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The Journal of Immunology, 2000, 164: 1002-1012.
Copyright © 2000 by The American Association of Immunologists

V{delta} Repertoire During Thymic Ontogeny Suggests Three Novel Waves of {gamma}{delta} TCR Expression

Julia Weber-Arden, Oliver M. Wilbert1, Dieter Kabelitz2 and Bernhard Arden3

Department of Immunology, Paul Ehrlich Institute, Langen, Germany


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Taking advantage of a PCR technique that allows amplification of all variable region genes with equal efficiency, we defined three novel waves of TCR {delta}-chain transcription during thymic ontogeny. The canonical DV101-D2-J2 rearrangement was confined to a narrow window from days 14 to 18 of gestation, indicating that the postulated two consecutive {gamma}{delta} precursor waves bearing this canonical DV101 rearrangement will coincide on day 16. Neonatal {delta}-chain transcripts used a second wave of diverse V{alpha} gene segments that are exclusively located in the {delta} locus-proximal gene cluster of intermingled single members of different V{alpha} subfamilies. In the adult, only expression of a clan of three homologous subfamilies, ADV7, DV104, and ADV17, persists. The members of the ADV7 subfamily are also scattered across the {alpha} locus, but their usage does not show the position-dependent bias of the other V{alpha}-to-{delta} rearrangements.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The {gamma}{delta} T cells can be detected in the thymus around day 13 of gestation, 3 days before {alpha}{beta} T cells are found. During fetal ontogeny successive waves of {gamma}{delta} cells arise bearing TCR composed of different variable (V{gamma} and V{delta}) regions (reviewed in Refs. 1 and 2). The first wave comprises cells bearing GV1 (V{gamma}3; for nomenclature, see Ref. 3) paired with DV101 (V{delta}1). On the basis of subtractive Ab staining, a second wave using GV2(V{gamma}4)/DV101 receptors has been postulated. Both fetal clonotypes, generated by canonical rearrangements of V, D (only in {delta}-chains), and J gene segments, colonize specific epithelia, where they persist in the adult mouse. The intraepithelial lymphocytes that populate the skin (s-IEL)4 bear the GV1/DV101 TCR (4), and both the intraepithelial cells of the female reproductive tract (r-IEL) (5, 6) and, during the perinatal period, the resident pulmonary lymphocytes (7) bear the GV2/DV101 TCR. The intestinal intraepithelial lymphocytes (i-IEL) exhibit some diversity in V{delta} usage, employing predominantly DV104, DV105, and ADV7 (ADV7 designating the V{alpha}7 subfamily that includes the homologous V{delta}6 gene segments; for nomenclature, see Ref. 3), whereas they express GV4 (V{gamma}5) almost exclusively (8, 9). GV3 (V{gamma}2) and GV5S1 (V{gamma}1.1) predominate in the adult thymus and spleen in combination with a variety of V{delta} gene segments. The {gamma}{delta} T cells that migrate to distinct epithelial tissues may be suited for different functions. The progenies of early fetal {gamma}{delta} thymocytes with restricted V gene usage may recognize autologous Ags from damaged cells rather than the agent inducing the damage, whereas {gamma}{delta} cells arising later in ontogeny and exhibiting junctional diversification may recognize foreign Ags associated with pathogens.

The first V{delta} repertoire study was performed by screening of a cDNA library from CD4-CD8- thymocytes with a constant (C{delta}) gene probe (10). This study revealed six V{delta} gene segments, originally designated V{delta}1–V{delta}6. Four of these defined novel subfamilies, whereas V{delta}3 and V{delta}6 were homologous to the previously described V{alpha}6 and V{alpha}7 subfamilies, respectively (75% identity at the nucleotide level being used as the cut-off between subfamilies) (11). Here we adhere to a nomenclature that has subsequently proposed to give the mixed subfamilies a common designation, ADV6 and ADV7, respectively, whereas the unique V{delta} subfamilies were designated DV101–DV105 (3). Ever since the initial repertoire study, subsequent studies of polyclonal {gamma}{delta} T cell populations were based on PCR primers specific for the already known V{delta} subfamilies and thus were confined to the originally characterized set of V gene segments. The {delta} locus is nested within the {alpha} locus on chromosome 14, between the germline V{alpha} and J{alpha} gene segments. This location should yield access to the large family of V{alpha} gene segments. However, the {delta}-chain repertoire appears to be limited to the classical V{delta} gene segments, DV101–DV105, ADV6, and ADV7. These map in vicinity to the D{delta} and J{delta} gene segments, with the exception of the ADV7 subfamily, which contains four or five different members that are scattered across the V{alpha} locus (12, 13). Furthermore, rearrangements of a few V{alpha} gene segments have occasionally been reported to occur in {gamma}{delta} T cell clones and hybridomas (summarized in Ref. 3). Moreover, Northern hybridizations of a large panel of neonatal thymocyte hybridomas with V{alpha} subfamily-specific probes revealed expression of V{alpha} gene segments from almost every V{alpha} subfamily in {gamma}{delta} thymocytes (14). It has remained unclear to date whether just a particular subset of V{alpha} gene segments from each subfamily was suited for {delta} rearrangement and expression in {gamma}{delta} receptors or whether in principle any V{alpha} gene segment was accessible for {delta} rearrangement.

