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ß-Positive, Tc1 and Tc0 CD8+ T Cell Clones Mediate the In Vivo Inhibition of Rat IgE1
Department of Immunology, King’s College School of Medicine and Dentistry, London, United Kingdom
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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(25–69 ng/ml) and IL-2 (7.6–22 U/ml), and occasionally
secrete IL-4 (68–81 U/ml IL-4), and when adoptively transferred into
CD8-depleted recipients, can effect a significant reduction in IgE (3-
to 50-fold). We also demonstrate that this in vivo inhibition of IgE is
dependent on the Ag-specific activation of the CD8+ T
cells, and that the activated CD8+ T cells will suppress
total/bystander IgE in an Ag-nonspecific manner. These data are
consistent with a growing literature demonstrating sensitization of MHC
class I-restricted CD8+ T cells by exogenous protein Ags
delivered to mucosal sites, and may represent a mechanism whereby a
selective pressure can be applied on the functional outcome of an
immunoglobulin response to environmental allergens. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, TNF-ß, or
IL-2. These cells support IgE production and effect eosinophilia
(8). We have reported previously that the i.p. immunization of Lister Hooded rats with the nonreplicating Ag OVA complexed to aluminum hydroxide results in the activation of a population of CD8+ T cells in the localized lymphoid tissue that suppress IgE when adoptively transferred to syngeneic animals (9). Moreover, in vivo depletion of CD8+ T cells at specific time points results in a marked increase in IgE (>46-fold). To further investigate these cells, we have developed a strategy for cloning OVA-specific CD8+ T cells that has made it possible to determine their precursor frequency, cell surface marker expression, cytokine profiles, cytotoxicity, and ability to regulate IgE in vivo.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Female PVG rats (125–150 g) were purchased from Harlan-Olac
(Bicester, U.K.). RPMI 1640 medium, anti-rat IFN- mAb (DB1) were
purchased from Life Technologies (Paisley, U.K.), and tissue culture
flasks and microtiter plates from Nunc (Roskilde, Denmark).
DOTAP3
(N-[1-(2, 3-dioleoxyloxy) propyl]
N,N,N-trimethyl ammonium
methylsulfate) transfection reagent was obtained from Boehringer
Mannheim Corp. (Manneheim, Germany). Rodent lymphoprep 1.077 was
purchased from Nycomed (Denmark). Human rIL-2 was purchased from
Euro-Cetus (Harefield, U.K.). Rat rIL-4 was obtained as a supernatant
from an IL-4-treated Chinese hamster ovary (CHO) cell line (kind gift
from Dr. N. Barclay, MRC Cellular Immunology Unit, Oxford, U.K.).
Dynabeads M450 sheep anti-mouse IgG were purchased from Dynal
(Wirral, U.K.), and phycoerythrin (PE)-labeled OX35 mAb (anti-rat
CD4), G4.18 (anti-rat CD3), and FITC-labeled OX-39 (anti-rat
CD25) were from AMS Biotechnology (Whitney, U.K.). OX-81 anti-rat
IL-4 was a kind gift from Dr. D. W. Mason (MRC Cellular Immunology
Unit, Dunn School of Pathology, University of Oxford, Oxford, U.K.).
DB-1 anti-rat IFN- was a kind gift from Dr. P. Van der Meide
(Leiden University, The Netherlands); OX8 anti-rat CD8 ascites
fluid, anti-
ß TCR PE (R73), anti-
TCR PE,
anti-CD25 PE, and OX8 FITC were purchased from Serotec (Oxford,
U.K.). [3H]thymidine and 51Cr were purchased
from Amersham International (Amersham, U.K.). FCS was purchased
from Globepharm (Surrey, U.K.). All other reagents were purchased
from Sigma Chemical Co. (Poole, Dorset, U.K.).
Immunization procedures
Age-, sex-, and batch-matched rats were separated into groups of four animals. Rats were immunized with alum-precipitated Ag prepared as follows. The Ag was made up to a concentration of 10 mg/ml in sterile saline. To 10 ml of the protein solution, 4.5 ml of 1 M NaHCO3 was added at 20°C for 20 min and then centrifuged at 3000 x g for 10 min. The precipitate was washed three times with sterile PBS before use and was stored with 0.01% thiomersal preservative. Rats were immunized with 100 µg of alum-precipitated Ag on day 0 and subsequently boosted on days 14, 21, and 35. CD8- depletion was achieved by one injection of 0.5 mg of OX-8 mAb on either days 10, 12, 14, or 16. Rats were bled from the tail at weekly intervals starting at day 0. The success of OX-8 depletion was determined cytofluorometrically by staining PBL and splenocytes.
