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*
Department of Immunology, Cleveland Clinic Foundation, Cleveland, OH 44195; 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
CD8+ T cells, whereas CD4+ T cells regulate the
magnitude and duration of the response. The requirement for CD40-CD154
engagement during CD8+ and CD4+ T cell priming
by hapten-presenting Langerhans cells (hpLC) is undefined and was
tested in the current study. Similar CHS responses to DNFB were
elicited in wild-type and CD154-/- animals. DNFB
sensitization of CD154-/- mice primed IFN--producing
CD8+ T cells and IL-4-producing CD4+ T cells.
However, anti-CD154 mAb MR1 given during hapten sensitization
inhibited hapten-specific CD8+, but not CD4+, T
cell development and the CHS response to challenge. F(ab')2
of MR1 failed to inhibit CD8+ T cell development and the
CHS response suggesting that the mechanism of inhibition is distinct
from that of CD40-CD154 blockade. Furthermore, anti-CD154 mAb did
not inhibit CD8+ T cell development and CHS responses in
mice depleted of CD4+ T cells or in CD4-/-
mice. During in vitro proliferation assays, hpLC from mice treated with
anti-CD154 mAb during DNFB sensitization were less stimulatory for
hapten-primed T cells than hpLC from either control mice or mice
depleted of CD4+ T cells before anti-CD154 mAb
administration. These results demonstrate that development of
IFN--producing CD8+ T cells and the CHS response are not
dependent on CD40-CD154 interactions. This study proposes a novel
mechanism of anti-CD154 mAb-mediated inhibition of CD8+
T cell development where anti-CD154 mAb acts indirectly through
CD4+ T cells to impair the ability of hpLC to prime
CD8+ T cells. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-producing cells induced by epicutaneous
sensitization with DNFB or oxazolone are hapten-specific
CD8+ T cells (12, 13). In contrast,
most of the hapten-specific CD4+ T cells develop
to type 2 cytokine (e.g., IL-4, IL-5, and IL-10)-producing cells.
CD4+ T cells also regulate the magnitude and
duration of the CHS response, although the mechanism of this regulation
remains undefined. In addition to recognition of specific hapten/MHC
complexes, development of the CD8+ effector and
the CD4+ regulatory T cell populations requires
engagement of B7-2 costimulatory molecules expressed by hpLC
(14). The requirement for other molecular interactions
between LC and hapten-specific T cells during priming for CHS remains
largely unknown. T cell engagement of the CD40 costimulatory molecule expressed by APCs is required for the development of many T cell responses. The ligand for CD40, CD154, is rapidly expressed on CD4+ T cells during cellular activation (15, 16). The use of CD154-/- mice has indicated that these interactions play a crucial role in the induction of CD4+ T cell-mediated immune responses and CD4+ T cell-dependent humoral responses (17, 18, 19). The requirement for CD40-CD154 interactions during the development of CD8+ T cell-mediated responses is not as clear as for CD4+ T cell-mediated responses. Whereas some studies have indicated the development of CD8+ T cell responses independently of CD40-mediated costimulation (20, 21, 22, 23), other studies have indicated that CD40-CD154 interactions are required for development of certain CD8+ T cell responses (24, 25).
Several recent studies have suggested that CD40-CD154 interactions might be required during the development of T cells mediating CHS responses (26, 27). This requirement has not been tested directly. In the present report we have tested the development of hapten-specific CD4+ and CD8+ T cell populations and the elicitation of CHS responses following DNFB sensitization of CD154-/- mice and in wild-type mice treated with anti-CD154 mAb during sensitization. The results indicate that CD8+ and CD4+ T cell development in CHS is not dependent on CD40-CD154 interactions. However, treatment with anti-CD154 mAb during sensitization inhibits the development of hapten-specific CD8+ T cells and the CHS response indirectly through CD4+ T cell-mediated effects. The results indicate a novel mechanism for the ability of therapeutic strategies using anti-CD154 mAb to inhibit CD8+ T cell responses.
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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 obtained through Dr. Clarence Reeder (National Cancer Institute, Frederick, MD). CD4-/- and CD154-/- mice on the C57BL/6 genetic background and IL4-/- 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
The following hybridomas were obtained from American Type
Culture Collection (Manassas, VA): GK1.5 (anti-mouse CD4), 2.43
(anti-mouse CD8), and 145.2C11 (anti-mouse CD3). mAb from the
hybridomas YTS191.1.2 (anti-mouse CD4) (28), KJ23a
(anti-V
17a) (29), and MR1 (anti-mouse CD154)
(30) were also used in these studies. mAb from the culture
supernatant of the IgG-producing hybridomas was purified by protein G
chromatography. PE-labeled mAbs specific for CD4 and CD8; capture and
detection mAbs for IL-4-, IL-10-, and IFN--specific ELISA; and
recombinant IFN-, IL-4, and IL-10 were purchased from PharMingen
(San Diego, CA). Polyclonal hamster IgG was purchased from Jackson
ImmunoResearch Laboratories (West Grove, PA).