In this study we have undertaken the first systematic V{delta} repertoire analysis throughout ontogeny. Sampling fetal thymocytes from days 14, 16, and 18 of gestation as well as thymuses from newborn and 4-wk-old mice, we have re-examined the thymic V{delta} expression waves at the transcriptional level. We used the technique of inverse PCR (iPCR), which permits the rapid amplification and identification of unknown V segments flanking the constant gene C{delta}. Moreover, this technique permits quantitative analysis of the V{delta} repertoire, because amplification of all V gene segments is performed with the same pair of specific primers. By contrast, using panels of V{delta} subfamily-specific primers that may differ in their amplification efficiency would result in biased representation of certain V gene segments. We were thus able to study the differentiation of the V{delta} repertoire during ontogeny and to define several novel waves of {delta}-chain transcription.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mice

Fetal thymuses were obtained from timed pregnancies, with day 0 being the day that a vaginal plug was observed. The newborn mice were used within 24 h of birth. Adult thymuses were obtained from 4-wk-old mice. Thymuses from day 16 of gestation and newborn and adult stages were prepared from BALB/c mice. Thymuses from day 14 of gestation were from C57BL/6 mice, and thymuses from day 18 of gestation were from (C57BL/6 x BALB/c)F1 mice. BALB/c mice were bred at the Max Planck Institute for Immunobiology (Freiburg, Germany). C57BL/6 mice were purchased from The Jackson Laboratory (Bar Harbor, ME). At all fetal stages analyzed as well as at birth, thymuses from several litters were pooled for isolation of RNA. At 4 wk of age, thymuses from two animals were pooled.

Inverse PCR

For amplification of flanking regions by iPCR, double-stranded cDNA is circularized such that the unknown V{delta}, D{delta}, and J{delta} gene segments at the 5' end of the molecule are ligated to the 3' end of the known C{delta} gene segment in a self-ligation step. Outwardly oriented C{delta}-specific primers (a 5' antisense and a 3' sense primer) were used to amplify around the circle from the C{delta} gene segment into the unknown flanking gene segments. The protocol of iPCR has been established for investigating TCR-{alpha}{beta} junctional diversity in human peripheral blood (15) and has been improved at certain steps to make up for the minimal amount of {gamma}{delta} message present in fetal murine thymus (16).

Briefly, RNA was extracted from thymocytes by the acid guanidinium thiocyanate phenol method (17). Five micrograms of total RNA was taken for oligo(dT)-primed double-stranded cDNA synthesis (Choice System, Life Technologies, Gaithersburg, MD). The cDNA was end-polished with 10 U of T4 DNA polymerase (Life Technologies) at 16°C for 5 min and extracted with phenol/CHCl3. DNA was ethanol-precipitated with 2.5 M NH4Ac. Circularization was performed in a total volume of 50 µl with 50 mM Tris-HCl, pH 7.5, 10 mM MgCl2, 1 mM DTT, 0.5 mM ATP, and 5 U of T4 DNA ligase (Life Technologies) overnight at room temperature. The whole cDNA was used to perform five iPCR reactions for each stage of development in parallel. PCR was performed in a total volume of 50 µl, containing 67 mM Tris-HCl (pH 8.8), 16.6 mM (NH4)2SO4, 0.1 mg/ml BSA, 2 mM MgCl2, 0.2 mM dNTP mix, and 0.5 µM of each of the following primers: DCG (antisense primer), 5'-CGA ATC TCC ATA CTG ACC-3' derived from C{delta} exon 1; and DCB (sense primer), 5'-TTA ATG CTC TCC AAG CAG-3' derived from C{delta} exon 4. The samples were overlaid with 30 µl mineral oil. Amplification took place in a Biometra Trio-Thermoblock under the following conditions: 5 min at 95°C, 85°C for addition of 2.5 U of AmpliTaq (Perkin-Elmer, Palo Alto, CA; hot start), 5 min at 95°C, 40 cycles of 1 min at 95°C, 1 min at 55°C, and 1 min at 72°C, followed by a final elongation step of 15 min at 72°C. Five iPCR samples per developmental stage were pooled, treated with 12.5 U of Klenow fragment (New England Biolabs, Beverley, MA), extracted with phenol/CHCl3, and ethanol precipitated with 2.5 M NH4Ac. After digestion twice with 40 U of HindIII (New England Biolabs), 1/10th of each pool was size-fractionated on an analytical 1% agarose gel (UltraPure Agarose, Life Technologies) and probed with the internal C{delta}-specific oligonucleotide GM11 (5'-CAT GAT GAA AAC AGA TGG-3'). The remaining pool was separated on a preparative 1% low melting agarose gel (SeaPlaque GTG; Biozym, Oldendorf, Germany), and the DNA was cut out. The one or two uppermost blocks were molten, extracted with phenol/CHCl3, and ethanol precipitated with 2.5 M NH4Ac.

Cloning and analysis of PCR products

Size-selected PCR products were ligated into HindIII-cut pBluescript II SK+ (Stratagene, La Jolla, CA). Transformed colonies (Escherichia coli strain XL2-Blue; Stratagene) were screened with the oligonucleotide probe GM11, and recombinant plasmid DNA from positive bacterial colonies were sequenced on an ABI automated sequencer with the Taq Dye-Deoxy Terminator Cycle sequencing kit (Applied Biosystems, Foster City, CA) using the C{delta}-specific oligonucleotide primer GM11.

