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Diversity and organization of human T cell receptor delta variable gene segments

Previous studies of the human TCR-delta gene identified a single commonly used V delta segment, denoted V delta 1. To better understand the extent of the human TCR-delta V gene repertoire, TCR-delta transcripts and gene rearrangements were examined in a new panel of cloned human TCR-gamma/delta lymphocytes. Through this analysis we identified and determined the structures of two new V delta segments, denoted V delta 2 and V delta 3. These V delta segments are different from previously characterized V alpha segments, supporting the notion that the human V delta and V alpha repertoires are distinct. Examination of V gamma gene segment usage in these cells reveals that the V delta 2 gene segment is used in conjunction with the V gamma 2 gene segment. Blot hybridization indicates that the V delta 2 gene segment lies between V delta 1 and D delta-J delta-C delta, and within 100 kb of the latter. Analysis of genomic clones indicates that the V delta 3 gene segment lies in an inverted orientation, approximately 2 kb 3' of C delta. This implies that rearrangement of V delta 3 to D delta-J delta-C delta occurs by inversion. Together with previous mapping studies, these results indicate that human V delta segments are dispersed, rather than clustered, within the TCR-alpha/delta locus. The analysis of rearrangements in polyclonal thymocyte DNA suggests that there may be a limited number of additional V delta gene segments yet to be characterized.

as nine murine Vs gene segments have now been identified (22) . Two of these are nearly identical to previously characterized Va gene segments, indicating the possibility that some Vs segments may function as Va segments as well .
To better understand the diversity of human TCRS gene segments and the relationship between the TCR-y/8 and TCR-a/p repertoires, we have examined TCR-S rearrangements in a new panel of cloned TCRy/S lymphocytes. Through this analysis we have identified and determined the structures of two new Vs segments, designated Vs2 and Vs3. These segments are distinct from known Va segments, and are widely separated within the TCR-a/8 locus both from Vs1 and from each other.

Materials and Methods
TCR-y/6 Cell Lines. The derivation and characteristics of peripheral blood-derived cell line IDP2 (4) and leukemic cell line Molt-13 (17,27) have been described previously. The cloned T cell line WM-14 was derived from umbilical cord blood cells (28) . The T cell clones LB117, LB207, LB210, LB213, and LB220 were obtained by stimulating the PBL from a healthy donor with Burkitt's lymphoma cells, followed by cloning using limiting dilution and feeder cell mixtures as described previously (29) . The derivation and properties of T cell clone F7 will be,described in a subsequent publication. The T cell clones were maintained in Yssel's medium and were expanded in IL-2 by weekly stimulation with feeder cells (29) .
Preparation and Blot Hybridization Analysis of RNA and DNA Samples. Total cellular RNA and high molecular weight genomic DNA were prepared from a single frozen pellet of N5 x 10' cells in each instance. Cells were lysed for RNA preparation by the urea-lithium chloride method (32) . 90% of the lysate was sheared and used for RNA preparation, whereas 10% of the lysate was diluted into 15 vol of 150 mM NaCl ; 10 mM NaEDTA ; 10 mM Tris HCl (pH 8.0); 0.4°Jo SDS; 100 ug/ml proteinase K, and was used for the preparation of genomic DNA (33) . Yields averaged 85 gg RNA and 50 ug DNA.
RNA was subjected to electrophoresis through 1 .5% agarose gels containing 2.2 M formaldehyde (34), and was then blotted onto and UV crosslinked to Hybond-N membranes (Amersham Corp., Arlington Heights, IL), according to the manufacturer's instructions . Genomic DNA was digested with the appropriate restriction enzyme, subjected to electrophoresis through 0.7% agarose gels, and was similarly blotted onto and UV crosslinked to Hybond-N membranes . Filters were prehybridized at 50°C and hybridized at 42°C in 50°70 formamide; 4.8 x SSC; 5 x Denhardt's ; 0.5% SDS; 200 wg/ml boiled salmon sperm DNA; 50 mM Hepes (pH 7 .0). High stringency washes were in 0.1 x SSC; 0.1% SDS at 50°C for Northern blots and at 60°C for Southern blots. Blots were stripped before reprobing by incubation for 1-2 h at 68°C in the prehybridization solution .
