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The Journal of Immunology, 2000, 165: 3695-3705.
Copyright © 2000 by The American Association of Immunologists

Development of Dendritic Epidermal T Cells with a Skewed Diversity of {gamma}{delta}TCRs in V{delta}1-Deficient Mice1

Hiromitsu Hara*, Kenji Kishihara3,*, Goro Matsuzaki*, Hiroaki Takimoto2,*, Tadasuke Tsukiyama{dagger}, Robert E. Tigelaar{ddagger} and Kikuo Nomoto*

Departments of * Immunology and {dagger} Molecular and Cellular Biology, Medical Institute of Bioregulation, Kyushu University, Fukuoka, Japan; and {ddagger} Department of Dermatology, Section of Immunobiology, Yale Skin Diseases Research Core Center, Yale University, New Haven, CT 06520


    Abstract
 Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
One of the most intriguing features of {gamma}{delta} T cells that reside in murine epithelia is the association of a specific V{gamma}/V{delta} usage with each epithelial tissue. Dendritic epidermal T cells (DETCs) in the murine epidermis, are predominantly derived from the "first wave" V{gamma}5+ fetal thymocytes and overwhelmingly express the canonical V{gamma}5/V{delta}1-TCRs lacking junctional diversity. Targeted disruption of the V{delta}1 gene resulted in a markedly impaired development of V{gamma}5+ fetal thymocytes as precursors of DETCs; however, {gamma}{delta}TCR+ DETCs with a typical dendritic morphology were observed in V{delta}1-/- mice and their cell densities in the epidermis were slightly lower than those in V{delta}1+/- epidermis. Moreover, the V{delta}1-deficient DETCs were functionally competent in their ability to up-regulate cytokines and keratinocyte growth factor-expression in response to keratinocytes. V{gamma}5+ DETCs were predominant in the V{delta}1-/- epidermis, though V{gamma}5- {gamma}{delta}TCR+ DETCs were also detected. The V{gamma}5+ DETCs showed a typical dendritic shape, {gamma}{delta}TCRhigh, and age-associated expansion in epidermis as observed in conventional DETCs of normal mice, whereas the V{gamma}5- {gamma}{delta}TCR+ DETCs showed a less dendritic shape, {gamma}{delta}TCRlow, and no expansion in the epidermis, consistent with their immaturity. These results suggest that optimal DETC development does not require a particular V{gamma}/V{delta}-chain usage but requires expression of a limited diversity of {gamma}{delta}TCRs, which allow DETC precursors to mature and expand within the epidermal microenvironment.


    Introduction
 Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
An ontogenic feature of murine fetal thymocyte development is the ordered and overlapping appearance of waves of cells expressing a {gamma}{delta} TCR composed of specific V{gamma}- and V{delta}-chains (1, 2, 3, 4, 5). The first two waves in thymic ontogeny are unique in that they express invariant {gamma}{delta}TCRs characterized by a lack of junctional diversity. The first wave appearing around gestation day (GD)4 14–16 expresses a canonical V{gamma}5 (GV1 by World Health Organization-International Union of Immunological Sciences nomenclature, see Ref. 6)-J{gamma}1-C{gamma}1 chain, and the second wave appearing around GD16–18 expresses a canonical V{gamma}6 (GV2)-J{gamma}1-C{gamma}1 chain. Both the V{gamma}5 and V{gamma}6 chains preferentially pair with a common canonical V{delta}1 (DV101)-D{delta}2-J{delta}2-C{delta} chain (5, 7, 8, 9). These V{gamma}5/V{delta}1 and V{gamma}6/V{delta}1 subsets home to epidermis and to mucosal epithelia of reproductive tract and tongue, respectively (7, 10). Later, {gamma}{delta} T subsets expressing variant V{gamma}1 (GV5S1), V{gamma}4 (GV3), and V{gamma}7 (GV4) chains in combination with multiple V{delta}-chains appear in neonatal and adult thymus and home to blood and peripheral lymphoid organs (1, 4, 11), as well as into other epithelial tissues such as the gastrointestinal tract (12, 13, 14).

Although the vast majority of T cells in the peripheral blood, lymph nodes, and spleen of adult mice express {alpha}ßTCRs, {gamma}{delta} T cells are a major T cell population in murine epithelia interfacing with external environment (e.g., skin, reproductive tract, gastrointestinal tract, and lung; Refs. 7, 11, 12 , and 15). One of the most intriguing features of these {gamma}{delta} T cells is the association of a specific V{gamma}/V{delta} usage with each epithelial tissue. Particularly, in the epithelia of skin and the reproductive tract, there are unique populations of {gamma}{delta} T cells with a highly restricted TCR repertoire as described above. Virtually all the reproductive tract epithelial T cells express the canonical V{gamma}6/V{delta}1-TCR, whereas the vast majority of the T cells in the epidermis, referred to as dendritic epidermal T cells (DETCs), express the canonical V{gamma}5/V{delta}1-TCR (16, 17, 18). The presence of such identical TCRs on T cells in these sites is thought to be related to the capacity of such cells to recognize an as yet identified stress-induced autologous Ag; DETCs have been reported to be stimulated in vitro by contact with stressed keratinocytes or a transformed keratinocyte line in a MHC-nonrestricted manner (16, 19). Production of various cytokines by DETCs after in vitro and in vivo activation and non-MHC-restricted cytotoxicity of DETC lines against tumor targets resembling lymphokine-activated killer cell activity has also been demonstrated (20, 21, 22, 23, 24, 25), consistent with a role for DETCs as effector cells in immune surveillance of the skin. In addition, it has been shown that activated V{gamma}5+ DETCs produce keratinocyte growth factor (KGF), consistent with a potential role for DETCs in wound healing (26, 27).

Transgenic {gamma}{delta}TCR-expressing T cells were found in the epidermis of KN6 (V{gamma}4/V{delta}5 (DV105)-TCR)-transgenic mice, suggesting that homing specificity of DETCs is independent of the TCR specificity (28). However, DETCs in the transgenic mice were smaller and were present in lower numbers/density than DETCs observed in normal mice. This observation was consistent with the possibility that a specific TCR usage might be required for complete development and/or maintenance of DETCs in epidermis. Recently, mice in which the V{gamma}5 gene was disrupted by gene targeting were generated (29); the morphology, density, and functional activity of the {gamma}{delta}TCR+ DETCs in such knockout mice was indistinguishable from that seen in littermate controls. A readily detectable, but variable proportion of the DETCs in the epidermis of V{gamma}5-deficient mice expressed a TCR in which V{gamma}1-J{gamma}4-C{gamma}4 chain was paired with a canonical V{delta}1-D{delta}2-J{delta}2-C{delta} chain. Furthermore, such V{gamma}1/V{delta}1 DETCs could be stained with a mAb, 17D1, previously felt to recognize only the canonical V{gamma}5/V{delta}1-TCR expressed by normal DETCs (30), suggesting that a specific TCR conformation rather than a simple linear epitope(s) composed of particular V{gamma}- and V{delta}-chains may be required for the normal development and maintenance of DETCs in epidermis.