Nomenclature

The designations used herein for TCR V genes follow the traditional numbering (3, 18) in compliance with the standard nomenclature (WHO-IUIS Nomenclature Subcommittee on TCR Designation) (19). DV101–DV105 designate V{delta} gene segments that lack similarity to V{alpha} gene segments. ADV refers to V{alpha} subfamilies that include homologous V{delta} gene segments. A/DV refers to germline V gene segments that are expressed in {alpha}- and/or {delta}-chain messages. GV1 and GV2 designate V{gamma} gene segments originally named V{gamma}3 and V{gamma}4 (1), or V{gamma}5 and V{gamma}6 (2), respectively. The numbering system for residues of the V segments was previously described (3).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Transcription of canonical DV101-D2-J2 rearrangements is preceded by heterogeneous DV101-D2-J1 rearrangements on day 14 and rapidly declines after day 16 of gestation

On day 14 of gestation, 27 C{delta}-containing cDNA clones were derived from transcripts of complete V-D-J rearrangements that exclusively contained the DV101 gene segment, and 22 (82%) of which were rearranged in-frame (Fig. 1Go). The majority, 16 of the productively rearranged clones, used the J{delta}1 gene segment, whereas J{delta}2 was found in only six clones. Half the J{delta}1-containing clones had the D{delta}2 and J{delta}1 gene segments joined in their germline configuration, mediated by a microhomology of 4 nt (AGCT) including two palindromic (P) nucleotides. These clones displayed a 7-nt homology (GATATCG; rather than 5 nt, as suggested in Ref. 20) at the V-to-D joint, with the germline 3' end of DV101 ending with ATC (3). This predominant rearrangement has previously been reported as the second most frequent rearrangement in s-IEL (8). The remaining J{delta}1-containing clones have heterogeneous rearrangements. Five of the six productive J{delta}2 rearrangements bear the canonical rearrangement that dominates in s-IEL (4, 8). It is mediated by a 3-nt homology between D{delta}2 and J{delta}2, resulting in deletion of the 3' half of the D{delta}2 gene segment and, at the V-D junction, by the same 7-nt homology described above. We found this canonical rearrangement in only 5 of 22 DV101 rearrangements (23%). This was unexpected, because it has been shown that the s-IEL precursors reside in the early fetal thymus (21).



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  		FIGURE 1. VDJ junctional sequences derived from TCR {delta}-chain transcripts from day 14 fetal thymocytes. Identical nucleotide sequences are shown only once; their frequency is indicated by individual clone numbers. They are derived from independent cDNA clones, because their sequences differ at the circular ligation site. Germline sequences are indicated at the top; the 3' end of the DV101 germline gene segment is as previously reported (3 ), resulting in a 7-nt homology with D{delta}2. Short regions of homology, possibly mediating homologous recombination, are underlined. Only in-frame sequences are shown, under which are presented deduced amino acid sequences in standard single-letter code. Only those residues are included that form the putative CDR3 that starts with the third residue after cystein 90 of the V segment and ends with the second residue before the Phe-Gly-X-Gly motif of the J segment.

 
In contrast, on day 16 of gestation, 10 of 27 in-frame DV101-containing clones (37%) carried the canonical J{delta}2 rearrangement (Fig. 2Go). This increase on day 16 indicates that the expression of this invariant {gamma}{delta} receptor is not the first event in thymic ontogeny. Rather, it is preceded by a frequent DV101-D-J{delta}1 rearrangement that is also present in the s-IEL population as well as heterogeneous J{delta}1 rearrangements. This canonical rearrangement persists on day 16 (5 of 14 productive rearrangements involving J{delta}1). On day 18, only 8 of 35 complete V-D-J in-frame rearrangements (23%) contained DV101. Of these, four had undergone the canonical rearrangement to J{delta}2, and two had undergone the canonical rearrangement to J{delta}1 (Table IGo; data not shown). Thus, the canonical DV101-D-J{delta}2 rearrangement increases to reach maximal levels only on day 16, but diminishes dramatically by day 18. In fact, it was undetectable in neonatal thymus, where none of the 64 V-containing clones analyzed carried DV101, casting doubt on the existence of a consecutive, late fetal wave of invariant receptors bearing DV101 paired with GV2.



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  		FIGURE 2. VDJ junctional sequences derived from TCR {delta}-chain transcripts from day 16 fetal thymocytes. The 3' ends of the V gene segments are in accordance with their germline configuration (See Footnote 5) (3 ). N nucleotides are centered, whereas P nucleotides are presented adjacent to the gene segment boundaries (see Fig. 1Go for other details).

 

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  		Table I. Time-dependent changes in the composition of the TCR {delta} repertoire1

 
Neonatal {delta}-chain transcripts use diverse, but distinct, V{alpha} gene segments from eight V{alpha} subfamilies

On day 16 of gestation, 39 of the 45 complete VDJ transcripts analyzed (87%) were derived from in-frame V-D-J rearrangements. About one-third of the productively rearranged transcripts used V gene segments other than DV101. Each three cDNA clones contained ADV7, the ADV17S3, and the DV104 gene segments. In addition, one cDNA clone each was found with a DV4S8-like gene segment, DV6S2, and DV105 (Fig. 2Go). By day 18, the majority (77%) of the productively rearranged (35 of 43, or 81%, of the V-D-J rearrangements were in-frame) cDNA clones had diverse non-DV101 V segments. Among these, several members of the ADV7 subfamily were found six times, ADV17S3 was found twice, and the DV104 gene segment was found 11 times (data not shown). In neonatal thymocytes, 43 of 64 V-bearing cDNA clones (67%) were rearranged in-frame. Among the productively rearranged cDNA clones, the ADV7 subfamily occurred most frequently (12 clones, or 28%). The members ADV7S1, ADV7S2, and DV7S4 each represented approximately one-third of this subfamily. Dominant expression of ADV7 was followed by DV104 (eight clones, or 19%) and ADV17S3 (five clones, or 12%; Fig. 3Go). These three subfamilies display a relatively high degree of sequence similarity (>=54% at the nucleotide level, whereas different V{alpha} subfamilies usually are <40% similar) (3). Together, these closely related subfamilies account for 59% of the productive V-D-J rearrangements. Thus, this homologous subset of subfamilies has increased from 23% on day 16 and 54% on day 18 to greater than half of all neonatal cDNA clones (Table IGo).