F(ab')2 of MR1 were prepared using the ImmunoPure
Fab' Preparation Kit (Pierce, Rockford, IL). The digestion was
performed according to the manufacturer’s instructions, and the purity
of F(ab')2 was evaluated by SDS-PAGE. The
activity of F(ab')2 was tested by the ability to
inhibit allo-reactive responses of mouse lymph node cells (LNC) in
vitro. Purified MR1 was FITC labeled using the QuickTag FITC
conjugation kit according to the manufacturer’s instructions (Roche,
Indianapolis, IN).
Hapten sensitization and elicitation of CHS
Mice were sensitized to DNFB by painting the shaved abdomen with 25 µl of 0.25% DNFB (Sigma, St. Louis, MO) and with 5 µl on each footpad on days 0 and +1. On day +5 hapten-sensitized and control unsensitized mice were challenged with 10 µl of 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 expressed in units of 10-4 in. as previously described (12). The ear swelling response is presented as the mean increase of each group of four sensitized or nonsensitized mice (i.e., eight ears) ± SEM.
CD4+ and CD8+ T cell depletion
For in vivo depletion of CD4+ T cells mice were injected with 200 µg of each anti-CD4 mAb, YTS 191 and GK1.5, i.p. on 3 consecutive days before hapten sensitization on days 0 and +1 as previously described (11, 12, 28). For in vivo depletion of CD8+ T cells 200 µg of anti-CD8 mAb 2.43 was injected on 3 consecutive days. In each experiment treated sentinel mice were used to evaluate the efficiency of CD4+ or CD8+ T cell depletion by Ab staining and flow cytometric analysis of spleen and LNC and was always >95% compared with cells from control, rat IgG-treated mice. Treated mice were rested 1–3 days before sensitization with DNFB. For in vitro depletion of CD4+ or CD8+ T cells, LNC from hapten-sensitized mice were incubated with specific Ab-coated magnetic beads (Dynabeads, Dynal, Oslo, Norway). The efficiency of this depletion was >95% for the target T cell population.
Isolation of hpLC
The isolation of hpLC was performed using the method described by Bigby and coworkers (31). Mice were painted with 0.25% DNFB as described above, and LNC were prepared 24 after the final painting (i.e., day +2 postsensitization). The cells were washed with RPMI 1640, resuspended at 107 cells/ml in RPMI 1640 supplemented with 5% FCS, layered over 3 ml of 14.5% metrizamide (Sigma) in PBS, and centrifuged at 600 x g for 15 min. The interface cells were collected and washed twice with complete medium. Microscopic and flow cytometry examination of cells from the interface indicated 60–80% surface Ig-/class II MHC+ cells with dendritic cell (DC) morphology.
Cell culture
Cells from skin draining lymph nodes from DNFB-sensitized and,
as a negative control, from unsensitized mice were prepared on day +4
and stimulated to produce cytokines by culture on anti-CD3
mAb-coated wells. The 96-well U-bottom tissue culture plates were
precoated 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 not expressed by BALB/c or C57BL/6 T cells, anti-V17a
mAb KJ 23a. The wells were washed, and 2 x
105 LNC were delivered to each well in 200 µl
of complete RPMI medium and cultured in a 7% CO2
humidified incubator at 37°C. After 48 h the culture
supernatants were harvested and assayed for cytokine production by
ELISA.
T cell allo-reactivity was assayed by standard MLR. As stimulator cells, BALB/c spleen cells were treated with 50 µg/ml of mitomycin C (Sigma) for 30 min at 37°C and washed twice in RPMI 1640, then 106 cells in 100 µl of complete RPMI 1640 medium were delivered to wells of a 96-well tissue culture plate. Responder LNC from naive C57BL/6 mice were added in 100 µl of complete RPMI 1640 medium at 2 x 105 cells/well (E:T cell ratio, 1:5). BALB/c LNC cultured with the BALB/c stimulator spleen cells were used as a negative control. Control hamster IgG, anti-CD154 mAb MR1, or F(ab')2 of MR1 was added to cell cultures at concentrations of 0.5, 5, and 50 µg/ml. Cells were cultured for 4 days in complete RPMI 1640 and were pulsed with 1 µCi/well [3H]thymidine. Cells were harvested 24 h later onto fiber filter mats, and 3H incorporation was determined by liquid scintillation counting.
