Cell, Vol. 55, 291-300, October 21, 1988. Copyright 0 1988 by Cell Press

The Joining of Germ-Line Va to Ja Genes Replaces the Preexisting Va-Ja Complexes in a T Cell Receptor a,p Positive T Cell Line Jean-Pierre Marolleau,’ Joseph D. Pondell,t Marie Malissen,* Jeannine Trucy,* Eliane Barbier: Kenneth B. Marcu,§ Pierre-Andre Cazenave,’ and Daniele Primi* l Unite d’lmmunochimie Analytique Departement d’lmmunologie CNRS 359 and lnstitut Pasteur 25 Rue du Dr. Roux 75724 Paris Cedex 15 France fGenetics Graduate Program SUNY at Stony Brook Stony Brook, New York 11794 *Centre d’lmmunologie de Marseille Luminy Case 906 13288 Marseille Cedex 9 France 5 Department of Biochemistry, Microbiology,

and Pathology SUNY at Stony Brook Stony Brook, New York 11794

Summary

To determine whether T cell receptor genes follow the same principle of allelic exclusion as B lymphocytes, we have analyzed the rearrangements and expression of TCRa and 8 genes in the progeny of the CD3+, CD4-/CD8- M14T line. Here, we show that this line can undergo secondary rearrangements that replace the pre-existing Va-Ja rearrangements by joining an up- stream Va gene to a downstream Ja segment. Both the productively and nonproductively rearranged al- leles in the M14T line can undergo secondary rear- rangements while its TCR8 genes an? stable. These secondary mcombinations are usually productive, and new forms of TCRa polypeptides are expressed in these cells in association with the original CS chain. Developmental control of this Va-Ja replacement phenomenon could play a pivotal role in the thymic se- lection of the T cell repertoire.

Introduction

Et and T cells recognize ligands through clonally dis- tributed receptors composed of variable (V), joining (J), constant (C), and, in some cases, diversity (D) gene seg- ments which are juxtaposed by specific DNA rearrange- ments during lymphocyte development (for review, see Tonegawa, 1983; Allison and Lanier, 1987). The sets of genes encoding the Tcell receptor (TCR) a and 8 subunits however, are different from those encoding the immuno- globulin (lg) receptor on 6 cells, and this reflects the differ- ence that exists between 6 and T cell recognition of anti- gen. Whereas E3 cells interact with free ligands, T

lymphocytes recognize antigens in the context of major histocompatibility complex (MHC) molecules (Zinkernagel and Doherty, 1975; Bevan, 1975; Gordon et al., 1975; -.. Shearer et al., 1975; Kappler and Marrak, 1976; Sprent, 1978); however, the exact mechanism by which this dual specificity operates remains elusive.

In the mouse, the variable region of the TCRp chain is assembled from VP-08 and Jf3 segments (Lai et al., 1987; Chou et al., 1987). There are 17 VP families, of which only three (VpS, 5, and 8) have multiple numbers (Patten et al., 1984; Barth et al., 1985; Behkle et al., 1985). In germ-line configuration, the TCR8 locus has two closely linked con- stant genes (Cpl and Cp2), each of which is associated with an upstream cluster of six functional Jp gene seg- ments and a single Dp segment (Lai et al., 1987; Chou et al., 1987).

The a chain locus is composed of more than 40 Vu genes and at least 50 Ja segments that are dispersed over 60 kb upstream of a single Ca gene (for review, see Davis et al., 1984; Kronenberg et al., 1986). The diversity of the TCR repertoire results from combinatorial joining of V-D and J segments, junctional diversity, and multiple transla- tional reading frames in the Dp segments. These mecha- nisms of diversity generate an extremely diversified reper- toire, but the selective forces that occur in the thymus during T cell development shape a peripheral repertoire that is self-MHC restricted and tolerant to self (Fink and Bevan, 1978; Zinkernagel et al., 1978).

To search for new mechanisms of TCR repertoire diver- sification, we analyzed the genomic contexts of lg and TCR genes in several subclones derived from the previ- ously described M14T C.620 thymoma that expresses a TCRa$ heterodimer (Primi et al., 1988). Here, we report that, even though these sublines maintain their original D-J” and Cp rearrangements, several subclones have undergone secondary rearrangements at the TCRa locus. Analysis of these rearrangements with Ja and Va probes revealed that these secondary rearrangements replace preexisting productive and nonproductive Va-Ja rear- rangements by joining an upstream Va segment to a downstream Ja gene on one and, in some cases, both al- leles. These secondary rearrangements are in most cases productive, and result in the expression of a new TCRa polypeptide that replaces the original receptor. Thus, in these cells, the expression of a TCRa polypeptide does not block further rearrangements in the TCRa locus.

