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*
Department of Immunology and
Howard Hughes Medical Institute, University of Washington, Seattle WA 98195
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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treatment of recombinase-activating gene 2
(Rag2-/-) mice to mimic pre-TCR signaling and drive
thymocyte development to the double positive stage, we have identified
murine GPR-9-6 as a chemokine receptor whose expression is strongly
induced following pre-TCR signaling. GPR-9-6 mRNA is present at high
levels in the thymus, and by RT-PCR analysis its expression is induced
as normal thymocytes undergo the double negative to double positive
transition. Furthermore we show that TECK (thymus-expressed chemokine),
a chemokine produced by thymic medullary dendritic cells, is a
functional ligand for GPR-9-6. TECK specifically induces a calcium flux
and chemotaxis of GPR-9-6-transfected cells. In addition, TECK
stimulates the migration of normal double positive thymocytes, as well
as Rag2-/- thymocytes following anti-CD3
treatment. Hence, GPR-9-6 has been designated as CC chemokine receptor
9 (CCR9). Our results suggest that TECK delivers signals through CCR9
important for the navigation of developing thymocytes. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-chain locus, the TCR
-polypeptide associates with the nonpolymorphic pre-T 
-chain and
the CD3 molecules to form the pre-TCR complex (1). In a
ligand-independent manner (2), the assembled pre-TCR then
delivers signals through non-receptor protein tyrosine kinases that
stimulate progression from the CD4-CD8- to
the CD4+CD8+ (double positive, DP) compartment.
This important early checkpoint ensures that only thymocytes that
express a functional TCR -chain polypeptide will continue to mature
(1). In addition to expression of the CD4 and CD8 genes,
several important phenotypic changes occur at this developmental
transition, including allelic exclusion of the TCR -chain locus,
induction of TCR -gene transcription and rearrangement, and marked
cellular proliferation, such that thymocyte numbers expand 100-fold
(1, 3). At a second major developmental checkpoint,
thymocytes survive or die according to signals for positive or negative
selection transmitted by the TCR -heterodimer (4).
Binding with too high an affinity results in negative selection,
whereas too low an affinity leads to death by neglect. In a minority of
DP thymocytes, the TCR binds MHC/peptide complexes present on the
surface of thymic stromal cells with the appropriate affinity and
signals further differentiation to mature CD4 or CD8 SP (single
positive) cells. Positively selected thymocytes reside as CD4 or CD8 SP
cells in the thymus for a time before emigrating to the periphery to
become mature peripheral T cells.
Induction of these developmental processes requires the close
approximation of T cell progenitors with thymic stromal elements, which
are thought to deliver signals for survival and differentiation through
cytokines and cell surface ligands (5). Although the
intracellular signals delivered by the pre-TCR in DN cells, or the TCR
-heterodimer in DP cells have been intensively studied, little is
known about the mechanisms that regulate the association of developing
thymocytes with specific thymic stromal elements. The composition of
thymic stromal cells has been shown to be highly complex and includes
several distinct subclasses of medullary and cortical thymic epithelial
cells, bone marrow derived dendritic cells (DC), macrophages, and
mesenchymal cells (5, 6). Different subpopulations of
developing thymocytes appear to interact with distinct stromal elements
(7, 8). More recently, association with specific types of
stromal cells has been shown to be required for the induction of
distinct stages of the thymocyte developmental program
(9, 10, 11). However, although early studies of T cell
maturation demonstrated that thymocytes migrate from the outer to the
inner cortex as they undergo the DN to DP transition, and to the
medulla as they mature to SP cells (5, 6, 12, 13), even
the signals that regulate this directional migration remain poorly
understood.
As in peripheral lymphoid organs, chemokines and their receptors likely
regulate the trafficking of developing T cells within the thymus
(14). Chemokines comprise a family of small basic
polypeptides that control leukocyte migration in a highly specific
manner (reviewed in Refs. 15 and 16). They stimulate chemotaxis by
activating pertussis toxin-sensitive G protein-coupled
seven-transmembrane receptors present on responding cells. With one
exception, all chemokines contain four cysteine residues linked by
intramolecular disulfide bonds. Four chemokine subfamilies have been
defined based on the spacing of the two NH2-terminal
cysteines with the majority falling into the CXC () or CC ()
family. Although there is some redundancy in the recognition of
chemokines by multiple receptors, distinct CXC and CC chemokine
receptor subfamilies (CXCR and CCR) have also been defined based on
structural homology and specific binding to the corresponding chemokine
class.
Developing thymocytes express multiple chemokine receptors and have
been shown to respond to chemokines that are produced within the
thymus. For example, thymocytes express CXCR4 and migrate in response
to its ligand SDF-1 (stromal cell-derived factor-1), which is
ubiquitously expressed but present at high levels in the thymus
(17, 18, 19, 20). Additional chemokine receptor-ligand pairs
present in the thymus include: CCR4, which binds TARC (thymus and
activation-regulated chemokine) and MDC (macrophage derived chemokine)
(21, 22, 23); CCR5 and MIP-1 (macrophage inflammatory
protein) (24); CCR7 and ELC (EBI-ligand chemokine or
MIP-3) (25, 26); and CCR8 and TARC (22, 27, 28). In some cases, these chemokine receptors have been shown to
be differentially expressed at specific stages of thymocyte
development. For example, DN and DP thymocytes are reported to express
higher levels of CXCR4 (relative to SP cells) and show greater
responsiveness to SDF-1 (17), whereas DP and SP thymocytes
express greater levels of CCR5 and are responsive to MIP-1
(24). Finally, CCR7 expression increases and chemotaxis to
ELC is acquired as thymocytes mature to SP cells (17, 26).
