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CD3+ T Cells upon CD226 Engagement
High Frequency and Induce Th2 Bias of
with Allergic Asthma Express CCR9 at
24-Invariant NKT Cells from Patients
Boquan and Tan Jinquan
Chunsong, Xuejun Zhang, Jin Youxin, Gong Feili, Jin
Xiong Jie, Deng Tao, Zhou Gang, Liu Junyan, Hu
Yang Sen, Bi Yongyi, He Yuling, Xie Luokun, He Li,
2005; 175:4914-4926; ;
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Print ISSN: 0022-1767 Online ISSN: 1550-6606.
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The Journal of Immunology
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V?24-Invariant NKT Cells from Patients with Allergic Asthma
Express CCR9 at High Frequency and Induce Th2 Bias of
CD3?T Cells upon CD226 Engagement1
Yang Sen,2*†Bi Yongyi,2§He Yuling,2‡Xie Luokun,‡He Li,¶Xiong Jie,‡?Deng Tao,#
Zhou Gang,‡Liu Junyan,‡Hu Chunsong,*†Xuejun Zhang,*†Jin Youxin,?Gong Feili,**
Jin Boquan,3††and Tan Jinquan3†‡
We have demonstrated that V?24?V?11?invariant (V?24?i) NKT cells from patients with allergic asthma express CCR9 at high
frequency. CCR9 ligand CCL25 induces chemotaxis of asthmatic V?24?i NKT cells but not the normal cells. A large number of
CCR9-positive V?24?i NKT cells are found in asthmatic bronchi mucosa, where high levels of Th2 cytokines are detected.
Asthmatic V?24?i NKT cells, themselves Th1 biased, induce CD3?T cells into an expression of Th2 cytokines (IL-4 and IL-13)
in cell-cell contact manner in vitro. CD226 are overexpressed on asthmatic V?24?i NKT cells. CCL25/CCR9 ligation causes
directly phosphorylation of CD226, indicating that CCL25/CCR9 signals can cross-talk with CD226 signals to activate V?24?i
NKT cells. Prestimulation with immobilized CD226 mAb does not change ability of asthmatic V?24?i NKT cells to induce
Th2-cytokine production, whereas soluble CD226 mAb or short hairpin RNA of CD226 inhibits V?24?i NKT cells to induce
Th2-cytokine production by CD3?T cells, indicating that CD226 engagement is necessary for V?24?i NKT cells to induce Th2
bias of CD3?T cells. Our results are providing with direct evidence that aberration of CCR9 expression on asthmatic V?24?i
NKT cells. CCL25 is first time shown promoting the recruitment of CCR9-expressing V?24?i NKT cells into the lung to promote
other T cells to produce Th2 cytokines to establish and develop allergic asthma. Our findings provide evidence that abnormal
asthmatic V?24?i NKT cells induce systemically and locally a Th2 bias in T cells that is at least partially critical for the
pathogenesis of allergic asthma. The Journal of Immunology, 2005, 175: 4914–4926.
invariant V?14-J?18 (mouse) or V?24-J?18 (human) rearrange-
atural killer T cells, originally characterized in mice as
cells that express both a TCR and NK1.1 (Refs. 1 and 2),
have more recently been defined as cells that have an
ment and reactivity to ?-galactosylceramide (?-GalCer)4pre-
sented by the CD1d (3). NKT cells play a critical role in modu-
lating the upcoming immune responses, involving in protection
against bacterial or parasitic infections and prevention of autoim-
mune diseases (4–6). The capacity of NKT cells to activate rapid
cytokine expression has been exploited to manipulate the out-
comes of autoimmunity and cancer (7). In an experimental asthma
model in mouse, V?14i NKT cells are required to participate in
allergen-induced Th2 airway inflammation through a CD1d-de-
pendent mechanism (8). Pulmonary V?14i NKT cells regulate cru-
cially development of airway eosinophilia, hyperresponsiveness,
Th2-cytokine production, and elevated levels of IgE Abs (8, 9). In
humans, CD1d-restricted TCR V?24?i NKT cells are important
regulators of immune responses through their efficient secretion of
Th1 and Th2 cytokines (10). However, it is not understood how
NKT cells can selectively regulate Th1 vs Th2 responses in vivo
when they can produce both IL-4 and IFN-?. Some of these dif-
ferences are thought to depend on the stimulatory conditions driv-
ing the response, including cell-cell interactions and cytokine bal-
ance in the microenvironment, as well as the nature of the Ag
(11–13). Up to now, the mechanism that NKT cells influence the
development of allergic inflammation through Th2 differentiation
of T cells in human remains nebulous.