In this study, we generated V{delta}1 gene-deficient (V{delta}1-/-) mice using a Cre/loxP gene targeting strategy (31, 32) in an attempt to clarify the requirement of V{delta}1 gene expression for the development of fetal thymocyte and of DETCs. In the V{delta}1-/- mice, we found markedly impaired development of V{gamma}5+ fetal thymocytes, which have been demonstrated to be DETC precursors; however, the DETCs in such mice have relatively normal morphology, density, and functional activity. The DETCs from V{delta}1-/- mice predominantly expressed the canonical V{gamma}5 chain paired with a fetal-type V{delta}6 (DV7, ADV7)-D{delta}2-J{delta}1-C{delta} chain with a limited junction, which was consistent with the possibility that they were derived from fetal thymocytes equally to normal DETCs. The V{gamma}5+ DETCs, but not V{gamma}5- DETCs, in V{delta}1-/- mice expressed a high level of TCRs and proliferate within the epidermis comparable to conventional V{gamma}5+ DETCs in control mice. These results suggest that optimal DETC development requires expression of a limited diversity of {gamma}{delta}TCRs, which allow DETC precursors to mature and expand within the epidermal microenvironment. In this report, the significance of TCR conformation in the development of DETCs is discussed.


    Materials and Methods
 Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Generation of V{delta}1 gene-deficient mice

An 11-kb mouse genomic DNA (BglII-BglII) fragment containing the V{delta}1 gene segments was obtained from the 129/SvJ mouse genomic library (Fig. 1GoA). A 0.9-kb DNA fragment between the 5'-flanking SacI site of the first exon and the EcoRV site located nearly at the 3' terminus of the second exon in the V{delta}1 gene was replaced with the loxP-flanked PGK-neo cassette. The final targeting construct consisted of a 0.9-kb short arm and a 6.3-kb long arm of homology linked by the loxP-flanked PGK-neo cassette. The targeting vector DNA linearized by SalI was electroporated into E14K embryonic stem (ES) cells, followed by selection in the presence of 300 µg/ml G418 for 7 days. ES cell clones with the expected homologous recombination event were screened by PCR and by Southern blot analysis of PstI-digested genomic DNA hybridized with a [32P]-labeled DNA fragment 3' flanking the targeting vector. To excise the PGK-neo cassette from the V{delta}1-targeted allele in the ES clones, 25 µg supercoiled Cre-encoding plasmid DNA (pMCcre-puro vector, constructed and kindly provided by Dr. J. Takeda, Osaka University, Suita, Osaka, Japan; cre gene was supplied by Dr. T. Yagi, National Institute of Physiological Sciences, Okazaki, Aichi, Japan) were transferred into 107 targeted ES cells by electroporation. After selection in 1 µg/ml puromycin-containing medium for 6–7 days, surviving ES colonies were isolated and the deletion event was identified by Southern blot analysis. ES clones with complete deletion of the PGK-neo cassette were microinjected into fertilized blastocysts from C57BL/6 mice. The resultant chimeric male mice with a high chimerity were crossed with C57BL/6 females to generate agouti mice with germline transmission. Following mating of heterozygously mutated mice, homozygotes were identified and distinguished from heterozygous and wild-type mice by Southern blot analysis.



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FIGURE 1. Generation of V{delta}1-deficient mice. A, Endogenous V{delta}1 gene locus and targeting construct (SI, SacI; RI, EcoRI; RV, EcoRV; PI, PstI; XI, XhoI; and LI, SalI). A 0.9-kb SacI-EcoRV fragment containing 2 exons encoding the V{delta}1 gene (solid boxes) was replaced with loxP(closed arrow heads)-flanked PGKneo cassette (open arrow, NEO) by homologous recombination. The direction of the NEO insertion corresponds to that of the open arrow. NEO was removed by transient expression of Cre recombinase (Cre). ES clones that survived each selection were screened by Southern blot analysis of PstI-digested genomic DNAs, using a genomic DNA fragment flanked as a probe displayed in the figure (thick bar). B, Southern blot analysis of ES clones and the V{delta}1-deficient mice. Genomic DNA was extracted from the parental E14K ES line (lane 1), ES clone 5C with the V{delta}1-targeted allele (lane 2), ES clone 5C.5 with the NEO-deleted allele (lane 3), and tails of V{delta}1+/+ mice (lane 4), V{delta}1+/- mice (lane 5) and V{delta}1-/- mice (lane 6) littermates. Genomic DNA samples were digested with PstI and hybridized with a probe indicated in A. The V{delta}1-targeted allele and the NEO-deleted allele were detected as a 1.8-kb and 4.3-kb band, respectively, in comparison to the wild-type allele (5.2-kb band).

 
Preparation and purification of epidermal cells

Epidermal cells were prepared as described previously (33). Briefly, both ventral and dorsal aspects of ear skin were separated from underlying cartilage using fine forceps, followed by flotation, dermal side up, on 1% trypsin/PBS solution for 45 min at 37°C. The epidermis was then separated from dermal tissue and epidermal single cell suspensions were prepared by mechanical agitation. The resulting epidermal cell suspensions were enriched and separated from dead cells by Lympholyte-M (Cedarlane Laboratories, Hornby, Ontario, Canada) density gradient centrifugation. Interface epidermal cells (IECs) were collected and cultured overnight in complete RPMI 1640 medium containing 10% FBS in the presence of 5U/ml human rIL-2 (R&D Systems, Minneapolis, MN). Before flow cytometric analysis, the cultured cells were separated from dead cells by centrifugation over Lympholyte-M.

