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INSERM Unite 80, Université Claude Bernard Lyon 1, Faculté Lyon Laennec, Lyon, France;
Life Sciences, L’Oréal Advanced Research Laboratories, Aulnay-Sous-Bois, France; and
INSERM Unite 404, Immunité et Vaccination, Lyon, France
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Contact sensitivity (CS) is a T cell-mediated immune reaction occurring after epicutaneous immunization and challenge with low m.w. chemicals, i.e., haptens, which covalently bind to discrete amino acid residues on self or exogenous proteins. Hapten-modified proteins could then be processed by APC into antigenic peptides, which are transported at the cell surface in association with class I or class II MHC molecules. Epidermal dendritic cells, i.e., Langerhans cells (LC) play a crucial role in the induction of CS. They capture the hapten (or haptenated proteins) in the skin and migrate to draining lymph nodes where they mature into functional APC endowed with the capacity to prime naive T cells (12). Hapten-specific presursor T cells recognize a conformational complex formed by the hapten-modified peptide within the groove of both MHC class I and class II molecules of the DC (13, 14, 15, 16).
Unlike classical delayed-type hypersensitivity (DTH) to protein or cellular Ags, which is mediated primarily by MHC class II-restricted CD4+ T cells (17), the T cell response to haptens appears more complex and is still not well understood. Previous studies reported that CS responses may involve CD4+ and/or CD8+ T cells depending on the hapten and the mouse strain used (18). We have recently demonstrated, using MHC class I- and class II-deficient mice, that the CS reaction to dinitrofluorobenzene (DNFB) in C57BL/6 mice was mediated by MHC class I-restricted CD8+ effector T cells and down-regulated by MHC class II-restricted CD4+ T cells (19). This suggested that the hapten presentation by MHC class I molecules is required for the induction of effector cells of CS, while presentation by MHC class II molecules results in the generation of regulatory cells. Several questions were raised by these data: 1) Do DC represent the APC type responsible for the priming of both CD8+ effector and CD4+ regulatory T cells? 2) Are DC able to present haptens on MHC class I and class II molecules simultaneously and independently? 3) Are DC involved in the induction of tolerance through hapten presentation by MHC class II/CD4 pathway?
In this study, we used hapten-pulsed DC from MHC class I- and class II-deficient mice to determine the relative contribution of the MHC molecules to the CS reaction. We also took advantage of the fact that hapten presentation through MHC class II molecules can generate cells able to down-regulate CS to test for the possibility of inducing hapten-specific tolerance in naive recipients. Our results demonstrate that DC can very efficiently immunize for CS reactions to DNFB but are not able to induce a long-lasting state of tolerance.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Mice with a mutation in the ß2-microglobulin (I-) (20) or Aß gene (II-) (21) have been created by gene-targeting techniques. Heterozygous (I- and II-) knockout mice were backcrossed for more than eight generations to C57BL/6 mice and used to generate the offspring of the present study. These mice were obtained from C. Benoist and D. Mathis (IBMG, INSERM Unite 184, Stasbourg, France) and bred in specific pathogen-free conditions in Iffa Credo/Transgenic Alliance, L’Arbresle, France. C57BL/6 (H-2b) mice were obtained from Iffa-Credo. Mice were used between 2 and 4 mo of age.
Allergens
The haptens 2,4-dinitro-fluorobenzene (DNFB; Sigma, St. Louis, MO) and picryl chloride (TNP; BDH Chemicals Ltd., Poole, England) were freshly prepared before CS assays. 2,4-Dinitrobenzenesulfonic acid (DNBS; Aldrich, Milwaukee, WI) and 2,4,6-trinitrobenzenesulfonic acid (TNBS; Sigma) were used for in vitro pulsing of dendritic cells. BSA and OVA, both from Sigma, were used in DTH assays.
