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Selective Expression of a Novel Surface
Molecule by Human Th2 Cells In Vivo
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of June 14, 2017.
Kinya Nagata, Kazuya Tanaka, Kazuyuki Ogawa, Kazumi
Kemmotsu, Toshio Imai, Osamu Yoshie, Hiroyuki Abe,
Kohtaro Tada, Masataka Nakamura, Kazuo Sugamura and
Shoichi Takano
J Immunol 1999; 162:1278-1286; ;
http://www.jimmunol.org/content/162/3/1278
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Copyright © 1999 by The American Association of
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Print ISSN: 0022-1767 Online ISSN: 1550-6606.
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References
Selective Expression of a Novel Surface Molecule by Human
Th2 Cells In Vivo
Kinya Nagata,1* Kazuya Tanaka,* Kazuyuki Ogawa,* Kazumi Kemmotsu,* Toshio Imai,†
Osamu Yoshie,2† Hiroyuki Abe,‡ Kohtaro Tada,‡ Masataka Nakamura,§ Kazuo Sugamura,‡ and
Shoichi Takano*
D41 effector Th cells can be divided into at least three
distinct subsets termed Th1, Th2, and Th0 in both mice
and humans, based on the profile of their cytokine production (1– 4). In humans, Th1 cells typically secrete IFN-g and
predominantly promote cell-mediated immune responses such as
delayed-type hypersensitivity reaction and macrophage activation.
Th2 cells are characterized by their production of IL-4, IL-5, and
IL-13, which promote strong humoral immunity including IgE production, and growth and differentiation of mast cells and eosinophils. Th0 cells secrete cytokines typical of both Th1 and Th2
cells, but Th0 function is not fully elucidated. Various pathological
conditions, such as infection, autoimmune diseases, and allergy,
exhibit the polarized Th1 and Th2 responses that are believed to be
closely implicated in the onset and outcome of these diseases (4, 5).
Since the discovery of Th1 and Th2 subsets there has been intense interest in finding their cell-surface markers that would be
useful not only in monitoring but also in manipulating the subsets
in vivo. Several surface molecules have been reported to be differentially expressed between Th1 and Th2 cells. LAG-3 (6), active ligands for P- and E-selectin (7), IL-12R b2 subunit (8, 9), and
CC chemokine receptor (CCR)3 5 (10) were shown to be domi-
C
nantly expressed on Th1 cells, whereas expression of CD30 (11),
IFN-gR b-chain (12), CCR3 (13), CCR4 (14), and ST2L (15) was
reported to be preferential to Th2 cells. All of these molecules
were originally identified with in vitro cell culture system. However, expression specificity of these molecules in human Th1 and
Th2 cells in vivo has not yet been fully established. Identification
of reliable markers in humans that are stably expressed in either
Th1 or Th2 subsets in vivo would facilitate our understanding of
functional involvement of the subsets in normal and disorderly
condition.
In this study, we took advantage of the gene expression screen
method (16, 17) to clone genes for molecules that are differentially
expressed between human Th1 and Th2 cells. One clone was finally selected that encodes a novel G protein-coupled receptor,
named CRTH2, selectively expressed in Th2 but not Th1 cells in
vitro and in vivo. Our results suggested that CRTH21 cells play
central roles in allergen-induced immune responses. Collectively,
CRTH2 could be a useful tool for the study of human Th2 in vivo
as well as in vitro.
Materials and Methods
Cell lines
*R & D Center, BioMedical Laboratories, Kawagoe, Saitama, Japan; †Shionogi Institute for Medical Science, Settsu, Osaka, Japan; ‡Department of Microbiology and
Immunology, Tohoku University School of Medicine, Sendai, Miyagi, Japan; and
§
Human Gene Sciences Center, Tokyo Medical and Dental University, Bunkyo-ku,
Tokyo, Japan
Received for publication June 22, 1998. Accepted for publication October 9, 1998.
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.
1
Address correspondence and reprint requests to Dr. Kinya Nagata, R & D Center,
BioMedical Laboratories, 1361-1 Matoba, Kawagoe, Saitama 350-1101, Japan. Email address: nagata@alk.co.jp
2
Current address: Department of Bacteriology, Kinki University School of Medicine,
Osakasayama, Osaka, Japan.
3
Abbreviations used in this paper: CCR, CC chemokine receptor; PPD, purified protein derivative of Mycobacterium tuberculosis; Der, extract of Dermatophagoides
Copyright © 1999 by The American Association of Immunologists
Human cell lines used in this study were: T cell lines Jurkat, Hut102,
Hut78, MT-2, TL-Mor, CCRF-CEM, and Molt-4; B cell lines Daudi,
BJAB, and an EBV-transformed lymphoblastoid cell line LCL-Nag; an
erythroid line HEL; a monocyte/macrophage line THP-1; a cervix carcinoma line HeLa; a hepatoma line Hep-G2; and an adenovirus-transformed
embryonic kidney line 293 obtained from the American Type Culture Collection (Manassas, VA). A mouse T cell line BW5147, mouse myeloma
line SP2/O-Ag8, rat T cell line TART-1, monkey kidney line COS7, and
transfectants of COS7 coexpressing human CD4 and the CCR5 or CXC
chemokine receptor (CXCR) 4 were also used. The expression of the transgenes in the COS7 transfectants were confirmed by HIV-1 infection. Cells
stably expressing CCR1, CCR2B, CCR3, CCR4, CCR5, CCR6, CCR7, or
fractalkine receptor CX3CR1 were described previously (18). Cell lines
were cultured in RPMI 1640 medium (except for HeLa, 293, and COS7
pteronyssinus; endo F, endoglycosidase F; PE, phycoerythrin; PerCP, peridinin chlorophyll protein.
