Change in antigen specificity of cytotoxic T lymphocytes ... - Europe PMC

Proc. Nati. Acad. Sci. USA Vol. 83, pp. 4441-4445, June 1986 Immunology

Change in antigen specificity of cytotoxic T lymphocytes is associated with the rearrangement and expression of a T-cell receptor fl-chain gene (aged killer celis/oligonucleotide probes/somatic mutation/N-region diversity)

JORG T. EPPLEN*, FRAUKE BARTELS, ANDREA BECKER, GABI NERZ, MARLOT PRESTER, AUGUSTINUS RINALDYt, AND MARKUS M. SIMON Max-Planck-Institut fur Immunbiologie, P.O. Box 1169, D-7800 Freiburg, Federal Republic of Germany

Communicated by Susumu Ohno, September 24, 1985

ABSTRACT Cloned H-Y-specific murine cytotoxic T lymphocytes, which alter antigen specificity in vitro ("aging"), simultaneously exhibit changes in the T-cell antigen receptor f-chain rearrangements and respective mRNAs expressed. fl-chain cDNA clones were isolated from a library prepared from mRNA of aged killer T cells. The sequence of the fchain variable region element (VAK) was found to be identical with germ-line DNA. Four bases at the f-chain diversity-joining region (Dp-Jp) junction cannot be explained by known germline Dp and Jp elements. These results illustrate that in T-cell clones altered antigen specificity correlates with a switch in productive fl-chain rearrangements of the T-cell receptor. When tested for its expression under physiological conditions, significant levels of VAK mRNA were found in normal lymphocyte populations. T lymphocytes recognize foreign antigen in the context of self antigens of the major histocompatibility complex via specific receptors on the cell surface (1). In studies with antibodies raised to the putative T-cell antigen receptor(s), these structures have been shown to be disulfide-linked heterodimers consisting of an a and a , polypeptide chain (2). Both a and ,3 chains are composed of variable (V) and constant (C) regions (3, 4). By using differential molecular cloning techniques, T-cell-specific cDNAs were substantially enriched (5). Thereby, candidates for the receptor-encoding molecules were identified exhibiting significant homology of the predicted amino acid sequence to immunoglobulin genes (5). Like immunoglobulin heavy-chain genes, the /3 chain of the T-cell antigen receptor has a V region commonly encoded by three distinct gene segments (Vi, D8, and Jo), which rearrange during T-cell maturation to generate the complete V gene (6, 7). The ,8-chain locus on chromosome 6 of the mouse (8, 9) contains two closely linked constant region genes (Ca1 and CA2), each associated with six potentially functional joining region (JO) gene segments (10, 11). Two diversity segments (Da31.1 and Dp32.1) have been localized 5' to the Cp1 and C,2 gene segments, respectively (11). Thus, in summary, the evidence that the T-cell antigen receptor molecules have been identified is compelling. We wanted to exploit long-term cultured T cells and derived variants to address questions on the correlation between specificities and expressed T-cell antigen receptor genes. Hereby the genetic evidence for the T-cell antigen receptor gene could be evaluated. Biochemical and molecular genetic analysis of the T-cell receptor proteins and genes has relied heavily on available cultured T cells. Much analysis has assumed that these cells are stable during extensive clonal proliferation in vitro. Recently, however, it has been demonstrated that long-term The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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cultured murine cytolytic T-cell clones (CTLL) can change their specificity and/or function (13). Cloned anti-H-Yspecific cytotoxic T lymphocytes (CTLL.1) lose their H-Y antigen specificity in culture and develop into variants [CTLL.3; aged killer (AK) cells] that gain a new specific cytolytic activity against a different target cell (P815 mastocytoma cells). This process of "aging" of T-cell clones (14) produces intermediate stages (CTLL.2), which express both specificities, anti-H-Y as well as anti-P815. Aging has been repeatedly observed in vitro. Because of this model characteristic of long-term cultured T-cell clones and their reproducible development in vitro, we investigated whether changes in the specificity of the CTLL had a genetic basis in the structure and/or expression of the T-cell antigen receptor. In CTLL.1, the p-chain locus on one chromosome 6 is rearranged and expressed. We report here on experiments that demonstrate that a new rearrangement event (in CTLL.3), resulting in the expression of a new 1-chain gene, parallels the switch in specificity. From a cDNA library, clones reflecting the newly activated gene were isolated, characterized, and compared to their germ-line sequence. The somatic mechanisms of generating diversity on the level of the T-cell antigen receptor 8 chains are discussed in the context of the expression of a specific V,3 gene segment in vivo.