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  		FIGURE 3. VDJ junctions of {delta}-chain transcripts from thymuses of newborn mice (see Figs. 1Go and 2Go).

 
The other half used diverse V{alpha} gene segments from six different V{alpha} subfamilies, including the larger subfamilies comprising 5–10 different members. Unexpectedly, the neonatal {delta}-chain transcripts preferentially used a single gene segment of these multimembered subfamilies. Thus, all three (two productive and one nonproductive) {delta} rearrangements of members of the V{alpha}4 subfamily contain the same gene segment (Fig. 3Go). Moreover, the same sequence was identified in the two V{alpha}4-to-{delta} rearrangements from days 16 and 18 of gestation and in one nonproductive rearrangement from adult thymocytes. The V exon sequence of this novel V{alpha}4 gene segment differs from DV4S8 by 13 nt, and we therefore designate it DV4S8A2. A similarity search of the nucleotide sequence databases revealed that our cDNA does not differ by a single nucleotide from the corresponding V exon of the genomic sequence of the mouse V{alpha}/{delta} locus. In this genomic sequencing project5 Lee and co-workers used cosmid clones that were derived from mouse strain 129/Sv. BALB/c and 129/Sv share the a haplotype of the TCR V{alpha} locus (22). The absence of allelic polymorphism between these two mouse strains permitted us to unequivocally identify the germline counterparts of our cDNA clones. Thus, the DV4S8A2-matching germline gene segment, among all V{alpha}4 subfamily members, maps the most proximal to the {delta} locus.

Similarly, just one member of the V{alpha}10 subfamily, DV10S7, was used in four neonatal and three day 18 V{alpha}10-to-{delta} rearrangements, including two nonproductive rearrangements. The one exception was a gene segment identical with ADV10S6 (clone NB55; Fig. 3Go). The DV10S7 segments of two cDNA clones (1839 and 1840; data not shown) from day 18 fetal thymus, which was derived from (BALB/c x C57BL/6)F1 mice are identical with that of cDNA KN25-D4, previously isolated as {delta}-chain message from C57BL/6 mice (23) and therefore designated DV10S7 (Table IGo) (3). The third day 18 cDNA (clone 1841, out-of-frame) differs from the former two by four nucleotide exchanges in the V segment. The latter bears a V segment identical with those of the four cDNAs isolated from BALB/c neonates. We therefore attribute these minor differences to strain polymorphism and tentatively designate the BALB/c allele DV10S7A2. In fact, the BALB/c-derived DV10S7A2 segment showed a perfect match with one V{alpha}10 germline gene segment from the genomic sequence of the V{alpha}/{delta} locus (see Footnote 5). Again, the matching member of the V{alpha}10 subfamily is located most proximal to the {delta} locus.

Among six neonatal V{alpha}2-to-{delta} rearrangements, including one nonproductive rearrangement (clone NB50; not shown), the ADV2S6 gene segment was used three times (Fig. 3Go and Table IGo). The dominance of ADV2S6 was further quantified by V{alpha}2 subfamily PCR using a V-specific primer conserved in all members of the V{alpha}2 subfamily and an antisense primer specific for C{delta}. The result was that 15 of 19 productive transcripts (79%) contained the ADV2S6 gene segment. The second most frequently used segment, which is identical with V{alpha}2.2 (24), was found in only two instances. Only one clone had an out-of-frame joint, also using ADV2S6 (data not shown). A database search identified ADV2S6 as the C{delta}-proximal V{alpha}2 subfamily member in the genomic sequence of the {alpha}/{delta} locus (see Footnote 5). This striking result raises the question of whether particular gene segments of this subfamily are targeted for {delta} rearrangement and others for {alpha} rearrangement. The V{alpha}2 subfamily PCR was, therefore, also performed with a C{alpha}-specific primer. The ADV2S6 gene segment was equally prevalent in {alpha}-chain message from neonatal thymocytes. Nine of fourteen in-frame rearrangements (64%) had joined ADV2S6 to diverse J{alpha} gene segments, followed by only three V{alpha}2.2 rearrangements (data not shown). These results demonstrate that the same V gene segment can be used for {alpha} and {delta} rearrangements. More importantly, the 3'-most gene segment of the larger V{alpha} subfamilies is preferentially used in neonatal thymocytes.

The usage of the smaller V{alpha} subfamilies containing a single or just two members further substantiates the bias for proximal V{alpha} in neonatal thymus. V{alpha}6 was found five times, four of five clones in-frame, each in neonatal (Fig. 3Go) and day 18 fetal thymus. BALB/c neonates exclusively expressed DV6S2, the first V{alpha} upstream of the classical V{delta} gene segments (see Footnote 5). The two members of the V{alpha}9 subfamily map to the distal and proximal ends of the {alpha} locus (13). Our cDNA from neonatal thymus differs from AV9S2 (clone HY-A1) (3) by 4 nt. We, therefore, designate it DV9S2A2. It matches the AV9 gene segment from the proximal end with the exception of 1 nt (see Footnote 5; Table IGo). This is the one exception where we did not find a perfect match. Finally, we found a new member or allele of the AV18 subfamily, which we named DV18S3 (out-of-frame clone NB60; data not shown). It is identical with the most proximal germline gene segment of the AV18 subfamily (see Footnote 5). Thus, almost all the classical V{alpha} gene segments that are located near the 3' end were identified in the neonatal {delta} repertoire.

