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*
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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-T cell development, and thymocyte selection
appeared unimpaired in adult CCR9-deficient (CCR9-/-)
mice. However, competitive transplantation experiments revealed that
bone marrow from CCR9-/- mice was less efficient at
repopulating the thymus of lethally irradiated Rag-1-/-
mice than bone marrow from littermate CCR9+/+ mice.
CCR9-/- mice had increased numbers of peripheral 
-T
cells but reduced numbers of TCR+ and
CD8+TCR+ intraepithelial
lymphocytes in the small intestine. Thus, CCR9 plays an important,
although not indispensable, role in regulating the development and/or
migration of both - and - T
lymphocytes. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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,
CCL4/MIP-1, CCL17/thymus and activation-regulated chemokine,
CCL19/EBI-1 ligand chemokine/MIP-3, CCL21/6-cysteine
chemokine/secondary lymphoid-tissue chemokine, CCL25/thymus-expressed
chemokine, and CXCL12/stromal cell-derived factor-1, suggesting
that chemokines may play an important role in thymopoiesis
(4, 5, 6, 7, 8). Although a great deal has been learned about the
function of chemokines and chemokine receptors in regulating the
migration of mature lymphocytes, little information is available
concerning their potential role in lymphocyte development. Expression of CCL25/thymus-expressed chemokine was initially detected in medullary dendritic cells (9), but recent experiments indicate that it is also expressed by thymic epithelial cells in both cortex and medulla (10, 11). Interestingly, CCL25 is also expressed in fetal thymus, raising the possibility that it may participate in recruiting T progenitor cells to the thymus (12). The only other known site of CCL25 production is the epithelial layer of the small intestine (11, 13, 14, 15). Thus, CCL25 may also be important for the development, homeostasis, and/or function of mucosal T lymphocytes.
CCR9 mediates chemotaxis in response to CCL25 (16, 17, 18, 19).
CCR9 is expressed on the majority of immature
CD4+CD8+ (double-positive
(DP)) thymocytes, is down-regulated during their transition to the
CD4+ or CD8+
(single-positive (SP)) stage, and is expressed on a minor subset of
CD8+ lymph node T cells (20, 21).
CD69+ thymocytes demonstrate enhanced
CCL25-induced migration compared with CD69-
thymocytes (21, 22), and thymocyte migration in response
to CCL25 is augmented by TCR signaling (21). These
findings suggest that CCR9 may be involved in regulating T cell
migration within the thymus, particularly during thymocyte selection.
Approximately half of all TCR+ thymocytes
and peripheral -T cells express CCR9, and these cells migrate to
CCL25 (21). The expression of CCR9 on specific -T
cell subsets (e.g., V2+, but not
V3+) indicates that CCR9 may also function in
the development and/or trafficking of -T cells (21).
Finally, pre-pro-B cells in the bone marrow respond to CCL25, raising
the possibility that CCR9 may regulate the early stages of B cell
development (23).
To investigate the role of CCR9 during lymphocyte development, we
generated CCR9-deficient (CCR9-/-) mice by
homologous recombination. Surprisingly, both -T cell and B cell
development appeared normal in adult CCR9-/-
mice. In addition, thymocyte selection of -lineage T cells was
unaffected in CCR9-/- mice, even through most
DP thymocytes express CCR9 at high levels and respond to CCL25.
However, the results of competitive bone marrow transplantation
experiments demonstrated that CCR9-/- bone
marrow cells had a reduced capacity to repopulate the thymus
compared with bone marrow cells from CCR9+/+
mice. These results suggest that CCR9 may be involved in regulating the
migration of progenitor cells to the thymus or the retention of T
progenitor cells in the thymus. CCR9-/- mice
also contained increased numbers of peripheral -T cells but
reduced numbers of TCR+ intraepithelial
lymphocytes (IEL) in the small intestine. Thus, CCR9 also plays an
important role in regulating the development and/or migration of
-T lymphocytes.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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An 
15-kb fragment containing the mouse CCR9 gene was cloned
from a 129 SvJ 
genomic library (Stratagene, La Jolla, CA). A 7-kb
EcoRV fragment and a 1.6-kb
HindIII-EcoRV fragment were then cloned into the
XpPNT (NEO/TK) vector (24). The targeting construct was
linearized with NotI and electroporated into 2 x
107 embryonic stem cells (ES cells). After
transfection, ES cells were selected in the presence of G418 and
gancyclovir and screened for homologous recombination. Chimeric mice
were generated from CCR9+/- ES cell clones by
injection into B6 blastocysts, and germline transmission of the mutant
allele was confirmed by Southern blot analysis of DNA obtained from
tail biopsies.