MATERIALS AND METHODS Murine CTLL and Con A-Activated T-Cell Lymphoblasts. H-Y-specific CTLL from C57BL/6 (B6) mice and their variants derived in vitro were grown in vitro as described (13). Functional activity and specificity were tested as described (14) in aliquots from the same cells that were harvested for extraction of nucleic acids. Con A blasts were obtained by stimulating 5 x 107 spleen or lymph node cells or 2 x 108 thymocytes in 18 ml of RPMI 1640 culture medium with 5 ,ug of Con A per ml. After 2-3 days, lymphoblasts were separated from other cells by centrifugation on a density gradient (Ficoll-Urovison, 4 = 1.077; Pharmacia) for 15 min at 2000 rpm. The interphase containing the enriched T-cell blast population was removed and washed three times in phosphate-buffered saline before RNA was extracted as described for CTLL. Extraction of RNA and DNA. RNA and DNA from CTLL were extracted by resuspending the cells in guanidinium thiocyanate followed by centrifugation through 5.7 M CsCl Abbreviations: V, C, D, and J, variable, constant, diversity, and joining regions of T-cell receptor; CTLL, cytolytic T-cell clones; kb, kilobase(s). *To whom reprint requests should be addressed. tPresent address: Department of Biochemistry, School of Medicine, Vanderbilt University, Nashville, TN 37232.

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Immunology: Epplen et al.

Proc. Natl. Acad. Sci. USA 83 (1986)

DNA Sequence Analysis. Dideoxy sequencing was carried out according to the procedure of Sanger et al. (23) using different specific oligonucleotide primers.

(15). RNA was recovered from the pellet. DNA was recovered from the interphase and treated with proteinase K, followed by phenol/chloroform extraction (16). DNA from mouse liver was prepared according to Blin and Stafford (17). Southern and RNA Blot Hybridization Analysis. High molecular weight DNA prepared from CTLL.1 to CTLL.3 was digested to completion with restriction enzymes according to the manufacturer's recommendations (Bethesda Research Laboratories). Digested DNAs were subjected to electrophoresis in 0.5% agarose gels, ethidium bromide stained, photographed under UV light, and transferred to nitrocellulose filters (18). Filters were hybridized in 6x SSC (lx SSC = 0.15 M NaCl/0.015 M Na citrate) (680C for 16 hr) with nick-translated probes (modified according to ref. 19) at 106 cpm/ml and washed in 2x SSC/0.1% NaDodSO4 at 65TC. For RNA blots, 10 ,ug of total cellular RNA was denatured in formaldehyde and electrophoresed in 1.4% agarose gels containing formaldehyde (20). After ethidium bromide staining and photography under UV light, RNA was blotted onto GeneScreen membranes (New England Nuclear) and hybridized with 5_-32P-labeled oligonucleotides according to Miyada et al. (21). Screening of the CTLL.3 cDNA Library and Screening of Two Genomic Mouse DNA Libraries. The cDNA library preparation from poly(A)+ RNA isolated from CTLL.3 (12, 14) has been described (22). The library was screened using T-cell receptor Cp3-specific 32P-labeled oligonucleotide (TCRfB; 5' CCATTCACCCACCAGCTCAGCT; for further description see Fig. 1). The B6 genomic libraries were prepared as partial Sau3AI and EcoRI digests of mouse liver DNA cloned in the Charon phages 4A and 28, respectively. Genomic libraries were screened with the nick-translated probe p/3AK1 (22) at 106 cpm/ml (specific activity, 1-2 x 108 cpm/,ug).