In experiments testing the proliferation of DNFB-immune T cells, cocultures of 2 x 105 CD4+ or CD8+ T cells and 2 x 104 hpLC given 2000 rad of gamma irradiation were established in triplicate in the wells of 96-well U-bottom culture plates. After 48 h cultures were pulsed with 1 µCi/well [3H]thymidine and harvested 18–20 h later, and 3H incorporation was determined by liquid scintillation counting.
Cytokine-specific ELISA
Polyvinylchloride ELISA plates were coated with capture
anti-IFN-, anti-IL-4, or IL-10 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 supernatants were tested
undiluted and in at least two dilutions. As a positive control, each
plate also included recombinant cytokine titrated to obtain a standard
curve for quantitation. Following incubation overnight at 4°C, each
plate was washed, and the biotin-labeled anti-cytokine mAb was
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 concentration of
cytokine in each test supernatant was calculated using the standard
curve on each plate.
Enzyme-linked immunospot (ELISPOT) assays for enumeration of
hapten-specific IFN-- and IL-4-producing cells
ELISPOT assays were performed as previously described (32, 33). Briefly, ELISPOT plates (Unifilter 350, Polyfiltronics,
Rockland, MA) were coated with 4 µg/ml IFN-- or IL-4-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 treated with control IgG or anti-CD154 mAb
were prepared on day +5 postsensitization and used as responder cells.
Syngeneic spleen cells from naive mice were treated with 50 µg/ml
mitomycin C and then labeled with 100 µg/ml DNBS before use as
stimulator cells as previously described (33, 34).
Responder and stimulator cells were cultured in serum-free HL-1 medium
(BioWhittaker, Walkersville, MD) supplemented with 1 mM
L-glutamine. Stimulator cells were plated at 5 x
105 cells/well with 2 x
105 responder cells/well. Responder cells plated
with unlabeled splenocytes were used as a negative (hapten-specificity)
control. After 24 h of cell culture at 37°C in 5%
CO2, cells were removed from the plate by
extensive washing with PBS/0.05% Tween 20. Biotinylated
anti-IFN- or anti-IL-4 mAb (4 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- or streptavidin-HRP for
IL-4 was added to each well. After 2 h at room temperature the
plates were washed with PBS/0.05% Tween-20, nitro blue
tetrazolium/5-bromo-4-chloro-30-indolyl substrate (Kirkegaard & Perry
Laboratories, Gaithersburg, MD) was added for detection of IFN-, and
3-amino-9-ethylcarbazole (Pierce) was added for detection of IL-4. The
resulting spots were counted with an ELISPOT image analyzer (developed
at Cellular Technology, Cleveland, OH) that was designed to detect
ELISA spots with predetermined criteria for size, shape, and
colorimetric density.
Stimulation and flow cytometric analysis of CD154 expression
LNC were prepared from naive mice or from hapten-sensitized mice
24, 48, or 72 h after sensitization and
CD4+- and CD8+-enriched
cell populations were prepared using Dynabeads. For anti-CD3
mAb-mediated activation, the cells were washed with RPMI 1640, and
2 x 105-cell aliquots were cultured in
96-well plates coated with anti-CD3 mAb 145.2C11 or, as a negative
control, anti-V17a mAb KJ23 for 16 h at 37°C. Following
the culture, the cells were washed twice with staining buffer
(Dulbecco’s PBS with 2% FCS/0.2% NaN3), and
5 x 105-cell aliquots were incubated on ice
in 100 µl of rat serum (Rockland, Gilbertsville, PA) diluted 1/1000
in the staining buffer. After 30 min the cells were washed twice and
stained with FITC-labeled anti-mouse CD154 mAb and PE-labeled
anti-mouse CD4 or CD8 mAb. After 30 min on ice the cells were
washed five times, resuspended in staining buffer, and analyzed by
two-color flow cytometry using a FACScan and CellQuest software (Becton
Dickinson, San Jose, CA). Sample data were collected on 10,000 ungated
cells. For analysis of CD154 expression, the entire
CD4+ or CD8+ T cell
population was gated, and the percentage of CD154-expressing T cells
was calculated by dividing the amount of positively stained gated cells
by the total number of gated cells.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-producing CD8+
and IL-4-producing CD4+ T cells to hapten sensitization in
CD154-/- mice
To begin to examine the requirement for CD40-CD154 interactions
for the induction and elicitation of CHS responses, wild-type C57BL/6
and CD154-/- mice were sensitized with DNFB,
and the ear swelling responses to hapten challenge were compared. The
magnitudes of CHS to DNFB in sensitized
CD154-/- and wild-type mice were similar (Fig. 1
). Ear swelling in unsensitized mice
challenged with DNFB was low for both sets of animals, indicating that
sensitization was required for the response. Depletion of
CD8+ T cells before hapten sensitization
abrogated the response to hapten challenge in each group. Depletion of
CD4+ T cells before DNFB sensitization resulted
in slightly elevated ear swelling responses at 24 h postchallenge
in each group. These results indicated that CD8+,
and not CD4+, T cells were required for CHS
responses to DNFB in wild-type and
CD154-/- mice.
