Distribution and development of the postnatal murine Vδ1 T-cell receptor repertoire

Abstract

Murine γ/δ T cells express canonical Vγ5Vδ1 chains in the epidermis and Vγ6Vδ1 chains at reproductive sites. Both subsets carry an identical Vδ1-Dδ2-Jδ2 chain which completely lacks junctional diversity. These cells are thought to monitor tissue integrity via recognition of stress-induced self antigens. In this study, we showed by reverse transcription–polymerase chain reaction (RT-PCR), complementarity determining region 3 (CDR3) spectratyping and sequencing of the junctional regions of Vδ1 chains from C57BL/6 mice (aged 1 day to 14 months) that the canonical Vδ1-Dδ2-Jδ2 chain is also consistently present at other sites such as the thymus, gut, lung, liver, spleen and peripheral blood. In addition, we found multiple Vδ1 chains with fetal type rearrangements which were also shared among organs and among animals. These Vδ1 chains were typically characterized by a conserved amino acid motif, ‘GGIRA’. Furthermore, by analysing the early postnatal period at days 10 and 16, we demonstrated that the diversification of the thymic Vδ1 repertoire is not paralleled by a diversification of extrathymic Vδ1+γ/δ T cells. This indicates that only fetal type rearrangements survive at extrathymic sites. In conclusion, γ/δ T cells expressing the canonical Vδ1-Dδ2-Jδ2 chain are not unique to the skin and reproductive sites. Furthermore, we found other γ/δ T cells expressing fetal type Vδ1 chains which were shared among different organs and animals. Thus, γ/δ T cells expressing conserved Vδ1 chains are likely to have important functions. We suggest a model in which this subset continuously recirculates throughout the organism and rapidly responds to stress-induced self antigens.

Introduction

γ/δ T cells are a major population in murine epithelia interfacing with the external environment, such as the skin and intestine. The γ/δ T cells of the skin, termed dendritic epidermal T cells (DETCs), almost exclusively express a ‘canonical’γ/δ T-cell receptor (TCR), composed of a Vγ5-Jγ1 and a Vδ1-Dδ2-Jδ2 chain lacking junctional diversity.^1,2 The gene nomenclature used here follows that of Asarnow et al. and Ito et al.^2,3 (Vδ1 equals TRDV4⁴). DETCs are derived from early fetal thymic cells.^2,5–7 During fetal thymus development, two consecutive waves of γ/δ T cells occur. The first wave expresses canonical Vγ5Vδ1 chains which home to the epidermis, and the second wave expresses canonical Vγ6Vδ1 chains which home to the mucosal epithelial of the uterus, vagina and tongue.⁸ These two Vγ chains share an identical canonical Vδ1-Dδ2-Jδ2 chain.^3,5,9,10 In contrast to γ/δ T cells of the skin and reproductive tract, intestinal γ/δ T cells are thought to express a relatively diverse repertoire as the junctional regions are diverse and different Vγ and Vδ chains are used.^11–13

Although the ligands recognized by these invariant TCRs have not been identified, they are believed to monitor tissue integrity via recognition of stress-induced self antigens expressed by epithelial cells.^6,14 In this regard, it has been demonstrated that DETCs play an important role in wound healing and the promotion of epithelial cell growth.⁷ Furthermore, in models of skin cancer, it was shown that DETCs can function as anti-epithelial tumour effector cells.¹⁵ In addition, γ/δ T cells are thought to down-regulate inflammatory responses to allergens or pathogens initiated by conventional α/β T cells.^16,17 However, γ/δ T cells can be also proinflammatory, and it has been suggested that their different functional roles depend on the expressed Vγ and Vδ chains.¹⁸