cDNA Library Construction andAnalysis. cDNA libraries containing 2-3 x 105 recombinants were constructed from LB117 RNA, WM14 RNA, and a mixture of LB207, LB210, and F7 RNA. Poly(A)`RNA prepared from -80 ug total RNA was converted to dscDNA using AMV reverse transcriptase, klenow, mung bean nuclease, and Eco RI methylase. After ligation of Eco RI linkers, size-fractionated cDNA was cloned into the Eco RI site of the vector ),gt10 (35). After screening with the appropriate probes, the 5' Eco RI fragments (VJCS) of positive clones and subfragments thereof were subcloned into either Bluescript (Stratagene) or M13mp18, and nucleotide sequences were determined on both strands by the dideoxy chain termination method (36,37) using modified T7 polymerase (38) (Sequencee ; United States Biochemical Corp., Cleveland, OH).
Analysis of Genomic Clones . An 11-kb Bam HI-Kpn I fragment carrying the 3' portion of Vg3, and a 3-kb Kpn I-Bam HI fragment carrying the 5' portion ofVs3 were isolated from cosmid K7A (20) and subcloned into Bluescript. Sequences were determined by the dideoxy chain termination method using double-stranded templates and exonuclease III-generated deletions (39, 40) (Erase-a-base System ; Promega Biotec, Madison, WI) according to the manufacturer.

Results
Two Novel Human Vs Segments . In previous studies it was shown that the TCRy/S peripheral blood T cell lines IDP2, PBL C1, and PBL Ll, and the leukemic T cell lines PEER and Molt-13, all displayed functional rearrangements using the Vsl and Js 1 gene segments (16)(17)(18) . To identify TCRy/S cell lines using distinct Vs segments, RNA samples from a panel of seven TCRy/S T lymphocyte peripheral blood clones were examined by blot hybridization using Vs1 and Cs probes . As in RNA from the previously studied IDP2 cell line (16), a Cs probe revealed TCRS mRNA species indicative of differentially polyadenylated transcripts arising from fully rearranged genes (2 .2 and 1.3 kb) in all cell lines ( Fig. 1 A). In addition, this probe detected differentially polyadenylated transcripts arising from partially or unrearranged genes (1.7 and 0.8 kb) in some cell lines. However, aside from IDP2 RNA, only a single cell line (F7) revealed transcripts (2.2 and 1.3 kb) detected by a Vsl probe ( Fig. 1 B). The remaining cell lines (LB117, LB207, LB210, LB213, LB220, and WM14), which expressed full-length Vsltranscripts of 2 .2 and 1 .3 kb, were presumed to be using distinct Vs gene segments .
To determine whether the Vs1cell lines all used the same novel Vs segment, Xba I digests of genomic DNA samples were analyzed by blot hybridization using Js1 and Js3 probes (Fig. 2, A and B). The Js1 probe detects a single germline fragment of 1.7 kb in SB (B cell) and HL60 (myeloid cell), and two rearrangements in the TCRy/S cell lines IDP2 and Molt-13 . Ofthese rearrangements, the 6.4-kb fragment shared by IDP2 and Molt-13 represents rearrangement to Vsl (18). The 2 .9kb rearrangement in IDP2 is quite common, and is thought to represent either D-j or D-D J, whereas the 7 .2-kb rearrangement in Molt-13 has not been detected elsewhere, and is unidentified. As expected, the Vsl+ cell line F7 shares the 6.4-kb V81-J8l rearrangement with IDP2 and Molt-13 . On the other hand, the Vslcell lines display three distinct Js1 rearrangements, two of which (4.2 and 1.9 kb) had not been observed previously. These were presumed to reflect rearrangement to two novel Vs segments, designated Vs2 (4.2 kb) and Vs3 (1.9 kb) .