Isolation of rat LN-derived CD8+ and CD4+ T cells
Rats were euthanized by a procedure approved by the United Kingdom Home Office, and their parathymic/posterior mediastinal LN were excised. Leukocytes were obtained by pressing tissue through 70-µm nylon filters (Becton Dickinson, Cowley, U.K.) into chilled HBSS. Mononuclear splenocytes/LN cells were purified on Ficoll-Hypaque. The mononuclear cells were then immediately washed twice in HBSS, and viable cell numbers were determined by trypan blue exclusion. CD8+ cells were purified by positive selection using magnetic beads. Anti-mouse IgG Dynabeads were coated overnight with OX8 (anti-CD8) or OX35 (anti-CD4) at a 1/50 dilution in PBS/0.1% BSA at 4°C. The beads were washed four times in PBS/0.1% BSA, and 4.8 x 107 beads were added to 1.5 x 108 washed mononuclear cells that had been resuspended in 2 ml of PBS/0.1% BSA and incubated at 4°C on a rolling mixer for 45 min. The attached CD8+/CD4+ T cells were collected using a magnetic particle concentrator (Dynal), and washed in culture medium. Aliquots of the CD8+/CD4+ T cells were retained and allowed to detach from the magnetic beads in RPMI/10% FCS overnight. The purity of these cells was then assessed by FACScan (Becton Dickinson). The CD8+ T cells were consistently greater than 95% pure, as assessed by staining with FITC-labeled OX8 (anti-CD8), displayed >95% staining with the anti-T cell marker OX19-FITC (anti-CD5) and less than 1% staining with anti-CD4 (OX35-PE), and are hereafter referred to as CD8+ T cells. CD4+ T cells were purified to an equivalent level.
Preparation of syngeneic APCs
Osmotic loading of target APCs with OVA or KLH was conducted as previously described (10). Briefly, the Ag was dissolved in serum-free sterile PBS and mixed with the cationic lipid transfection reagent, DOTAP, to a final concentration of 200 to 400 µg/ml in a total volume of 800 to 1000 µl for 20 min at room temperature. This mixture was then added to naive syngeneic splenocytes in rat growth medium at a concentration of 2 x 106/ml in a total volume of 30 ml, and incubated overnight at 37°C/5% CO2. OVA-pulsed splenocytes were then removed and washed twice in PBS before being irradiated at 4000 rad. Following irradiation, the splenocytes were then suspended in growth medium to a concentration of 2 x 106/ml, and used as APC syngeneic feeder cells. Trypsinization of OVA and KLH was conducted using 5 U of enzyme activity/µg of protein at 37°C for 2 h. The trypsin was inactivated following incubation of mixture at 56°C for 2 h. The trypsinated OVA/KLH was then layered onto syngeneic splenocytes in growth medium at a concentration of 100 µg/ml and incubated overnight at 37°C/5% CO2. Following irradiation, these cells were used as syngeneic APCs, as above.