Between the neonatal and adult periods, DETCs expand greatly in the epidermis and are thought to be maintained for the life of the animal.¹⁹ It is likely that the local environment is important in this regard. Targeted disruption of the Vγ5 or Vδ1 genes did not result in a loss of DETCs in the epidermis.^20,21 Furthermore, in Vδ1-deficient mice, DETCs were functionally competent and exhibited a typical dendritic morphology. Thus, these studies suggested that homing and development of DETCs are independent of the canonical Vγ5Vδ1 TCR.¹⁹ However, the DETCs were smaller and lower in density compared to those in normal mice, indicating that the expression of the canonical Vγ5Vδ1 chain is not essential for homing to the skin but greatly enhances maintenance and proliferation in the skin.^7,22

In adult mice, γ/δ T cells expressing Vδ1 or the canonical Vδ1-Jδ2 TCR are thought to be restricted to the skin and reproductive sites, as such rearrangements have rarely been detected in other organs such as the thymus, lymph nodes, spleen and blood, or in other epithelial sites which are populated by their own distinctive subsets.^7,21 One study showed that γ/δ T cells expressing the canonical Vδ1-Jδ2 chain are frequent in the late fetal and newborn gut and liver.²³ However, these Vδ1+ γ/δ T cells are thought to be lost during postnatal development. In contrast, our data suggest that Vδ1 T cells, expressing the canonical Vδ1 rearrangement, ‘survive’ in low numbers as we could detect such cells in multiple organs of adult mice. In addition, they were detected in the peripheral blood, indicating that they continuously recirculate. These Vδ1 cells might recognize and respond to common ‘danger signals’. This hypothesis is supported by numerous studies which found a high frequency of Vγ6Vδ1 γ/δ T cells in response to inflammation.^18,24–30

Our data clearly show that the thymus is capable of producing highly diverse Vδ1 rearrangements in the early postnatal period. However, these diverse rearrangements were not found in the skin or other organs, indicating that only canonical and fetal type rearrangements ‘survive’ at extrathymic sites. These data further support the notion that γ/δ T cells expressing canonical Vδ1 rearrangements are positively selected.

Materials and methods

General comments

We analysed the δ chain of the γ/δ TCR as rearranged γ chains may also be transcribed in α/β T cells whereas the δ genes are deleted during the α-chain gene rearrangement. Therefore, δ transcripts are derived solely from γ/δ T cells whereas γ chains are also transcribed by α/β T cells. This makes it impossible to judge whether a particular Vγ transcript is derived from γ/δ or α/β T cells.

The methods we used do not allow us to determine which Vδ transcript was paired with which Vγ chain. Thus, the canonical Vδ1-Jδ2 chain could be derived from γ/δ T cells expressing different Vγ chains. However, the analysis of cell lines or hybridomas has the disadvantage that only a small number of cells can be analysed. In addition, during the process of cloning and in vitro manipulation, the TCR could be skewed. Using our technique, tissue was immediately snap-frozen in liquid nitrogen and analysed without any prior manipulations, allowing a real-time picture of the TCR repertoire to be obtained.

Mice

Pregnant and non-pregnant C57BL/6 mice were obtained from Charles River Laboratories (Sulzfeld, Germany). Mice from two litters were killed: one mouse at day 1, two at day 10 (a and b) and two at day 16 (a and b) after birth. In addition, mice were killed at 11 weeks and at 6 and 14 months. Mice were raised under standard breeding conditions. Small tissue samples were obtained from the small and large intestines (gut), skin, thymus, liver and spleen at necropsy and immediately snap-frozen in liquid nitrogen and stored at −80° until further use. Peripheral blood mononuclear cells (PBMC) were isolated using a Ficoll density gradient.

Tissue samples were homogenized in RNAlater and RNA was extracted according to the manufacturer's protocol (Ambion, Austin, TX). RNA (1 to 2 μg) was reverse-transcribed into cDNA in a 20-μl reaction mix. A polymerase chain reaction (PCR) was performed with 2 μl of this reaction mix. Thus, ∼100 ng of cDNA was utilized in each PCR reaction. Vδ1 and Vδ5-Vδ7 transcripts were amplified with Taq polymerase using Vδ- and Cδ-specific primers (Table 1). After an initial hot start, amplification of TCRδ rearrangements consisted of 37 cycles of 40 seconds at 94°, 50 seconds at 61°, and 1 min at 72°, followed by a final extension for 10 min at 72°.