The recruitment of leukocytes into tissues is dependent on a
series of adhesion and activation steps mediated by adhesion mol-
ecules and chemokine receptor interactions (10). Distinct subsets
of T cells have been characterized that are targeted to skin
*Department of Dermatology and†Department of Immunology, The First Affiliated
Hospital, Anhui Medical University, Hefei, People’s Republic of China;‡Department
of Immunology,§Department of Public Health, and¶Department of Parasitology,
Institute of Allergy and Immune-Related Diseases, Wuhan University School of Med-
icine and Public Health, Wuhan, People’s Republic of China;?The State Key Labo-
ratory of Molecular Biology, Institute of Biochemistry and Cell Biology, Shanghai
Institutes for Biological Sciences, Chinese Academy of Science, Shanghai, People’s
Republic of China;#Department of Internal Medicine, Renmin Hospital, Wuhan Uni-
versity, Wuhan, People’s Republic of China; **Department of Immunology, Tongji
Medical College, Huazhong University of Science and Technology, Wuhan, People’s
Republic of China; and††Department of Immunology, Fourth Military Medical Uni-
versity, Xian, People’s Republic of China
Received for publication February 14, 2005. Accepted for publication August 8, 2005.
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 is supported by the National Key Basic Research Program of China from
the Ministry of Science and Technology of People’s Republic of China (Grants
2001CB510004 and 2001CB510008), National Science Foundation of China (Grants
39870674 and 30030130), a special grant from the Personnel Department of Wuhan
University, Science Foundation of Anhui Province (98436630), Education and Re-
search Foundation of Anhui Province (Grant 98JL063), Research Foundation of
Health Department of Hubei Provincial Government (Grant 301140344), and Science
Foundation of Education Bureau of Hubei Province (Grant 2001A14009).
2Y.S., B.Y., and H.Y. contributed equally to this work.
3Address correspondence and reprint requests to Dr. Tan Jinquan, Department of
Immunology, Anhui Medical University, Hefei, or Department of Immunology, Wu-
han University School of Medicine, Dong Hu Road 115, Wuchang 430071, Wuhan,
People’s Republic of China; E-mail address: firstname.lastname@example.org; or Dr. Jin
Boquan, Department of Immunology, Fourth Military Medical University, Xian,
People’s Republic of China; E-mail address: email@example.com
4Abbreviations used in this paper: ?-GalCer, ?-galactosylceramide; DN, double
negative; NML, normal subject; AST, asthmatic patient; MCNC, migrating cell on
negative control; CI, chemotactic index; QD, quantum dot; DC, dendritic cell; Tr,
regulatory T cell.
The Journal of Immunology
Copyright © 2005 by The American Association of Immunologists, Inc.0022-1767/05/$02.00
by guest on June 18, 2013
(CLA?CCR4?), small intestine (?4?7highCCR9?), and mucosal
sites (CCR10?) (reviewed in Ref. 10). Several chemokine recep-
tors, such as CCR1, CCR4, CCR6, and CXCR6, are differentially
expressed by CD4?, CD8?, and double-negative (DN) NKT cell
subsets (14–16). CCR9, mainly on T cells in human small intestine
(17), is expressed at a very low level on normal human NKT cell
subsets (14, 18). Currently, there are very few data on the role of
chemokines and their receptors in the recruitment of NKT cells to
different tissue sites in vivo, particularly in pathogenesis in humans
(10). It will be important to determine the chemokine receptor
interactions that mediate both homeostatic trafficking of NKT cells
and recruitment into different sites of inflammation.
Adhesion molecule CD226 belongs to an Ig superfamily (19–
27) with ligands CD112 and CD155. This molecule has very im-
portant biological functions, such as mediating human NK cell
cytotoxicity and signaling transduction of T cell activation and
differentiation (19–27). We have reported the high sensitivity of
NKT cells from active systemic lupus erythematosus patients to
apoptosis because of CD226 deficiency (28).
In the present study, we have documented that asthmatic
V?24?i NKT cells selectively express CCR9. Th1-biased asth-
matic V?24?i NKT cells are inducers of Th2 bias of CD3?T
cells. CCL25/CCR9 signals can cross-talk with CD226 to activate
V?24?i NKT cells for induction of Th2 bias of CD3?T cells.
Materials and Methods
Reagents and Abs
PE- anti-human V?11 mAb, FITC-anti-human V?24 mAb, anti-CD112
(R2.477.1), and anti-CD155 (D171) were purchased from Beckman
Coulter. All recombinant chemokines CCL20, CCL25, CXCL9, and mouse
monoclonal anti-CCR6 (53103.111), anti-CCR9 (112509), anti-CXCR3
(3010.211), anti-IL-13 (31606), anti-IL-10 (23738.111), and anti-TGF-1?