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17a mAb-coated culture
wells. After 48 h the supernatants were collected and tested for
IFN- and IL-4 (Fig. 2). During in
vitro stimulation with anti-CD3 mAb, immune
CD8+ T cells from
CD154-/- mice produced readily detectable
levels of IFN-, although this production was consistently lower
(e.g., by 
30%) compared with production by immune
CD8+ T cells from wild-type mice. Although low
levels of IFN- were produced by immune CD4+ T
cells from wild-type mice, this was not observed by
CD4+ T cells from
CD154-/- mice. Similar levels of IL-4 were
produced by anti-CD3 mAb-stimulated CD4+ T
cells from sensitized CD154-/- and wild-type
mice. As previously reported (12, 13, 14), culture of naive T
cells on anti-CD3 mAb coated wells produced low to undetectable
levels of IFN- and IL-4 (Fig. 2), and culture of immune T cells from
C57BL/6 mice on anti-V17a mAb coated wells produced undetectable
levels of the cytokines (data not shown). These results indicated that
CD40-CD154 interactions were not required for, but may augment, the
development of IFN--producing CD8+ and
IL-4-producing CD4+ T cells during sensitization
for CHS.
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The results presented above indicated the ability of DNFB
sensitization to induce effector CD8+ T cells and
CHS responses in CD154-/- mice, but
anti-CD154 mAb has been reported to inhibit CHS responses
(26). To begin to investigate the effect of anti-CD154
mAb on the induction and elicitation of CHS in our hands, groups of
BALB/c mice were injected with different doses of anti-CD154 mAb
MR1 or 500 µg of polyclonal hamster IgG at the time of DNFB
sensitization, and ear swelling responses were measured 24 h after
hapten challenge. Administration of anti-CD154 mAb MR1 during
hapten sensitization inhibited CHS responses in a dose-dependent
manner. Greater than 70% inhibition of CHS was achieved in BALB/c mice
with 250 µg of MR1 (Fig. 3) and in
C57BL/6 mice given 500 µg of MR1 (data not shown).
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and IL-4 production by immune
CD4+ and CD8+ T cells from
control and MR1-treated mice was compared. CD4+
and CD8+ T cell-enriched populations from LNC of
mice treated with control Ig or MR1 during DNFB sensitization were
cultured on anti-CD3 mAb-coated wells, and after 48 h the
supernatants were collected and tested for IFN-, IL-4, and IL-10.
CD8+ T cells from mice treated with control Ig
during DNFB sensitization were stimulated to produce IFN- by culture
on anti-CD3 mAb-coated wells (Fig. 4). CD8+ T cells
from mice treated with MR1 during DNFB sensitization produced much
lower levels of IFN-, correlating with the inhibition of the CHS
response by MR1. Immune, but not naive, CD4+ T
cells produced lower levels of IFN- than the
CD8+ T cells, and this production was also
inhibited by MR1 treatment.
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production, CD4+ T cells
from mice treated with control Ig or MR1 during DNFB sensitization
produced IL-4 and IL-10 in anti-CD3 mAb-coated wells. It was
noteworthy that IL-4, but not IL-10, production by
CD4+ T cells from MR1-treated mice was
significantly increased compared with production by immune
CD4+ T cells from control Ig-treated mice (Fig. 4
). T cells from naive mice produced low to undetectable levels of
IFN-, IL-4, and IL-10. Culture on control anti-V17a
mAb-coated wells did not stimulate detectable cytokine production by
immune CD4+ or CD8+ T cells
(not shown).
