of June 13, 2013.
This information is current as
Exposure to Inhaled Ovalbumin
T Cells Changes with
The Influence of IgE-Enhancing and
Willi K. Born
and Aydintug, J. M. Wands, Hua Huang, Rebecca L. O'Brien
Yafei Huang, Niyun Jin, Christina L. Roark, M. Kemal
2009; 183:849-855; Prepublished online 19 June
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The Journal of Immunology
by guest on June 13, 2013
The Influence of IgE-Enhancing and IgE-Suppressive ?? T
Cells Changes with Exposure to Inhaled Ovalbumin1
Yafei Huang,*†Niyun Jin,*†Christina L. Roark,*†M. Kemal Aydintug,*†J. M. Wands,*†
Hua Huang,*‡Rebecca L. O’Brien,*†and Willi K. Born2*†
It has been reported that the IgE response to allergens is influenced by ?? T cells. Intrigued by a study showing that airway
challenge of mice with OVA induces in the spleen the development of ?? T cells that suppress the primary IgE response to
i.p.-injected OVA-alum, we investigated the ?? T cells involved. We found that the induced IgE suppressors are contained within
the V?4?subset of ?? T cells of the spleen, that they express V?5 and CD8, and that they depend on IFN-? for their function.
However, we also found that normal nonchallenged mice harbor IgE-enhancing ?? T cells, which are contained within the larger
V?1?subset of the spleen. In cell transfer experiments, airway challenge of the donors was required to induce the IgE suppressors
among the V?4?cells. Moreover, this challenge simultaneously turned off the IgE enhancers among the V?1?cells. Thus, airway
allergen challenge differentially affects two distinct subsets of ?? T cells with nonoverlapping functional potentials, and the
outcome is IgE suppression. The Journal of Immunology, 2009, 183: 849–855.
heightened as ever-increasing proportions of the world’s popula-
tion suffer from allergies (2). In healthy mammals, IgE Abs ac-
quired via the gastrointestinal tract by the newborn may serve as a
first line of defense (3). IgE is synthesized and functions in the
normal adult largely in the mucosal tissues where the IgE concen-
trations are high, whereas concentrations of IgE in the circulation
remain low by comparison with other Igs (1). Mechanisms re-
sponsible for this biased anatomical distribution include the dis-
tribution and longevity of cells that express the receptors for
IgE, as most of the IgE in the tissues is cell bound and thus
protected from degradation, and local IgE synthesis, which is
favored by the Th2 environment of the mucosal tissues that
maintains local IgE levels (1).
IgE Abs are also induced during vaccination. Aluminum adju-
vants (alum), currently the most widely used adjuvants in human
and animal vaccines, stimulate the innate system and can favor
Th2-biased reactivity (4). Immunization of previously untreated
laboratory animals with soluble inert protein Ags using alum typ-
ically elicits Th2-type responses, accompanied by the development
of IgE Abs. However, the outcome of this type of immunization
also depends on prior exposure. Mucosal exposure to the same Ag
may result in nonresponsiveness and the failure of the immuniza-
tion to elicit Th2 reactivity (5). For example, repeated airway
ntibodies of the IgE class are prominent in the host re-
sponse to parasitic infections and in allergic responses to
many nonpathogenic Ags (1). The interest in IgE has
challenge of rodents with OVA without adjuvant leads to non-
responsiveness to a subsequent i.p. injection of OVA-alum,
which otherwise would induce Th2 reactivity and a strong OVA-
specific IgE response (6). How airway exposure alters the outcome
of OVA-alum immunization is not yet fully understood (7).
Several groups have provided evidence that ?? T cells can mod-
ulate the OVA-alum-induced IgE response. Investigating the de-
velopment of tolerance to inhaled OVA, McMenamin et al. (8)
found that ?? T cells from tolerized mice efficiently and selectively
suppressed primary OVA-alum-induced IgE responses. In appar-
ent contrast, others reported that ?? T cells are required for the
development of IgE responses to OVA and other Ags (9, 10). The
underlying mechanisms responsible for these apparently opposing
observations remained unresolved.
That ?? T cells can exert both Th1-like and Th2-like effects
on the immune responses to pathogens has been recognized
(11), and later studies revealed the surprising circumstance that
these different and sometimes opposed functional effects on the
host responses segregate with TCR-V?-definable subsets of ??
T cells, such as V?4?and V?1??? T cells (12, 13). Specific
functional contributions of these and other TCR-defined subsets
suggested that the ?? TCR determines not only ligand speci-
ficity of ?? T cells but also their functional potential (14). Con-
sistent with this concept, a recent study showed that the ability
of ?? T cells with specificity for the T22 molecule to express
IL-17 and IFN-? depends on TCR-ligand interactions during
their development (15).
Given the divergent observations regarding the role of ?? T cells
in the IgE response, we were interested in determining whether
different TCR-V?-definable subsets of ?? T cells also exert op-
posed effects on IgE production. In the current study, we took
advantage of the observation that normal mice immunized with a
single i.p injection of OVA-alum make a primary IgE response to
OVA. The results of these experiments indicate that V?1??? T
cells are able to enhance the primary IgE response induced by
OVA-alum, whereas V?4?cells in contrast are able to suppress it.
Moreover, we found that in addition to their different functional
potentials, the overall effect of the IgE-modulating ?? T cells crit-
ically depends on the exposure history of the animal. Whereas the
*Integrated Department of Immunology, National Jewish Health, Denver, CO 80206;
†University of Colorado, Denver, CO 80206; and‡Department of Medicine, National
Jewish Health, Denver, CO 80206
Received for publication December 9, 2008. Accepted for publication May 6, 2009.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1This work was supported by National Institutes of Health Grants AI40611 and
HL65410 (to W.K.B.) and AI44920 and AI063400 (to R.L.O.).