Culture medium
The culture medium was RPMI 1640 (Life Technologies, Paisley, Scotland) supplemented with 10% heat-inactivated FCS (Boehringer-Mannheim, Meylan, France), 100 U/ml penicillin, 100 µg/ml streptomycin, 2 mM L-glutamine, and 5.10-5 M 2-ME (all from Sigma); this was referred to as complete medium.
LC-enriched epidermal cell suspensions
Single epidermal cell (EC) suspensions were prepared from ear skin. Epidermal sheets were obtained by 1-h incubation of the ear in 0.05% trypsin (Life Technologies, Grand Island, NY) in PBS at 37°C. The resulting EC suspension (2–4% LC) was centrifuged onto Nycoprep gradient (Nycoprep mixer; Nycomed, Oslo, Norway), and cells of the interface containing 8 to 14% LC were collected. To further enrich for LC, the cells of the interface were incubated for 24 h at 37°C in complete medium supplemented with 50 ng/ml recombinant murine GM-CSF (Schering-Plough, Kenilworth, NJ). Nonadherent cells were then harvested, and viable cells, containing 20 to 30% MHC class II-positive cells (LC), as assessed by FACS analysis, were recovered by Nycoprep density centrifugation.
DC generation from bone marrow cultures
Large numbers of DC were generated in vitro from bone marrow progenitors as previously described (22, 23). Briefly, bone marrow was flushed from the long bones of the hind limbs and depleted of RBC with ammonium chloride, 0.83%. The cells were extensively washed in HBSS and incubated at 37°C in 24-well culture plates (106 cells/ml, 1 ml/well) in complete medium supplemented with 50 ng/ml of recombinant murine GM-CSF (Schering-Plough). To deplete nonadherent granulocytes without dislodging clusters of developing DC, half of the medium containing floating cells was aspirated every other day and replaced by fresh medium supplemented with GM-CSF. At day 7 of culture, nonadherent cells were harvested for phenotypic analysis and transfer experiments, and these are referred to as bone marrow-derived DC (BM-DC).
Ag pulsing of APC
Cell pellets (24-h cultured LC, 7-day cultured BM-DC, or mitomycin-treated splenocytes) were resuspended in HBSS without FCS containing DNBS (100 µg/ml), TNBS (2 mM), or BSA (0.1 mg/ml), respectively (pH 8). Cells were incubated at 37° for 10 min with TNBS, for 30 min with DNBS, and for 24 h with BSA.
Immunization with DC
105 DNBS- or TNBS-derivatized epidermal DC or BM-derived DC from either I+/II+, I+/II-, or I-/II+ mice were injected s.c. in 200 µl of saline into naive C57BL/6 (I+/II+) recipient mice to prime for CS. 106 BSA-pulsed I+/II+, I+/II-, and I-/II+ BM-DC were similarly injected s.c. into naive C57BL/6 mice (I+/II+) to sensitize for DTH.
Assay for CS: the mouse ear swelling test
The procedure for the mouse ear swelling test has been described in details elsewhere (24). For sensitization to DNFB, mice were painted once (day -5) on the shaved dorsal skin with 25 µl of 0.5% DNFB in acetone:olive oil (4:1, v:v) or with 7% TNP in acetone. Five days later (day 0), mice were challenged by the application of either DNFB (0.15% in acetone:olive oil, 4:1) or TNP (1% in olive oil) as control, on each side of the right ear, while the left ear received the vehicle alone. Ear thickness was assessed before and at various intervals after challenge using a spring-loaded micrometer (J15, Blet SA, Lyon, France). Ear swelling was calculated by subtracting the initial value from the values recorded on the corresponding day and further subtracting any swelling recorded for the vehicle-painted ear from the swelling recorded for the hapten-challenged ear. In the present study, mice were injected s.c. with hapten-derivatized DC, and the responses were compared with those elicited in mice sensitized by direct epicutaneous hapten application. A group of mice injected with the same number of unmodified DC served as a negative control. We tested for down-regulation of the inflammatory response by injecting hapten-derivatized BM-DC into mice sensitized via the epicutaneous route.