A

RESULTS cDNA Clones. To determine whether the change in specificity observed in the variants derived from H-Y-specific CTLL.1 involved changes in the T-cell antigen receptor, cDNA clones for the 13-chain mRNA were isolated from AK cells (CTLL.3) that had lost their original specificity and expressed specific cytolytic activity for P815 target cells instead (14). We have previously described the isolation and sequence of one such cDNA clone, p/AK1 (22). Additional 13-chain cDNA clones were identified by oligonucleotide (see Fig. 1A) as well as by nick-translated conventional probes (pl3AK1 insert DNA). Four different clones were isolated (Fig. LA; p3AK1, pBAK17, pAK18, pAAK20) from 15,000 independent colonies. All cDNA clones were sequenced and shown to be identical in the overlapping regions. Together, the four clones cover 1.1 kilobases (kb) containing >50 bases each of 5' and 3' untranslated sequences. A single discrepancy to the published sequence of the C,92 region in the B1OWR7 strain (11) was found in pA3AK20 in the segment coding for the intracytoplasmic domain of the Ca-chain protein. Instead of a stretch of eight adenines, there are only seven adenines in pf3AK20. No additional cDNA clone overlapping this region was found. To exclude a strain-specific polymorphism, two genomic B6 liver libraries in Charon phages were screened with the probe p3AK1. Phages containing C91 and C132 germ-line sequences were isolated from the genomic Sau3AI library. The analysis of B6 Cp32 germ-line DNA [bases 1840-2788 according to Malissen et al. (11)] yielded complete identity cDNA clones

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FIG. 1. Schematic diagram of cDNA clones representing the /3-chain mRNA of the CTLL.3 T-cell antigen receptor (A) and genomic organization of its germ-line V region sequence (B). Restriction enzyme cleavage sites are represented by the vertical lines with the following abbreviations: A, Asu II; B, BamHI; E, EcoRI; H, HindIII; K, Kpn I; N, Nco I; S, Sau3AI; T, Taq I. 5'UT and 3'UT refer to 5' and 3' untranslated regions of the mRNA; L is the leader sequence. The position of the oligonucleotide hybridization probes is indicated below the composite representation of the cDNA clones; the sequences of individual oligoprobes are given in the text. In the partial restriction maps of the germ-line clones XVAK16 and XVAK18, the leader (L) and VAK gene segments are indicated by open boxes. The 1.4-kb BamHI fragment was sequenced according to Sanger et al. (23), and its sequence is shown in Fig. 3.

Immunology: Epplen et al. with the

sequence


reported from E310.WR7 liver DNA. In

contrast to pf3AK20, a stretch of eight adenines is also present in B6 Cp2 germ-line DNA. T'his discrepancy prompted us to design two oligonucleotide s (A7, 5' CTCAGGAA-

llI--TTTCTTGACC; A8, 5' CTCAIGGAATl--l--l---TTT CTTGACC) specific for either the gerrm-line or the pJ3AK20 sequence. By RNA blot hybridizaLtion, it was found that CTLL.3 mRNA contains only the A,8 sequence, as expected in transcripts from the genomic genoe (data not shown). The A7 sequence of pBAK20 is thus am artefact, presumably produced during molecular cloning. Gene Rearrangements and Expres sion in CTLL.1 and Derived Variants. The germ-line orgaLnization of the 3-chain locus of the murine T-cell antigen r eceptor has been determined with respect to the J and C reg;ion elements (10). It has been previously shown that each 'T-cell clone potentially manifests different gene rearrangebments (24). These rearrangements presumably depend ori the specific V, D, and J region elements selected for expre ssion. Using the cDNA clone pf3AK1 from the AK CTLL.34as a probe, the genomic organization of the T-cell receptor f3-chain genes was compared in (i) the H-Y-specific CTL]L.1, (ii) a variant that expressed cytolytic activity on both Xmale B6 and P815 target cells (CTLL.2), and (iii) CTLL.3. Si ince this probe contains both V (VAK, DO, and Jp2.6) and C (C#2) regions, it serves to reveal rearrangements in both ge ne segments. Southern blot hybridization of BamHI-digested DNAs (Fig. 2A) shows that CTLL. 1 has only one C#1 gene remaining (11-kb band) and presumably one C,62 gene rearrm tnged (6-kb band). The rearrangement of C92 is not revealed tby this digest, but it was confirmed by other digests (not showwn) and implied by the expression data (see below and Fig. 2B). In additione the V In In contrast, region VAK (1.4-kb band) is not re-,arrand. arranged. CTLL.3 has no C131 gene remainin g. The V region VAK shows rearrangement (1.4 kb -2. kb), and there iS no germ-line copy of VAK remaining. C'TLL.2 representing an intermediate stage between CTLL.1 and CTLL.3 showed a mixed pattern with respect to VA K rearrangement and probably contained a mixed popul;ation of CTLL.1 and CTLL.3 cells.

