CCR10 Is Important for the Development of
Skin-Specific γδT Cells by Regulating Their
Migration and Location
This information is current as
of June 18, 2017.
Yan Jin, Mingcan Xia, Allen Sun, Christina M. Saylor and
Na Xiong
J Immunol published online 11 October 2010
http://www.jimmunol.org/content/early/2010/10/11/jimmun
ol.1001612
http://www.jimmunol.org/content/suppl/2010/10/12/jimmunol.100161
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Supplementary
Material
Published October 11, 2010, doi:10.4049/jimmunol.1001612
The Journal of Immunology
CCR10 Is Important for the Development of Skin-Specific
gdT Cells by Regulating Their Migration and Location
Yan Jin, Mingcan Xia,1 Allen Sun,1 Christina M. Saylor, and Na Xiong
U
nlike conventional ab T cells, which preferentially reside in secondary lymphoid organs for adaptive immune
responses, various subsets of unconventional T cells,
such as the gd T cells with innate properties, preferentially reside
in epithelial tissues covering the external and internal surface of
the body, including the skin, reproductive tract, lungs, and intestines, where they function as the first line of defense (1).
The gd T cells of different epithelial tissues use different gd
TCRs and originate from thymi of specific ontogenic stages (2). In
mice, skin intraepithelial gd T lymphocytes (sIEL; also referred
to as dendritic epidermal T cells) express canonical Vg3/Vd1+
gdTCRs, and their precursors are generated only in early fetal
thymi. Vg4+ cells of later fetal thymi contribute as the dominant
gd T cell population in other epithelial tissues, such as the reproductive tract, tongue, and nasal mucosa (2, 3). In contrast, gd
T cells located in the secondary lymphoid organs (SLO) are
preferentially Vg2 or Vg1.1+ and originate from the adult thymus.
Although it is well established that the waved generation of gd
Center for Molecular Immunology and Infectious Diseases, Department of Veterinary
and Biomedical Sciences, The Pennsylvania State University, University Park, PA
16802
1
M.X. and A.S. contributed equally to this work.
Received for publication May 14, 2010. Accepted for publication September 5, 2010.
This work was supported by grants from the National Institutes of Health (to N.X.)
and, in part, under a grant with the Pennsylvania Department of Health using Tobacco
Settlement Funds (to N.X.). The department specifically disclaims responsibility for
any analyses, interpretations, or conclusions.
Address correspondence and reprint requests to Dr. Na Xiong, The Pennsylvania
State University, Department of Veterinary and Biomedical Sciences, 115 Henning
Building, University Park, PA 16802. E-mail: [email protected]
The online version of this article contains supplemental material.
Abbreviations used in this paper: EGFP, enhanced GFP; ES cell, embryonic stem cell;
neo+, recombinant CCR10-knockout ES cells with the neo cassette; neo2/neo2, homozygous CCR10-knockout/EGFP-knockin mice with the neo cassette deleted; +/neo2,
heterozygous CCR10-knockout/EGFP-knockin mice with the neo cassette deleted;
2RT, no reverse transcription; S, SacI; S1PR1, sphingosine 1-phosphate receptor 1;
sIEL, skin intraepithelial Vg3+ T lymphocyte; SLO, secondary lymphoid organ.
Copyright Ó 2010 by The American Association of Immunologists, Inc. 0022-1767/10/$16.00
www.jimmunol.org/cgi/doi/10.4049/jimmunol.1001612
T cell subsets is primarily due to the genomically programmed
rearrangement of specific Vg genes at different ontogenic stages
(4), mechanisms regulating their tissue-specific development are
poorly understood.
Recent studies found that a selection process is involved in the
tissue-specific development of Vg3+ sIELs, the dominant epidermal T cell population in mice. The Vg3+ sIELs play an important
role in protection of the skin through various functions such as
immune surveillance against tumors (5), regulation of local inflammatory responses (6), and promotion of wound healing (7).
In genetically modified mice whose production of the Vg3+ gd
T cells is impaired in the fetal thymus, the Vg3+ gd T cells are still
the dominant subset of sIELs in adults, suggesting that the Vg3+
cells are selected over other T cell subsets to develop into sIELs
(8). In the absence of native Vg3/Vd1+ sIELs, such as in Vg3 or
Vd1 knockout mice, other gd T cell subsets could substitute in
the skin. However, the substitute sIELs have a restricted TCR configuration (9–11). In TCRd6.3 transgenic mice, transgenic sIELs
were absent unless an endogenously encoded TCRd chain, preferentially TCRd1, was coexpressed (11), supporting the involvement of selection.
The selection process for sIEL development starts within the
fetal thymus. We previously reported that fetal thymic gd T cell
populations that display activated or memory phenotypes correlated with their development into sIELs (12). In wild-type mice,
the fetal thymic Vg3+ sIEL precursors are a predominant population that displays the activated phenotypes, including the upregulation of CD122 (IL-15Rb), suggesting that they are selected. In
a substrain of FVB mice (Taconic Farms, Germantown, NY) that
bears the mutated Skint1 molecule, a selecting ligand for the Vg3+
sIEL precursors, the Vg3+ fetal thymic gd T cells were found to
remain at an immature status and could not develop into sIELs
efficiently (13, 14), confirming that positive selection is critical
for the development of sIELs. Furthermore, a subset of transgenic
fetal thymic gd T cells developed into sIELs if they were positively selected as the Vg3+ cells (12, 15).