One CC chemokine hypothesized to play a role in the trafficking of developing thymocytes is TECK (thymus-expressed chemokine). Originally identified by random sequencing of a recombinase-activating gene 1 (Rag1-/-) thymus cDNA library, TECK is specifically expressed in the thymus and small intestine, both sites of T cell development (29). TECK efficiently stimulates the chemotaxis of thymocytes and thymic DC (29, 30). Furthermore, in situ hybridization studies for TECK mRNA or protein indicate that this chemokine is specifically produced by medullary DC of the adult thymus (29). In addition, recent RT-PCR analysis indicates that TECK is expressed by fetal thymic epithelial cells, suggesting that it may also function to direct the immigration of early T cell progenitors to the thymic rudiment (30). Thus it has been proposed that TECK is likely to regulate the migration of developing thymocytes (29).
To identify regulatory molecules important for thymocyte development,
we have performed a genetic screen to identify transcripts induced by
pre-TCR stimulation. Rag2-/- mice (which lack
recombination activating genes required for TCR locus rearrangement) do
not express the pre-TCR and therefore contain only immature DN
thymocytes (31). However, development of DN thymocytes to
the DP stage can be experimentally induced in a highly synchronous
manner by treatment with anti-CD3 mAb in vivo
(31, 32, 33). cDNA from stimulated and unstimulated
Rag2-/- thymocytes was isolated and applied to a
PCR-based subtractive hybridization procedure. From this screen we
isolated a cDNA clone corresponding to the murine homologue of an
orphan chemokine receptor, human GPR-9-6. Interestingly, GPR-9-6 is
most homologous to CCR7, known to be important for the trafficking of
peripheral T cells and DC in the lymph node and spleen
(34). Here, we report that murine GPR-9-6 mRNA is strongly
induced (>10-fold) following anti-CD3 treatment of
Rag2-/- thymocytes and is expressed at high levels in
normal murine thymocytes. Analysis of sorted thymocyte subsets has
confirmed that GPR-9-6 expression is up-regulated at the DN to DP
transition. Furthermore, we have determined that the chemokine TECK is
a functional ligand for GPR-9-6, and that DP and SP thymocytes and
anti-CD3-stimulated Rag2-/- thymocytes migrate in
response to TECK. Another group also recently reported the cloning of
murine and human GPR-9-6 cDNAs and the identification of TECK as a
specific ligand (35). Our results are consistent with
theirs, but include a detailed analysis of the expression pattern of
GPR-9-6 in murine thymocytes, as well as the migratory response of
specific thymocyte subpopulations to TECK. As TECK is produced by
thymic DC and fetal thymic epithelial cells, the results reported below
suggest that CCR9 (GPR-9-6) may transmit signals to T cell progenitors
that are important for their trafficking during development in the
thymus. Thus, our analysis supports the emerging view (36)
that the controlled expression of different chemokine receptors at
specific stages of thymocyte differentiation serves to regulate the
trafficking of developing T cells.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Rag2-/- mice were housed in a specific pathogen
free (SPF) facility. Four-week-old Rag2-/- mice were
injected i.p. with 150 µg of anti-CD3 mAb (145-2C11) diluted
in 300 µl PBS or PBS alone. 145-2C11 was affinity purified on protein
G sepharose (Pharmacia Biotech, Piscataway, NJ). Sixteen hours after
mAb treatment, pooled thymocytes from each group were isolated by
disaggregation through a wire mesh. Total RNA was isolated by CsCl
gradient centrifugation, and poly(A)+ RNA prepared using
the FastTrack 2.0 kit (Invitrogen, Carlsbad, CA). cDNA was synthesized
using an oligo(dT)-based primer and preamplified for 19 cycles using
the SMART kit (Clontech, Palo Alto, CA) under conditions designed to
give full-length cDNA products and to minimize distortion of the
relative abundance of individual clones. The preamplified cDNA was used
for suppression subtractive hybridization with the Clontech PCR Select
kit according to the manufacturer’s instructions and screened
essentially as described (37), except that the resulting
difference products were subcloned into pBluescript (Stratagene, La
Jolla, CA). Sequencing reactions were performed with primers specific
to the T3 and T7 promoters, using the dye Rhodamine PCR mix and
analyzed on an ABI model 377 (Applied Biosystems, Foster City, CA)
automated sequencer. Following homology searches against the National
Center for Biotechnology Information (NCBI) Nonredundant and EST
databases using the BLAST 2.0 algorithm, one clone (2B7) matched two
nonoverlapping murine thymus-derived ESTs (accession nos. AA110666 and
AA863796) and was chosen for further study. As the assembled contig
(Sequencher 3.0 software, Gene Codes, Ann Arbor, MI) lacked an open
reading frame, rapid amplification of cDNA ends (RACE) PCR was
performed using the Marathon cDNA amplification kit (Clontech)
according to the manufacturer’s instructions. 5'-RACE using the primer
5'-GTAGCAGGGAGCCTCAGAAGGAAAGGCC-3' produced an 
2.5-kb fragment;
sequencing revealed it to be the murine homologue of the orphan human
chemokine receptor GPR-9-6 (accession no. U45982). A cDNA fragment
containing the entire open reading frame of murine GPR-9-6 was then
generated using KlenTaq polymerase (Clontech) to amplify normal thymus
cDNA with the following forward and reverse primers:
5'-CAGGCAGCTGCAGTGGTCCTCTCCC-3' and 5'-TGTGCAAGGCTGGGCTGTCTTTGC-3'.
Six independent clones were subcloned into pBluescript using internal
PstI sites and sequenced completely to obtain a consensus
sequence.
Northern blot analysis
Total RNA was isolated from thymi of 4-wk-old PBS or
anti-CD3 treated Rag2-/- mice, or tissues of
6-wk-old C57BL/6 mice using RNA Stat-60 (Tel-Test, Friendswood, TX).