To further test the effects of anti-CD154 mAb treatment on the
development of CD8+ and
CD4+ T cell populations during hapten
sensitization for CHS, ELISPOT assays were performed on T cells from
naive or DNFB-sensitized mice treated with either control IgG or
anti-CD154 mAb. Enriched CD4+ or
CD8+ T cells were used as responder cells, and
DNBS-labeled syngeneic splenocytes were used as stimulator cells. After
24 h of culture, the cells were removed from the plates, and the
numbers of IFN-- and IL-4-producing cells in each group were
quantified using an ELISPOT assay. In contrast to cells from naive
mice, cells from DNFB-sensitized mice contained many IFN--producing
CD8+ T cells and IL-4-producing
CD4+ T cells when cultured with DNBS-labeled
stimulator cells (Fig. 5). Background
numbers of spots were observed when the immune
CD4+ and CD8+ T cell
populations were cultured with unlabeled splenocytes (not shown).
Consistent with the results observed in the cytokine ELISA experiments,
the number of hapten-specific IFN--producing
CD8+ T cells was reduced to background levels,
and the number of IL-4-producing CD4+ T cells was
increased by treatment with anti-CD154 mAb during DNFB
sensitization. Collectively, these data indicated that MR1 treatment
during DNFB sensitization inhibited the development of
IFN--producing CD8+ and
CD4+ T cells, but had an enhancing effect on the
development of the IL-4-producing CD4+ T cell
population.
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The augmentation of hapten-specific CD4+ T
cells responses by MR1 treatment during DNFB sensitization suggested
that anti-CD154 mAb might not be blocking CD40-CD154 interactions
during T cell priming for CHS. To examine this more closely,
F(ab')2 of MR1 were prepared and tested. The
function of these fragments was first demonstrated by the ability to
inhibit the induction of alloreactive T cell proliferation in mixed
lymphocyte cultures (Fig. 6). Groups of
mice were given 500 µg of control Ig, MR1, or the
F(ab')2 during sensitization with DNFB. In
contrast to MR1, administration of the F(ab')2 of
MR1 during DNFB sensitization did not inhibit ear swelling responses or
the induction of IFN--producing CD8+ T cells
(Fig. 7). These results suggested that
anti-CD154 mAb did not mediate inhibition of CHS through blockade
of CD40-CD154 interactions.
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The results indicating that anti-CD154 mAb inhibited the
development of CD8+ T cells in CHS suggested that
hapten-primed CD8+ T cells might express CD154
during hapten priming. T cell expression of CD154 was tested following
anti-CD3 mAb or PMA/ionomycin stimulation of
CD4+ and CD8+ T
cell-enriched populations from naive or DNFB-primed mice. In addition,
several different times of culture from 6–16 h were tested at which
optimal expression of CD154 on CD4+ T cells had
been previously reported (15, 16). Both naive and
hapten-primed CD4+ T cells taken 24 h after
sensitization expressed easily detectable levels of CD154 following
stimulation by anti-CD3 mAb (Fig. 8, activated cells). Culture on anti-V17a mAb-coated wells did not
stimulate CD4+ T cells to express CD154 (Fig. 8, resting cells). In contrast to CD4+ T cells,
expression of CD154 on CD8+ T cells following
culture of naive or hapten-primed T cells on anti-CD3 mAb-coated
wells for 6–16 h was minimal to undetectable. Detectable expression of
CD154 following a 6-h culture with PMA/ionomycin was also observed on
naive and immune CD4+, but not
CD8+, T cells (data not shown).
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The expression of CD154 on activated CD4+,
but not CD8+, T cells suggested that the
inhibitory effect of MR1 on the development of IFN--producing
CD8+ T cells in CHS might be mediated indirectly
through CD4+ T cells. Two approaches were used to
test this possibility. First, groups of mice were treated with control
rat IgG or anti-CD4 mAb on days -3 through -1 before hapten
sensitization and MR1 treatment on days 0 and +1. The efficiency of
CD4+ T cell depletion in anti-CD4 mAb-treated
mice was >95% as monitored by flow cytometry (not shown). Consistent
with our previous observations (12, 13), depletion of
CD4+ T cells before hapten sensitization
increased the magnitude and duration of CHS (Fig. 9A). Although MR1 treatment
during DNFB sensitization inhibited CHS in control IgG-treated mice,
this treatment did not inhibit the CHS response in CD4-depleted
mice.