Culture of rat-purified CD8+ T cells and generation of clones
Purified rat LN-derived CD8+ T cells were cultured at 5 x 105/ml in complete medium (RPMI adjusted to 310 mOsm + 2.2 g/L sodium carbonate, 0.3 g/L L-glutamine, 110 mg/L sodium pyruvate, 0.05 mM 2-ME, anti-mycotic/anti-biotic soln (100 U penicillin/100 µg streptomycin/0.25 µg ampotericin B), 1% nonessential amino acids, and 10% FCS) in 25-cm2 culture flasks at 37°C, 5% CO2. For proliferation assays, 200 µl of 5 x 105/ml CD8+ T cells were cultured in flat-bottom microtiter plates for 4 days with 2 x 106/ml of OVA-DOTAP-treated or trypsin-OVA-pulsed, syngeneic splenic feeders before addition of 0.5 µCi of [3H]thymidine per well. Eighteen hours later, the CD8+ T cells were harvested and tritium incorporation was measured using a Canberra Packard Matrix 96 direct beta counter. In cloning experiments investigating the precursor frequency of OVA-specific CD8+ T cells, the purified CD8+ T cells were placed in culture at a range of concentrations from 1000 to 0.1 cells/well in 20 µl of growth medium with 2 x 104/well OVA-DOTAP-treated feeder cells in Terasaki plates. The wells were scored for clonal growth 8 days later. All other clones were generated by culturing the purified CD8+ T cells initially at 1 x 106/ml with 2 x 106/ml OVA-DOTAP-treated feeder cells. These cells were subjected to three cycles of OVA stimulation before being placed at limiting dilution. The growth medium was changed every 5 days, and IL-2 at a concentration of 50 U/ml was given in between OVA stimulations. After the third OVA stimulation, these cells were placed in 200-µl cultures in microtiter plates at limiting dilution of 3, 1, 0.3, and 0.1 cells/well with 2 x 106/ml OVA-DOTAP-pulsed feeder cells. After 5 days of culture, the growth medium was changed and 50 U of rIL-2 was added. The wells were scored for clonal growth 8 to 10 days later. Positive wells at concentrations of 3 and 1 were discarded. Positive wells at limiting dilution concentrations of 0.3 and 0.1 were expanded by repeated stimulation in 8-day cycles with OVA-DOTAP-treated feeders plus fresh growth medium and 50 U/ml of IL-2.
IFN-, IL-4, and IL-2 secretion and cytolytic activity
IFN- was measured in supernatants from cells stimulated for
48 h with OVA-DOTAP-treated APCs by ELISA. Nunc MaxiSorp
microtiter plates were coated overnight with anti-rat IFN- mAb
(3 µg/ml in 0.1 M carbonate/bicarbonate buffer, pH 9.6). Plates were
washed in PBS/0.05% Tween-20, and samples were added for 2 h at
room temperature. Rabbit anti-IFN- antiserum was added at 1/1000
in PBS/1.5% rat serum, 0.5% Tween for 1 h, followed by
anti-rabbit IgG mAb-ALP (1/10,000) in the same diluent for 1
h. Color was developed using 0.5 mg/ml p-nitrophenyl
phosphate substrate in 0.05 M diethanolamine buffer, pH 9.8. IFN-
levels were calculated by reference to commercial IFN- standards.
The limit of detection of the assay was 0.25 ng/ml. IL-4 activity in
the 48-h supernatants was assayed using an in-house bioassay (32). The
bioassay utilizes the co-mitogenic effect that IL-4 has on naive
PMA-stimulated rat CD8+ T cells. Rat rIL-4 CHO cell
supernatant was used as a standard. The specificity of this assay was
confirmed by the ability of anti-rat IL-4 Ab supernatant (OX-81) to
block proliferation of CD8+ T cells stimulated with 10
ng/ml of PMA and positive control rIL-4 CHO cell supernatants by
greater than 90%. Briefly, unprimed purified PVG splenocytes were
placed in triplicate culture for 48 h at a concentration of 5
x 104/ml with 50, 25, and 12.5 µl of sample supernatant
+/- OX-81 (anti-rat IL-4 supernatant) at a 1/100 dilution +
10 ng/ml of PMA. rIL-4 CHO cell supernatant was used as a positive
control. Cells were pulsed with 0.5 µCi of
[3H]thymidine per well after 48 h, and the plates
were harvested and counted 18 h later. The difference in mean
proliferation - OX-81 was used to express IL-4 activity as an
IL-4-induced proliferative index.
IL-2 activity was measured in supernatants from cells using a CTLL cell line (ECACC 87031904). Human rIL-2 was used as a standard. The lower detection limit was 0.2 U/ml. The specificity of this assay was confirmed by the failure of rat rIL-4 to stimulate the CTLL cells at any concentration, and the ability of anti-mouse IL-2R Ab to block proliferation by more than 85%. Overall homology between rat and mouse IL-4 protein sequences is relatively low (61%), and there is no reported functional homology.
