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*
Department of Immunology, Guy’s, King’s, and St. Thomas’s School of Medicine, Kings College, London, United Kingdom; and
Section of Allergy and Clinical Immunology, Department of Medicine, Yale University School of Medicine, New Haven, CT 06510
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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-/-,
and OVA257–264-specific V
2V
5 TCR-transgenic mice, we
have identified the key steps involved. OVA-specific
IFN--/- CD8 T cells inhibited IgE responses equivalent
to wild-type CD8 T cells (up to 98% suppression), indicating that CD8
T cell-derived IFN- was not required. However, OVA-specific CD8 T
cells could not inhibit IgE in IFN--/- recipients
unless reconstituted with naive, wild-type CD4 T cells, suggesting that
CD4 T cell-derived IFN- did play a role. Transfer of either Tc1 or
Tc2 V2V5 TCR-transgenic CD8 T cells inhibited IgE and
OVA-specific Th2 cells while promoting OVA-specific Th1 cell responses,
suggesting a potential role for a type 1 inducing cytokine such as
IL-12. CD8 T cells were shown to induce IL-12 in
OVA257–264-pulsed dendritic cells (DC) in vitro.
Furthermore, CD8 T cells were unable to inhibit IgE responses in
IL-12-/- recipients without the addition of naive,
wild-type DC, thus demonstrating a pivotal role for IL-12 in this
mechanism. These data reveal a mechanism of IgE regulation in which CD8
T cells induce DC IL-12 by an IFN--independent process that
subsequently induces Th1 and inhibits Th2 cells. Th1 cell IFN- is
the final step that inhibits B cell IgE class switching. This
demonstrates a novel regulatory network through which CD8 T cells
inhibit allergic sensitization. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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(1) and IL-10
(2) produced by specific subsets of regulatory T cells
also inhibit inflammatory immune responses (3). The
mechanism for immune regulation of IgE Ab responses has yet to be fully
defined. Following parenteral immunization with soluble Ags, it has
been demonstrated that IgE responses require T cell help
(4). CD4 Th2 T cells provide this help in the form of CD40
ligand, which activates B cells, and IL-4, which induces Ig class
switching to IgE (5, 6).
CD8 T cells also can inhibit IgE responses (7, 8). CD8 T
cells that inhibit IgE are sensitive to the toxic lectin ricin
(9). Depletion of these cells in vivo using ricin or
anti-CD8 mAb increases the capacity of CD4 T cells to produce IL-4
and decreases IFN- production (10, 11). Recently, these
IgE inhibitory CD8 T cells have been cloned and found to express the
TCR and to be MHC class I restricted (12). Their
capacity to inhibit IgE is unrelated to their ability to produce
IFN-, but paradoxically their effect on IgE can be blocked by
anti-IFN- mAb (12). CD8 T cells also regulate
airway hyperresponsiveness in rats (13, 14). Presentation
of soluble Ag (OVA) via MHC class I runs counter to the predominant APC
pathway for soluble exogenous proteins in which Ag peptides are
presented to CD4 T cells complexed with surface MHC class II molecules.
However, there now is clear evidence that small but important amounts
of such exogenous Ag are directed intracellularly to be presented via
MHC class I (15, 16), and derived peptide MHC class I
complexes can activate CD8 T cells. We have previously demonstrated
that OVA-specific CD8 T cells can suppress IgE responses when
adoptively transferred into wild-type recipients responding to OVA-alum
immunization. We also determined that activating the regulatory
potential of CD8 T cells was Ag specific, whereas their regulatory
effects could influence IgE responses to coimmunized irrelevant Ag
(17). This suggests a suppressive mechanism that, once
activated to a single allergen, could have an impact on the
generation of IgE in response to other harmful allergens.
In the current study, we have investigated the mechanisms by which
OVA-specific CD8 T cells inhibit IgE responses. Using
IFN--/- mice, we have shown that CD8 T cells
do not need to produce IFN- to inhibit IgE, but that IFN- is
required and is supplied by participating OVA-specific CD4 Th1 T cells.