2Address correspondence and reprint requests to Dr. Willi K. Born, Integrated De-
partment of Immunology, National Jewish Health, 1400 Jackson Street, GB K409,
Denver, CO 80206. E-mail address: email@example.com
Copyright © 2009 by The American Association of Immunologists, Inc. 0022-1767/09/$2.00
The Journal of Immunology
by guest on June 13, 2013
IgE-enhancing ?? T cells lose this ability upon airway allergen-
exposure, the IgE-suppressive ?? T cells gain theirs under the
Materials and Methods
Female C57BL/6 mice and several mutant strains of the same genetic back-
ground (B6.TCR-??/?, B6.TCR-??/?, B6.TCR-??/???/?) were obtained
from The Jackson Laboratory. TCR-V?4?/?6?/?mice deficient in V?4?
and V?6?T cells were a gift from Dr. K. Ikuta (Kyoto University, Kyoto,
Japan). They were backcrossed to the C57BL/6 genetic background and
used after 11 backcross generations. B6.TCR-??/?IFN-??/?mice were
generated by crossing the single mutants and breeding double mutants
identified in the F2 generation. B6.TCR-V?1-transgenic mice were a gift
from Dr. Pablo Pereira (Institut Pasteur, Paris, France). All mice were 8–12
wk old at the time of the experiments. Mice were maintained on an OVA-
free diet and were cared for at National Jewish Health (Denver, CO), fol-
lowing guidelines for immune deficient animals. All experiments were con-
ducted under a protocol approved by the Institutional Animal Care and Use
Ag exposure and immunization
The animals were exposed to 1% OVA (w/v) (5? crystalline; EMD Bio-
sciences) in saline aerosol inhalation for 30 min daily, 5 days/wk for up to
2 wk, and subsequently once per wk (designated 10N in this paper), fol-
lowing a method described by others (8). To induce OVA-specific IgE,
mice were immunized by i.p. injection of 10 ?g of OVA in aluminum
hydroxide (AlumImject; Pierce Scientific) (8).
Treatment with Abs against the TCR
Hamster pan anti-TCR-C? mAbs (clone GL3), anti-V?4 mAb (clone UC3),
and anti-V?1 mAb (clone 2.11) were purified from hybridoma culture su-
pernatants using a protein G-Sepharose affinity column (Amersham Phar-
macia Biotech). T cells were targeted by injection of 200 ?g of hamster
anti-TCR-?, anti-V?4 or anti-V?1 mAbs into the tail veins of mice 4 days
before the i.p. immunization with OVA. The effect of these treatments on
the targeted T cells was monitored as previously described (13). This ap-
proach, which is based on staining with non-cross-blocking anti-TCR
mAbs, allows an assessment of the effect of treatment on TCR expression,
but it does not assess the fate of the targeted T cells. The antibody treat-
ments transiently reduce TCR expression by ?90%. Sham Ab treatments
were performed with nonspecific hamster IgG (The Jackson Laboratory).
Throughout this article, we use the nomenclature for murine TCR-V?
genes introduced by Heilig and Tonegawa (16).
T cell purification from spleen and lung
Total spleens and lungs were harvested from naive or 10N mice at the time
of the experiments. Lungs were dissected into small pieces and exposed to
an enzymatic digestion mixture containing 0.125% dispase II (Roche),
0.2% collagenase II (Sigma-Aldrich), and 0.2% collagenase IV (Sigma-
Aldrich) for 90 min at 37°C. After enzymatic digestion, a single-cell sus-
pension was prepared by pushing the lung tissue fragments through a 70-
?m-diameter nylon mesh (BD Falcon). A suspension of splenocytes was
prepared by mechanical dispersion. Cell suspensions were treated with
Gey’s RBC lysis solution and passed through nylon wool columns to ob-
tain T lymphocyte-enriched cell preparations containing ?75% T cells as
previously described (13). Total cell counts were determined using a
Adoptive transfer of ?? T cells
Splenic nylon wool-nonadherent (NAD) cells from naive and 10N mice
(B6.TCR-??/?, B6.TCR-??/?IFN-??/?) were incubated with biotinylated
anti-V?4 mAb (clone UC3) or anti-V?1 mAb (clone 2.11) for 15 min at
4°C, washed, and incubated with streptavidin-conjugated magnetic beads
(Streptavidin Microbeads; Miltenyi Biotec) for 15 min at 4°C and passed
through magnetic columns to purify V?4?or V?1?cells as previously
described in detail (17). This produced a cell population containing ?90%
V?4?or V?1?viable cells as determined by two-color staining with anti-
TCR-? and anti-V?4 or anti-V?1 mAbs. These splenic V?4?or V?1?
cells were washed in PBS and resuspended to a concentration of 1.5 ? 105
cells/ml PBS, and 3 ? 104cells/mouse were injected in 200 ?l of PBS
via the tail vein into B6.TCR-??/?mice directly before the OVA
In some experiments, subpopulations of V?4?or V?1?cells were pu-
rified using the MoFlo cell sorter. NAD cells were incubated with FITC-
conjugated anti-V?4 mAb (clone UC3) or FITC-conjugated anti-V?1 mAb
(clone 2.11) and PE-conjugated anti-CD8? (clone 53-6.7; BD Pharmingen)
or biotinylated anti-V?5 (clone F45-152), followed by PE-conjugated
streptavidin (20 min at 4°C), and then washed. Cells were next sorted based
on their expression of V? chain and V?5 or CD8? using a MoFlo cell
sorter (Dako Cytomation). Purified cells were washed in PBS and resus-
pended to a concentration of 1.5 ? 105cells/ml of PBS, and 3 ? 104
cells/mouse injected in 200 ?l of PBS via the tail vein into B6.TCR-??/?
mice directly before the OVA immunization. In general, the cell sorter-
purified cells were less effective than those prepared by magnetic bead
selection and therefore have only been compared with each other.
