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*
Division of Dermatology, Sunnybrook and Women’s College Health Science Centre, and
Amgen Institute, Departments of Medical Biophysics and Immunology, University of Toronto, Toronto, Ontario, Canada; and
Department of Dermatology, University of Tsukuba, Tsukuba, Ibaraki, Japan
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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, indicating
that both CD4+ and CD8+ T cells are able to
secrete IFN-. LNCs from CD8, but not CD4, KO mice were able to
produce IL-4 and IL-10, suggesting that IL-4 and IL-10 are mainly
derived from CD4+ T cells. Intracellular cytokine staining
of LNCs confirmed that IFN--positive cells consisted of
CD4+ (Th1) and CD8+ (type 1 cytotoxic T) T
cells, whereas IL-10-positive cells were exclusively CD4+
(Th2) T cells. Collectively, these results suggest that both
CD4+ Th1 and CD8+ type 1 cytotoxic T cells are
crucial effector cells in CHS responses to dinitrofluorobenzene and
oxazolone in C57BL/6 mice. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and IL-2, thereby initiating the cutaneous
inflammatory reaction (efferent phase) (4, 6).
Traditionally, it is believed that CHS represents the prototype of
delayed-type hypersensitivity, which is mediated by
CD4+ T cells and down-regulated by
CD8+ T cells. Circumstantial evidence supporting
this view has come from a number of studies. First, in nickel-allergic
patients, the presence of nickel-specific CD4+
Th1 cells has been demonstrated in the peripheral blood and skin
(7, 8, 9, 10). Additionally, it has been shown that T cell lines
from FITC-sensitized mice possess a CD4+ Th1-like
phenotype, producing large amounts of IFN- (11). In
vivo depletion of CD4+, but not
CD8+, T cells significantly inhibited CHS
responses to dinitrofluorobenzene (DNFB) (12, 13). In
vitro depletion of CD4+, but not
CD8+, T cells from LN cells (LNCs) resulted in a
loss of CHS transfer (14). In vitro positively selected
CD4+ T cells have been shown to be capable of
adoptively transferring CHS to naive mice (15). Priming of
naive T cells in vitro with haptenated LCs could induce
CD4+ hapten-specific T cells that mediate CHS
following injection into naive syngeneic mice (16). CD4
knockout (KO) mice mounted a reduced CHS response (17).
CD4+ T cells positively selected from
hapten-activated LNCs secreted significant amounts of IFN-, whereas
CD8+ T cells produced only very low or
undetectable levels of this cytokine (18).
Contrary to the traditional understanding of CHS, however, some studies
suggest that CHS responses are mediated by CD8+ T
cells and down-regulated by CD4+ T cells. For
example, clones of CD8+ T cells have been
isolated from lesional skin of patients with allergy to urushiol
(19). Nickel-specific CD8+ T cells
have been detected in patients with allergy to nickel, and these cells
possess a type 1 cytotoxic T (Tc1) cell phenotype (20). In
vivo depletion of CD8+ T cells resulted in
reduction of CHS responses to DNFB and dimethylbenzanthracene
(13, 21), whereas deletion of CD4+ T
cells led to an exaggeration of the CHS response to DNFB
(21). MHC class I-deficient mice were unable to mount a
CHS response to DNFB, whereas MHC class II-deficient mice developed an
exaggerated CHS response (22). The enhanced CHS responses
in MHC class II-deficient mice were decreased by treatment with
anti-CD8 Ab, or by injection of wild-type (WT)
CD4+ T cells (23). MHC class
I+/II- dendritic cells
(DCs) induced hapten-specific immune responses in vitro and in vivo
(24), whereas MHC class
I-/II+ DCs down-regulated
the CHS response (25). T cells in the skin-draining LNs of
hapten-sensitized mice showed polarized patterns of cytokine
production, with IFN--secreting CD8+ T cells
and IL-4/IL-10-secreting CD4+ T cells
(26).