The cytolytic activity of the clones was tested by 51Cr release. OVA-DOTAP-pulsed target cells were radiolabeled with 200 µCi (7.4 MBq) of Na251CrO4 at 37°C for 1 to 2 h and washed four times in rat growth medium. Triplicate microtiter cultures at various CD8+ T cell E:T ratios were set up in round-bottom microtiter plates using 1 x 105 51Cr-labeled targets/well in 200 µl of rat growth medium. Following incubation of targets with effectors for 4 h at 37°C, 5% CO2, the plates were centrifuged at 200 x g for 5 min. Fifty microliters of supernatant were removed from each well, and 51Cr release was determined according to standard methods (11).
Serum IgE ELISA
Serum IgE was measured by ELISA. Throughout, 50-µl volumes
were used, and the assay temperature was 4°C. Microtiter plates were
coated with anti-rat IgE (MARE-1) at 0.5 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, and duplicate
serum samples diluted 1/10 or greater in assay diluent (PBS/0.5% horse
serum/0.05% Tween-20) were added. After 2 h at room temperature,
the plates were washed as before, and labeled anti-rat Ig 
light
chain (MARK-1-peroxidase) Ab at 0.5 µg/ml in assay diluent was added.
After 2 h at room temperature, the plates were washed as before,
and TMB (3,3',5,5'-tetramethyl benzidine) substrate (ImmunoPure
Microwell peroxidase substrate kit Pierce & Warriner, Chester, U.K.)
was added according to the manufacturer’s instructions at 25°C. The
absorbance was read at 450 nm in a Titertek Multiskan plate reader (ICN
Flow, High Wycombe, U.K.), and the results were expressed as ng/ml by
reference to the standard curve constructed using dilutions of rat IgE
myeloma protein IR162 from 250 to 1 ng/ml. The interassay coefficient
of variation was 4.94% over 32 separate assays.
Ag-IgG ELISA
Specific IgG Ab to OVA was determined by ELISA. Microtiter plates were coated with Ag at 30 µg/ml in coating buffer overnight at 4°C and washed three times, as above. Test and control serum samples diluted at 1/100 or greater in assay diluent were added in duplicate. After 2 h at 4°C, the plates were washed three times, as above, and alkaline phosphatase-conjugated rabbit anti-rat IgG diluted at 1/300 was added. After 1 h, the plates were again washed three times, as above, and a 1 mg/ml solution of p-NPP in diethanolamine buffer (pH 9.8, 0.1 M) was added at 100 µl/well and incubated at 25°C for 1 h. Absorbance was read at 405 nm, and the results were expressed as arbitrary U/ml by reference to a standard curve constructed using a positive serum pool at dilutions of 1/30 to 1/30,000. The interassay coefficient of variation was 6.9% over 32 separate assays.
Statistical analysis
All results were expressed as means ± SD from cohorts of four rats. All analyses have been conducted on an Apple Macintosh computer using the statistical package Stat view 512+, using the Mann-Whitney U test.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To determine whether CD8+ T cells are important
during the early part of the immune response to OVA-alum, PVG rats were
depleted of CD8+ T cells by a single injection of 0.5 mg
OX-8 mAb at different times during the primary immune response that
coincided with the appearance of CD25-positive CD8+ T cells
(data not shown) in the lymph nodes (parathymic/mediastinal) that drain
the peritoneal cavity (days 10, 12, 14, and 16). Control animals were
given isotype-matched IgG. The percentage of CD8+ T cells,
CD4+ T cells, and NK cells was determined by double
labeling of the cells for CD3/CD8, CD3/CD4, and CD3/NKRP.1. In
OX-8-treated animals, the proportion of CD8+ T cells in the
peripheral blood was reduced significantly (0.24 ± 0.2%) as
compared with control animals (17.36 ± 3.2%). In the spleen, the
proportion of CD8+ T cells remaining was 1.3 ± 0.6%
as compared with the control animals (14.3 ± 2.1%) corresponding
to more than 92% depletion. The proportion of CD4+ T cells
in the spleen and peripheral blood was increased in the OX-8-treated
rats, as predicted by the loss of the CD8+ T cells. Total
IgE was monitored until day 28. All rats depleted after day 10 showed a
massive enhancement in total IgE (Fig. 1
); the maximal response was seen in day
16 depleted rats, in which titers of 16,480 ng/ml were found, as
compared with undepleted controls of 220 ng/ml, representing a 46-fold
increase. OVA-specific IgG Abs increased in all immunized animals, but
were unaffected by CD8 depletion.