H-Y (25), P14 (26), and AND (27)
TCR transgenes were bred into the CCR9-/-
background. The phenotype of thymocytes and peripheral T cells was
analyzed by staining with PE-labeled anti-CD4, CyChrome-labeled
anti-CD8, and FITC-labeled anti-H-Y clonotypic receptor
(T3.70), anti-TCR-V 2 (P14), or anti-TCR-V 11 (AND) mAb
as previously described (28). Mice were maintained in the
H-2Db background by mating with C57BL/6
mice.
Cell preparation and flow cytometry
Thymus, spleen, lymph node, and bone marrow were excised from
mice, and single-cell suspensions were prepared. IEL were prepared from
small and large intestines according to conventional methods
(29). Standard flow cytometry was performed as previously
described using a FACSCalibur and CellQuest software (BD Biosciences,
Mountain View, CA) (21). Anti-CD3
, anti-CD4,
anti-CD8, anti-CD8, anti-CD25, anti-CD19,
anti-CD44, anti-B220, anti-CD45.2, anti-TCR,
anti-TCR, anti-TCR-V2, anti-TCR-V4,
anti-TCR-V2, anti-TCR-V11, and anti-DX5 mAb were
purchased from BD PharMingen (San Diego, CA). The polyclonal
anti-CCR9 Ab has been described previously (21).
Anti-H-Y clonotypic receptor mAb (T3.70) was purified from cell culture
supernatants in our laboratory. Anti-TCR-V1 (2.11) (30)
and anti-TCR-V5 (GL1) (31) mAbs were provided by
Dr. L. Lefrancois (University of Connecticut Health Center, Farmington,
CT). Unconjugated anti-FcRII (2.4G2) was used to block
nonspecific binding of the labeled Ab. PE-conjugated streptavidin and
CyChrome-conjugated streptavidin were also purchased from BD
PharMingen.
Chemotaxis assays
Chemotaxis assays were performed as previously described (18, 21) with modifications, using 6.5-mm Transwell tissue culture inserts with a 5-µm pore size (Corning, Cambridge, MA). Thymocytes were suspended at 1 x 107 cell/ml in RPMI 1640 plus 0.5% BSA, and 100 µl cell suspension was added to an insert in a well with 600 µl 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. Murine CXCL12 and CCL25 were obtained from PeproTech (Rocky Hill, NJ) and R&D Systems (Minneapolis, MN), respectively.
Northern blotting
Total RNA was isolated from various organs from B6 and
Rag-1-/- mice using TRIzol (Life Technologies,
Gaithersburg, MD). Twenty micrograms of total RNA was fractionated on a
1% agarose/formaldehyde gel and transferred onto a GeneScreen Plus
nylon membrane (New England Nuclear, Boston, MA). The membrane was
hybridized with 32P-labeled cDNA fragments
encoding mouse CCR9, mouse CCL25, or human EF-1.
Bone marrow chimeras
Bone marrow cells were isolated from femurs of CCR9+/+ (CD45.2), CCR9-/- (CD45.2), and B6.SJL-Ptprca/BoAiTac mice (B6.CD45.1; Taconic Farms, Lexington, KY). A total of 2 x 106 cells consisting of various ratios of CCR9+/+ (CD45.2) and B6.CD45.1 bone marrow cells or CCR9-/- (CD45.2) and B6.CD45.1 bone marrow cells were injected i.v. into lethally irradiated (9.5 Gy) Rag-1-/- B6 mice (CD45.2). One to 2 mo after the transplantation thymocytes and lymph node B cells were analyzed by FACS for the presence of CD45.2+ cells.
Statistical analysis
Data from mice of the different phenotypes were analyzed using Student’s t test.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To investigate the biological significance of CCR9 in vivo,
CCR9-/- mice were generated by homologous
recombination. The gene-targeting vector was designed to delete exon 4,
which contains most of the CCR9-coding sequence (Fig. 1
A). The expected structure of
the targeted CCR9 locus was confirmed by Southern
blotting with the indicated 3' probe (Fig. 1B). CCR9 mRNA
was undetectable in the thymus, spleen, lymph node, and small intestine
from CCR9-/- mice (data not shown). FACS
analysis confirmed that thymocytes from CCR9-/-
mice lacked CCR9 surface expression (Fig. 1C).