Flow cytometric analysis
For flow cytometric analyses, NAD cells (2 ? 105/well) in 96-well plates
(Falcon; BD Biosciences) were stained for TCR expression using a PE-labeled
pan C? Ab (clone GL3) and FITC-conjugated anti-V?1 (clone 2.11), or anti-
V?4 (clone UC3) Ab followed by biotinylated anti-V? mAbs: anti-V?4 (clone
GL-2), anti-V?5 (clone F45-152), anti-V? 6.3 (clone 17-C), anti-V?6?12
(clone F4.22) and anti V?8 (clone B20.1), plus streptavidin-allophycocyanin.
All samples were analyzed on a FACScan flow cytometer (BD Biosciences)
counting a minimum of 25,000 events per gated region, and the data were
processed using FlowJo 6.4.1 software (Tree Star).
Determination of serum IgE levels
Sera were harvested on day 14 after the i.p. immunization with OVA-alum.
For OVA-specific serum IgE determinations, plates were coated overnight
at 4°C with 2 ?g/ml rat anti-mouse IgE Ab (clone R35-72; BD Bio-
sciences). The serum samples were then added, and biotin-labeled OVA
subsequently added to the wells. Before biotinylation, OVA was first dia-
lyzed at 4°C overnight against 0.1 M borate buffer (pH 8.4). Biotinylated
OVA was then prepared by reacting 1 ml of OVA in PBS (1 mg/ml) with
150 ?l of N-hydroxysuccinimidobiotin in DMSO (1 mg/ml) for 4 h at room
temperature, followed by overnight dialysis against PBS at 4°C. The bound
OVA-biotin was detected with streptavidin-conjugated HRP (BD Bio-
sciences) followed by 100 ?l/well of TMB substrate solution. OVA-spe-
cific IgE levels in the samples were compared with an internal standard
obtained from pooled sera of hyperimmunized BALB/c mice, which was
arbitrarily assigned to equal 1000 ELISA U. Total IgE levels were mea-
sured by sandwich ELISA using rat anti-mouse IgE at 2 ?g/ml (clone
R35-72; BD Biosciences) as a capture Ab followed by biotinylated rat
anti-mouse IgE H chain mAb (clone R35-118; BD Biosciences) at 2 ?g/ml,
and detected as described above. All samples were read using a VERSA-
max tunable microplate reader and processed using a SoftMax Pro 4.7.1
Data are presented as means ? SEM. The unpaired t test was used for two
group comparisons, and ANOVA was used for analysis of differences in
three or more groups. Statistically significant levels are indicated as fol-
lows: ?, p ? 0.05; ??, p ? 0.01; ???, p ? 0.001; NS, not significant.
Altered IgE response in ?? T cell genetically modified mice and
in normal mice treated with Abs against the ?? TCR
To test the concept that ?? T cells are capable of modulating the
IgE response, we initially examined genetically modified mice,
including mice lacking all T cells (B6.TCR-??/???/?), all ?? T
cells (B6.TCR-??/?), V?4?and V?6??? T cells only (B6.TCR-
V?4?/?6?/?), and mice expressing a rearranged V?1J?4C?4
transgene (B6.TCR-V?1 Tg), plus wild-type controls (C57BL/6).
We also examined mice lacking all ?? T cells (B6.TCR-??/?).
Total serum IgE levels were measured by ELISA, in adult age- and
sex-matched untreated mice, and in mice injected i.p. with OVA-
alum, 14 days after the injection (Fig. 1a). Without treatment,
basal serum IgE levels were low in comparison with wild-type
controls in mice lacking all ?? T cells (B6.TCR-??/?vs C57BL/6,
p ? 0.01), but high in mice deficient in V?4?and V?6??? T cells
(B6.TCR-V?4?/?6?/?vs C57BL/6, p ? 0.0001) and in mice ex-
pressing the V?1J?4C?4 transgene (B6.TCR-V?1 transgenic vs
C57BL/6, p ? 0.001). Mice lacking ?? T cells did not express IgE
at significant levels. At day 14 after OVA-alum injection, all mice
except those lacking ?? T cells exhibited substantially increased
850IgE REGULATION BY ?? T CELLS
by guest on June 13, 2013
serum IgE levels, and the relative increases in IgE resembled the
pattern of the untreated mice.
In a follow-up experiment with wild-type C57BL/6 mice
only, we again examined serum IgE levels on day 14 after the
i.p. OVA-alum injection, comparing groups that had been
treated with Abs against TCR-?, TCR-V?4, and TCR-V?1 or
with nonspecific Ig, 4 days before the OVA-alum sensitization
(Fig. 1b). We have shown previously that the i.v.-injected anti-
TCR mAbs inactivate the targeted T cells (13). Although the
treatments with anti-TCR-? and TCR-V?1 mAbs did not sig-
nificantly decrease serum IgE levels by comparison with the
controls, the treatment with anti-TCR-V?4 mAb significantly
increased IgE levels, and there were significant differences be-
tween the different Ab-treated groups (Fig. 1b), consistent with
the findings in the genetically modified mice (Fig. 1a). Taken
together, the data shown in Fig. 1 suggested that the overall
effect of ?? T cells in untreated mice, and in mice sensitized
with OVA-alum, is to increase the primary IgE response but
also suggested that different V?-defined subsets are capable of
modulating the IgE response in opposite directions: whereas
V?1?cells might enhance it, V?4?cells might suppress it.
Adoptively transferred V?4?cells from OVA-challenged but not
untreated mice, inhibit the primary anti-OVA IgE response in
To further investigate the effect of V?4??? T cells on the IgE re-
sponse, we positively selected V?4?cells from the spleen of un-
treated B6.TCR-??/?mice (using magnetic beads), and transferred
3 ? 104of the purified cells to B6.TCR-??/?mice by i.v. injection,
directly before the i.p. OVA-alum sensitization. We examined both
serum total IgE (Fig. 2a) and OVA-specific IgE in the cell transfer
recipients (Fig. 2b). V?4?cells derived from the untreated donors did
not significantly alter serum IgE levels in the OVA-alum-sensitized
recipients. However, others have reported that mice exposed to aero-
solized OVA by inhalation on at least 10 consecutive days (10N-
treated mice) lose their ability to mount an IgE response upon i.p.