Only transcripts from three closely related subfamilies persist in the adult

In the adult, among 50 V-bearing cDNA clones 35 (70%) were rearranged in-frame (Fig. 4Go). There was a striking dominance of the ADV7 subfamily, which was found in 14 (40%) of the productively rearranged transcripts. Whereas the second wave of diverse neonatal V{alpha}-to-{delta} rearrangements is totally diminished in 4-wk-old thymus, the contribution of productive ADV7 transcripts steadily increases from 8% (3 of 39) on day 16 of gestation to 17% on day 18 (6 of 35), 28% (12 of 43) at birth, and 40% (14 of 35) in 4-wk-old thymocytes (Table IGo). On day 16, DV7S6 was found twice. It most likely represents the BALB/c allele of ADV7S1, and we refer to it as DV7S1A2. Another member DV7S4 was found in one clone. On day 18, DV7S1A2 was found once, ADV7S3 was found three times, and DV7S4 was found once. ADV7S3 was exclusively found in day 18 fetal (BALB/c x C57BL/6)F1 mice, suggesting that it may represent the C57BL/6 allele of DV7S4. In neonates, we found four DV7S1A2, three ADV7S2, three DV7S4, and one DV7S5 clone. Based on sequence similarity, the ADV7 subfamily can be subdivided into two subsets, ADV7S1/2 and ADV7S3/4/5, with mean nucleotide sequence similarities of 95% within and 80% between subsets. Including nonproductive rearrangements, the subfamily members ADV7S1/2 and ADV7S3/4/5 were represented with equal frequency, whereas among the in-frame transcripts the S1/2 to S3/4/5 ratio was 7:5. By contrast, in day 18 fetal (BALB/c x C57BL/6)F1 thymocytes the in-frame transcript S1/2 to S3/4/5 ratio was 1:5 (Table IGo). Thus, there is selective expression of different subsets in the two mouse strains. All members of the ADV7 subfamily are used, in contrast to the preferential usage of members from the proximal cluster in the neonatal wave of diverse V{alpha} subfamilies. Moreover, the overexpressed ADV7S1/2 gene segments map to the more distal V{alpha} clusters (12), whereas DV7S4 is identical in nucleotide sequence to the most proximal member in the genomic sequence of the {alpha}/{delta} locus (see Footnote 5). The ADV7 subfamily is thus unique in that even at early stages in ontogeny its proximal member is not preferentially used.



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  		FIGURE 4. VDJ junctions of {delta}-chain transcripts from thymuses of 4-wk-old mice (see Figs. 1Go and 2Go).

 
The two subfamilies, DV104 and ADV17, which share a high degree of similarity with ADV7, closely follow the expression pattern of the ADV7 subfamily. On day 16, each of these starts with a frequency of 8% (3 of 39 productive transcripts). In neonatal thymus DV104 has increased to 19% (8 of 43), reaching 23% (8 of 35) in the adult thymus. Of interest, in (BALB/c x C57BL/6)F1 mice, DV104 has already reached 31% (11 of 35) by day 18. Thus, whereas the ADV7 subfamily dominates in BALB/c mice, DV104 appears to be overselected in C57BL/6 mice. Using a V region-specific mAb to DV104, variation in the relative levels of {gamma}{delta} splenocytes expressing DV104 was reported for these strains of mice (25). Selection of DV104+ cells appears to be tissue specific and is linked to the particular GV/DV104 pair expressed at the surface of these cells (26). In neonatal thymus we found two productive transcripts of a hitherto undescribed second functional member of the DV104 subfamily, which we refer to as DV104S2. It is located 10 kb upstream of the DV104S1 gene segment (see Footnote 5). ADV17 is the least frequent of the three homologous subfamilies. The exclusively used ADV17S3 segment amounts to 6% of the total productive rearrangements (2 of 35) on day 18, 12% (5 of 43) in newborn, and 9% (3 of 35) in 4-wk-old thymus. It may be worth noting that we found one pseudogene, DVX, transcribed at significant rates. Its 3' end shares 68% identity with the ADV7S1 gene segment. DVX extends the sequence of the previously described cDNA Z78 (10). The major portion of the V gene segment, including Cys22, appears to be deleted, but its leader exon and 5'-flanking region are conserved. It shows an expression pattern similar to that of the clan subfamilies. It cannot be excluded that minor differences in gene expression levels or RNA stability will lead to pattern distortions. Therefore, eventual confirmation by cell surface staining will be required to confirm the observed pattern of TCR {delta}-chain transcription.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
First wave: canonical DV101-D2-J2 rearrangements are transcribed with a single maximum on day 16 of gestation

