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TCR+ Thymocytes Preferentially Respond to CCL251
*
Laboratory of Mammalian Genes and Development, National Institute of Child Health and Human Development, and
Laboratory of Clinical Investigation, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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TCR+ thymocytes and peripheral TCR+
T cells expressed CCR9 on their surface, and these cells migrated in
response to CCL25. These findings suggest that CCR9 may play an
important role in the development and trafficking of both
TCR+ and TCR+ T
cells. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To characterize more precisely the expression of CCR9 during T cell
ontogeny, we generated polyclonal Ab that recognizes murine CCR9, and
we examined the relationship between CCR9 surface expression and the
specific chemotactic activity to CCL25 during T cell development. Our
results demonstrate that CCR9 is expressed on the cell surface of both
and TCR+ thymocytes and that these
cells are specifically responsive to CCL25. Moreover, in T
lineage cells, CCR9 expression is developmentally regulated, and CCL25
responsiveness is influenced by activation through the TCR.
Collectively, these data support the idea that CCR9 plays an important
role in T cell development in both the and T
lineages.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Female C57BL/6 (B6) mice were bred within our facility. Embryos
at various stages of gestation were obtained from time-mated pregnant
mice. The date on which a vaginal plug was observed was designated
gestation day 0.5. Mutant strains of mice used in this study included
Rag-1-/- (14), TCR-
chain-/- (15), and MHC class
I x II-/-
(2-microglobulin-/- x
A-/-) (16).
Generation of anti-murine CCR9 Ab
Rabbit polyclonal Ab against murine CCR9 was prepared according to standard methods. A 16-aa NH2-terminal peptide of murine CCR9 (CMFDDFSYDSTASTDD) was coupled to keyhole limpet hemocyanin. Immune serum was produced in New Zealand White rabbits, and Ab was purified using the immunizing peptide coupled to normal human serum-activated Sepharose (Amersham Pharmacia Biotech, Piscataway, NJ). Control rabbit IgG was purified from preimmune serum by affinity to protein A (Pierce, Rockford, IL).
Validation of anti-murine CCR9 Ab
Mouse CCR9 cDNA was amplified from mouse thymocyte cDNA by PCR using the following EcoRI site containing primers: forward primer, 5'-CCGGAATTCTGAATAGCCCTCCTGAAGCTGATTGGC-3'; reverse primer, 5'-CCGGAATTCCCAAAAAGGACCATATGCCCT-3'. The PCR fragment was inserted into the EcoRI site of the retroviral vector hCD4.RV (17), which was kindly provided by T. Murphy, Washington University School of Medicine (St. Louis, MO). hCD4.RV contains a multiple cloning site, followed by an internal ribosome entry site, followed by sequences encoding a truncated human CD4. The plasmids containing CCR9 in sense and antisense orientations and control plasmids without insert were used to transfect Phoenix-Eco cells, obtained from G. Nolan, Stanford University (Stanford, CA), through the American Type Culture Collection (Manassas, VA), using the protocol on G. Nolan’s web site at http://www.stanford.edu. Two days after transfection, the cells were stained for CD4 and with the affinity-purified biotinylated anti-murine CCR9 and control Abs, followed by PE-conjugated streptavidin (av-PE).3
Abs and reagents
Abs used for flow cytometric analysis were purchased from BD
PharMingen (San Diego, CA) and included FITC, PE, or
CyChrome-conjugated anti-CD3, anti-CD4, anti-CD8,
anti-CD25, anti-CD44, anti-B220, anti-TCR,
anti-TCR, anti-V2TCR, and anti-V3TCR mAbs.
Unconjugated anti-FcRII (2.4G2) was used to block nonspecific
binding of the labeled Ab. av-PE and CyChrome-conjugated streptavidin
(av-CyChrome) were also purchased from BD PharMingen. Murine CXCL12 and
CCL25 were obtained from PeproTech (Rocky Hill, NJ) and R&D Systems
(Minneapolis, MN), respectively.
