both CD4+ and
CD8+ T cell subsets following exogenous IL-12 treatment
To further examine the role of IL-12 during the development of T
cells for CHS, the expression of the inducible component of the IL-12R
(i.e., IL-12R2) was tested on CD4+ and
CD8+ T cells from DNFB-sensitized and naive mice.
Lysates of enriched CD4+ and
CD8+ T cell populations obtained from LNC of
naive or hapten-sensitized mice contained detectable amounts of
IL-12R1 mRNA (Fig. 4). However, the
expression of the IL-12R2 component that is up-regulated during Th1
cell development (16) was not detected on either cell
population from the sensitized mice. In contrast, administration of
IL-12 during hapten sensitization resulted in detectable amounts of
IL-12 R2 mRNA in lysates of both CD4+ and
CD8+ T cells (Fig. 4).
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Recent studies from this laboratory demonstrated the development
of hapten-specific CD4+ and
CD8+ T cells as well as CHS responses following
sensitization of CD154-/- mice
(17). Since IL-12 production is stimulated by CD40-CD154
engagement (18, 19), the ability of exogenous IL-12 to
enhance CHS responses in the absence of CD40-/CD154-mediated
costimulation was tested. Groups of wild-type C57BL/6 and
CD154-/- animals were sensitized to DNFB with
or without IL-12 treatment, and ear swelling responses to DNFB
challenge were compared (Fig. 5). The
magnitude of CHS in sensitized CD154-/- mice
was slightly lower than the magnitude in sensitized wild-type animals.
Treatment with IL-12 during sensitization amplified CHS in each
group.
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-producing
CD4+ and CD8+ T cells in
wild-type and CD154-/- mice treated with
or without IL-12 during DNFB sensitization was compared by ELISPOT
assay. The number of IFN--producing CD8+ T
cells in response to hapten-labeled stimulator cells was slightly
reduced in sensitized CD154-/- mice compared
with the number in sensitized wild-type animals (Fig. 6). The development of IFN--producing
CD4+ T cells was at very low to undetectable
levels in both groups of sensitized mice. IL-12 treatment increased the
number of IFN--producing CD8+ T cells in both
wild-type and CD154-/- mice. IL-12 treatment
also promoted the development of IFN--producing
CD4+ T cells in sensitized wild-type mice, but
had a marginal to undetectable effect on the development of
IFN--producing CD4+ T cells in
CD154-/- mice (<10 spots/2 x
105 cells). These results indicated that
IFN--producing CD8+ T cell development
occurred independently of CD40-CD154 interactions during hapten
sensitization, but the development of type 1 CD4+
T cells required CD40-CD154 costimulation, and the deficiency in this
costimulatory pathway was not reversed by exogenous IL-12.
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-producing CD8+ T cell
development and CHS are observed in CD154-/-
mice following sensitization, the administration of anti-CD154 mAb
during sensitization of wild-type mice inhibits both these events
through a CD4+ T cell-mediated mechanism
(17). The ability of IL-12 treatment to overcome the
anti-CD154 mAb-mediated inhibition of CHS was next tested. Groups
of C57BL/6 mice were given IL-12 with or without anti-CD154 mAb MR1
during hapten sensitization with DNFB. Whereas MR1 treatment inhibited
CHS in the control group, administration of both IL-12 and MR1 during
sensitization resulted in responses similar to those in the control
group (Fig. 7). The CHS responses in
IL-12- plus MR1-treated animals were, however, clearly lower than the
responses in animals given IL-12 alone during DNFB sensitization.
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17a mAb-coated wells produced nondetectable
amounts of the cytokines (data not shown), and T cells from
nonsensitized mice produced low to nondetectable levels of IFN- and
IL-4 following culture on anti-CD3 mAb-coated wells (Fig. 8). CD8+ T cells
from the control group of hapten-sensitized mice produced IFN-, and
the CD4+ T cells produced IL-4 and low amounts of
IFN- (Fig. 8). As recently reported, administration of MR1 during
hapten sensitization inhibited IFN- production by
CD4+ and CD8+ T cells and
enhanced IL-4 production by CD4+ T cells
(17). Both CD4+ and
CD8+ T cell populations isolated from mice
treated with IL-12 during DNFB sensitization produced high levels of
IFN-, and CD4+ T cell production of IL-4 was
absent. IL-12 treatment during DNFB sensitization completely restored
the production of IFN- by CD4+ and
CD8+ T cells from MR1-treated mice to the levels
observed in the control group. However, IL-12 treatment completely
inhibited the exaggerated CD4+ T cell production
of IL-4 observed in mice treated only with anti-CD154 mAb during
sensitization. Furthermore, IFN- production by
CD4+ T cells from these mice was lower than
production by CD4+ T cells from mice treated with
only IL-12 during sensitization.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-producing
CD8+ T cells and CD4+ T
cells producing type 2 cytokines, such as IL-4 and IL-5
(11). However, in our experience hapten-primed
CD4+ T cells often produce very low, but
detectable, amounts of IFN- following in vitro stimulation
(17). These results suggest that hapten sensitization
generally skews CD4+ T cell development to the
Th2 phenotype, but that a small number of hapten-specific Th1
CD4+ T cells may also develop. The cytokine and
cellular interactions required for the development of these individual
T cell populations in the CHS response remain under investigation in
several laboratories.