(1D11) mAbs were purchased from R&D Systems. Anti-CD161 (N-20)
pAb and anti-NKG2D (1D11) mAb were purchased from Santa Cruz Bio-
technology. ?-GalCer was purchased from Sigma-Aldrich. We had gener-
ated a mouse anti-human mAb against CD226.
Patients, normal subjects (NMLs), and cell purification
In- or outpatients with allergic asthma at Departments of Internal Medicine
in the university hospitals of Anhui Medical University and Wuhan Uni-
versity were voluntary recruited. All asthma patients had had at least two
typical consecutive onsets and specific IgE ? CAP class 3 (CAP system;
Pharmacia). The group of untreated asthmatic patients (AST) were ages
18–56 years (14 males and 18 females); the group of asthmatic patients
with corticosteroid treatment (AST-t) were ages 16–51 years (8 males and
10 females); and the group of asthmatic patients in remission (AST-r) were
ages 17–48 years (9 males and 9 females). The general statuses of all
patients were stable without any severe cardiopulmonary and other vital
organ complication. The NMLs were ages 19–50 years (8 males and 10
females) without history of atopy (hayfever, asthma, and eczema) and with
undetectable levels of specific IgE. In the experimental procedure, the asth-
matic patients in each group were randomly selected for carrying out each
assay with sufficient numbers for statistically analysis. All samples were
collected with patients’ consent and local research ethics committee ap-
proval. PBMCs were isolated from heparinized peripheral blood of patients
and healthy volunteers. V?24?i NKT cells were positively isolated using
V?24-biotin mAb together with MACS streptavidin-labeled magnetic mi-
crobead cell sorting (Miltenyi Biotec), according to the manufacturer’s
instructions. CD3?CD56?CD161?T cells were obtained using first a ny-
lon wood column assay to negatively select CD3?T cells to avoid preac-
tivation, followed by a depletion of CD56?or CD161?cells by CD56 and
CD161 MACS multisort microbeads (Miltenyi Biotec) (29). For all mag-
netic separations, FcRs were blocked with Fc Block (Miltenyi Biotec). The
purities of cell populations were analyzed on a flow cytometer. The sam-
ples of normal bronchi mucosa from lung tissue were anonymously from
the patients without atopy background undergoing partial lung resection for
accidental chest trauma. The samples of bronchi mucosa from allergic
asthma patients were obtained through fiber-optic bronchoscopy.
For detection of chemokine receptors (CCR6, CCR9, or CXCR3), purified
CD3?T cells either isolated from patients with allergic asthma or from
normal controls were incubated with FITC-labeled anti-human V?24 mAb
and PE-labeled anti-human V?11 mAb and PerCP-labeled mouse anti-
human chemokine receptor mAb or CD226 (secondary PerCP-labeled)
mAb at 5 ?g/ml or 5 ?g/ml matched isotype mouse Ab (DakoCytomation).
The PerCP-labeled third Ab in PBS containing 2% BSA and 0.1% sodium
azide was incubated for 20 min, followed by washing twice in staining
buffer as described previously (30). All procedures were conducted at 4°C.
The analyses were performed with a flow cytometer (COULTER XL;
Coulter). For intracellular cytokine immunofluorescence staining, as de-
scribed elsewhere (31), the cells were washed twice in PBS and then fixed
and permeabilized using IntraPrep (Beckman Coulter), according to man-
ufacturer’s instructions. The cells were then incubated with the primary
mouse anti-human cytokine mAb for 15 min at room temperature. Cells
were washed twice and stained with PerCP-labeled goat anti-mouse Abs
for 15 min at room temperature. Cells were washed twice and stained with
FITC-labeled anti-human V?24 mAb and PE-labeled anti-human V?11
mAb for 15 min at 4°C. Cells were washed and resuspended in PBS con-
taining 0.5% formaldehyde for flow cytometry.
Real time quantitative RT-PCR assay
All real-time quantitative RT-PCR were performed as described elsewhere
(32–34). Briefly, total RNA from purified CD4?or CD8?T cells (1 ? 106,
purity ? 99%) was prepared by using Quick Prep total RNA extraction kit
(Amersham Biosciences), according to the manufacturer’s instructions.