The immunoregulatory potential of CD8 T cells to inhibit IgE is also
dependent on their ability to stimulate IL-12 production by APC
that, in turn, activates Th1 cells. Thus, CD8 T cells are unable
to inhibit IgE in IL-12-/- mice but can do so
if IL-12-/- mice are reconstituted with
wild-type (IL-12 competent) dendritic cells
(DC).3
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Wild-type C57BL/6 mice (6–8 wk) were obtained from Harlan Olac
(Bicester, U.K.). Several breeding pairs of OVA peptide-specific, class
I-restricted V2V5 TCR-transgenic mice (OT-I) were a kind gift
from Dr. M. Merkenschlager (Royal Postgraduate Medical School, Imperial
College, London, U.K.). Breeding colonies of IL-12 and IFN- knockout
mice were obtained from The Jackson Laboratory (Bar Harbor, ME). RPMI
1640 and AIM V culture medium were purchased from Life
Technologies (Paisley, U.K.), and tissue culture flasks and microtiter
plates were from Nunc (Roskilde, Denmark). Sterile PBS and HBSS were
purchased from Life Technologies. FCS was purchased from Globepharm
(Surrey, U.K.). Purified OVA (grade V) was purchased from Sigma-Aldrich
(Poole, U.K.). Rodent lymphoprep 1.077 was purchased from Nycomed
(Birmingham, U.K.), and FCS was purchased from Globepharm. Anti-mouse
IgE heavy chain (LO-ME-3), anti-IgE 
L chain (OX-20), and
recombinant mouse IL-4 were purchased from Serotec (Oxford, U.K.), and
anti-IgG1 alkaline phosphatase was purchased from The Binding Site
(Birmingham, U.K.). mAbs and recombinant proteins for cell culture flow
cytometry, and ELISAs for IL-12, IFN-, IL-4, IL-10, and 70-µm
nylon filters were from BD Biosciences (Oxford, U.K.). Anti-CD4
and -CD8 microbeads for MACS were purchased from Miltenyi Biotec
(Bisley, U.K.). Complete medium comprised an equal mixture of AIM
V serum-free medium and RPMI 1640 supplemented with
L-glutamine (2 mM), nonessential amino acids (1%),
streptomycin (100 ng/ml), penicillin (100 U/ml), sodium pyruvate (1
mM), and 2-ME (5 µM), which were purchased from Life Technologies.
[3H]Thymidine was purchased from Amersham
Biosciences (Little Chalfont, U.K.). All other reagents were
purchased from Sigma-Aldrich.
Immunization procedure
Groups of five age-, sex-, and batch-matched mice were immunized with alum-precipitated OVA prepared as follows. OVA was dissolved at 10 mg/ml in sterile saline. To 10 ml of this protein solution, 4.5 ml of 1 M NaHCO3 and 10 ml of KAlSO4 were added at 20°C for 20 min. The mixture was then centrifuged at 3000 x g for 10 min. The precipitate was washed three times with sterile PBS, resuspended in 10 ml PBS, and stored at 4°C. Recipient mice were immunized i.p. with 100 µg of OVA-alum diluted in 0.1 M Al(OH)3.
Isolation of murine LN and spleen-derived CD8 T cells
OVA-specific CD8 T cells were obtained from mice immunized 21
days previously with 100 µg of OVA-alum. Mice were euthanized in
CO2 and their parathymic/posterior mediastinal
lymph node (LN) and spleens were excised. Leukocytes were obtained by
pressing tissue through 70-µm nylon filters (BD Biosciences) into
chilled PBS. Mononuclear splenocytes and LN cells were purified on
lymphoprep. The cells were then immediately washed twice in PBS,
and viable cell numbers were determined by trypan blue exclusion. CD8 T
cells were purified using MACS separation. Cells were resuspended in
MACS buffer to a concentration of 
108 cells/ml
and then incubated with anti-CD8 microbeads at a concentration of 4
µl/107 total cells for 30 min at 4°C. VS+
MACS columns were prepared for use by flushing 5 ml of buffer through
while in the magnetic field. The cells were then added to the column
and washed three times with 5 ml of buffer. To elute the CD8-positive
fraction, the column was removed from the magnetic field and a plunger
was used to force through 10 ml of buffer. The CD8 cells were then spun
at 200 x g for 10 min, counted to assess yield, and
stained for purity using PE-labeled anti-CD3 with either
CyChrome-labeled anti-CD8 or anti-CD4 for flow cytometric
analysis. Purified CD8 T cells (>98%) were then resuspended in PBS at
5 x 106 cells/ml for adoptive transfer in
200 µl (106 cells/mouse) i.p.