OVA-alum injection, and give rise to suppressive ?? T cells (8). We
therefore prepared V?4?cells from the spleen of 10N-treated
B6.TCR-??/?mice. Unlike those from the untreated donors, these
cells markedly suppressed both total IgE and OVA-specific IgE levels
b). These data confirmed that IgE-suppressive ?? T cells are induced
by airway allergen challenge (8) and showed that this occurs inde-
pendently of ?? T cells. In an earlier study, we made essentially the
sive ?? T cells and found that V?4??? T cells derived from
3Abbreviations used in this paper: AHR, airway hyperresponsiveness; DC, dendritic
ground serum IgE levels and primary OVA-alum-induced IgE responses in
genetically altered mouse strains. Total serum IgE concentrations in
C57BL/6, B6.TCR-??/?, B6.TCR-??/???/?, B6.TCR-??/?, B6.TCR-
V?4?/?6?/?, and B6.TCR-V?1 Tg mice was measured using sandwich
ELISA 14 days after i.p. injection of OVA-alum and in nontreated controls.
Results for each group are expressed as the mean ? SEM (n ? 18, 4, 4, 20,
15, 6, in the order shown). NT, Nontreated; ???, Significant difference (p ?
0.001) compared with OVA-challenged C57BL/6 mice. Significant differ-
ences in basal IgE levels between the mouse strains shown in a are listed
in the text. b, Effect of treatment with anti (?)-TCR mAbs on the primary
OVA-alum-induced IgE response in C57BL/6 mice. Normal C57BL/6
mice were injected i.v. with nonspecific hamster IgG (HIgG), anti-TCR-?,
anti-TCR-V?4, and anti-TCR-V?1 mAbs on day ?4, and total serum IgE
concentrations were determined on day 14 after i.p. injection of OVA-
alum. Mice that received the OVA-alum injection but no Ab treatment
were included as controls. Results for each group are expressed as the
mean ? SEM (n ? 4 in each group). ?, Significant difference compared
with untreated or hamster IgG-treated controls (p ? 0.05).
Altered IgE response in ?? T cell-modified mice. a, Back-
mice is inhibited following adoptive transfer of V?4?cells from airway
OVA-challenged B6.TCR-??/?mice. Purified V?4?cells (3 ? 104/inoc-
ulum) from naive or 10N-treated B6.TCR-??/?mice, or from 10N-treated
B6.TCR-??/?IFN-??/?mice, were adoptively transferred i.v. to B6.TCR-
??/?recipients, just before i.p. OVA-alum injection. Serum total IgE (a)
and OVA-specific IgE (b) levels were measured 14 days after the OVA-
alum treatment. Results for each group are expressed as the mean ? SEM
(n ? 4 in each group). Significant differences between mice that received
cells and those that received no cells are indicated. ??, p ? 0.01; NS, not
significant; NT, not treated; EU, ELISA units.
Primary OVA-alum-induced IgE response in B6.TCR-??/?
851The Journal of Immunology
by guest on June 13, 2013
wild-type C57BL/6 mice and B6.TCR-??/?mice are functionally
equivalent (17). Finally, on the basis of the data of others suggesting
that IgE-suppressive ?? T cells depend on IFN-? (8), we tested
whether V?4?cells from the spleens of 10N-treated B6.TCR-??/
?IFN-??/?mice had suppressive activity. In clear contrast to their
wild-type counterparts, these cells failed to suppress the IgE response
(Fig. 2). These data confirmed that IFN-?-dependent ?? T cells can
become potent inhibitors of the IgE response (8) and identified V?4?
cells as the source population of the suppressors.
IgE-suppressive V?4?cells express V?4V?5-TCRs and CD8
To further investigate the properties of IgE-suppressive V?4???
T cells, we examined the V? usage of V?4?and V?1?cells in
spleen and lung of untreated and 10N-treated B6.TCR-??/?mice
(Table I). After 10N treatment, the overall numbers and relative
frequency of V?4?cells remained stable or slightly decreased in
spleen and increased in lung, whereas V?1?cells more generally
decreased in spleen and less prominently increased in lung. Certain
V?4?subpopulations (V?4V?5?cells and V?4V?8?cells) showed
relative and absolute or at least relative increases in lung and spleen
suggesting that these cells are selected in the 10N-treated mice and
hence that they might be enriched in IgE suppressors.
Of the two subpopulations of V?4?cells, V?4V?5?cells are
more accessible because they are present in larger numbers in both
organs. Therefore, to test the idea that IgE suppressors among
V?4?cells are enriched during the 10N treatment, we purified
V?4?V?5?and V?4?V?5?cells by FACS sorting of splenocytes
V?5?V?4 cells were purified from the spleen of 10N-treated B6.TCR-
??/?mice by FACS sorting. Purified V?4?V?5?or V?4?V?5?cells (3 ?
104/inoculum) were adoptively transferred i.v. to B6.TCR-??/?recipients
just before i.p. injection of OVA-alum. Serum total IgE (a) and OVA-
specific IgE (b) levels were measured 14 days after the OVA-alum injec-
tion. Results for each group are expressed as the mean ? SEM (n ? 4 in
each group). Significant differences between mice that received cells and
those that received no cells are indicated. ???, p ? 0.001; NS, not signif-
icant; NT, not treated; EU, ELISA units.
IgE-suppressive V?4??? T cells coexpress V?5. V?5?and
CD8??V?4 cells were purified from the spleen of 10N-treated B6.TCR-
??/?mice by FACS sorting. Three ? 104cells of each type were adop-
tively transferred i.v. to B6.TCR-??/?recipients just before i.p. injection
of OVA-alum. Serum total IgE levels were measured 14 days after the
OVA-alum injection. Results for each group are expressed as the mean ?
SEM (n ? 4 in each group). Significant differences between mice that
received cells and those that received no cells are indicated. ???, p ?