Chemotaxis assays
Chemotaxis assays were performed as described (10), with modifications, using 6.5-mm Transwell tissue culture inserts with a 5-µm pore size (Costar, Cambridge, MA). Thymocytes were suspended at 1 x 107 cells/ml in RPMI 1640 plus 0.5% BSA, and 100 µl of cell suspension was added to an insert in a well with 600 µl of medium. After equilibration at 37°C for 1 h, chemokines were added to the wells and the plates were incubated for an additional 2 h before cells were harvested, collected by centrifugation, and counted. Duplicate wells were used for each condition.
Cell preparation
Thymi, lymph nodes, and spleens were excised from mice, and
single cell suspensions were prepared. Intestinal intraepithelial
lymphocytes (iIEL) were isolated as previously described
(18). Briefly, small intestines were cut longitudinally
and then into 5-mm pieces, and washed three times with CMF
(Ca2+-, Mg2+-free HBSS with
1 mM HEPES, 2.5 mM NaHCO3, pH 7.3) containing 2%
calf serum. Washed intestinal pieces were stirred at 37°C for 20 min
in CMF containing 10% calf serum and 1 mM dithioerythritol. This step
was repeated, the resultant supernatants were rapidly filtered through
nylon wool, and the filtrate was centrifuged through a 44/67%
Percoll gradient (Amersham Pharmacia Biotech). The cells at the
interface of the gradient were collected. For purification of
TCR+ and TCR+
iIEL, cells were stained with biotinylated anti-TCR or
anti-TCR, and with anti-FcR to prevent nonspecific
binding of the labeled mAb, followed by incubation with
streptavidin-coupled microbeads. Stained cells were applied to a MACS
column (Miltenyi Biotec, Auburn, CA), and adherent cells were
recovered.
Immunofluorescence analysis
Standard flow cytometry was performed, as described previously (19), using a FACSCalibur and CellQuest software (BD Biosciences, San Jose, CA).
Enrichment of DN cells from thymus
Double-negative (DN) thymocytes were enriched by MACS. Briefly,
thymocytes were stained with a mixture of biotinylated anti-CD4,
anti-CD8, anti-B220, anti-TCR, and anti-FcR to
prevent nonspecific binding of the labeled mAb, followed by incubation
with streptavidin-coupled microbeads. Stained cells were applied to a
MACS column, and nonadherent cells were recovered.
Thymocyte stimulations
In vivo CD3 cross-linking. Rag1-/- mice were injected i.p. with 100 µg of anti-CD3 mAb (2C11). Mice were sacrificed 1, 3, or 5 days after injection, and thymocytes were harvested and stained as described above.
In vitro CD3 cross-linking. Twenty-four-well plates were coated with 10 µg/ml anti-CD3 (2C11) in PBS overnight at 4°C and subsequently washed with hybridoma serum-free medium (Life Technologies, Rockville, MD). A total of 4 x 106 thymocytes was resuspended in 2 ml of hybridoma serum-free medium and plated in uncoated or anti-CD3-coated wells for 20 h at 37°C, 5% CO2. After incubation, thymocytes were pelleted, washed, and resuspended at 1 x 107 cells/ml in RPMI 1640 with 0.5% BSA, and chemotaxis assays were performed as described above.