0022-1767/99/$02.00
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The search for reliable marker molecules discriminating between human Th1 and Th2 cells identified a gene encoding a novel
member of the G protein-coupled leukocyte chemoattractant receptor family, which is selectively expressed in Th2 but not Th1
lineage cells, thereby named CRTH2 (chemoattractant receptor-homologous molecule expressed on Th2 cells). Studies with antiCRTH2 mAbs demonstrated that CRTH2 was expressed in a small population (0.4 – 6.5%) of CD41 T cells in fresh PBMCs of
healthy adults, but no remarkable expression was seen in B cells and NK cells. In some cases, CD81 T cells (;3.5%) expressed
CRTH2. Phenotypes of CD41 T cells expressing CRTH2 were CD45RA2, CD45RO1, and CD251, similar to those of Ag-activated
effector/memory T cells. Freshly isolated CRTH21 CD41 T cells produced Th2- but little or no Th1-type cytokines upon stimulation with PMA and ionomycin. In addition, an allergen-induced proliferative response in fresh PBMCs was significantly and
selectively reduced by subtracting CRTH21 cells. Together, these results indicate that CRTH2 is selectively expressed in vivo in
an activated state of Th2 cells including allergen-responsive Th2 cells, suggesting its pivotal roles in ongoing Th2-type immune
reactions. The Journal of Immunology, 1999, 162: 1278 –1286.
The Journal of Immunology
cells, which were cultured in DMEM with the same addition) and supplemented with 10% FCS and antibiotics at 37°C under 5% CO2 in air.
Generation of Th1 and Th2 lines and clones from PBMC
Southern and Northern blot analyses
Total RNAs were extracted from cells with Trizol reagent (Life Technologies, Gaithersburg, MD). Southern and Northern blot analyses were conducted with Hybond N1 nylon membranes (Amersham, Buckinghamshire,
U.K.) under stringent hybridization condition according to the manufacturer’s instructions (Version 2). 32P-labeling of probes was performed with
a random primer DNA-labeling kit (Takara Shuzo, Shiga, Japan). A 246-bp
RT-PCR product of b-actin mRNA was used as a control probe (see below). The radioactivity on the membranes was visualized by BAS 1000
Image Analyzing System (Fuji Photo Film, Tokyo, Japan).
Preparation of a subtracted Th2 cDNA library and isolation of
Th2-specific cDNA fragments
A subtracted Th2 cDNA library was prepared by the gene expression
screen method (16, 17) using total RNAs from a typical Th1 clone 2P15
and a typical Th2 clone 2P26, both of which were obtained from the same
donor (K.T.). Three rounds of subtractive hybridization were performed
between 2P15 and 2P26 cDNA fragments as described (17). The final
products were used as subtracted Th1 and Th2 probes. A portion of the
subtracted Th2 probe was digested with XbaI and cloned into the XbaI site
of pBluescript SK2 (Stratagene, La Jolla, CA), generating a subtracted
Th2 cDNA library. The subtracted Th2 cDNA library was screened by
differential hybridization with the 32P-labeled subtracted Th1 and Th2
probes. Clones that were selectively hybridized with the subtracted Th2
probe were selected. DNA sequences were determined by the dye-terminator cycle sequencing method on ABI 377 automated sequencer (PerkinElmer Japan, Chiba, Japan).
Cloning of full-length cDNA
cDNA was synthesized from the 2P26 poly(A)1 RNA with a SuperScript
Choice System (Life Technologies) and cloned into the EcoRI site of
Lambda ZAP II phage vector (Stratagene). The resultant phage library was
screened by plaque hybridization with the selected Th2-specific cDNA
fragment B (see text).
Expression of CRTH2 in mammalian cell lines
The full-length CRTH2 cDNA (clone B19) was subcloned into three mammalian expression vectors: pCXN2 (19), pRc/CMV (Invitrogen, San Diego, CA), and pcDL-SRa296 (20). In brief, the cDNA insert was excised
separately at the EcoRI site in the cloning adapter, HindIII/XbaI sites in the
multicloning site of the phagemid vector, or EcoRI site in 59-side adapter
and EcoRV site within the cDNA (nucleotide 1365), then subcloned into
the EcoRI site of pCXN2, HindIII/XbaI sites of pRc/CMV, or EcoRI/KpnI
(blunted) sites of pcDL-SRa296, respectively, generating pCXN2/B19,
pRc/B19, and pcDL-SRa/B19. These expression plasmids were introduced
in various cell lines by electroporation, and stable transfectants were selected in the presence of geneticin (Sigma, St. Louis, MO). Transfectants
used in this study were pCXN2/B19-transfected TART-1 (TART/B1912.10) and BW5147 (BW/B19-3.4), pRc/B19-introduced 293 (293/B19-1),
and pcDL-SRa/B19-transfected Jurkat (Jurkat/B19-1).
Generation of mAbs and anti-peptide Abs
mAbs were generated by i.p. immunization of Wistar rats with 107 TART/
B19-12.10 cells once a week. Three days after the fifth immunization,
sensitized spleen cells were fused with SP2/O-Ag8 cells. Culture supernatants of the hybridomas were screened by indirect membrane immunofluorescence method using TART-1, TART/B19-12.10, and phycoerythrin
(PE)-conjugated goat anti-rat IgG (Biosource International, Camarillo,
CA). Anti-peptide Abs to CRTH2 were generated by immunization of New
Zealand White rabbits with peptide corresponding to the deduced sequence
of the first (MSANATLKPLCPILEQMSRLQSHSNTSIRYIDH), the third
(RDTISRLDGRIMCYYNVLLLNPGPDRDATCNSRQ), or the fourth
(PYHVFSLLEARAHANPGLRP) extracellular domain of CRTH2, which
had been conjugated to keyhole limpet hemocyanin with m-maleimidobenzoyl-N-hydroxysuccinimide ester (Pierce, Rockford, IL) (21). The antipeptide Ab was affinity-purified using corresponding peptide coupled to an
Affi-Gel10 (Bio-Rad Laboratories, Richmond, CA).