Isolation of murine bone marrow-derived DC
DCs were isolated from bone marrow. Femurs from euthanized naive mice were placed in a petri dish with PBS and any remaining muscle tissue was removed. Holding the bone with forceps, one of the epiphyses was removed using scissors. A 5-ml syringe filled with PBS and a 21-gauge needle were used to flush out the marrow. Cells were resuspended at 2 x 105/ml in complete medium containing 20 ng/ml GM-CSF and 1 ng/ml IL-4. After 3 days, cells were stained for MHC class II (I-Ad) (PE) and CD11c (PE) to assess the number of DC present. Cells were also stained for CD3 (FITC), CD4 (CyChrome), CD8 (CyChrome), and CD22 (PE) to assess T and B cell contamination. Based on high levels of both MHC class II and CD11c staining, DCs were up to 80% pure (data not shown).
Generation of OVA-specific Tc1 and Tc2 cell populations
OVA-specific CD8 T cells were isolated from peripheral LN and
spleen of V2V5 TCR-transgenic mice and cultured at
106 cells/ml for 3 days in complete medium. To
generate T cytotoxic (Tc)1 cells, activating anti-CD3 (4 µg/ml)
was bound to the culture plate and recombinant mouse IL-12 (5 ng/ml)
was added. To skew the cells to Tc2, IL-12 was omitted and, in addition
to anti-CD3, PMA (10 ng/ml), anti-CD28 (1 µg/ml), IL-4 (100
U/ml), and blocking anti-IL-12 (10 µg/ml) were added. Phenotypic
assessment was by stimulation with OVA257–264
for 5 h followed by intracellular cytokine staining for IFN-
and IL-4 and ELISA analysis of 24-h culture supernatant for secreted
IFN-, IL-4, and IL-10.
Intracellular cytokine staining
CD8 T cell cultures were washed thoroughly with PBS/1% FCS then
resuspended in complete medium containing 5 µg/ml OVA peptide and 3
µM monensin at 106/ml for 5 h at 37°C.
Cells were then washed and stained for FACS at
106/tube. Anti-CD4 or CD8 CyChrome-labeled
surface marker Abs were then added and incubated for 15 min. Cells then
were washed, incubated for 15 min with 250 µl of Perm/Fix solution
(BD PharMingen, San Diego, CA), then washed twice with 2–4 ml of
Perm/Wash buffer (PBS/0.5% BSA/0.1% saponin; BD PharMingen).
Anti-IFN- FITC and anti-IL-4 PE were added at 1 µl/tube,
mixed, and then incubated for 30 min at 18°C. Cells were then washed
with Perm/Wash buffer as before and resuspended in 500 µl of 1% PFA.
Cells were then analyzed using a BD Biosciences FACSCalibur flow
cytometer.
Cytokine ELISAs
CD8 T cell cultures were washed thoroughly with PBS then
resuspended in complete medium containing 5 µg/ml
OVA257–264 at 106/ml for
24 h at 37°C. Supernatant from each well was stored at -30°C
for ELISA analysis. Throughout, 50-µl volumes were used, and the
assay was performed at 25°C. IFN-, IL-4, IL-10, and IL-12 (p40
chain) were measured using capture and detector Ab pairs. Microtiter
plates (Maxisorb; Nunc) were coated with detector Ab at 1 µg/ml in
carbonate/bicarbonate (pH 9.6, 0.1 M) coating buffer overnight at 4°C
and washed three times with PBS/0.05% Tween 20. Duplicate supernatant
samples diluted 1/50 or greater in assay diluent (PBS/1% rat
serum/0.5% Tween 20) were added. After 2 h, the plates were
washed and biotinylated detector Ab at 1 µg/ml was added. After
2 h, the plates were washed and streptavidin-conjugated alkaline
phosphatase was added at 1 µg/ml. After 2 h, the plates were
washed, and p-nitrophenyl phosphate substrate diluted to 1
mg/ml in diethanolamine buffer (0.1 M) was added. After 1 h,
absorbance was read at 405 nm in a plate reader (Molecular Devices,
Crawley, U.K.), and the results were expressed as nanograms per
milliliter by reference to a standard curve constructed using dilutions
of recombinant cytokine.