CCR9-/- mice did not exhibit any developmental
abnormalities, were produced in Mendelian ratios, and were
indistinguishable from CCR9+/+ or
CCR9+/- littermates on the basis of size,
activity, or fertility (data not shown).
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-lineage T cell development in adult
CCR9-/- mice
We initially focused our analysis on T cell development in
CCR9-/- mice, because CCR9 is expressed on the
surface of most thymocytes, especially
CD4+CD8+ (DP) cells
(20, 21). The numbers and distribution of the major thymic
and peripheral T cell subsets, as defined by the expression of CD4 and
CD8, were normal in CCR9-/- mice (Fig. 2). In addition, surface expression of
CD3, TCR, CD5, and CD69 were indistinguishable on thymocytes and
lymph node T cells from CCR9-/- and
CCR9+/+ littermates (data not shown).
Histological studies revealed normal thymus architecture in
CCR9-/- mice (data not shown). Peripheral T
cells from CCR9-/- mice demonstrated normal
proliferative responses after CD3 cross-linking, and
CCR9-/- mice had normal Ab responses to T
cell-dependent Ags (data not shown). Finally, there were no differences
in the number or phenotype of thymocytes from newborn
CCR9-/- and CCR9+/+ mice
(data not shown). Collectively, these data indicate that -T cells
can develop normally in the absence of CCR9.
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). CCR9+/-
thymocytes expressed CCR9 at lower levels than
CCR9+/+ littermates (Fig. 1
C) and
exhibited a reduced chemotactic response to CCL25 (Fig. 3). These data
indicate that CCR9 expression is essential for chemotaxis of thymocytes
to CCL25.
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CCR9-/- mice contain increased numbers of -T
lymphocytes in spleen and lymph node
Approximately half of all TCR+ cells
in thymus, spleen, and lymph node express CCR9 and can respond to CCL25
(21). Therefore, we next compared the number and phenotype
of -T cells in different organs from
CCR9-/- and CCR9+/+ mice.
The number and percentage of TCR+
thymocytes were similar in CCR9-/- and
CCR9+/+ mice (Fig. 4A). However, both the
absolute number and the percentage of
TCR+ cells were increased 2-fold in
spleen and lymph nodes of CCR9-/- mice compared
with CCR9+/+ mice (Fig. 4A). We
previously observed that TCR+ cells that
resemble recent thymic emigrants (i.e.,
CD44lowCD45RBlow)
preferentially express CCR9 (21). However, no significant
differences in CD44, CD45RB, CD62L, or IEL
integrin expression were observed on peripheral -T cells from
CCR9-/- and CCR9+/+ mice
(data not shown). To ascertain whether -T cell subsets were
different in CCR9-/- and
CCR9+/+ mice, we analyzed the percentages of
TCR-V1, -V2, -V5, or -V4-expressing cells among total
CD3+ T cells. TCR-V1+,
-V2+ -V5+, or
-V4+ cells appeared to be increased in the
lymph node (Fig. 4B) and spleen (data not shown) of
CCR9-/- mice. On average,
TCR-V1+, TCR-V5+, and
TCR-V4+ cells were increased more than
TCR-V2+ cells
(TCR-V1+, 3.1-fold;
TCR-V2+, 1.8-fold;
TCR-V5+, 2.9-fold;
TCR-V4+, 2.6-fold). In contrast, no
significant difference in TCR-V1, -V2, -V5, or -V4 usage
was detected in thymocytes from CCR9-/- and
CCR9+/+ mice (data not shown).