The positive selection of fetal thymic Vg3+ sIEL precursors
might endow them with a unique homing property to migrate in
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Unlike conventional ab T cells, which preferentially reside in secondary lymphoid organs for adaptive immune responses, various
subsets of unconventional T cells, such as the gd T cells with innate properties, preferentially reside in epithelial tissues as the first
line of defense. However, mechanisms underlying their tissue-specific development are not well understood. We report in this
paper that among different thymic T cell subsets fetal thymic precursors of the prototypic skin intraepithelial Vg3+ T lymphocytes
(sIELs) were selected to display a unique pattern of homing molecules, including a high level of CCR10 expression that was
important for their development into sIELs. In fetal CCR10-knockout mice, the Vg3+ sIEL precursors developed normally in the
thymus but were defective in migrating into the skin. Although the earlier defect in skin-seeding by sIEL precursors was partially
compensated for by their normal expansion in the skin of adult CCR10-knockout mice, the Vg3+ sIELs displayed abnormal
morphology and increasingly accumulated in the dermal region of the skin. These findings provide definite evidence that CCR10 is
important in sIEL development by regulating the migration of sIEL precursors and their maintenance in proper regions of the
skin and support the notion that unique homing properties of different thymic T cell subsets play an important role in their
peripheral location. The Journal of Immunology, 2010, 185: 000–000.
2
the skin. Compared with unselected fetal thymic gd T cells,
positively selected Vg3+ sIEL precursors had a coordinate switch
in the expression of multiple homing molecules, including the upregulation of CCR10 (G protein-coupled receptor 2) and sphingosine 1-phosphate receptor 1 (S1PR1), as well as the downregulation of CCR6, which might be important for their peripheral
location (12). S1PR1 is known to be critical for thymus exiting of
mature T cells (16). In contrast, considering the high-level expression of chemokine CCL27, a ligand for CCR10, in the skin
(17), CCR10 may serve as a skin-homing receptor for positively
selected Vg3+ cells. However, a recent publication found no apparent sIEL defect in adult CCR10-knockout mice, leaving the
role of CCR10 in sIEL development unclear (18).
Using a newly generated strain of CCR10-knockout mice with
a knocked-in enhanced GFP (EGFP) as a reporter for CCR10, we
systematically analyzed the regulation of CCR10 expression in
cells at different stages of sIEL development and its role in sIEL
development. We report in this paper that CCR10 is involved in
multiple aspects of sIEL development.
Generation of CCR10-knockout/EGFP-knockin mice
CCR10-knockout/EGFP-knockin mice were generated as outlined in Fig. 1.
First, a targeting construct was assembled that included a 3.6-kb DNA
fragment exactly 59 of the start codon of the CCR10 gene (59 arm), followed by a coding sequence for EGFP, a loxP-flanked neo gene cassette,
and a 0.5-kb DNA fragment consisting of a 0.2-kb 39 portion of the CCR10
coding region and a 0.3-kb 39 noncoding region (39 arm). The 3.6-kb 59
and 0.5-kb 39 arms were PCR amplified from genomic DNA from 129 svJ
mice and confirmed by sequencing. The linearized targeting construct was
then transfected into mouse embryonic stem (ES) cells (the J1 line) (19), of
which the G418/gancyclovir-resistant clones were screened for knockout
recombinants by genomic PCR with a primer set that amplifies a 0.9-kb
band from the 39 end of the targeted CCR10 allele (Fig. 1A, 1B). The
knockout clones were further confirmed by a Southern blot with a probe
specific for a region 59 upstream of the targeted region (Fig. 1A, 1C).
The targeted allele (neo+) deletes a 1.7-kb DNA fragment coding for Nterminal extracellular and all of the seven trans-membrane domains of the
CCR10 gene and replaces it with an EGFP coding sequence and a loxPflanked neo cassette. The neo+ CCR10-knockout clones were microinjected into blastocysts of C57BL/6 (B6) mice to generate chimera mice.
The male chimera mice were crossed with female EIIa-Cre transgenic
mice to delete the loxP-flanked neo gene cassette from the CCR10 targeted
allele to generate heterozygous CCR10-knockout/EGFP-knockin mice
(CCR10+/EGFP) (Fig. 1A, 1C). The CCR10+/EGFP mice were backcrossed to
wild-type B6 mice for eight to nine generations and intercrossed to generate
homozygous CCR10-knockout/EGFP-knockin (CCR10EGFP/EGFP) mice.
B6 and transgenic EIIa-Cre mice were purchased from The Jackson Laboratory (Bar Harbor, ME). All animal experiments were approved by Pennsylvania State University Institutional Animal Care and Use Committee.
Abs and reagents
Anti-CD3, CD122, CD62L, and gdTCR Abs were purchased from eBioscience (San Diego, CA); anti-CD24, CCR7, and aE from BioLegend (San
Diego, CA); anti-Vg2, Vg3, b7, and a4b7 from BD Biosciences (San
Diego, CA); and anti-CCR9 from R&D Systems (Minneapolis, MN). The
17D1 Ab was previously described (9). CCL27 was purchased from
PeproTech (Rocky Hill, NJ).