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The optimum stimulus for inducing OVA-specific proliferation of
the day 14 LN-derived CD8+ T cells purified to >95%
(see Materials and Methods) was determined. Figure 2 illustrates the proliferation of
purified lymph node-derived CD8+ T cells cultured with
irradiated APCs treated with DOTAP-OVA complexes (see Materials
and Methods) compared with cells cultured with trypsinized
OVA-pulsed APCs or whole OVA-pulsed APCs. The stimulator cells were
prepared as above and used at a concentration of 1 x
106/ml. KLH was used under identical conditions as
irrelevant Ag controls. The CD8+ T cells were purified as
above from the posterior mediastinal/parathymic LN on day 14
postimmunization. These cells were used at a concentration of 1 x
106/ml cells and proliferated vigorously (27,000 cpm)
compared with control cultures of purified cells with
OVA-peptide-pulsed APCs (2000 cpm) or with whole OVA-pulsed APCs (800
cpm). The KLH-irrelevant Ag controls were significantly lower than the
OVA-DOTAP cultures (2,900 cpm), but were not significantly lower in
either the peptide or whole OVA cultures.
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The responder frequency of OVA-specific CD4+ and CD8+ T cells was determined at limiting dilution utilizing Taswell’s algorithm (12). The cells were removed from the posterior mediastinal and parathymic LN, purified as above, and cultured at various concentrations (1000–0.1 cells/well) with irradiated OVA-DOTAP-treated syngeneic spleen cells at a concentration of 2 x 106 cells/ml in a total volume of 20 µl in Terasaki plates. The wells were scored for clonal growth 8 days later, and the number of positive/negative wells compared with the cell concentration and the responder frequency was calculated. The frequency of responders shows good linearity for both CD8+ and CD4+ T cells, with a mean precursor frequency of OVA-specific CD8+ T cells in cloning experiments of 1 cell in 3371 ± 611, which compares with an OVA-specific CD4+ T cell responder frequency of 1 cell in 526 ± 158.5.
Proliferation of CD8+ T cell clones with OVA-DOTAP-treated APC stimulators vs KLH-DOTAP APC-irrelevant Ag controls
The antigenicity of the CD8+ T cell clones was
tested by culturing 1 x 105 clonal cells with an
equal number of OVA-DOTAP (black bars)- or KLH-DOTAP (white
bars)-treated APCs (see Materials and Methods) for
48 h, and the proliferation was tested by
[3H]thymidine incorporation. All of the six clones tested
proliferated at a significantly higher rate with the OVA-treated APCs
than with the KLH-treated APCs, with clone OACD8:24 proliferating at
the highest level (12,900 cpm ± 3,600 cpm in OVA cultures vs
2,300 ± 760 cpm in KLH cultures). This suggests that these cells
are OVA specific (see Fig. 3).
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The MHC restriction of the OVA-specific CD8+ T
cells was determined using anti-MHC class I and II blocking Abs.
CD8+ T cell clones were placed in culture with increasing
concentrations of anti-MHC I RT1A (0.01–1 m/ml), which is targeted
to a monomorphic determinant on rat MHC class I molecules (Fig. 4a), and anti-MHC
II RT1B (0.01–10 mg/ml), which is directed to a monomorphic
determinant on rat MHC class II (Fig. 4b). The
anti-MHC I significantly reduced the OVA-specific proliferation of
all of the responder CD8+ T cells (approximately 57%
inhibition over six clones tested) at the highest concentration. The
addition of anti-MHC II mAb had no significant effect on the
DOTAP-OVA-treated APC-induced CD8+ T cell clonal
proliferation.