In the present study, we examined CHS responses in gene KO mice deficient in CD4+ or CD8+ T cells to directly clarify the requirement for the two T cell subsets in CHS. In addition, we depleted CD8+ T cells from CD4 KO mice or CD4+ T cells from CD8 KO mice in vivo using respective Abs, and then examined alterations of the CHS response. To further elucidate the effector role of CD4+ vs CD8+ T cells in CHS, we performed adoptive transfer experiments using LNCs lacking CD4+ T cells or CD8+ T cells, or both T cell subsets. Finally, we determined cytokine production patterns in LNCs of hapten-sensitized mice by ELISA and intracellular cytokine staining. Our data suggest that both CD4+ Th1 and CD8+ Tc1 cells significantly contribute to the full development of CHS responses to DNFB and oxazolone (OXZ) in C57BL/6 mice.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The CD4 KO and CD8 KO mice were generated by gene disruption, and the mutation was interbred into a C57BL/6 background, as described previously (27, 28). These mutant mice were maintained under a specific pathogen-free environment. C57BL/6 mice were obtained from the Charles River Breeding Laboratories (Saint Constant, Quebec, Canada) and used as a WT control. All mice were used at 8–12 wk of age. Each experimental group consisted of four to six mice. Animal protocols were approved by the Institutional Animal Care and Use Committee.
Reagents and Abs
DNFB and OXZ were purchased from Sigma (St. Louis, MO). Rat
anti-mouse Ia, anti-CD4 (clone YTS 191.1.2), anti-CD8
(clone YTS 169.4), anti-CD4/PE, anti-CD4/FITC,
anti-CD8/FITC, goat anti-rat IgG/PE, goat anti-rat
IgG/FITC, goat anti-rat IgG/biotin, streptavidin/FITC, NLDC-145,
and isotype controls were purchased from Cedarlane (Hornby, Ontario,
Canada). Rat anti-mouse IFN- (XMG1.2), anti-IL-10
(JES5-2A5), rat IgG1 isotype control (R3-34), anti-IFN-/PE,
anti-IL-10/PE, anti-CD32/CD16 (2.4G2, Fc block), and hamster
anti-mouse CD3
(145-2C11) were purchased from PharMingen Canada
(Mississauga, Ontario, Canada).
Immunolabeling of epidermal sheets
The density of epidermal LCs was examined by anti-Ia in situ immunolabeling, as previously described (29). Epidermal sheets were obtained from mouse ears using 0.5 M of ammonium thiocyanate, and labeled with anti-Ia Ab in a three-step immunolabeling procedure. The Ia+ cells (LCs) were counted from coded samples using a micrometer grid.
Assay for CHS
Induction of CHS was conducted using the methods described previously (30, 31). The shaved mouse abdomen skin was painted with 25 µl of 0.5% DNFB in acetone/olive oil (4:1) or 150 µl of 3% OXZ in alcohol/acetone (3:1). Five days later, mice were challenged with 10 µl of 0.2% DNFB or 1% OXZ on each side of the right ear. As a control, the left ear was painted with an identical amount of vehicle. The ear thickness was measured at 24, 48, and 72 h after challenge. Results were expressed as net ear swelling, which was calculated by subtracting the thickness of the vehicle-treated ear from the thickness of the hapten-challenged ear. The percentage response was calculated according to the following formula: response = (net ear swelling in KO mice/net ear swelling in WT mice) x 100%.
In vivo Ab-blocking studies
Immediately before DNFB challenge, mice were treated with either
anti-IFN- Ab (XMG1.2) at a dose of 20 µg or anti-IL-10 Ab
(JES5-2A5) at a dose of 40 µg in 40 µl PBS. The neutralizing Abs
were injected intradermally into one side of each ear of the mouse
using a 30-gauge needle under pentobarbital anesthesia. As a control,
an equal volume and concentration of nonimmune rat IgG1 (R3-34) were
injected into the ear of the control groups. It has been shown that up
to 40 µg of rat IgG induces no change in ear swelling
(32).
In vivo depletion of T cell subpopulations
For depletion of CD4+ T cells from CD8 KO mice, 100 µg of anti-CD4 Ab (YTS 191.1.2) was injected i.p. for 3 consecutive days. For depletion of CD8+ T cells from CD4 KO mice, rat anti-CD8 Ab (YTS 169.4) was employed. This Ab treatment has been shown to eliminate >95% of the targeted T cell subpopulation (21, 26). The mice were sensitized with DNFB the day following the last injection, and then challenged 5 days later, as described above.