Flow cytometry
Cells were incubated with fluorescently labeled Abs for 30 min at 4˚C (or
37˚C for CCR7 staining) and analyzed on the flow cytometer FC500
(Beckman Counter, Miami, FL).
Cell sorting
E17 fetal thymocytes of CCR10+/EGFP mice were stained and sorted for
EGFP+ and EGFP2 Vg3+ cells with an Influx sorter (Cytopeia, San Jose,
CA). E17 fetal thymocytes of wild-type mice were stained and sorted for
CD122+ and CD1222 Vg3+ cells. Vg3+ sIELs were purified by the sorter
from epidermal cell preparations based on Vg3/CD3 expression. Purities
of the sorted populations were $95%.
Immunofluorescent microscopy of epidermal sheets
The experiment was performed similarly, as described (22). The epidermal
sheets were peeled from the ear skin of adult mice or the back skin of 2- to
3-d-old newborns after the skins were incubated in a 20 mM EDTA/PBS
solution for 1 h. The sheets were fixed with acetone and then stained
with FITC-conjugated anti-Vg3 Abs (or biotin-conjugated anti-Vg3 Abs/
streptavidin-Alexa 568) overnight and analyzed on a fluorescence microscope—Olympus BX61 (Olympus, Melville, NY) or Nikon Eclipse TE300
(Nikon, Melville, NY).
Immunofluorescent microscopy of skin sections
In this procedure, 10-mm cryosections of the back skin were fixed in
methanol for 30 min at 220˚C and costained with Alexa 647-conjugated
anti-CD3 and FITC-conjugated anti-Vg3 Abs in the Antibody Amplifier
Eclipse (ProHisto, Columbia, SC) at 4˚C overnight. The stained sections
were covered with Vectashield mounting medium containing DAPI (Vector
Laboratories, Burlingame, CA) and analyzed on an Olympus BX61 fluorescence microscope or an Olympus Fluoview 1000 confocal microscope
(Olympus America, Center Valley, PA).
In vitro chemotaxis assay
The experiment was performed as described (12). Briefly, 5 3 105 E17 fetal
thymocytes of wild-type or CCR10-knockout mice suspended in DMEM/
10% FBS were placed into the upper chamber of a Transwell plate containing 5-mm pore filters (Corning Costar, Corning, NY) and incubated
with CCL27 (R&D Systems) or a conditioned medium of E18 fetal skin in
the bottom chamber for 4 h. Cells migrating into the bottom chamber were
collected and analyzed by flow cytometry for Vg3+ gdT cells.
Semiquantitative RT-PCR
The experiment was performed as previously described (12). Primer sequences are CCR7f: 59-CTCGGCAACGGGCTGGTGATACTG-39 and
CCR7r: 59-CGTGTCCTCGCCGCTGTTCTTCTG-39 for CCR7; CCR1059: 59-AGAGCTCTGTTACAAGGCTGATGTC-39 and CCR10-39: 59-CAGGTGGTACTTCCTAGATTCCAGC-39 for CCR10; S1PR1-59: 59-GTACTTCCTGGTTCTGGCTGTGC-39 and S1PR1-39: 59-CGTTTCCAGACGACATAATGG-39 for SIPRI; L3: 59-CCAGCAGCCACTAAAATGTC-39 and
J1: 59-CTTACCAGAGGGAATTACTATGAG-39 for TCRg3; GAPDH-59:
59-CTGACGTGCCGCCTGGAGAAA-39 and GAPDH-39: 59-TGTTGGGGGCCGAGTTGGGATAG-39 for GAPDH; EGFP-59: 59-ACTACCTGAGCACCCAGTCCGCCCTG-39 and EGFP-39: 59-GCTCTAGATTTACTTGTACAGCTCGT-39 for EGFP. Primers for tubulin have been described (23).
Statistical analyses
All data are expressed as means 6 SD. Statistical significance was determined by two-tailed student t tests; p , 0.05 is considered significant.
Results
Cell preparation
Generation of CCR10-knockout mice with a knocked-in EGFP
as a reporter for CCR10 expression
Thymocytes were isolated from mice as described (12). To isolate lymphocytes from epidermal and dermal regions, the dorsal and ventral skin
was treated with 20 mM EDTA, to be separated into the epidermis and
dermis. The epidermis was minced and digested with a 0.3% trypsin solution for 20 min at 37˚C to dissociate the cells (20). The dermis was
minced and digested with collagenase for 1–2 h with gentle shaking to
dissociate the cells (21). Mononucleocytes were enriched from the cell
preparations, using Percoll gradients (40%/80%), and then subjected to
flow cytometric analysis.