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TCR+ IEL in
CCR9-/- mice
Mucosal lymphocytes are composed of IEL and lamina propria
lymphocytes. IEL consist of TCR+ and
TCR+ cells, with
TCR+ cells making up 50% of the total
population in the small intestine and 20% of the total population
in the large intestine (32). CCL25 and CCR9 mRNAs are
detectable in duodenum and small intestine, but not in esophagus,
stomach, appendix, and large intestine (Fig. 5A). In addition, CCR9 mRNA is
expressed in both TCR+ and
TCR+ small intestinal IEL
(21). The number of small intestinal IEL or large
intestinal IEL was not statistically different in
CCR9-/- and CCR9+/+ mice
(data not shown). However, in CCR9-/- mice the
percentage of TCR+ IEL was decreased in
small intestine but increased in large intestine (Fig. 5, B
and C). There was no difference in CD2 and CD8 expression
on large intestinal IEL from CCR9-/- and
CCR9+/+ mice (data not shown). In addition, small
intestinal TCR+ IEL from
CCR9-/- mice resembled those from
CCR9+/+ mice in that they were uniformly
CD8+ and expressed high levels of
IEL integrin (data not shown).
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/V repertoire is diverse in
TCR+ IEL, the TCRs expressed by small
intestinal IEL consist predominantly of TCR-V1 or -V5 paired with
TCR-V4, -V5, -V6, or -V7 (33, 34). To assess
whether there were any differences in the TCR-V/V repertoire of
TCR+ IEL in small intestine, we analyzed
TCR+ IEL in CCR9-/-
and CCR9+/+ mice for the expression of TCR-V1,
-V2, -V5, and -V4 by flow cytometry. The percentages of
TCR-V1, -V2, and -V5-bearing cells among
TCR+ IEL were similar in
CCR9-/- and CCR9+/+ mice
(Fig. 5, D and E). However, the percentage of
TCR-V4-bearing IEL was markedly decreased in
CCR9-/- mice (Fig. 5, D and
E). Most TCR-V4+ IEL coexpress
TCR-V5 and, to a lesser extent, TCR-V1. Consistent with this
observation, both TCR-V5/V4+ and
TCR-V1/V4+ IEL subsets were decreased in
CCR9-/- mice (Fig. 5E).
Collectively, these data indicate that CCR9 is involved in the
generation or maintenance of TCR+ IEL and,
in particular, TCR-V4+ IEL in small
intestine.
We next evaluated TCR+ IEL in the small
intestine of CCR9-/- and
CCR9+/+ mice. TCR+
IEL subsets can be distinguished by the expression of CD8, CD8,
and CD4. The percentage of TCR+ IEL that
were CD4+CD8- and
CD4+CD8+ was not
consistently different in CCR9-/- and
CCR9+/+ mice (Fig. 5F). However, the
percentage of CD8+ IEL was reduced in
CCR9-/- mice (Fig. 5F). No
significant differences were observed in the number or subset
distribution of small intestinal lamina propria lymphocytes in
CCR9-/- and CCR9+/+ mice
(data not shown).
Normal B lymphopoiesis in CCR9-/- mice
Northern blot analysis revealed that CCR9 mRNA is
expressed in bone marrow from B6 mice (Fig. 6A).
Rag-1-/- bone marrow cells, which lack mature T
and B cells, had an equivalent level of CCR9 mRNA expression,
indicating that CCR9 is expressed on lymphoid progenitor cells and/or
myeloid cells. In contrast to CCR9 expression, CCL25 mRNA was
undetectable in the bone marrow (Fig. 6A). Bowman et al.
(23) described an immature population of bone marrow cells
that migrates in response to CCL25. This population is phenotypically
similar to
DX5-CD19-B220+
bone marrow cells (35, 36).
DX5-CD19-B220+
cells can be further subdivided on the basis of CD4 surface expression
into B cell precursors
(CD4-DX5-CD19-B220+)
and cells of unknown lineage and potential
(CD4+DX5-CD19-B220+)
(37). Significantly, staining of bone marrow cells with
anti-CCR9 Ab revealed high level expression of CCR9 on
CD4+DX5-CD19-B220+
cells, but only very low levels of CCR9 on
CD4-DX5-CD19-B220+
cells (Fig. 6B). Both populations migrated in response to
CCL25 (data not shown). No statistically significant differences in the
number or the percentage of
CD4+DX5-CD19-B220+
and
CD4-DX5-CD19-B220+
bone marrow cells were observed in CCR9-/- and
CCR9+/+ mice (Fig. 6C). In addition,
CCR9-/- mice contained normal numbers of bone
marrow cells and peripheral B cells and contained normal proportions of
pro-B
(CD43+IgM-B220low),
pre-B
(CD43-IgM-B220low),
immature B
(CD43-IgM+B220low),
and recirculating bone marrow
(CD43-IgM+B220high)
B cells and exhibited normal IgM and IgD surface profiles on splenic B
cells (Fig. 6C and data not shown). Thus, CCR9 is not
essential for normal B cell development.