In some experiments, IECs from V{delta}1-/- mice were separated into V{gamma}5+ cells and V{gamma}5- cells using a magnetic cell sorting system (Vario MACS; Miltenyi Biotec, Auburn, CA). Briefly, IECs were stained with FITC-conjugated anti-V{gamma}5 (F536; PharMingen, San Diego, CA). After washing, the cells were labeled magnetically with anti-FITC Multisort Microbeads (Miltenyi Biotec). Labeled cells were applied onto a separation column (Type RS+; Miltenyi Biotec) placed in a magnetic field (Vario MACS), and the column was washed with PBS/BSA. The flow-through was collected as the V{gamma}5- cell fraction. Bound cells were eluted from the column with PBS/BSA after removing it from the magnetic field and were used as V{gamma}5+ cells. The purity of each V{gamma}5+ or V{gamma}5- fraction among CD3+ T cells was >95%.

Flow cytometric analysis

For four-color flow cytometric analysis, cells were stained with FITC-conjugated, PE-conjugated, allophycocyanin (APC)-conjugated, or biotin-conjugated mAb, followed by staining them with RED670-conjugated streptavidin (Life Technologies, Gaithersburg, MD). In this study, the following dye-conjugated mAbs were used: FITC-conjugated anti-C{delta} (GL3; PharMingen), anti-V{gamma}5 (F536; PharMingen), anti-V{gamma}1 (2.11; generously provided by Dr. P. Pereira, Institut Pasteur, Paris, France), anti-V{gamma}5/V{delta}1 (17D1) mAbs, PE-conjugated anti-C{delta} (GL3; PharMingen) mAbs; biotin-conjugated anti-V{gamma}5 (F536; PharMingen), anti-Thy-1.2 (Meiji Health Center, Tokyo, Japan) mAbs; and APC-conjugated anti-CD3{epsilon} (2C11; PharMingen) mAb. Anti-V{gamma}1 mAb and anti-V{gamma}5/V{delta}1 mAbs were conjugated with FITC using a standard technique. Flow cytometric analysis was performed using a FACScalibur flow cytometer with CellQuest analysis software (Becton Dickinson, Sunnyvale, CA).

Preparation of epidermal sheets and immunohistochemistry

EDTA-separated epidermal sheets were prepared from ears as previously described (34). Briefly, epidermal sheets were fixed in cold acetone for 15 min and then extensively washed with PBS. Two-color immunofluorescence was performed as follows: sheets were initially incubated overnight at 4°C with APC-conjugated anti-CD3{epsilon} (2C11; PharMingen) and either FITC-conjugated anti-C{delta} (GL3; PharMingen), anti-Cß (H57; PharMingen), or anti-V{gamma}5 (F536; PharMingen) mAb. After thorough rinsing with PBS, the specimens were mounted in PBS/glycerol, coverslipped, and viewed under a fluorescence microscope (Axiovert 100; Zeiss, Oberkochen, Germany) with CellScan image analyzer system (Scanalytic, Billerica, MA). To aquire images of the epidermal sheets stained with FITC- and APC-conjugated mAbs, filters for 546 nm and 470 nm were selected, respectively. The black and white images were incorporated and analyzed. For quantification of DETC densities, the number of DETC was counted in seven fields of 0.256 mm2 per specimen; each specimen was prepared from a different distinct mouse, and four to six mice were used for each group. Data were expressed as mean (± SD) density of positive cells per square millimeter.

Analysis of V{gamma} and V{delta} usage and junctional diversities of fetal thymocytes and DETCs

Total RNA was extracted from IECs, fetal thymocytes, or sorted V{gamma}5+ or V{gamma}5- cells with TRIzol reagent (Life Technologies) according to the manufacturer’s instructions. The first strand cDNA was synthesized and amplified by RT-PCR. In brief, total RNA was reverse transcribed with 20 pmol of random hexamer primer (Life Technologies) using reverse transcriptase SuperScript II (Life Technologies) according to the manufacturer’s instructions. The cDNA aliquots were amplified by PCR with various combinations of V{gamma}/C{gamma} or V{delta}/C{delta} primers and 2.5 U AmpliTaq Gold DNA polymerase (Perkin-Elmer/Cetus, Norwalk, CT) in a total volume of 50 ml. The reaction buffer consisted of 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl2, and 0.01% gelatin. The conditions of thermal cycling were as follows: 94°C for 1 min, 54°C for 1 min, and 72°C for 30 s. PCR samples were heated at 94°C for 7 min before the first cycle and the final extension was prolonged to 4 min. PCR primers used in this study were identical with those previously described (35). Southern blot analysis of V{gamma}/C{gamma}-PCR products was performed with C{gamma}2 cDNA probe (MNG8), which binds to all members of Cg genes (data not shown). The C{gamma}2 probe was labeled with [{alpha}-32P]dCTP using the Megaprime DNA Labeling System (Amersham, Arlington Heights, IL) according to the manufacturer’s instructions. Southern blot analysis of V{delta}/C{delta}-PCR products was performed using as a probe an oligonucleotide common either to J{delta}1 (5'-TTCCACAGTCACTTGGGTCCCCA-3') or to J{delta}2 (5'-CTCCACAAAGAGCTCTATGCC-3') segments and labeled with [{gamma}-32P]ATP using Ready-To-Go T4 polynucleotide kinase (Pharmacia Biotech, Uppsala, Sweeden) according to the manufacturer’s instructions.

To analyze the N region diversities of TCR {gamma}- and {delta}- chains, the RT-PCR products were gel purified using a Qiaex II gel extraction kit (Qiagen, Chatsworth, CA) and subcloned into T vector pCR II (Invitrogen, San Diego, CA). The subclones were sequenced by the dideoxy chain-termination method using a Dye Deoxy Terminator Cycle Sequencing Kit (Amersham) and an Applied Biosystems model 377 DNA sequencer (Foster City, CA).