Table 1.

Primers used in this study.

Vδ1	5′ tta tac tcg aca ttc aga agg c 3′
Vδ5	5′ ctt cca tct ggt gat ctc tcc 3′
Vδ6	5′ cat cag cct tgt cat ttc agc c 3′
Vδ7	5′ tct ttg cac att tcc tcc tcc c 3′
Cδ1	5′ aac aga tgg ttt ggc cgg agg 3′
Cδ2	5′ gta gaa atc ttt cac cag aca agc 3′

Complementarity determining region 3 (CDR3) spectratyping

For analysis of CDR3 lengths, 2–3 μl of each PCR mixture was added to formamide-containing loading buffer. PCR products were heat-denatured for 2 min at 95°. PCR products were then size separated on a 6% denaturing polyacrylamide gel and visualized by silver staining (Silver Sequence DNA staining reagents) as recommended by the manufacturer (Promega, Mannheim, Germany). Bands were photographed by exposing polyacrylamide gels for 8–20 seconds to an automatic processor-compatible film.

Cloning and sequencing of amplified Vδ1 transcripts

PCR-amplified Vδ1 transcripts were cloned into the linearized T-vector pCR2.1 using the TA cloning kit according to the conditions recommended by the manufacturer (Invitrogen BV, Leek, The Netherlands). Recombinant plasmid DNA from individual colour-selected colonies of Escherichia coli strain INVaF’ was reamplified by PCR and sequenced using the ABI 310 automatic sequencer and the ABI prism dye terminator cycle sequencing ready reaction kit with AmpliTaq DNA Polymerase, FS (Perkin-Elmer, Weiterstadt, Germany), according to the conditions recommended by the manufacturer. In addition, dominant bands from 16-day- and 11-week-old mice were selected and directly sequenced without cloning, as previously described.³¹ We consistently noted double bands corresponding to a CDR3 length of 12. When both bands were eluted from the gel and directly sequenced we always obtained the canonical Vδ1-Jδ2 sequence (#8897-11). Thus, these double bands are likely to be secondary to different migration properties of the two DNA strands. We calculated the CDR3 length according to Rock et al.³² and our previous work.^33,34

Control experiment

As we found the canonical Vδ1-Jδ2 sequence frequently in all tissues analysed, we controlled for possible PCR contamination by amplifying cDNA from the same animals with a Cδ-specific primer (Table 1; Cδ2) which annealed outside of previously amplified PCR products (using the same Vδ primer). Using this approach we confirmed our findings, as identical CDR3 profiles and the canonical Vδ1 sequence could be obtained (data not shown).

Results

CDR3 length analysis suggests a similar Vδ1 repertoire in different organs

We were interested to determine if Vδ1 transcripts could be detected at extradermal sites and to what extent the canonical Vδ1-Jδ2 sequence was present. Therefore, we analysed the Vδ1 repertoire from different organs and from mice of different ages by RT-PCR. CDR3 length analysis was performed by running PCR products on a denaturing polyacrylamide gel. We were especially interested in the intestine and lungs, as these organs also interface with the external environment. As can be seen in Fig. 1, Vδ1 transcripts could be amplified from all organs analysed, with the exception of the liver in old mice, where only a weak or no Vδ1 band could be observed. From all other organs, strong PCR products could be obtained, suggesting that Vδ1 transcripts are not a rarity in organs such as the intestine, lung, spleen and peripheral blood. Furthermore, the CDR3 spectratyping of Vδ1 transcripts from different organs resembled that of the skin, as the band patterns were very similar.

Figure 1.