RNA (8 µg) from Rag2-/- thymi, Rag2-/-
TAP-/- OT-1 transgenic (nonselecting thymi; see 37),
or Rag2-/- OT-1 transgenic (selecting) or were separated
on a 1% agarose formaldehyde gel, transferred to a Zeta-Probe GT
membrane (Bio-Rad, Hercules, CA), and analyzed according to the
manufacturer’s instructions. For multi-tissue Northern blots, 10 µg
RNA from the indicated tissues were loaded per lane. To analyze mCCR9
(murine CCR9) expression in activated splenocytes, C57BL/6 splenocytes
were cultured at 2 x 106 cells/ml in RPMI 1640 10%
FCS (Gemini-BioProducts, Calabasas, CA) alone or in the presence of 2.5
µg/ml Con A (Calbiochem, La Jolla, CA), or 5 µg/ml LPS (Difco,
Detroit, MI), and analyzed at 0, 24, and 48 h. Murine cDNA probes
included the 1.4-kb PstI fragment of mCCR9, an 450-bp
fragment from the 5' coding region of mCCR7, CD69, and EF1 as a
loading control. These were radiolabeled by random priming (Life
Technologies, Gaithersburg, MD) using [-32P]dATP
(DuPont, Boston, MA).
RT-PCR analysis of sorted thymocyte subsets and fetal thymi
Thymocytes from 6 to 8-wk-old C57BL/6 mice were sorted using a flow cytometer into DN, DP, CD4 SP, and CD8 SP fractions. RNA was prepared from sorted cells and used to synthesize cDNA as previously described (38). Three-fold serial dilutions of cDNA from each population were normalized according to the hypoxanthine phosphoribosyltransferase (HPRT) signal. Amplification of HPRT was performed using similar conditions with primers specific to exons 7/8 and 9 (38), except that an annealing temperature of 55°C was used. PCR of CCR9 was performed for 30 cycles (94°C for 1 min, 68°C for 45 s, and 72°C for 1 min) using the forward primer 5'-CCCAGTTTCTGTTACTTCAGATCTG-3', with the reverse primer being the same reverse primer listed above for RACE PCR of mCCR9. PCR products were separated on a 1% agarose gel, transferred to Zeta-Probe GT membrane, and visualized by Southern blotting. For mCCR9, the 270-bp 2B7 probe was used; for HPRT a probe was generated by PCR using the primers listed above. For RT-PCR analysis of fetal thymi, timed matings were conducted, and the day of gestation was calculated based on the plug date being designated as day 1. RT-PCR was performed as for sorted thymocyte subsets, except that 28 cycles were used.
Generation of mCCR9 and mCCR7 expression constructs
PCR amplification was performed using PFU Turbo polymerase (Stratagene), with the resulting constructs verified by DNA sequencing. An HA (hemagglutinin) epitope tag was added to the NH2 terminus of mCCR9 at two different sites: the initiator methionine present in our mCCR9 cDNA clone, and the methionine at amino acid 13, predicted to be the start site in the reported human genomic GPR-9-6 (hCCR9) clone. These constructs were generated using 5' primers designed to contain a BamHI restriction enzyme site, and a 3' primer in the mCCR9 coding sequence. The 5'-tagged cDNA fragments of the resulting PCR products were ligated to 3' mCCR9 coding sequences in pBluescript at the NcoI site for HA-CCR9.1 or the NsiI site for HA-CCR9.2 (because this 5' primer contained an NcoI site). The initiator methionine for these constructs is indicated by bold lettering: HA-CCR9.1 forward primer; 5'-AAGGGATCCGCTATGGCTTATCCTTATGATGTTCCTGATTATGCTGCTTTTGATGACTTCAGCTATGACTCC-3'; HA-CCR9.2 forward primer; 5'-AAGGGATCCACCATGGCTTATCCATACGATGTTCCAGATTATGCTATGCCCACAGAACTCACAAGCC-3'; and reverse primer; 5'-GCTTCAAGCTACCCTCTCTCCTTG-3'.
A construct spanning the coding region of mCCR7 (accession no. L31580) was generated by PCR amplification of overlapping 5' and 3' fragments that were then joined using a unique HindIII site. For the 5' fragment an EcoRI restriction site was introduced. PCR primers used were: 5' CCR7, 5'-AAGGAATTCTACAGCCCCCAGAGCACC-3' and 5'-GATGCATAGGAGCAGCAGCATCC-3'; 3' CCR7, 5'-GCCAAGTCCTGGATCTTTGGCG-3' and 5'-GCTTGAGTGTTAACTTCTCAGCC-3'.
The 1.4-kb untagged mCCR9 cDNA, each HA-tagged mCCR9 cDNA, and mCCR7 were then subcloned from pBluescript into the retroviral expression vector pMI (39, 40). Subcloning of mCCR9 constructs utilized BamHI and EcoRI sites, whereas mCCR7 was subcloned using BamHI and XhoI/SalI sites.
Isolation and flow cytometry of L1.2 cells expressing mCCR9 and mCCR7
The murine pre-B cell line L1.2 (41) was maintained in RPMI 1640 containing 10% FCS, 2 mM glutamine, 25 mM HEPES, 50 µM 2-ME, 100 U/ml penicillin, and 100 µg/ml streptomycin. L1.2 cells were retrovirally infected with each chemokine receptor-pMI construct. pMI contains an internal ribosome entry site (IRES), allowing cells expressing the chemokine receptor transgene to be identified by the IRES-driven human CD2 marker (hCD2). Generation of retrovirus, infection of cells, and enrichment for infected cells by panning for hCD2-positive cells was performed as previously described (39, 40). Following panning, L1.2 clones expressing each chemokine receptor construct were obtained by limiting dilution. For flow cytometry, retrovirally infected L1.2 cells were stained using PE-anti-hCD2 (PharMingen, San Diego, CA), or the anti-HA mAb HA.11 (Berkeley Antibody, Berkeley, CA) followed by FITC-goat anti-mouse IgG (PharMingen). L1.2 cells were stained in the presence of the rat anti-Fc receptor mAb 2.4G2 (42). Flow cytometry was performed on a FACScan or FACSCalibur machine, and analyzed using CellQuest software (Becton Dickinson, San Jose, CA).