In vitro culture of DETCs and semiquantitative RT-PCR of cytokine and KGF gene expression

IECs (2.5 x 105 cells; 5–10% DETC) were cultured in 96-well round-bottom plates at 37°C for 40 h in 5 µg/ml Con A. The cultured or freshly isolated IECs were analyzed for cytokine mRNA expression using semiquantitative RT-PCR method. Procedures for total RNA extraction and cDNA synthesis were identical with those described above. PCR conditions were identical with those previously described in (36) but using pairs of primers for hypoxanthine phosphoribosyltransferase (HPRT; control), IL-2, IL-4, IFN-{gamma}, and KGF. PCR primer sequences were as follows: HPRT sense 5'-GTTTGTTGTTGGATATGCCCTTGAC-3', antisense 5'-GGGGACGCAGCAACTGACATTTCTA-3'; IL-2 sense 5'-TGATGGACCTACAGGAGCTCCTGAG-3', antisense 5'-GAGTCAAATCCAGAACATGCCGCAG-3'; IL-4 sense 5'-CGAAGAACACCACAGAGAGTGAGCT-3', antisense 5'-GCTCATTCATGGTGCAGCTTATCG-3'; IFN-{gamma} sense 5'-AGCGGCTGACTGAACTCAGATTGTAG-3', antisense 5'-GTCACAGTTTTCAGCTGTATAGGG-3'; KGF sense 5'-CGGAATTCATGCGCAAATGGATACTGACACGG-3', antisense 5'-CGGAATTCTTAGGTTATTGCCATAGGAAG-3'. RT-PCR products were electrophoresed on 1.8% agarose gels and stained with ethidium bromide.


    Results
 Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Generation of ES cell clones with a defined deletion at the V{delta}1 locus and V{delta}1-deficient mice

To avoid possible interference of the PGK-neo cassette at the V{delta}1 locus with TCR{delta} gene rearrangement and transcription, we used the Cre-loxP system (31, 32) to delete the V{delta}1 gene segment. In the construction of a targeting vector, the loxP-flanked PGK-neo gene was replaced with two exons coding the V{delta}1 gene (Fig. 1GoA). The targeting construct was electroporated into E14K ES cells; of the 600 G418-resistant ES colonies screened, 3 PCR-positive ES clones were obtained. To remove the PGK-neo cassette from the targeted allele of the ES clones, pMCcre-puro was electroporated into each ES cell clone; transient expression of Cre recombinase allowed the PGK-neo cassette to be excised from the genome of the targeted ES cells. Homologous recombination and deletion of the PGK-neo cassette was confirmed by Southern blot analysis of PstI-digested genomic DNA from the ES cells using a genomic DNA fragment flanked to the 3' terminus of the targeting construct as a probe (Fig. 1Go, A and B). Autoradiography showed that the targeted V{delta}1 allele and the neo gene-deleted allele were detected as a 1.8-kb band and a 4.3-kb band, respectively, in comparison to a 5.2-kb band from the wild-type allele (Fig. 1GoB, lanes 1–3). Chimeric mice were generated from the V{delta}1-deficient ES cells, and germline-transmitting mice were obtained by repetitive mating of the chimeric mice with C57BL/6 mice. PstI-digested genomic DNA from tails of offspring from the germline-transmitting mice was analyzed by Southern blot analysis (Fig. 1GoB, lanes 4–6). Wild-type (V{delta}1+/+), heterogyzously mutated (V{delta}1+/-) and homozygously mutated (V{delta}1-/-) mice from littermates generated by mating V{delta}1+/- mice or V{delta}1+/- with V{delta}1-/- mice were used in the following experiments. V{delta}1-/- mice were born at the expected Mendelian ratios and appeared healthy with no apparent anatomical abnormalities.

Markedly impaired development of V{gamma}5+ as well as {gamma}{delta}TCR+ fetal thymocytes in V{delta}1-/- mice

Because V{delta}1 gene rearrangement and transcription predominates in early fetal thymocyte development (1, 5, 8, 9), we first analyzed the kinetics of fetal thymocyte development in V{delta}1+/+ and V{delta}1-/- mice by flow cytometry. The total number of Thy-1+ thymocytes of V{delta}1+/+ and V{delta}1-/- mice were comparable with each other during the fetal to newborn period (GD15 to newborn (GD20); Fig. 2Go, A and B). However, both the percentages and numbers of {gamma}{delta}TCR+ and V{gamma}5+ thymocytes were markedly lower in V{delta}1-/- fetuses during the early fetal period (GD15-GD18), compared with those in V{delta}1+/+ fetuses (Fig. 2Go, C–F). The numbers of the {gamma}{delta}TCR+ thymocytes in V{delta}1-/- mice were nearly 20 times less on GD15 and GD16, and 4 times less on GD18 than those in V{delta}1+/+ mice (Fig. 2GoG). The numbers of the V{gamma}5+ thymocytes in V{delta}1-/- mice were nearly 60 times less on GD15, 30 times less on GD16, and 10 times less on GD18 than those in V{delta}1+/+ mice (Fig. 2GoH). But while the number of {gamma}{delta}TCR+ thymocytes in newborn V{delta}1-/- mice was indistinguishable from that observed in newborn V{delta}1+/+ mice (Fig. 2GoC), the numbers of V{gamma}5+ thymocytes remained low in newborn V{delta}1-/- mice compared with newborn V{delta}1+/- mice (Fig. 2GoD). These results indicate that in the absence of a functional V{delta}1 chain, development of control levels of {gamma}{delta}TCR+ thymocytes is notably delayed until the late fetal/newborn period; in contrast, the development of the V{gamma}5+ subset of thymocytes is markedly impaired throughout the entire fetal/newborn period.



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FIGURE 2. Markedly impaired development of V{gamma}5+ as well as {gamma}{delta}TCR+ fetal thymocytes in V{delta}1-/- mice. Fetal thymocytes were obtained from V{delta}1+/+ and V{delta}1-/- mice at gestation day (GD) 15, 16, 17, and 18, and newborn (NB) mice. Thymocyte suspensions were triple-stained with either FITC-conjugated anti-V{gamma}5 or FITC-conjugated anti-C{delta} in combination with both APC-conjugated anti-CD3 and biotin-conjugated anti-Thy1.2 mAbs, followed by staining them with RED670-conjugated streptavidin, and then flow cytometric analysis. {gamma}{delta}TCR+ (C) and V{gamma}5+ (D) cells in the Thy-1+ cell population shown. The absolute numbers of Thy-1+ (B), {gamma}{delta}TCR+ (E), and V{gamma}5+ (F) cells per thymus were calculated from the total number of unstained thymocytes (A), while the percentages of {gamma}{delta}TCR+ (C) and V{gamma}5+ (D) cells were calculated as proportions of Thy-1+ cells. Relative cell numbers of {gamma}{delta}TCR+ (G) and V{gamma}5+ (H) cells in a V{delta}1-/- fetal thymus were calculated based on the absolute numbers of the corresponding cells in a V{delta}1+/- fetal thymus (set as 100). Open symbols and closed symbols represent the data from V{delta}1+/+ and V{delta}1-/- mice, respectively. Similar results were obtained from three independent experiments and representative data are shown here.