0.001. NS, not significant; NT, not treated; EU, ELISA units.
IgE-suppressive V?4??? T cells express CD8. CD8??and
Table I. Sizes of ?? T cell subpopulations and their relative percentage in spleen and lung of naive and 10N-treated B6.TCR-??/?micea
Naive spleen143 ? 23
(11.5 ? 0.6)
128 ? 33
(14 ? 0.6)
296 ? 69
(24 ? 1.5)
285 ? 81
(30.7 ? 3.7)*
20 ? 8
(1.6 ? 0.5)
14 ? 4
(1.6 ? 0.6)
21 ? 5.6
(1.7 ? 0.4)
14 ? 5.3
(1.6 ? 0.8)
15 ? 4.5
(1.2 ? 0.4)
21 ? 13
(1.4 ? 0.6)
163 ? 45
(7.4 ? 0.1)
115 ? 67
(8.7 ? 2.1)
568 ? 141
(25.9 ? 0.5)
319 ? 89
(25.8 ? 3.0)
114 ? 50
(5.2 ? 1.7)
84 ? 11
(7.0 ? 1.4)
199 ? 15
(9.4 ? 1.7)
101 ? 22
(8.3 ? 1.0)
23 ? 7.5
(1.1 ? 0.4)
14 ? 5.8
(1.1 ? 0.1)
aNylon wool nonadherent (NAD) cells derived from spleen and lung were counted. The percentages of individual ?? T cell subpopulations in NAD cells were determined
cytofluorimetrically. Absolute cell numbers (?1000) were calculated as percentage ? NAD cell numbers. Numbers in parentheses, percentages of ?? T cell subpopulations in
total ?? T cells. Four mice were used in each group, and data for spleen cells were expressed as means ? SEM. Lung cells were pooled, and the average numbers are shown.
Significant differences between naive and challenged mice are indicated. ?, p ? 0.05.
852IgE REGULATION BY ?? T CELLS
by guest on June 13, 2013
from 10N-treated B6.TCR-??/?mice. Cells of either type (3 ?
104/inoculum) were then adoptively transferred to B6.TCR-??/?
recipients by i.v. injection directly before i.p. OVA-alum sensiti-
zation (Fig. 3). Overall, the FACS-sorted cells were less effective
than the cells enriched by magnetic bead selection. However, the
FACS-selected V?4?V?5?cells significantly decreased both se-
rum total IgE (Fig. 3a) and OVA-specific IgE (Fig. 3b), whereas
V?4?V?5?cells had a lesser effect, and only serum total IgE was
significantly reduced. These data confirm that V?4?V?5??? T
cells can suppress IgE. Our experiment leaves open the possibility
that other V?4?cells, e.g., V?4?V?8??? T cells, also might
function as IgE suppressors.
It has been proposed that IgE-suppressive ?? T cells express
CD8 (8); in addition, V?4??? T cells express CD8 more fre-
quently than other ?? T cells (18). To examine the possibility that
the V?4?IgE suppressors coexpress CD8, both V?4?CD8??and
V?4?CD8??cells were purified by FACS sorting of splenocytes
from 10N-treated B6.TCR-??/?mice, and subsequently tested in
the cell transfer assay (Fig. 4). Whereas the V?4?CD8??cells
completely suppressed the OVA-specific IgE response, V?4?
CD8??cells had no effect whatsoever. Taken together, these data
suggest that a subset of ?? T cells expressing V?4V?5 TCRs and
CD8 contains most if not all IgE suppressors in our model.
Adoptively transferred V?1?cells from untreated but not
OVA-challenged mice enhance the primary anti-OVA IgE
response in TCR-??/?mice
To further investigate the effect of V?1??? T cells on the IgE
response, we positively selected V?1?cells from the spleens of
untreated B6.TCR-??/?mice, and transferred 3 ? 104purified
cells to B6.TCR-??/?mice, shortly before the i.p. OVA-alum sen-
sitization. V?1?cells derived from untreated donors significantly
increased serum total IgE (Fig. 5a) and OVA-specific IgE levels
(Fig. 5b) in the OVA-alum-sensitized recipients, in marked con-
trast to the V?4?cells, which required OVA challenge of the
donor mice to become functional IgE suppressors (Fig. 2). How-
ever, V?1?cells from 10N-treated B6.TCR-??/?mice no longer
had any significant effect on the IgE level (Fig. 5). This is remi-
niscent of our earlier observation that V?1?cells from untreated
mice enhance AHR (19), and it suggests that V?1??? T cells in
untreated mice already exert a Th2-like influence which enables
them to promote both AHR and the IgE responses.
We had also found that AHR-enhancing V?1??? T cells express
V?5 (19). To test whether the IgE enhancers also are V?1V?5?cells,
we purified both V?1?V?5?and V?1?V?5?cells by FACS sorting
of splenocytes from nontreated B6.TCR-??/?mice, and adoptively
transferred either type to B6.TCR-??/?recipients by i.v. injection
shortly before i.p. OVA-alum sensitization (Fig. 6). However, both
but not airway OVA-challenged B6.TCR-??/?mice enhance the primary
OVA-alum-induced IgE response in B6.TCR-??/?mice. Purified V?1?
cells (3 ? 104/inoculum) from nontreated (NT) or 10N-treated B6.TCR-
??/?mice were adoptively transferred i.v. to B6.TCR-??/?recipients just
before i.p. OVA-alum injection. Serum total IgE (a) and OVA-specific IgE
(b) levels were measured 14 days after the OVA-alum injection. Results for
each group are expressed as the mean ? SEM (n ? 4 in each group).
Significant differences between mice that received cells and those that re-
ceived no cells are indicated. ?, p ? 0.05; ??, p ? 0.01. NS, not significant;
NT, not treated; EU, ELISA units.