Chemokines and calcium fluorimetry
Recombinant murine 6Ckine (SLC), Mip-3 (ELC), and TECK were
from R&D Biosystems (Minneapolis, MN), while MDC was a gift from Dr.
Jason Cyster (University of California, San Francisco). For calcium
fluorimetry, L1.2 cells were loaded with 20 µg/ml Indo-1-AM
(Molecular Probes, Eugene, OR, or Sigma, St. Louis, MO) in the dark at
37°C in the presence of DMEM, 3% FCS, and 4 mM Probenecid (Sigma)
using standard procedures (43). Mobilization of calcium in
response to a dose range of chemokines or ionomycin was measured using
a FACStarPlus flow cytometer; listmode files were collected
using Lysis II Software (Becton Dickinson). Data were later analyzed
using ReproMan (True-Facts Software, San Diego, CA) and Mtime 2.0
software (Phoenix Flow Systems, San Diego, CA).
Chemotaxis assays
Migration assays were performed using transwell polycarbonate membranes (24-well format, 6.5 mM diameter, 5 µM pore size) from Costar (Cambridge, MA). Transwells were preincubated overnight in assay medium consisting of RPMI 1640, 0.5% FCS, and 1% BSA. For assays using Rag2-/- thymocytes, 4-wk-old mice were treated with mAb 2C11 or PBS as described above, with their thymocytes isolated 20 h or 3 days post-mAb stimulation. In chemotaxis assays, 5 x 105 L1.2 cells or 1 x 106 thymocytes in 100 µl were added to the upper chamber and allowed to migrate to chemokines diluted in 600 µl assay medium in the lower chamber. Following incubation for 4 h at 37°C and 8% CO2, the number of transmigrated cells in the lower chamber was determined by counting with a hemocytometer. Migrated C57BL/6 thymocytes or thymocytes of Rag2-/- mice treated with 2C11 mAb for 3 days were stained with anti-CD4-PE and anti-CD8-FITC (PharMingen). Rag2-/- thymocytes 20-h post-2C11 treatment were stained with anti-CD44-PE and anti-CD25-FITC mAb. All of these mAb were from PharMingen. Statistical significance was assessed using the Mann-Whitney U test as described (37). The percentage of each thymocyte subpopulation (DN, DP, CD4 SP, or CD8 SP) that responded to TECK or ELC was calculated as follows: the absolute number of cells migrating in the absence of chemokine was subtracted from the absolute number of cells from that population migrating in the presence of chemokine. This number was then divided by the absolute number of input cells for a given population.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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stimulation of Rag2-/- thymocytes
To identify regulatory molecules induced during the DN to DP
transition of thymocyte development, we performed subtractive
hybridization using cDNA from anti-CD3-stimulated and
unstimulated Rag2-/- thymocytes. We and others have found
that thymocytes of Rag2-/- mice treated with a single
i.p. injection of the anti-CD3 mAb 2C11 undergo the DN to DP
transition in a synchronous and ordered fashion. Stimulated thymocytes
start to acquire surface expression of the activation marker CD69
within 2 h, decrease CD25 within 16 h, and start to express
CD8 by 2–3 days, with 50% of cells becoming
CD4+CD8+ by day 4 (32, 33, 44).
Cellular proliferation is also rapidly induced, such that cell numbers
increase 2-fold by 16 h, 10-fold in 4 days, and 100-fold in 1
wk.
We performed subtractive hybridization using cDNA from pooled
Rag2-/- thymocytes 16 h post-anti-CD3 mAb
treatment. At this time point, >80% of thymocytes become
CD69+ and progress from being
CD44-CD25+ to
CD44-CD25- (data not shown), as normally
occurs following pre-TCR signaling (1). Among the cDNAs
obtained following subtractive hybridization, one cDNA clone (clone
2B7) was of interest because it only matched two overlapping murine
thymus-derived ESTs, although it lacked an open reading frame. Northern
blot analysis showed that this clone was derived from an 3-kb mRNA
whose expression was strongly induced following anti-CD3
stimulation of Rag2-/- thymocytes, and showed high level
expression in normal murine thymus. RACE PCR to identify the
full-length coding sequence revealed that 2B7 corresponded to the
murine homologue of the orphan human chemokine receptor GPR-9-6. As
discussed above, GPR-9-6 recently has been designated as CCR9
(14, 35), and we will refer to it as such for the rest of
this manuscript.
Sequence analysis of our 1.4-kb cDNA mCCR9 fragment revealed that it encodes an open reading frame of 369 aa that is highly homologous to the hCCR9 genomic clone (hGPR-9-6; accession no. U45982) listed in the GenBank database. The predicted amino acid sequence we obtained was identical to that recently reported by Zaballos et al. (Ref. 35; EMBL accession no. AJ132336) showing an 86% amino acid identity between mCCR9 and hCCR9. We too found that mCCR9 contains an additional 12 NH2-terminal amino acid residues not found in the original hGPR-9-6 clone due to the presence of an additional upstream coding exon. While chemokine receptors are generally encoded by a single large exon, it is interesting that like CCR9, the two chemokine receptors most homologous to CCR9, CCR6 and CCR7, are exceptions to this rule (Refs. 34 and 35, and our own unpublished results).