 
Generation of {gamma}{delta}TCR+ DETCs with normal phenotype in the epidermis of V{delta}1-/- mice

To examine the effects of the deletion of the V{delta}1 gene on the development of DETCs, we analyzed the density of DETCs in the ear skin from 1-, 8-, and 16-wk-old V{delta}1+/+, V{delta}1+/-, and V{delta}1-/- mice by immunohistochemistry. Epidermal sheets were double-stained with APC-conjugated anti-CD3 mAb in combination with either FITC-conjugated anti-C{delta}, anti-Cß, or anti-V{gamma}5 mAb. DETCs detected in the epidermal sheets from V{delta}1+/+ and V{delta}1+/- mice were comparable to each other in density and phenotype of {gamma}{delta}TCR (data not shown). Interestingly, CD3+ and {gamma}{delta}TCR+ DETCs with a typical dendritic morphology characteristic of normal DETCs were observed in V{delta}1-/- mice (Fig. 3Go, E and e). Unexpectedly, despite the profound deficiency in V{gamma}5+ fetal thymocyte precursors of DETCs in V{delta}1-/- mice, the densities of CD3+ DETCs in epidermal sheets from 1-wk-old V{delta}1-/- mice were indistinguishable from those in V{delta}1+/- littermates, and by 8 and 16 wk of age, CD3+ DETC densities in V{delta}1-/- epidermal sheets were only 14 and 22% lower than in V{delta}1+/- controls (Table IGo). As previously reported in normal mice (16, 17, 18), virtually all CD3+ DETCs from V{delta}1+/- mice were {gamma}{delta}TCR+ and V{gamma}5+ (Table IGo; Fig. 3Go, A, C, a, and c). In contrast, while all the CD3+ DETC from V{delta}1-/- mice were {gamma}{delta}TCR+ (Fig. 3Go, E and e), V{gamma}5- DETCs were frequently observed in the CD3+ DETCs from V{delta}1-/- mice (Fig. 3Go, G and g). Interestingly, V{delta}1-/- mice showed an age-associated increase of percent V{gamma}5+ DETCs (Table IGo); while the density of V{gamma}5- DETCs in V{delta}1-/- mice changed minimally between 1 wk and 16 wk (36% increase over 1-wk level at 8 wk, and 10% increase over 1-wk level at 16 wk; Fig. 4Go), the density of V{gamma}5+ DETCs almost doubled between 1 and 8 wk (191% increase) and almost tripled between 1 and 16 wk (275% increase; Table IGo, Fig. 4Go), suggesting that the expansion potential of V{gamma}5+ DETCs in the epidermis is higher than that of V{gamma}5- DETCs. Notably, the V{gamma}5- DETCs in V{delta}1-/- mice were localized as clusters distinct from the clusters of V{gamma}5+ DETCs. Many of the V{gamma}5- DETCs showed a less dendritic shape (Fig. 3Go, I and i; a cluster located in lower part of Fig. 3Go, G and g), while most of the V{gamma}5+ DETCs showed a highly dendritic morphology typical of DETCs of normal mice (Fig. 3Go, H and h; a cluster located in upper part of Fig. 3Go, G and g). Additionally, some V{gamma}1+ cell clusters were detected within the V{gamma}5- DETC clusters in the epidermal sheets from V{delta}1-/- mice (data not shown), whereas no V{gamma}1+ cells could be found in V{delta}1+/- mice (data not shown). In epidermal sheets from either V{delta}1+/- or V{delta}1-/- mice, we could detect no {alpha}ßTCR+ cells (Fig. 3Go, B, F, b, and f).



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FIGURE 3. Immunohistochemical analysis of {gamma}{delta} T cells in epidermal sheets from V{delta}1+/- and V{delta}1-/- mice. Epidermal sheets prepared from 8-wk-old V{delta}1+/- mice (A–D, a–d) and V{delta}1-/- mice (E–I, e–i) were double-stained with APC-conjugated anti-CD3 (A-I) in combination with FITC-conjugated anti-C{delta} (a,e), anti-Cß (b and f), or anti-V{gamma}5 (c, d, g, h, i) mAb. A V{gamma}5+ cluster and a V{gamma}5- cluster were focused on in H and h and I and i, respectively. Original magnification: x200 (A, a, B, b, C, c, E, e, F, f, G, g), x400 (D, d, H, h, I, i). Representative photographs are shown here.

 

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Table I. Cell density of DETCs in epidermal sheets from V{delta}1+/- and V{delta}1-/- mice

 


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FIGURE 4. Preferential expansion of V{gamma}5+ DETCs within the epidermis of V{delta}1-/- mice. Epidermal sheets from 1-, 8-, and 16-wk-old V{delta}1+/- and V{delta}1-/- mice were stained with APC-conjugated anti-CD3{epsilon} and FITC-conjugated anti-V{gamma}5 mAbs. The densities of CD3+V{gamma}5+ and CD3+V{gamma}5- cells were counted and calculated as described in Table IGo. Data are expressed as mean ± SD (cell density in square millimeters; n = 4–6) .