To study the role of CCR10 in sIEL development, we generated
CCR10-knockout/EGFP-knockin mice. The CCR10 coding sequence was replaced with an EGFP coding sequence that serves as
a reporter for CCR10 expression (Fig. 1A–D). The neo gene
cassette was removed from the targeted CCR10 allele by the Cre/
LoxP-mediated deletion, to get “clean” CCR10-knockout/EGFPknockin mice because the neo gene cassette, with its own strong
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Materials and Methods
ROLES OF CCR10 IN SKIN gd T CELL DEVELOPMENT
The Journal of Immunology
3
promoter, interfered with regulation of knocked-in EGFP gene
expression by the endogenous regulatory machinery (24).
The knocked-in EGFP in CCR10-knockout/EGFP-knockin mice
faithfully reports the expression of CCR10. Of the E17 fetal
thymocytes of heterozygous CCR10+/EGFP mice, significantly
higher percentages of CD122+ Vg3+ gd T cells expressed much
higher levels of EGFP than did CD1222 Vg3+ gd T cells or earlier
progenitors (Fig. 1E). This correlates with our previous findings
that the positively selected Vg3+ fetal thymic sIEL precursors had
higher CCR10 transcripts than did the unselected gd T cell population (12). Furthermore, in purified EGFP+ and EGFP2 populations of the E17 fetal thymic Vg3+ gd T cells of CCR10+/EGFP
mice, levels of CCR10 and EGFP transcripts were correlated (Fig.
1F). EGFP expression was also correlated with CCR10 transcription between different thymic T cell and sIEL populations, as
demonstrated in several experiments throughout this paper (Fig.
2A, 2B). In addition, nearly all IgA+ cells, but few other cells, of
the intestine had high EGFP signals (not shown), also correlating
with previous findings (25). These results indicate that the
knocked-in EGFP is a reliable reporter for CCR10 expression and,
to the best of our knowledge, provide the first direct evidence that
CCR10 is expressed on the majority of positively selected CD122+
Vg3+ fetal thymic gd T cells.
Unique patterns of expression of CCR10 and other homing
molecules on different thymic T cell populations
Taking advantage of the knocked-in EGFP reporter, we analyzed
the expression of CCR10 (EGFP) in fetal thymic Vg3+ and other
thymic T cell subsets that are known to have different preferential
peripheral locations. First, we compared the expression of CCR10
(EGFP) between the Vg3+ sIEL precursors and conventional
thymic CD4+ and CD8+ ab T cells that preferentially localize in
SLOs. In contrast to fetal thymic CD122+ Vg3+ cells, few conventional thymic ab T cells expressed CCR10 (EGFP) (Fig. 2A).
Instead, the conventional ab T cells expressed higher levels of
CCR7 and CD62L, molecules involved in their localization to
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FIGURE 1. Generation of CCR10-knockout mice with a knocked-in EGFP reporter. A, Targeting and screening strategies for generation of CCR10knockout/EGFP-knockin mice. B, The screening for CCR10-knockout ES cell clones by a genomic PCR with primers P1 and P2 (A) that would amplify
a 0.8-kb band from the CCR10-knockout allele, but not the wild-type CCR10 allele or CCR10-knockout construct inserted into the genome. C, Southern
blot analysis of CCR10-knockout ES cells or mice. Genomic DNAs of the cells and mice were digested with restriction enzyme SacI and probed with
a 0.8-kb DNA fragment located 59 upstream of the 59 arm in the CCR10 allele (A, 59 Probe). The Southern blot gave bands of 6.9, 7.9, and 10 kb of the
wild-type, neo+ CCR10 targeted allele (neo+), and neo-deleted targeted CCR10 allele (neo2), respectively. D, Identification of heterozygous and homozygous CCR10-knockout mice by a genomic PCR with primers P3, P4, and P5 (A), which amplifies a 330-bp wild-type and a 280-bp knockout band.
E, Flow cytometric (FACS) analysis of EGFP expression in fetal thymic Vg3+ gd T cells before and after the positive selection. A total of 31 CCR10+/EGFP
fetuses were analyzed. F, Correlated expression of the knocked-in EGFP and endogenous CCR10 genes. Different fetal thymic Vg3+ cell populations were
purified from CCR10+/EGFP and CCR10+/+ mice based on their EGFP or CD122 expression (as indicated) by cell sorters. Levels of CCR10 and EGFP
transcripts in the sorted populations were analyzed by semiquantitative RT-PCR. GAPDH was used as loading controls. n = 2. +/+, wild-type ES cells and
mice; neo+, recombinant CCR10-knockout ES cells with the neo cassette; neo2/neo2, homozygous CCR10-knockout/EGFP-knockin mice with the neo
cassette deleted; +/neo2, heterozygous CCR10-knockout/EGFP-knockin mice with the neo cassette deleted; 2RT, no reverse transcription; S, SacI.
4
ROLES OF CCR10 IN SKIN gd T CELL DEVELOPMENT
SLOs (26, 27), than did the Vg3+ sIEL precursors (Fig. 2A). The
differential expression of CCR10 versus CCR7 between the positively selected CD122+ Vg3+ and conventional CD4+ ab T cell
populations was further confirmed by semiquantitative RT-PCR,
whereas both populations expressed similar levels of S1PR1 (Fig.
2B), a molecule involved in T cell egress from the thymus (16).
The majority of adult thymic gdT cells expressed patterns of
homing molecules similar to those of the abT cells (Fig. 2A,
right panels), consistent with their preferential location in SLOs.