Complementarity determining region 3 (CDR3) profiles of Vδ1 transcripts from different sites and from mice of different ages are similar. Sequence analysis (see Figs 2 and 3) confirmed that the canonical Vδ1-Jδ2 sequence (corresponding to the arrows on the right of the gels) dominated all Vδ1 repertoires irrespective of the organ analysed. Thus, there is little compartmentalization among different sites. PBMC, peripheral blood mononuclear cells.

The canonical Vδ1-Jδ2 chain dominates the Vδ1 repertoire of extradermal sites

We next cloned and sequenced the Vδ1 transcripts from different organs of selected mice. As can be seen in Fig. 2, the canonical Vδ1-Jδ2 sequence (8897-11), which is normally used by DETCs, dominated all organs analysed. This sequence corresponded to the dominant band shown in Fig. 1 (arrows; see also materials and methods). In addition, we found several Vδ1 transcripts which joined to Jδ1 and also completely lacked N-region additions. Thus, these data suggest that there is no or very little compartmentalization of the Vδ1 repertoire within different sites.

Figure 2.

Extradermal Vδ1 junctional sequences. Postnatal VDJ junctional sequences were derived from multiple organs of mice aged 1 day to 14 months. Germline sequences are shown at the top of the figure. P nucleotide additions and complementary genomic sequences are underlined. Numbers on the right refer to the fraction of transcripts that carry the indicated junctional regions. As expected from the complementarity determining region 3 (CDR3) profiles (Fig. 1), the canonical Vδ1-Jδ2 sequence (8897-11) dominated the gut [small intestine (si) and colon (co)], the lungs (lu), the liver (li), the spleen (sp) and the peripheral blood mononuclear cells (PB). In 16-day- and 11-week-old mice, the canonical sequence was also obtained by direct sequencing of ‘double bands’ (see arrows in Fig. 3) as indicated by an ‘X’ on the right of the figure. In addition, several Vδ1 sequences, which all lacked N-region additions, could be recovered. Almost all of these sequences (‘*’) were also found in the thymus or skin of these animals (Fig. 3), further supporting the notion that there is no significant difference in the Vδ1 repertoires at different sites. These sequence data are available from EMBL/GenBank/DDBJ under accession numbers GU121507–GU121550.

The early postnatal dermal Vδ1 repertoire resembles that of extradermal sites

The canonical Vδ1-Jδ2 chain is almost exclusively expressed by DETCs of adult mice.¹ However, there are no data on the early postnatal period, and it is possible that the repertoire is more diverse at this time-point and becomes more restricted with age. This would support the hypothesis that the canonical Vδ1-Jδ2 sequence is positively selected and maintained in the skin.

As shown in Fig. 3, we found, as expected, the canonical Vδ1-Jδ2 sequence which dominated the skin of all mice analysed (sequence 8897-11, highlighted in grey). However, in addition we detected several Vδ1 transcripts in the skin at days 1, 10 and 16 which completely lacked N-region additions and which were shared among different mice. These sequences were identical with those found at extradermal sites (Fig. 2). In the skin of adult mice, analysed at week 11 and at 6 and 14 months, we exclusively found the canonical Vδ1-Jδ2 sequence, indicating that γ/δ T cells expressing the canonical Vδ1-Jδ2 sequence have a survival advantage and may be positively selected. The lack of N-region additions was a surprise, as terminal deoxynucleotidyltransferase (TdT) is fully expressed at day 10 and the thymocytes of the same mice had diverse Vδ1 rearrangements with multiple N-region additions (see below). TCR and immunoglobulin genes from fetal and neonatal repertoires lack N regions, as murine TdT expression is regulated during ontogeny and complex junctional regions appear approximately 3–5 days after birth.^35,36 Thus, newborn mice exclusively express a fetal type TCR repertoire at extrathymic sites with relatively simple junctional sequences.

Figure 3.