Results

Characteristics of the M14T Subclones The M14T line is a T cell lymphoma obtained by retroviral transformation of C.B20 thymic lymphocytes (Primi et al. 1988; see Experimental Procedures). M14T cells were found to be CD4-/CD8- and to express a CDS-associated surface heterodimer containing a TCRa chain possibly in association with a TCR8 chain.

To determine whether immunodifferentiation events oc-

Cdl 292

NIM REDUCED

REDUCED

Figure 1.

(A) Biochemical analysis of antiCa reactive proteins expressed on the indicated M14T subclones. 1251-labeled cells were immunoprecipitated with antiCa antiserum and run under reducing conditions on 10% SDS-PAGE gels. (6) Western blot ‘analysis of TCRa chain production in M14T sub- clones. Fifty microliters of lysates from 5 x lo6 cells was fractionated by electrophoresis over a 10% acrylamide gel under reducing and non- reducing conditions, and the proteins were transferred to nitrocellulose and tested for bound a-chains by sequential incubations with an anti- Ca antiserum and i251-labeled protein A.

cur during propagation of M14T cells, we derived a series of subclones by limiting dilutions on thymocyte feeder cells. Four independent isolates were selected, surface- labeled with 1251, lysed in NP-40 buffer, and immunopre- cipitated with a rabbit anti-Ca antiserum. Analysis of the precipitates by SDS-polyacrylamide gel electrophoresis

(SDS-PAGE) under reducing conditions revealed that, al- though all subclones tested express the TCRa$ heterodi- mer, these polypeptides displayed a different electrophor- etic mobility than the a and 8 chains precipitated.from the original M14T line (Figure 1A). These differences were reproducibly observed in several independent experi- ments. The results suggested that modifications of the TCR dimer had occurred during the in vitro propagation of the M14T clones. It was, therefore, essential to confirm that each of these sublines was derived from the same pa- rental cell. Total cellular DNA from each subclone was digested with EcoRl and Pvull and analyzed by South- ern blot hybridization to 32P-labeled C8 and JH specific probes. We previously determined that both M14T Igh al- leles had undergone D-J joining (Primi et al., 1988). Fig- ure 2 shows that all subclones display the same two Dn-JH rearrangements as the parental line. Both Cp al- leles exist as 9.0 kb rearranged bands in M14T and in each of the four sublines, and their C82 alleles remain in germ- line configuration (Figure 2). These results establish that each of the M14T sublines was derived from the same cloned, parental cell line. One possible explanation for the differences in the TCR electrophoretic mobility observed in some M14T subclones is that structural modifications have occurred in the TCRa chain component of these samples. To test this hypothesis directly, cells from several clones were lysed in NP40 buffer, fractionated by SDS-PAGE under reducing and nonreducing conditions, transferred to nitrocellulose filters, and tested for hybridi- zation with a rabbit antiCa antiserum and 1251-Protein A. The results of this Western blot analysis are shown in Fig- ure 1B. Under reducing and nonreducing conditions, a protein of relative molecular mass of 41 kd could be seen in all samples, which probably represents incompletely processed, free cytoplasmic TCRa chains. Under non- reducing conditions, high molecular weight bands of 90 kd (M14T, M14T-2) 92 kd (M14T-3, M14T-4, M14T-5), and 95 kd (M14T-6 and M14T-7) were also present. However, no additional higher molecular weight band was detected in clone M14T-1. The latter bands represent the TCRa chain complexed with the TCR8 polypeptide. Under re- ducing conditions, TCRa subunits migrated at 44 kd (M14T and M14T-2) 46 kd (M14T-4 and M14T-5) and 49 kd (M14T-3 and M14T-6). No TCRa chain was detected in the M14T-1 subline. A third, band of 47 kd was also expressed by clones M14T-3 and M14T-6. The size heterogeneity of the TCRa chains suggested that DNA rearrangements af- fecting the TCRa locus are involved.

Secondary Ja Rearrangemente Molecular analysis of the TCRa locus is complicated by the presence of 50 Ja segments that encompass close to 60 kb of DNA (Winoto et al., 1985; Arden et al., 1985). To analyze the organization of the TCRa alleles in our clones, we performed Southern blots with genomic probes that span about 50 kb of the Ja region (Malissen et al., sub- mitted). Total cellular DNA from M14T was digested with BamHl and sequentially hybridized to 32P-labeled Pr3 and Pr2 Ja probes (Figure 3). No hybridization was ob- served with probes located 5’ to probe Pr3 (Prll to Pr4)

Secondary Va-Ja Rearrangements in T Cells 293

kb e3.7-

S.S-

4.3-

w PVUII

Figure 2. Cg and JH Rearrangements in M14T Subclones

Approximately 5 pg of DNA from each subclone was digested with Pvull or EcoRI, electrophoresed through a 1% agarose gel, transferred to filters, and, respectively, assayed for hybridization to C!3 and JH specific DNA probes.