However, a small but notable portion of adult thymic gd T cells
expressed CCR10 (EGFP) (Fig. 2A); but the CCR10 (EGFP)+
adult thymic gd T cells displayed expression profiles of many
other homing molecules that were different from those of the
CCR10 (EGFP)+ Vg3+ sIEL precursors that, apart from expressing low CD62L and CCR7, were also low in the expression of
CCR9 and integrin a4b7, molecules involved in homing to the
gut, and high in aEb7, an integrin involved in epidermal localization of sIELs (Fig. 2C) (28).
We also analyzed the expression of CCR10 (EGFP) on Vg32 gd
T cells in fetal thymi of CCR10+/EGFP mice. Compared with the
Vg3+ fetal thymic cells, a smaller portion of the Vg32 fetal
thymic gd T cells expressed lower levels of CCR10 (Fig. 2D).
The CCR10 (EGFP)+ Vg32 fetal thymic gd T cells did not
express CD122, indicating that they were not selected as the Vg3+
sIEL precursors, and, like the CCR10 (EGFP)+ adult thymic gd
T cells, expressed other homing molecules differently than did the
CCR10+ Vg3+ sIEL precursors (Supplemental Fig. 1).
A sizable portion of NK1.1+ adult thymic T cells, similar to
the thymic gd T cells, also expressed CCR10 (EGFP) (Fig. 2E).
However, the CCR10 (EGFP)+ NKT cells were mainly ab T cells
(Supplemental Fig. 2), suggesting that they are a distinct population. Interestingly, the CCR10 (EGFP)+ NKT cells displayed an
expression pattern of homing molecules that was more similar to
patterns of CCR10+ Vg3+ fetal thymocytes than to those of other
CCR10+ gd T cells, including high-level integrin aE but low-level
a4b7 expression (Supplemental Fig. 2).
Together, these data demonstrate that the Vg3+ sIEL precursors
and other thymic T cell populations express different patterns of
homing molecules that might determine their specific peripheral
localizations. In particular, CCR10 (EGFP) is expressed on various thymic gd T cell subsets known to localize preferentially into
different epithelial tissues that abundantly express ligands for
CCR10, including CCL27 and CCL28 (29, 30, 31), but not on the
conventional ab T cells that preferentially localize into SLOs. In
addition, the expression of CCR10 (EGFP)+ on one NKT subset
might be associated with their preferential localization in nonlymphoid peripheral tissues, including the skin (32). Most notably,
the high level of CCR10 (EGFP) expression on the majority of
positively selected Vg3+ fetal thymic T cells suggests a potentially
important role of CCR10 in their development into sIELs.
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FIGURE 2. Differential expression of CCR10 and
other homing molecules among positively selected
fetal thymic Vg3+ and other thymic T cell subsets. A,
FACS analysis of CCR10 (EGFP), CD62L, and CCR7
expression on positively selected E16 fetal thymic
Vg3+ (n = 8) versus adult (n = 3) thymic CD4+, CD8+,
or gd T cells. The CCR10 (EGFP) analysis was on
CCR10+/EGFP mice (n $ 4). B, Semiquantitative RTPCR determination of CCR7, CCR10, and S1PR1 expression in mature E16 fetal thymic CD122+ Vg3+ and
adult thymic CD24lowCD62LhighNK1.12 CD4+ cells of
C57BL/6 mice. The experiments were repeated twice.
C, FACS analysis of a4b7, CCR9, CCR7, CD62L, aE,
and b7 expression on CCR10 (EGFP)+ E16 fetal (n =
10) thymic Vg3+ versus adult (n = 2) thymic gdT cells.
D, Comparison of CCR10 (EGFP) and CD122 expression on E16 fetal thymic Vg3+ versus Vg32
gdT cells of CCR10+/EGFP mice. More than five mice
were analyzed. E, Expression of CCR10 (EGFP) on the
gated adult NK1.1+ CD3+ thymocytes. The experiments were repeated twice.
The Journal of Immunology
Normal developmental and selection processes of Vg3+ sIEL
precursors in fetal thymi of CCR10-knockout mice
To dissect the involvement of CCR10 in sIEL development, we first
analyzed the generation and selection of the Vg3+ sIEL precursors
in fetal thymi of CCR10-knockout mice. Compared with wildtype littermates, homozygous CCR10EGFP/EGFP mice had essentially the same number of fetal thymic Vg3+ cells that also expressed the positive selection and maturation markers CD122 and
CD24 (Fig. 3A). In addition, there was only a slight, if any, difference in percentages of EGFP+ Vg3+ fetal thymocytes between
CCR10EGFP/EGFP and CCR10+/EGFP mice (Fig. 3B). These results
indicate that CCR10 is not involved to any great extent in the
intrathymic development processes of Vg3+ sIEL precursors or
their egress from the thymus, which is likely mediated by different
homing molecules, such as S1PR1 (16).
