Immunological Reviews 1991, No. 120 Published by Munksgaard. Copenhagen. Denmark No pan may be reproduced by any process without written permission from the author(s)

Biology of the Human yd T-Cell Receptor STEVEN PORCELLI, MICHAEL B . BRENNER & HAMID BAND

INTRODUCTION The specificity of responses to foreign antigens is a hallmark of both humoral and cellular immunity in vertebrates. Although a complex interaction of many distinct cell types and a host of biologically active soluble mediators contribute to the inflammatory response, only two cell types interact in a highly specific manner with foreign antigens to initiate the immune response. B lymphocytes and T lymphocytes accomplish this through the use of highly diverse protein complexes on their cell surfaces which act as antigen receptors. After the identification of membrane-bound immunoglobulin as the B-cell receptor for antigen, it was several decades before a specific T-cell antigen receptor, the a^ TCR, was discovered (AlHson et al. 1982, Meuer et al. 1983, Haskins et al. 1983). This heterodimer was found to be associated with a complex of invariant chains called CD3 based on co-modulation (Meuer et al. 1983), co-immunoprecipitation (Borst et al. 1982) and by chemically cross-linking the subunits on the cell surface (Brenner et al. 1985, Allison & Lanier 1985). The genes encoding the a^ TCR reveal immunoglobulin-like sequences composed of separate variable (V), diversity (D) and joining (J) gene segments that are brought together into a contiguous gene during development (Yanagi et al. 1984, Hedrick et al. 1984, Chien et al. 1984, Saito et al. 1984a, Sim et al. 1984, Hannum et al. 1984, Fabbi et al. 1984). That the a^ TCR is the receptor carrying specificity for both foreign antigen and the major histocompatibility (MHC) self-restricting element was demonstrated by conferring this dual specificity to recipient cells by transfection of the genes (Dembic et al. 1986, Saito et al. 1987). IDENTIFICATION OF THE yS T-CELL RECEPTOR Following the cloning of the TCR^ gene, Tonegawa and colleagues (Saito et al. 1984b) isolated a distinct T cell-specific rearranging gene which at first was thought to encode the TCRo subunit. When the true TCRa gene was isolated Laboratory of Immunochemistry, Dana-Farber Cancer Institute, and Department of Rheumatology and Immunology, Harvard Medical School, Boston, MA. USA 02115.

138

PORCELLI ET AL.

(Chien et al. 1984, Saito et al. 1984a, Sim et al. 1984), it became clear that this other gene was a third T cell-specific rearranging gene and it was renamed TCRy. Subsequent studies revealed that many of the y genes were nonfunctional due to out-of-frame V-J joining (Helig et al. 1985, Rupp et al. 1986, Reilly et al. 1986, Iwamoto et al. 1986, Yoshikai et al. 1987) and transcripts were found in the thymus primarily during the early stages of development {Raulet et al. 1985, Snodgrass et al. 1985). Based on these features, it appeared that the rearranging y gene might be important only as a developmental rudiment and no specific function could be ascribed to it. In 1986, however, the y gene was demonstrated to encode a protein expressed on the surface of a distinct subset of T cells as part of a novel TCR heterodimer (Brenner et al. 1986, Weiss et al. 1986, Bank et al. 1986, Moingeon et al. 1986). This second TCR was identified when pan-reactive mAb against the a/9TCR, ^ Framework 1 (Brenner et al. 1987a) and WT31 (Spits et al. 1985), revealed the presence of CD3 ^ cells in human peripheral blood which lacked surface expression of the afi heterodimer (Brenner et al. 1986, Weiss et al. 1986). Further study of these cells showed that they also lacked transcripts capable of cross-hybridizing with TCRa and p cDNA probes, confirming that they could not express a/f Tcell receptors. Chemical cross-linking and immunoprecipitation techniques revealed the expression of two polypeptide chains in association with CD3 on the surface of these cells, one of which was demonstrated to be the product of the rearranging y gene (Brenner et al. 1986). The second CD3-associated radiolabeled species was thought to be another TCR subunit, and it was designated TCR^ (Brenner et al. 1986). Thus, this second receptor became known as the yS TCR. its identification led to a host of additional questions regarding the structure of the TCR^ gene, the specificity of this TCR and the function of the cells which bear it. Here, we summarize our current understanding of the biology of yS T cells in the htiman.