(Malissen et al. submitted and data not shown), suggest- ing that rearrangements on both TCRa alleles of the pa- rental M14T line had deleted these sequences. Pr3 and Pr2 hybridized to the same 7.3 kb BamHl germ-line frag- ment and to a 10.5 kb BamHl rearranged band, but an ad- ditional 4.6 kb BamHl rearranged species was only de- tected with Pr3 (Figure 4). These results establish that the M14T line has two rearranged Ja segments. One of these Ja segments is located within a 10.5 kb germ-line BamHl fragment spanning the 5’ end of the Pr3 while the other Ja segment is positioned within the 7.3 kb BamHl frag- ment that encompasses the S’end of Pr3 and Pr2 (see Fig- ure 4).

Hybridization of BamHI-digested DNAs of five M14T subclones with Pr2 and Pr3 revealed the existence of new, rearranged Ja fragments. In M14T-1, secondary Ja rear- rangements occurred on both alleles. One allele lost the original M14T 10.5 kb rearranged species and contains a new 3.5 kb Pr2 positive band. The other M14T-1 allele lost both its germ-line 7.3 kb fragment and the 4.8 kb rear- ranged band and now contains a 16 kb rearrangement de- tected by Pr2 and Pr3. The latter observation implies that one of these alleles has rearranged to a more 3’ Ja seg- ment The presence of the 10.5 kb fragment in clone M14T- 2 suggests that a secondary recombination has occurred on only one allele in this subline. The 7.3 kb germ-line and the 4.8 kb rearranged bands have been replaced by a 7.7

kb Pr2 and Pr3 positive fragment, implying that the rear- ranged Ja gene is now located in a DNA fragment be- tween Ja6-19 and the BamHl site 3’ to Ja6-19. M14T-2 and M14T-3 have both undergone identical secondary rearrangements. Weaker rearranged BamHl fragments of 8.0 kb and 5.0 kb are also present in M14T-2 and M14T-3, respectively, suggesting that minor fractions of these sub- lines contain different secondary rearrangements. Since the only Pr3 hybridizing fragment remaining in M14T-6 is the 10.5 kb band originally present in the M14T line, we conclude that a secondary rearrangement has only oc- curred on one allele in this clone. In M14T-6, the disap- pearance of the M14T 7.3 and 4.8 kb Pr3 positive frag- ments and the presence of a new Pr2 positive 4.5 kb rearranged band strongly suggests that a new Ja recom- bination occurred downstream of Ja6-19 on one allele. Novel secondary Ja rearrangements were detected on both M14T-7 alleles. One M14T-7 allele deleted the Pr3 positive 4.8 and 7.3 kb fragments of M14T, replacing them with a novel Pr2/Pr3 positive 17 kb band. A new Pr2 reac- tive 5 kb fragment is also present in the M14T-7 subline. Thus, in a manner similar to M14T-1, M14T-7 has under- gone new Ja rearrangements on both alleles.

TCRa Gene Expression To understand the molecular events that occurred in our subclones better, we constructed a cDNA library of M14T

Cell 294

kb 23.7-

7 9.5- T

6.7-

4.3-

Pr2 8amHl

Pr3 Bamtll

Figure 3. Ja Rearrangements in M14T Subclones

Ten micrograms of DNAfrom each subclone was digested with BarnHI, electrophoresed through a 1% agarose gel, transferred to filters, and assayed for hybridization to Pr2 and Pr3. Arrows indicate fragments that hybridize with both Pr2 and Pr3 probes.

ceils and screened it with a Ca probe. Sequence analysis of 12 cDNA clones revealed that two different Va genes are transcribed in M14T. The sequences of two represen- tative cDNA clones are shown in Figure 4. The Al3 clone contains a member of the Va8 family joined to an un- known Ja element (Jal4T), but as shown in Figure 4, the sequence of this clone is not in the correct translational reading frame. The A26 clone, on the other hand, represents a functional message with a member of the Va4 family joined in-frame with the Ja6-19 (Malissen et al., unpublished data) element, demonstrating that this al- lele codes for the M14T a-chain polypeptide expressed in M14T cells.