Measurement of OVA-specific IgE by PCA
Passive cutaneous anaphylaxis (PCA) was used to measure IgE Abs, as this is the most reliable and reproducible method. It has been demonstrated that the only reaginic Ab active at the site of injection 48 h after skin transfer is IgE (18). Furthermore, the method correlates well with in vitro sensitization using rat basophil leukemia cells and monoclonal IgE. In addition, it provides evidence that the Abs measured are functional. PCA is not affected by high IgG Ab titers or changing Ab affinity as can be ELISA. Mouse serum OVA-specific IgE titers were measured by PCA in Wistar rats (Harlan Olac). Serial 4-fold dilutions of serum from 1/8 to 1/2048 in PBS were made, and 50 µl of up to 50 diluted samples were injected intradermally into the shaved back of an anesthetized rat. After 48 h, the rat was again anesthetized and 500 µl of 10 mg/ml OVA/1% Evans blue dye was injected into the tail vein. After 30 min, without regaining consciousness, the rat was euthanized and the skin response was recorded. OVA-specific IgE Ab titers were measured at day 7 and are represented as the mean ± SE of the highest dilution of test serum that produced a positive mast cell-dependent PCA reaction (skin blueing).
Statistics
Group comparisons were made using a two-tailed Student’s t test for independent samples.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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+) and Tc2
(IL-4+) V2V5 TCR CD8 T cell lines
Because IgE responses are inhibited by IFN- and because CD8 T
cells are strongly biased to produce IFN-, we investigated the
contribution of IFN- to CD8 T cell inhibition of IgE Ab responses by
adoptive transfer of polarized Tc1 and Tc2 OVA-specific cell lines. Tc1
and Tc2 lines were generated in vitro with CD8 T cells from C57BL/6
(H2Kb) mice that have a transgenic V2V5 TCR
that specifically binds the OVA peptide 257–264 when complexed to MHC
class I H2Kb (19). Tc1 lines were
polarized by culture of V2V5 TCR CD8 T cells for 3 days with
plate-bound anti-CD3 and IL-12, Tc2 lines with plate-bound
anti-CD3, anti-CD28, PMA, IL-4, and anti-IL-12. When
analyzed for intracellular cytokine production following stimulation
with OVA257–264 for 5 h, 56% of Tc1 cells
were found to produce IFN- alone, 0.2% produced both IFN- and
low levels of IL-4, and no cells were detected that produced IL-4
alone. Conversely, 36% of Tc2 cells produced IL-4 alone, 0.3%
produced both IL-4 and low levels of IFN-, and 0.1% produced
IFN- alone (data not shown). The cytokine profile of both subsets
was confirmed by stimulation of the polarized Tc1 and Tc2 cells with
OVA257–264 for 24 h and analysis of
secreted cytokines in supernatants by ELISA. Tc1 cells produced 276
ng/ml IFN- with no detectable IL-4 or IL-10, whereas Tc2 cells
produced 5.5 ng/ml IL-4, 14 ng/ml IL-10, and very low levels of IFN-
at 1.4 ng/ml.
Tc1 cells are more effective inhibitors of IgE responses than Tc2 cells
To determine the relative IgE inhibitory potential of OVA-specific
Tc1 vs Tc2 cells, numbers varying from 102 to
106 were adoptively transferred into wild-type
recipients immunized simultaneously with OVA in alum (Fig. 1
). Control groups received naive,
unpolarized V2V5 CD8 T cells or OVA-alum immunization alone. Both
control groups made an OVA-specific IgE response that peaked at day 7
with a titer of over 1/512, declining to basal levels (1/8) by day 21.
However, transfer of in vitro polarized 106
OVA257–264-specific Tc1 or Tc2 cells inhibited
the IgE response 64- and 32-fold at day 7 (Fig. 1a)
(p < 0.01). Dose response showed that
transfer of 10-fold less (105) Tc1 or Tc2 cells
also inhibited IgE by 12- (p < 0.01) and
8-fold (p < 0.05) at day 7. A difference in
the inhibitory abilities of Tc1 and Tc2 cells was observed when
104 cells were given (Fig. 1b). At
this cell number, Tc1 cells still inhibited IgE responses 16-fold
(p < 0.01), whereas Tc2 cells could only exert
an 2-fold reduction in IgE (p > 0.05).
Adoptive transfer of 103 and
102 anti-OVA Tc1 or Tc2 cells did not result
in significant inhibition (p > 0.05).