RNA was reverse transcribed by using oligo (dT)12–18and Superscript II
reverse transcriptase (Invitrogen Life Technologies). The real-time quan-
titative PCR was performed in special optical tubes in a 96-well microtiter
plate (Applied Biosystems) with an ABI PRISM 7700 Sequence Detector
Systems (Applied Biosystems). By using SYBR Green PCR Core Reagents
kit (Applied Biosystems), fluorescence signals were generated during each
PCR cycle via the 5? to 3? endonuclease activity of AmpliTaq Gold (33) to
provide real-time quantitative PCR information. The sequences of the spe-
cific primers were as follows: V?24 sense, 5?-AAGCAAAGCTCTCTGC
ACATCACA-3?, and V?24 antisense, 5?-GTCACTGGATTTAGAGTCT-
3?; CD226 sense, 5?-TCAAATAGCCACATTGTTTCGGA-3?, and CD226
antisense, 5?-AGGGTATATTGGTTATCGGTTTTACC-3?; CCR6 sense,
5?-CCTGGGGAATATTCTGGTGGTGA-3?, and CCR6 antisense, 5?-
CATCGCTGCCTTGGGTGTTGTAT-3?; CCR9 sense, 5?-CATTGACGC
CTATGCCATGT-3?, and CCR9 antisense, 5?-GACCTGGAAGCAGAT
GTCAATGT-3?; CXCR3 sense, 5?-GGAGCTGCTCAGAGTAAATCAC-
3?, and CXCR3 antisense, 5?-GCACGAGTCACTCTCGTTTTC-3?; IFN-?
sense, 5?-GCTAAAACAGGGAAGCGAAAAA-3?, and IFN-? antisense,
5?-GGACAACCATTACTGGGATGCT-3?; IL-2 sense, 5?-TGCAAGGGA
CTCAGGTGATG-3?, and IL-2 antisense: 5?-TGCTGCTTATTTAGGAT
ACCTATTAACTCA-3?; IL-4 sense, 5?-CACAGGCACAAGCAGCT
GAT-3?, and IL-4 antisense, 5?-GCCAGGCCCCAGAGGTT-3?; IL-13
sense, 5?- GAGTGTGTTTGTCACCGTTG-3?, and IL-13 antisense, 5?-
TACTCGTTGGCTGAGAGCTG-3?; IL-10 sense, 5?-GTGATGCCCCA
AGCTGAGA-3?, and IL-10 antisense, 5?-TCCCCCAGGGAGTTCACA-
3?; and TGF-? sense, 5?-TCAGAGCCACAAATCCTGAAAG-3?, and
TGF-? antisense, 5?-CACCAAGTGTACCCCGAAAGA-3?.
All unknown cDNAs were diluted to contain equal amounts of ?-actin
cDNA. The standards, “no template” controls, and unknown samples were
added in a total volume of 50 ?l/reaction. PCR retain conditions were 2
min at 50°C, 10 min at 95°C, 40 cycles with 15 s at 95°C, and 60 s at 60°C
for each amplification. Potential PCR product contamination was digested
by uracil-N-glycosylase because dTTP is substituted by dUTP (32). Uracil-
N-glycosylase and AmpliTaq Gold (Applied Biosystems) were applied ac-
cording to the manufacturer’s instructions (33, 34).
Western and Northern blotting
For mRNA detection (Northern blot), as previously described (35), 5 ?g of
total RNA from each sample were electrophoresed under denaturing con-
ditions, followed by blotting onto Nytran membranes, and cross-linked by
UV irradiation as described previously (35). The target cDNA probes, la-
beled by ?-[32P]dCTP, were obtained by PCR amplification of the se-
quences mentioned above from total RNA from PBMCs from normal
adults (for CCR6, CXCR3, V?24, and CD226) or thymocytes from the
specimen of thymusectomy (for CCR9). The membranes were hybridized
overnight with 1 ? 106cpm/ml of32P-labeled probe, followed by inten-
sively washing with 0.2? SSC (1? SSC ? 0.15 M NaCl, 0.015 M sodium
citrate (pH 7.0)) and 0.1% SDS before being autoradiographed. For protein
detection (Western blot analysis), the cells were lysed in lysis buffer (0.4%
4915 The Journal of Immunology
by guest on June 18, 2013
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4926CCR9 AND CD226 ON ASTHMATIC NKT CELLS
by guest on June 18, 2013
The Journal of Immunology
Letter of Retraction
We wish to retract the article titled “Va24-Invariant NKT Cells from Patients with Allergic Asthma Express CCR9 at High Frequency
and Induce Th2 Bias of CD31T Cells upon CD226 Engagement” by Yang Sen, Bi Yongyi, He Yuling, Xie Luokun, He Li, Xiong Jie,
Deng Tao, Zhou Gang, Liu Junyan, Hu Chunsong, Xuejun Zhang, Jin Youxin, Gong Feili, Jin Boquan, and Tan Jinquan, The Journal
of Immunology, 2005, 175: 4914–4926.
Wuhan University School of Medicine
Copyright ? 2012 by The American Association of Immunologists, Inc. 0022-1767/12/$16.00
by guest on June 18, 2013