Immunology 1993 78 252-259

Diversity of functional T-cell receptor 6-chain transcripts from bonemarrow cells of athymic nude mice

S. SPIEB, A. KUHROBER, R. SCHIRMBECK, B. ARDEN* & J. REIMANN Institute of Microbiology,University of Ulm, Ulm, Germany and *Max-Planck-Institute for Immunobiology, Freiburg, Germany

Accepted for publication 19 August 1992

SUMMARY

CD3 + cells are detectable in bone marrow of athymic mice homozygous for the nude mutation. Aspreviously shown, cells expressing the y6 T-cell receptor (TcR) represent 30-40% of this T-cellpopulation. Using VW-specific, Vi4-specific, and Cb-specific primers, TcR 6-chain transcripts were

reverse transcribed and polymerase chain reaction (PCR)-amplified from total RNA prepared frombone marrow cells (BMC) of 6-month-old NMRI nu/nu mice. Amplified TcR 6-chain eDNA was

cloned, and 49 randomly selected clones derived from seven amplification reactions were sequenced.Sequence analyses showed: (1) more than 80% of the sequenced clones represented in-frametranscripts of the TcR 6-chain; (2) in-frame transcripts containing V61-, V62-, V63-, V64-, Vb5-, V66-and Va4-gene segments were detectable in nude BMC; (3) V62-, V64- and V65-containing transcriptswere more abundant and more diverse than V61- and V63-containing transcripts; (4) extensive N-region diversity was present in the V-D62 (N 1), D62-D6 1 (N2) and D6 1 -J6 I (N3) junctional regions;(5) P nucleotide additions were present in many transcripts; and (6) unusual truncated, in-frametranscripts with deleted D- and J-region genes were detected. A large potential TcR 6-chain repertoireis thus present in nude BMC.

INTRODUCTION

Lymphoid stem cells in bone marrow (BM) give rise to thedifferent developmental lineages of T and B lymphocytes. Themainstream of T-cell development goes through the thymuswhere functional maturation of T cells and the selection of theT-cell receptor (TcR) repertoire for self-tolerance and self-restriction takes place (reviewed in ref. 1). An additionalextrathymic pathway of T-cell differentiation is suggested by theobservation that T cells develop in athymic nude mice. Thy- I +CD3 + T lymphocytes are detectable in spleen, lymph nodes,liver and BM of adult nude mice.2-22 These T cells express eitherthe 'single-positive' (CD4+ CD8- or CD4- CD8+) or the'double-negative' (CD4- CD8-), but not the 'double-positive'(CD4+ CD8+) phenotype.3'68'9"1222 A relative deficiency ofCD4+ T cells is observed in most analyses of lymphoid organsfrom nude mice.9 '3 Surface expression of CD3- or TcR-associated determinants on peripheral T cells from athymic miceis lower than that found in peripheral T-cell subsets fromeuthymic mice.'4"16 The extremely variable TcR-V/3 usage pat-

Abbreviations: BM, bone marrow; BMC, bone marrow cells; SC,spleen cells; LN, lymph node; LNC, lymph node cells; PCR, polymerasechain reaction; TcR, T-cell receptor for antigen.

Correspondence: Dr J. Reimann, Institute of Microbiology, Univer-sity of Ulm, Oberer Eselsberg, Albert-Einstein-Allee 11, D-79 Ulm,Germany.

terns of T-cell populations obtained from individual nude miceis an indication of oligoclonality of TcRcf3+ T cells in nudemice.'6'23 Peripheral T cells from nude mice express TcR-VJJchain gene segments with self-reactive specificity,'6'24'25 which aredeleted in the thymus of euthymic mice. In vitro clonalexpansion of CD4+ and CD8+ nude T cells seems impairedalthough these cells display substantial interleukin-2 (IL-2)secretion and IL-2 receptor expression.5"2"3 Cloned CD4+ andCD8+ T-cell lines have been established in long-term culturefrom T-cell populations obtained from nude spleen or lymphnodes.2" 13,17,20 The recognition specificity of nude-derivedcytotoxic T cells seems degenerate.2'7