Analysis of mCCR9 expression during T cell development and in murine tissues
Using the full-length cDNA as a probe, we performed Northern blot
analysis to determine the time course of CCR9 up-regulation following
anti-CD3 stimulation of Rag2-/- mice (Fig. 1
A). CCR9 mRNA was strongly
induced within 16 h of anti-CD3 stimulation, and was
further up-regulated by 48 h, to a level comparable to that
observed for normal C57BL/6 thymocytes. Some up-regulation (5-fold) was
also observed in RNA from Rag2-/- thymocytes only 7
h after anti-CD3 treatment, a time point at which the majority
thymocytes express CD69 (data not shown). To confirm that mCCR9 is
induced as thymocytes mature to the DP compartment, we performed
Northern blot analysis using RNA from OT-1 TCR transgenic thymocytes on
a nonselecting (Rag2-/- TAP1-/-) or
selecting (Rag2-/-) background. In the former, thymocytes
are arrested at the DP stage due to lack of the MHC class I/peptide
ligand (37). As shown in Fig. 1B, mCCR9
expression is markedly up-regulated in a nonselecting background
containing mostly DP cells, relative to Rag2-/- DN
thymocytes. In addition, Northern blot analysis of RNA from multiple
tissues revealed that mCCR9 expression was restricted to the lymphoid
system (Fig. 1C), with very high level expression in the
thymus and much lower levels in lymph node, spleen, and small
intestine, another site of T cell development (45).
Finally, by Northern blot analysis we failed to detect any CCR9
expression following stimulation of murine splenocytes with Con A or
LPS, suggesting that CCR9 expression is not induced by activation of
peripheral T or B cells (data not shown).
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For comparison, we evaluated the expression of CCR7 under the same
conditions. Although CCR7 mRNA expression was also induced at low
levels by anti-CD3 treatment of Rag2-/- thymocytes
(data not shown), it was unlike CCR9 in that it was present at higher
levels in spleen and lymph node, relative to the thymus (Fig. 1
C), consistent with previous reports (51, 52).
Strong up-regulation of CCR7 expression and responsiveness to its
ligand ELC have been shown to occur as DP thymocytes mature to SP cells
(17, 26) and following peripheral T cell activation
(53). Although some increased CCR7 expression appears to
occur at the DN to DP transition (17), the CCR7 expression
pattern differs significantly from CCR9, suggesting that the latter may
play a role in earlier stages of thymocyte development.
To confirm that CCR9 expression is induced as normal murine thymocytes
undergo the DN to DP transition, we analyzed mCCR9 expression using
cDNA from sorted thymocyte subsets (Fig. 2A). Quantitative RT-PCR
analysis showed that CCR9 expression was 5- to 10-fold higher in DP
thymocytes relative to DN cells, and then decreased with maturation to
the CD4 SP or CD8 SP compartment. This result was confirmed using cDNA
from two independent thymocyte sorts. Some amount of CCR9 mRNA
expression in DN cells was expected, because this population also
includes thymocytes that have received a pre-TCR signal and have
initiated the DN to DP transition (1, 54). This result is
consistent with our Northern blot analysis which showed that CCR9
expression is rapidly induced by anti-CD3 treatment of
Rag2-/- mice (Fig. 1A), and that CCR9 mRNA is
present at high levels in nonselecting thymi in which development is
arrested at the DP stage (Fig. 1B). Our results differ from
those reported by Zaballos et al. (35), who did not
observe differential expression of CCR9 in different thymocyte
subpopulations. However, their analysis did not include a quantitative
titration of input cDNA, making it quite possible that they could have
missed the differential expression that we observed.
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B). mCCR9 mRNA was detected in fetal thymi as early as day
15 of gestation, when only immature DN thymocytes are present. An
increase in CCR9 expression was observed in two independent experiments
using cDNA from day 17 fetal thymi, or pooled thymi from day 17–19,
consistent with up-regulation following pre-TCR stimulation as
significant numbers of DP cells are present in day 18 fetal thymus
(data not shown). In addition, we detected low levels of mCCR9 in cDNA
isolated from day 15 and 16 fetal liver (data not shown). Together
these results suggest that in addition to playing a role at the DN to
DP transition of thymocyte development, CCR9 might also function in the
immigration of thymocyte progenitors into the thymus. TECK stimulates a calcium flux in mCCR9-transfected cells
The results described above strongly suggest that CCR9 is up-regulated by pre-TCR stimulation of murine thymocytes. In addition, CCR9 is detectable in early thymocyte progenitors. Interestingly, the pattern of CCR9 expression is quite similar to that reported for the chemokine TECK (29), with TECK present at a high level in the thymus and in small intestine, but not in the spleen. TECK is also present in Rag2-/- thymus and fetal thymus as early as gestational day 14. Shown to be produced by DC in the medullary compartment of adult murine thymi (29) or fetal thymic MHC class II+ epithelial cells (30), TECK has been implicated as playing a role in thymocyte development. Similar to TECK, the expression pattern of CCR9 suggests a possible role in thymocyte development.
As a first step toward identifying a functional ligand for mCCR9, we
generated L1.2 cells stably expressing two different
NH2-terminal HA-tagged versions of mCCR9. The HA tag was
either placed upstream of the initiator methionine encoded by the mCCR9
cDNA clone (mCCR9.1) or at amino acid 13, the homologous methionine
residue predicted to be the translational start from the human genomic
sequence (CCR9.2). Thus the HA tag allowed us to analyze the level of
mCCR9 expression at the cell surface and to determine which methionine
start codon would generate a stable protein. As shown in Fig. 3A, flow cytometry analysis
demonstrated that L1.2 cells expressed significant levels of either
HA-tagged version of mCCR9 at the cell surface, indicating that either
methionine could initiate the translation of a stable protein.