Preparation of LNCs
Skin-draining LNs (i.e., axillary and inguinal LNs) were collected from sensitized mice (5 days post-DNFB sensitization) and naive mice, and single cell suspensions were prepared. Briefly, LNs were gently disrupted by rubbing between the frosted ends of two microscope slides, and then filtered through a nylon mesh cell strainer. LNCs were washed with HBSS and resuspended in complete RPMI 1640 medium (33).
Isolation of migratory hapten-modified LCs
The migratory hapten-modified LCs were isolated from LNCs using metrizamide gradient, as described by Bigby et al. (34). Briefly, WT mice were painted with 0.5% DNFB, and 24 h later LNCs were obtained. LNC suspensions (5 ml) at 5 x 106 cells/ml were layered onto 2 ml of 14.5% metrizamide and gradient centrifuged at 1200 rpm for 15 min. Cells at the interface were collected. Consistent with previous reports, microscopic examination demonstrated that 50–80% of this fraction was Ia+/DEC-205+ DCs (34, 26).
Preparation of CD4+ T cell and CD8+ T cell double-deficient LNCs
To prepare CD4+ T cell and CD8+ T cell double-deficient LNCs, either CD4+ T cells were depleted in vitro from the CD8 KO LNCs or CD8+ T cells were depleted from the CD4 KO LNCs. The CD8 KO and CD4 KO LNCs were suspended at 2.5 x 107 cells/ml with 25 µg/ml of anti-CD4 or anti-CD8 Ab, respectively, for 60 min on ice. After washing, the cells were resuspended at 2.5 x 107 cells/ml in a 1/9 dilution of Low-Tox-M rabbit complement (Cedarlane) in HBSS/10% FCS and incubated at 37°C for 60 min. These cells were then washed extensively before use in culture or adoptive transfer.
FACS analysis of LNCs
Anti-CD4/PE vs anti-CD8/FITC double staining was performed in LNCs. Briefly, LNCs were preincubated with anti-CD32/CD16 for 5 min, and then incubated with anti-CD4/PE for 30 min on ice. After washing, cells were incubated with anti-CD8/FITC for 30 min. The cells were subjected to FACS analysis. To confirm the efficiency of in vivo depletion of CD8+ T cells in CD4 KO mice, LNCs were first incubated with rat anti-mouse CD8, then reacted with goat anti-rat IgG/FITC, and finally labeled with anti-CD4/PE. To confirm the efficiency of in vivo depletion of CD4+ T cells in CD8 KO mice, LNCs were first incubated with rat anti-mouse CD4 and then reacted with goat anti-rat IgG/PE, and finally labeled with anti-CD8/FITC.
Adoptive transfer of CHS immune response
Four groups of LNCs were prepared from donor mice (5 days post-DNFB sensitization): CD4+ T cell deficient, CD8+ T cell deficient, CD4+ T cell and CD8+ T cell double deficient, and WT LNCs. Naive WT mice were used as recipient mice. LNC suspensions (5 x 107 cells/200 µl HBSS) were injected i.v. into each recipient mouse via the tail vein. The recipient mice were challenged 1 h later by epicutaneous application of DNFB on the ear, and ear swelling was evaluated 24 h later.
Quantitation of in vitro cytokine production
LNCs obtained from sensitized mice (5 days post-DNFB
sensitization) and naive mice were stimulated with either plate-bound
anti-CD3 or hapten-modified LCs. For anti-CD3 stimulation,
96-well U-bottom plates were precoated with hamster anti-mouse
CD3 (25 µg/ml), or hamster IgG isotype control for 90 min at
37°C. LNCs were seeded at 5 x 105
cells/200 µl/well, and incubated at 37°C for 48 h. For
stimulation with LCs, 5 x 105 LNCs were
cocultured with 104 LCs in 200 µl/well for
48 h (26). Next, culture supernatants were collected,
and the concentrations of IFN-, IL-4, and IL-10 proteins were
quantitated by a sandwich ELISA using ELISA kits (Genzyme, Cambridge,
MA) (35). Absorbance was read at 450 nm. Each supernatant
was analyzed in duplicate, and each experiment was repeated three
times.