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C82 CB1 FIG. 2. (A) Genomnic Southern (18) hybPridization of DNAs from an anti-H-Y cytotoxic T-lymphocyte clone (CTLL.1) and its in vitro derivatives (CTLL.2 and CTLL.3), which h,ave anti-P815 specificity. DNAs were digested with BamHI, electrop)horesed in 0.5% agarose gel, blotted, and hybridized to the probe pq3AK1 as described. (B) RNA blot hybridization of total cellular:RNAs from CTLL.1 to CTLL.3. RNA was isolated from cultured ccells and 10,ug of each was electrophoresed (20) and blotted. The satme blot was hybridized at the indicated temperatures consecutiv to the oligonucleotide probes VAK (5' GGGCATCCCTGAAK rCATCTAT; 570C), C,02 (5' TATAGGGTGGCCTTCCCCAGTAG 67°C), and Ci (5' TACAGGGTGGCTTTCCCTAGCAG; 670C'; according to Mivada et al. (21). The probe was stripped after each eexposure by washing the filter in 0.1x SSC at 680C for 20 min (expo,sure for 1 day using one PBAK1

PROBE -VAK

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intensifying screen).

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In the case of CTLL.3, it is not possible to distinguish between two situations. (i) One C,2 gene segment is deleted and the other rearranged. (ii) Both C,92 genes are rearranged to the same VO. Southern blot hybridizations using five different restriction enzymes and several independent probes representing parts ofthe C,9 genomic region never yielded any signals in addition to the ones expected from functional VAK rearrangement. Cytogenetic analysis of CTLL.1 and its variants has previously revealed gross numerical as well as extensive structural aberrations in CTLL.3 (12). Since individual restructured chromosomes could not be identified in CTLL.3, it is also impossible to determine by standard cytogenetic analysis how many T-cell antigen receptor chain loci remain in CTLL.3. Germ-Line Gene Encoding VAK. From a Charon 4A genomic library, two independent clones, XVAK16 and XVAK18, were isolated that contain the germ-line V region of p,3AK1 (Fig. 1B). A 1.4-kb BamHI fragment hybridizing to pP3AK1 was subcloned into the plasmid pUC8, and both strands were sequenced from various secondary subclones in different M13 phages (25). The XVAK sequence is depicted in Fig. 3. Typical "CAAT"S and "TATA" boxes are present, and the leader sequence (L) is separated from the next exon by 1.5 years. Junctional and N-Region Diversity. T cells exhibit two possible mechanisms to generate diversity in T-cell receptor P-chain genes (26).end (i) Junctional the in the which V, arising J gene flexibility D, andfrom points at variability segments are joined. (ii) N-regional diversity arising from the