Defective sIEL development in CCR10-knockout mice
Temporally regulated CCR10 expression in sIELs and their
precursors
Surprisingly, despite the defects in sIEL development in the absence of CCR10, we could not detect EGFP signals in the sIELs of
adult CCR10+/EGFP or CCR10EGFP/EGFP mice under the fluorescent
microscope (Fig. 4C, right panel; and data not shown). This was
not due to leakage of the highly soluble EGFP proteins out of the
sIELs during the preparation process of epidermal sheets because
we did not treat the epidermal sheets with any permeabilizing
reagents. In addition, with the more quantitative flow cytometric
analysis, we detected only very weak EGFP signals of Vg3+ sIELs
isolated from adult CCR10+/EGFP mice, in clear contrast to those
of CCR10+/EGFP fetal thymic Vg3+ sIEL precursors (Fig. 5A).
These results suggest that the expression of CCR10 on sIELs of
adult mice is downregulated from the expression level of their
fetal thymic precursors. In direct confirmation of this, the level of
CCR10 transcripts in purified Vg3+ sIELs of wild-type adult mice
was much lower than that of purified CD122+ Vg3+ fetal thymocytes (Fig. 5B). Considering that keratinocytes are in direct
contact with sIELs and express high levels of the ligand CCL27
for CCR10, the temporally regulated CCR10 expression on sIELs
and their precursors is likely involved in calibration of the CCR10/
ligand interaction strength for the migration and distribution of
sIELs within the skin.
Increased accumulation of Vg3+ gd T cells in dermal regions
of the skin in CCR10-knockout mice
To test whether CCR10 is involved in maintaining the proper
distribution of sIELs within the skin, we isolated cells from epidermal and dermal regions of the skin separately and analyzed them
for Vg3+ T cells by flow cytometry. There were ∼2-fold lower
percentages of epidermal Vg3+ T cells in CCR10EGFP/EGFP mice
than in wild-type controls (Fig. 6A), confirming that sIEL development is impaired in CCR10-knockout mice. In contrast, there
was more than a 3-fold increase in the percentage of Vg3+ gd
T cells in cells isolated from the dermis of CCR10EGFP/EGFP
mice compared with wild-type mice (Fig. 6B), demonstrating that
CCR10 is important for the proper distribution of Vg3+ cells
within the skin. In addition, we consistently observed lower percentages of Vg32 gd T cells in the dermis of CCR10-knockout
mice ‘compared with wild-type mice, suggesting that the absence
of CCR10 might affect other T cell populations within the dermis.
We then performed immunofluorescent microscopic analysis of
skin sections to directly visualize Vg3+ T cells in the epidermis
and dermis. In wild-type mice, a majority of Vg3+ T cells reside in
the epidermis, although many were also found in the dermis,
specifically in the epithelia surrounding hair follicles (Fig. 6C, 6D;
Supplemental Fig. 5). Compared with wild-type controls of corresponding compartments, CCR10-knockout mice showed fewer
Vg3+ cells in the epidermis but more Vg3+cells in the dermis (Fig.
6C, 6D). As a result, CCR10-knockout mice, on average, had
many more Vg3+ cells in the dermis than in the epidermis (Fig.
6D), confirming that CCR10 is indeed important in maintaining
the proper distribution of Vg3+ cells within the skin. In addition,
a majority of Vg3+ cells in the dermis of CCR10-knockout mice
still resided in the epithelia surrounding the hair follicles, although
some could be found outside the hair follicles (Fig. 6C).
Defective migration of CCR10-knockout Vg3+ sIEL precursors
toward the skin
FIGURE 3. Normal development of Vg3+ sIEL precursors in fetal
thymi of CCR10-knockout mice. A and B, Comparison of CD24 and
CD122 expression on E16 fetal Vg3+ thymocytes of CCR10EGFP/EGFP
(CCR102/2) and wild-type (CCR10+/+) littermates by flow cytometry.
More than five mice of each genotype were analyzed. C, FACS analysis of
EGFP expression on gated Vg3+ gd T cells of CCR10EGFP/EGFP mice
versus CCR10+/EGFP (CCR10+/2) mice. The staining of wild-type cells was
used as a negative control for EGFP expression.
Considering the dominant expression of the CCR10 ligand
CCL27 by keratinocytes, the most likely underlying mechanism
for the number, morphology, and distribution of Vg3+ gd T cells
in the skin of CCR10-knockout mice is due to the defective
homing and positioning of the Vg3+ sIEL precursors in(to) the
skin in the absence of the CCR10/ligand interaction. In contrast,
the CCR10/ligand interaction is unlikely to be involved in the
survival/proliferation of sIELs in the skin, because sIELs expanded in CCR10-knockout mice as much as in wild types from
the newborn to adult stages and the reduction in numbers of sIELs
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Although there was no defect in the generation and selection of
Vg3+ sIEL precursors in the fetal thymus of CCR10EGFP/EGFP
mice, their seeding into the skin was severely impaired. Transcripts for Vg3+TCRs were much lower (.10-fold) in the skin of
E18 fetal CCR10EGFP/EGFP mice than in their wild-type littermates
(Fig. 4A). In addition, the direct immunofluorescent microscopic
analysis of epidermal sheets for Vg3+ cells revealed that there
were significantly fewer (2.2-fold reduction) sIELs in 2- to 3-d-old
newborn CCR10EGFP/EGFP mice than in their wild-type littermates
(Fig. 4B, 4E). Compared with their corresponding wild-type littermates, CCR10-knockout mice also showed significantly reduced numbers of sIELs in young (5- to 6-wk-old) and mature
(10- to 12-wk-old) adults, although the extent of reduction was
slightly smaller in the mature than in the young adult mice (1.6versus 2-fold reduction) (Fig. 4C, 4E; Supplemental Fig. 3). This
finding differs from a recent report (18). The epidermal layers
of adult CCR10-knockout mice had an uneven distribution of
sIELs (Fig. 4C; Supplemental Fig. 3), consistent with reduced
skin-seeding by sIEL precursors at earlier stages. In addition,
sIELs in the adult CCR10EGFP/EGFP mice did not have the typical
dendritic morphology seen in normal sIELs (Fig. 4D), suggesting
a role of CCR10 in sIEL morphogenesis. As a control, Langerhans cells, the other major type of epidermal immune cells, developed normally in CCR10EGFP/EGFP mice (Supplemental Fig. 4).