The development of CD4+ T cells in response to
IFN--producing cells is largely, although not exclusively, dependent
upon the presence of IL-12 during Ag priming (12). The
development of CD4+ T cells to type 2
cytokine-producing cells during sensitization for CHS responses
suggests that IL-12 is absent or at suboptimal levels during priming of
hapten-specific CD4+ and
CD8+ T cells by hapten-presenting Langerhans
cells (hpLC). Previous results from this laboratory demonstrated that
administration of rIL-12 during sensitization with DNFB or Ox skewed
hapten-specific CD4+ T cell development to
IFN--producing cells (13). This altered development
resulted in the ability of hapten-primed CD4+ T
cells to mediate ear swelling responses (e.g., in the absence of
CD8+ T cells) to hapten challenge. Another
consequence of this altered development was the elimination of a major
regulatory component of the response resulting in ear swelling
responses of increased magnitude and extended duration compared with
responses in animals not given IL-12 during sensitization. As shown in
the current report, IFN--producing CD4+ T cell
development and CHS responses were absent in
CD8
-/- mice unless exogenous IL-12 was given
during sensitization. Despite similar magnitudes of CHS in
CD8-/- and CD4-/- mice
treated with IL-12 during sensitization, the number of hapten-specific
CD4+ T cells producing IFN- was almost 3-fold
lower than the number of IFN--producing CD8+ T
cells from these respective mice. This result may be indicative that
CD4+ T cells produce more IFN- on a per cell
basis than the CD8+ T cells during elicitation of
the response. An alternative explanation is that IFN--producing
CD4+ and CD8+ T cells may
have differences in the ability to traffic to the hapten challenge
site.
Equivalent CHS responses were also observed in IL-12
p40-/- mice and wild-type animals following
sensitization and challenge with DNFB. These results are consistent
with the proposal that IL-12 is either absent or at low levels and has
little influence on CD4+ and
CD8+ T cell development during priming for CHS.
In contrast, results from two other laboratories have indicated the
ability to inhibit the induction of CHS responses by treating mice with
anti-IL-12 mAb during hapten sensitization (20, 21).
In our hands, treatment with these Abs did not alter T cell development
or the magnitude of CHS responses following sensitization with DNFB.
However, we have recently observed that sensitization with supraoptimal
doses of hapten promotes the development of IFN--producing
CD4+ T cells (manuscript in preparation). The
possible role of endogenous IL-12 in the development of these
CD4+ T cells is under investigation.
In contrast to the role of IL-12 in CD4+ T cell
development, the effect of IL-12 on the development of hapten-specific
CD8+ T cells during sensitization for CHS has not
been previously tested. Results from the current report indicate that
the expression of the IL-12R2 component of the IL-12R is absent on
CD8+ and CD4+ T cells from
sensitized mice, but is up-regulated by administration of rIL-12.
Recent in vitro studies by Wu and co-workers showed that IL-12R2
expression on human CD4+ and
CD8+ T cells is up-regulated by IL-12
(22). Administration of rIL-12 during sensitization also
increased the number of hapten-specific, IFN--producing
CD8+ T cells in the skin draining lymph nodes 2-
to 3-fold. This increased development was much greater than that
observed for hapten-specific, IFN--producing
CD4+ T cells from mice treated with IL-12 during
sensitization. The ability of exogenous IL-12 to enhance
CD8+ T cell responses has been reported by other
laboratories (23, 24, 25). In one study the ability of IL-12
to enhance Ag-driven CD8+ T cell proliferation in
vitro was dependent on exogenous IL-2 (26). During
sensitization for CHS IL-12-mediated enhancement of
CD8+ T cell development occurred in
CD4-/- mice, indicating that this enhancement
was not dependent on CD4+ T cells. Our previous
studies have indicated that hapten-primed CD4+ ,
but not CD8+, T cells produce IL-2
(27). In this light, the current results may be indicative
that IL-2 is not the growth factor promoting the IL-12-mediated
enhanced growth of CD8+ T cells. Several points
are worth considering. First, IL-12 stimulation may alter the phenotype
of hapten-specific CD8+ T cells to produce IL-2.