TCR y GENES The human y genes were identified by cross-hybridization with murine TCR y gene probes. The human y gene locus, on the short arm of chromosome 7 (Rabbitts et al. 1985, LeFranc & Rabbitts 1985, Murre et al. 1985), consists of a V gene cluster upstream of two J gene clusters each associated with a distinct C region gene segment (LeFranc & Rabbitts 1985, Murre et al. 1985, Quertermous et al. 1986b, 1986a, 1987, LeFranc et al. 1986c, 1986b, 1986a, Dialynas et al. 1986, Forster et al. 1987, Huck et al. 1988). Fourteen Vy genes have been described and their exact order in the genome is known (Fig. lA, please note alternative nomenclatures). Six of these appear to be pseudogenes, while the other 8 are potentially functional. Based on sequence comparisons, the most upstream nine Vy segments are highly homologous to each other and have been grouped as the

139

BIOLOGY OF yd TCR

1.1 I.E 1.3 1.4 1.5 •

^

«j

^

a

1.5 I.e a

\j

1.7 1.8 I

D

n

«

Jy

Cy

1.1 V2 1.3

C1

Oy

I V D 1 1

Figure lA. Genomic organization of the human TCR y locus on chromosome 7. The Iwo prevalent nomenclatures according to Seidman and colleagues (Strauss et at. 1987) (upper) or Lefranc and colleagues (Huck et al. 1988) (lower) are shown. Throughout this paper the first nomenclature is used. The TCR y locus contains 8 potentially functional V gene segments (filled boxes) upstream of two J clusters each with a Cy segment. Pseudogenes are shown as open boxes denoted by (i//). Adapted from (Huck et a!. 1988, Hochslenbach & Brenner 1990).

Vyl family (Vyl.1-1.8). In addition, Vy2-4 are three non-cross-hybridizing Vgene segments that lie downstream from the Vyl family (Fig. lA). The Vy genes show an overall sequence homology to other TCR V gene segments. For example, Vyl shows 33% amino acid identify with a V^ff consensus sequence (Hochstenbach& Brenner 1990). Among the two clusters of J segments, the proximal and distal J segments are homologous to each other and cross-hybridize on Southern blots (LeFranc et al. 1986c, 1986b, Quertermous et al. 1986a, 1987, Huck & LeFranc 1987). Thus,

Figure IB. Genomic organization of the human TCR ajS locus on chromosome 14. The nomenclature is according to (Takihara et al. 1989, Hata et al. 1989). The TCR 3 locus is located between Va (on 5' side) and ia segments (on 3' side). The Va"s and Vd], 5 and 2 segments lie upstream of three D^ and JS segments. \S3 is located on 3' side of CS in an inverse orientation. The locations of VM and 6 have not been determined. Adapted from Hochstenbach & Brenner (1990).

140

PORCELLI ET AL. K R ^ JUHCTIOHAI

J! GERHIIHE

Ifl

ion PBL Cl NOLT-13

fil TeTGCcneTeGGAGG V7Z TeTeCCTTGTGGGWe V T 1 . 3 TerccCACaeGGACAM

tgtgncttgtsggiggtg

GERMLWE

V7I.3

atttittataigiHc 6 GM CCCCCCa

^^2.3

TTATTATMUAHC 3^2.3 ATTATAAGAAAC J 7 1 . 3 TAAGAAAC Jii.S

tgtgcc(CCtg99*C*9g

Figure 2A. TCR y junctional sequences of IPD2. PBL Cl (Krangel et al. 1987a) and MOLT13 (Hochsteobach et al. 1988) are depicted to illustrate the incoqjoration of N/P-nucIeotides, and deletion of germline nucleotides to generate diversity. Germlioe gene sequences are shown in lowercase tetters for comparison.