Based upon the above assignments, neither LB207 nor LB213 carries a functional Jsl rearrangement . We have previously described a distinct Js segment (Js2) that is homologous to the murine Js2 element and is located N10 kb 3' ofJ8 1 . Since it is now clear that an additional Js segment lies between these two J segments (19,23), it seems appropriate to rename the most 3' Js segment as Js3 . Our Js3 probe detects a 5.0-kb germline Xba I fragment that carries both Js2 and Js3, and therefore, should detect rearrangements to either of these Js segments . Notably, the Js3 probe detects a 3.4-kb rearrangement specific to LB207 and LB213, indicating the Blot hybridization analysis of TCR-s and TCR-y transcripts . 5-4g samples of total RNA was subjected to electrophoresis through 1 .5% agarose containing 2 .2 M formaldehyde, transferred to Hybond-N membranes, and serially probed with the indicated 32 P-labeled DNA fragments . likelihood of functional rearrangement to either Js2 orJs3 in these cell lines. Based upon the disposition of Xba I sites flanking Js l, Js2, and Js3, we predicted that in these instances Js3 had rearranged to the same V segment (Vs2) identified by the 4.2-kb Js1 rearrangement.
To confirm these interpretations and characterize the novel V segments, we generated cDNA libraries from LB117 and from WM14 poly(A)+ RNA. Using a Cs probe we identified cDNA clones representing the transcripts of the functionally rearranged TCRS gene in each cell line. The nucleotide and deduced amino acid sequences of these cDNA clones are presented in Fig. 3, A and B, and the deduced amino acid sequences for the V segments are compared both with each other and with that of V61 in Fig. 3 C. LB117 (Vs2) and WM14 (Vs3) clearly use Vs segments distinct from the Vs1 segment and from each other. Both cDNAs display potential contributions from the Ds1 and Ds2 elements as well as from N region nucleotides within the junctional region, both utilize the Js1 segment, and both maintain an open reading frame across the VD-DJ junction .
The Vsl, Vs2, and Vs3 gene segments are all distinct from previously reported Va segments, further supporting the notion that the human Va and Vs repertoires Samples of genomic DNA were digested with Xba I, subjected to electrophoresis through 0.7% agarose, transferred to Hybond-N membranes, and serially probed with the indicated 32 plabeled DNA fragments. The amount of DNA loaded was 5 pg in most instances, but was -2 .5 wg for LB213 and -1 ug for LB220. The data for LB220 in A derives from a longer exposure of the autoradiograph than the data for the remaining samples. SB and HL60 served as germline controls . A phage X Hind III digest served as molecular weight markers. are distinct . Furthermore, they display only low levels of amino acid sequence identity with each other, matching at 23-33°jo of the residues in various pairwise comparisons ( Fig . 3 C) . Nevertheless there are striking homologies with particular murine Vs segments (Fig . 3 C) . For example, human V81 matches a member of the murine Vs6 family at 58% of the residues, and human Vs3 matches murine V65 at 66% of the residues (22) . Such high levels of interspecies sequence conservation despite low levels of interspecies sequence conservation most probably indicate that these pairs of V segments have evolved from distinct ancestral V segments whose existence predates the divergence of mouse and man. Similar observations have been made for particular human and murine V5 (41) and Vy (42) gene segments .
Rearrangement and Expression of MR-6 and TCR-y Gene Segments. To further characterize the TCR-8 rearrangements in the remaining TCR-y/8 cell lines in the panel, Vs2-and V83-specific probes were generated and were used to probe both Northern blots (Fig. 1, C and D) and Southern blots (Fig . 4, A and B) . All Val -cell lines expressed either Vs2 or Vs3 transcripts, although one cell line, LB210, expressed both . The Vs2 gene segment was found on a 3 .7-kb germline Xba I fragment that was rearranged either to a 4.2-kb fragment (corresponding to rearrangement to Js l) or to a 3.4-kb fragment (corresponding to rearrangement to J82 or J83) . The V83 gene segment was found on a 9.4-kb germline Xba I fragment that was rearranged to a 1.9-kb fragment (corresponding to rearrangement to Jsl) in two cell lines. As  To determine which of the two rearrangements (Vs2 or Vs3 to J8 1) in LB210 was productive, and which of the Js segments (Js2 or Js3) was rearranged to Vs2 in LB207, a cDNA library generated from RNA of both cell lines was screened with a Vs2 cDNA probe, as well as withJ8 I-and J83-specific oligonucleotide probes . Two cDNA clones that used the Vs2 segment along with either Jsl (LB210) or Js3 (LB207) were characterized. Their nucleotide and deduced amino acid sequences are presented in Fig. 3 A . Both cDNA clones maintain an open reading frame across the VD-DJ junction, indicating that they represent the functional transcripts in the respective cell lines. Thus, in LB210, which displays both Vs2 and Vs3 rearrangements and transcripts, the Vs2 rearrangement is productive . Further, in LB207, and likely in LB213, productive rearrangements of Vs2 to Js3 have occurred . This is the first evidence for functional utilization of the human JO element.