Intracellular cytokine staining
The frequency of IFN-- or IL-10-producing cells in the LNs of
DNFB-sensitized or naive C57BL/6 mice was determined using
intracellular cytokine staining with Cytofix/Cytoperm Plus kits
(PharMingen) (36, 37). LNCs were cultured in 96-well
U-bottom plates precoated with anti-CD3 for 24 h, and 2
µM monensin was added for the final 5 h. As a control, LNCs were
cultured in media alone. The cells were harvested, preincubated with
anti-CD32/CD16, and then stained for the cell surface Ag by
incubation with anti-CD4/FITC or anti-CD8/FITC for 30 min on
ice. After washing, cells were fixed with 2% paraformaldehyde,
permeabilized with 0.5% saponin, and subsequently stained for
intracellular cytokines by incubation with anti-IFN-/PE or
anti-IL-10/PE, according to the instructions of the manufacturer.
As a control, the fixed/permeabilized cells were preincubated with
unconjugated anti-IFN- or anti-IL-10 before staining. The
percentage of cytokine-positive cells was determined by FACScan flow
cytometry.
Statistical analysis
A minimum of three experiments was performed for each assay. All data are expressed as the mean and SEM. The statistical significance of differences between the means was determined by applying a two-tailed Student’s t test. A difference was considered statistically significant with p < 0.05.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To determine the requirement for the two T cell subpopulations in
CHS responses, we performed CHS assays using gene KO mice lacking
CD4+ or CD8+ T cells.
Before performing the CHS assay, we examined the density of LCs in the
epidermis, and demonstrated that normal numbers of LCs were present in
CD4 KO and CD8 KO mice (data not shown). Mice were sensitized with DNFB
or OXZ, and challenged 5 days later. Ear-swelling responses to DNFB and
OXZ were significantly depressed in CD4 KO mice and CD8 KO mice, as
compared with WT mice (Fig. 1
). The
depression of CHS response was more significant in CD8 KO mice than CD4
KO mice (p < 0.05). Moreover, the duration of
the CHS response was shortened in CD4 KO and CD8 KO mice. Ear-swelling
responses returned to baseline in CD4 KO and CD8 KO mice 72 h
after challenge, whereas an obvious ear swelling remained in WT mice at
this time.
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The demonstration that CHS response was depressed, but not
completely abrogated in the absence of CD4+ or
CD8+ T cells alone raised the possibility that
either T cell subset could function as effector cells for CHS. To
further address this issue, we in vivo depleted either
CD8+ T cells from CD4 KO mice or
CD4+ T cells from CD8 KO mice using respective
Abs, and then examined CHS responses. FACS analysis on LNCs confirmed
that treatment with Ab for 3 consecutive days resulted in elimination
of >95% of the targeted T cell subsets (Fig. 2). The resulting
CD4+ T cell and CD8+ T cell
double-deficient mice were virtually unable to mount hapten-specific
ear-swelling responses (Fig. 3).
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Ab suppresses CHS in CD4 KO and CD8 KO mice, whereas
anti-IL-10 Ab enhances CHS in CD8, but not in CD4, KO mice
Before performing Ab-blocking studies in CD4 KO and CD8 KO mice,
dose titration tests were performed in WT mice. We found that CHS
responses were significantly suppressed by anti-IFN- Ab, and
enhanced by anti-IL-10 Ab in WT mice in a dose-dependent manner.
Maximal effects were obtained when 20 µg of anti-IFN- or 40
µg of anti-IL-10 Ab was injected (data not shown). When CD4 KO or
CD8 KO mice were injected with 20 µg of anti-IFN- Ab, CHS
responses were reduced by 
50% at 24 h, and abrogated
completely at 48 h in both CD4 KO and CD8 KO mice. On the other
hand, injection of 40 µg of anti-IL-10 Ab resulted in a
significant enhancement of CHS in CD8, but not in CD4, KO mice (Fig. 4).