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B). Production of
IFN- by CD8+ T cells from CD4 mAb-depleted
(Fig. 10) or
CD4-/- (not shown) mice was also not inhibited
by MR1 treatment during DNFB sensitization. These data indicated that
MR1-mediated inhibition of CD8+ T cell
development and the CHS response required CD4+ T
cells.
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Preliminary experiments indicated that MR1-mediated inhibition of
CHS and CD8+ T cell development was not abrogated
by anti-IL-4 mAb or in IL-4-/- mice (data
not shown). Because these results indicated that the increased
CD4+ T cell production of IL-4 induced by MR1 was
not the mechanism by which the anti-CD154 mAb inhibited the
induction of CHS, we then asked whether the effect might be mediated
during the initial stage of T cell priming with hapten. To begin to
examine this, we tested the ability of hpLC isolated from mice treated
with control IgG or with anti-CD154 mAb during DNFB sensitization
to stimulate immune CD8+ T cell proliferation. As
previously observed (12, 13), hpLC obtained from control
IgG-treated mice induced DNFB immune, but not naive,
CD8+ T cells to proliferate (Fig. 11A, group 1 vs group 5).
Culture of immune CD8+ T cells without hpLC
resulted in low levels of proliferation similar to naive cells (not
shown). Consistent with the inhibited development of
CD8+ T cell development in animals treated with
anti-CD154 mAb during DNFB sensitization, coculture of these
CD8+ T cells and hpLC from control IgG-treated
mice resulted in low levels of T cell proliferation (group 2). In
contrast to immune CD8+ T cell proliferation in
cocultures with hpLC from control IgG-treated mice, reduced
proliferation of immune CD8+ T cells was observed
in cultures with hpLC from mice treated with anti-CD154 mAb during
DNFB sensitization (group 3 vs group 1). Coculture of
CD8+ T cells from mice treated with
anti-CD154 mAb during DNFB sensitization and hpLC from mice treated
with anti-CD154 mAb during sensitization also resulted in low
levels of T cell proliferation (group 4). Whereas hpLC from wild-type
mice treated with anti-CD154 mAb had reduced capacity to stimulate
the proliferation of immune CD8+ T cells, this
reduced stimulation was not observed in cocultures of immune
CD8+ T cells and hpLC isolated from the lymph
nodes of CD154-/- mice treated with
anti-CD154 mAb (Fig. 11B).
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C). However, hpLC
isolated from mice depleted of CD4+ T cells
before MR1 treatment and DNFB sensitization induced higher
proliferation of immune CD8+ T cells than hpLC
from mice treated with control Ig during sensitization. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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As shown in the current report, CD40-CD154 interactions are not
required for the development of the effector CD8+
or the regulatory CD4+ T cells during DNFB
sensitization for CHS. The development of IFN--producing
CD8+ T cells and IL-4-producing
CD4+ T cells and the magnitude of the CHS
response were similar in CD154-/- and wild-type
animals. Furthermore, CHS responses in CD154-/-
mice depleted of CD4+ T cells before
sensitization were of greater magnitude than responses in control
CD154-/- mice, indicating the presence of
regulatory CD4+ T cell activity during CHS in
CD154-/- mice. A low level of IFN--producing
CD4+ T cells was observed in sensitized
wild-type, but not CD154-/-, mice. Other
laboratories have also reported that the induction of
IFN--producing, but not IL-4-producing, CD4+ T
cells requires CD40-CD154 interactions (40, 41). In
contrast to our results, Moodycliffe and coworkers (27)
recently reported the absence of CHS responses to FITC sensitization
and challenge in CD154-/- mice. The results
also indicated the inability of LC to migrate from the skin
sensitization site to the draining lymph nodes, accounting for the
absence of responses in these mice. At this time it is difficult to
reconcile these results with those presented in the current report. A
possible explanation may lie in the different haptens tested in each
study. In support of this, we have previously observed that responses
to FITC are considerably weaker than responses to DNFB
(33). Although the results in
CD154-/- animals may be dependent upon the
sensitizing hapten under study, the results in the current report
clearly demonstrate that CD154 is not required for the induction of T
cells and CHS responses to at least some haptens.