The first fetal thymic wave expresses GV1 paired with a {delta}-chain composed of DV101, D{delta}2, and J{delta}2 segments. Both chains are encoded by canonical rearrangements. Using an mAb directed to an epitope on the GV1 gene product, it has been shown that the total number of GV1+ cells peaks on day 16, and the cells have disappeared by day 18. By subtraction of this population from the total number of CD3+ cells, a second wave of {gamma}{delta} T cells using V{gamma} segments other than GV1 with a maximum on day 18 became apparent (27). However, the lack of GV2-specific Abs prevents a direct comparison between the GV1 and GV2 waves (28). Our results revealed that the transcription of the canonical DV101-D{delta}2-J{delta}2 rearrangement is confined to a narrow time window between days 14 and 18 of gestation, with a maximum on day 16 (Fig. 5Go). Thus, there is a good concordance between the extent of canonical DV101 rearrangement and the abundance of GV1+ cells. Unexpectedly, we did not observe a late fetal wave of DV101 expression that could provide the {delta}-chain for pairing with GV2. We cannot rule out the possibility that the changes in the DV101 expression level observed by us during fetal development might reflect strain-dependent differences. Thus, strain-specific developmental changes in the thymic environment may play a role in shaping the GV1/DV101 TCR repertoire (29). Yet, our data are in good agreement with more recent quantitative PCR analyses of genomic DNA rearrangements. GV1 and GV2 rearrangements were shown to be relatively infrequent on day 14, and their abundance increases about 20-fold by day 15. Both GV1 and GV2 rearrangements decrease by day 18, whereas GV3 rearrangements increase to reach a maximum on day 18 (30). Moreover, this study shows that the relative frequencies of the different V{gamma} rearrangements correspond reasonably well with the frequencies of V{gamma}+ cells at the different time points. Thus, our observation of a decline in canonical DV101 expression on day 18 is corroborated by the reported coincident decline in GV1 and GV2 rearrangements. It has also been demonstrated by others that all GV2+, but none of the GV2-, hybridomas carried GV1 rearrangements (31), indicating that GV1+ and GV2+ T cells belong to a common, distinct lineage that does not give rise to other {gamma}{delta} T cells. Most of the GV2+ cells carried out-of-frame GV1 rearrangements and, thus, appear to have a second chance to rearrange GV1 or GV2 on the other allele. These data are consistent with a major first wave consisting of two subsets expressing GV1 or GV2 and the same DV101 rearrangement. The onset of canonical DV101-D-J{delta}2 rearrangement is on day 14. Yet, it is outnumbered by heterogeneous rearrangements to J{delta}1 with one canonical DV101-D-J{delta}1 rearrangement predominating (Fig. 5Go). Thus, the canonical DV101-D-J{delta}2 rearrangement is not the first event in thymic ontogeny. In comparison, the canonical GV1 rearrangement amounts to 81% of the productive GV1 rearrangements on day 14 (32). Similarly, 87% of the productive GV2 rearrangements are of the canonical type (32), suggesting that the onset of canonical GV2 expression occurs as early as that of GV1. Taken together, we conclude that the previously postulated two consecutive waves of canonical GV1 and GV2 expression completely overlap and coincide with the short wave of canonical DV101 expression. This suggests that on day 16 of gestation the thymus harbors the maximal number of precursors of both s-IEL and r-IEL.



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  		FIGURE 5. Schematic diagram of the three thymic waves of {delta}-chain transcription during ontogeny. ----, Early fetal wave of DV101 expression; ——, neonatal wave of diverse V{alpha} expression; -·-, third wave of limited V gene usage, increasing until 4 wk of age.

 
Second wave: preferential usage of the proximal cluster of duplicated V{alpha} gene segments in neonatal {gamma}{delta} thymocytes

The genomic organization of the {alpha}/{delta} locus with the {delta} locus nested between the V{alpha} and J{alpha} gene segments raises the question of whether the V{alpha} repertoire is completely or partially accessible for functional {delta} rearrangement and, consequently, expression in {gamma}{delta} receptors. We have shown for three large V{alpha} subfamilies that just a single member of each subfamily preferentially undergoes {delta} rearrangement in neonatal thymocytes. Previously, occasional V{alpha}-to-{delta} rearrangements have been described (for review, see Ref. 3). The prevalent V{alpha} gene segments of our study precisely match those described in the literature. Thus, the exclusively used DV4S8A2 gene segment differs from DV4S8 by only five amino acid residues (3). This limited extent of variation may be ac- counted for by allelic polymorphism between the BALB/c mouse strain used in this analysis and the C57BL/6 strain from which the CD4-CD8- {gamma}{delta} thymocyte hybridoma expressing DV4S8 was derived (33). Similarly, DV10S7A2 from BALB/c neonates in this study differs by four amino acid replacements from DV10S7 (3), previously isolated from a CD4-CD8- {gamma}{delta} thymocyte hybridoma from C57BL/6 mice. Moreover, the occurrence of both forms in fetal thymus on day 18 of gestation in (BALB/c x C57BL/6)F1 mice further argues for allelic variation and, therefore, prevalence of one V{alpha}10 gene segment in seven of eight clones (Table IGo). Finally, the predominantly used V{alpha}2 gene segment is identical with AV2S6 previously isolated from BALB/c mice (3). It differs from DV2S8 by four residues, indicating that these may represent allelic counterparts rather than two different subfamily members. DV2S8 was previously found to be expressed in {gamma}{delta} thymocyte hybridomas from C57BL/6 neonates (34). Taken together, if one takes into account allelic variation, our systematic analysis combined with occasional previous findings reveals that just a single V gene segment from a given V{alpha} subfamily is used in {gamma}{delta} receptors.