Northern blot analysis of total cellular RNA with TCRa and TCR6 probes revealed the presence of full-length TCRa and TCR6 RNAs in all samples (Figure 5). M14T-1 expressed a low level of TCRa transcripts, which was also reflected in the absence of TCRa polypeptide in this sub- clone (see above). Comparable amounts of mature-sized Va8 transcripts were only found in M14T and M14T-7. A low level of Va8 hybridization was seen in M14T-1 cells, and no signals were detected in the other sublines. Thus, the expression of full-length TCRa transcripts and the ab- sence of Va8 messages in clones M14T-2, M14T-3, and M14T-6 strongly suggest that the secondary Ja rearrange- ments observed in some subclones reflect Va-Ja gene replacement on the nonproductively rearranged allele of the M14T parental line.

Secondary Va Gene Rearrangements Southern blots of BamHI-digested DNAs were hybridized to a Vu8 probe and to a fragment of the A26 cDNA clone containing a Va4 variable region. Liver DNA contained four Vu8 positive bands with sizes of 15,8,6.2, and 4.8 kb. The 8 and 6.2 kb bands were absent in M14T DNA, while the 15 and 4.8 kb bands appeared to remain in germ-line context (Figure 6). However, the latter 4.8 kb band repre- sents the expressed Va8 gene in M14T since it comigrates with a novel Ja rearrangement (Jal4T) detected by the Pr3 probe (compare Figures 3 and 5). The existence of a Vu8 rearrangement in M14T was also confirmed by digest- ing M14T DNA with EcoRl (Figure 6). We also established that the remaining 15 kb germ-line Va8 gene is located on the nonproductively rearranged allele since M14T-6 cells have deleted all of the Vu8 genes and have retained the expressed Va4 (A28) allele in its original context (compare Figures 3 and 6). Thus, in M14T, productive joining of a Va4 gene to Ja6-19 on one allele results in the deletion of all members of the Vu8 family (Figure 7).

M14T-1 and M14T-7 contain novel rearranged Vu8 genes of 16 and 17 kb, respectively, which comigrate with a novel Ja rearrangement detected by the Pr2 and Pr3 probes (Figures 3 and 6). Thus, the secondary rearrange- ments that occur in these subclones involve the joining of the same 15 kb germ-line Vu8 gene, to two different 3’Ja segments with deletion of the intervening nonproductively rearranged Va8-Jal4T genes (Figure 7). All other sub- clones have deleted all of their Vu8 genes by joining other Va genes upstream of the Va8 family to a new down- stream Ja element. This interpretation is further sup- ported by the absence of Vu8 transcripts in these cells (Figure 5).

Analysis of liver DNA with the Va4 (A26) probe revealed the existence of five bands of 15, 8, 6.7, 5.8, and 4 kb. The 4, 6.7, and 8 kb fragments were absent in M14T DNA, but a new fragment of 10.5 kb was detected (Figure 6). This latter band comigrates with the Ja rearrangement (Ja6- 19) detected by the Pr2 probe (Figure 3), identifying this 10.5 kb Va4 rearrangement as the functional M14T allele. M14T-6 DNA contained the same functional Va4 allele as the parental M14T line, implying that the secondary Va-Ja rearrangement had occurred on the nonproductively rear- ranged allele in M14T-6.

Both M14T-1 and M14T-7 have deleted the 10.5 kb Va4 positive band, but retain the 5.8 and 15 kb germ-line frag- ments, suggesting that, in these clones, a secondary rear- rangement has joined Va genes 5’ to Va4 to Ja elements 3’ to Ja6-19 (Figure 7). M14T-2 and M14T-3 DNAs, on the other hand, retain the original rearranged 10.5 Va4 frag- ment but delete the germ-line 5.8 kb band. This particular pattern strongly suggests that the 5.8 kb fragment in M14T contains a germ-line Va4 gene which has been deleted from the functional allele but remains on the nonfunction- ally rearranged chromosome (see Figure 7).

TCRa Chain Replacements The TCRa gene rearrangements in some M14T subclones are likely to lead to structural modifications of the TCRa

%rxtdary Va-Ja Rearrangements in T Cells

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Figure 4.

A26: Nucleotide sequence of a portion of the A26 cDNA clone that specifies the functionally rearranged Va allele of the Ml4T line. The A26 sequence is shown in comparison with a member of the Va4 gene family (Becker et al., 1985). Variable, joining, and constant region sequences are indicated, and amino acids are given above each codon. Dots represent identical nucleotides. A13: Nucleotide sequence of a portion of the Al3 cDNA clone that specifies the nonfunctionally rearranged Va allele of the M14T line. The A13 se- quence is shown in comparison with a functionally rearranged member of the Va8 gene family, 82 (Becker et al., 1985). Variable, joining, and constant region sequences are indicated, and amino acids are given above each codon. Dots represent identical nucleotides. The asterisk between amino acids 93 and 94 of the Al3 sequence represents the site of V-J recombination and the amino acid in brackets is not present in either the Vu or Ja sequences. The Al3 Va-Ja join is not in the correct translational reading frame.

chain polypeptides. This is the case for M14T-1 and M14T- 7 cells in which Va-Ja gene replacement has occurred on both alleles (Figures 3 and 6). The M14T-6 clone has re- tained the original functional Va4-Ja6-19 rearrangement but has recombined the nonfunctional allele. Surprisingly, the a-chain polypeptide expressed by this line has a differ- ent electrophoretic mobility than the one in the parental M14T cells (Figure 1B).