Assay for DTH response to purified protein Ags
DTH response to protein Ags was measured by a conventional
footpad swelling assay. Mice were sensitized by the s.c. injection of
200 µl of either 50 µg of BSA emulsified with CFA (1:1, v:v) or
106 BSA-treated or unmodified BM-DC as control. Seven
days later, the mice were challenged by the s.c. injection in the left
hind foodpad of 0.6 mg of BSA, or OVA as control, diluted in saline.
The right footpad was injected with saline alone. BSA-specific DTH was
calculated by subtracting the swelling of the right (control) footpad
from the swelling of the left footpad induced by BSA, according to the
formula: 
e (BSA) - e (saline), where e (BSA) = footpad
thickness after BSA - before BSA, and e (saline) = footpad
thickness after saline - before saline. Mice injected with the
corresponding number of unmodified BM-DC served as a negative
control.
Hapten-specific T lymphocyte proliferation assay
T cells from the spleen and the inguinal and axillary lymph
nodes of immunized mice were pooled together and partially purified by
negative selection using anti-Ig columns (Biotex, Edmonton,
Alberta, Canada). The resulting cell suspensions contained >98%
viable Thy-1.2+, CD3+ cells.
CD4+ and CD8+ T cells were enriched by negative
selection of CD4+ and CD8+ cells, respectively
(Biotex), using columns coated with goat anti-mouse IgG and either
a rat mAb anti-mouse CD8
(clone YTS 169.4) or a rat mAb
anti-mouse CD4 (clone YTS 191.1). The residual CD4+ and
CD8+ cell populations in the nonadherent effluent cells
represented <0.5%. In vivo hapten-primed T cells (2.5 x
105/well) were cocultured for 3 days at 37°C in 96-well
microtiter plates with 106 mitomycin-treated syngeneic
spleen cells that were either hapten-derivatized with DNBS and TNBS or
untreated. T cell proliferation was determined on day 3 of culture by
[3H]thymidine incorporation (1 µCi/well) during the
last 6 h of culture. The results are expressed as cpm/culture.
Flow cytometric analysis
A panel of mAbs was used to identify and characterize
single-cell suspensions obtained from draining lymph nodes and spleens
of sensitized animals, as well as the cells in epidermal cell
suspensions and in BM-DC cultures. mAbs used included: FITC- and
phycoerythrin-conjugated rat anti-mouse CD4 (IgG2a, clone CT-CD4),
FITC-conjugated rat anti-mouse CD8 (IgG2a, clone CT-CD8a),
FITC-conjugated Thy 1.2 (mouse IgG2b, clone 5a-8), FITC-conjugated
anti-Ia(b,d) (mouse IgG2a, clone 28–16–8S),
mouse anti-mouse FITC-conjugated H-2Db (IgG2a, clone
CTDb), FITC-conjugated rat IgG2b anti-mouse CD11b (Mac-1, rat
IgG2b, clone M1/70.15), and FITC-conjugated rat anti-mouse Ly-5
(B220, IgG2a, clone RA3–6B2), all purchased from Caltag Laboratories
(Burlingame, CA). Phycoerythrin-conjugated
anti-Ia(b,d,p,q) (rat IgG2b, M5/114) was purchased from
Boehringer Mannheim. The hamster anti-mouse CD11c (clone N418) and
polyclonal hamster IgG Abs were purchased from American Type Culture
Collection (Rockville, MD). Rat IgG2a anti-mouse CD80 (B7.1, clone
16-10A1) and CD86 (B7.2, clone GL1) were from PharMingen (San Diego,
CA). Rat IgG2b anti-mouse Ia (clone CD311) and rat IgG2b
anti-mouse NLDC-145 (DEC 205) were kindly provided by A. Glasebrook
(25) and George Kraal (Amsterdam, The Netherlands), respectively.