5
6
ROLES OF CCR10 IN SKIN gd T CELL DEVELOPMENT
in CCR10-knockout mice is, in fact, less evident when the mice
become older (Fig. 4E). This observation suggests that the
CCR10-deficient sIEL precursors are capable of a homeostatic
expansion. Therefore, we performed in vitro migration assays to
test directly whether the CCR10-deficient Vg3+ fetal thymocytes were defective in migration to the skin. As expected, the
CCR10EGFP/EGFP Vg3+ thymocytes were unable to migrate toward
CCL27 at all, indicating that CCR10 is the only chemokine receptor expressed by sIEL precursors for CCL27 (Fig. 7A). Importantly, the CCR10EGFP/EGFP Vg3+ thymocytes also had defects
FIGURE 5. Regulated CCR10 expression in sIELs and their thymic
precursors. A, FACS analysis of CCR10 (EGFP) expression on adult Vg3+
sIELs and fetal thymic CD122+ Vg3+ T cells of CCR10+/EGFP mice. B,
Comparison of levels of CCR10 transcripts in adult sIELs and positively
selected fetal thymic sIEL precursors. RNAs of purified adult Vg3+ sIELs
and E16 fetal thymic CD122+Vg3+ cells of wild-type mice were analyzed
by semiquantitative RT-PCR for CCR10 transcripts. The experiments were
repeated twice.
in the migration toward culture media of the fetal skin (Fig. 7B),
suggesting that they have impaired ability in CCL27-mediated
skin homing.
Discussion
Complementing the SLO-residing conventional T cells, various
populations of nonclassical, innate-like T cells predominantly localize in specific nonlymphoid tissues as the first line of defense.
However, development of tissue-specific T cells is not well understood. Our study on murine sIELs found that compared with
other thymic T cell subsets, the fetal thymic Vg3+ sIEL precursors
had a unique skin-homing property (CCR10highaEb7highCCR7low
CD62LlowCCR9lowa4b7low) that plays an important part in their
peripheral localization. Using a newly generated strain of CCR10knockout/EGFP-knockin mice, we demonstrate that the positive
selection-associated upregulation of CCR10 on the fetal thymic
Vg3+ sIEL precursors is important for their seeding into the skin.
In addition, we also show that CCR10 is important for maintaining the proper morphology and epidermal distribution of Vg3+ gd
T cells within the skin.
The skin-seeding, dendritic morphology, and proper epidermal
distribution of sIELs are likely linked events mediated by the
CCR10/ligand interaction. In the skin, keratinocytes of the epidermis highly express CCL27, a ligand for CCR10 (17, 33). In the
absence of CCR10/CCL27 signals, fetal thymic Vg3+ T cells
could not migrate into the epidermis efficiently and might end up
in other regions of the skin and possibly other tissues. However,
we did not find Vg3+ cells in several tissues we tested in adult
CCR10-knockout mice, including the spleen, lymph node, and
female reproductive tract (data not shown). This finding suggests
that even if the CCR10-knockout fetal thymic Vg3+ cells migrate
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FIGURE 4. Impaired sIEL development in
CCR10EGFP/EGFP mice. A, Comparison of TCRg3
transcripts in fetal skin of CCR10EGFP/EGFP and wildtype mice by semiquantitative RT-PCR. Skin RNAs of
E18 fetal CCR10EGFP/EGFP and wild-type mice were
subjected to semiquantitative RT-PCR analysis for
rearranged TCRg3. GAPDH was used as a loading
control. The experiments were repeated twice. B, Immunofluorescent microscopic analysis of epidermal
sheets of the 2- to 3-d-old newborn CCR10EGFP/EGFP
and wild-type mice for Vg3+ sIELs. Epidermal sheets
of the back skin were stained with biotin-conjugated
anti-Vg3 Abs followed with Alexa 647-conjugated
streptavidin. C, Immunofluorescent microscopic analysis of epidermal sheets of 5- to 6-wk-old adult
CCR10EGFP/EGFP and wild-type mice for Vg3+ sIELs.