To understand the significance of these differences bet- ter, the TCRs of M14T and M14T-6 were immunoprecipi- tated from lz51 surface-labeled cells with a rabbit anti-Ca antiserum. In a first series of experiments, the a-chain of these complexes was analyzed by equilibrium isoelectric focusing (IEF) in the first dimension, followed by SDS-PAGE in the second dimension. As shown in Figure 6, the a-chain of M14T has an isoelectric point in the range of 5.6-6.2, with an M, of 43,000, whereas M14T-6 has a more acidic a-chain with a M, of 49,000, confirming that structural changes have occurred in these two polypep- tides. Since the genomic contexts of the functional a locus of M14T and M14T-6 are identical, our finding indicates that the membrane form of the a-chain of the M14T paren- tal line is encoded by the Va4 (A26) allele, while the M14T-

6 TCRa chain is encoded by a functional transcript from a new Va-Ja rearrangement that occurred on the original nonfunctional M14T allele. To test this hypothesis directly, membrane as well as cytoplasmic a-chains of M14T-6 were isoiated by immunoprecipitating unlabeled cell lysates with antiCa serum. The isolated material was isoelec- trofocused in the first dimension and run on SDS-PAGE in the second dimension. Proteins were then transferred to nitrocellulose filters, and revealed with the antiCa anti- serum and 1251-labeled protein A. Figure 8 shows that an a-chain with M, and isoelectric points identical to the a-chain detected on M14T-6 membranes and a second a-chain with M, and isoelectric points identical to the one originaliy expressed by M14T are coexpressed in M14T-6 cells. These data establish that, at least in the cells stud- ied here, the product of a functional a gene does not pre- vent further recombinations and that this secondary mo- lecular event may lead to the existence of two u-chain polypeptides in the same cell.

Discussion

Allelic exclusion, which is central to the process of clonal

Cdl 296

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w

Figure 5. Northern Blot Analysis of mRNA from M14T Subclones

Ten micrograms of total cellular RNA was electrophoresed and trans- ferred to a,nylon membrane. The same filler was sequentially hybrid- ized to Cp, Ca, and Va8 specific cDNA probes. The filter was auto- radiographed for 18 hr after Cf3 and Ca and for 60 hr after Va8 hybridization.‘The positions of migration of 28s and 18s rDNAs are in- dicated.

selection in B cells, occurs because only one light chain gene and one heavy chain gene are productively rear- ranged. The other alleles are not rearranged or are non- productively rearranged (Perry et al., 1980; Coleclough et al., 1981; Walfield et al., 1981; Ritchie et al., 1984). The presence of a productively rearranged gene, however, is not sufficient to signal allelic exclusion, which may be in part controlled by the membrane form of the u chain (Nus- senzweig et al., 1987) (for review, see Alt et al., 1986).

Here, we show that a T cell line that already expresses the product of a functionally rearranged TCRa gene can undergo secondary Vu-Ja recombinations accompanied

by deletion of the intervening DNA sequences, thereby resulting in the expression of new TCRa polypeptides.