Despite the induction of IFN--producing CD8+ T
cells and CHS responses in DNFB-sensitized
CD154-/- mice, administration of anti-CD154
mAb during sensitization inhibited the development of IFN--producing
CD8+ T cells and the CHS response to hapten
challenge. Inhibition of CHS responses to DNFB by anti-CD154 mAb
was previously reported by Tang and coworkers (26). During
these studies decreased IFN- production by in vitro stimulated LNC
was also observed in mice treated with MR1 during DNFB sensitization,
but the effect of Ab treatment on the development of the individual
CD4+ and CD8+ T cell
populations in CHS was not tested. This and many other studies
demonstrating the inhibitory effects of anti-CD154 mAb on T cell
priming have assumed that the inhibitory mechanism was mediated through
blockade of the CD40-CD154 interactions required for T cell priming
(26, 35, 36, 37, 38, 39). In the current report we have demonstrated
that MR1-mediated inhibition of CHS is not due to direct blockade of
CD40-CD154 interactions during CD8+ T cell
priming for CHS, but is due to an active process requiring
CD4+ T cells. First, CD8+ T
cell development and the CHS response were not inhibited when either
CD4-/- mice or wild-type mice depleted of
CD4+ T cells were treated with anti-CD154 mAb
during hapten sensitization. Because CD4+ T cells
are not required for the development of the effector
CD8+ T cells in CHS, the anti-CD154
mAb-mediated inhibition observed in the presence of
CD4+ T cells is not due to the blockade of
CD4+ T cell development. Furthermore,
administration of anti-CD154 mAb augmented the development of
hapten-specific (i.e., IL-4-producing) CD4+ T
cells. Second, administration of F(ab')2 during
sensitization did not inhibit CD8+ T cell priming
or CHS responses. Although these fragments were active in inhibiting
alloreactive T cell responses in vitro, it is difficult to assess their
activity in vivo. Administration of the fragments, however, did result
in extended CHS responses compared with those in control Ig-treated
mice (A. Gorbachev, unpublished observations). The reasons for this
enhancing effect are under investigation.
Several recent studies have indicated that CD40-CD154 engagement is required for CD4+ T cells to deliver signals to DC, which then enable the DC to prime functional cytolytic CD8+ T cells responses (42, 43, 44). The ability of agonist anti-CD40 mAbs to replace CD4+ T cells in this maturation suggests the absence or lack of sufficient CD154 expression by CD8+ T cells to direct DC maturation during this priming. Low level CD154 expression on CD8+ T cells following in vitro stimulation by various methods has been previously reported, although this expression may be restricted to CD8+ T cells producing type 2 cytokines and having the ability to provide helper signals to B cells for Ig production (45, 46). Lefrancois and coworkers (25) recently reported the CD154 blockade-mediated inhibition of mucosal CD8+ T cell expansion in response to OVA and suggested that these T cells may express CD154 during Ag priming. Thus, in contrast to CHS, CD40-CD154 interactions appear to play a crucial role in the development of CD8+ T cell-mediated mucosal immune responses. Consistent with reports from other laboratories (15, 16), CD154 expression was easily detectable on DNFB-primed CD4+ T cells following TCR-mediated stimulation in vitro. In conjunction with the lack of observed effects of anti-CD154 mAb directly on CD8+ T cells during priming for CHS, the results of the current study indicate that CD8+ T cells do not express CD154 during the development of this immune response.
The mechanism by which anti-CD154 mAb treatment promotes
immunoregulatory CD4+ T cell activity and
inhibits the development of effector CD8+ T cells
is not entirely clear at this time. It is also not clear whether the
anti-CD154 mAb enhances natural CD4+ T cell
immunoregulation in the response or generates an entirely different
form of CD4+ T cell immunoregulation.
IL-4-producing, but not IL-10-producing, CD4+ T
cell development was amplified by anti-CD154 mAb treatment during
sensitization. In contrast, Blair and coworkers (47)
demonstrated increased production of IL-10 by human
CD4+ T cells stimulated in vitro with both
anti-CD3 and anti-CD154 mAb. Initially, the increased
CD4+ T cell production of IL-4 appeared to be an
obvious potential mechanism for the immunoregulation induced by
anti-CD154 mAb. A regulatory role for IL-4 in CHS has been reported
in one, but not another, study (48, 49). However, the
inhibition of IFN--producing CD8+ T cell
development also occurred in IL-4-/- mice
treated with MR1 during sensitization (A. Gorbachev, unpublished
observations). In conjunction with the absence of increased
CD4+ T cell production of IL-10, these results
suggested that inhibition of CHS by anti-CD154 mAb was not mediated
through up-regulated CD4+ T cell production of
immunoregulatory cytokines.
These results led us to consider the effects of anti-CD154 mAb
directly on the CD4+ T cells during interaction
with the hapten-presenting LC, rather than blocking these interactions.