Immunoblotting and endoglycosidase F (endo F) treatment
Cells (;5 3 106) were labeled with an anti-CRTH2 mAb (10 –20 mg/ml)
at 4°C for 30 min, washed, and lysed in 1 ml of lysis buffer (1% sucrose
monolaurate (Dojindo Laboratories, Kumamoto, Japan), 25 mM Tris/HCl
(pH 7.5), 150 mM NaCl, 5 mM EDTA, and protease inhibitors) at 4°C for
1 h. The cell lysate was clarified by centrifugation, and the immune complex was immunoprecipitated with 107 anti-rat IgG-coupled magnetic
beads (Dynal). The precipitates were washed five times with the lysis
buffer, then were subjected to SDS-PAGE or endo F treatment followed by
SDS-PAGE as previously described (22). The proteins in the polyacrylamide gel were electrically transferred onto a BA-S85 nitrocellulose membrane (Schleicher & Schüll, Dassel, Germany), and CRTH2 was visualized
with a rabbit anti-CRTH2 Ab (5 mg/ml) and a horseradish peroxidaselabeled goat anti-rabbit IgG (Zymed Laboratories, San Francisco, CA) followed by chemiluminescent detection using Western blot chemiluminescence reagent (New England Nuclear, Boston, MA).
Flow cytometry
The following materials were obtained from Becton Dickinson (San Jose,
CA): FITC-conjugated mAbs to CD3 (clone Leu-4), CD4 (Leu-3a), CD8
(Leu-2a), CD19 (Leu-12), HLA-DR (L243), and IFN-g (25723.11); PEconjugated mAbs to IL-4 (3010.211) and IFN-g; peridinin chlorophyll protein (PerCP)-conjugated mAb to CD4; and appropriate isotype-matched
controls. FITC-conjugated mAbs to CD16 (3G8), CD45RO (UCHL-1), and
CD45RA (HI100); PE-conjugated mAbs to CD25 (M-A251), IL-5
(TRFK5), and IL-13 (JES10-5A2); and control conjugates were purchased
from PharMingen (La Jolla, CA). PE- and RED670-labeled streptavidins,
FITC-conjugated streptavidin, and FITC-labeled anti-CD62L mAb (Dreg
56) were obtained from Life Technologies, Biomedia (Foster City, CA),
and Immunoteck (Marseille Cedex, France), respectively. Control rat
IgG2a was purchased from Zymed. To biotinylate mAbs, a long-arm Nhydroxysuccinimidyl-biotin (Vector Laboratories, Burlingame, CA) was
used. Staining of cell surface Ags was performed in accordance with the
manufacturer’s instructions. Intracellular cytokines were stained according
to the method of Picker et al. (23). The stained cells were analyzed on
FACScan flow cytometer using CellQuest software (both from Becton
Dickinson).
RT-PCR
Total RNA was treated with RNase-free DNaseI (Promega, Madison, WI)
and was reverse transcribed in 20 ml of reaction mixture using Superscript
II RT (Life Technologies) according to the manufacturer’s recommended
method. A portion of the reaction product was subjected to PCR using an
AmpliTaq DNA polymerase (Perkin-Elmer). Sense and antisense primers
used in this study were as follows: for CRTH2, 59-CCTCTGTGCCCA
GAGCCCCACGATGTCGGC and 59-CACGGCCAAGAAGTAGGT
GAAGAAG; for b-actin, 59-TGAAGTCTGACGTGGACATC and 59ACTCGTCATACTCCTGCTTG (24). For each PCR reaction (25 ml), the
sample was first denatured at 95°C for 2 min and amplified by 30 cycles of
PCR (94°C, 1 min; 67°C, 1 min; and 72°C, 2 min) for CRTH2 or by 23
cycles of PCR (94°C, 1 min; 65°C, 1 min; and 72°C, 2 min) for b-actin.
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PBMCs were isolated from heparinized blood by density gradient centrifugation using Ficoll-Paque (Pharmacia Biotech, Uppsala, Sweden) and cultured in RPMI 1640 medium containing 10% FCS and antibiotics with
appropriate additives at 37°C under 5% CO2 in air. Th1 cells were induced
from PBMCs of healthy volunteers by stimulation with 1 mg/ml of PHA
(PHA-P; Wako Pure Chemical, Osaka, Japan) or 5 mg/ml of a purified
protein derivative of Mycobacterium tuberculosis (PPD) (Japan BCG Laboratory, Tokyo, Japan) in the presence of 5 ng/ml of human rIL-12 (R&D
Systems, Minneapolis, MN) and 100 ng/ml of human rIFN-g (Genzyme,
Cambridge, MA) (Th1 line). Th2 cells were induced from PBMCs from
normal adults by stimulation with PHA or an extract of Dermatophagoides
pteronyssinus (Der; 1% (v/v)) (Torii Pharmaceutical, Tokyo, Japan) in the
presence of 50 ng/ml of human rIL-4 (Genzyme) and 10 mg/ml of neutralizing anti-IFN-g mAb (K1) (Genzyme) (Th2 line). PBMC cultures were
supplemented with 50 U/ml of human rIL-2 (Shionogi, Osaka, Japan) on
day 4 – 6 and further cultured for several days to expand cells. Th1 and Th2
clones were established by limiting dilution. Briefly, CD41 cells were purified from the Th1 or Th2 line with anti-CD4 Ab-coupled magnetic beads
(Dynal, Lake Success, NY) and plated in a 96-well round-bottom plate at
0.5–10 cells per well in the presence of PHA (1 mg/ml) and IL-2 (100
U/ml) with 1–5 3 104 LCL-Nag cells/well, which had been previously
treated with 50 mg/ml of mitomycin C (Kyowa Hakko Kogyo, Tokyo,
Japan) at 37°C for 30 min. One half of the medium was replaced with fresh
medium containing IL-2 (100 U/ml) twice a week. Clones were examined
for their production of cytokines to specify their property.