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To further elucidate the effector role of
CD4+ vs CD8+ T cells in
CHS, we performed adoptive transfer experiments. The donor cells
consisted of four groups of DNFB-primed LNCs:
CD4+ T cell deficient, CD8+
T cell deficient, CD4+ T cell and
CD8+ T cell double deficient, and WT LNCs. These
LNCs were separately injected into recipient naive C57BL/6 mice and
then DNFB was painted onto the ear. As shown in Fig. 5, WT, CD4+ T
cell-deficient and CD8+ T cell-deficient LNCs
were all able to induce a CHS response. However, the capacity of
transferring CHS was significantly lower for CD4+
T cell-deficient LNCs and CD8+ T cell-deficient
LNCs. Interestingly, transfer of CD4+ T cell and
CD8+ T cell double-deficient LNCs did not induce
CHS responses in naive mice.
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, whereas only CD8+, but not CD4+, T
cell-deficient LNCs secrete IL-4 and IL-10
To determine cytokine profiles in skin-draining LNs of CD4 KO and
CD8 KO mice, DNFB-primed LNCs were stimulated with immobilized
anti-CD3 or hapten-modified LCs. As shown in Fig. 6, significant amounts of IFN- were
detected from anti-CD3-stimulated CD4+ T
cell-deficient LNCs and CD8+ T cell-deficient
LNCs, implicating that both CD8+ and
CD4+ T cells were able to produce the type 1
cytokine. In contrast, IL-4 and IL-10 were only detected from
CD8+, but not from CD4+, T
cell-deficient LNCs, suggesting that type 2 cytokines were exclusively
produced by CD4+ Th2 cells. Significant amounts
of IFN- or IL-4/IL-10 were not detected from the naive LNCs, or the
DNFB-primed LNCs stimulated with control hamster IgG. Similarly, when
hapten-modified LCs were used as Ag-specific stimuli, both CD4 KO LNCs
and CD8 KO LNCs produced IFN-, whereas CD8 KO, but not CD4 KO, LNCs
produced IL-4 and IL-10 (Fig. 7).
However, the amounts of cytokines induced by LCs were considerably
lower compared with those induced by immobilized anti-CD3.
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-positive cells consist of CD4+
Th1 and CD8+ Tc1 cells, whereas IL-10-positive cells are
exclusively CD4+ Th2 cells
To confirm the cellular source of cytokines and determine the
frequency of cytokine-secreting cells in LNCs, we performed
intracellular cytokine staining. After 24 h of culturing LNCs in
media alone, significant numbers of cytokine-positive cells were not
detected from naive or DNFB-primed LNCs. However, following stimulation
of DNFB-primed LNCs with immobilized anti-CD3, significant
numbers of CD4+ and CD8+ T
cells were shown to be IFN- positive. In addition, a significant
number of IL-10-positive cells were detected from DNFB-primed
CD4+, but not CD8+, T cells
(Table I). In control experiments, when
LNCs were preincubated with unconjugated anti-IFN- or
anti-IL-10 Ab, no positively labeled cells were detected. As shown
in Fig. 8, in a representative
experiment, 9.4% of CD4+ T cells (or 3.2% of
total LNCs) and 21.4% of CD8+ T cells (or 4.1%
of total LNCs) were IFN- positive, and 8.2% of
CD4+ T cells (or 2.9% of total LNCs) were IL-10
positive. However, significant amounts of IL-10-positive cells were not
detected from the CD8+ T cells.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Over the last two decades, a large number of studies have focused on
the respective role of CD4+ vs
CD8+ T cells in CHS; however, conflicting results
have been obtained (Table II). The
discrepancy may be due to the great diversity in CHS responses. CHS in
mice is genetically controlled and differs considerably among strains
having different alleles at the H-2 loci. The response is
profoundly modified by non-MHC genes as well. In addition to mouse
genetic background, CHS responses vary depending on the hapten
(40). Moreover, different types of allergens may even
result in qualitatively different immune responses characteristic of
selective Th1 and Th2 activation, respectively. Contact allergens such
as DNFB, OXZ, and 2,4-dinitrochlorobenzene (DNCB) preferentially induce
Th1-predominant responses, whereas respiratory allergens such as
trimellitic anhydride induce Th2-predominant responses
(41, 42, 43). Exceptionally, the contact allergen FITC may
also induce a Th2 response (43).
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production and 2) production
of the down-regulatory Th2 cytokines IL-4 and IL-10 in these mutant
mice. Nevertheless, our data suggest that both
CD4+ T cells and CD8+ T
cells play an effector role in CHS responses. Our adoptive transfer
experiments using LNCs from CD4 KO and CD8 KO mice support this notion.