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ß, and TCR-; production
of IL-2, IL-4, and IFN-; and levels of cytotoxicity of OVA-specific
CD8+ T cell clones
Cell surface expression of CD8, CD4, CD3, TCR-ß, and
TCR- was determined by FACS using 1 x 106
cells from each of the six CD8+ T cell clones. The cloned
cells were labeled with OX8 FITC (anti-CD8), OX35 PE
(anti-CD4), anti-CD3 FITC, anti-ß TCR FITC, and
anti- TCR FITC. All clones expressed CD8, CD3, and TCR-ß
to varying degrees, and all clones tested were found to be CD4
negative, TCR- negative. Hence, the OVA-specific CD8+
T cell clones generated in this system are
CD8+CD3+ß TCR+. To assess the
capacity of the CD8+ T cell clones to produce Th1 and Th2
cytokines, the secretion of IL-4 and IFN- from the OVA-specific
CD8+ T cell clones was determined following stimulation for
48 h by OVA-pulsed APCs. Supernatants were removed and stored at
-20°C until use. IL-4 and IL-2 were measured by bioassay (33), and
IFN- by ELISA. All of the CD8+ T cell clones secreted
IFN- (range 25.42–69.05 ng/ml) and IL-2 (range 22–37 U/ml), and
four also made IL-4 (range 68–81 U/ml). These six clones were tested
for their ability to lyse 51Cr-labeled OVA-pulsed
splenocyte targets. The efficiency of killing was assessed using a
51Cr release assay. Clones were rested in growth medium
supplemented with 50 U/IL-2 for 14 days. Five of the six
CD8+ T cell clones tested displayed cytolytic activity over
a range of target APCs:CD8+ T cell effector ratios. Peak
activity was seen at an E:T ratio of 1:500, and it is the percentage
lysis at this E:T ratio that is shown above (Table I).
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The ability of the six OVA-specific CD8+ T cell
clones to regulate IgE in vivo was tested (Fig. 5). A substantial IgE response was
induced in syngeneic PVG rats by the day 14 CD8+ T cell
depletion method described above. On day 21 postimmunization, 1 x
106 cloned CD8+ T cells in sterile saline
solution were adoptively transferred i.v. to recipient rats. Control
animals received a similar volume of sterile saline. All clones tested
significantly reduced the resulting IgE response when compared with
saline-treated animals (range 3- to 50-fold). OVA-specific IgG was also
increased following OVA-alum immunization. However, the magnitude and
duration of this response were unaffected by CD8 depletion or by the
adoptive transfer of the CD8+ T cell clones (data not
shown). The SE bars have been removed for clarity, with the maximum
variation being no greater than 21% for each sample tested.
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We titrated the in vivo IgE-inhibitory effects of clones OACD8.45
and OACD8.53 by adoptively transferring these clones at a range of
concentrations into CD8-depleted recipients (Fig. 6, a and b)
on day 21 postimmunization. The resulting effects on the
OVA-alum-induced IgE response are expressed as serum total IgE from day
35 postimmunization; KLH-alum was used as the irrelevant Ag control.
Clone OACD8.45 displayed significant IgE immunoregulatory activity at
concentrations of 1 x 106 cells down to 1 x
104 cells (15- to 2-fold inhibition) per animal (Fig. 6a); it also significantly reduced the response to
KLH-alum when 1 x 106 cells were transferred, but not
at lower concentrations. The IgE immunoregulatory activity of clone
OACD8.53 on OVA-alum-induced IgE was lost at concentrations of less
than 1 x 106 cells per animal (3- to 2-fold) (Fig. 6b). The response to KLH-alum was unaffected by the
adoptive transfer of this clone at all concentrations tested. The
IFN- secretion and cytolytic activity of these two clones are
comparable.
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We tested the effect of OVA-specific CD8+ T cell
clones on a bystander IgE response in vivo. This was achieved by
adoptively transferring 1 x 106 cells of clones
OACD8.45 and OACD8.53 into animals immunized i.p. with OVA-alum,
KLH-alum, or both OVA-alum and KLH-alum following CD8 depletion, as
above (Fig. 7). In animals
given OVA-alum only, the IgE response was reduced by the adoptive
transfer of both of the clones (17-fold for clone OACD8.45 and 5-fold
for OACD8.53 in this experiment). As before, clone OACD8.45 induced a
significant decrease in KLH-alum-induced IgE in animals given KLH-alum
only, but the effect was reduced greatly when compared with the
OVA-alum-induced response (3-fold reduction). Clone OACD8.53, as
before, had no effect on the animals given KLH-alum only. Both of the
clones tested induced a significant reduction in total IgE in animals
given both KLH-alum and OVA-alum, which was comparable with that of
OVA-alum alone (14-fold for clone OACD8.45 and 4-fold for
OACD8.53).