Within the T-cell system, mutually exclusive T-cell subsetsexpress alternative types of TcR. The large majority of humanor murine T cells express the heterodimeric TcRo/3.26 MostTcR43+ T cells recognize peptides in association with majorhistocompatibility complex (MHC)-encoded glycoproteins. Adifferent T-cell subset expresses a TcR composed of y6 hetero-dimers.2" Recognition specificities and MHC restriction patternof this T-cell subset are uncertain. The TcR repertoire of foetalthymus-dependent yb T cells, and of some y6 T-cell populationsresiding in particular anatomical sites is unusually restricted.28 Tcells expressing the TcR4,B or the TcRy6 develop in nudemice.'8'2' y6 T cells are over-represented in BM, liver, spleen andlymph nodes of nude mice. 18"1921"22 The functional role ofextrathymic y6 T-cell populations is uncertain. 'Double-nega-tive' TcRyb+ T cells from spleen or lymph node of nude miceseem to be able to abrogate oral tolerance."'

252

Diversity of TcR 6-chain transcripts

Table 1. Sequences of synthetic primers used to amplify TcR 5-chaincDNA. Sequences of the primers for V6 I, V62, V53, V65 and Vax4were taken from ref. 38. The sequences of the primers for V64, V66

and C6 were designed by the authors

V61 CACGTCGACCAGTTGCCAAAACTTTTACTGTV62 CACGTCGACAAGAAGGAGATGAAGTCACCAV63 CACGTCGACCTCTTCAGGGTCCAGAATAGV64 CACGTCGACAAGTCTGTGCAGGTGGCAGV65 CACGTCGACCCCAGATTTATTTTGGTATCGCAV66 CACGTCGACAACAAGCAGGCAGGAGGG

C6 CTGGGGGAGATGACTATAGC

Va4 CTTACGATACACTGCAACTAC

Extrathymic differentiation of TcRacf+ and TcRyb+ T cellsmay take place in the liver. T cells of both subsets are present inmouse liver. 19,29-31 Substantial numbers of T cells accumulate inthe liver of adult nude mice.'9 The T-cell populations in liver areincreased in tumour-bearing hosts,29 during bacterial infec-tions,30 and in autoimmune MRL-Ipr/lpr mice.3' Alternatively,T cells may develop in situ in BM, gut or spleen from lymphoidstem cells. The in vitro generation of cytotoxic Thy- 1 + CD3 + Tcells from Thy- I - CD3 - non-functional precursor cells in BMCpopulations has been described.3233 Progressing TcR rearrange-ment patterns were detected in Southern analyses of T-cell-depleted bone marrow cell (BMC) cultures suggesting that earlysteps of T-cell differentiation can be induced in vitro in BMC.34Induction of the 'single-positive' CD4- CD8+ phenotype hasbeen demonstrated in in vitro clonally expanded nude BM-derived 'double-negative' CD3+ T cells.'8 Expression of theCD8 molecule in extrathymic T cells may represent an acti-vation but not a lineage marker.2535-37 It is therefore of interestto define the emerging TcR repertoire in nude BM. The diversityof TcR 6-chain transcripts expressed in nude BMC was

determined in the present study. The obtained sequence dataindicate a large diversity of the potentially functional TcR 6-

chain repertoire.

MATERIALS AND METHODS

MiceOutbred NMRI nu/nu mice were obtained from the Zentralinsti-tut fur Versuchstierzucht (Hannover, Germany). Mice were

kept under specific pathogen-free (SPF) conditions in theanimal colony of the University of Ulm (Ulm, Germany). Six-month-old female mice were used.