1279
1280
SELECTIVE EXPRESSION OF A NOVEL SURFACE MOLECULE BY TH2 IN VIVO
FIGURE 1. Expression of CRTH2 mRNA in human Th1 and Th2 cells.
Total RNAs (10 mg) from Th1 clones 1P04 and 2P15 (lanes 1 and 2,
respectively), Th2 clones 2P26 and KND4 (lanes 3 and 4), PPD-induced
Th1 lines (lanes 5–9), and Der-induced Th2 lines (lanes 10 –14) were analyzed for relative level of CRTH2 mRNA by Northern blot analysis using
cDNA fragment B as a probe. The relative level of b-actin mRNA was
monitored to check the amount of total RNA applied to each lane.
Fractionation of fresh PBMC and cultured cells
Results
Cloning of a novel G protein-coupled receptor from a Th2
cDNA library
After screening of 3 3 103 cDNA fragments from the subtracted
Th2 cDNA library, we obtained 45 independent cDNA fragments
that were differentially hybridized with the subtracted Th2 (2P26derived) but not Th1 (2P15-derived) probe. Northern blot analyses
using total RNAs of 2P15 and 2P26 cells showed that 13 of the 45
cDNA fragments were actually expressed at detectable levels in a
2P26-specific manner. Search of the EMBL, GenBank, and DDBJ
databases revealed that 6 of the 13 cDNA fragments were novel,
whereas the others are known genes. After analyzing mRNA levels
using a large panel of Th1 and Th2 cells, we selected a cDNA
fragment called fragment B as a candidate for the Th2-specific
gene.
The cDNA fragment B (272 bp) was hybridized with a 3.0-kbp
mRNA species that was selectively expressed in all Th2 clones and
lines but not in any Th1 clones and lines (Fig. 1). The significant
expression of this gene was not observed in human cell lines derived from various tissues in Northern blot analysis.
After screening of a 5 3 105 phage Th2 (2P26) cDNA library
with the cDNA fragment B as a probe, we selected two cDNA
clones, B6 (2884 bp) and B19 (2911 bp). The DNA sequencing
revealed that their nucleotide sequences overlap completely. The
nucleotide sequence data of cDNA clone B19 will appear in the
DDBJ, EMBL, and GenBank nucleotide sequence databases with
the accession number AB008535. The cDNA fragment B corresponds to nucleotide 1908 –2179 in cDNA clone B19.
The longest open reading frame of 1185 nucleotides of cDNA
clone B19 starts from the first ATG (nucleotide 113), which
roughly conforms to the Kozak rule (25), and encodes a protein of
395 amino acids with a calculated molecular mass of 43 kDa. Fig.
2 shows the deduced amino acid sequence of the protein encoded
by B19. A hydropathy analysis indicated seven putative transmembrane domains, which is a characteristic feature of the G proteincoupled seven transmembrane receptor (STR) superfamily. In-
deed, the highest amino acid sequence identity is found with
members of the leukocyte receptor for the “classical” chemoattractants, such as FMLP receptor (32%) (Ref. 26 and Fig. 2), C3a
receptor (30%) (27), and C5a receptor (29%) (28), which belong to
the STR superfamily (29). On the other hand, the amino acid sequence identities of this protein with receptors for CC- or CXCchemokines, such as CCR3 (25%) (30) and IL-8 receptor (22%)
(31), are slightly lower. In addition, the B19-encoded protein lacks
some common features characteristic to chemokine receptors, such
as four conserved cysteine residues in each of the extracellular
domains and an amino acid motif DRYLAIVHA within the second
intracellular domain (30). For these circumstances, we named this
protein CRTH2 (chemoattractant receptor-homologous molecule
expressed on Th2). CRTH2 has two potential N-glycosylation sites
at the first extracellular domain and has a unique long cytoplasmic
tail in which four consensus sites for protein kinase C phosphorylation are found (Fig. 2).
Production of rat anti-CRTH2 mAbs
To confirm the Th2-specificity of CRTH2 expression at the protein
level, we generated five mAbs named BM6 (IgG2b), BM10
(IgG2b), BM16 (IgG2a), BM17 (IgG2a), and BM18 (IgG2a).
CRTH2-specificity of these mAbs was confirmed using a large
panel of CRTH2 mRNA-positive and -negative cell lines. All of
these mAbs reacted with CRTH2-transfected cell lines (TART/
B19-12.10, BW/B19-3.4, 293/B19-1, and Jurkat/B19-1), but they
were not seen on their parent lines (TART-1, BW5147, 293, and
Jurkat) and other CRTH2 mRNA-negative cell lines (Hut102,
Hut78, TL-Mor, CCRF-CEM, MT-2, Molt-4, Daudi, BJAB, LCLNag, THP-1, HEL, HeLa, Hep-G2, and COS7) in flow cytometry.