There are several possibilities to explain the observed bias in V{alpha} usage in {gamma}{delta} thymocytes. Regulatory sequences may dictate {alpha}-ness vs. {delta}-ness of individual subfamily members. Comparative analysis of the 5'- and 3'-flanking sequences of the highly homologous V{alpha}2 subfamily genes, however, did not reveal any significant differences between ADV2S6 and its counterparts (24) (ADV2S6 is identical with the segment designated Tcra V2.6 herein). Therefore, differential activation of promoter regions or targeting of recombination signal sequences in the vicinity of the coding sequence can be excluded. This possibility was unlikely, given the fact that the same V gene segment, ADV2S6, undergoes {alpha} and {delta} rearrangements with equal relative frequencies. Instead, positive thymic selection may choose certain specificities of {gamma}{delta} receptors encoded by particular V{alpha} subfamily members. The occurrence of the same V{alpha} gene segments in nonproductive rearrangements, however, argues against positive selection being solely responsible for overexpression. Based on our comprehensive sequence analysis, we, rather, conclude that proximity determines V{alpha} rearrangement to genes in the {delta} locus. A similar mechanism is thought to control the transcription of the globin genes that are closest to the locus control region early in development (35). Comparison of our {delta}-chain clones expressed in BALB/c mice with their genomic counterparts (see Footnote 5) in all instances revealed a perfect match, with only one exception. The absence of polymorphic nucleotide substitutions permitted us to identify their genomic location. Adjacent to the classical V{delta} gene segments DV101, 102, 104, and 105 that appear to be exclusively associated with C{delta} is located the {delta}-proximal V{alpha} cluster, one of multiple duplicated clusters, each containing intermingled single representatives of different V{alpha} subfamilies. Among the 10 furthest proximal V{alpha} gene segments, pseudogenes not included, seven are represented in the neonatal thymic {delta} repertoire: DV6S2, DV9S2A2, ADV2S6, DV10S7A2, ADV17S3, DV18S3, and DV4S8A2, from 3' to 5' (ADV designating V segments that undergo {alpha} and {delta} rearrangement; Fig. 6Go). ADV11S5 was not identified in this study, but has previously been isolated from a cytolytic CD4-CD8- {gamma}{delta} T cell clone from peripheral lymph nodes of BALB/c nu/nu mice (36). We thus observed an almost perfect coincidence of V{alpha} subfamily members mapping to the proximal cluster and their expression in newborn thymus {delta}-chain message. By contrast, if one compares V{alpha} usage in TCR-{alpha}{beta} repertoire development in human thymus and hemopoietic organs from 14- and 15-wk-old fetuses (37) with their genomic location in the human V{alpha}/{delta} locus (see Footnote 6), V{alpha} gene segments from the 5' half of the V{alpha}/{delta} locus appear to be preferentially used, in particular all V{alpha} gene segments from the distal end. Together, this may reflect a bidirectional readout mechanism, from the 5' end for {alpha}-chain rearrangements and from the 3' end for {delta}-chain rearrangements, at distinct stages in ontogeny.



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  		FIGURE 6. Schematic representation of the {delta}-proximal cluster of V{alpha} gene segments. The distances between the V gene segments are not drawn to scale. Their genomic order is revealed by sequence analysis of the {alpha} locus (see Footnote 5). Filled boxes represent V genes that were found to be expressed in the neonatal {delta}-chain repertoire; open boxes indicate V genes that were not identified in the present repertoire study. A/DV denotes V gene usage in {alpha}-chain and/or {delta}-chain messages.

 
The functional significance of this shared repertoire of V genes is not known. The mouse {alpha} locus has evolved through several rounds of duplication, such that the proximal duplication unit includes a subset of genes representative of most subfamilies. Preferential readout of the proximal cluster may thus permit the neonate, despite its limiting number of T cells, to build up a diverse spectrum of specificities against a broad range of pathogens. This would require extensive cross-reactivity to fill the holes in the repertoire. In fact, the mean CDR3 length of the {delta} rearrangements from the proximal V{alpha} cluster is nine amino acids, three residues shorter than that of the other neonatal {delta} rearrangements (Fig. 7Go; see below), which could reflect a low affinity repertoire of this subset. Increased promiscuity for antigenic peptides has been demonstrated in the neonatal {alpha}{beta} T cell repertoire (38). Amplification of {alpha}-chain transcripts from neonatal thymus with a V{alpha}2 subfamily-specific primer also yielded a mean CDR3 loop length of nine amino acids (data not shown). Recently, crystal structure analysis of a {delta}-chain with a CDR3 length of 10 amino acids revealed that in terms of its relative position in the TCR combining site, its CDR3 resembles that of V{alpha} and forms a flat binding surface, as observed in {alpha}{beta} TCRs (39). We have previously provided evidence that the neonatal {delta}-chain CDR3 regions, displaying limited junctional diversity, may be more flexible, changing conformation to associate with various Ags (40).



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  		FIGURE 7. CDR3 length distribution of translated transcripts from neonatal thymus. The CDR3 lengths of {delta}-chains containing V genes from the proximal V{alpha} cluster (gray bars) are compared with those containing V genes from the clan subfamilies ADV7, ADV17, and DV104 (filled bars). The mean length of {delta}-chains with the proximal V{alpha} sequences (9 aa residues) was significantly shorter than that of {delta}-chains with the clan V sequences (12 residues). Bars represent the frequency of clones with a defined CDR3 length. The CDR3 loop length corresponds to the length of the amino acid sequences displayed in Fig. 3Go and is defined in Fig. 1Go.