Cells (106) were incubated with Abs for 30 min at 4°C and
washed twice with PBS containing 1% FCS and 0.1% sodium azide. Cell
suspensions were preincubated with 50 µg/ml of anti-mouse
CD16/CD32 (rat IgG2b, clone 2.4G2) from PharMingen to block
Fc
III/IIR, whenever mouse, hamster, or directly conjugated rat Abs
were used. FITC-conjugated donkey anti-rat IgG (H + L)
(Jackson Immunoresearch Laboratories, West Grove, PA) and
FITC-conjugated goat anti-hamster IgG (H + L) (Caltag) were
used as secondary Abs.
The appropriate isotype-matched Abs were used as controls of specific staining. Fluorescence was measured with a FACScan (Becton Dickinson, Mountain View, CA) flowcytometer, and data were analyzed with LYSYS II software (Becton Dickinson). A minimum of 20,000 events were scored.
Immunohistochemical analysis of epidermal sheets
Epidermis from the ears was peeled off the connective tissue by incubation for 1 h at 37°C in PBS supplemented with 20 mM EDTA (Sigma). The sheets were cut in small pieces, rinsed, and incubated overnight at 4°C with specific Abs or isotype-matched irrelevant Abs. After three washes in PBS/1% BSA, the sheets were incubated for 30 min at room temperature with biotinylated F(ab')2 fragment of goat Abs specific for rat IgG (H + L) (Pierce, Interchim, Montluçon, France), washed, and then further incubated with streptavidin-conjugated to peroxidase (ABC kit, Dako, Capinteria, CA). The reaction was developed using 3-amino-9-ethylcarbazole (AEC) substrate and H2O2 (Dako).
Statistical analysis
Panels consisted of five mice each, and all experiments were performed at least twice. The statistical significance of differences between mean values of experimental groups was evaluated using the two-tailed Student’s t test (p < 0.05).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We analyzed the ability of LC from C57BL/6 mice or from syngeneic
MHC class I- and MHC class II-deficient mice to immunize for CS to
DNFB. Immunohistochemical analysis of epidermal sheets using the
DC-specific anti-CD11c Ab (N418) revealed that the network of LC
was comparable in all three types of mice. In addition, we verified
that N418+ epidermal LC from
I+/II- mice lack expression of MHC class II
(Fig. 1
A). EC
suspensions from either C57BL/6 mice or from syngeneic
I+/II- or I-/II+ mice
were enriched in LC by Ficoll gradient centrifugation followed by 24-h
culture in the presence of GM-CSF. EC suspensions from C57BL/6 (Fig. 1B) and I-/II+ mice (data
not shown) contained 20 to 30% LC-expressing MHC class II
molecules.
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, mice receiving DNBS-derivatized LC
from either C57BL/6 or MHC class II-deficient (class
I+/II-) mice, developed a strong
hapten-specific CS response (Fig. 2). The intensity and magnitude of
the inflammatory reaction was similar to that observed in mice
immunized by the epicutaneous application of DNFB (ear swelling of mice
sensitized by skin painting shown in Fig. 3A). In contrast, s.c.
injection of DNBS-derivatized I-/II+ EC from
MHC class I-deficient mice was unable to prime naive mice for CS to
DNFB, indicating that the presence of MHC class I but not the presence
of MHC class II molecules was mandatory for the induction of CS
effector T cells.
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Since EC contain only 20–30% of LC, the contribution of DC in priming for CS effector T cells was addressed more directly using highly enriched DC suspensions obtained by in vitro culture of bone marrow progenitors for 7 days in the presence of GM-CSF. These in vitro-differentiated DC (referred to as BM-DC), were then tested for their ability to induce CS in naive recipient mice. BM-DC, established from either MHC I+/II+, MHC class I-, or MHC class II-deficient mice, comprised 40 to 50% of cells exhibiting a dendritic morphology with slender cytoplasmic processes. FACS analysis revealed that BM-DC cultures from C57BL/6 mice contained 40 to 50% of MHC class II+ cells coexpressing CD11c, DEC 205, B7.1, B7.2, CD11b, and F4/80 and lacking B220, Thy1.2, CD4, and CD8 molecules (data not shown). These data confirmed that BM-DC had a phenotype characteristic of mature interdigitating DC.