Northern blotting and semiquantitative RT-PCR
Poly(A)+ RNA was isolated from small
intestines of B6 and Rag1-/- mice using the
MicroFast Track kit (Invitrogen, San Diego, CA), fractionated on a 1%
agarose/formaldehyde gel, and transferred onto GeneScreen Plus nylon
membrane (NEN, Boston, MA). The membrane was hybridized with
32P-labeled cDNA fragments encoding mouse CCR9,
mouse CCL25, or human elongation factor-1. For quantitative RT-PCR
analysis, total RNA was isolated from purified iIEL subsets
using TRIzol reagent (Life Technologies), and first-strand cDNA
template was synthesized using SuperScript II reverse transcriptase
(Life Technologies) with random hexamers. Serial dilutions of these
cDNA templates were subjected to PCR amplification using sets of
primers of CCR9 (forward primer, 5'-ATTGCACAAGAGTGAAGACC-3';
reverse primer, 5'-GTCAACAGCCTGCACTACAA-3') or CD3
(forward
primer, 5'-GCCTCAGAAGCATGATAAGC-3'; reverse primer,
5'-AGACTGCTCTCTGATTCAGG-3'). Cycling parameters were 30 s at
94°C, 45 s at 56°C, and 1 min at 72°C for 35 cycles to
detect CCR9 mRNA, and 32 cycles for CD3
. PCR products were separated
by electrophoresis on a 2% agarose gel and visualized by staining with
ethidium bromide.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We and others have found that CCR9 mRNA is selectively expressed in the thymus and small intestine, as assessed by Northern blot and RT-PCR analysis (Refs. 6, 7 , and 9 and data not shown). However, the surface expression of CCR9 on murine T cells subsets has not been examined. To investigate the role of CCR9 and its ligand, CCL25, during T cell development, we generated polyclonal Ab against murine CCR9. Rabbits were immunized with a keyhole limpet hemocyanin-coupled peptide consisting of a 16-aa NH2-terminal fragment of murine CCR9. Within this 16-aa peptide, only six amino acid residues are conserved between mouse and human CCR9 gene. Immune serum was affinity-purified on a CCR9 peptide column, biotinylated, and used for staining. Biotinylated IgG, purified from preimmune serum, was used as control Ab.
The specificity of the anti-peptide Ab was confirmed by staining a
human embryonic kidney cell line transfected with murine CCR9 cDNA.
Affinity-purified Ab against CCR9 peptide reacted with cells
transfected with vector DNA containing mouse CCR9 sequences in the
sense orientation, but not with cells transfected with vector without
insert (Fig. 1
A), or with
vector containing CCR9 sequences in the antisense orientation (data not
shown). Similar results were obtained with NIH3T3 cells transduced
using murine CCR9-encoding retrovirus (data not shown). In addition,
the anti-CCR9 Ab did not react with mouse CCR6-, CCR8-, or
CXCR3-expressing cells (data not shown).
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B).
Incubation with CXCL12 at 37°C or with CCL25 at 4°C had no effect
on CCR9 staining (Fig. 1B). These results suggest that CCL25
may induce down-modulation of CCR9 on thymocytes. CCR9 expression on adult and fetal thymocytes and T cell subsets
Using affinity-purified anti-CCR9, we studied the expression
of CCR9 on thymocytes and mature T cell subsets. Surface staining
demonstrated that CCR9 is expressed on most double-positive (DP)
thymocytes and is down-regulated on transitional single-positive (SP;
CD4+CD8low and
CD4lowCD8+) and mature SP
(CD4+CD8- and
CD4-CD8+) thymocytes (Fig. 2A). Most DN cells did not
express CCR9 (Fig. 2A), and analysis of immature DN
thymocyte subsets (distinguished on the basis of CD25 and CD44
expression) failed to reveal detectable CCR9 surface expression (Fig. 3A). These data are consistent
with previous results obtained using RT-PCR analysis (9, 10). In the periphery, CCR9 was expressed on a small subset of
CD8+ T cells but was not detectable on
CD4+ T cells (Fig. 2B). Chemotactic
assay of lymph node cells showed preferential migration of
CD8+ T cells (
8-fold) relative to
CD4+ T cells in response to CCL25 (data not
shown). Among CD8+ T cells, naive
(CD44lowCD62Lhigh) cells
preferentially migrated to CCL25 (data not shown), supporting the idea
that recent thymic CD8+ emigrants still express
CCR9 and can respond to CCL25. CCR9 expression was undetectable on
CD3-activated mature T cells, NK cells, and
NK1.1+ T cells (data not shown).
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B).