These mAbs were shown to compete with each other for binding
to CRTH2 transfectants, suggesting that they recognize a common
or neighboring epitope on the protein. Hence, a representative
mAb, BM16, which showed the highest binding affinity to CRTH2
transfectants, was mainly used for further examination. The flow
cytometric profiles of the transfectants stained with BM16 are presented in Fig. 3A. Similar results were obtained with other antiCRTH2 mAbs. Because several chemokine receptors have been
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For purification of CRTH21 or CRTH22 cells, fresh PBMCs or cultured
cells were heavily labeled with BM16 at 50 mg/ml for 30 min at 4°C in the
presence of 10% normal human serum. After extensive washing, Ab-labeled cells were either positively isolated by labeling with anti-rat IgGcoupled microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany) followed by two rounds of purification on MS1 positive selection column
(Miltenyi) attached with 26-gauge needle for flow regulation, or they were
removed from unlabeled cells with excess amounts of anti-rat IgG-coupled
magnetic beads (Dynal).
FIGURE 2. Primary structure of CRTH2. The deduced amino acid sequence of CRTH2 is aligned with that of human N-formyl peptide receptor
(FMLPR) (26). The identical amino acids are indicated by dots. Putative
transmembrane (TM) segments are overlined and numbered as TM1-TM7.
The conserved proline and cysteine residues are indicated by inverted triangles. Potential sites for N-glycosylation and protein kinase C phosphorylation are presented by asterisks and stars, respectively.
The Journal of Immunology
1281
reported to be expressed on T cells (29, 32), we carefully examined
the possible cross-reactivity of anti-CRTH2 mAb BM16 with
known chemokine receptors. But no positive reaction was observed with transfectants stably expressing CCR1, CCR2B, CCR3,
CCR4, CCR5, CCR6, CCR7, CX3CR1, or CXCR4 in flow cytometric analysis (data not shown).
Characterization of CRTH2 protein
Expression of CRTH2 on the cultured Th clones and lines
Using mAb BM16, CRTH2 expression in Th clones was examined
at the single cell level. As shown in Fig. 3C, most cells of each Th2
clone expressed significant levels of CRTH2. All eight typical Th2
clones showed similar results. In contrast, all five typical
Th1 clones expressed little or no visible CRTH2. Th2-selective
expression of CRTH2 was also confirmed in polyclonal Th2 lines
(Fig. 3C).
To further verify the Th2-specificity of CRTH2 expression, we
examined the cytokine profile of CRTH21 cells in polyclonal
PBMC cultures in which both Th1 and Th2 cells were growing
concurrently. As shown in Fig. 4, isolated CRTH21 cells produced
typical Th2 cytokines IL-4, IL-5, and IL-13, but they produced
little or no Th1 cytokine IFN-g. These results clearly indicate that
CRTH2 is selectively expressed in Th2 but not Th1 cells. The
results also indicate that most Th0 cells do not express CRTH2. On
the other hand, a significant number of Th2 cells remained in
CRTH2-depleted cell fraction (Fig. 4), suggesting CRTH2 was
expressed in not all but a large population of Th2 cells in such
mixed Th cultures.
CRTH2 expression in fresh PBMCs
FIGURE 3. Selective expression of CRTH2 on the surface of Th2 cells.
A, BM16 staining of CRTH2 transfectants. Cells indicated in each panel
were stained with BM16 (solid line) or control rat IgG2a (dotted line) (10
mg/ml) at 4°C for 30 min followed by PE-labeled goat anti-rat IgG (1:200
dilution) at 4°C for 30 min. B, Detection of CRTH2 by Far-Western blotting. Jurkat (lane 1), Jurkat/B19-1 (lanes 2, 3, 6, and 7), and Th2 clone
6L21 (lanes 4, 5, 8, and 9) cells (5 3 106/lane) were subjected to FarWestern blotting using the indicated Abs and a rabbit anti-peptide Ab
against the fourth extracellular domain of CRTH2 as described in Materials and Methods. Positions of CRTH2 are indicated by arrows. C, Selective expression of CRTH2 in cultured Th2 cells. Upper panels, Cytokine
pattern in Th clones and lines. Cells were stimulated with PMA (20 ng/ml;
Wako) and ionomycin (1 mg/ml; Sigma) in the presence of brefeldin A (10
mg/ml, Wako) at 37°C for 4 h, then intracellular cytokines were stained
with FITC-labeled anti-IFN-g (x-axis) and PE-labeled anti-IL-4 (y-axis).
Quadrants were set according to the staining of control mAbs. Lower panels, CRTH2 expression. CRTH2 expression was examined as described in
panel A. Percentages of positive cells are shown in each panel. The cells
used in this experiment were MID3 (Th1 clone), 6L21 (Th2 clone), PPDTan (Th1 line), and Der-Tan (Th2 line).
We next examined the CRTH2 expression in fresh PBMCs from
several healthy adults. The representative results are presented in
Fig. 5. For all donors, gradual but distinct expression of CRTH2
was observed in a small population (0.4 – 6.5%) of CD41 T cells.
In some cases, CRTH2 expression was also detected in a small
portion (;3.5%) of CD81 T cells (data not shown) and in CD32
undefined cell population (Fig. 5A), whereas it was not remarkable
in B cells (CD191) and NK cells (CD161) in any donors examined. A three-color analysis showed that the majority of CRTH21
cells in CD41 lymphocytes have phenotypes of CD45RA2,
CD45RO1, and CD251 for all individuals (Fig. 5B), indicating
that CRTH2 is mainly expressed in an activated state of effector/
memory CD41 T cells. CRTH2-specificity of the staining with
BM16 was confirmed by the dominant expression of CRTH2
mRNA in the sorted BM16-bound cells as compared with BM16unbound cells (Fig. 5C).