Alternatively, IL-12 administration may enhance the production of a
growth factor used for clonal expansion of the
CD8+ T cells or positively affect growth factor
receptor expression on the CD8+ T cells. For
example, IL-15 is required for CD8+ T cell
expansion in many immune responses (28, 29, 30), and the
effects of IL-12 on IL-15 production or on CD8+ T
cell expression of the IL-15R -chain have not been thoroughly
investigated.
The development of IFN--producing CD4+ T cells
also is dependent upon interactions between CD40 expressed by APC and
CD154 expressed by the T cell (31, 32). The skewing of
hapten-specific CD4+ T cell development to type 2
cytokine production might be indicative of the absence of these
interactions during T cell priming for CHS. In support of this, recent
studies from this laboratory revealed that the development of
hapten-specific IL-4-producing CD4+ T cell and
IFN--producing CD8+ T cell populations as well
as CHS responses were similar in DNFB-sensitized wild-type and
CD154-/- mice (17). One
consequence of CD40 ligation during T cell priming is the stimulation
of APC to produce IL-12 (18, 19). In studies by Stuber and
colleagues (33), administration of IL-12 circumvented the
anti-CD154 mAb-mediated inhibition of effector, IFN--producing
CD4+ T cell development and restored the
induction of hapten-induced colitis. Similarly, administration of
rIL-12 to CD154-/- mice restored
IFN--producing CD4+ T cell development and
protection from leishmaniasis (34). In contrast to
wild-type animals, however, administration of rIL-12 during DNFB
sensitization of CD154-/- animals did not
rescue the development of hapten-specific CD4+ T
cells producing IFN-. This indicates that exogenous IL-12 cannot
substitute for CD40-mediated signaling and that both IL-12 and
CD40-CD154 interactions are required for the development of Th1
CD4+ T cells able to mediate CHS responses.
CD40-CD154 interactions during T cell priming also up-regulate many
costimulatory molecules on the dendritic cell surface
(35). We previously reported that T cell engagement of
B7-2, but not B7-1, on hpLC was necessary for priming of the
IL-4-producing CD4+ and IFN--producing
CD8+ T cell populations during sensitization for
CHS (36). It is conceivable that the induction of
hapten-specific CD4+ T cell development to an
IFN--producing phenotype through administration of IL-12 during DNFB
sensitization requires additional costimulatory signals, such as B7-1,
that are initiated through CD40-CD154 interactions during T cell
priming by hpLC. In contrast to CD4+ T cells,
CD8+ T cell development was enhanced by giving
IL-12 during sensitization of CD154-/- mice,
again indicating that IFN--producing CD8+ T
cell development is not dependent upon CD40-mediated stimulation.
Although development of IFN--producing CD8+ T
cells is similar in wild-type and CD154-/-
animals, administration of anti-CD154 mAb during hapten
sensitization of wild-type mice inhibits the development of these T
cells and the CHS response (17). By contrast, the
development of hapten-specific CD4+ T cells
producing IL-4 is enhanced by the treatment of wild-type mice with
anti-CD154 mAb during sensitization. We have recently shown that
the inhibitory effects of the anti-CD154 mAb treatment are
abolished by depleting mice of CD4+ T cells
before hapten sensitization and anti-CD154 mAb administration
(17). Together these findings indicate that the inhibition
of CD8+ T cell development for CHS by
anti-CD154 mAb is mediated through direct effects on
CD4+ T cells, rather than through blockade of
CD40-CD154 interactions. Since administration of rIL-12 during hapten
sensitization inhibits regulatory CD4+ T cell
development, we tested whether exogenous IL-12 would reverse the
inhibitory effects of anti-CD154 mAb treatment. The development of
ear swelling responses in mice treated with both anti-CD154 mAb and
IL-12 was restored to the level in the control group, although not to
the level in mice treated with IL-12 alone. Administration of IL-12
with the anti-CD154 mAb had two notable effects on the development
of hapten-specific CD4+ and
CD8+ T cells. First, IL-12 rescued the
development of hapten-specific CD8+ T cells
producing IFN-. Thus, IL-12 circumvented the inhibitory effect of
anti-CD154 mAb mediated through CD4+ T cells.