We assessed Vy and Jy segment usage within this panel by analyzing Northern blots with Vyl family, Vy2, Vy3, and Vy4 probes (Fig. 1, E, F, G, and H, respectively) and Southern blots of Kpn I-digested genomic DNA with Jy1.3/2 .3 and Vy2 probes (Fig. 5, A and B, respectively ; nomenclature according to references 12 and 14). As shown previously by Huck and Lefranc (15), the linkage of human Jy segments predicts that a Jy1.3/2 .3 probe can detect all TCR-y gene rearrangements in Kpn I digests of human genomic DNA. Additional analysis using a Vy2 probe served to specifically identify Vy2 rearrangements, and further, to distinguish between rearrangements to Vy 1 and Vy3, since the germline Vy2 gene segment would be deleted upon rearrangement of Vy 1 gene segments, but retained upon rearrangement of Vy3.
All cell lines within the panel displayed two Jy rearrangements (Fig. 5 A) . In some cases Northern blots allowed an assessment ofwhich rearrangement was productive, since only one transcript was detected (Fig. 1, E, F, G, and H). Further, cells that had productively rearranged the Vy2 gene segment could be identified using the mAb anti TiyA, which specifically stains the surface of lymphocytes using this V segment (43). All cells that displayed a Vy2 to Jy rearrangement were found to be TiyA+ by surface fluorescence (data not shown) . As a result, unambiguous assignment of the productive rearrangement could be made in most instances.
A summary of the Vy and Vs transcripts and Jy and Js rearrangements detected in all of the clonal cell lines that we have examined so far is presented in Table I . All ofthe cell lines studied appear to productively rearrange one of the three characterized human Vs segments, and either a Vyl family member or Vy2 . A striking finding ofthis analysis was that all cell lines within the panel that display productive rearrangement of the Vs2 segment also display productive rearrangement of the Vy2 gene segment. This relationship is not reciprocal, in that the Vy2 gene segment is used in conjunction with either Vs1 or Vs2. The Vy2Vs2 cell lines within the panel use either Jy1.2 (JyP ; 12.0-kb rearrangement) or Jyl.3/2 .3 (7.5-kb rearrangement) ( Fig . 5 and Table I), and either Jsl or JO, indicating no specific restrictions on J segment utilization. Since the panel of cell lines examined is small, any conclusions must be tentative. However, these data may suggest that TCR-8 chains using the Vs2 gene segment may only be correctly assembled and expressed on the cell surface in association with TCR-y chains using the Vy2 gene segment. Alternatively, the data may reflect either positive selection for this particular receptor, or negative selection for receptors expressing Vs2 in conjunction with other Vy segments . Fur- FIGURE 5 . Blot hybridization analysis of TCR-y gene rearrangements . Samples of genomic DNA (5 ug) were digested with Kpn I, subjected to electrophoresis through 0.7% agarose, transferred to Hybond-N membranes, and serially probed with the indicated 32P-labeled DNA fragments. (A) TheJy 1.3/2 .3 probe detects both theJy l and Jy 2 clusters (germline fragments [GL] of 9 and 16 kb, respectively). The migration of Hind III fragments of phage ?, DNA are noted on the left border of the figure, whereas the predicted sizes (in kb) and assignments of rearrangements according to Huck and Lefranc (15) are shown on the right border. (B) The Vy2 probe detects two apparent germline fragments in Kpn I digests (GL) . The explanation for this is unclear, although the analysis of Xba I digests with this probe reveals that Molt-13, LB117, LB207, and WM14 retain no germline copies of Vy2, a result that correlates with the presence or absence of the larger germline Kpn I fragment (data not shown) . It is on this basis that distinction is made between Vy l and Vy3 rearrangements on the nonproductive chromosome (Table I) . The 11-kb fragment in F7 DNA correspond to neither of the Jy rearrangements in these cells. Whether this represents an unusual rearrangement or a polymorphism is unclear. ther studies will be necessary to determine the significance of the relationships observed in this panel, and to determine the mechanisms by which these relationships are maintained .