DNBS-derivatized I+/II- BM-DC could prime
naive mice for CS as efficiently as I+/II+
BM-DC (Fig. 3
A). Unmodified
I+/II+ and I+/II-
BM-DC failed to sensitize mice for a DNFB-specific CS. The CS response
induced by DNBS-pulsed BM-DC was hapten specific. Indeed, mice
immunized with DNBS-pulsed DC and challenged with TNP did not develop a
CS reaction. Similarly, mice immunized with TNBS-treated DC did not
develop a CS reaction upon challenge with DNFB (Fig. 3B).
Alternatively, mice injected with DNBS-modified
I-/II+ BM-DC were unable to develop a CS
response upon DNFB challenge (Fig. 3A). This was not
due to impaired Ag processing and presentation function, since these
cells, when pulsed with native BSA delivered in FCS-containing medium,
could induce a specific DTH of a magnitude comparable with that
generated by immunization with BSA emulsified in CFA (Fig. 3C). Thus, MHC class I but not class II molecules
expressed by BM-DC were necessary and sufficient for priming MHC class
I-restricted CS effector T cells in vivo.
MHC class I+ DC can prime class I-restricted CD8+ T cells in vivo
To confirm that class I-restricted CD8+ T cells
could be primed by haptenated DC in vivo, we tested the ability of
CD8+ T cells isolated from draining lymph nodes to
proliferate in response to in vitro restimulation by haptenated spleen
cells from either I+/II+,
I+/II-, or I-/II+
mice. As shown in Table I,
CD8+ T cells, purified from spleen and lymph nodes on day 5
after DC transfer, proliferated in response to restimulation by
I+/II+ and I+/II- but
not I-/II+ DNBS-pulsed DC. No proliferation
was ever observed in response to TNBS-modified spleen cells. Thus,
hapten-specific class I-restricted CD8+ T cells were primed
by DC in vivo and were present in secondary lymphoid organs at the time
of hapten challenge.
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We next examined whether the lack of CS response to DNFB in mice
immunized with DNBS-treated I-/II+ BM-DC
was due to induction of class II-restricted CD4+ T cells,
which down-regulate CS (19). We tested for the presence of
hapten-specific CD4+ T cells in lymphoid organs of C57BL/6
mice after s.c. immunization with DNBS-treated
I-/II+ BM-DC. We assayed the ability of
unfractionated or purified CD4+ T cells to proliferate in
response to in vitro restimulation by DNBS-modified spleen cells from
either normal (MHC class I+/II+) mice or MHC
class I- or MHC class II-deficient mice. DNBS-specific T cell
proliferation was only found in either unfractionated or
CD4+-enriched but not in the CD8+-enriched T
cell suspensions, upon in vitro restimulation with DNBS-derivatized
spleen cells from either C57BL/6 (class I+/II+)
or MHC class I-deficient (class I-/II+) mice,
both expressing MHC class II molecules (Table II). Proliferation was never observed
when the T cells were stimulated with TNBS-haptenated spleen cells.
These results demonstrated that immunization with DNBS-modified MHC
class I-/II+ BM-DC induced hapten-specific
class II-restricted CD4+ T cells.