The experimental results revealed that both the
CD4+ T cell-deficient LNCs and
CD8+ T cell-deficient LNCs had a decreased
capacity of transferring CHS. Additionally, CD4+
T cell and CD8+ T cell double-deficient LNCs were
unable to transfer CHS at all. Although CHS responses were depressed in mutant mice deficient in CD4+ or CD8+ T cells, the ear-swelling response was not completely lost. Therefore, we hypothesized that CD8+ T cells may mediate the remaining CHS response seen in CD4 KO mice, and CD4+ T cells may be responsible for the CHS responses seen in CD8 KO mice. To test this hypothesis, we performed in vivo CD4+ or CD8+ T cell depletion studies. Abs were used to remove CD4+ T cells from CD8+ KO mice or CD8+ T cells from CD4 KO mice. We found that the CHS response was virtually abolished in these CD4+ T cell and CD8+ T cell double-deficient mice. These results suggest that both CD4+ T cells and CD8+ T cells contribute to the full development of CHS responses.
Previous studies using gene KO mice and neutralizing Abs have
demonstrated that cytokines play important effector and regulatory
roles in CHS responses. The type 1 cytokine IFN- promotes CHS,
whereas the type 2 cytokines, IL-10 and to a lesser extent IL-4,
down-regulate CHS responses (44, 45, 46, 47). Our in vivo
Ab-blocking studies in CD4 KO and CD8 KO mice support this notion.
Administration of anti-IFN- Ab before challenge resulted in a
reduction of CHS in both CD4 KO and CD8 KO mice, whereas anti-IL-10
Ab significantly enhanced ear swelling in CD8, but not in CD4, KO
mice.
It has been demonstrated that type 1 cytokines can be produced by
CD4+ Th1 cells as well as
CD8+ Tc1 cells, whereas type 2 cytokines can be
derived from Th2 and Tc2 cells (48, 49). Determination of
the cellular sources of IFN- and IL-4/IL-10 in the hapten-primed
LNCs was very helpful for determining the relative role of T cell
subpopulations in CHS responses. Several groups have examined the
cytokine profiles in LNCs of mice following hapten sensitization. Xu et
al. (26) demonstrated that in vivo depletion of
CD8+ T cells in mice resulted in a decrease of
IFN- production by DNFB-primed LNCs, whereas depletion of
CD4+ T cells led to an increasing IFN-
secretion. This suggests that IFN- is mainly produced by
CD8+ Tc1 cells. In contrast, Moussavi et al.
(18) found that CD4+ T cells
positively selected from DNCB-primed mouse LNCs produced IFN-,
whereas CD8+ T cells secreted only very low or
undetectable levels of IFN-. This discrepancy may be accounted for
by the differences in the experimental protocol. For example, in one
case DNFB was used as the contact sensitizer, while in the other case
DNCB was used. Moreover, Ab treatments are not able to completely
remove all CD4+ or CD8+ T
cells, and may even trigger other responses from T cells.
In the present study, we examined cytokine production patterns
utilizing LNCs from CD4 KO and CD8 KO mice, which are completely
deficient in CD4+ or CD8+ T
cells. LNCs were stimulated with either immobilized anti-CD3 or
hapten-modified LCs. ELISA analysis revealed that both
CD4+ T cell-deficient LNCs and
CD8+ T cell-deficient LNCs were capable of
producing IFN-, and that CD8+, but not
CD4+, T cell-deficient LNCs could produce IL-4
and IL-10. These results suggest that IFN- is derived from both
CD4+ T cells and CD8+ T
cells, while IL-4 and IL-10 are produced only by
CD4+ T cells. Although the observation that type
2 cytokines are exclusively derived from CD4+ T
cells in LNCs is consistent with the data of Xu et al.
(26), the results concerning the main cellular source of
type 1 cytokine are different. One explanation for this discrepancy is
that different sensitization protocols were employed. Instead of two
daily DNFB paintings, we used a single painting for DNFB sensitization
(50, 51).
To confirm the observations from the ELISA study, we performed
intracellular cytokine staining on the DNFB-primed LNCs from WT mice.