Importantly these results suggested that we had obtained a functional
mCCR9 cDNA clone.
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). Like the HA-tagged mCCR9 constructs, the untagged mCCR9 or
mCCR7 cDNA inserts were expressed using the retroviral vector pMI,
which contains an IRES followed by a cDNA encoding a tail-less form of
human CD2 (hCD2). This makes it possible to identify transgene
expressing cells by flow cytometry for the hCD2 reporter (39, 40). As shown, the mCCR9 and mCCR7 constructs were expressed at
comparable levels in L1.2 cells (Fig. 3B). Because mCCR9 is
expressed at high levels in developing thymocytes, we focused on
chemokines reported to be present in the thymus. mCCR9 failed to
mediate a significant calcium flux to ELC or SLC (over a concentration
range of 1–50 nM), whereas mCCR7 transfectants responded well to both
of these previously established ligands (Refs. 25 and 26; Fig. 3, D and E, and data not shown). Consistent with
this observation, it has been reported by others that the human GPR-9-6
clone (hCCR9) fails to respond to ELC in calcium flux assays
(25). Similar results have been reported for TARC
(22). In addition mCCR9 transfectants were unresponsive to
MDC (Fig. 3D). Remarkably, TECK stimulated a strong and
specific calcium flux in mCCR9 L1.2 cells, but not in untransfected or
mCCR7-transfected L1.2 cells. This rapid response was dose sensitive,
with over 90% or 50% of the transfected cells responding to 10 nM or
1 nM TECK, respectively (Fig. 3C). It has also been reported
that TECK does not stimulate CCR6-transfected cells (55).
Our results are consistent with the calcium flux analysis performed by
Zaballos et al. (35) using CCR9 transfected HEK 293 cells,
although we observed a somewhat more robust calcium flux in
mCCR9-transfected L1.2 cells at lower concentrations of TECK. In
addition, we have determined that L1.2 cells expressing either
HA-tagged version of mCCR9 (Fig. 3A) mobilize intracellular
calcium in response to TECK, achieving levels quite comparable to that
of L1.2 cells expressing the full-length untagged mCCR9 cDNA (data not
shown). Thus, the first 12 aa of mCCR9 are not essential for ligand
binding. Together, the results described above confirm that mCCR9 is a
specific and functional receptor for TECK, a chemokine proposed to play
a role in the migration of developing thymocytes (29). TECK stimulates chemotaxis of mCCR9-transfected cells
We next tested the ability of mCCR9 to stimulate the chemotaxis of
L1.2 cells in response to TECK. As shown in Fig. 4, we observed a robust migration such
that 90% of CCR9 positive L1.2 cells migrated in response to TECK
(left panel), but not to ELC (right
panel). Conversely, CCR7 positive L1.2 cells responded to ELC, but
not to TECK. Chemotaxis was distinguished from chemokinesis by
inclusion of TECK in both the upper and lower compartment of the
chemotaxis chamber (CCR9 L1.2 control, left panel). In
contrast to previous studies reporting a maximal chemotactic response
of thymocytes to TECK at 10 ng/ml (1 nM; Refs. 29 and 30), we
observed that mCCR9 transfectants and thymocytes (see below) required
significantly higher concentrations of TECK (50 nM) to undergo
chemotaxis. While Zaballos et al. (35) did not analyze
chemotaxis in response to mTECK, they observed that a relatively high
dose of hTECK (100 nM) was also required to stimulate efficient
migration of hCCR9 HEK 293 cells. These results, together with the
strong calcium flux observed in mCCR9 transfectants (even in the
presence of 1 nM TECK, Fig. 3C), establish that TECK is a
specific and functional ligand for mCCR9.
|
stimulated Rag2-/-
thymocytes and normal DP and SP thymocytes
We next analyzed the chemotactic activity of murine thymocytes in
response to TECK. As shown in Fig. 5A, normal C57BL/6 thymocytes
responded to TECK with 10-fold more thymocytes migrating to the bottom
transwell chamber containing TECK, relative to the control. Like the
L1.2 CCR9 transfectants, a lower concentration of TECK failed to
stimulate the chemotaxis of normal thymocytes, consistent with the
response being mediated by CCR9. Because we had identified CCR9 as a
chemokine receptor up-regulated following anti-CD3 treatment of
Rag2-/- thymocytes, we wished to determine whether
responsiveness of Rag2-/- thymocytes to TECK could be
similarly induced. We tested the chemotactic activity of
Rag2-/- thymocytes 20 h after in vivo treatment with
anti-CD3 or PBS as control. Flow cytometry of stimulated
thymocytes has consistently shown that at this time point >90% of
thymocytes have received a pre-TCR-like signal and down-regulated CD25
(data not shown), whereas Northern blot analysis has revealed that
mCCR9 mRNA is present at high levels (Fig. 1A). We noted
that even in the absence of chemokine, anti-CD3-stimulated
thymocytes showed significantly more migratory activity than the PBS
control, consistent with the notion that the migration of developing
thymocytes is induced following pre-TCR signaling (Fig. 5A).