Jyl.l is highly homologous to Jj'2.1, and }yi3 and 2.3 are identical in amino acid sequence. However, Jyl.2 has no homologue in Jy2 cluster and is distinct from the other Jy gene segments, Diversity for y genes is therefore limited since only 8 functional Wy genes and only 5 functional Jy germline genes exist, in contrast to Va, V^ and Ja germline gene segments for which nearly 100 of each exists. Some additional diversity is realized, however, through the incorporation of small numbers of random nucleotides at the V-J joining ends and because of deletions in nucleotides at the 3' ends of V segments and at the 5' ends of J segments (Fig. 2A). Each of the two J clusters is followed by a downstream constant (C) gene segment, such that rearrangement to a Jyl gene will result in splicing to the Cyl gene segment when the mRNA transcript is produced, and vice versa for Sy2 and Cy2 (LeFranc et al. 1986a, Dialynas et al. 1986). The Cy genes are each composed of from three to five exons. The first constant region exon (CI) encodes the immunoglobuUn-hke extracellular domain. The transmembrane region and cytoplasmic tail are encoded by the third exon (CIII). The second exon (CII), which may be present as one, two or three copies, encodes the connecting peptide region between the Cl and CIII encoded segments. Cyl genes have a single copy of the CII exon which contains a cysteine that is presumed to be utilized for disulfide linkage to TCR 3. In contrast, two allelic forms of the Cy2 genes have been

t JUHCTltWAL DIVERSITY piz IDP2 n i Cl HOLT-IJ

TCTTG TCnCGCGMC TtTTGGGGAAC

GERHLINE tcttggggtut

CTGTACGGG G A M CCGGCrC s***t*gt

AC

H/p

TCCIA GAAAGGAA CCTAC AG ccttuctac

TGGGGGATACG CGGICTTTCCA1 TGGGGG TGGGGTCETGGGATAGSTGG tiGGGGG GT actgggggiticg

CCGATAAAC AC ACACCUTMAC «eaccgattMC

Figure 2B. TCR S junctional sequences of IDP2 (Hata et al. 1987), PBL Cl and MOLT13 (Hata et al. 1988) are shown to illustrate the basis of extensive junctional diversity through usage of one (MOLT-13), two (PBL Cl) or three (IDP2) DS segments, incorporation of N/P-nucleotides, and deletion of germline nucleotides at the V-D, D-D and D-J junctions. Germline gene sequences are shown in lowercase letters for comparison.

141

BIOLOGY OF yS TCR

found; one of these has two highly homologous but not identical copies of the CII exon (called copies 'b' and 'c') and the other contains these two copies ('b' and 'c') and an additional related copy ('a') of the CII exon. The C>'2 allele containing three copies, referred to as allele 2abc, occurs less frequently in the population compared to the allele 2bc, with frequencies of 32 and 68%, respectively (Li et al. 1988). Although all copies of the CII exon are highly homologous, none of the copies in the C>'2 genes contains a cysteine residue. Thus while the connector region in Cy2 genes is extended by the duplication or triplication of the CII exons, they cannot encode subunits that participate in disulfide-linkage to the 6 chain. The three structural forms of the yd TCR resulting from these differences in constant region structure are depicted schematically {Fig. 3A). TCR yS PROTEINS These differences in the TCR y constant gene segments result in three structurally distinct protein forms, observed on individual human T-cell clones when examined

FDRM1

FORM 2abc

F0RM2bc

r PBL Cl (Cyl)

IDP2 {Cy2)

8

MOLT-13 (Cy21

Figure 3A. Schematic representation oiyS TCR constant region exon usage in disulfide and non-disulfide-linked structural forms. Note the single copy of the CII exon in the Cyl structure encodes a cysteine allowing disulfide-linkage of the y and d subunits (Form 1). Two (Form 2bc) or three (Form 2abc) copies of the CII exon are present in Cyl alleles. These do not contain cysteines and encode non-disulfide-linked TCRs. Compare these schematic forms with the visualized proteins in Fig. 3B. (Adapted from (Brenner et al. 1988).