Importantly, significant inhibition was still observed from
105 Tc2 cells that barely produced detectable
levels of IFN-. Further, levels of OVA-specific IgG1 were unaffected
by adoptive transfer of all doses of Tc1 or Tc2 CD8 T cells, showing
that CD8 cell inhibition was isotype specific for these two
Th2-dependent Ab classes (data not shown).
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Because CD8 T cells inhibited IgE, we also determined their effect
on the concomitant CD4 T cell response using a similar adoptive
transfer protocol using 106 Tc1 or Tc2 cells. CD4
T cells and APC were purified from day 7 mice immunized with OVA-alum
to induce IgE that had received V2V5 Tc1 (Fig. 2a) or V2V5 Tc2 CD8 T
cells (Fig. 2b), or no CD8 T cells (Fig. 2c), and
were cultured with 100 µg/ml OVA. The generation of intracytoplasmic
cytokines (IL-4 and IFN-) in the CD4 T cells was determined 6 days
later following restimulation with PMA and ionomycin. In the positive
control animals that received OVA-alum without CD8 T cells, the
percentage of OVA-specific LN CD4 Th2 cells
(IFN--IL-4+) and Th1
cells (IFN-+IL-4-)
following culture with OVA were 23 and 0.4% respectively (Fig. 2c). In recipients of Tc1 cells, the percentage of Th2 cells
was reduced to 1.1%, while Th1 cells were increased to 35% (Fig. 2a). Similarly, although not as dramatically, the percentage
of Th2 cells was reduced to 3.7% in recipients of Tc2 cells, while the
percentage of Th1 cells was increased to 11% (Fig. 2b).
Thus, both Tc1 and Tc2 OVA-specific CD8 regulatory T cells, when given
at the induction of the IgE response with OVA-alum, promoted
development of specific Th1 cells in recipients that may have been
involved in the inhibition of the CD4 Th2 (IL-4)-dependent responses,
perhaps by producing IFN-, that lead to a decrease in IgE production
by B cells.
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-/- mice
Although IFN- is noted for its ability to inhibit IgE
responses, the capacity of Tc2 CD8 T cells to suppress IgE suggests
that IFN- may not be required for inhibition by CD8 T cells because
Tc2 cells made practically no IFN-. However, this small amount of
IFN- could have inhibited IgE, or IL-12 that was produced by APC
could have induced IFN- in the Tc2 CD8 T cells after transfer.
Therefore, we determined whether CD8 T cells from
IFN--/- mice could inhibit the OVA-specific
IgE response. We previously showed that CD8 T cells collected at day 21
from mice immunized with 100 µg of OVA-alum inhibited IgE responses
when adoptively transferred to naive mice that were then similarly
immunized (17). These will henceforth be called
"OVA-primed day 21 CD8 T cells." OVA-primed day 21 CD8 T cells from
IFN- mice inhibited the OVA-specific IgE response as effectively as
CD8 cells from wild-type mice (Fig. 3a). However, promotion of
OVA-specific Th1 and inhibition of OVA-specific Th2 cells suggested
that there might be a role for IFN- in the regulatory process.
Indeed, a requirement for IFN- was demonstrated when day 21
OVA-primed CD8 T cells were unable to inhibit IgE in
IFN--/- mice (Fig. 3b), but could
do so in IFN--/- mice that were constituted
with CD4 T cells isolated from naive wild-type mice that may have
produced essential IFN- (Fig. 3c). Thus, CD8 T cell
inhibition of IgE is independent of CD8 T cell-derived IFN- but does
require IFN- production by CD4 T cells.
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Because CD8 T cell-derived IFN- was not required for the
inhibition of IgE responses but Th1 IFN- was involved, and because
IL-12 is known to favor Th1 responses, we postulated that CD8
regulatory T cells could enhance production of IL-12. We first
established whether OVA-specific CD8 T cells could induce IL-12 in APCs
by culture of naive OVA257–264-specific
V2V5 TCR CD8 T cells with DC pulsed with either
OVA257–264, an irrelevant OVA peptide, or with
medium alone for 72 and 144 h. Supernatants were collected and
IL-12 (p40 chain) content was determined by ELISA (Fig. 4). OVA257–264
peptide-pulsed DC cultured with V2V5 TCR CD8 T cells caused a
3-fold increase in IL-12 p40 levels to 6 and 7 ng/ml at 72 and 144
h respectively. No IL-12 p40 was detected above the limit of detection
of the assay (0.8 ng/ml) in supernatants from unpulsed DC, and <2
ng/ml was detected at either time point in supernatants from V2V5
T cells cultured with irrelevant peptide-pulsed DC. Thus, inhibitory
Ag-specific CD8 T cells can induce IL-12 production in DC following
stimulation with specific Ag.