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on OVA-alum-induced
IgE responses
All six clones tested produced IFN- on in vitro Ag stimulation.
A blockade of IFN- by the administration of 0.5 mg of the mAb DB.1
(anti-rat IFN-) was effected on day 21 post-OVA-alum
immunization (Fig. 8). The study groups included animals that were CD8
depleted on day 14, and animals that were CD8 depleted on day 14
postimmunization, then reconstituted on day 21. The anti-IFN-
was introduced i.p. at the same time as 1 x 106
cells of the IgE-inhibitory clone OACD8.45 were administered i.v. The
blockade of IFN- significantly increased the production of IgE in
all of the subject groups tested, including naive unimmunized control
animals (23 ± 16 ng/ml to 3980 ± 551 ng/ml). The
anti-IFN- also greatly reduced IgE-inhibitory activity of clone
OACD8.45 (5-fold to 1-fold). The only group not significantly affected
by the administration of anti-IFN- was the OVA-alum-immunized
group CD8+ depleted on day 14.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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secretion by MHC I-restricted CD8+ T cells,
resulting in a cytokine milieu in the lymphoid microenvironment
unfavorable to Th2 T cell development (23). It was proposed previously that rat CD8+ T cells were important in IgE regulation in studies of airway responses to OVA (24). An investigation into castor bean sensitization provided further evidence for the importance of CD8+ T cells in controlling the magnitude and duration of IgE responses (25). However, the mechanisms by which these CD8+ T cells are recruited and activated, the way in which they interact with accessory cells and recognize Ag, as well as the way that they mediate their effects remained unclear. The present study sought to define the nature of these CD8+ T cells by generating CD8+ T cell clones from the posterior mediastinal and parathymic lymph nodes of day 14 OVA-alum-immunized PVG RT7b rats. At this time point, in vivo CD8 depletion is known to enhance the IgE response, and an activated population of CD3+ T cells could be distinguished that were CD8high and CD25high (data not shown). When purified, these cells exhibited a moderate proliferative response to trypsinized OVA-pulsed APCs and a strong proliferative response to OVA-DOTAP-treated APCs. Following three cycles of Ag stimulation, the responding CD8+ T cells were placed in culture at limiting dilution with irradiated OVA-pulsed APCs. This approach facilitated the single cell analysis of the CD8+ T cells of interest.
The responding CD8+ T cells were MHC class I restricted: a mAb specific for a monomorphic determinant on MHC class I effected a significant decrease in the OVA-specific proliferative response of the purified CD8+ T cells. A mAb specific for a monomorphic determinant on MHC class II had no effect, even at 10-fold higher concentration.
Inherent in our understanding of these data is the concept that the exogenous OVA introduced into the peritoneal cavity is able to activate CD8+ T cells in the context of MHC class I. The production of most peptides destined for the presentation by MHC class I begins in the cytosol with the limited hydrolysis of antigenic proteins. Until recently, it was thought that this pathway was reserved solely for endogenously synthesized Ag. However, it is now clear from studies of murine macrophage and dendritic cells that exogenous protein Ags can gain entry to the cytosol of potential APCs, viz the MHC class I pathway (26), and that this occurs quite naturally in vivo as part of an important immunologic defense against pathogens that reside in the phagosomal intracellular compartment (27). There are also additional reports that suggest that CD8+CTLs can be primed by exogenous Ags (28, 29, 30) and of in vivo activation of CD8+ T cells to soluble OVA that mediate the adoptive transfer of oral and nasal tolerance (31, 32).
The frequency of OVA-specific CD8+ T cells was much lower
than for the corresponding CD4+ T cells purified at the
same time and cultured under comparable conditions (approximately 1:6
responding CD4:CD8 cells). This suggests that most of the exogenous OVA
introduced into the peritoneum is directed via the conventional MHC
class II pathway, to OVA-specific CD4+ T cells. The
OVA-specific CD8+ T cell clones generated could be
separated into two groups based upon their cytokine secretion following
Ag stimulation. All of the clones secreted IFN- and IL-2, and a
small number also secreted IL-4. The existence of functionally distinct
subsets within the CD8+ T cell pool is well recognized,
with evidence from transgenic mouse and rat systems (33, 34, 35). It has
been proposed from Mycobacterium leprae studies that
noncytotoxic human CD8+ T cell clones secrete IL-4 (36).