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Since we had previously reported that MHC class II-restricted
CD4+ T cells down-regulate CS to DNFB in C57BL/6 mice
(19), we next examined whether DNBS-modified
I-/II+ BM-DC could block the induction
(afferent phase) or the expression (efferent phase) of CS. For this
experiment, mice were injected s.c. with haptenated
I-/II+ DC either at the time of skin
sensitization (day -5) or 1 day before ear challenge (day -1). As
shown in Figure 4, the inflammatory
response generated by DNFB painting was not affected in mice receiving
s.c. unmodified I-/II+ BM-DC. Identical
results were obtained with DC injected i.v. (data not shown).
Alternatively, inhibition of the ear swelling in response to DNFB
challenge was observed in mice that received DNBS-modified
I-/II+ BM-DC either at the time of
epicutaneous sensitization or 1 day before ear challenge. In both
cases, the magnitude of this inhibition was correlated with the number
of injected DC. These data strongly suggest that the inhibition of the
CS response to DNFB induced by DNBS-pulsed
I-/II+ DC is due to priming of hapten-specific
class II-restricted CD4+ T cells, which interfere with the
priming of CS effector T cells. DNBS-pulsed
I+/II+ and I+/II-
BM-DC injected at the time of epicutaneous DNFB sensitization or 1 day
before challenge had no significant effect on the ear swelling response
(data not shown).
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We next tested whether MHC class
I-/II+ DC injection could tolerize
C57BL/6 mice for hapten-specific CS induced by epicutaneous DNFB
sensitization. For this experiment, mice were pretreated by either one,
two, or three s.c. injections of haptenated BM-DC 5 days before
sensitization by hapten painting. As shown in Figure 5A, a single injection of
either unmodified or DNBS-modified I-/II+
BM-DC 5 days before DNFB sensitization (day -10) failed to tolerize
C57BL/6 mice for a CS response to DNFB. Likewise, three injections of
DNBS-pulsed I-/II+ BM-DC performed on 3
consecutive days (day -12, -11, and -10), could not inhibit the
inflammatory response generated by subsequent DNFB sensitization (Fig. 5B). Thus, DNFB sensitization was efficient at
inducing a CD8+ CS effector population in C57BL/6 recipient
mice, despite the presence of preactivated CD4+ regulatory
T cells. These data show that DC, although capable of inducing class
II-restricted CD4+ cells, which down-regulate the magnitude
and duration of the CS reaction, are unable to generate a state of
Ag-specific tolerance.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Epidermal DC are very efficient in immunizing naive animals for CS to various haptens (28, 29, 30). Our results using DNFB demonstrate that the magnitude of the inflammatory reaction obtained with hapten-pulsed DC recovered from normal mice and injected s.c. was always equal to or greater than that obtained in mice immunized by the epicutaneous route. This level of response probably relates to the fact that in vitro pulsing of DC with the hapten generates a much higher number of DC carrying the hapten as compared with that resulting from in vivo hapten application onto the skin. Alternatively, the DC preparations may be enriched for APCs responsible for the induction of effector cells and may lack APCs that could induce the down-regulatory cells of CS. BM-DC were as efficient as epidermal LC cultured for 24 h with GM-CSF, confirming that DC can capture the hapten and subsequently prime for effector cells of CS (31, 32).