Interestingly, although thymocytes from gestation day 17.5 contain DP
cells, these cells did not express CCR9 (Fig. 3B). Injection
of anti-CD3 mAb into Rag1-/- mice mimics
pre-TCR signals promoting cell proliferation and transition of DN
thymocytes to the DP stage. It was previously reported that CCR9 mRNA
expression is strongly induced following anti-CD3 treatment of
Rag2-/- thymocytes (9). To examine
the role of pre-TCR signaling in regulating surface expression of CCR9,
Rag1-/- mice were injected with 100 µg of
anti-CD3, and CCR9 expression was examined 3 and 5 days later.
Untreated Rag1-/- thymocytes did not express
surface CCR9 (Fig. 4) and could not
respond to CCL25 (data not shown). Three days after stimulation with
anti-CD3, Rag1-/- thymi contained DP cells;
however, these cells remained CCR9- (Fig. 4B). Detectable CCR9 surface expression was only observed at
day 5 when >90% of thymocytes were DP (Fig. 4B). Taken
together, these data indicate that although CCR9 mRNA is rapidly
induced by pre-TCR engagement (9), CCR9 surface expression
begins only after thymocytes have developed to the DP stage.
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To determine whether TCR engagement affects CCR9 expression,
CCR9 levels were examined on
CD3lowCD69-,
CD3lowCD69+, and
CD3highCD69+ thymocyte
subsets, because CD69 and CD3 are up-regulated on DP thymocytes after
engagement of the TCR by positively or negatively selecting ligands in
the thymus (20, 21). Comparison of gated
CD3lowCD69- and
CD3lowCD69+ cells did not
reveal any difference in the level of CCR9 surface expression; however,
CCR9 expression was slightly lower on
CD3highCD69+ thymocytes
(data not shown). When the chemotactic response of thymocytes was
examined, CD69+ thymocytes exhibited enhanced
migration to CCL25 relative to CD69- thymocytes
(Fig. 5). Analysis of the surface
phenotype of migrating cells revealed that
CD3highCD69+ cells were
especially responsive to CCL25 (Fig. 5), and most of these cells were
CD4/CD8 SP thymocytes (data not shown). CXCL12 also induced
preferential migration of CD69+ thymocytes, but
these cells were predominantly CD3low DP (Fig. 5
and data not shown).
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-/- mice. MHC class
I/II-/- and TCR--/-
mice contain DP thymocytes, but these cells fail to undergo positive
selection and lack CD69+ cells due to the absence
of TCR engagement or TCR expression, respectively (15, 16). Although CCR9 surface expression levels were similar on DP
thymocytes from control (B6), MHC class
I/II-/-, and TCR--/-
mice, thymocytes from MHC class I/II-/- and
TCR--/- mice exhibited reduced CCL25-induced
migration as compared with B6 mice (Fig. 6). To determine whether TCR-mediated
signaling enhances CCL25-induced chemotaxis, MHC class I/II-deficient
thymocytes were cultured with or without plate-bound anti-CD3 in
serum-free media for 20 h and then assayed for chemotaxis. TCR
stimulation significantly enhanced migration in response to CCL25 (Fig. 7A). Migration to CXCL12 was
unchanged by TCR stimulation. Although migration to CCL25 was enhanced
in cells stimulated by TCR cross-linking, surface levels of CCR9 were
down-regulated (Fig. 7B). Thus, although TCR cross-linking
results in down-regulation of CCR9 surface expression, it augments
chemotactic activity to CCL25.
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T lineage cells
Total populations of DN thymocytes exhibited a weak but
significant chemoattractant activity to CCL25 (Ref. 10 and
data not shown). DN thymocytes are heterogeneous and contain mature
TCR+ cells in addition to immature
lineage cells. To determine which subsets of DN thymocytes respond to
CCL25, we performed chemotaxis assay on total DN thymocytes after
depletion of CD4+, CD8+,
B220+, and TCR+
cells. Migrated cells were stained with anti-CD3 and
anti-TCR and examined by FACS (Fig. 8B). In the absence of
chemokine, <1% of DN cells migrated in this assay (Fig. 8A). CCL25 induced the chemotaxis of 60% of
TCR+ thymocytes but only 3% of
TCR- DN cells.