 
In an attempt to confirm and extend the above immunohistochemical data, flow cytometric analyses were performed using IECs isolated from ear skin of 8-wk-old V{delta}1+/- and V{delta}1-/- mice. In each flow cytometric analysis, forward and side scatters were set to gate on the lymphoid cell population. As shown in Fig. 5GoA, in IECs from V{delta}1+/- mice ~93% of the CD3+ cells were {gamma}{delta}TCR+ cells and ~95% of the {gamma}{delta}TCR+ cells were V{gamma}5+ cells. Thus, DETCs from V{delta}1+/- mice were almost exclusively V{gamma}5+ {gamma}{delta}TCR+ cells. In contrast, the CD3+ IECs from V{delta}1-/- mice were 80–90% {gamma}{delta}TCR+ cells and only approximately half of the {gamma}{delta}TCR+ cells were V{gamma}5+. Furthermore, while V{gamma}1+ cells were present in very small numbers in V{delta}1+/- mice, ~20% of the CD3+{gamma}{delta}TCR+ cells in V{delta}1-/- mice were V{gamma}1+ (Fig. 5GoB). It is notable that CD3+ V{gamma}5- cells in V{delta}1-/- mice were apparently of {gamma}{delta}TCRlow phenotype whereas the V{gamma}5+ cells were relatively of {gamma}{delta}TCRhigh phenotype in both V{delta}1+/- and V{delta}1-/- mice (Fig. 5GoA). There were some differences in the data from flow cytometric and immunohistological analyses. A small number of CD3+ {gamma}{delta}TCR- cells (equivalent to {alpha}ßTCR+ cells, data not shown) was detected in IECs from both V{delta}1+/- and V{delta}1-/- mice. They may be represent a "contamination" of the epidermal cell with dermal {alpha}ß T cells, because no {alpha}ßTCR+ cells were observed in the epidermal sheets (Fig. 3GoB). In addition, the composition of V{gamma}5+ cells in CD3+ DETCs from V{delta}1-/- mice, instead of accounting for 70–80% of the CD3+ DETCs as seen by immunohistochemical analysis, were <50% by flow cytometric analysis. This result may be due to a greater sensitivity of flow cytometric analysis compared with immunohistological analysis; i.e., perhaps the V{gamma}5+ {gamma}{delta}TCR+ cells in V{delta}1-/- mice were not stained brightly enough to be detected by immunohistochemical analysis because many of the V{gamma}5- {gamma}{delta}TCR+ cells detected by flow cytometry were {gamma}{delta}TCRlow. Collectively, although DETCs could develop without marked impairment in V{delta}1-/- mice, a significant number of unusual DETCs expressing V{gamma}5- {gamma}{delta}TCRs (including V{gamma}1+ cells) were detected in V{delta}1-/- mice.



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FIGURE 5. Flow cytometric analysis of DETCs from V{delta}1+/- and V{delta}1-/- mice. A, IECs prepared from 8-wk-old V{delta}1+/- and V{delta}1-/- mice as described in Materials and Methods were stained with FITC-conjugated anti-V{gamma}5, PE-conjugated anti-C{delta}, and APC-conjugated anti-CD3{epsilon} mAbs, or with FITC-conjugated anti-Cß, PE-conjugated anti-C{delta} and APC-conjugated anti-CD3{epsilon} mAbs, and analyzed by flow cytometry with gating on CD3+ cells or CD3+TCR{gamma}{delta}+ cells, respectively. Values in the panels indicate percentages of cells within the quadrants and are representative of four separate experiments. B, IECs prepared from 8-wk-old V{delta}1+/- and V{delta}1-/- mice were stained with FITC-conjugated anti-V{gamma}1, PE-conjugated anti-C{delta}, biotin-conjugated anti-V{gamma}5, and APC-conjugated anti-CD3 mAbs, followed by staining with RED670-conjugated streptavidin and analysis by flow cytometry with gating set on CD3+TCR{gamma}{delta}+ cells. The values in the panels indicate percentages of cells within each quadrant, and are representative of 3 distinct experiments. C, IECs prepared from 8-wk-old V{delta}1+/- and V{delta}1-/- mice were stained with either FITC-conjugated anti-17D1 or control mAb in combination with PE-conjugated anti-C{delta} and APC-conjugated anti-CD3 mAbs; Staining is shown as histograms gated on CD3+TCR{gamma}{delta}+ cells. Values in the histograms indicate percentages of 17D1+ cells and are representative of results obtained in four separate experiments.

 
No requirement for the 17D1-defined {gamma}{delta}TCR conformation for DETC development

Monoclonal Ab 17D1 was initially characterized as recognizing {gamma}{delta}TCRs containing canonical V{gamma}5-J{gamma}1-C{gamma}1/V{delta}1-D{delta}2-J{delta}2-C{delta} sequences expressed by DETCs but not by any other {gamma}{delta} T cells (30). Recently, Mallick-Wood et al. (29) reported that a TCR composed of V{gamma}1-J{gamma}4-C{gamma}4/V{delta}1-D{delta}2-J{delta}2-C{delta}-chains and expressed on DETCs from V{gamma}5-/- mice could be also recognized by 17D1. They suggested that such V{gamma}1/V{delta}1-TCRs from V{gamma}5-/- mice conserved the same TCR conformation as formed by the canonical V{gamma}5/V{delta}1-TCRs of normal mice. Furthermore, they also raised the possibility that such a specific TCR conformation rather than simple linear epitopes composed of the particular V{gamma}- and V{delta}-chains might be required for optional development and maintenance of DETCs in the epidermis. To test this hypothesis, IECs from V{delta}1-/- mice were stained with 17D1 and analyzed by flow cytometry. As shown in Fig. 5GoC, virtually no DETCs from V{delta}1-/- mice were 17D1+, although 70–80% of {gamma}{delta}TCR+ DETCs from V{delta}1+/- mice were 17D1+. Thus, the {gamma}{delta}TCR(s) on DETCs from V{delta}1-/- mice did not express an epitope recognized by 17D1, implying that an essential portion of the 17D1 epitope involves the V{delta}1-D{delta}2-J{delta}2 region. Furthermore, these results indicate that the 17D1 epitope is not essential for development and localization of DETC within the skin.

Normal capacities of in vitro cultured DETCs from V{delta}1-/- mice for cytokine and KGF production

It has been demonstrated that the production of KGF and cytokines including IL-2 and IFN-{gamma} is induced or enhanced in normal DETCs in response to Con A or stressed keratinocytes (19, 25, 26) To examine the capacities of DETCs from V{delta}1-/- mice for cytokine and KGF production, freshly isolated IECs from V{delta}1+/+ and V{delta}1-/- mice were cultured in the presence or absence of the mitogen, Con A. The purity of DETCs in the IECs was ~10%; while keratinocytes accounted for the vast majority of the remaining cells, small numbers (<5%) of Langerhans cells were also present. In freshly isolated IECs from both V{delta}1+/+ and V{delta}1-/- mice, IL-2 and IFN-{gamma} gene expression was weakly detected before culture while KGF and IL-4 gene expression was undetectable (Fig. 6Go). Con A stimulation enhanced IL-2 gene expression in IECs from both V{delta}1+/+ and V{delta}1-/- mice (Fig. 6Go). Similarly, IL-2 production enhanced by dish-bound anti-{gamma}{delta}TCR mAb (UC-7) in DETCs from V{delta}1-/- mice was also comparable to that detected in DETCs from V{delta}1+/+ mice, indicating that {gamma}{delta}TCR-mediated signaling is not impaired in DETCs from V{delta}1-/- mice (data not shown). Notably, IL-2, KGF, and IFN-{gamma} gene expression was induced or enhanced simply by culture in medium alone (i.e., no Con A), implying that DETCs from V{delta}1-/- mice can respond to unknown Ags possibly expressed on stressed keratinocytes during the in vitro culture as previously observed in freshly isolated DETCs and DETC lines from normal mice (16, 19). These results indicate that the DETCs from V{delta}1-/- mice are functionally competent at least in their capacity to up-regulate cytokine and KGF expression in response to mitogen or to other epidermal cells. The cytokine and KGF gene expression were not significantly detected in CD3+ cell-depleted IECs from V{delta}1+/+ and V{delta}1-/- mice (data not shown). Thus their gene expression was mostly derived from DETCs.