Additional Ja1 Rearrangements in Newborn Thymus DNA . Since there was no evidence for the utilization of additional Vs segments within this panel of cell lines, we sought to determine whether additional Js rearrangements could be detected in Xba I digests of newborn thymus DNA (Fig. 6) . A total of sevenJs l rearrangements Nomenclature used to denote TCR-y gene segments is that of references 12 and 14 . The assignments reflect the data obtained in this study as well as in references 17 and 18 . The assignment of rearrangements and transcripts as either (p) productive or (n) nonproductive results from a combination of blot hybridization data, cDNA sequences, and staining with mAb anti-Ti yA. Unambiguous assignment of the productive rearrangement could be made in each case, except for the TCR-y rearrangements in WM14, which are bracketed. Vy l family segments are denoted explicitly when the information is available from sequence analysis (e .g ., Vy l .8), or simply as Vy l when the assignment relies solely on hybridization data that does not distinguish Vy l family segments from one another. Transcripts detected at very low levels are in parentheses .  were detected in each of two DNA samples. Those of 6.4, 4.2, and 1.9 kb correspond to rearrangement to Val, Vs2, and Vs3, respectively, whereas that of 2 .9 kb corresponds to the common rearrangement (thought to be partial D-J or D-DJ) shared by many cell lines. Of the three remaining J81 rearrangements (8 .2, 3.4, and 2.4 kb ; Fig. 6, open triangles), one might reflect the second partial rearrangement (D -J or D-DJ) . Thus, the J8 1 probe may detect rearrangements to two or three as yet unidentified V segments . The low frequency of rearrangements detected by the Js3 probe precluded an analysis of rearrangements to the Js2 or Js3 gene segments in total thymocyte populations. However, these data suggest strongly that the number of frequently rearranged human Vs segments will not be large.
Dispersed Organization ofHuman Vs Segments within the TCR-aM Locus. Our initial studies of the genomic organization of the TCRa/S locus revealed that the V81 genomic segment was a large distance from the C6 locus, with one Va segment (Va 13 .1) immediately 5' of Val, and at least one Va segment (Va 17 .1) between Val and Ds-J6-C8 (20). These data indicated that Va and Vs segments were interspersed within the locus. To determine whether Vs segments were clustered or dispersed, we determined the locations of the newly identified Vs2 and Vs3 segments . As seen in Fig. 2 C, the F7 cell line, which has rearranged Vs l to Jal on both chromosomes (Fig . 2, A and B and reference 20), has deleted both copies of the Vs2 gene segment . This places the Vs2 gene segment in the region between V81 and the Cs locus . However, whereas previous studies have indicated that the Va 17 .1 gene segment maps by field inversion gel electrophoresis to the Sal I and Sfi I fragments carrying V81 (20), Vs2 maps to the Sfi I and Sal I fragments carrying D8-J8-C8 (data not shown) . Thus, Vs2 lies between Va 17 .1 and Ds Js-Cs, and must be within 100 kb 5' of the latter (Fig . 7) . It is intriguing that like F7, Molt-13 also has deleted both copies of the Vs2 gene segment . Molt-13 displays a productive Vs l to Js l rearrangement on one chromosome, and an unidentified Js 1 rearrangement on the other chromosome, with one FIGURE 7 . Dispersed genomic organization of Vg gene segments. Sfi I fragments of 190 and 180 kb carrying the Vsl and Cg segments are shown. The linkage between these fragments has notbeen established. The order of the gene segments mapping onto these fragments has been established in genomic Southern blotting experiments and by the mapping of cosmid clones (in the regions covered by the bold horizontal lines) . How ever, the precise distances between the segments mapping outside the regions covered by cosmid clones are uncertain. The nomenclature for V segments is according to Klein et al . (46). Since we have only attempted to map a limited number of Va segments, additional unidentified Va segments may also map to these fragments. A more detailed restriction map of two adjacent subcloned Barn HI-Kpn I fragments that carry the Jg3, Cg, and Vg3 segments is also presented. Transcriptional orientations are denoted by arrows . The orientation of the Jg3 and Vg3 segments with respect to mapped restriction sites has been directly established by nucleotide sequence analysis. The orientation of the Cg segment has been established elsewhere. copy of Vsl in the germline configuration (18) . This, together with field inversion gel electrophoresis mapping (data not shown), suggests that the nonproductive Js l rearrangement in Molt-13 involves an unidentified region mapping between Va17.1 and Vs2 .