Postnatal VDJ junctional sequences derived from dermal and thymic Vδ1 transcripts from mice aged 1 day to 14 months. Two mice (day 16a and b) were killed at the same age. The previously described canonical Vδ1-Jδ2 chain (8897-11) (highlighted in grey) was the most dominant sequence in the skin and thymus. All other sequences derived from the skin also lacked N-region additions. In contrast, thymic sequences at days 10 and 16 had complex junctional sequences with extensive N-region usage. Several invariant sequences were shared by different animals (‘*’) and were also detected in other organs (Fig. 2). ‘—’ indicates sequences that are out of frame. These sequence data are available from EMBL/GenBank/DDBJ under accession numbers GU121507–GU121550.

Vδ1 transcripts with diverse junctional regions are not detected at extrathymic sites

Next we compared the thymic with the dermal Vδ1 repertoire from the same mice (Fig. 3). The canonical Vδ1-Jδ2 sequence (8897-11) was also detected in the thymus, but at a lower frequency, and in the 10- and 16-day-old mice the thymic Vδ1 repertoire consisted of multiple additional Vδ1 transcripts which were characterized by multiple N-region additions. This was in contrast to the dermal and extrathymic Vδ1 transcripts (Fig. 2), which completely lacked N-region additions irrespective of the age of the mice. Furthermore, whereas several thymic Vδ1 transcripts were out of frame, all Vδ1 rearrangements of the skin were productive. Thus, with the exception of the canonical Vδ1-Jδ2 sequence and several Vδ1 transcripts which completely lack N-region additions, there is little overlap between the two sites, and thymic Vδ1 sequences with N-region additions do not populate (survive in) the skin or extradermal sites.

The thymic Vδ1 repertoire is highly restricted in old mice, whereas the repertoires of the other Vδ families are highly diverse

We next wished to determine whether the thymic repertoire of other Vδ families is more diverse than that of Vδ 1 and if the complexity of rearrangements is similarly restricted in the thymus of old mice irrespective of the V region analyzed. As expected from the sequence analysis (Fig. 3), CDR3 length analysis of thymic Vδ1 transcripts showed a diverse Vδ1 band pattern until day 16 which became highly restricted in the older mice (Fig. 4). In contrast, CDR3 spectratyping of Vδ6 transcripts of the same mice suggested a highly diverse repertoire with increasing CDR3 length with age. This was also the case for Vδ5 and Vδ7 transcripts (data not shown). Thus, the thymus is also capable of producing a highly diverse γ/δ TCR repertoire in old mice. However, this capacity is not used for the Vδ1 repertoire.

Figure 4.

Complementarity determining region 3 (CDR3) length analysis of Vδ1 (top) and Vδ6 (bottom) transcripts in the murine thymus. As expected from the sequencing data of Fig. 3, the CDR3 profiles of thymic Vδ1 transcripts from mice aged 1 to 16 days were more diverse than those from the older mice, which almost exclusively expressed the canonical Vδ1-Jδ2 sequence (see arrow on the right and sequence 8897–11 in Fig. 3). In contrast to Vδ1, the CDR3 profiles for Vδ6 transcripts were highly diverse. Similar data were obtained for Vδ5 and Vδ7 transcripts (data not shown).

The amino acid sequences of the CDR3 region suggest preferential rearrangement and/or selection

Figure 5 shows the translated amino acid sequences of all Vδ1 transcripts that could be recovered from the different tissues of mice of different ages. Almost all sequences that lacked N regions were found in different organs and shared among mice. Interestingly, most of these sequences have previously been published.^{23,24,28,37–42} Thus, Vδ1 sequences, which lack N regions, might be preprogrammed (as identical sequences are found in different mice), but are likely to be further selected by recognition of a limited set of antigens which are shared among different mice and organs. With the exception of the canonical sequence 8897-11, Jδ2 was rarely used (in six of 39 sequences). The junctional regions almost exclusively used the Dδ2 Gen segment. In 25 out of 39 different sequences, the first reading frame of the Dδ2 gene segment was used, resulting in the amino acid sequence ‘GGIR’.

Figure 5.