Expression of MHC class I molecules by DC is mandatory for the induction of the CS reaction in our experimental system and for the activation of hapten-specific CD8+ T cells in the lymph nodes, since only I+/II+ and I+/II- DC were able to induce a CS reaction. Alternatively, no CS reaction occurred in mice immunized with DC obtained from MHC class I-deficient mice, although these cells expressed normal levels of MHC class II molecules. This lack of sensitizing properties was not due to a functional blunt in Ag presentation inasmuch as I-/II+ DC were functional for in vivo priming of C57BL/6 mice for a CD4+ T cell-mediated DTH response to BSA. Thus, induction of CD8+ effector T cells of CS is dependent upon the presentation of the hapten by MHC class I molecules and does not require the presence of MHC class II molecules, implying that DC can directly prime CD8+ T cells in the absence of CD4+ T cell help. The requirement for CD4+ helper signals during the generation of CD8+ T cell responses remains a matter of debate and depends on several factors among which are the avidity of the TCR for the peptide/class I MHC ligand (33), the nature of the APC responsible for the CD8+ T cell priming (34, 35), and the contribution of the costimulatory molecules CD80 and CD86 (36, 37). Since the first observation by Inaba et al. showing that DC were able to prime CD8+ T cells in the absence of T cell help (38), several studies have confirmed this unique functional property of DC for the development of CD8+ T cells specific for alloantigens as well as for tumor and viral Ags. MHC class I+/II- epidermal LC lines derived from fetal skin, and lacking constitutive and inducible expression of class II proteins and mRNA, can activate naive allogeneic CD8+ T cells in vitro (37) and are able to prime for transplantation immunity in vivo (39) and to generate cross-priming of class I-restricted CD8+ CTLs with exogenous viral Ags (40). Our data are in line with these observations and show that hapten presentation by MHC class I molecules on mature DC expressing CD80 and CD86 is necessary and sufficient for the priming of naive CD8+ T cells in vivo.
An important finding in this study is the observation that DC represent the APC type responsible for priming of hapten-specific class II-restricted CD4+ T cells, which have been reported to control the magnitude of the inflammatory response to DNFB in C57BL/6 mice (19). These results highlight the differences in the mechanisms involved in CS and DTH, since DTH to BSA was shown to be mediated by MHC class II presentation by DC to CD4+ T cells. Indeed, transfer into naive mice of haptenated DC, which express MHC class II but not class I molecules, generates peripheral CD4+ T cells, which proliferate upon hapten restimulation in vitro. Moreover, these hapten-pulsed I-/II+ DC decrease the magnitude of the inflammatory response in CS when they are injected into naive recipient mice either at the time of epicutaneous DNFB sensitization or 1 day before DNFB challenge. It is unlikely that inhibition of CS results from a nonspecific blockade of the class I/CD8 interaction by an excess of DC inasmuch as injection of equivalent numbers of unmodified DC does not affect the CS response. Alternatively, it may be proposed that DC-induced down-regulation is due to a quantitative increase in the number of CD4+ T cells, since the down-regulation cannot be detected if the transferred haptenated DC coexpressed class I and class II molecules. In this case, simultaneous priming of CD8+ and CD4+ T cells occurs and is induced by hapten presentation on MHC class I and class II present on the transferred DC as well as on the recipient’s own DC. Such masking of the down-regulatory effect of the CD4+ T cells is reminiscent of classical studies on suppression and contrasuppression of CS (41) and could be due to a higher proliferation capacity of CD8+ T cells compared with CD4+ T cells or to a lower threshold of hapten required to activate CD8+ vs CD4+ T cells. This hypothesis is supported by the observation that down-regulation of CS induced by DC transfer is proportional to the number of I-/II+ DC injected. Thus, CD4+-mediated inhibition of CS seems to be dependent on a balance between the respective sizes of CD8+ and CD4+ subsets responding to the hapten. The present data confirm our previously reported observation that CD4+ T cells can limit the intensity and duration of the the CS response to DNFB in C57BL/6 mice and demonstrate that activation of this down-regulatory pathway is initiated by hapten presentation by MHC class II molecules on the DC.
However, CD4+ T cells appear unable to induce tolerance for CS. First, inhibition of the CS response to skin sensitization with DNFB induced by DC transfer can be overcome by a second DNFB challenge (data not shown). Second, class I-/II+ DC are unable to inhibit the CS response to DNFB when injected 5 days before skin sensitization. Thus, class I-/II+ DC can prime or reactivate hapten-specific T cells but are unable to induce memory-tolerogenic T cells. Another possibility is that the CD4+ T cells could dampen the inflammatory reaction only during a transient state of activation, which is the case in the coinjection experiments but which is not the case when CD4+ T cell activation has been achieved 5 days before.