Cytokine production in CRTH21 PBMCs
It was of great interest to ask whether CRTH2 is also selectively
expressed in Th2 cells in vivo. To ascertain this, we first compared
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The Far-Western blotting method was performed to analyze the
CRTH2 protein. As shown in Fig. 3B, BM16 specifically precipitated a 55- to 70-kDa protein, which was recognized by a rabbit
anti-CRTH2 peptide (the fourth extracellular domain) Ab from cell
lysates of CRTH2-transfected Jurkat and a Th2 clone, 6L21. This
protein band was further confirmed to be CRTH2 itself by using
two other independent anti-peptide Abs against the first and third
extracellular domains of CRTH2. Specificity of the binding of
these anti-peptide Abs was corroborated by blocking tests with
each peptide. As a result of the treatment with endo F, the CRTH2
decreased its molecular mass from 55–70 kDa to 35– 40 kDa (Fig.
3B, lanes 7 and 9), indicating the presence of N-linked sugar on the
CRTH2 as predicted from amino acid sequence.
1282
SELECTIVE EXPRESSION OF A NOVEL SURFACE MOLECULE BY TH2 IN VIVO
the cytokine patterns of unfractionated, CRTH2-depleted, and
CRTH2-enriched cell fractions derived from the same fresh
PBMCs of several healthy adults. As shown in Fig. 6, Th2 cells
having the ability to produce IL-4, IL-5, or IL-13, but not IFN-g,
were greatly purified by the positive selection with BM16. Contrarily, a significant reduction of Th2 cells was observed in
CRTH21 cell-depleted fraction as compared with unfractionated
cell population. On the other hand, the proportion of Th1 cells was
not significantly affected by the negative selection. However, the
isolation procedure employed in this study seemed to somewhat
affect the total ability of T cells to produce cytokines. Hence, we
next stimulated cells in a whole blood to minimize cell damage
according to instructions of the FastImmune cytokine system (Becton Dickinson), then directly observed for a correlation between
the expression levels of CRTH2 and intracellular cytokines. The
Downloaded from http://www.jimmunol.org/ by guest on June 14, 2017
FIGURE 4. Cytokine production in isolated CRTH21 cells. A, Purity of
CRTH21 cells in unfractionated, CRTH21, and CRTH22 cell fractions. A
Th line (unfractionated (uf)), which had been generated with Der and IL-2
without Th1- and Th2-oriented cytokines, was incubated with BM16 (solid
line) or control rat IgG2a (dotted line), washed, and fractionated into
BM16-unbound (ub) and BM16-bound (b) cell fractions as described in
Materials and Methods. The cell-bound rat IgG was visualized by incubating cells with PE-labeled goat anti-rat IgG (1:200 dilution) at 4°C for 30
min. CD41 lymphocytes gated by staining of FITC-labeled anti-CD4 were
analyzed. B, Cytokine production in each fraction. Cytokine production in
uf (left panels), ub (central panels), and b (right panels) cell fractions
described in panel A was examined with FITC-labeled anti-IFN-g (x-axis),
and PE-labeled mAb (y-axis) to IL-4 (upper panels), IL-5 (central panels),
or IL-13 (lower panels) as described in Fig. 3C. Normal rat serum (10%)
was supplemented to all staining buffers to block anti-rat IgG Ab on the
cell-bound magnetic beads. CD41 lymphocytes gated by staining with
PerCP-labeled anti-CD4 were analyzed.
FIGURE 5. CRTH2 expression on various populations of PBMC of
healthy adults. A, CRTH2 expression on various subsets of PBMC. PBMCs
from a healthy adult were preincubated with normal rat IgG (positive staining, CRTH2 PE) or unlabeled BM16 (control staining, CRTH2 PE (block))
(500 mg/ml) at room temperature for 30 min, then biotinylated BM16 (10
mg/ml) was added to the cells. The cells were incubated at 4°C for 30 min,
washed, and incubated with PE-labeled streptavidin (1:100 dilution) and
FITC-labeled leukocyte subset markers at 4°C for 30 min. Lymphocytes
gated by side and forward light scatters were analyzed. B, CRTH2 expression on subpopulations of CD41 lymphocytes. PBMCs from a healthy
donor were stained with biotinylated BM16 and PE- or FITC-labeled
streptavidin, PerCP-labeled anti-CD4, and FITC- or PE-labeled mAb to
surface markers as described in panel A. CD41 lymphocytes gated by
PerCP staining were analyzed. C, Preferential expression of CRTH2
mRNA in BM16-bound PBMCs. PBMCs (uf) from a healthy adult were
separated into BM16-unbound (ub) and BM16-bound (b) cell fractions in
which percentages of BM16-bound cells were 5.4%, 1.4%, and 91.6%
respectively. DNaseI-treated total RNAs (0.2 mg) from the uf (lanes 1–3),
ub (lanes 4 – 6), and b (lanes 7– 8) cell populations were analyzed for relative levels of CRTH2 mRNA by RT-PCR as described in Materials and
Methods. The amounts of RT reaction product subjected to PCR were 2 ml
(lanes 1, 4, and 7), 0.2 ml (lanes 2, 5, and 8), and 0.02 ml (lanes 3, 6, and
9). DNA was stained with ethidium bromide.