142

PORCELLI ET AL.

by '"I labeling and SDS-PAGE analyses (Brenner et al. 1987b, Hochstenbach et al. 1988) (Fig. 3B). A disulfide-linked yS heterodimer of about 70-80 kDa is observed from WM-14 or PBLCl cells, and this resolves as two '"I labeled species {y at 40 kDa and 6 at 43 kDa) after reduction of the complex (see WM-14 in Fig. 3B). Cloning and sequencing of the TCR y cDNA clone from a T-cell displaying a disulfide-hnked yS receptor revealed usage of the Cyl gene, consistent with the idea that the single copy of the CII exon in the Cyl gene encodes a cysteine that mediates disulfide-linkage to TCR S (Krangel et al. 1987a). In contrast, two non-disulfide-linked yS TCRs were observed, one composed of a larger TCRy species (55 kDa) as on IDP2 cells (Brenner et al. 1987b), and the other composed of a smaller TCRy species (40 kDa) as on MOLT-13 cells

Cr2

• Cr2

(be)

(abc) Mr

i

-69

^"^•^-46

'•¥ - 30

-'•

•

•

, N R •• W M - 1 4

N R MOLT-13

Figure 3B. Three protein forms of the yd TCR. '^'I surface labeled T cells were solubilized in digitonin, immunoprecipitated with anti-CD3 mAb, resolved by SDS-PAGE and visualized by autoradiography. WM-14 cells express a disulfide-linked y6 TCR (Form 1) with a 70 kDa radiolabeled species visualized under nonreducing conditions (N) and two predominant species of smaller size are seen under reducing conditions (R). The two non-disuifide-linked forms are detected with TCRy species of 40 kDa (Form 2bc. MOLT-13 cells) and 55 kDa (Form 2abc, IDP2 cells). Note that under nonreducing conditions the TCR 6 subunit in these non-disulfide-linked forms migrates faster than under reducing conditions. Open triangles point to the TCR(5 subunits and solid triangles depict TCRy chains. Compare these visualized proteins with their schematic representation in Fig. 3A. (Adapted from (Hochstenbach et al. 1988)).

BIOLOGY OF y'2 gene segments. It was still unexplained why such a large size difference existed between tbe Iwo non-disulfide-linked TCRy polypeptides. Although form 2abc has one copy more than form 2bc of the CII exon, this only predicts an additional 1.5 kDa in size. Yet 2abc is 15 kDa larger than 2bc (55 versus 40 kDa). This was likely to be the result of differential glycosylation, but since no N-Unked glycans are encoded by copy 'a' of the CII exon, both chains contain the same number of potential N-linked glycan acceptor sites in the constant region (four potential sites). This issue was resolved when transfection of a 2abc cDNA clone yielded a 55 kDa polypeptide in MOLT-13 cells which also express the endogenous 2bc 40 kDa polypeptide. Deglycosylation studies revealed the that while both 2abc and 2bc forms had the same number of potential glycan acceptor sites, it appeared that only two of these sites were used on form 2bc. The absence of the 'a' copy of the CII exon appears to alter the secondary or tertiary structure of the polypeptide so that several of the glycan acceptor sites are not used (Hochstenbach et al. 1988, Band et al. 1989). All three of the human y3 structural forms appear in the peripheral immune system, but to date no specific functional differences between them have been noted. However, a recent report found that y3 T cells expressing C}'2-encoded TCRs displayed distinct cytoskeletal organization and morphology (Grossi et al. 1989), suggesting that some functional differences may eventually be appreciated. Moreover, a comparable situation to these three forms may exist in mice where, although all y3 receptors are disulfide-linked, connector regions in murine Cyl, Cy2 and Cy4 are 15, 10, and 33 amino acids in length, respectively (Hayday et al. 1985, Garman et al. 1986, Iwamoto et al. 1986). The TCR y3 complex is expressed on the cell surface in association with the

144

PORCELLI ET AL.