Isolation ofRNA, cDNA synthesisSingle-cell suspensions were prepared from BM ofNMRI nudemice. Total RNA was isolated from 6 x 107 BMC obtained fromfemurs and tibiae of four nude mice by the guanidiniumisothiocyanate/CsCl method. Isolated RNA was purified byphenol/chloroform extraction and precipitated. The quality ofthe RNA was checked by electrophoresis in a 1% formamide-containing agarose gel run in I x MOPS buffer.

cDNA was synthesized from 5 pg total RNA using theMMLV- reverse transcriptase kit (BRL, Eggenstein, Ger-many).

Amplification ofcDNA by polymerase chain reaction (PCR)The TcR 6-chain transcripts were selectively amplified fromnude BMC cDNA by PCR using the oligonucleotide primerslisted in Table 1. Six V-specific primers (V6 1-6) and one Va-specific primer (Va4) were used. The murine Va4 gene segment isknown to rearrange in-frame with C6,27 and was included as afurther control. All oligonucleotide primers were synthesized onthe 38-lA DNA synthesizer (Applied Biosystems, Foster City,CA). The PCR reaction buffer was composed of 10 mm Tris-HCl, pH 8-3 at 250, 50 mM KCl, 0-01% (w/v) gelatin, 0-1IUm ofeach primer, 0-2 mm dNTP and 1 U of Taq DNA Polymerase I(Perkin-Elmer/Cetus, Munich, Germany) in a reaction volumeof 100 Ml. For primer combinations V64/Cb, V65/Cb and Va4/C6, the MgCl2 concentration was 1 5 mM; for the V61/C6, V62/C6, V63/Cb and V66/C6 primer combinations, the MgCl2concentration was 3 mm. The mixture was overlaid with 80 pl ofparaffin oil (Merck, Darmstadt, Germany). PCR were subjectedto 30 cycles of amplification. Each cycle consisted of 45 secondsdenaturation at 92°, 15 min annealing at 56° and 15 minextension at 72°. A further extension step of 1 hr ensured that thereaction was completely terminated. One-tenth of each PCR-amplified DNA was analysed by electrophoresis in a 1-8%agarose gel run in 1 xTBE buffer. Subsequently, gels wereblotted onto nitrocellulose filters and hybridized overnight to a5' Cb-probe labelled by random priming. The 5' Cb probe used,a 210 base pair (bp) HgiAI/EcoRI cDNA fragment containingJ61 and the first 126 bp of C6 (B. Arden, unpublishedobservation) was 5' ofthe C6 primer used. Control PCR withouttemplate were included in all experiments. No bands weredetected in this negative control group on ethidium bromide-stained gels or after Southern hybridization.

Sequencing of PCR-amplified TcR 6-chain transcriptsPCR products were digested with the following restrictionenzymes: Vb/Cb-amplified DNA was digested with SalI/EcoRI,the Vcx/Cb-amplified DNA was digested by Smal/EcoRI. Thedigested DNA was cloned into SalI/EcoRI or SmaI/EcoRIdigested Ml3mpl9 RF (Boehringer, Mannheim, Germany).Escherichia coli cells DHSa were transformed with recombinantMl 3mp19 and white plaques were picked from IPTG/X-gal/LB-plates. Single-strand (ss) DNA from recombinant phageswas spotted onto nitrocellulose filters and hybridized to the 5'C6 probe described above. Clones giving a positive signal inSouthern analyses were sequenced using a modification of theSanger dideoxy chain-termination method.

RESULTS

PCR-amplified TcR 6-chain transcripts from nude BMC

As shown previously,' non-adherent, low-density cells repre-sent 3-8% of non-fractionated BMC populations from nudemice. In this subset 5-10% of the cells display the CD3+low CD4-CD8 - phenotype. About 60% ofthese T cells express the TcRac/and 40% express the TcRy6. This was demonstrated in flowcytometric analyses of fractionated nude BMC populations,and in Northern analyses of TcR6 and TcRa transcriptsexpressed by BMC of this subset. After clonal expansion underlimiting dilution conditions in vitro, these T cells generatedTcRafl+ or TcRyb+ clones which expressed the CD4- CD8+phenotype.' In the present study the diversity and rearrange-

253

S. Spiefi et al.

VSI VS2 V83 V84a b a b a b a b

V85 V86 Va4a b a b a b

Figure 1. Expression of TcR 3-chain genes by bone marrow cells from nude mice. (a) Agarose gel; (b) Southern hybridization to C3probe.