The Journal of Immunology
FIGURE 7. Correlation between expression levels of CRTH2 and cytokines in CD41 peripheral blood lymphocytes. Whole blood of a healthy
adult was stimulated for 4 h as described in Fig. 3C. Leukocytes were
purified with standard erythrocyte-lysing solution, stained with FITC-labeled anti-CD4 and with biotinylated BM16 followed by RED670-conjugated streptavidin as described in Fig. 5 (x-axis), fixed, permeabilized, and
finally stained with PE-conjugated mAbs to IFN-g, IL-4, IL-5, and IL-13
as indicated on the left of each panel. CD41 lymphocytes gated by FITC
staining were analyzed.
Effect of cytokines on the expression of CRTH2
multiple staining revealed that most CRTH21 CD41 cells (.85%)
produced at least one of three typical Th2 cytokines, IL-4, IL-5,
and IL-13, but they produced little IFN-g (Fig. 7). Together, these
results indicate that CRTH2 is selectively expressed in Th2 but not
Th1 cells, even in vivo. Similar results were obtained in all donors
examined, although significant down-regulation of CRTH2 by T
cell activation was observed in some cases.
CRTH2 expression in allergen-responsive T cells
Most allergen-responsive Th cells were shown to exhibit Th2 phenotype (5, 11). Therefore, we next examined whether CRTH2 is
also expressed in such cells. PBMCs from adults susceptible to
pollen allergens were examined for their proliferative responses
against the allergens with cell populations negatively selected with
BM16 or control IgG2a. As shown in Table I, proliferative responses against the pollen allergens were markedly reduced by
subtracting CRTH21 cells, whereas those against a typical Th1type Ag PPD (2) were not significantly affected by the negative
selection. When purified CD41 cells were used as responder cells,
nearly complete depletion of the allergen-specific response was
observed (Table I, Expt. 2), which consequently suggested that the
majority of CD41 cells responsive to the pollen allergens also
expressed CRTH2.
The most potent environmental factors that influence Th1 and Th2
differentiation are known to be cytokines IL-12 and IL-4, respectively (3, 33, 34). Therefore, we next investigated the effect of
these cytokines on the expression of CRTH2. In PHA-stimulated
primary PBMC cultures, addition of IL-4 or IL-12 resulted in
marked enhancement or complete repression of the development
of CRTH21 cells, respectively. However, in these cultures, it was
difficult to verify the direct effect of IL-4 or IL-12 upon CRTH2
expression because proportions of Th2 cells in the cultures also
considerably changed in response to these cytokines; so, we examined this with Th2 clones. Among five Th2 clones used, two
clones (TKD21 and 6L21) showed slight enhancement in CRTH2
expression levels (26 –28% increase in relative mean fluorescence
intensity) in response to IL-4, while three clones (TKD23, TKD24,
and 6L21) indicated reduced CRTH2 expression (22–50% reduction) after treatment with IL-12. Table II shows results with one of
the responder clones, 6L21. These changes in CRTH2 expression
were considered to be directly induced by the cytokines because
the proportion of Th2 cells in the culture was not significantly
affected by these treatments (Table II). When both cytokines were
simultaneously added, the effect of IL-12 was dominant over that
of IL-4. This inhibitory effect of IL-12 on CRTH2 expression was
nearly completely cancelled by an anti-IL-12 p40 subunit mAb
(clone C8.6; Genzyme).
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FIGURE 6. Cytokine production in freshly isolated CRTH21 CD41
lymphocytes. PBMCs from a healthy adult were fractionated with BM16,
and cytokine production in each fraction was examined as described in Fig.
4. A, Purity of CRTH21 CD41 lymphocytes in unfractionated (uf), BM16unbound (ub), and BM16-bound (b) cell populations. B, Cytokine production in each cell population described in A. Only CD41 lymphocytes gated
by PerCP staining were analyzed.
1283
1284
SELECTIVE EXPRESSION OF A NOVEL SURFACE MOLECULE BY TH2 IN VIVO
Table I. Selective elimination of allergen-responsive cells from PBMCs by subtracting CRTH21 cells
Donor
Ab Used for
Subtraction
% of CRTH21
Cellsc
Expt. 1a
T.Y.
Rat IgG2a
2.1
BM16
K.H.
0.1
Rat IgG2a
3.9
BM16
Expt. 2b
K.H.
0.1
Rat IgG2a
2.9
0.2
2
Pollend
PPDe
2
Pollen
PPD
2
Pollen
PPD
2
Pollen
PPD
39.5 6 4.7
150.0 6 10.4
732.1 6 101.5
29.4 6 7.4
56.4 6 16.2
534.1 6 27.9
24.8 6 4.6
113.6 6 19.2
949.7 6 32.6
26.5 6 4.9
48.1 6 19.2
872.7 6 60.1
2
Pollen
PPD
2
Pollen
PPD
3.6 6 1.1
41.2 6 9.1
159.3 6 58.2
3.4 6 0.5
5.9 6 2.8
112.4 6 26.1
Stimulation
Index f
3.8
18.5
1.9
18.2
4.6
38.3
1.8
32.9
11.4
44.3
1.7
33.1
a
Monocytes and B cells were isolated from PBMCs of each donor with a mixture of anti-CD14-coupled and anti-CD19coupled microbeads (Miltenyi Biotec) and were used as APCs (5 3 104 cells/well), in which percentages of CD141 and CD191
cells were 49.5% and 24.4% (donor, T.Y.), and 43.5% and 25.8% (donor, K.H.), respectively. The remaining PBMCs were
negatively selected with BM 16 (or rat IgG2a) and anti-rat IgG-coupled magnetic beads (Dynal), then unbound cells (105
cells/well), in which proportions of CD41 lymphocytes were 21% (donor, T.K.) and 29% (donor, K.H.), were cultured with
APCs for 5 days in the absence (2) or presence of the indicated Ag. Cell proliferation was evaluated by [3H]thymidine uptake.