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As shown in Fig. 2, no obvious defects in -T cell
development were observed in adult CCR9-/-
mice. Bleul et al. (12) reported previously that CCL25 is
expressed in the early thymic anlage of the mouse fetus, and that fetal
blood prothymocytes
(Thy1+c-kitlow)
respond to CCL25. These findings suggest that CCL25/CCR9 may be
involved in the migration of prothymocytes into the thymus. To explore
this possibility further, we examined CCR9 expression on immature
thymocyte populations from adult mice. CCR9 expression was undetectable
on
CD3-CD4-CD8-
(triple-negative (TN)) thymocytes from adult mice, including the most
immature (CD44+CD25- TN)
subset, and adult TN thymocytes cells failed to migrate in response to
CCL25 (21) (data not shown). In addition, the number and
distribution of TN thymocyte subsets in CCR9-/-
and CCR9+/+ mice, as defined by the expression of
CD44 and CD25, were similar (data not shown). Thymus size and
cellularity can be normal even if the number of immature T progenitor
cells in the thymus is reduced, presumably because these cells are
capable of expanding (1). Consequently, examination of
thymocytes in the adult steady state condition may not reveal a
potential defect in the progenitor cell population. To determine
whether the loss of CCR9 affects the migration of bone marrow
progenitor cells into the thymus or the establishment or retention
of T progenitor cells in the thymus, we performed a competitive
transplantation experiment. Total bone marrow cells from
CCR9-/- mice (CD45.2) and B6.CD45.1
(CCR9+/+) mice were mixed in different ratios and
injected into lethally irradiated Rag-1-/-
(CD45.2) mice. As a control, identical mixtures of bone marrow cells
from littermate CCR9+/+ (CD45.2) and B6.CD45.1
mice were injected into irradiated Rag-1-/-
(CD45.2) mice. One to 2 mo after the bone marrow transplantation, the
number and percentage of CD45.2+ thymocytes and
peripheral (lymph node) B cells were determined. Significantly, when
mixtures of CCR9-/- and B6.CD45.1 bone marrow
were injected, the percentage of thymocytes derived from
CCR9-/- bone marrow cells was consistently
lower than the expected ratio (Fig. 7, A and C). In contrast, the percentage of lymph
node B cells derived from CCR9-/- bone marrow
cells was consistently close to the expected ratio (Fig. 7D). The reduction in CCR9-/- bone
marrow-derived thymocytes was evident at both the
CD44+/-CD25+ TN and the
CD44-CD25- TN stage (Fig. 7B), indicating that cells from
CCR9-/- mice were competitively disadvantaged
at or before the
CD44+/-CD25+ TN stage.
Taken together, these data indicate that under competitive conditions,
bone marrow cells from CCR9-/- mice are
impaired in their ability to reconstitute T cell, but not B cell,
development in irradiated Rag-1-/-
mice.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-lineage thymocytes and
approximately half of all TCR+ thymocytes
and T cells (20, 21). Mucosal T cells also express CCR9,
and fetal blood prothymocytes and pre-pro-B cells migrate in response
to CCL25 (11, 12, 13, 14, 15, 21, 23). Analysis of
CCR9-/- mice revealed that 1) -T cells
develop normally in adult CCR9-/- mice, but
CCR9-/- bone marrow cells exhibit a reduced
capacity to repopulate the thymus of irradiated
Rag-1-/- mice under competitive conditions
compared with CCR9+/+ bone marrow cells; 2)
CCR9-/- mice contain increased numbers of
peripheral -T cells, but reduced numbers of small intestinal
TCR+ IEL; and 3) B cell development is
unaffected in CCR9-/- mice.
The finding that -T cell development and thymocyte selection
appear unperturbed in adult CCR9-/- mice was
unexpected given the fact that most thymocytes express high levels of
CCR9 and migrate to CCL25. The inability of
CCR9-/- thymocytes to respond to CCL25
demonstrates that these cells do not express another receptor for
CCL25. Several different chemokines are expressed in the thymus, and it
is possible that they may share overlapping targets and therefore
compensate for the loss of CCR9. Histological examination of the thymus
revealed no obvious abnormalities in CCR9-/-
mice; however, more detailed localization studies of specific thymocyte
subsets may be required to detect subtle intrathymic migration
defects.