TCR+ thymocytes were less responsive to
CXCL12, as only 10% of TCR+ thymocytes
migrated to this chemokine. Thus, CCL25 appears to be an especially
effective chemoattractant for TCR+
thymocytes.
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T cells from adult and fetal
thymus. Approximately one-half of T cells from adult thymus
express CCR9 (Fig. 9A). CCR9
was also expressed on gestation day 17.5
TCR+ thymocytes, but not on gestation day
14.5 TCR+ thymocytes (Fig. 9B).
Moreover, V3+ thymocytes, which preferentially
migrate to the skin, did not express CCR9, whereas most
V2+ thymocytes expressed CCR9 (Fig. 9B). Approximately 30–40% of lymph node and splenic
T cells expressed CCR9 (Fig. 9A). Most
CCR9+TCR+ lymph node
T cells were CD44low and
CD45RBlow (Fig. 9C), suggesting that
they were recent thymic emigrants (21, 22).
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As previously reported, both CCR9 and CCL25 mRNA are also
expressed in the small intestine (Fig. 10A) (11, 12, 13).
To determine the origin of CCR9 and CCL25 expression in the small
intestine, we examined mRNA levels in B6 and
Rag1-/- mice by Northern blotting (Fig. 10A). In Rag1-/- small intestines,
which lack mature T and B lymphocytes, CCR9 expression was decreased,
but CCL25 expression was constant as compared with B6 mice (Fig. 10A). These data suggest that CCR9 is mainly expressed by
mature lymphocytes, whereas CCL25 is produced by nonlymphoid cells.
Interestingly, we could not detect CCR9 surface expression on either
TCR+ or TCR+
iIEL (Fig. 10B), or on lamina propria lymphocytes (data not
shown). We next purified TCR+ and
TCR+ iIEL and examined CCR9 expression by
semiquantitative RT-PCR analysis. CCR9 mRNA expression was observed in
both TCR+ and
TCR+ iIEL subsets, although
TCR+ iIEL expressed less CCR9 mRNA as
compared with TCR+ iIEL (Fig. 10C).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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lineage T cells, CCR9 surface expression is first
observed on DP thymocytes and is down-regulated during the transition
of DP thymocytes to the mature CD4+ or
CD8+ SP stage (Fig. 2). Interestingly, although
thymocytes from gestation day 17.5 and Rag1-/-
thymi 3 days after anti-CD3 stimulation contain DP cells, these
cells did not express CCR9 on their surface (Figs. 3B and
4B). We observed that CCR9 mRNA is expressed in gestation
day 14.5 thymocytes, and that thymocytes from gestation day 17.5 and
adult mice express CCR9 mRNA at equivalent levels by Northern blot
analysis (data not shown). These results indicate that CCR9 gene
expression is induced at an earlier stage than we are able to detect
using our Ab. One possible explanation for this discrepancy is that
surface expression of CCR9 is regulated posttranscriptionally and does
not correlate with mRNA levels.
Our results also indicate that the response of
CCR9+ cells to CCL25 is not simply dictated by
the level of CCR9 surface expression.
CD3highCD69+ thymocytes
demonstrated enhanced CCL25-induced migration as compared with
CD3lowCD69+ thymocytes,
even though they express lower levels of CCR9 (Fig. 5). In contrast,
thymocytes from MHC class I/II-/- and
TCR--/- mice, which fail to undergo positive
selection and lack CD69+ cells, showed reduced
CCL25-induced migration, although they expressed normal levels of CCR9
(Fig. 6). In vitro TCR stimulation of MHC class
I/II-/- thymocytes significantly enhanced
migration in response to CCL25, even though CCR9 surface expression was
down-regulated (Fig. 7). Down-regulation of CCR9 expression was also
observed by Zabel et al. (11) after activation of human
PBLs by anti-CD3 stimulation. Collectively, these data indicate
that the response of CCR9+ thymocytes to CCL25 is
enhanced by TCR signals during positive selection. Thus, CCL25
responsiveness is regulated by the expression of its receptor, CCR9,
and by TCR signaling. These findings are consistent with previous
results demonstrating that the activation state of T cells can
influence their response to chemokines (23).