The mechanisms underlying down-regulation of inflammatory reactions are
not well understood, although a role for Th2 cytokines, especially IL-4
and IL-10, has been demonstrated in the dampening of various DTH
reactions (42, 43, 44) including CS (45, 46, 47). Xu et al. recently reported
that hapten-specific CD4+ T cells produce IL-4 and IL-10,
whereas CD8+ T cells produce IFN- (48). This is in line
with our observation that hapten-primed CD4+ T cells, which
proliferate upon in vitro restimulation with the specific hapten,
produce IL-4 (data not shown). It is thus tempting to speculate that
the regulatory effect of CD4+ T cells is mediated by the
synthesis of anti-inflammatory cytokines in the lymph nodes and/or
in the skin, upon activation through presentation of the hapten by MHC
class II molecules. Alternatively, killing of effector T cells by
CD4+ T cells through a Fas-dependent mechanism could
contribute to control the induction and/or the expression of CS. In
this respect, Fas-L+ CD4+ T cells were recently
reported to induce apoptosis in Fas+ CD8+ T
cells in vitro (49). The precise mechanisms involved in the control of
both the afferent and the efferent limb of CS are currently under
investigation.
Our data provide evidence that the hapten DNFB can be presented independently by both MHC class I and class II molecules, raising the question of the mechanisms by which a foreign molecule could be taken up by DC and processed for T cell priming. Haptens can bind to either extracellular or cell surface proteins that are internalized and processed into peptides in the endosomal/lysosomal compartments where they bind to the MHC class II groove. Several mechanisms may contribute to hapten binding to class I. The hydrophobic liposoluble hapten may penetrate through the cell membranes allowing binding to self proteins in the cytoplasm (50, 51), which can then be processed through class I. Alternatively, haptens may bind to extracellular proteins, which may have a particulate conformation adequate for uptake in phagolysosomes and may subsequently be processed through class I (52). This possibility seems unlikely for DNFB, which binds covalently to amino acids residues. Finally, since haptens can covalently bind to discrete AA residues (lysine residues for DNFB), it is also possible that part of the hapten may bind directly to peptides within the grooves of MHC class I and class II molecules (50). Our data showing that DC can present haptenated peptides on both Ag-presenting pathways confirm previous observations in allergic contact dermatitis to urushiol (50) and are in line with recent studies that emphasize the property of mature DC to present exogenous Ags on both MHC class I and MHC class II molecules (53, 54).
In conclusion, our study indicates that DC are instrumental in the priming of both MHC class I-restricted CD8+ effector cells and MHC class II-restricted CD4+ down-regulatory cells, concurrently and independently.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Jean-François Nicolas, INSERM U. 80, Faculté Lyon Laennec, rue Guillaume Paradin, 69372 Lyon Cedex 08, France. E-mail address:
3 Abbreviations used in this paper: DC, dendritic cell; CS, contact sensitivity; DNFB, dinitrofluorobenzene; LC, Langerhans cell; TNP, trinitrophenyl; DNBS, 2,4-dinitrobenzenesulfonic acid; TNBS, 2,4,6-trinitrobenzenesulfonic acid; DTH, delayed type hypersensitivity; EC, epidermal cell; BM-DC, bone marrow-derived dendritic cells; GM-CSF, granulocyte/macrophage colony-stimulating factor.
Received for publication July 17, 1997. Accepted for publication October 21, 1997.
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), 5.105 (
),
106 (
) of DNBS-derivatized or unmodified (
)
BM-derived I-/II+ DC were administered s.c. to
syngeneic C57BL/6 mice at the time of epicutaneous DNFB sensitization
(day -5) (A) or 24 h before challenge
(day -1) (B). The CS response was determined
at various time points after ear challenge with DNFB. Controls include
normal unsensitized C57BL/6 mice receiving DNFB on the ear only (•).
The data represent ear swelling (mean ± SD) of five mice per
group and are representative of four experiments.