Previous results indicate that prothymocytes in fetal blood respond to
CCL25 (12). This observation together with the finding
that CCL25 is not expressed in bone marrow (Fig. 6A)
suggested that CCR9 may participate in the migration of T-progenitor
cells from bone marrow to thymus. Although we were unable to detect
CCR9 surface expression on immature TN thymocytes from adult mice, and
these cells did not respond to CCL25, CCR9+ cells
were present in adult bone marrow (Fig. 6, A and
B).
CD4-DX5-CD19-B220+
bone marrow cells, which include early B progenitors (37, 38), expressed low levels of CCR9 and could respond to CCL25
(Fig. 6B and data not shown). In addition,
CD4+DX5-CD19-B220+
cells were found to express high levels of CCR9 and could respond to
CCL25. The lineage affiliation and differentiation potential of
CD4+DX5-CD19-B220+
cells remain unclear, although phenotypically similar cells from bone
marrow that respond to CCL25 contain both B and T cell progenitors
(36, 37, 39, 40). To investigate whether CCR9 is involved
in regulating the migration of T-progenitor cells to thymus, we
performed competitive bone marrow transplantation experiments. The
results demonstrated that CCR9-/- bone marrow
cells are competitively disadvantaged compared with
CCR9+/+ bone marrow cells in their ability to
repopulate the thymus of irradiated Rag-1-/-
mice (Fig. 7). These findings suggest three nonmutually exclusive
possibilities: 1) that CCR9 regulates the generation of prothymocytes
in the bone marrow, 2) that CCR9 regulates the migration of
prothymocytes into the thymus or their migration or retention within
the thymus, and 3) that CCR9 regulates the proliferation of early
thymocytes in the thymus. Because immature TN thymocytes do not express
surface CCR9 and do not respond to CCL25 (21), it appears
unlikely that CCR9 is directly involved in the expansion of early
thymocytes. Bleul et al. (12) reported that three
chemokines (CCL25, CXCL12, and CCL21) are expressed in the thymic
anlage, and both CCL25 and CXCL12 attract fetal blood prothymocytes.
Similar to CCR9-/- mice, mice deficient in
CXCL12 or its receptor, CXCR4, also showed no obvious abnormality in T
cell development (41, 42, 43). On the basis of these findings,
we speculate that CCR9 in addition to other chemokine receptors such as
CXCR4 may play an important and partially redundant role in regulating
the migration of prothymocytes into the thymus. Our inability to detect
surface expression of CCR9 on immature TN thymocyte subsets is not
necessarily in conflict with this idea, as the population of
CCR9+ prothymocytes may be extremely small, or
CCR9 may be down-regulated when prothymocytes enter the thymus.
-T cell development and/or homeostasis were clearly altered in
CCR9-/- mice. Although
CCR9-/- mice contained normal numbers of
TCR+ thymocytes, the number of -T
cells was increased in secondary lymphoid organs (spleen and lymph
nodes; Fig. 4A). All TCR-V/V pairs examined were
increased in lymph nodes and spleen of CCR9-/-
mice (Fig. 4B and data not shown). In addition, we could not
detect any significant difference in the expression of CD44, CD45RB,
and other surface markers (e.g., CD62L and IEL
integrin) on peripheral -T cells from
CCR9-/- and CCR9+/+ mice
(data not shown), suggesting that the increased number of peripheral
-T cells in CCR9-/- mice is not due to
the accumulation of one particular subpopulation of -T cells. The
kinetics of thymocyte development are much more rapid for - than
-T cells, and -T cells appear to be dependent on the thymic
environment for a relatively brief period during their development
(44). Thus, in the absence of CCR9, -T cells may be
generated in higher numbers and immigrate more rapidly into the
periphery.
In contrast to lymph nodes and spleen, we observed that the percentage
of small intestinal TCR+ IEL was
consistently decreased in CCR9-/- mice. In the
gastrointestinal tract, CCL25 expression is restricted to the small
intestinal epithelium (Fig. 5A) (11, 13, 14, 15).