b
Monocytes were purified from PBMCs of donor K.H. and used as APCs (2.5 3 104 cells/well). CD41 cells were isolated
from the remaining PBMCs with CD4 MultiSort Kit (Miltenyi Biotec) and negatively selected with biotinylated BM 16 (or
biotinylated rat IgG2a) and streptavidin-coupled magnetic beads (Dynal). Then, unbound cells (5 3 104 cells/well) were cultured
for 6 days with APCs and the indicated Ag. Purity of CD141 monocytes and CD4† lymphocytes was 94.8% of APCs and 97.1%
of responder cells, respectively.
c
Percent of CRTH21 cells in CD41 lymphocytes.
d
An extract of pollen of Japanese cedar Cryptomeria japonica (Torii), 1% (v/v).
e
A total of 5 mg/ml.
f
Mean cpm in Ag (1) cultures/mean cpm in Ag (2) cultures.
Discussion
Our main goal in this study was to isolate surface molecules by
which we can easily distinguish Th1 or Th2 cells from other subsets of CD41 Th cells in humans in vivo. In the present study, we
isolated CRTH2 as a candidate for the Th2-specific gene from a
subtracted Th2 library. Studies with a specific mAb clearly demonstrated that CRTH2 is selectively expressed on the surface of
Th2 cells in vivo as well as in vitro. Little or no CRTH2 expression
was observed on naive T cells, Th1 cells, and most Th0 cells in
normal peripheral bloods. Surface phenotype (CD45RA2/
Table II. Effect of cytokines on the expression of CRTH2 in established
Th2 cellsa
Expression of CRTH2
Treatment
2
IL-4
IL-12
IL-4 1 IL-12
%
RMFIb
99.4 6 0.1 117.8 6 7.0
99.4 6 0.1 150.9 6 7.0*
93.4 6 0.3 55.4 6 0.8*
91.4 6 0.3 58.3 6 1.2*
Cytokine Production (%)c
Th1
Th2
Th0
,0.5
,0.5
,0.5
,0.5
43.8
44.9
43.1
42.8
,0.5
,0.5
,0.5
,0.5
a
A Th2 clone, 6L21, was cultured in the presence of IL-2 alone (2) or IL-2 plus
indicated cytokine(s), which were used at 100 ng/ml (IL-4) and 10 ng/ml (IL-12).
After 4 days, CRTH2 expression and cytokine production were examined as described
in Fig. 3. A mean (6SD) value of triplicate cultures (expression of CRTH2) and a
value in a 1:1:1 mixture of cells from the triplicate cultures (cytokine production) are
presented. * p , 0.01 (Student’s t test).
b
Relative mean fluorescence intensity (RMFI) of total cells. RMFIs of cells
stained with control IgG2a were 5.1– 6.5.
c
Percentages of Th1, Th2, and Th0 represent percentages of IFN-g-single positive, IL-4-single positive, and double-positive cells, respectively.
CD45RO1) of CRTH21 CD41 lymphocytes is consistent with
that of Th2, known as a subset of effector T cell. The CD45RO1
phenotype also agrees with a previous observation that most IL4-producing cells, including Th2 cells, are CD45RO1 (23). Furthermore, the results from the depletion experiments strongly suggest that CRTH2 is actually expressed on naturally developing
Ag-specific Th2 cells, such as pollen allergen-responsive T cells.
Thus, as yet, CRTH2 appears to be one of the most possible markers for human Th2 cells in vivo.
In some Th2 clones, CRTH2 was demonstrated to be up-regulated by the major Th2 inducer, IL-4, while down-regulated by the
powerful Th1 inducer, IL-12. These results suggest involvement of
autocline and/or paracline regulation mechanisms by such cytokines in Th2-specific expression of CRTH2, at least in some cell
conditions, although effects of these cytokines were not consistent
among Th2 clones and were not so striking even in responder
clones as compared with those on stimulated primary PBMC cultures. Here, absence or weakness of response to IL-12 in Th2
clones may be explained by the lack or poor expression of functional IL-12R b2 subunit as previously reported (8, 9). On the
other hand, IL-4 signaling might be no longer effective in such Th2
clones in which CRTH2 expression was maintained at high levels
by unknown mechanisms. Thus, another kind of approach is required to clarify the possible implication of these cytokines.
The present study also showed that the expression of CRTH2 in
PBMCs is highly restricted to an activated state (CD251) of T cells
(35). This is consistent with our observation that the majority of
Th2 cells in PBMCs exhibited low to intermediate levels of CD25
Ag (data not shown). However, we also observed a small number
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BM16
Ag
Mean (6SD)
[3H]Thymidine Uptake
(cpm 3 1022)
The Journal of Immunology
In conclusion, we have cloned a novel surface molecule by
which we can easily distinguish Th2 cells from naive T cells, Th1
cells, and most Th0 cells among CD41 lymphocytes of peripheral
blood. The protein enables us to highly purify or remove rare Th2
cells from PBMCs or cultured Th cells. Thus, this protein will be
useful for Th2 study and may also be a possible target for therapeutic intervention.
Acknowledgments
We thank Dr. Y. Yaoita (Tokyo Metropolitan Institute for Neuroscience,
Tokyo, Japan) for instruction of the gene expression screen method, Drs.
T. Kimura and N. Yamamoto (Tokyo Medical and Dental University, Tokyo, Japan) for COS7 transfected cells, and Drs. J. Miyazaki (Osaka University, Osaka, Japan) and Y. Takebe (National Institute of Infectious Disease, Tokyo, Japan) for expression vectors.
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might not be expressed in some populations of Th2 cells in vivo,
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