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IL-12-/- mice were used (20)
to determine the possible contribution of IL-12 in the inhibition of
IgE responses by CD8 T cells in vivo. Adoptive transfer of
106 Tc1 and Tc2 V2V5 TCR CD8 T cells failed
to inhibit IgE responses in IL-12-/- compared
with wild-type mice (Fig. 5, group B).
The importance of DC-derived IL-12 was indicated further by
reconstitution of IL-12-/- mice in a
dose-dependent fashion with DC
(102–105 DC per mouse)
from naive (IL-12-competent) mice (Fig. 5b, groups C–F). An
important control was adoptive transfer of 105 DC
from naive (IL-12-competent) mice with 106 naive
CD8 T cells (Fig. 5b, group A) to
IL-12-/- mice. No inhibition was observed in
this group, demonstrating that DC alone could not inhibit the IgE
response. These data show that IL-12 is an essential element of the
regulatory CD8 T cell pathway.
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Although we had shown that OVA-specific CD8 T cells induced IL-12
and that a shift from a Th2 to a Th1 dominant response was associated
with inhibition of IgE, the relationship among DC, CD4 T cells, and the
cytokines they produce in CD8 T cell suppression of IgE was unclear.
For this purpose, IFN--/- and wild-type mice
were used to generate Th1-like cells. Intracellular cytokine analysis
of Th1 cells from wild-type mice revealed that 50% stained positive
for IFN-, 1.2% stained positive for IL-4, and 19% stained positive
for IL-2 (Fig. 6a). In
contrast, of Th1-like cells from IFN--/-
mice, only 0.4% stained positive for IFN- and 1.7% stained
positive for IL-4, but more (32%) were positive for IL-2 (Fig. 6a). We then adoptively transferred
IFN--/- and IL-12-/-
recipient mice with either naive CD4 T cells (Fig. 6b,
groups C and D), OVA-primed day 21 wild-type (Fig. 6b,
groups E and F), or IFN--/- (Fig. 6b, groups G and H) Th1 cells. As before (Fig. 3
c), coadoptive transfer of naive wild-type CD4 T cells,
with day 21 OVA-primed CD8 T cells, into
IFN--/- recipient mice reconstituted
inhibition of the IgE response by up to 16-fold (Fig. 6b,
groups C and D). However, inhibition of IgE did not occur in
IL-12-/- recipients. This finding demonstrated
that the mechanism by which CD8 T cells inhibit the CD4 Th2 response is
dependent on IL-12. Coadoptive transfer of OVA-specific Th1 cells with
day 21 OVA-primed CD8 T cells resulted in suppression in both
IFN--/- and IL-12-/-
recipient mice by between 8- and 16-fold (Fig. 6b, groups E
and F), demonstrating that, if generated before transfer, Th1 cell
inhibition of IgE was independent of IL-12. In other words, IL-12 was
only required to generate Th1 cells. However, suppression was not
observed in either IFN--/- or
IL-12-/- recipient mice adoptively transferred
with day 21 OVA-primed CD8 T cells together with
IFN--/- Th1 cells (Fig. 6b,
groups G and H), suggesting that DC-derived IL-12 was the induced
factor that was vital for IgE suppression.
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-/-mice (Fig. 6c, groups C
and D) transferred to wild-type recipients were unable to suppress the
IgE response compared with control animals (Fig. 6c, group
A) or mice that received day 21 OVA-primed CD8 T cells (Fig. 6c, group B), thereby confirming that the IgE regulatory
pathway described here is a unique property of the CD8 T cell. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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2V5
TCR-transgenic T cells and IFN--/-and
IL-12-/- mice. At the outset, CD8 T cell
IFN- was considered the most likely candidate for CD8 T cell
inhibition of IgE, because IFN- inhibits Th2 T cell growth
(21) and B cell IgE class switching (6).
However, the experiments described in this work suggest that this is
not the case. Our results show that OVA-specific CD8 inhibitory T cells
interact with DC independently of IFN- to induce IL-12 that, in
turn, promotes the generation of OVA-specific Th1 cells that inhibit
Th2-dependent IgE class switching via production of IFN-. The
ability of CD8 T cells to activate this pathway is unique and is not
shared by CD4 T cells.