Adoptively transferred V?1??? T cells from nontreated
from nontreated mice to enhance the primary OVA-alum-induced IgE re-
sponse. Purified V?1?V?5?or V?1?V?5?cells (3 ? 104/inoculum) from
nontreated (NT) B6.TCR-??/?mice were adoptively transferred i.v. to
B6.TCR-??/?recipients directly before Ag challenge. Serum total IgE (a) and
OVA-specific IgE (b) levels were measured 14 days after the OVA-alum in-
jection. Results for each group are expressed as the mean ? SEM (n ? 4 in
received no cells are indicated. ?, p ? 0.05; ??, p ? 0.01. EU, ELISA units.
Co-expression of V?5 is not required for V?1??? T cells
853The Journal of Immunology
by guest on June 13, 2013
V?1?V?5?and V?1?V?5?cells significantly increased serum total
IgE and OVA-specific IgE in the B6.TCR-??/?cell transfer recipi-
the AHR enhancers, do not have to coexpress V?5.
Net effect of nonseparated ?? T cells on the primary anti-OVA
IgE response in TCR-??/?mice
Because our findings with transferred purified cells representing the
?? T cell subsets suggested that the net effect of all ?? T cells might
be a switch from IgE enhancement to IgE suppression, we tested this
idea by transferring total nonseparated ?? T cells derived from donors
that either remained untreated or were OVA challenged (10N; Fig. 7).
Because the total number of transferred cells (3 ? 104i.v.) remained
the same, in this experiment fewer cells from either ?? T cell subset
were transferred by comparison with the purified cells representing
the individual subsets. As predicted, the nonseparated ?? T cells from
challenged donors suppressed the primary IgE response. However,
nonseparated ?? T cells from untreated donors did not significantly
enhance the IgE response (see Discussion).
In this study, we confirmed the earlier observation of others that ??
T cells exert a regulatory influence on the IgE response (8–10, 20).
We extended these findings to show that there are IgE-suppressors
and IgE enhancers among IgE-regulatory ?? T cells, which belong
to different subsets of ?? T cells and are distinguished by their
TCR expression. Furthermore, we demonstrate that although the
suppressors require induction by airway exposure to allergen, the
enhancers do not, and finally that airway allergen challenge, pre-
viously reported to induce ?? T cell-dependent suppression of the
IgE response (8, 20), actually has a dual effect. It induces the IgE
suppressors and inactivates the IgE enhancers, thereby mediating
?? T cell-dependent IgE suppression in the challenged mice.
These observations raise several new questions. If ?? T cells
indeed regulate the IgE response, when are they likely to play a
role and how far-reaching might their influence be? In this study,
we investigated the effect of ?? T cells on serum IgE levels after
a single i.p. injection of OVA-alum. After transfer of nonseparated
?? T cells derived from untreated or challenged donors, the change
in the primary IgE response was roughly 2-fold, and after transfer
of purified ?? T cells representing the regulatory subsets, the range
of changes in total and OVA-specific IgE levels increased to ?10-
fold. Considering that IgE levels in circulation tend to be small (1),
and that only 3 ? 104?? T cells were transferred, the regulatory
potential of ?? T cells may be substantial. We chose to investigate
serum IgE and to transfer small numbers of splenic ?? T cells
mainly for experimental convenience. Conceivably, the regulatory
effect of ?? T cells on IgE in the mucosal tissues, where ?? T cells
are more concentrated (21) and where IgE Abs are present at much
higher levels (2), might be greater. Our data also reveal differences
in background serum IgE levels of ?? T cell genetically altered
mice, and here the range of differences reaches ?100-fold. It
seems clear, therefore, that ?? T cells profoundly influence the IgE
response and that their influence is not limited to the special case
of immunization with OVA-alum.
What might be the significance of the correlation between TCR
expression and IgE-regulatory function? We found that the effect
of V?4?cells on the IgE response ranged from none to suppres-
sion, whereas that of V?1?cells ranged from enhancement to
none. These nonoverlapping functional effects are reminiscent of
other studies on which the two types of ?? T cells also had non-
overlapping and opposed functional effects (12). In particular,
V?1?cells also enhanced AHR whereas V?4?cells suppressed it
(13, 17, 19, 22). Our recent studies have shown that the V?1???
T cell subset in normal untreated mice contains AHR-enhancing
cells (19, 23) but the V?4?subset does not. However, the AHR
and IgE-regulatory ?? T cell populations do not appear to be iden-
tical. Whereas the AHR enhancers seem to be limited to those cells
that express V?1V?5 (19), IgE enhancers were also found among
V?1?V?5?cells. Alternatively, most of the IgE suppressors ap-
pear to express V?4V?5 whereas the AHR suppressors which also
express V?4 might be less biased with regard to V? expression
(17). Moreover, we found that the IgE suppressors tend to express
CD8, unlike the AHR suppressors which may be CD8?or CD8?
(22). At least for those functional populations that depend on de-
fined V?V? pairs (the AHR enhancers and the IgE suppressors), it
seems likely that the TCR is somehow involved with their func-
tion, either because TCR-ligand interactions help determine cellu-
lar differentiation or because they define function itself through
ligand specificity (14, 24). Consistent with the former possibility,
a recent study showed that TCR-ligand interactions in the thymus
determine the ability of peripheral ?? T cells to express certain
cytokines (15). However, possible ligands for the IgE-regulatory
?? T cells remain unknown.
How can airway challenge mediate IgE suppression? Having
examined both IgE suppressors and enhancers, it is clear that air-
way challenge does more than simply change the ratio of these
induced IgE response. Purified ?? T cells (3 ? 104/inoculum) from non-
treated (NT) or OVA-challenged (10N) B6.TCR-??/?mice were adop-
tively transferred i.v. to B6.TCR-??/?recipients directly before Ag
challenge. Serum total IgE (a) and OVA-specific IgE (b) levels were mea-
sured 14 days after the OVA-alum injection. Results for each group are
expressed as the mean ? SEM (n ? 4 in each group). Significant differ-
ences between mice that received cells and those that received no cells are
indicated. ?, p ? 0.05; ??, p ? 0.01, ???, p ? 0.001. EU, ELISA units.