Both TCRa alleles in the M14T line undergo secondary rearrangements while its TCR8 genes are stable. The pa- rental M14T clone had Vu-Ja rearrangements on both chromosomes. One allele contains a functional Va4- Ja6-19 join while a nonfunctional rearrangement of a Vu8 gene resides on the other allele. This type of receptor gene organization resembles that of immunoglobulin genes in many 6 cells, in which only one of the two avail- able alleles can encode a functional polypeptide. How- ever, contrary to B cells, the expression of a functional Vu-Ja complex did not prevent further joining of an up- stream Vu to a downstream Ja gene in several M14T sub- clones. The nonproductively rearranged Va8-Jal4T com- plex was replaced in all subclones analyzed while the functional Va4-Ja6-19 allele was retained in three out of the five subclones. Many of the secondary rearrange- ments in M14T subclones resulted in the generation of Ja positive rearranged fragments of different sizes. More- over, we were able to determine that different Vu genes are used in the subclones. These results are not consis- tent with the selective cloning of populations of cells predi- sposed to a defined secondary recombination, but rather suggest that these secondary molecular events can utilize several of the still available Va and Ja germ-line elements. The secondary recombinations on the nonfunctional al- leles of M14T-1 and M14T-2 use different Vu genes. Simi- larly, M14T-1 and M14T-7 join the remaining member of the Vu8 family in the parental line to different Ja segments. The genomic context of M14T and its derivatives suggests that members of the Vu4 family are located 5’ to the Vu8 genes. Since the secondary rearrangements in M14T-2 re- sult in the deletion of the Va8B, Va8A, and Vu4 genes, the relative distance of a Vu gene to the primary rearrange- ment is not a limiting factor for its usage in secondary recombinations. The molecular mechanism responsible for these Vh gene replacements will await the structural analysis of their recombination sites. This phenomenon would seem to differ in at least one respect from lg locus Vu gene replacements described for several immature B cell lines as the J gene segments did not participate in this latter phenomenon (Reth et al., 1986; Kleinfield et al., 1986).

There is one important question that concerns the phys- iological significance of our findings. We recently detected secondary Vu-Ja rearrangements in another immortal- ized cell line with a CD4+/CD8+, CD3+ phenotype that had been obtained independently from M14T. The phe- nomenon, therefore, is not restricted to the properties of one particular transformed cell nor to cells with a particu- lar surface matter phenotype. It can be argued, however, that these secondary recombinations can only occur in transformed cells in which the normal regulatory signals may be altered. However, two lines of evidence strongly suggest that Vu-Ja replacements are important physio- logical events. Recently, the analysis of DNA clones iso- lated from circularized DNA extracted from normal thymo- cytes has been reported (Okazaki and Sakano, 1988). The sequence of some of these clones revealed the presence

.%&ondary Va-Ja Rearrangements in T Cells

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Figure 6. Vu8 and Vu4 Rearrangements in M14T Subclones

Ten micrograms from each subclone was di- gested with BamHl or EcoRI, electrophoresed. transferred to filters, and analyzed for hybrid- ization either to a Va8 or to a Va4 specific cDNA probe.

Figure 7. Schematic Representation of the Molecular Events Occurring at the TCRa Locus of M14T and Its Derivatives

Arrow-capped lines represent the sizes of the restriction fragments detected with the indi- cated probes, and rearranged Va-Ja genes are indicated as black boxes. B indicates the BamHl sites. Circles represent Bamtil sites that have been deleted, while squares indicate the positions of the new restriction sites. Germ- line DNA segments are indicated as dotted lines. The Va8 and Vu4 family members have been arbitrarially named Va8A. VaBB, and Va4A. All other nonidentified Va and Ja genes are indicated with other capital letters.

Pr3 Pr2 Vi8

4 I

PH

6

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se-

Ml4T (membrane)

M14T-6 (membrane)

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M14T-6 (cell &sate)

Figure 8. lsoelectrofocusing of the TCRa Subunit from M14T and M14T-8

(Top) lmmunoprecipitates of 1251-labeled membranes with antiCa an- tiserum were subjected to equilibrium isoelectric focusing in a tube gel followed by SDS-PAGE in a 10% slab gel. The pH gradient was estab- lished in a separate tube gel, run in parallel with the experimental gels. (Sottom) lmmunoprecipitates of M14T-8 total cell lysates with anti& antiserum were treated as above. The gel was electroblatted on a nitrocellulose filter, and the TCRa chains were revealed by sequential incubations with anti-Ca antiserum and r251-protein A.

of Vu genes functionally rearranged to Ja segments. Since circularized DNA represents deleted sequences fol- lowing rearrangements, the most likely interpretation of these findings is that some normal thymocytes have un- dergone secondary rearrangements that have deleted the preexisting functional Vu-Ja complexes. Moreover, since these studies involved a relatively limited number of clones, the results further suggest that Vu-Ja replace- ment occurs at a high frequency in normal T cells.

One implrcation of our findings is that some fully differ- entiated immunocompetent T cells should permanently retain a detectable imprint of the secondary genomic events that occurred in the early stages of differentiation, i.e., the presence of two functionally rearranged TCRa al- leles in the same cell. Indeed, the presence of two func- tionally rearranged TCRa alleles has been described in a helper T cell clone (Malissen et al., submitted). Taken to- gether, these observations are compatible with our find- ings, and further suggest that our experimental model represents a valuable tool for dissecting the molecular events that occur in the early stages of differentiation of normal thymocytes.