Unlike Vs2, the Vs3 gene segment is not deleted upon rearrangement of V81 to J81 (Fig . 4 B), indicating that it does not lie between these gene segments . Further, Vs3 rearrangements to Js I were detected by a Cs probe (Fig . 4, B and C), indicating that Vs3 lies extremely close to Cs . The analysis of cosmid clones spanning the Cs region demonstrated that Vs3 lies in an inverted orientation, roughly 2-3 kb 3' of the Cs segment (Fig. 7). After subcloning and fine mapping of the Kpn I-Bam HI fragments spanning this region, the nucleotide sequence of the germline Vs3 segment was determined . This analysis revealed a structure typical of other TCR V segments, including separate exons encoding the leader peptide and the main body of the V segment, and heptamer and nonamer recombination signals flanking the 3' end of the second exon, thereby establishing its boundary (Fig. 8).
The location and orientation of the Vs3 segment relative to Cs is analogous to that described for murine Vp14 relative to Cp2 (44), and implies that rearrangement of Vs3 occurs by inversion of Ds Js-C8 . These data provide further support for the notion that a limited number of human Vs segments are dispersed within the human TCR-a/8 locus.

Discussion
The present study represents an extension of previous work carried out by ourselves and others detailing the diversity and organization of human TCR-8 gene segments . Previous studies have identified two Ds segments, three Js segments, and one Cs segment, nested within the TCR-a locus, and located just upstream of the Ja region (20,(23)(24)(25)(26)) . An estimated 50-100 Va segments are situated in the region 5' of the Ds, Js, Cs, Ja, and Ca segments (2,26). Despite the large number of V segments available for recombination, all productive TCR8 gene rearrangements in previously examined TCR-y/8 cell lines involve a single V segment, denoted V81 (16)(17)(18). This V segment is distinct from previously characterized Va segments, yet is situated among Va segments, at least 180 kb 5' of Jsl (20) . These observations Nucleotide and deduced amino acid sequence of the V83 genomic segment. Splice donor and acceptor sequences, as well as hepamer and nonamer recombination signals, are underlined . These sequence data have been submitted to the EMBL/GenBank Data Libraries under the accession number Y00792 .
suggested a limited repertoire of germline TCR-8 gene segments, and a highly controlled rearrangement process that is able to segregate TCR8 and TCR-a recombinational events and repertoires, despite the interspersion of TCR-8 and TCR-a gene segments .
Our analysis of a larger panel of TCR-y/8 cell lines has now allowed the identification of two additional Vs segments, termed Vs2 and Vs3. Like Vs1, these Vs segments are distinct from previously characterized Va segments . Blot hybridization experiments indicate that Vs2 lies within 100 kb 5' of the Cs region, and analysis of çosmid clones reveals that Vs3 lies ti2-3 kb 3' of Cs . These results clearly demonstrate that Vs segments are not present as a discrete cluster, but rather are dispersed within the locus. It follows that the segregation of Va and Vs rearrangements may be mediated by local signals marking particular V segments, rather than long range signals marking large segments of the locus. Such signals might include unique promoter or other elements that could help to modulate the accessibility of particular V segments to recombinases at a specific time in development (45), but probably do not include the heptamer and nonamer recombination signals flanking the V segments, since they are indistinguishable .