Ear epidermal sheets were costained with FITCconjugated anti-Vg3 Ab and DAPI. The tissue in the
image on the right was stained with DAPI and used as
a control for endogenous EGFP signals. D, Highmagnification immunofluorescent microscopy of ear
epidermal sheets of adult CCR10EGFP/EGFP and wildtype mice for morphology of Vg3+ sIELs. Images of
three pairs of littermates are shown. Original magnification 3200. E, Quantitative comparison of numbers
of Vg3+ sIELs in CCR10EGFP/EGFP and wild-type mice
of different ages. The number of Vg3+ sIELs was
calculated from the immunofluorescent microscopy of
epidermal sheets, with at least five fields counted for
each mouse. One dot represents the average number of
Vg3+ sIELs per field from one mouse.
The Journal of Immunology
7
into tissues other than the skin, they might not be able to survive
and proliferate.
Because sIELs are in direct contact with keratinocytes in the
epidermis, CCL27 secreted by keratinocytes likely acts on CCR10
expressed on sIELs in a concentrated fashion to help maintain the
morphology and location of Vg3+ cells within the epidermal region. Therefore, in the absence of CCR10/CCL27 signals, the
Vg3+ cells could neither maintain their dendritic shape nor attain
the epidermal location properly, resulting in their redistribution in
the dermal region of the skin. Interestingly, the Vg3+ cells in
dermal regions of CCR10-knockout mice were still predominantly
FIGURE 7. Defective migration of CCR10EGFP/EGFP fetal thymic Vg3+
cells toward CCL27 and skin in the in vitro chemotaxis assay. The migration index is calculated as the ratio of numbers of Vg3+ cells migrating
into the bottom chamber in the presence of CCL27 (A), conditioned medium of fetal skin culture (B) versus medium only. The experiments were
repeated twice.
localized in the hair follicle regions, as in wild-type mice (34),
suggesting that they are specifically attracted to these sites. It will
be interesting to determine what other molecules are involved in
their positioning in the follicles. It was previously reported that
sIELs rounded up in response to the local stimulation and moved
away from the epidermis (35). It is possible that the morphological
changes of sIELs due to absence of CCR10-mediated signals are
analogous to the induced morphological changes during their
activation.
The effects of CCR10 on the migration, morphology, and in-skin
distribution of Vg3+ sIELs are associated with the regulated expression of CCR10 in the sIELs and their precursors. This is likely
involved in the calibration of the interaction strength between
sIELs and neighboring keratinocytes for their proper distribution
and functions. A high level of CCR10 expression is likely needed
for the efficient migration of sIEL precursors in(to) the skin, but
not for their retention once they are inside. In contrast, continuous
strong CCR10/ligand interaction could break the homeostasis
balance of sIELs and other skin cells and might affect functions of
sIELs, especially considering that the skin can upregulate the
expression of CCL27 during inflammation (36). Although the
sIELs had downregulated CCR10 expression from the level
expressed by their precursors, they seemed to upregulate the expression of other chemokine receptors. A recent report found that
although very few fetal thymic sIEL precursors expressed CCR4,
nearly all the adult sIELs of the skin did, suggesting that CCR4 is
upregulated on the sIELs or that CCR4+ sIELs selectively expanded in the skin (18). These findings indicate that there is
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FIGURE 6. Abnormal accumulation of Vg3+
gd T cells in dermal regions of CCR10-knockout
mice. A, FACS analysis for Vg3+ sIELs in cells
isolated from epidermis of CCR10EGFP/EGFP and
wild-type mice. B, FACS analysis for Vg3+
T cells in cells isolated from the dermal regions of
the CCR10EGFP/EGFP and wild-type mice. A set of
representative plots of at least three experiments
is shown. C, Immunofluorescent microscopy of
skin sections of CCR10EGFP/EGFP and wild-type
mice costained with FITC-conjugated anti-Vg3
and Alexa 647-conjugated anti-CD3 Abs. The
dashed lines run along borders of the epidermis
and dermis. Isotype controls are shown at the
bottom. Original magnification 3100. D, Quantification of epidermal and dermal Vg3+ cells of
CCR10EGFP/EGFP and wild-type mice based on
the immunofluorescent microscopic analyses in
C. Five mice of each genotype were analyzed,
with more than 10 fields counted for each mouse.
8
Acknowledgments
We thank Drs. Joonsoo Kang, David Wiest, David Raulet, Jianke Zhang, and
Avery August for discussion and comments and Thomas Salada for the
ES cell microinjection.
Disclosures
The authors have no financial conflicts of interest.
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a dynamic switch in the expression of chemokine receptors on
sIELs from their fetal thymic precursors that might be important
for their proper maintenance and functions in the skin.
Despite the severely impaired skin-seeding of CCR10-deficient
sIEL precursors at the fetal stage, the number of sIELs in adult
CCR10EGFP/EGFP mice was only mildly reduced from that in wildtype mice, whereas the number of Vg3+ gd T cells in the dermal
region was even higher in CCR10EGFP/EGFP mice. This finding
suggests that besides CCR10, other homing molecules are able to
direct their localization in the skin. Possible candidate homing
molecules for this include E- and P-selectin ligands (18). In addition, the results also suggest that CCR10 is not critical for
survival or proliferation of sIELs in the skin. In fact, the severe
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9