Initial studies showed that CCL25 is produced by thymic dendritic cells
located at the corticomedullary junction (5). Subsequent
in situ hybridization studies demonstrated that CCL25 mRNA is expressed
by thymic epithelial cells in both the cortex and the medulla
(10). In contrast, CXCL12 mRNA is expressed in the cortex,
especially in the subcapsular region (24). During positive
selection, DP thymocyte development proceeds from the
CD69+CD3low to the
CD69+CD3high and then the
CD69-CD3high stage. The
response of thymocytes to CCL25 is enhanced by TCR stimulation, is
maximal at the CD69+CD3high
stage, and is down-regulated at the
CD69-CD3high stage,
whereas the response to CXCL12 is down-regulated at
CD69+CD3high SP stage (Fig. 5). Based on these data, we speculate that CCL25 may regulate the
intrathymic trafficking of thymocytes in collaboration with other
thymus-expressed chemokines including CXCL12.
CCL25 is also expressed by fetal thymic MHC class
II+ epithelial cells (25), and in
situ hybridization studies indicate that CCL25 is expressed in the
thymic anlage in day 12.5 embryos (26). A bone marrow cell
subset of pre/pro-B cell phenotype
(B220lowCD24-AA4.1+NK1.1-),
which may contain thymocyte precursors, migrates to CCL25
(27). CCL25 attracts fetal blood prothymocytes and
CD44+CD25- DN newborn
thymocytes (26). Based on these observations, it has been
suggested that CCL25/CCR9 interaction could play a role in the
recruitment of T progenitors to thymus. However, we were unable to
detect CCR9 surface expression on
CD44+CD25- DN thymocytes
from fetal and adult mice (Figs. 3A and 4A), and
CD44+CD25- DN thymocytes
from Rag1-/- mice did not respond to CCL25
(data not shown). It is possible that
CD44+CD25- DN cells from
newborn mice express CCR9 at levels that are not detectable by our Ab.
Alternatively, CCR9 expression on prothymocytes may be down-regulated
rapidly after these cells enter the thymus.
Campbell et al. (28) proposed that CCL25 might function to
retain cells in the thymus until they have fully matured because
CD4+ SP
CD69-L-selectinhigh
thymocytes lose responsiveness to CCL25. However, we observed that a
subset of peripheral CD8+ T cells and
CD44lowCD45RBlow T
cells expresses CCR9 (Figs. 2B and 9C).
Furthermore, a subset of naive
(CD44lowCD62Lhigh)
CD8+ T cells preferentially responds to CCL25
(data not shown). These results indicate that CCR9 expression and CCL25
responsiveness are not sufficient to inhibit CD8+
SP and TCR+ thymocyte emigration. Moreover,
SP thymocytes accumulate in the thymus of pertussis toxin transgenic
mice, suggesting that pertussis toxin-sensitive signaling responses are
not essential for retention of thymocytes but instead may be required
for thymocyte emigration (29).
Recent data suggest that chemokines and their receptors may play a role
in the development and trafficking of lineage as well as
lineage T cells (30). We found that a high percentage of
TCR+ thymocytes and peripheral T
cells expresses CCR9 and migrates in response to CCL25 (Figs. 8A and 9A). These data suggest that chemokines
may regulate the development and function of lineage T cells as
well as lineage T cells. CCR9 was expressed on 80% of
thymocytes bearing the V2-TCR, which is the major population in
adult thymus and peripheral lymphoid tissues (Fig. 9B).
Notably, V3-bearing fetal thymocytes, which are known to be the
precursors of dendritic epidermal T cells, do not express CCR9 (Fig. 9B). In humans, CCR9 is not expressed on cutaneous
lymphocyte Ag-positive memory CD4+ and
CD8+ lymphocytes, which traffic to skin
(11). The restricted expression pattern of CCR9 within
different T lineage subsets suggests that CCR9 may regulate the
migration of selected T lineage populations to specific sites
such as the small intestine, in which CCL25 is known to be highly
expressed.