Amino acid sequences from translated Vδ1 transcripts shown in Figs 1 and 4. The ‘X’ on the right side of the figure indicates the site from which a given sequence could be obtained. Most of the non-canonical sequences (‘*’) were shared by different mice, as highlighted in bold; some have previously been published by other groups, as indicated on the right (ref). Please note that, within the junctional region, the motive ‘GGIRA’ was often found (see Fig. 6).

Non-canonical Vδ1 sequences that lack N regions share a common amino acid motif ‘GGIRA’

As shown in Fig. 5, 15 out of 25 Vδ1 chains expressed an alanine ‘A’ at the junction between Dδ2 and Jδ1. This amino acid was in part generated by a degenerative code (Fig. 6). Furthermore, we could identify two pairs of Vδ1 transcripts which encoded identical Vδ1 amino acid chains (8898-4 and 8883-9; M392-2 and M468-12). Thus, these data strongly suggest selection of Vδ1 chains carrying the motif ‘GGIRA’. However, the observation that the canonical Vδ1-Jδ2 chain was almost exclusively found in old mice suggests that the other fetal type Vδ1 chains are lost during murine development. This may happen because the recognized antigens disappear, or γ/δ T cells expressing the canonical Vδ1-Jδ2 chain are preferentially selected by the same antigens.

Figure 6.

Different Vδ1 nucleotide sequences code for identical amino acid sequences. When individual Dδ2-Jδ1 junctions were analysed, several different junctional nucleotide sequences were noted to result in the identical translated amino acid motif ‘GGIRA’. Furthermore, different Vδ1 transcripts (8883-9 and 8898-4; M392-2 and M468-12) encoded identical Vδ1 chains (see also Figs 3 and 5).

Discussion

The major finding of this study is that the canonical Vδ1-Jδ2 rearrangement was consistently detected by RT-PCR in multiple organs such as the thymus, intestine, liver, spleen and PBMC of young and old mice. Previously it was thought that γ/δ T cells expressing canonical Vδ1-Jδ2 chains are limited to the prenatal period and survive after birth only in the skin, in the lungs and at reproductive sites.^28,43,44 In other organs, including the thymus, they were rarely found or even undetectable.^{22,28,40,45,46} Although our technique did not allow us to determine the frequency of such Vδ1+ cells, the consistent finding of Vδ1 transcripts in multiple tissues from mice of different ages strongly suggests that Vδ1+ cells are present at different sites throughout murine development. This conclusion is supported by the results of studies by other groups, who occasionally found canonical Vδ1-Jδ2 rearrangements in multiple organs of normal adult mice.^{27,44,47–50} Therefore, it is likely that Vδ1+ γ/δ T cells have a significant function in tissues other than the skin and reproductive sites. Otherwise, these cells would have been lost during development.

What could be the function of these Vδ1+ γ/δ T cells? We propose that these cells are present at multiple sites and screen for conserved danger signals. This proposal is supported by our finding that the canonical Vδ1-Jδ2 chain was also detected in the peripheral blood. Although Vδ1+ γ/δ T cells form a minor population outside the skin and reproductive sites, they are likely to be capable of expanding and/or recruiting other immune cells when danger occurs. This notion is supported by several studies which found that Vδ1+ γ/δ T cells, most of them expressing the canonical Vδ1-Jδ2 chain which pairs with Vγ6, preferentially expanded after tissue injury or under inflammatory conditions.^18,24–29 Furthermore, Vδ1+ γ/δ T cells controlled the infiltration of neutrophils and macrophages after Escherichia coli infection via chemokines.^30,51 In studies of Listeria monocytogenes-induced orchitis, infiltrates of Vγ6Vδ1+ γ/δ T cells were also seen in the non-infected testes, indicating that the activating antigen is derived not from the infectious agent but rather from self antigen. Our observation that γ/δ T cells with identical canonical Vδ1-Jδ2 chains are found in different organs and in different mice suggests that these cells recognize an antigen that is not tissue or mouse specific and is present throughout murine development.