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FIGURE 6. Gene expression of cytokines and KGF in freshly isolated and cultured V{delta}1+/- and V{delta}1-/- DETCs. IECs (2.5 x 105 cells; 5–10% DETCs) from V{delta}1+/- or V{delta}1-/- mice were cultured in 96-well round-bottom plates at 37°C for 40 h in complete RPMI 1640 medium containing 10% FBS, with or without 5 µg/ml Con A. The cultured or freshly isolated epidermal cells (the majority of which were keratinocytes) were analyzed for cytokine and KGF mRNA expression using semiquantitative RT-PCR as described in the Materials and Methods. The amount of cDNA for each sample was adjusted using a pair of HPRT primers. Similar results were obtained from two independent experiments.

 
Change of V{delta} usage in fetal thymocytes and DETCs from V{delta}1-/- mice

To characterize differences in the {gamma}{delta} T cell repertoires in fetal thymocytes and adult DETCs from V{delta}1-/- and V{delta}1+/+ mice, V{gamma}/V{delta} usage was first analyzed by semiquantitative RT-PCR. No obvious differences in V{gamma} usage were observed in DETCs from V{delta}1-/- vs V{delta}1+/+ mice (data not shown). As shown in Fig. 7GoB, V{delta}-J{delta}2 expression in early fetal thymocyte (GD15–16) from V{delta}1+/+ mice was almost exclusively confined to V{delta}1, although V{delta}6 was weakly expressed by GD17. Similarly, adult DETC from V{delta}1+/+ mice displayed only V{delta}1-J{delta}2 expression. As expected, V{delta}1-J{delta}2 expression was undetectable in both fetal thymocytes and adult DETC from V{delta}1-/- mice; and while no V{delta}-J{delta}2 expressionwas seen in adult DETC from V{delta}1-/- mice, V{delta}6-J{delta}2 and V{delta}8-J{delta}2 were weakly expressed in fetal thymocytes. V{delta}-J{delta}1 expression is shown in Fig. 7GoA. In V{delta}1+/+ mice, V{delta}1-J{delta}1 expression predominated in both fetal thymocytes and adult DETC. However, V{delta}6-J{delta}1 and V{delta}7-J{delta}1 expression was apparent in GD15 fetal thymocytes, and this expression, along with that of V{delta}2-J{delta}1 and V{delta}3-J{delta}1 was up-regulated atGD16–17. Weak expression of V{delta}6-J{delta}1 is also seen in adult DETC from V{delta}1+/+ mice. In V{delta}1-/- mice, no V{delta}1-J{delta}1 expression was seen. Adult DETC from V{delta}1-/- mice expressed predominantly V{delta}6-J{delta}1, with detectable expression of V{delta}7-J{delta}1. V{delta}6-J{delta}1 and V{delta}7-J{delta}1 expression also predominated in GD15 fetal thymocyte, while at later times, expression of V{delta}2, V{delta}3, and V{delta}8 increase. In summary, the expression of V{delta} genes in adult DETC from both V{delta}1+/+ and V{delta}1-/- mice most closely resembles that of GD15 fetal thymocytes, and in the absence of a functional V{delta}1 gene, expression of other previously minor V{delta} gene products now predominates.



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FIGURE 7. V{gamma}/V{delta} gene usage in fetal thymocytes and adult DETCs from V{delta}1+/+ and V{delta}1-/- mice. A and B, Total RNA extracted from fetal thymocytes and IECs of 8-wk-old V{delta}1+/+ or V{delta}1-/- was reverse transcribed into cDNA and amplified by PCR with primers for C{delta} and various V{delta} segments. Southern blot analysis of V{delta}-C{delta} PCR products was performed using an oligonucleotide probe common to the J{delta}1 (A) or J{delta}2 (B) segment to examine separately the J{delta}1- or J{delta}2-linked repertoires, respectively. Representative results from two separate experiments are shown. C, Total RNA extracted from sorted V{gamma}5+ or V{gamma}5- DETCs from 8-wk-old V{delta}1-/- mice was reverse transcribed into cDNA and amplified by PCR with primer sets for various V{gamma}-C{gamma} or V{delta}-C{delta} genes. Southern blot analysis of V{gamma}-C{gamma} PCR products was performed using MNG8 as a probe. The Southern blot analysis of the V{delta}-C{delta} PCR products was conducted using a common oligonucleotide probe for J{delta}1 or J{delta}2 segments. Similar results were obtained from two independent experiments and the representative data are shown.

 
To analyze potential differential V{gamma}/V{delta} usage in V{gamma}5+ and V{gamma}5- DETCs from V{delta}1-/- mice, populations were purified using immunomagnetic beads before RT-PCR analysis (Fig. 7GoC). V{gamma}5- DETCs preferentially expressed V{gamma}1, V{gamma}2, and V{gamma}4. The expression of V{delta} gene was relatively similar in V{gamma}5+ and V{gamma}5- DETCs, with V{delta}6-J{delta}1 transcripts predominating in both populations; V{delta}5-J{delta}1 transcripts were only detectable in V{gamma}5- DETCs.