Interestingly, although the percentage of
TCR+ IEL in the small intestine of
CCR9-/- mice was decreased, we found that the
percentage of TCR+ IEL in the large
intestine was increased (Fig. 5, B and C). The
large intestinal TCR+ IEL in
CCR9-/- mice were phenotypically similar to
those in CCR9+/+ mice (i.e., they did not
resemble small intestinal IEL; data not shown), indicating that the
increase in large intestinal TCR+ IEL is
not due to migration of cells from the small intestine to the large
intestine.
All IEL from human small intestine express CCR9 and respond to CCL25
(13, 14, 15), and in mice both
TCR+ and TCR+
IEL have been shown to express CCR9 mRNA (21). Thus, CCR9
might be important for the recruitment of mature -T cells to the
intestinal mucosa. Indeed, the observation that large intestinal IEL
were not decreased in CCR9-/- mice localizes
the defect to the site of CCL25 production (Fig. 5, A and
B). Consistent with this idea is the finding that small
intestinal
TCR+CD8+ IEL,
which are thought to be thymically derived and would therefore migrate
from the periphery to the small intestine, are also reduced in
CCR9-/- mice (Fig. 5F). Another
possibility is that in the absence of CCR9,
TCR+ IEL fail to be retained in the small
intestine. However, the preferential loss of
TCR-V5/4+ IEL and our inability to find
these cells in lymph node or spleen (Fig. 5E and data not
shown) suggest that there may be a direct role for CCR9 in
V5/4+ IEL development or that these cells
may fail to survive if they are unable to home to the proper site. The
accumulation of diverse subsets of -T cells in the periphery of
CCR9-/- mice could also reflect the pool of
cells that initially migrate to the intestine and may differ
significantly from the population that ultimately becomes established
as IEL. Finally, the increase in peripheral -T cells may reflect
a shift in a dynamic equilibrium between the small intestine and the
peripheral pool. The lower numbers of TCR+
IEL in CCR9-/- mice even in the face of what
may be a compensatory increase in these cells in the periphery suggests
an important role for CCR9 in establishing this equilibrium.
CCR9 and CCL25 mRNA are also detected in the small intestine of
Rag-1-/- mice, which lack mature lymphocytes
(21), raising the possibility that CCR9 plays a role in
early mucosal T cell development and/or recruitment of IEL precursors
to the small intestine. Cryptopatches are multiple clusters of
c-kit+IL-7R+Thy1+
lymphocytes located in the crypt lamina propria of the murine intestine
(45). CCR9 may be involved in cryptopatch formation and/or
extrathymic TCR+ IEL development, perhaps
by regulating the migration of progenitor cells from the fetal liver,
fetal thymus, or adult bone marrow to the small intestine.
TCR+ IEL are greatly reduced in nude
mice and neonatal thymectomized mice (46). Transplantation
of fetal or neonatal thymus, but not adult thymus, into nude mice
results in the generation of a substantial number of thymically derived
TCR+ IEL (46, 47). These data
indicate that the thymus is involved in the development of
TCR+ IEL, but the mechanism by which the
thymus participates is still unclear. The thymus may provide IEL
precursors or a thymus-derived factor that is required for the
differentiation and/or expansion of IEL (48), and CCR9
could be important for these functions.
In summary, these results identify a role for CCR9 in - and
-T cell development that may include regulating the migration of
progenitor cells to specific sites of T lymphopoiesis. The selective
expression of CCL25 in the small intestine and the deficiency of small,
but not large, intestinal -T cells also suggest the existence of
distinctive mechanisms of lymphocyte recruitment that may permit
functional specialization of immune responses in different segments of
the gastrointestinal tract.
|
Acknowledgments |
|---|
1 and anti-TCR-V5 mAbs. We thank S. Hayes, C. Feng, and
K. Laky for valuable advice and D. El-Khoury for excellent technical
assistance. |
Footnotes |
|---|
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, Bethesda, MD 20892. E-mail address: pel{at}helix.nih.gov
3 Abbreviations used in this paper: MIP, macrophage-inflammatory protein; DN, double negative; DP, double positive; IEL, intraepithelial lymphocyte; TN, triple negative; ES cell, embryonic stem cell.
Received for publication November 27, 2001. Accepted for publication January 18, 2002.
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, Data from CCR9+/+ mice;
•, data from CCR9-/- mice. D, Reduction
in TCR-V