Since the quantities of cytokines induced by hapten-modified LCs were
considerably lower compared with those induced by immobilized
anti-CD3, we used anti-CD3-stimulated LNCs for intracellular
cytokine staining. Consistently, Xu et al. (26)
demonstrated that anti-TCR or anti-CD3 Ab was able to stimulate
LNCs to produce higher levels of cytokines than LCs. Furthermore,
Matyszak et al. (52) recently demonstrated that the amount
of cytokines produced during primary Ag-specific immune responses was
very small, which could be enhanced by restimulation with anti-CD3.
They also demonstrated (by intracellular staining) that none or very
little IFN--, IL-4-, or IL-10-positive T cells was detected from
DC-stimulated Ag-specific CD4+ T cells in the
absence of anti-CD3. However, when restimulated with anti-CD3,
significant amounts of cytokine-positive T cells were detected. Our
intracellular cytokine staining confirmed that immobilized anti-CD3
was able to induce significant amount of cytokine-producing cells in
LNCs. We demonstrated that IFN--positive cells in the skin-draining
LNs of sensitized mice consisted of both CD4+
(Th1) T cells and CD8+ (Tc1) T cells, whereas
IL-10-positive cells were exclusively CD4+ (Th2)
T cells.
Collectively, the present study provides evidence that both CD4+ T cells and CD8+ T cells are required for the full development of CHS. CD4+ Th1 and CD8+ Tc1 cells are both effector cells, while CD4+ Th2 cells are negative regulatory cells for the CHS responses to DNFB and OXZ in C57BL/6 mice.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Daniel N. Sauder, Division of Dermatology, Sunnybrook and Women’s College Health Science Center, University of Toronto, 2075 Bayview Avenue, Toronto, Ontario, Canada M4N 3 M5.
3 Abbreviations used in this paper: CHS, contact hypersensitivity; DC, dendritic cell; DNCB, 2,4-dinitrochlorobenzene; DNFB, dinitrofluorobenzene; KO, knockout; LC, Langerhans cell; LN, lymph node; LNC, LN cell; OXZ, oxazolone; Tc, cytotoxic T; WT, wild type.
Received for publication January 27, 2000. Accepted for publication September 15, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-producing (Tc1) effector CD8+ T cells and interleukin (Il) 4/IL-10-producing (Th2) negative regulatory CD4+ T cells. J. Exp. Med. 183:1001.RI. J. Exp. Med. 175:1353. production in Th1 CD4+ T cells: evidence for two distinct pathways for promoter activation. Eur. J. Immunol. 29:548.[Medline]
receptor 2 gene results in severe immune defects in mice. Proc. Natl. Acad. Sci. USA 95:8233.-producing CD8+ 
+ T lymphocytes and IL-2-producing CD4+ 
ß+ T lymphocytes during contact sensitivity. J. Immunol. 158:2567.[Abstract]
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 J. M. Escandell, M. C. Recio, S. Manez, R. M. Giner, M. Cerda-Nicolas, R. Gil-Benso, and J.-L. Rios Dihydrocucurbitacin B Inhibits Delayed Type Hypersensitivity Reactions by Suppressing Lymphocyte Proliferation J. Pharmacol. Exp. Ther., September 1, 2007; 322(3): 1261 - 1268. [Abstract] [Full Text] [PDF]
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M. Matsumoto, A. Shigeta, Y. Furukawa, T. Tanaka, M. Miyasaka, and T. Hirata CD43 Collaborates with P-Selectin Glycoprotein Ligand-1 to Mediate E-Selectin-Dependent T Cell Migration into Inflamed Skin J. Immunol., February 15, 2007; 178(4): 2499 - 2506. [Abstract] [Full Text] [PDF]
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K. Ishizaki, A. Yamada, K. Yoh, T. Nakano, H. Shimohata, A. Maeda, Y. Fujioka, N. Morito, Y. Kawachi, K. Shibuya, et al. Th1 and Type 1 Cytotoxic T Cells Dominate Responses in T-bet Overexpression Transgenic Mice That Develop Contact Dermatitis J. Immunol., January 1, 2007; 178(1): 605 - 612. [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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 W.-K. Suh, S. Wang, G. S. Duncan, Y. Miyazaki, E. Cates, T. Walker, B. U. Gajewska, E. Deenick, W. Dawicki, H. Okada, et al. Generation and Characterization of B7-H4/B7S1/B7x-Deficient Mice. Mol. Cell. Biol., September 1, 2006; 26(17): 6403 - 6411. [Abstract] [Full Text] [PDF]