proposed random trimming and repair of sequences on either side of the D gene segments during joining. To examine these possibilities, germ-line and rearranged gene segments from the Dp-J joining regions were compared. As depicted in Fig. 4, four bases at the Dp-J,0 junction in pBAK1 (and pBAK17) cannot be accounted for by germ-line sequences. Expression of (-Chain Sequences in CTLL. Specific oligonucleotide probes were used to investigate the pattern of ,pchain expression in CTLL.1 and its variants. The rearrangements of the p-chain genes and their differential expression are reflected in RNA blot hybridization data (Fig. 2B). As revealed by specific oligonucleotide probes (for description see Fig. LA), there were no Cp1 transcripts demonstrable in CTLL.1 to CTLL.3, whereas positive signals representing Cs2 mRNA were observed. A probe (VAK) that specifically detects V9 mRNA in CTLL.3 (and CTLL.2) revealed no such message in CTLL.1 demonstrating the functional switch from CTLL.1 to CTLL.3. To further investigate some aspects of the mechanisms Generating diversity on the p-chain level, the oligonucleotide DJAK was synthesized (5' GAAGTACTGTTCCCAACCCCCAG; see Figs. LA and 4). This probe spans part of the Dp and Jp elements used, as well as the non-germ-lineencoded Dp-J junction. As expected, the DJAK probe did not hybridize to CTLL.1 RNA, whereas CTLL.2 show specific hybridization signals. In addition, this probe gave an equivalent signal intensity to the VAK oligonucleotide probe on RNA blots of three independently derived CTLL.3 (data not shown). Expression of the CTLL.3-Speciflc VAK Sequences in Vivo. We wanted to determine whether the CTLL.3-specific V region gene was expressed and detectable in vivo. Con A-activated murine T cells from thymus, spleen, and peripheral lymph nodes indeed harbor various amounts of VAK mRNA. As shown in Fig. 5, the expression of the VAK gene can be demonstrated in the thymus of young B6 mice, where VAK mRNA is more abundant than in lymphocytes of the peripheral lymphatic organs. From the relative signal inten-

Immunology: Epplen et al.

4444


GGATCCCAGGTGGCATTCCTTGGATGGCCCTCCCTTCAGTTTCTGTATTTGTTTCTTTCTGTTGTCTTGACACAACAGAAACCAAGTTATTCTCTTATGC TCCAGTAGACGAACCCCAACCTCTGCAGATGGTAAGTTTTGATTAACTCACTGAGATAACAACAACAGTAGTAGTACACTAAAGGAGGATATGATTCTAG TAGGAAGAAGAGTGAGTCGGGGCTCTAACATCAGAGTTATTGGTGGGTGTGGATAAGACCAAAGTACACTGAACACATATATAAAAATTCAAAGAAAAAG TAAAAATTACTACTAAAAGAAGTGTCCAATAGGTAATTTGATATGTAATATTTCAGGAAAAGTAGAATGTGTGTGTTTTGGAGTGTGGGGAGATGGGATA ATCATTGAAGCACAAAGGATTTGGTAAATAAATATGTGTAGGGATACTTTAAGTCTTTGGATGAACCACCCATGCCTGTACCAGAACAATTACTTATTTC ATGGTTTTATTTTTTTCTTATAAAGGAACAGTGATCACTTTTGAAAGGTGATCCTTTGAGCAGTGACGGAGACAGTGGTGTCATCACAAGCCCATACACC m

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FIG. 3. Germ-line nucleotide sequence of (-chain V region gene (VAK) expressed in CTLL.3. The predicted amino acid sequence encoded by the leader and V region exons is identified by the single-letter code. The sequence was determined according to the dideoxy method (23). CAAT and TATA boxes are underlined.

sities, it is estimated that VAK mRNA is less abundant in the lymphatic organs of older B6 mice (data not shown).

DISCUSSION The availability of cloned T cells allows us to investigate the regulation of antigen receptor expression in mature functional T cells. Here we show de novo rearrangement and expression of a previously silent Vp gene in a variant (CTLL.3) of H-Y-specific CTLL (CTLL.1), which correlates with the appearance of a new specificity. The finding that T-cell populations with a new specificity arise from differentiated T cells in vitro indicates that the T-cell specificity repertoire continues to evolve during an ongoing immune response in vitro and poses considerable constraints on the clonal selection theory (27). Since expression of the V region gene VAK is demonstrable in thymus, spleen, and lymph node mRNA, functional VAK rearrangements cannot simply be accounted for by mere cell culture artefacts. Whether the switch in antigen specificity observed in vitro is also occurring in vivo remains to be shown. However, the principal possibility exists that mature functional T cells are able to change their recognition structures, resulting in the expression of a new specificity. VAK