Of the three human Vs segments already characterized, two display quite high amino acid sequence identity with particular murine Vs segments . Identity is 58% in a comparison of human Vsl with a murine Vs6 family member, and 66% in a comparison of human Vs3 with murine Vs5. Conservation extends to genomic organization as well, since murine Vs5, like human Vs3, lies 3' to the Cs segment, in an inverted orientation (A . Korman, personal communication), whereas murine Vs6, like human Vsl, lies at some distance 5' to Cs . This conservation strengthens the emerging close relationship between the global organizations of the human and murine TCRa/8 loci.
The analysis ofJsl rearrangements in newborn thymus DNA supports the likelihood that there may be two or three additional Vs segments utilized within this polyclonal population that are not represented within the clonal cell lines that we have examined to date . Since the rearrangements in question occur at low frequencies within the thymus, their characterization probably awaits the identification of clonal cell lines displaying these rearrangements. It is also possible that distinct and novel Vs segments may be rearranged at a selective time in thymic development and/or in TCRy/8 cells populating distinct sites in the periphery and hence might not be represented in the polyclonal samples we have analyzed .
It is important to note that the vast majority ofJ81 rearrangements detected in newborn thymocyte DNA must occur in cells that do not express functional TCRy/S on their surfaces, since only 0.5-1 .0% of the thymocytes in these samples stained with the mAb antiTCR-S1 (V. Groh, personal communication) . Based upon the intensity of the rearrangements relative to control single copy examples in clonal cell lines, we assume that the fraction of thymocytes displaying J81 rearrangements is much higher. This would argue that the detection of a limited number ofJs1 rearrangements could not reflect selection for the expression offunctional receptors, but rather must reflect specificity in the rearrangement process per se.
Current information indicates that the TCR-y/8 V segment repertoires are both limited and of similar size in man and mouse. In man there are at most seven functional Vy segments (in four families), and at least three functional Vs segments . In mouse there are six functional Vy segments (in five families) and at least nine functional VS segments (in six families) (7,22) . These numbers are significantly lower than those estimated for TCRa/ß, which range from 30 to 100 for each V segment (2,3). Based upon our limited panel analysis, it may be inappropriate to assume combinatorial association of Vy and Vs segments, since five of five examples of TCR-y/8 cell lines using the Vs2 segment also used the Vy2 segment. Whether this might reflect constraints imposed on chain pairing or on receptor specificity is unclear, but it nevertheless suggests that the total number of VyVs pairs may be limited . Such data could be interpreted to indicate that TCR-y/8 might interact with a discrete set of putative restricting elements or antigen-presenting molecules . The resolution of this issue clearly awaits the derivation and study of antigen-specific TCRy/S T cell lines and clones .

Summary
Previous studies of the human TCR-S gene identified a single commonly used Vs segment, denoted Vsl . To better understand the extent of the human TCR-S V gene repertoire, TCR-S transcripts and gene rearrangements were examined in a new panel of cloned human TCR-y/8 lymphocytes . Through this analysis we identified and determined the structures of two new Vs segments, denoted Vs2 and Vs3 . These Vs segments are different from previously characterized Va segments, supporting the notion that the human Vs and Va repertoires are distinct . Examination of Vy gene segment usage in these cells reveals that the Vs2 gene segment is used in conjunction with the Vy2 gene segment. Blot hybridization indicates that the Vs2 gene segment lies between Vs1 and D8-J8-Cg, and within 100 kb of the latter. Analysis of genomic clones indicates that the Vs3 gene segment lies in an inverted orientation, ti2 kb 3' of Cs . This implies that rearrangement of Vs3 to Ds-Js-Cs occurs by inversion . Together with previous mapping studies, these results indicate that human Vs segments are dispersed, rather than clustered, within the TCRa/S locus . The analysis of rearrangements in polyclonal thymocyte DNA suggests that there may be a limited number of additional Vs gene segments yet to be characterized .