CD3 complex (Brenner et al. 1986). However, the CD3 complex is subtly different from that on cells bearing the a^ TCR. This is shown by the slower relative mobility of the CD3(5 chain on SDS-PAGE when in association with the yd TCR, compared to its mobility when associated with the a/? TCR (Brenner et al. 1986). This difference results frotn the processing of the two N-glycans attached lo the CD3^ subunit to the complex form wben associated with the y3 TCR, while only one of these two glycans is normally so processed on a^ TCR-bearing T lymphocytes (Krangel ct al. 1987b). In most other respects the yS TCR-CD3 and afi TCR-CD3 associations appear similar and botb complexes transduce a signal resulting in [Ca+^], flux and subsequent activation of the cells (Krangel et al. 1987b, Bank et al. 1986, Wu et al. 1988, Patel et al. 1989, Ciccone et al. 1988). No example of y3 TCR expression in the absence of tbe CD3 complex has been observed, suggesting that tbe physical association of the two complexes is required for cell surface expression.

TCR 6 GENES Human TCR 3 cDNA clones were isolated first by the subtractive hybridization approach using mRNA derived from the IDP2 y6 T-cell line (Hata et al. 1987). These cDNA clones represented genes that were rearranged and expressed in yd T cells, and deleted in most a^ T cells. These cDNA clones were shown to encode the TCR d protein since they directed in vitro transcription and translation of a polypeptide that was recognized in immunoprecipitation studies witb a TCR 6specific monoclonal antibody, anti-TCR^l (Band et al. 1987). Separately, Chien and Davis (Chien et al. 1987b) isolated a rearranging gene upstream of Ca in the mouse which was called Cx. This gene was shown to encode the murine TCR(5 chain based on partial amino acid sequence infonnation (Bom et al. 1987), and by immunoprecipitation of a human TCR d chain with an anti-peptide serum based on tbe deduced sequence of the putative murine TCR d gene (Loh et al. 1987). TCR d transcripts reveal the presence of segments that by homology to immunoglobulin and TCR a, ^ and y genes correspond to V, D, and J gene sequences (Hata et al. 1987, Chien et al. 1987b, Elliott et al. 1988) (Fig. 4). Both the murine and human TCR d genes are located within the TCRa locus, between the Va and Ja gene segments (Chien et al. 1987b, Hata et al. 1989) (Fig. IB). Tbe human TCR d locus consists of a single Cd gene segment, three iS segments immediately upstream of C3, and three D^ segments 5' of tbe J(5 genes (Hata et al. 1989, Takihara et al. 1989, Loh et al. 1988). Three commonly used \d genes, V^l, Vd2 and V^3, were first identified on yd T cells. Wdl and 2 arc located 5' of D^ segments, while V^3 is found in an inverse orientation about 5 KB downstream from the Cd gene segment (Hata et al. 1989) suggesting that it must rearrange by inversion to the oppositely oriented Dd gene segments. V^l shows substantial amino acid sequence homology with a Va consensus sequence

BIOLOGY OF y(5 TCR

TCRr

iH

V M c s—s s—s

145

i i i 3'UT|AAAA

DDD

TCRS

fM V \m I

S

I

S

I

c

I

;AAA

3 U T AAAA

I

S—S SH

Figure 4. Comparative structures of the TCR y and S transcripts as exemplified by the receptor expressed on IDP2 cells (Krangel et al. 1987a, Hata et al. 1987). Leader (L), variable (V), diversity (D), joining (J), constant (C), and 3'-untranslated (3'-UT) portions are indicated. Note that the TCR S transcript contains up to three D segments in tandem. Locations of intrachain disulfide loops are noted. A cysteine in TCR S that mediates interchain disulfide linkage with TCR y polypeptides encoded by the Cyl gene is shown. (Adapted from (Brenner et al. 1988)).