In both mice and humans, CCL25 is highly expressed in the epithelial
cells lining the small intestine, and, in humans, CCR9 is selectively
expressed on the surface of intestinal homing T lymphocytes and mucosal
lymphocytes in the small intestine (6, 11, 12, 13). We also
observed that CCR9 is expressed in murine small intestine, as assessed
by Northern blot analysis, and in both TCR+
and TCR+ iIEL subsets by RT-PCR (Fig. 10, A and C). However, we did not detect CCR9 surface
expression with our Ab on murine iIEL and lamina propria lymphocytes
from the small intestine (Fig. 10B and data not shown).
Furthermore, in our experiments, iIEL did not migrate in response to
200 nM CCL25, a concentration that induces chemotaxis of thymocytes and
peripheral CD8+ T cells (data not shown). Based
on these observations, we speculate that surface expression of CCR9 on
iIEL may be down-regulated by CCL25 binding or cell activation, and/or
the level of CCR9 expression may be too low to detect by our Ab.
Rag1-/- small intestines lack mature
lymphocytes but contain
CD3-CD8+ iIEL
(31) and cryptopatches (32). Fig. 10A shows that small amounts of CCR9 mRNA are expressed in
Rag1-/- small intestine. These findings
indicate that CCR9 may be expressed in the progenitor cells for mucosal
lymphocytes and could potentially play a role in early mucosal T cell
development and/or recruitment of precursor T cells to the
intestine.
In conclusion, we have shown that during thymocyte development, CCR9
surface expression starts and is maximal at the DP stage and is
down-regulated on mature SP thymocytes. DP thymocytes that have
received activating signals through their TCRs exhibit enhanced
CCL25-induced migration, suggesting that CCR9 may function in the
process of intrathymic trafficking during positive selection. Among
T cells, CCR9 is expressed on selective subsets, and about
one-half of TCR+ thymocytes express CCR9
and migrate in response to CCL25. Thus, CCR9/CCL25 may be important for
regulating the migration of specific subsets of T cells to
particular sites. Collectively, these data suggest that CCR9
may play an important role in the development and trafficking of both
and T cells.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Paul E. Love, Laboratory of Mammalian Genes and Development, National Institute of Child Health and Human Development, National Institutes of Health, 9000 Rockville Pike, Building 6B, Room 2B210, MSC 2780, Bethesda, MD 20892. E-mail address: pel{at}helix.nih.gov
3 Abbreviations used in this paper: av-PE, streptavidin-PE; av-CyChrome, streptavidin-CyChrome; DN, double-negative; DP, double-positive; iIEL, intestinal intraepithelial lymphocyte; SP, single-positive.