DETCs from V{delta}1-/- mice express fetal-type TCR {gamma}- and {delta}-chains with restricted junctional diversities

To examine the diversity of {gamma}{delta} T cell repertoire in DETCs from V{delta}1-/- mice, we sequenced V(D)J junctional regions amplified by PCR of cDNAs prepared from V{gamma}5+ and V{gamma}5- DETCs. As shown in Fig. 8GoA, all in-frame RT-PCR clones amplified at the V{gamma}5-J{gamma}1 junction in DETCs from V{delta}1-/- mice showed the canonical sequence observed in normal DETCs (30/30). In-frame RT-PCR clones amplified at the V{delta}6-J{delta} junction from both V{gamma}5+ (Fig. 8GoB) and V{gamma}5- (Fig. 8GoC) DETCs predominantly expressed V{delta}6-D{delta}2-J{delta}1 products, and while P nucleotides were present in over 60% of the sequences, N nucleotides were present in <20% of the junction; such junctions are common in fetal thymocytes (1, 37). Notably, an amino acid sequence ((I/V/L)GGIRA) at the V{delta}6-D{delta}2-J{delta}1 junctions was very frequently observed in both V{gamma}5+ and V{gamma}5- DETC transcripts (10/17 and 11/20, respectively; data not shown). Such a junctional sequence was previously observed in neonatal thymic hybridomas and DETC lines spontaneously secreting cytokines (38). Only a few V{delta}6+ clones used D{delta}1, D{delta}2, and J{delta}1 genes, which are typically detected in adult thymocytes (1, 37). Collectively, these data suggest that the diversity of {gamma}{delta} T cell repertoire of DETCs in V{delta}1-/- mice is biased toward relatively simple joints, consistent with the majority of such cells being derived from fetal, rather than adult, sources.



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FIGURE 8. Junctional sequences of TCR {gamma}- and {delta}-chains expressed in DETCs from V{delta}1-/- mice. RT-PCR products prepared for the analysis of V{gamma} and V{delta} usage in IECs from 8-wk-old V{delta}1+/+ or V{delta}1-/- mice were subcloned into T-vector pCRII (Invitrogen), followed by sequencing the subclones. Nucleotide sequences of V{gamma}5-J{gamma}1 junctions in whole DETCs from V{delta}1-/- mice (A) and those of V{delta}6-J{delta} junctions in V{gamma}5+ (B) and V{gamma}5- (C) DETCs from V{delta}1-/- mice. The sequences are aligned with germline sequences of TCR {gamma} and {delta} segments. The percentage (proportion) of in-frame joints in the V{gamma}5-J{gamma}1 (A), V{gamma}5+ V{delta}6-J{delta} (B), and V{gamma}5- V{delta}6-J{delta} (C) sequences were 94% (30/32), 89% (17/19), and 100% (20/20), respectively. Only sequences with in-frame joints are shown here.

 

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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
In this report, we examined the influence of the V{delta}1 gene deletion on fetal thymocyte and DETC development. Because fetal thymocytes expressing V{gamma}5-bearing TCRs were previously reported to be precursors of DETCs (10, 18, 39, 40), we first examined the development of fetal thymocytes in V{delta}1-/- mice. The development of {gamma}{delta}TCR+ and V{gamma}5+ fetal thymocytes of V{delta}1-/- mice was markedly impaired during the early-middle fetal period (GD15-GD18); thereafter, the number of {gamma}{delta}TCR+, but not V{gamma}5+, thymocytes recovered in newborn mice (Fig. 2Go). It has been reported that V{delta}1 gene rearrangement and transcription predominate in early fetal thymocyte development and that the V{delta}1 chain preferentially pairs with V{gamma}5 or V{gamma}6 chains (1, 5, 8, 9). Our results confirm and extend these reports by demonstrating that the sequence of rearrangement and expression of {gamma}{delta}TCR genes is coordinately regulated during ontogeny and that V{delta}1-bearing {gamma}{delta} T cells represent a major subset of fetal thymocytes during early to middle phases of fetal thymocyte development. Thus, we suggest that the V{delta}1-bearing TCR expression is crucial for development of {gamma}{delta}TCR+ as well as V{gamma}5+ fetal thymocytes.

Because the development of V{gamma}5+ fetal thymocytes was markedly impaired in V{delta}1-/- mice, we expected a similarly impaired development of DETCs in these mice. However, {gamma}{delta}TCR+ DETCs with a typical dendritic morphology were readily observed in V{delta}1-/- mice, and their density in adult V{delta}1-/- epidermis was only about 20% lower than in V{delta}+/- epidermis (Fig. 3Go, Table IGo). Moreover, the {gamma}{delta}TCR+ DETCs from V{delta}1-/- mice were capable of up-regulating mRNA for various cytokines and KGF (26) in response to keratinocytes (Fig. 6Go). These results strongly suggest that the V{delta}1 structure is not essential to generate the TCR conformation required for relatively normal DETC development and function. Rather similar results have been shown in V{gamma}5-/- mice, where V{gamma}5- {gamma}{delta}TCR+ DETCs displayed morphologically and functionally normal development (Mallick-Wood et al., Ref. 29). Although these results strongly suggest that neither canonical V{gamma}5- nor V{delta}1-bearing TCRs are essential for DETC development and localization, the possibility remains that TCRs expressing either canonical V{gamma}5 and/or V{delta}1 may constitute more preferential conformations than other TCRs for optimal DETC development/localization, ligand recognition, and/or function. This possibility is consistent with the following observations: 1) canonical V{gamma}5+ DETCs or canonical V{delta}1+ DETCs are a dominant population in V{delta}1-/- mice or V{gamma}5-/- mice, respectively (Table IGo; Ref. 29); 2) the proportion, and even more strikingly, the absolute numbers of V{gamma}5+ DETC increased in an age-associated manner in V{delta}1-/- mice (Table IGo); 3) V{gamma}5- DETCs displayed less dendritic morphology than V{gamma}5+ DETCs in V{delta}1-/- mice (Fig. 3Go); 4) the V{gamma}5+ DETCs, but not V{gamma}5- DETCs, in V{delta}1-/- mice retained the ability to expand and develop normally in the epidermis comparable to conventional V{gamma}5+ DETCs in normal mice (Fig. 4Go); and 5) expression levels of TCR on V{gamma}5- DETCs in V{delta}1-/- mice are apparently lower than those of V{gamma}5+ DETCs in V{delta}1-/- mice and normal mice (Fig. 5GoA). The latter three observations are consistent with the possibility that the V{gamma}5- DETCs in V{delta}1-/- mice may be less mature than V{gamma}5+ DETCs, as are both our and others’ observations that DETCs in normal mice from birth to 2 wk of age show a round, less dendritic shape and low-level TCR expression compared with adult DETCs (data not shown; Refs. 41 and 42).

The V{gamma}5+ DETCs in V{delta}1-/- mice predominantly expressed TCRs composed of the canonical V{gamma}5 chain paired with various V{delta}6-D{delta}2-J{delta}1 chains containing relatively few N nucleotide additions (Fig. 8