The findings reported here also give rise to important questions concerning the functionality of the T cell reper- toire. The random assembly of TCR gene segments is essential for amplifying the otherwise limited germ-line repertoire of these genes, but on the other hand, may pro- duce T cell antigen receptors that recognize self-deter- minants. Thus, proper selection is essential for coordinat- ing the function of the immune system. Additionally, the T cell receptor must also be selected to recognize anti- gens in the context of the major histocompatibility com-

plex (MHC). Although recent experiments suggest that the unselected T cell repertoire is already biased toward the recognition of self-MHC antigens (Kappler et al., 1987a, 1987b), MHC restriction is not determined by the T cell genotype but by the MHC products expressed in the thy- mus. Random alterations of TCR specificities imposed by the Vu gene replacement mechanism described here would change the repertoire of the clones selected in the thymus. One possible solution to this paradox can be found in the particular phenotype of the clones studied here, which belongs to a subset of recently characterized T cells (Fowlkes et al., 1987; Budd et al., 1987). However, we have recently observed the same phenomenon of Vu-Ja replacements in a CD4+/CD8+ line, which sug- gests that this is a general mechanism in early thymo- cytes. We would like, therefore, to suggest that in more mature T cells, further secondary rearrangements are prevented either by a decrease of the recombinase activ- ity or by a mechanism that renders the TCR locus inacces- sible to the enzyme. This hypothesis is supported by the fact that the genomic context of the M14T line has re- mained stable for more than one year of culturing, while secondary recombinations have occurred at high fre- quency if these cells were cloned in the presence of thymocytes. Moreover, Vu replacement, was also ob- served after in vivo transfer of M14T (Primi et al., unpub- lished data). These observations suggest that environ- mental factors in the thymus are responsible for the progressive series of TCRa gene rearrangements in the M14T line.

In conclusion, our results demonstrate that the rules that govern TCRa chain expression in at least some T cells are different from those that control the expression of immunoglobulin and TCR8 genes. A complete under- standing of the physiological significance of our findings awaits a more extensive analysis of TCRa genes and their products in T cell subsets representative of different stages of T lymphocyte differentiation.

Expsrlmental Procedures

Cell Lines The M14T line is a T cell lymphoma obtained by retroviral transforma- tion of CB20 thymocytes (Primi et al.. 1986). This line was cloned by limiting dilution soon after it adapted in vitro. One clone, (M14T) was selected and was continuously grown in vitro for 9 months. (M14T) sub- clones were obtained by limiting dilutions on SALBlc thymocytes feeder cells (3 x lrZ@/ml) in 0.2 ml volume.

Antlbodies The antiCa antiserum used in these experiments was obtained by in- jecting rabbits with a chimeric protein containing the Ig Vk and TCR Ca domains (kindly provided by Dr. K. Karjalainen) (Traunecker et al., 1986).

1251-Labeling, Immunopreclpitstlons, and Gel Electmphoresis Cells were either 1251-labeled by the lactoperoxydase catalyzed reac- tion or washed three times in phosphate buffer saline (PBS). The radio- iodinated or unlabeled cells were then lysed by resuspension in ice- cold lysis buffer containing 05% Triton X100,0.15 M NaCI, 10 mM Tris (pH 8.2) EDTA 5 mM, 100 mM iodoacetamide, and 1 pm phenyl- methylsulfonyl fluoride. After 30 min on ice, insoluble material was re- moved by centrifugation for 50 min at 18,000 x g.

For immunoprecipitation, radiolabeled cell lysates were precleared

Secondary Va-Ja Rearrangements in T Cells 299

with protein A and with rabbit immunoglobulins. The precleared cell ly- sates were incubated with 10 ul of rabbit antiCa antiserum and 25 pl of protein ASepharose beads for 1 hr at room temperature and washed four times with lysis buffer containing 1 mM EDTA and 0.1% sodium dodecyl sulfate (SDS). The immunoprecipitated material was analyzed on one-dimensional SDS 10% polyacrylamide gels. For two- dimensional electrophoresis, immunoprecipitated material was run in an equilibrium isoelectric focusing (IEF) tube gel as a first dimension followed by SDS-PAGE on a 10% slab gel in the second dimension. For Western blot analysis, unlabeled cell lysates were separated on SDS 10% polyacrylamide gels. Proteins were electroblotted to nitrocel- lulose and revealed by sequential incubations with the rabbit anti& antiserum and ‘*+labeled protein A. For analysis of intracellular and membrane-bound Ca chains, unlabeled cell lysates were immunopre- cipitated with rabbit antiCa antibodies. The immunoprecipitated pro- teins were subjected to two-dimensional electrophoresis, electroblot- ted to nitrocellulose filters, and revealed by sequential incubations with the rabbit antiCa antiserum and 1251-labeled protein A.