Received for publication August 27, 2001. Accepted for publication October 25, 2001.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-15 that differ in their sensitivities to ligand. J. Immunol. 164:1293. and block thymocyte development at different stages. Nature 360:225.[Medline]
RI can support T cell development and function in mice lacking endogenous TCR 
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+ T cells from the adult murine thymus. Int. Immunol. 5:331./ T cells. J. Exp. Med. 187:357. and T lymphocytes to chemokines. Eur. J. Immunol. 28:104.[Medline]
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H. Li, Z. Gang, H. Yuling, X. Luokun, X. Jie, L. Hao, W. Li, H. Chunsong, L. Junyan, J. Mingshen, et al. Different Neurotropic Pathogens Elicit Neurotoxic CCR9- or Neurosupportive CXCR3-Expressing Microglia J. Immunol., September 15, 2006; 177(6): 3644 - 3656. [Abstract] [Full Text] [PDF]
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 F Annunziato, L Cosmi, F Liotta, E Lazzeri, P Romagnani, R Angeli, L Lasagni, R Manetti, F Marra, C Gerard, et al. CXCR3 and {alpha}E{beta}7 integrin identify a subset of CD8+ mature thymocytes that share phenotypic and functional properties with CD8+ gut intraepithelial lymphocytes Gut, July 1, 2006; 55(7): 961 - 968. [Abstract] [Full Text] [PDF]
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S. Uehara, S. M. Hayes, L. Li, D. El-Khoury, M. Canelles, B. J. Fowlkes, and P. E. Love Premature Expression of Chemokine Receptor CCR9 Impairs T Cell Development J. Immunol., January 1, 2006; 176(1): 75 - 84. [Abstract] [Full Text] [PDF]
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S. Vielkind, M. Gallagher-Gambarelli, M. Gomez, H. J. Hinton, and D. A. Cantrell Integrin Regulation by RhoA in Thymocytes J. Immunol., July 1, 2005; 175(1): 350 - 357. [Abstract] [Full Text] [PDF]
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 D. P. Widney, Y. Hu, A. K. Foreman-Wykert, K. C. Bui, T. T. Nguyen, B. Lu, C. Gerard, J. F. Miller, and J. B. Smith CXCR3 and Its Ligands Participate in the Host Response to Bordetella bronchiseptica Infection of the Mouse Respiratory Tract but Are Not Required for Clearance of Bacteria from the Lung Infect. Immun., January 1, 2005; 73(1): 485 - 493. [Abstract] [Full Text] [PDF]
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V. E. Mick, T. K. Starr, T. M. McCaughtry, L. K. McNeil, and K. A. Hogquist The Regulated Expression of a Diverse Set of Genes during Thymocyte Positive Selection In Vivo J. Immunol., November 1, 2004; 173(9): 5434 - 5444. [Abstract] [Full Text] [PDF]
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 C. M. Witt and E. A. Robey The Ins and Outs of CCR7 in the Thymus J. Exp. Med., August 16, 2004; 200(4): 405 - 409. [Abstract] [Full Text] [PDF]
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T. L. Staton, B. Johnston, E. C. Butcher, and D. J. Campbell Murine CD8+ Recent Thymic Emigrants are {alpha}E Integrin-Positive and CC Chemokine Ligand 25 Responsive J. Immunol., June 15, 2004; 172(12): 7282 - 7288. [Abstract] [Full Text] [PDF]
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 N. Hosoe, S. Miura, C. Watanabe, Y. Tsuzuki, R. Hokari, T. Oyama, Y. Fujiyama, H. Nagata, and H. Ishii Demonstration of functional role of TECK/CCL25 in T lymphocyte-endothelium interaction in inflamed and uninflamed intestinal mucosa Am J Physiol Gastrointest Liver Physiol, March 1, 2004; 286(3): G458 - G466. [Abstract] [Full Text] [PDF]
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J. Plotkin, S. E. Prockop, A. Lepique, and H. T. Petrie Critical Role for CXCR4 Signaling in Progenitor Localization and T Cell Differentiation in the Postnatal Thymus J. Immunol., November 1, 2003; 171(9): 4521 - 4527. [Abstract] [Full Text] [PDF]
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A. Lugering, T. Kucharzik, D. Soler, D. Picarella, J. T. Hudson III, and I. R. Williams Lymphoid Precursors in Intestinal Cryptopatches Express CCR6 and Undergo Dysregulated Development in the Absence of CCR6 J. Immunol., September 1, 2003; 171(5): 2208 - 2215. [Abstract] [Full Text] [PDF]
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S. Hue, R. C. Monteiro, S. Berrih-Aknin, and S. Caillat-Zucman Potential Role of NKG2D/MHC Class I-Related Chain A Interaction in Intrathymic Maturation of Single-Positive CD8 T Cells J. Immunol., August 15, 2003; 171(4): 1909 - 1917. [Abstract] [Full Text] [PDF]
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S. Uehara, A. Grinberg, J. M. Farber, and P. E. Love A Role for CCR9 in T Lymphocyte Development and Migration J. Immunol., March 15, 2002; 168(6): 2811 - 2819. [Abstract] [Full Text] [PDF]
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