Surprisingly, Tc2 V2V5 TCR CD8 T cells also inhibited IgE. That a
type 2 cell could induce a type 1 response runs counter to our view of
immune regulation. Tc1 and Tc2 cells both promoted Th1 cell development
and inhibited generation of Th2 CD4 T cells. Thus, this is the first
time that a type-2 T cell has been shown to promote a Th1-type
response. The significance of these observations is that activation,
possibly of any CD8 T cell possessing the appropriate TCR, has the
potential to influence the differentiation of co-responding CD4 T
cells, and therefore the development of the ensuing immune response.
This finding may have potential application to targeted vaccination to
prevent allergies.
OVA257–264-specific V2V5 TCR-transgenic
mice (15) had the same down-regulatory function as day 21
OVA-primed CD8 T cells, confirming that these inhibitory CD8 T cells
operate via the same rules of peptide/MHC recognition as wild-type
CD8 T cells. Interestingly, although these V2V5 cells were
specific for a single OVA epitope, they inhibited the IgE response to
whole OVA. This agrees with previous experiments in rats
(12) and mice (17) where IgE inhibitory CD8 T
cells from OVA-primed mice inhibited the keyhole limpet
hemocyanin or BSA-specific IgE response, provided that these
animals had been immunized with both Ags, thus allowing OVA-specific
activation of the regulatory CD8 T cell.
Because Tc2 cells made small amounts of IFN- and might have been
induced to make IFN- in vivo, we determined whether CD8 T cell
IFN- was important in IgE inhibition by using
IFN--/- mice (22). Our results
clearly show that CD8 T cells do not need to secrete IFN- to inhibit
IgE responses in vivo. However, IFN- was involved, because CD8 T
cells were unable to inhibit IgE responses when adoptively transferred
to IFN--/- mice. This finding
suggests that CD8 T cells do not directly suppress IgE via their
production of IFN-, but indirectly via IFN- produced by Th1
cells. Accordingly, inhibition of IgE could be restored by
reconstitution of IFN--/- mice with naive,
IFN--competent, CD4 T cells, confirming that IgE inhibitory CD8 T
cells indirectly inhibit IgE responses by promoting Th1 cells.
The relationship of CD8 T cell activation and prevention of IgE responses is consonant with the infection model of allergic sensitization (23) in which exposure to pathogens in early life reduces the risk of subsequent allergy. CD8 T cells are activated early in the immune responses by many pathogens. For example, infection with Listeria monocytogenes (24, 25) rapidly induces CD8 T cells, and even heat-killed Listeria can stimulate a protective CD8 response (26). CD8 T cells have been shown to promote Th1 responses in response to respiratory syncytial virus where F-protein activates CD8 T cells, leading to a protective Th1 response (27). The model described in this paper may serve as a useful system to study CD8 regulatory mechanisms relevant to infection and has the advantage that it is free of pathogen-induced effects.
A crucial effect of regulatory CD8 T cells that leads to
counter-regulation by OVA-specific CD4 T cells to inhibit IgE responses
was enhancement of IL-12 p40 production from DC. It is well recognized
that IL-12 promotes Th1 responses (28, 29, 30) and that this
can be induced by IFN- (31). Indeed, drugs such as
sulfasalazine that inhibit Th1 responses do so by inhibiting IL-12
(32). The mechanism for CD8 cell induction of IL-12 is
presently unknown, but a potential candidate is macrophage-inflammatory
protein-1, a CD8 cell chemokine that induces IL-12 synthesis by
stimulation of the CCR5 on DC (33). CD8 T cells too
produce RANTES during allograft rejection (34). We used
IL-12-/- mice (20) to investigate
the importance of IL-12 in IgE down-regulation. A crucial finding was
that neither Tc1 nor Tc2 CD8 T cells could inhibit IgE responses in
IL-12-/- mice. Further, transfer of titrated
doses of naive, IL-12-competent DC, together with OVA-primed day 21 CD8
T cells, into IL-12-/- mice restored their
capacity to inhibit IgE. In addition to IL-12, IFN--inducing factor
(IL-18) may also be involved. The IFN--inducing activity of IL-18
requires expression of the IL-18R, and IL-18R expression depends on
IL-12. Thus, IL-12-/- mice are de facto also
IL-18 deficient. Both IL-18-dependent (LPS-induced shock) and
-independent (in vivo Staphylococcus aureus,
enterotoxin-B) induction of IFN- have been described
(35).