Second, in contrast to CD8+ T cells,
CD4+ T cell development to IFN--producing
cells was inhibited in mice given both IL-12 and anti-CD154 mAb
during sensitization. We interpret these results as indicating that
exogenous IL-12 primes the CD4+ T cells during
interaction with hpLC to become IFN--producing cells, but that
CD40-CD154 interactions are required to complete this process. In the
absence of these interactions, CD4+ Th1 cells
receiving only IL-12 signal do not develop at optimal levels. The
synergistic effects of IL-12 and CD40-CD154 interactions on the
development of human CD4+ T cells to
IFN--producing cells during stimulation with anti-TCR mAb have
been recently reported (37). In contrast to
CD4+ T cells, the effect of IL-12 on the
development of CD8+ T cells for CHS is
independent of CD40-CD154 engagement.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Anton Gorbachev, NB3-30, Department of Immunology, Cleveland Clinic Foundation, 9500 Euclid Avenue, Cleveland, OH 44195-0001. E-mail address: tosha79{at}hotmail.com
3 Abbreviations used in this paper: CHS, contact hypersensitivity; DNFB, 2,4-dinitrofluorobenzene; hpLC, hapten-presenting Langerhans cells; LNC, lymph node cells; Ox, oxazolone.
Received for publication February 27, 2001. Accepted for publication April 24, 2001.
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References |
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  P. Gurung, T. A. Kucaba, T. A. Ferguson, and T. S. Griffith Activation-Induced CD154 Expression Abrogates Tolerance Induced by Apoptotic Cells J. Immunol., November 15, 2009; 183(10): 6114 - 6123. [Abstract] [Full Text] [PDF]
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 S. F. Martin, J. C. Dudda, E. Bachtanian, A. Lembo, S. Liller, C. Durr, M. M. Heimesaat, S. Bereswill, G. Fejer, R. Vassileva, et al. Toll-like receptor and IL-12 signaling control susceptibility to contact hypersensitivity J. Exp. Med., September 1, 2008; 205(9): 2151 - 2162. [Abstract] [Full Text] [PDF]
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D. He, L. Wu, H. K. Kim, H. Li, C. A. Elmets, and H. Xu CD8+ IL-17-Producing T Cells Are Important in Effector Functions for the Elicitation of Contact Hypersensitivity Responses J. Immunol., November 15, 2006; 177(10): 6852 - 6858. [Abstract] [Full Text] [PDF]
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H. Riemann, K. Loser, S. Beissert, M. Fujita, A. Schwarz, T. Schwarz, and S. Grabbe IL-12 Breaks Dinitrothiocyanobenzene (DNTB)-Mediated Tolerance and Converts the Tolerogen DNTB into an Immunogen J. Immunol., November 1, 2005; 175(9): 5866 - 5874. [Abstract] [Full Text] [PDF]
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A. V. Gorbachev and R. L. Fairchild CD40 Engagement Enhances Antigen-Presenting Langerhans Cell Priming of IFN-{gamma}-Producing CD4+ and CD8+ T Cells Independently of IL-12 J. Immunol., August 15, 2004; 173(4): 2443 - 2452. [Abstract] [Full Text] [PDF]
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M. L. Salem, A. N. Kadima, Y. Zhou, C. L. Nguyen, M. P. Rubinstein, M. Demcheva, J. N. Vournakis, D. J. Cole, and W. E. Gillanders Paracrine Release of IL-12 Stimulates IFN-{gamma} Production and Dramatically Enhances the Antigen-Specific T Cell Response after Vaccination with a Novel Peptide-Based Cancer Vaccine J. Immunol., May 1, 2004; 172(9): 5159 - 5167. [Abstract] [Full Text] [PDF]
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H. Akiba, J. Kehren, M.-T. Ducluzeau, M. Krasteva, F. Horand, D. Kaiserlian, F. Kaneko, and J.-F. Nicolas Skin Inflammation During Contact Hypersensitivity Is Mediated by Early Recruitment of CD8+ T Cytotoxic 1 Cells Inducing Keratinocyte Apoptosis J. Immunol., March 15, 2002; 168(6): 3079 - 3087. [Abstract] [Full Text] [PDF]
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) or 200 µg rIL-12 (
). On day
5 DNFB-sensitized and control, unsensitized (•) mice were ear
challenged with 0.2% DNFB, and ear thickness was measured at 24-h
intervals. The mean increase in ear thickness following hapten
challenge is shown in units of 10-4 in. ± SEM. The
results shown are representative of three individual experiments.
) for each group represents the mean number detected in
triplicate wells using an immunospot image analyzer. The results are
representative of two experiments. *, p < 0.05
(compared with values in the control untreated group).
), 200 µg rIL-12 (•), or
anti-CD154 mAb plus IL-12 (
). Naive mice (