Table 2. Sequence analysis of PCR-amplified nude BM-derived TcR 3-chain cDNA clones

Vb,a n* Clonest orft Nl D61 N2 D32 N3 J61 J32

V61 11 2 2 1 1 1 1V62 7 7 7 4 4 6 7 3 7V33 6 2 1 2 2 1 2 2V54 10 5 4 2 2 2 4 5 5V35 6 6 3 4 5 5 6 3 6V36 7 4 4 1 1 2 1 3

Va4 2 1 1 1 1 1 1

* The number (n) ofcDNA clones sequenced.t The number of distinct TcR rearrangements (clones) detected.The number of clones with in-frame rearrangements.

ment patterns of TcR 3-chain transcripts obtained from nudeBMC were investigated.RNA was prepared from non-fractionated BMC of four 6-

month-old NMRI nu/nu donor mice. Total RNA was reversetranscribed into cDNA, and TcR 3-chain transcripts wereselectively amplified by PCR from aliquots ofthis cDNA using aCb-primer, and either of six Vb (Vb 1, V32, V63, V64, V65, V66)primers, or the Va4 primer listed in Table 1. All seven PCRyielded DNA fragments of the expected 0 5 kb length in at leastfour independent amplifications (representative examples areshown in Fig. 1, left lane). Southern analyses with a 5' C3 probeshowed all seven types ofamplified DNA fragments to hybridizewith varying intensities with this probe (Fig. 1, right lanes). TcR3-chain transcripts are thus present in nude BMC and all sixdescribed V3 gene segments are rearranged in cells ofnude bonemarrow origin.

Sequence analysis of PCR-amplified TcR &chain transcriptsDNA fragments amplified in PCR were cloned into theM13mpI9 vector. A total of 49 randomly selected clones fromseven PCR were sequenced. In these 49 sequences, 27 distinctrearrangement events were detected (Table 2).

In some amplification reactions, every individual clonesequenced was different, e.g. in the Cb/V32 PCR (seven out ofseven clones sequenced) or in the Cb/V65 PCR (six out of six

clones sequenced). In contrast, only two distinct rearrangementevents were found in 11 sequences obtained from the C3/V31PCR, or in six sequences obtained from the Cb/V33 PCR.Although all six known V3 gene segments can be rearranged innude BMC, the diversity patterns and/or the frequency of usageof particular TcR 3-chain-expressing clones in these cells seemunequally distributed.

Nucleotide sequences of the 27 different TcR 3-chaintranscripts are presented in Fig. 2 (and see Fig. 4). In addition tothe diversity in V3 gene segment usage, extensive junctionaldiversity was evident in most sequenced transcripts. Insertionsof one to 15 nucleotides not encoded in the germline werepresent in all clones in the V3/Db 1 (N 1), the D3/D32 (N2) and/or the D32/J3 1 (N3) junctional regions. In each of the three N-regions, about 50% of the transcripts showed insertions. TheD32 gene segment was preferentially rearranged in more than80% of the transcripts, while the D31 gene segment wasrearranged in only 50% of the transcripts. In clones NuVDl .1and NuVD2.4, four nucleotides listed as N-region nucleotides,GGAG in N2 and AGGG in N3 respectively, could alterna-tively be interpreted as in tandem rearrangement of twofragments of the D32 gene segment, not unprecedented ineuthymic mice (B. Arden, unpublished observation). Exonuc-leolytic digestion extensively reduced the 11 bp germline D31sequence such that only three to nine nucleotides of thissequence were detectable in the TcR 3-chain transcripts. Incontrast, a more extensive part of the 16 bp germline D32sequence was retained in most transcripts. All 26 clonessequenced contained J3 1 rearrangements. J32 was not found inany of these clones. Two unusual transcripts lacking D and Jgene segments are described below. In contrast to most foetaland some organ-specific TcR 3-chain rearrangement patterns,the data on nude BMC TcR 3-chain expression indicate anextensive potential diversity of the repertoire of this T-cellsubset.