Rag2-/- thymi likely contain a significant amount of TECK
(29), that could stimulate the migratory activity of CCR9
positive cells. Furthermore, anti-CD3-treated
Rag2-/- thymocytes acquired the ability to specifically
respond to TECK in chemotaxis assays. This result was reproducible, and
the difference in the migration of anti-CD3 treated
Rag2-/- thymocytes in the presence or absence of TECK was
statistically significant (p < 0.01). We also
tested the chemotactic activity of Rag2-/- thymocytes 3
days post-anti-CD3 treatment, a time point when >90% of cells
are CD25-, with 5% DP cells (data not shown). At this
time point, the number of cells migrating (±SEM) in the absence of
chemokine was similar to unstimulated Rag2-/- thymocytes
(2.3 x 104 ± 0.26), but was increased in
response to TECK (1.0 x 105 ± 0.09). Hence, as
for CCR9 L1.2 cells, normal C57BL/6 and anti-CD3-stimulated
Rag2-/- thymocytes showed specific migration in response
to 100 nM TECK. Because TECK and CCR9 show a similar expression pattern
at sites of T cell development, these results are consistent with CCR9
mediating the chemotaxis induced by TECK in developing thymocytes.
|
B). A difference in the
populations of C57BL/6 thymocytes migrating to TECK in comparison to
ELC was readily apparent. While the flow cytometry profile of cells
migrating to ELC showed an enrichment of CD4 SP and CD8 SP cells
(right panel), consistent with previous reports (17, 26), both DP and SP thymocytes migrated in response to TECK
(middle panel). We also calculated the percent of input
cells (±SEM) specifically migrating for each thymocyte subset in
response to TECK or ELC, and thus confirmed that increased
responsiveness to TECK is acquired with thymocyte maturation to DP
cells (Table I). This correlates well
with up-regulation of CCR9 expression at the DN to DP transition.
However, although RT-PCR analysis showed that SP thymocytes express a
lower amount of CCR9 mRNA relative to DP cells (Fig. 2A),
both populations migrated similarly to TECK. It is possible that a
significant amount of CCR9 protein may still be present in a
subpopulation of SP thymocytes, which can only be determined with
development of staining reagents for CCR9. After submission of this
manuscript, Campbell et al. (36) reported a detailed
analysis of the migration of specific populations of DP and SP
thymocytes to several chemokines expressed in the thymus, including
TECK. Consistent with our own results (Fig. 5 and Table I), DP and SP
thymocytes responded to TECK (36). Interestingly,
responsiveness to TECK was lost in the most mature population of CD4 SP
thymocytes (CD69-L-selectinhigh), while
migration to ELC and SLC was strong, suggesting that CCR9 is
specifically down-regulated just prior to emigration from the thymus.
Peripheral T cells (29, 36) and activated T cells
(30) are unresponsive to TECK, consistent with a loss in
CCR9 expression once thymocytes fully mature and emigrate to the
periphery. This conclusion is supported by our Northern blot analysis
of Con A-stimulated splenocytes, in which we failed to detect
up-regulation of CCR9, although expression of CD69 mRNA was induced
(data not shown). Together with the results of our chemotaxis assays,
these results support the notion that CCR9 delivers signals to
thymocytes in response to TECK that are important for T cell
development.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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(57) fail to emigrate to the periphery. Finally, it has
also been suggested that recirculation of mature peripheral T cells to
the thymus may also be driven by chemoattractants (17).
Despite the biologic importance of these processes, little is known
about the specific chemokines or the receptors that regulate the
trafficking of developing thymocytes at these different stages.
In this report we have identified murine GPR-9-6 as a CC family
chemokine receptor whose expression is up-regulated at the DN to DP
transition of thymocyte development. We have shown that GPR-9-6 mRNA
expression is rapidly induced following anti-CD3 stimulation of
Rag2-/- thymocytes (which mimics pre-TCR signaling), and
that it is significantly increased as normal thymocytes mature to DP
cells. In addition we have determined that GPR-9-6 is a specific and
functional receptor for the chemokine TECK, and that
anti-CD3-stimulated Rag2-/- thymocytes, as well as
normal DP and SP cells, migrate in response to TECK. Based on sequence
homology to other CC chemokine receptors and its specific ability to
mediate a calcium flux and chemotaxis in response to TECK, we agree
with the proposal by Zaballos et al. (35) to designate
GPR-9-6 as CCR9. It has also been proposed that the chemokine receptor
D6 should be designated as CCR9 (14, 58, 59), while the
rat and human equivalents have also been published as CCR10 (60, 61). However, this receptor is highly promiscuous in its binding
and expression pattern, and the human D6 equivalent fails to signal in
transfected cells (59). Hence, GPR-9-6 has been designated
here and by Zaballos et al. (35) as CCR9.
In contrast to our studies, Zaballos et al. (35) failed to
observe differential expression of CCR9 by RT-PCR analysis of sorted
thymocyte subsets. Notably, they evaluated CCR9 expression in
CD25+ DN cells, which comprise the majority of thymocytes
in Rag2-/- mice due to absence of the pre-TCR (1, 31). This discrepancy from our results is likely explained by
nonlinear amplification of a small amount of CCR9 mRNA present in their
sorted DN thymocytes. In support of this, by Northern blot analysis we
detected low level expression of CCR9 mRNA in Rag2-/-
thymocytes, which was strongly up-regulated following anti-CD3
stimulation (Fig. 1A), as well as strong expression of CCR9
mRNA in thymocytes arrested at the DP stage of development (Fig 1B). Moreover, we performed a quantitative RT-PCR analysis
of normal thymocyte subsets using titrated cDNA from two independent
sorts. We have also recently performed in situ hybridization studies
which indicate that CCR9 mRNA is expressed in the majority of cortical
thymocytes, and to a lesser extent in the medulla (in collaboration
with T. Gurney and N. Landau, unpublished results).
Because TECK is produced by medullary DC in the adult thymus (29), our results suggest that up-regulation of CCR9 following pre-TCR assembly may be an important mechanism regulating the cortico-medullary migration of developing thymocytes. Another possibility proposed by Campbell et al. (36) is that TECK may function to retain cells in the thymus until they have fully matured, since CD4 SP CD69-L-selectinhigh thymocytes lose responsiveness to TECK. Finally, TECK has also been shown to be expressed by fetal thymic MHC class II+ epithelial cells, suggesting that it could also play a role in the recruitment of early thymocyte progenitors (30). In support of this notion, we have detected CCR9 expression in fetal liver, as well as in fetal thymus tissue as early as day 15 of gestation. Thus, CCR9 and its ligand TECK may regulate the trafficking of developing thymocytes at two different stages: during emigration of hematopoietic precursors to the fetal thymus, and later following induction of the DN to DP transition.