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 K. Viswanathan, C. Daugherty, and F. S. Dhabhar Stress as an endogenous adjuvant: augmentation of the immunization phase of cell-mediated immunity Int. Immunol., August 1, 2005; 17(8): 1059 - 1069. [Abstract] [Full Text] [PDF]
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D. A. Carlow, M. J. Williams, and H. J. Ziltener Inducing P-Selectin Ligand Formation in CD8 T Cells: IL-2 and IL-12 Are Active In Vitro but Not Required In Vivo J. Immunol., April 1, 2005; 174(7): 3959 - 3966. [Abstract] [Full Text] [PDF]
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K. Takeshita, T. Yamasaki, S. Akira, F. Gantner, and K. B. Bacon Essential role of MHC II-independent CD4+ T cells, IL-4 and STAT6 in contact hypersensitivity induced by fluorescein isothiocyanate in the mouse Int. Immunol., May 1, 2004; 16(5): 685 - 695. [Abstract] [Full Text] [PDF]
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S. C. Barlow, H. Xu, C. T. Weaver, J. R. Lindsey, T. R. Schoeb, and D. C. Bullard Development of dermatitis in CD18-deficient PL/J mice is not dependent on bacterial flora, and requires both CD4+ and CD8+ T lymphocytes Int. Immunol., February 1, 2004; 16(2): 345 - 351. [Abstract] [Full Text] [PDF]
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S. Nakae, A. Nambu, K. Sudo, and Y. Iwakura Suppression of Immune Induction of Collagen-Induced Arthritis in IL-17-Deficient Mice J. Immunol., December 1, 2003; 171(11): 6173 - 6177. [Abstract] [Full Text] [PDF]
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 H. V. L. N. Swamy, T. K. Smith, E. J. MacDonald, N. A. Karrow, B. Woodward, and H. J. Boermans Effects of feeding a blend of grains naturally contaminated with Fusarium mycotoxins on growth and immunological measurements of starter pigs, and the efficacy of a polymeric glucomannan mycotoxin adsorbent J Anim Sci, November 1, 2003; 81(11): 2792 - 2803. [Abstract] [Full Text] [PDF]
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T. Hirata, B. C. Furie, and B. Furie P-, E-, and L-Selectin Mediate Migration of Activated CD8+ T Lymphocytes into Inflamed Skin J. Immunol., October 15, 2002; 169(8): 4307 - 4313. [Abstract] [Full Text] [PDF]
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B. Wang, C. Feliciani, B. G. Howell, I. Freed, Q. Cai, H. Watanabe, and D. N. Sauder Contribution of Langerhans Cell-Derived IL-18 to Contact Hypersensitivity J. Immunol., April 1, 2002; 168(7): 3303 - 3308. [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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D. A. Carlow, S. Y. Corbel, M. J. Williams, and H. J. Ziltener IL-2, -4, and -15 Differentially Regulate O-Glycan Branching and P-Selectin Ligand Formation in Activated CD8 T Cells J. Immunol., December 15, 2001; 167(12): 6841 - 6848. [Abstract] [Full Text] [PDF]
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 B. Wang, C. Feliciani, I. Freed, Q. Cai, and D. N. Sauder Insights into molecular mechanisms of contact hypersensitivity gained from gene knockout studies J. Leukoc. Biol., August 1, 2001; 70(2): 185 - 191. [Abstract] [Full Text] [PDF]
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N. Yawalkar, R. E. Hunger, C. Buri, S. Schmid, F. Egli, C. U. Brand, C. Mueller, W. J. Pichler, and L. R. Braathen A Comparative Study of the Expression of Cytotoxic Proteins in Allergic Contact Dermatitis and Psoriasis : Spongiotic Skin Lesions in Allergic Contact Dermatitis Are Highly Infiltrated by T Cells Expressing Perforin and Granzyme B Am. J. Pathol., March 1, 2001; 158(3): 803 - 808. [Abstract] [Full Text] [PDF]
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