(Hata et al. 1987), and in fact can rearrange to Ja gene segments where it is expressed as part of an a^ TCR (Miossec et al. 1990). In addition, some V gene segments known to rearrange to Ja segments also rearrange functionally to Dd segments, such that they can encode part of either type of T-ceU receptor. For example, Val7.1 sometimes rearranges to a Dd-id segment where it encodes part of a yd TCR (and then is called V^5) (Takihari et al. 1989, Hata et al. 1989). Other V genes, such as the Vrf4 which is closely related to Va6.1, are probably members of Va gene families that are also interchangeably used in D3-JS gene rearrangements (Guglielmi et al. 1988). To data, V(52 and V^3 have been found only as part of y3 TCRs, and a majority of the Vos have not been observed rearranged to D^s. Thus while some interchangeability of V genes occurs for a and d, the two repertoires are largely separate. The molecular basis for whether a particular V gene will be used exclusively or interchangeably is not known. In general, most of the V genes used on TCR 3 chains are clustered nearest to Di5 and J^ in the germline. It has not been possible to distinguish V^s from Vas based on coding sequence or based on their site-specific recombination signal sequences. Like other TCR and immunoglobulin genes, TCR 3 genes are generated by rearrangement of the dispersed germline gene segments during T-cell development. Thus a particular Vd segment is recombined to one or more of the Dd segments and one of the three Jd segments. TCR^ gene rearrangements frequently incorporate relatively large numbers of N-nucleotides (and some P nucleotides) (Lafaille et al. 1989) at the junctions of rearranging gene segments. In addition, deletion of nucleotides from the joining ends of germline sequences and/or imprecise joining appear to contribute to junctional diversity for this chain. One of the most striking aspects of TCR d diversity Is tbe usage of up to three D segments

146

PORCELLI ET AL.

in tandem (Hata et al. 1988) (Figs. 2B and 4). The combination of diversity at the joining ends and the tandem use of multiple D segments results in unprecedented potential diversity for TCR 3. This extensive junctional diversity contrasts vfith the limited repertoire of germline V^ segments available. Three Vd gene segments account for the majority of the y3 T cells and only 6 different V genes have been reported to date as part of D3-33 gene rearrangements. TCR CHAIN PAIRING Sequence similarities are also noticeable between the Ca and Cd regions and betvi'een C^ and Cy regions, respectively (Hata et al. 1987, Hochstenbach & Brenner 1990). For example, Ca and Cd contain 49 and 50 amino acid residues, respectively, between the two cysteines that are likely to form the intradomain disulfide bond as part of the Ig CH3-like domain. Similarly, Cp has 64 and Cy 55 residues between the comparable cysteines. Both Ca and C3 lack a tryptophan in the third (C) P strand, a conserved feature of other Ig-like constant domains, while both Cy and C^ contain the corresponding tryptopTian. In addition, both Cjff and Cy contain an asparagine-linked glycan acceptor site at an identical location in the loop betu-een the fourth (D) and fifth (E) p strands. The greater similarity between the Ca and the C^ versus the C^ and the Cy is underscored by comparisons of their sequences in the membrane-flanking and presumed transmembrane regions (Hata et al. 1987). The putative TCR a and 3 transmembrane and intracytoplasmic regions display a pair of basic amino acids, with a four-amino acid spacer, that are probably buried within the membrane. These are followed by a 10-amino acid spacer, an additional basic residue, and a short uncharged carboxy-terminal tail of three or four residues. In contrast, the corresponding regions of C0 and Cy display a single basic residue predicted to be within the transmembrane region, and they contain longer and more highly charged intracytoplasmic tails (Fig. 5). Overall, Cd and Ca are 30-35% identical in transmembrane region, while Cd shows only limited homology to C^ (8%) and Cy (13%) in this region (Hata et al. 1987). The Cp and Cy segments, however, are 33% identical (Hochstenbach & Brenner 1990). These structural features in the constant domain, and those noted above in the V domains, indicate that TCR a and d are closely related, as are TCR ^ and y. This suggests that each member of the pair complements its partner in a similar fashion. This idea raises the possibility that cross-chain pairing might occur. Yet, the available data suggest that chain pairing is governed largely by a physical preference, although not absolute, for TCRy to pair with 3 and for TCRa to pair with ^. For example in PEER cells, TCRy, d and 0 genes are productively rearranged and transcribed abundantly. However, only a yd TCR appears on the cell surface, suggesting that these two subunits pair preferentially, even in the presence of TCR^ (IConing et al. 1987). When this cell line is transfected with a functional TCRa chain, it now