In the family of chemokine receptors CCR9 is phylogenetically grouped
with CCR6 and CCR7, suggesting a similarity in function (34, 35). In this regard it is interesting that CCR6 and CCR7 are
predominantly expressed in lymphoid tissues, and that they share the
feature of having introns in their 5' coding sequence, which is
uncharacteristic of most chemokine receptors (34, 52, 62).
Both CCR6 and CCR7 appear to play important roles in regulating the
trafficking of different subsets of T cells and DC in the periphery
(63). For example, liver and activation-regulated
chemokine (LARC; MIP-3), a unique ligand for CCR6, specifically
stimulates the migrational arrest of memory CD4+ T cells
under rolling flow conditions (64). Recent evidence
indicates that CCR7 may be essential for the homing of naive peripheral
T cells to lymph nodes and the splenic white pulp (26, 63). In particular, T cells of mice with the spontaneous Plt
mutation (paucity of lymph node T cells) fail to migrate to the T
cell-rich zones of peripheral lymphoid organs (65, 66), a
phenotype attributed to the absence of the CCR7 ligand, SLC, in these
mice (67). CCR6 and CCR7 are also expressed by specific
subsets of DC. While CCR6 is present on immature DC derived from
hematopoietic but not monocytic precursors, DC activation and
maturation is accompanied by a loss of CCR6 expression with sharp
up-regulation of CCR7 (68, 69). Thus it has been suggested
that CCR7 may be important for the colocalization of T cells and
Ag-presenting DC in peripheral lymphoid tissues (63).
Interestingly TECK has also been reported to have chemotactic activity
for activated DC and macrophages (29), suggesting that
CCR9 could play a role in the association of these cell types with
developing thymocytes.
While several chemokines and their receptors are expressed within the thymus, their specific contribution to T cell development remains unclear. Studies of mice deficient in SDF-1 or its receptor, CXCR4, demonstrated that chemokines can play essential roles in lymphoid development, as either of these mouse mutants shows a severe defect in early B cell development prior to the B220+CD43+ stage (70, 71, 72). However, despite high level expression of SDF-1 and CXCR4 in the thymus, thymocyte development proceeds normally in their absence (72). In addition CCR5 is expressed on developing thymocytes, but its absence does not appear to impair T cell development. Humans with homozygous CCR5 mutations have the benefit of being resistant to HIV infection, but appear to have normal immune function (73). Finally, TECK has been shown to specifically stimulate the migration of fetal thymic precursors in chemotaxis assays and to be produced by MHC class II+ fetal thymus epithelial cells (30). Together with our observation that CCR9 is expressed in the fetal liver and in day 15 fetal thymus, these results implicate a role for TECK and CCR9 in the recruitment of fetal thymocyte precursors. However, in one report an anti-TECK antiserum (which neutralized chemotaxis of thymic precursors to TECK) failed to inhibit colonization of fetal thymus lobes (30). The role of TECK later in thymocyte development was not addressed in this study. Although it cannot be excluded that the amount of anti-TECK antiserum present in the fetal thymus lobes was insufficient to fully neutralize chemokine activity, it is also possible that TECK has some redundancy of function with other chemokines. As for other important regulatory molecules, it is likely that some chemokines and their receptors may serve redundant functions in T cell development. However, the tight regulation of chemokine receptor expression at discreet stages of thymocyte development strongly suggests that different chemokines serve specific functions during T cell differentiation (36).
In addition to stimulating migration, there is evidence that chemokine
receptors may function to deliver signals for lymphocyte proliferation
and survival (14). For example, the CC chemokine RANTES
has been shown to stimulate T lymphocyte activation and proliferation
(74), whereas SDF-1 was initially cloned by Nagasawa et
al. (75) as a pre-B-cell growth stimulating factor (PBSF).
Studies of the ability of SDF-1 to drive the proliferation of immature
DN thymocytes have not been reported. A role for chemokine receptors in
promoting cell survival was suggested by the observation that the
chemokine I-309, (which binds CCR8), protects murine T cell lymphomas
from dexamethasone-induced apoptosis (76). Our studies
show that CCR9 expression is strongly induced following pre-TCR
stimulation, a time of intense cellular proliferation (Figs. 2 and 3,
and 1). Having identified TECK as a specific ligand, we are now in
a position to analyze the function of CCR9 with regard to its ability
to support thymocyte proliferation or survival, and to analyze the
function of CCR9 in the regulating the trafficking of developing
thymocytes.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Anne M. Norment, Department of Immunology, University of Washington, Seattle, WA 98195-7370. E-mail address:
3 Abbreviations used in this paper: DN, double negative; DC, dendritic cell; DP, double positive; ELC, EBI-ligand chemokine; EF, elongation factor; EST, expressed sequence tag; h, human; m, murine; MDC, macrophage-derived chemokine; MIP, macrophage inflammatory protein; SDF-1, stromal cell-derived factor; SLC, secondary lymphoid tissue expressed chemokine; SP, single positive; TARC, thymus and activation-regulated chemokine; TECK, thymus-expressed chemokine; RACE, rapid amplification of cDNA ends; HA, hemagglutinin; HPRT, hypoxanthine phosphoribosyltransferase; IRES, internal ribosome entry site; Rag, recombinase-activating gene.
Received for publication August 13, 1999. Accepted for publication October 28, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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