 
Third wave: a clan of three homologous subfamilies dominates in the adult thymus

The neonatal wave of diverse V{alpha}-to-{delta} rearrangements has totally vanished in 4-wk-old thymocytes (Fig. 5Go). Just a few V gene segments dominate in the adult thymus. Thus, the frequency of productively rearranged ADV7 transcripts increases continuously from day 16 of gestation, reaching a plateau of 40% at 4 wk of age (Fig. 5Go). In-frame rearrangements of the subfamily members of ADV7S1/2 outnumber those of ADV7S3/4/5 (see Results). We have previously demonstrated that positive selection plays a role in the overexpression of the ADV7S1/2 subset (40). The DV7S4 gene segment is located most proximal of all ADV7 subfamily members (see Footnote 5; Fig. 6Go). Its location in the proximal V{alpha} cluster should favor preferential utilization in the {delta} repertoire and is thus in striking contrast with the observed counterselection. The other two members, ADV7S1 and ADV7S2, are expressed with equal frequency. They are interspersed with different subfamilies throughout the {alpha} locus (12). Thus, neonatal expression of the ADV7 subfamily members does not show the position-dependent bias of the other V{alpha} subfamilies. A similar transcription pattern of the ADV7-like pseudogene DVX indicates that preferential expression of the clan subfamilies may be regulated at the DNA level before selection at the protein level. More studies need to be performed regarding the role of their flanking sequences in promoting preferential {delta} rearrangement. Biased expression of IgH J-proximal V genes has been demonstrated in neonatal mice (41), although the mouse model is controversial (42, 43). Interestingly, not all human VH segments preferentially expressed during early stages of ontogeny map in the proximity of the JH segments (44). A defect specific to the rearrangement of the JH-distal VH gene segments has been described in mice that lack the {alpha}-chain of the IL-7R (45). The defect correlates with reduced expression of Pax-5, a transcription factor that is required for V-to-DJH recombination in B cells (46). Furthermore, differential targeting of TCR V{gamma} genes for rearrangement has recently been shown to be controlled by sequences immediately upstream of the V{gamma} gene segments (47). The three subfamilies, ADV7, DV104, and ADV17, display high sequence similarity with one another (>54%) and clearly represent a distinct subset of V{alpha}/{delta} subfamilies. Expression of this clan of homologous subfamilies increases from 23% on day 16 of gestation to 59% in neonatal and 72% in adult thymus (Fig. 5Go).

A comparison of the mouse and human V{alpha}/{delta} subfamilies reveals a number of human homologues of the mouse clan subfamilies (hDV101, hADV6, hAV12, and hADV14) (48). The human clan counterparts are equally scattered across the V{alpha} locus (see Footnote 6). All these are expressed in {gamma}{delta} thymocytes and in {gamma}{delta} cells of the intestinal mucosa, with prevalence of hDV101 (49, 50). The CDR1 and CDR2 lengths of the clan subfamilies are each increased by one residue, on the average, compared with the other V{alpha} subfamilies. An extension of the 3' ends of the clan germline V gene segments, encoding two additional residues, results in increased CDR3 length. In neonatal thymus, the clan members have an average CDR3 length of 12 residues as opposed to nine residues for the {delta} rearrangements from the proximal V{alpha} cluster (Fig. 7Go). PCR with primers specific for the ADV2 and ADV7 subfamilies, which dominate the proximal V{alpha} and clan repertoires, respectively, also yielded mean CDR3 lengths of 9 and 12 residues, respectively (data not shown). This is in part due to usage of the D{delta}1 gene segment, while the neonatal {delta} rearrangements from the proximal V{alpha} cluster lack D{delta}1 and also to the presence of N-nucleotides and their virtual absence in the V{alpha}-to-{delta} rearrangements. The shorter CDR3 of the V{alpha}-to-{delta} rearrangements may establish a low affinity repertoire useful for a first defense, whereas the longer clan CDR3 may provide a more specific response against common pathogens. Within the highly variable CDR2 of most V{alpha} subfamilies, there is a functionally conserved motif of residues with alternating charge, called the KEK motif (51). It has been proposed that the positive net charge of the KEK motif, which is located on the lateral surface of the V{alpha} domain, may be responsible for binding to the CD8 coreceptor (51). The motif is absent in all clan V gene segments, with the exception of ADV7S1 (3). Instead, in the clan subfamilies ADV17 and DV104 (as in their human homologues hADV14 and hADV6) (18) this putative protein-protein interaction motif is replaced by a site (NXT) for potential N-linked glycosylation. A carbohydrate moiety would prevent interaction with the CD8 coreceptor. It is, therefore, conceivable that usage of the clan subfamilies defines a lineage of {gamma}{delta} T cells that lack CD8 coreceptors. Of note, {gamma}{delta} i-IEL expressing the human clan homologue DV101 were recently shown to recognize the MHC class I-related molecules MICA and MICB, which lack a CD8 binding site (52). We are currently investigating whether usage of the clan and the proximal V{alpha} subfamilies correlates with the absence or the presence, respectively, of the CD8 coreceptor in {gamma}{delta} thymocytes as well as in {gamma}{delta} i-IEL.


   Acknowledgments
 
We thank Martin Selbert for his invaluable help with automated sequencing, and Constanze Taylor for help with the manuscript. We also thank Profs. D. Petzoldt and H. Näher (Department of Dermatology, University of Heidelberg) for their support.


   Footnotes
 
1 Current address: Department of Bio- and Chemoinformatics, Merck KGaA, Darmstadt, Germany. Back

2 Current address: Institute for Immunology, University of Kiel, Kiel, Germany. Back

3 Address correspondence and reprint requests to Dr. Bernhard Arden at the current address, Department of Dermatology, Universitätsklinikum, Voss Strasse 2, D-69115 Heidelberg, Germany. Back

4 Abbreviations used in this paper: i-IEL, r-IEL, and s-IEL, intraepithelial lymphocytes of the intestine, female reproductive organs, and skin, respectively; h, human; iPCR, inverse PCR; C{delta}, TCR-{delta} constant gene; CDR, complementarity-determining region; N, N-region nucleotides; P, palindromic. Back

5 I. Y. Lee, K. Wang, A. F. Smit, J. Yu, G. K.-S. Wong, S. P. Iadonato, C. L. Magness, P. Green, M. V. Olson, and L. Hood. Submitted (January 14, 1998) to the EMBL/GenBank/DDBJ databases under accession numbers AC003995–AC003997, AC004096, AC004102, and AC005938. Back

6 C. Boysen, I. Lee, T. M. Smith, A. Smit, K. Wang, L. Rowen, and L. Hood. Submitted (July 20, 1997) to the EMBL/GenBank/DDBJ databases under accession numbers AE000658–AE000661. Back

Received for publication July 20, 1999. Accepted for publication October 26, 1999.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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