BIOLOGY OF yiS TCR

147

TRANSMEMBRANE AND FLANKING REGION CHARGED RESIDUES

Figure 5. TCR transmembrane and flanking region charged residues. The approximate relationships of the basic {-\-) and acidic ( —) residues located within and adjacent to the putative membrane spanning regions of the TCR constant domains are shown. (Adapted from (Hata et al. 1987)). pairs with TCR^ and results in expression of the ap TCR on the cell surface (Saito et al. 1988). In other cells containing all four TCR chains as a result of hybridoma fusion and transfection, both afi and yd heterodimers are noted on the cell surface, but cross-paired receptors are not detected (Saito et al. 1988). These results strongly suggest that a physical preference for pairing when alternative chains are available exists for a with 0 and for y with d. However, the question remains whether cross-chain pairing could occur if the preferred partner is not available. This, in fact, could correspond to a physiologically relevant circumstance in development. TCR^ and y gene expression have been shown to occur early in murine fetal thymic development (beginning on about day 14) and this is followed by the appearance of TCR^ transcription and rearrangement on about d 16, while a transcription does not become substantial for another day. It is thus possible that some thymocytes might rearrange TCRy nonproductively, but have productive TCRd and ^ gene rearrangements available, prior to TCRa rearrangement. Such cells might pair TCR p and d and express this as a CD3-associated TCR on the cell surface. We have identified one cell line on which this appears to have occurred (Hochstenbach & Brenner 1989). Thus cross-chain pairing is physically possible and may even occur lo some extent in development. However, it must be a rare occurrence, or if frequent it may yield cells that cannot efficiently survive thymic development since ^(5 TCR-bearing cells have not been found with frequency in the periphery. No examples of ay heterodimers have been reported, although the possibility that these chains could pair in the absence of their preferred partner chains has not been rigorously excluded.

DEVELOPMENT AND LINEAGE RELATIONSHIPS Studies in mice first showed that y3 TCR-bearing lymphocytes appear in development in the thymus before ay? TCR cells (Pardoll et al. 1987, Havran & Allison

148

PORCELLI ET AL.

1988). Moreover, V-(D)-J gene rearrangements for y and 3 follow an ordered sequence, and diversity at the V-D-J junctional region is influenced markedly by the stage of development in which rearrangement takes place (Havran & Allison 1988, Houlden et al. 1988, Chien et al. 1987a, Elliott et al. 1988). In humans, expression of TCRy and /f transcripts occurs in immature tumor cells and thymocytes in the absence of a-gene transcription, which only appears later in ontogeny (Collins et al. 1985, van Dongen et al. 1987, Royer et al. 1985). This suggested that TCR rearrangement and expression might follow an ordered course in the human, yd T cells appear in the human thymic region by 9.5 weeks of gestation, while a^ T cells dominate soon thereafter, based on staining human fetal abortuses with mAb anti-TCR^l and y?Fl (B. F. Haynes, M. E. Martin, M. B. Brenner, unpublished). Comprehensive studies on gene segment usage and diversity of fetal and postnatal thymus-derived T-cell clones and polyclonal cell populations have been carried out. They reveal that initial rearrangements at the TCR(5 loci join Vd2 to D^3, and initial rearrangements at the TCRy locus join downstream \y gene segments (VyL8 and Vy2) to upstream Sy gene segments which associate with Cyl (Krangel et al. 1990, McVay et al. 1991). These genes encode disulfidelinked yd TCR proteins. A switch then occurs such that later in development V^l genes are preferentially rearranged and join to the more upstream Dd\ and T>d2 gene segments. Similarly in the later periods of development, the more upstream Vy gene segments (Vyl family members) join to the downstream Sy2 segments which splice to C72 to yield non-disulfide-linked cell surface proteins (Krangel et al. 1990, McVay et al. 1991). Studies of gene rearrangements on the chromosome not encoding the expressed protein in adult yd T-cell clones are consistent with this formulation of ordered rearrangements (Triebel et al. 1988). In addition to the sequential use of germline gene segments, striking differences are seen in the degree of diversity since the earliest y and 3 gene rearrangements are often simple germline gene segment joints, while later in development two or three D

FH. P35

c

27/3 .62

m •

s