Net effect of total ?? T cells on the primary OVA-alum-
854 IgE REGULATION BY ?? T CELLS
by guest on June 13, 2013
cells in favor of the suppressors but rather affects the two regula- Download full-text
tory populations separately. The experiments shown in this paper
do not formally rule out that ?? T cells migrate from the chal-
lenged lung to the spleen. However, it would be difficult for pul-
monary ?? T cells to change the composition of splenic ?? T cells
substantially because the splenic population of ?? T cells is much
larger (25, 26). More likely perhaps, ?? T cells in the spleen are
influenced by other signal carriers from the lung, e.g., pulmonary
dendritic cells (DC). Our preliminary studies with ?? T cells ex-
posed to transferred non-T cells from challenged mice are consis-
tent with such a mechanism (Y. Huang, unpublished observations).
Moreover, we found both V?1?and V?4??? T cells in the spleen
in close proximity to CD8?DC in the splenic periarteriolar lym-
phoid sheaths (22), a known destination of the peripheral signal
carriers or shuttles. Furthermore, the splenic ?? T cells required
the presence of CD8?splenic DCs or their functional development
in another model (22). Therefore, we envision that the splenic ??
T cells might be compelled to change their function under the
influence of CD8?splenic DC, which must have received signals
from the challenged lung (27). Indeed, such a mechanism has al-
ready been described, where CD8?DC, which are known to re-
main in the lymphoid tissues (28), depend on shuttle cells to re-
ceive stimulatory signals and Ags from the peripheral tissues
including the lung (29–32).
Despite the clear IgE-enhancing effect of purified V?1?cells de-
rived from nonchallenged donors, total ?? T cells derived from such
donors enhanced IgE only weakly (detected in the assay for total
serum IgE, but not for OVA-specific IgE). This simply might be due
to the smaller number of transferred V?1?cells in this experiment
(only approximately one-half of total splenic ?? T cells are V?1?) or
it might be caused by interactions among the IgE-regulatory ?? T cell
subsets. In any case, the net effect of ?? T cells in untreated mice
therefore might be only slightly supportive of the primary IgE re-
sponse, whereas their effect in challenged mice is clearly suppressive.
In sum, we found that airway challenge brings about coordi-
nated and opposite functional changes of the IgE-enhancing and
-suppressive ?? T cells. This coordinated change of two antago-
nistic cell-types appears to represent an efficient mechanism in the
regulation of the primary IgE response.
We acknowledge the advice and support of Dr. Katja Aviskus and the expert
help of Shirley Sobus and William Townend with cell analysis and sorting.
The authors have no financial conflict of interest.
1. Gould, H. J., B. J. Sutton, A. J. Beavil, R. L. Beavil, N. McCloskey, H. A. Coker,
D. Fear, and L. Smurthwaite. 2003. The biology of IgE and the basis of allergic
disease. Annu. Rev. Immunol. 21: 579–628.
2. Gould, H. J., and B. J. Sutton. 2008. IgE in allergy and asthma today. Nat. Rev.
Immunol. 8: 205–217.
3. Thornton, C. A., J. A. Holloway, E. J. Popplewell, J. K. Shute, J. Boughton, and
J. O. Warner. 2003. Fetal exposure to intact immunoglobulin E occurs via the
gastrointestinal tract. Clin. Exp. Allergy 33: 306–311.
4. Eisenbarth, S. C., O. R. Colegio, W. O’Connor Jr., F. S. Sutterwala, and
R. A. Flavell. 2008. Crucial role for the Nalp3 inflammasome in the immunos-
timulatory properties of aluminium adjuvants. Nature 453: 1122–1127.
5. Mowat, A. M. 1987. The regulation of immune responses to dietary protein
antigens. Immunol. Today 8: 93–98.
6. Sedgwick, J. D., and P. G. Holt. 1985. Down-regulation of immune responses to
inhaled antigen: studis on the mechanism of induced suppression. Immunology
7. Seymour, B. W. P., L. J. Gershwin, and R. L. Coffman. 1998. Aerosol-induced
immunoglobulin (Ig)-E unresponsiveness to ovalbumin does not require CD8?or
T cell receptor (TCR)-?/??T cells or interferon (IFN)-? in a murine model of
allergen sensitization. J. Exp. Med. 187: 721–731.
8. McMenamin, C., C. Pimm, M. McKersey, and P. G. Holt. 1994. Regulation of
IgE responses to inhaled antigen in mice by antigen-specific ?? T cells. Science
9. Zuany-Amorim, C., C. Ruffie, S. Haile, B. B. Vargaftig, P. Pereira, and
M. Pretolani. 1998. Requirement for ?? T cells in allergic airway inflammation.
Science 280: 1265–1267.
10. Svenson, L., B. Lilliehook, R. Larsson, and A. Bucht. 2003. ?? T cells contribute
to the systemic immunoglobulin E response and local B-cell reactivity in allergic
eosinophilic airway inflammation. Immunology 108: 98–108.
11. Ferrick, D. A., M. D. Schrenzel, T. Mulvania, B. Hsieh, W. G. Ferlin, and H. Lepper.
1995. Differential production of interferon-? and interleukin-4 in response to Th1-
and Th2-stimulating pathogens by ?? T cells in vivo. Nature 373: 255–257.
12. Huber, S. A., D. Graveline, M. K. Newell, W. K. Born, and R. L. O’Brien. 2000.
V?1?T cells suppress and V?4?T cells promote susceptibility to coxsackievirus
B3-induced myocarditis in mice. J. Immunol. 165: 4174–4181.
13. Hahn, Y.-S., C. Taube, N. Jin, L. Sharp, J. M. Wands, M. Kemal Aydintug,
M. Lahn, S. A. Huber, R. L. O’Brien, E. W. Gelfand, and W. K. Born. 2004.
Different potentials of ?? T cell subsets in regulating airway responsiveness:
V?1?cells, but not V?4?cells, promote airway hyperreactivity: Th2 cytokines,
and airway inflammation. J. Immunol. 172: 2894–2902.