As discussed above, CD4+ T cells deliver positive
signals through CD154 to DC, which then render the DC competent to
generate CD8+ cytolytic responses
(42, 43, 44). It is conceivable that Ab ligation of CD154
expressed by CD4+ T cells during these
interactions may induce the delivery of negative signals to the DC,
which undermine DC function during CD8+ T cell
priming. In support of this, in vitro studies have indicated that
ligation of CD154 on CD4+ T cells delivers
signals affecting cytokine production and other immune functions
(47, 50, 51). On this basis, we postulated that Ab
ligation of CD154 expressed by CD4+ T cells
during priming by hpLC may result in the delivery of negative signaling
to the hpLC, and this signaling renders the hpLC incompetent for
stimulation of hapten-specific CD8+ T cell
maturation, resulting in inhibition of the CHS response (Fig. 12). This
model is supported by the observations in this
report. The inhibitory activity of the
anti-CD154 mAb is dependent on CD4+ T cells
and apparently requires Ab Fc-Fc receptor ligation. Although wild-type
CD4+ T cells express CD154 during priming, the
CD4+, but not the CD8+, T
cell population develops in wild-type mice treated with anti-CD154,
indicating that the Ab does not block CD4+ T cell
priming. The hpLC isolated from the anti-CD154 mAb treated animals
have reduced capacity to stimulate immune CD8+ T
cell proliferation in vitro, and this reduced hpLC function requires
CD4+ T cells during anti-CD154 mAb
administration. Other consequences of anti-CD154 mAb treatment on
the hpLC have not yet been identified. However, there is no apparent
decrease in the number of hpLC in the skin-draining lymph nodes of mice
treated with control Ig and anti-CD154 mAb during hapten
sensitization, and hpLC isolated from mice treated with MR1 during
hapten sensitization express levels of class II MHC and B7-2 equivalent
to those of hpLC from control treated mice (A. Gorbachev, unpublished
observations).
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Robert L. Fairchild, NB3-79, Department of Immunology, Lerner Research Institute, Cleveland Clinic Foundation, 9500 Euclid Avenue, Cleveland, OH 44195-0001.
3 Abbreviations used in this paper: CHS, contact hypersensitivity; DC, dendritic cells; DNFB, 2,4-dinitrofluorobenzene; LNC, lymph node cells; hpLC, hapten-presenting Langerhans cells; ELISPOT, enzyme-linked immunospot.
Received for publication August 16, 2000. Accepted for publication November 28, 2000.
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inducible protein (IP-10) expression is mediated by CD8+ T cells and is regulated by CD4+ T cells during the elicitation of contact of hypersensitivity. J. Invest. Dermatol. 107:360.[Medline]
producing (Tc1) effector CD8+ T cells and Il-4/Il-10 producing (Th2) negative regulatory CD4+ T cells. J. Exp. Med. 183:1001.This article has been cited by other articles:
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A. V. Gorbachev, H. Kobayashi, D. Kudo, C. S. Tannenbaum, J. H. Finke, S. Shu, J. M. Farber, and R. L. Fairchild CXC Chemokine Ligand 9/Monokine Induced by IFN-{gamma} Production by Tumor Cells Is Critical for T Cell-Mediated Suppression of Cutaneous Tumors J. Immunol., February 15, 2007; 178(4): 2278 - 2286. [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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L. Nagelkerken, I. Haspels, W. van Rijs, B. Blauw, J. L. Ferrant, D. M. Hess, E. A. Garber, F. R. Taylor, and L. C. Burkly FcR Interactions Do Not Play a Major Role in Inhibition of Experimental Autoimmune Encephalomyelitis by Anti-CD154 Monoclonal Antibodies J. Immunol., July 15, 2004; 173(2): 993 - 999. [Abstract] [Full Text] [PDF]
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S. Nakae, C. Naruse-Nakajima1, K. Sudo, R. Horai, M. Asano, and Y. Iwakura IL-1{alpha}, but not IL-1{beta}, is required for contact-allergen-specific T cell activation during the sensitization phase in contact hypersensitivity Int. Immunol., December 1, 2001; 13(12): 1471 - 1478. [Abstract] [Full Text] [PDF]
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)
and CD8+-enriched (
) cell populations were prepared from
lymph nodes of the sensitized and naive mice, and 2 x
105-cell aliquots were cultured on anti-CD3 or
anti-V
) or
anti-CD4 mAb (• and 
) and 2 days later (i.e., days 0 and +1)
were sensitized with 0.25% DNFB. Groups of control and
CD4+ T cell-depleted mice were given 500 µg of
anti-CD154 mAb MR1 (