A very important question is whether blood contamination in tissues contributed to the appearance of canonical sequences. We cannot entirely rule out this possibility; however, we believe that contaminating γ/δ T cells only played a minor role. For example, in contrast to other Vδ families (data not shown), the amplification of Vδ1 transcripts from the liver samples was difficult and resulted frequently in a faint Vδ1 PCR signal. This suggests that Vδ1+ γ/δ T cells are rare in the liver and that contaminating Vδ1+ cells may indeed have contributed to the repertoire, as the liver has a very good blood supply. However, we obtained strong Vδ1+ PCR products from the other organs, implying that contaminating Vδ1+ γ/δ T cells did not play a major role in these samples. Furthermore, multiple additional CDR3 bands were detected which were not present in the peripheral blood (Fig. 1). In addition, we rarely saw any overlap when the repertoires of human γ/δ T cells from the peripheral blood and the intestine were compared.⁵² Finally, others have also identified the canonical sequence in several organs.^{27,44,47–50}

Although the canonical Vδ1-Jδ2 sequence clearly dominated the Vδ1 repertoires in all organs analysed, we discovered additional fetal type sequences after birth, which were also shared among different organs and different animals. Interestingly, other groups have also described identical sequences.^{23,24,28,37–42} Furthermore, the nucleotide sequences of different junctional regions translated into identical amino acid sequences, which were typically characterized by the amino acid motif ‘GGIRA’. However, these fetal type sequences were not found in the skin of adult mice, further supporting the notion that γ/δ T cells expressing the canonical Vδ1-Jδ2 chain are preferentially maintained and selected in the epidermis based on the specificity of the TCR.^7,19,22,53 Nevertheless, there is a possibility that the other fetal type Vδ1 chains recognize a different antigen which disappears during murine development.

Another major finding was the observation that the postnatal diversification of the thymic Vδ1 repertoire is not paralleled by a diversification of DETCs or other Vδ1-expressing γ/δ T cells. Our data showed that, during the early postnatal period (days 10 and 16), the thymic Vδ1 repertoire expresses, in addition to the canonical Vδ1-Jδ2 chain, multiple Vδ1 chains with highly complex junctional regions. However, these Vδ1 chains were not detected at extrathymic sites, suggesting that γ/δ T cells with fetal type sequences are preferentially selected or that γ/δ T cells with diverse Vδ1 junctional regions do not leave the thymus. In contrast, as shown herein for Vδ5, Vδ6 and Vδ7, the murine thymus is capable of producing and exporting γ/δ T cells with highly diverse junctional regions when other Vδ families are expressed.

Studies on other species also identified TCR δ sequences expressed during fetal development that were shared among different subjects.^33,54 However, these sequences, which typically carried simple junctional regions, are usually lost later in life when TdT is active. Furthermore, in pigs and humans the TCR δ repertoire is highly compartmentalized and no identical junctional regions of TCR δ chains are shared between different adult individuals.^31,52 Thus, of the species investigated to date, mice are unique in that they carry a population of γ/δ T cells expressing a canonical Vδ1-Jδ2 chain which is found not only in multiple organs and throughout development but also in different mice.

In conclusion, γ/δ T cells, expressing the canonical Vδ1-Jδ2 chain, are not unique to the skin and reproductive sites. It is highly likely that they continuously recirculate throughout the murine body in order to recognize and rapidly respond to conserved antigens which are expressed at extradermal sites, for example under inflammatory conditions.¹⁴ Thus, these cells may play an important roles, for example in wound healing and promotion of epithelial growth^7,55 in organs other than the skin. Other functions have been suggested by studies using Vδ1^−/− mice, which demonstrated a protective response against infections through the production of proinflammatory cytokines^30,51,56, and Vδ1+ T cells have been reported to be crucial for repertoire formation in the lungs.⁴⁴ The different functional roles of Vδ1+ cells may depend in part on the coexpressed Vγ chain and its receptor specificity.^18,28

Disclosures

The authors declare that there is no conflict of interest.