14. O’Brien, R. L., C. L. Roark, N. Jin, M. K. Aydintug, J. D. French, J. L. Chain,
J. M. Wands, M. Johnston, and W. K. Born. 2007. ?? T cell receptors: functional
correlations. Immunol. Rev. 215: 77–88.
15. Jensen, K. D. C., X. Su, S. Shin, L. Li, S. Youssef, S. Yamasaki, L. Steinman,
T. Saito, R. M. Locksley, M. M. Davis, N. Baumgarth, and Y.-H. Chien. 2008.
Thymic selection determines gammadelta T cell effector fate: antigen-naive cells
make interleukin-17 and antigen-experienced cells make interferon ?. Immunity
16. Heilig, J. S., and S. Tonegawa. 1986. Diversity of murine ? genes and expression
in fetal and adult T lymphocytes. Nature 322: 836–840.
17. Jin, N., C. Taube, L. Sharp, Y.-S. Hahn, X. Yin, J. M. Wands, C. L. Roark, R. L.
O’Brien, E. W. Gelfand, and W. K. Born. 2005. Mismatched antigen prepares ?? T
cells for suppression of airway hyperresponsiveness. J. Immunol. 174: 2671–2679.
18. Lahn, M., A. Kanehiro, K. Takeda, J. Terry, Y.-S. Hahn, M. K. Aydintug,
A. Konowal, K. Ikuta, R. L. O’Brien, E. W. Gelfand, and W. K. Born. 2002.
MHC class I-dependent V?4? pulmonary T cells regulate ?? T cell-independent
airway responsiveness. Proc. Natl. Acad. Sci. USA 99: 8850–8855.
19. Jin, N., N. Miyahara, C. L. Roark, J. D. French, M. K. Aydintug, J. L. Matsuda,
L. Gapin, R. L. O’Brien, E. W. Gelfand, and W. K. Born. 2007. Airway hyper-
responsiveness through synergy of ?? T cells and NKT cells. J. Immunol. 179:
20. McMenamin, C., M. McKersey, P. Ku ¨hnlein, T. Hu ¨nig, and P. G. Holt. 1995. ??
T cells down-regulate primary IgE responses in rats to inhaled soluble protein
antigens. J. Immunol. 154: 4390–4394.
21. Hayday, A., and R. Tigelaar. 2003. Immunoregulation in the tissues by ?? T cells.
Nat. Rev. Immunol. 3: 233–242.
22. Cook, L., N. Miyahara, N. Jin, J. M. Wands, C. Taube, C. L. Roark, T. A. Potter,
E. W. Gelfand, R. L. O’Brien, and W. K. Born. 2008. Evidence that CD8?
dendritic cells enable the development of ?? T cells that modulate airway hy-
perresponsiveness. J. Immunol. 181: 309–319.
23. Jin, N., C. L. Roark, N. Miyahara, C. Taube, M. K. Aydintug, J. M. Wands,
Y. Huang, Y. S. Hahn, E. W. Gelfand, R. L. O’Brien, and W. K. Born. 2009.
Allergic airway hyperresponsiveness-enhancing ?? T cells develop in normal
untreated mice and fail to produce IL-4/13, unlike Th2 and NKT cells. J. Immu-
nol. 182: 2002–2010.
24. O’Brien, R. L., M. Lahn, W. Born, and S. A. Huber. 2001. T cell receptor and
function cosegregate in ?-? T cell subsets. Chem. Immunol. 79: 1–28.
25. Hahn, Y.-S., C. Taube, N. Jin, K. Takeda, J.-W. Park, J. M. Wands,
M. K. Aydintug, C. L. Roark, M. Lahn, R. L. O’Brien, E. W. Gelfand, and
W. K. Born. 2003. V?4?T cells regulate airway hyperreactivity to methacholine
in ovalbumin-sensitized and challenged mice. J. Immunol. 171: 3170–3178.
26. Wands, J. M., C. L. Roark, M. K. Aydintug, N. Jin, Y.-S. Hahn, L. Cook, X. Yin,
J. Dalporto, M. Lahn, D. M. Hyde, et al. 2005. Distribution and leukocyte con-
tacts of ?? T cells in the lung. J. Leukocyte Biol. 78: 1086–1096.
27. Lambrecht, B. N., and H. Hammad. 2003. Taking our breath away: dendritic cells
in the pathogenesis of asthma. Nat. Rev. Immunol. 3: 994–1003.
28. Shortman, K., and S. H. Naik. 2007. Steady-state and inflammatory dendritic cell
development. Nat. Rev. Immunol. 7: 19–30.
29. Carbone, F. R., G. T. Belz, and W. R. Heath. 2004. Transfer of antigen between
migrating and lymph node-resident DCs in peripheral T-cell tolerance and im-
munity. Trends Immunol. 25: 655–658.
30. Allan, R. S., C. M. Smith, G. T. Belz, A. L. van Lint, L. M. Wakim, W. R. Heath,
and F. R. Carbone. 2003. Epidermal viral immunity induced by CD8a?dendritic
cells but not by Langerhans cells. Science 301: 1925–1928.
31. Mount, A. M., C. M. Smith, F. Kupresanin, K. Stoermer, W. R. Heath, and
G. T. Belz. 2008. Multiple dendritic cell populations activate CD4?T cells after
viral stimulation. PLoS ONE 3: 1–10.
32. Belz, G. T., C. M. Smith, L. Kleinert, P. Reading, A. Brooks, K. Shortman,
F. R. Carbone, and W. R. Heath. 2004. Distinct migrating and nonmigrating
dendritic cell populations are involved in MHC class I-restricted antigen presen-
tation after lung infection with virus. Proc. Natl. Acad. Sci. USA 101:
855The Journal of Immunology
by guest on June 13, 2013