RNA and Poly(A)- RNA Preparation Total cellular RNA was prepared by the guanidinium thiocyanate/hot phenol method (Feramisco et al., 1962). Poly(A)+ RNA was isolated by two cycles of binding to oligo (dT) cellulose (Maniatis et al., 1982). The integrity of the poly(A)+ RNA was verified by Northern hybridization with an [a32P]dCTP labeled GAPDH (glyceroldehyde 3phosphate de- hydrogenase) (Piechaczyk et al., 1964) probe.

Preparation and Scmening of the M14T cDNA Library Ten micrograms of poly(A)+ RNA was converted into double-stranded cDNA using a BRL cDNA synthesis kit (BRL). 5’ and 3’ ends of the cDNA were flushed with T4 polymerase (BRL). Internal EcoRl sites were methylated with EcoAl methylase (New England Biolabs) prior to the addition of EcoRl linkers. The cDNAs were ligated into the EcoRl site of S-ZAP (Stratagene).

Recombinant phage DNAs were packaged using the Gigapack gold in vitro packaging extract (Stratagene). I-ZAP recombinant phages were plated on E. coli 884 at a density of 20,000 plaques/l50 mm plate. Plaques were lifted onto nitrocellulose filters and prehybridized for 4 hr at 65OC in 3x SSC, 5x Denhardt’s solution, 5 mM EDTA, and 0.1% SDS. Hybridization was carried out in the same solution containing 10 &ml-’ poly A, 50 uglml sheared salmon sperm DNA, and 3 x lo5 cpmlml 32P-labeled probe for 20 hr. Positive phages were cloned and their cDNA inserts subcloned into p-Bluescript SK(-) (Stratagene).

DNA Sequencing Sequencing of double-stranded p-Bluescript SK(-)/cDNA clone tem- plates was carried out according to the dideoxy chain termination method (Sanger et al., 1977) using Sequenase (United States Bio- chemical Corp.). Sequencing reactions were carried out using [32P] dATP and a synthetic oligo primer, 5’-CTGTCCTGAGACCGAGGATC-3’. which is complementary to a portion of the first domain of the murine TCRa constant region gene.

Southern and Northern Blot Analysis Genomic DNAs were digested with the indicated enzymes and electro- phoresed in 0.8% agarose gels and transferred to Hybond filters (Amersham). Hybridizations were performed for 18 hr at 65OC. Total cellular RNAs (10 Kg) were electrophoresed, transferred to Hybond filters. and hybridized overnight at 65OC.

Probes The probes used in this work were: a BamHI-EcoRI fragment of the JH locus (JH) containing JH3 and JH4 (Marcu et al., 1980). a 618 bp BamHI-EcoRI fragment of the 66TI clone (C,) (Hedrick et al., 1964) (kindly provided by Dr. M. Davis), a 1.1 kb EcoRl fragment of plasmid T 1-2 (Ca) (DembiC et al., 1965) (a gift from Dr. M. Steinmetz), a 400 bp Rsa-Spel fragment of cDNA clone A26, which contains 375 bp of a Vu4 variable region and 25 bp of L-ZAP poly linkers, and a 400 bp EcoRI-Hindlll fragment containing the Va6.4 variable region (a gift from Dr. E. Palmer). The Ja probes used were: Pr4, Pr3, Pr2. and Pr2 (Malissen et al., submitted).

Acknowledgments

We thank Dr. J. Levy for advice on cDNA cloning, and Drs. D. Ott, E. Jouvin-Marche, and B. Malissen for helpful comments and stimulating discussion. We thank Dr. K. Karjalainen for his generosity in providing invaluable reagents, and Dr. A. M. Drapier for excellent technical as- sistance. We would also like lo thank M. Berson for her terrific secretar- ial and editorial help.

This work was supported by a grant from the Association pour la Re- cherche sur le Cancer (ARC), the Fondation pour la Recherche MBdicale, the CNRS, the INSERM, and, in part, by National Institutes of Health grant GM26939 to K. B. M. J. D. F. was supported by an NIH training grant (GM97964) awarded to the Genetics Graduate Program, SUNY at Stonybrook.

The costs of publication of this article were defrayed in part by the payment of page changes. This article must therefore be hereby marked “‘advertisemen? in accordance with 18 USC. Section 1734 solely to indicate this fad.

Received May 31, 1988; revised July 21, 1968.

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Note Added in Proof

The data referred to as Malissen et al., submitted, is now published: M. Malissen, J. Trucy, F. Letourneur, M. Rebai’, D. E. Dunn, F. W. Fitch, L. Hood, and B. Malissen. Cell 55, 49-59.