Our results demonstrate that Ag-specific, cognate communication between
the CD8 T cell and DC is essential (17) for inhibition of
IgE. The molecules induced by this cognate interaction that actually
inhibit the IgE B cell response (IL-12 and IFN-) have yet to be
defined but are not Ag specific. This contrasts with earlier
descriptions of IgE regulatory factors before the TCR was
discovered, which appeared to be Ag specific (36, 37). CD8
T cells require OVA peptide-MHC activation on DC to stimulate IL-12,
but because IL-12 acts nonspecifically, if other Ag also are present
and are stimulating a primary IgE response, they too will be inhibited
(17). This has important consequences for therapy of
allergic disease, because it should only be necessary to stimulate CD8
T cells that recognize a single peptide to inhibit the IgE response not
only to this peptide but also to other peptides generated from that Ag,
and even to other Ag that are also present.
In contrast to CD4 T cells, CD8 T cells can be activated by TCR ligation alone (38, 39) and do not require costimulation for priming (40, 41). Indeed, CD8 T cells primed in vivo with low affinity peptides could kill efficiently in vitro (42). Strategies for facilitating such presentation of Ag to TCR, for example as an approach to treating allergies, could include DNA immunization (43, 44) and cationic lipid encapsulation of antigenic peptide that directly fuses with the APC cell membrane, thus introducing Ag into the cytosol and therefore the MHC class I processing pathway. Cross-priming in which soluble Ag enters the MHC class I pathway has been well established by a number of investigators (15, 45, 46, 47). Indeed, as whole OVA was able to induce IgE inhibitory CD8 T cells in both rats (12) and in mice as shown in this study, it is possible that one of the contributing factors to the genetic predisposition of an individual to allergies (atopy) are defects in MHC class I cross-priming, leading to a reduced ability to activate IgE-inhibiting CD8 T cells. The pathway of IL-12 activation described may be crucial for determining the apparent constitutive set point of the immune response that ensures dominance of protective immunity to newly encountered Ag by promoting Th1 over Th2 responses. This would prevent allergic sensitization by inhibiting Th2 responses and thus consequent IgE production. This pathway may contribute to outgrowing allergies in childhood. We predict that this pathway may be involved in ongoing allergies. Thus, targeting this pathway might inhibit allergic sensitization in infants and could attenuate allergic immune responses in adults.
Our findings on the CD8 T cell-DC IL-12 pathway of inhibiting IgE
responses bring molecular definition to the important processes that
down-regulate IgE. Investigation of immune regulation has long focused
on mechanisms of inducing IgE responses, such as Th2 cells, their
cytokines IL-4 and IL-13, and cell surface costimulatory
CD40:CD40-ligand-dependent signaling processes. By contrast,
down-regulation of IgE, which has immense potential clinical
application, has received less attention. Although it was known that
CD8 T cells and IFN- were involved, the precise steps were unclear.
This study details a novel mechanism of such immune down-regulation,
explaining the role of the inhibitory CD8 T cell, which stimulates DC
to produce IL-12 that activates CD4 Th1 cells to produce IFN-, which
inhibits IgE-producing B cells.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. David M. Kemeny, Department of Immunology, Guy’s, King’s, and St. Thomas’s School of Medicine, Kings College, Rayne Institute, 123 Coldharbour Lane, London, SE5 9NU, U.K. E-mail address: david.kemeny{at}kcl.ac.uk
3 Abbreviations used in this paper: DC, dendritic cell; Tc, T cytotoxic; PCA, passive cutaneous anaphylaxis; LN, lymph node.
Received for publication August 22, 2001. Accepted for publication November 1, 2001.
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) or Tc2 (
) cells were
transferred to groups of five wild-type (C57BL/6) mice (106
per recipient) immunized i.p. concurrently with 100 µg of OVA-alum.
Control mice (n = 5) were given 106
unstimulated OVA257–264-specific V
) or were
immunized with OVA-alum alone (•). The results are representative of
three independent experiments. b, Different numbers
(102–106) of in vitro primed V
) or from
IFN-
) were transferred to wild-type mice
i.p. This experiment is representative of three independent
experiments. b, A total of 106 OVA-primed
day 21 CD8 T cells from wild-type mice (
and
, respectively) or wild-type mice (