However, when the cytolytic potential of six of the OVA-specific clones
was tested with OVA-treated splenocyte targets, there was no
correlation between IL-4 secretion and a lack of cytotoxic activity,
with five of the six clones tested displaying efficient killing of
targets. Thus, the relationship between cytokine secretion and
functional heterogeneity remains ill defined.
The OVA-specific CD8+ T cell clones generated following the
peritoneal introduction of OVA-alum were TCR-ß+,
TCR-, CD8+, CD4-, and
CD3+. This is in contrast to models based on the inhalation
of Ag, in which there is evidence to suggest that IgE immunoregulatory
CD8+ T cells produced following inhalation of soluble OVA
express the TCR- chains (37). The IgE immunoregulatory potential
of six of the clones was tested by adoptive transfer of 1 x
106 cells into CD8-depleted syngeneic recipients. All of
the clones transferred effected a significant decrease in
OVA-alum-induced IgE in vivo without affecting the levels of
OVA-specific IgG, and in the case of clone OACD8.45, this effect could
be titrated down to as few as 1 x 104 cells. This
clone also displayed some nonspecific inhibition of KLH-alum-induced
IgE in control animals, but this effect was only evident at the highest
concentration of cells used (1 x 106), suggesting
that there may be both Ag specific and Ag-nonspecific aspects to the
inhibitory mechanisms displayed by these clones. Additional experiments
have suggested that the in vivo inhibition of IgE by OVA-specific
CD8+ T cell clones may require the activation of these
cells in vivo by OVA. However, following triggering by OVA, the clones
inhibit IgE in an Ag-nonspecific manner, as displayed by their
reduction of KLH-alum-induced IgE in animals given both KLH and OVA.
Several studies have shown similar effects with Ags delivered via the
oral route (38), and hence, this could be a general mechanism
associated with the delivery of Ags to mucosal sites. We looked for a
correlation between the cytolytic activity of the clones and their
immunoregulatory potential, as several recent reports have intimated
that cytotoxic lymphocytes elicited during an ongoing immune response
against a soluble Ag may reduce the magnitude of the antigenic stimulus
by killing APC targets (14, 39). However, we could find no significant
correlation between IgE levels and cytotoxicity. We also determined
whether there were direct links between in vitro IFN- production and
IgE immunoregulatory potential, as there have been precedents for this
thesis in recent reports detailing immune deviation of the atopic T
cell response away from the pathogenic Th2 phenotype (23). Neutralizing
anti-rat IFN- monoclonal administered i.p. increased IgE in
animals that received OVA-specific CD8+ T cell clones, but
also increased IgE to a great extent in control animals. This
demonstrates that IFN- is an extremely important mediator of IgE
responses in vivo. However, these data are inconclusive with respect to
the CD8+ T cell clones, as the IFN- monoclonal did not
completely ablate the IgE-inhibitory activity and would of course block
CD4+ and NK cell-derived IFN-. Hence, it seems likely
that the mechanisms underlying the IgE-specific immunoregulatory
activity of CD8+ T cells involve several factors, such as
cytokine secretion, cytotoxicity, cell motility, and survival, and
could combine to mediate the in vivo effect. These mechanisms are
currently under investigation in our laboratory.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. D. M. Kemeny, Department of Immunology, King’s College School of Medicine and Dentistry, Bessemer Road, London SE5 9PJ, U.K.
3 Abbreviations used in this paper: DOTAP, N-[1-(2, 3-dioleoxyloxy) propyl] N,N,N-trimethyl ammonium methylsulfate; CHO, Chinese hamster ovary; KLH, keyhole limpet hemocyanin; LN, lymph node; PE, phycoerythrin.
Received for publication December 19, 1996. Accepted for publication September 24, 1997.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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T cells. Science 265:1869. and IL-4 regulate the growth and differentiation of CD8+ T cells into subpopulations with distinct cytokine profiles. J. Immunol. 155:2928.[Abstract]
T cells down-regulate primary IgE responses in rats to inhaled soluble protein antigens. J. Immunol. 154:4390.[Abstract]
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