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Newly acquired anti-P815 specificity has been observed to develop independently in CTLL, also with preexisting specificities (14). The specificities in all functionally active CTLL (1-3) are strictly correlated with the data on mRNA expression. The AK (anti-P815) phenotype of CTLL.3 was found to be always reflected in VAK (and DJAK) positive mRNA expression. The fact that the functional switch is correlated with changes in T-cell receptor expression provides further evidence that the , chain, at least, encodes a component of the antigen-specific receptor. At present, it is not known by what means new rearrangements are induced in in vitro cultured T-cell clones. It is possible that CTLL.2 is a diversifying cell clone with dual antigen specificity, probably due to different subpopulations. Gross numerical chromosome aberrations have been demonstrated in the majority of metaphases (13), ranging from a hypodiploid (33) to quasitetraploid (85) chromosomal complement. Thus, the functionally active P-chain locus was probably lost in some of the cells, allowing the remaining locus to rearrange. A selective advantage of these AK cells would best explain their preponderance in culture. Due to the lack of appropriate models for early T-cell differentiation, little is known about the molecular elements that mediate the ordered assembly and expression of the T lymphocyte antigen receptor genes. In B-cell differentiation,

PROBE

3'

GAACAGTACTTC

GACCCCCAACCCTTGTCATGAAG

REARRANGED 5'

FIG. 4. 3-chain sequences of the V, D, and J gene segments in germ-line configuration and after in vitro rearrangement in CTLL.3 illustrating junctional and N-region diversity. The cDNA clones pA3AK1 and p,8AK17 are compared with germ-line gene segments XVAK, D,92.1, and J,2.6. Four nucleotides in the cDNA clones are encoded by neither the D nor the J segment (box).

Immunology: Epplen et al. D20 T LN S

D40 T LN S


CTLL.3

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FIG. 5. RNA blot hybridization of total RNA from Con Aactivated thymus-, lymph node-, and spleen-derived T lymphoblasts from B6 mice of different ages and total RNA from CTLL.3. RNA was isolated from cultured cells and 10 ,ug of each sample (corresponding to 107 cells) was electrophoresed (20) and blotted (18). The blot was hybridized to the oligonucleotide probe VAK (see legend to Fig. 2; exposure for 6 days using two intensifying screens).

analyzed by virus-transformed pre-B cells, productive heavy-chain gene assembly and expression generally precedes that of light chains (28) and may serve also a regulatory function-signaling the cessation of heavy-chain gene assembly (29). Functional de novo rearrangement and expression of V region genes in B cells has not been reported so far. Studies on the genes encoding antigen receptors on B and T lymphocytes have revealed striking similarities in their organization and DNA rearrangements (28, 30). Both V gene regions of the B-cell antigen receptor light and heavy chains and the T-cell antigen receptor a and chains are encoded by multiple germ-line DNA segments that can be assembled to generate complete functional genes. Molecular genetic analysis of the B-cell antigen receptor has revealed that in addition to multiple germ-line genes and combinatorial joining of the segments, somatic mutation of rearranged V region genes plays a key role in its diversification. In contrast, similar studies on T-cell antigen receptor genes indicate a small repertoire of V,3 gene segments (30-32) and rare cases of somatic mutations of rearranged genes (33). Thus, ,(3chain diversity is mainly arising from variable joining events and N-region diversification. Comparing the sequences of germ-line VAK and cDNA clones, not a single point mutation was detected in CTLL.3 mRNA even after >18 months of continuous cell culture. This finding is in contrast to the situation in B cells (28) but agrees with previous sequence data in T cells (12), making hypermutability of (chain V genes unlikely as a means of generating diversity at the T-cell antigen receptor level. The repeated occurrence of the DJAK complementary sequence in CTLL.3 mRNA, including four nucleotides not deducible from presently known germ-line DNA, necessitates the search for additional Do elements. as

We thank U. Hochgeschwender and D. L. Woodland for reading, and Mrs. R. Schneider for typing, the manuscript. The valuable contributions of R. B. Wallace are gratefully acknowledged.

1.

Zinkernagel, R. M. & Doherty, P. C. (1975) J. Exp. Med. 141, 1427-1436.

2.

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