Most nude BMC-derived TcR 8-chain transcripts are potentiallyfunctional

About 80% of the different sequenced transcripts (21 out of 27)were rearranged in-frame (Fig. 3). In some rearrangements, oneof three possible open reading frames of D32 was preferentiallyused, e.g. translations of five out of seven sequenced V62rearrangements used the same D32 reading frame. In otherrearrangements, all three open reading frames were used at

20 -16-

0 . ..

0-40*350.3

254

Diversity of TcR 6-chain transcripts 255

clone V61 N1 D61 N2 D62 N3 J61.___________________________________________________________________________________________________germline TGGGTCAGATAT GTGGCATATCA ATCGGAGGGATACGAG CTACCGACAAANuVD1.1 TGGGTCAGATAT GGAG CGGAGGG GCC TAGCTACCGACAAA

____________________________________________________________________________________________________

V62 N1 D61 N2 D62 N3 J61.___________________________________________________________________________________________________germline TGTGGAGGGAAA GTGGCATATCA ATCGGAGGGATACGAG CTACCGACAAANuVD2.1 TGTGGAGGGAAA GATG GGG CTACCGACAAANuVD2.2 TGTGGAGGG CCTCTT CAT ATCGGAGGGA CTACCGACAAANuVD2.3 TGTGGAGGGAAA G GTG CCGGG GGGATACGA A CTACCGACAAANuVD2.4 TGTGGAGG TCT TATCGGAGGGATA AGGG CCGACAAANuVD2.5 TGTGGAGGGAAA G GTGGC GCC ATCGGAGGGATACGAG CTACCGACAAANuVD2.6 TGTGGAGGGAAA GCCCTA GGAG CTACCGACAAANuVD2.7 TGTGGAGGGAAA CCGGG ATC GT CGGAGGGATACGAG GG CGACAAA

____________________________________________________________________________________________________

V63 N1 D51 N2 D62 N3 JE1.__

germline TATCCTGAGAGT GTGGCATATCA ATCGGAGGGATACGAG CTACCGACAAANuVD3.1 TATCCTGAGAGT TC CAT ATCGGAGGGAT ACCGACAAANuVD3.2 TATCCTGAGAGTA GCAGA GGCAT TCC ATATCGGAGGGATACG CTACCGACAAA

____________________________________________________________________________________________________

V64 N1 D61 N2 D62 N3 J61.___________________________________________________________________________________________________germline TCTCATGGAGCG GTGGCATATCA ATCGGAGGGATACGAG CTACCGACAAANuVD4.1 TCTCATGGAGCGCG TC CAT ATCGGAGGGATACGAGCT GC CAAANuVD4.2 TCTCATGGAG ATATCGGAGGGATACG CCCGCTC CAAANuVD4.3 TCTCATG AGCGTTGCT CCGACAAANuVD4.4 TCTCATGGAG A GCATA CGTGTCCTCCCCCCT ATACGAGCTC CCGACAAANuVD4.5 TCTCATGGAGCG GGAGGGATACG GTTCCCT TACCGACAAA

____________________________________________________________________________________________________

V65 N1 D61 N2 D62 N3 Jc1----------------------------------------------------------------------__-----__---------------------

germline GCCTCGGGGTAT GTGGCATATCA ATCGGAGGGATACGAG CTACCGACAAANuVD5.1 GCCTCGGGGTAT TG CGGAGGGATA AA AGCTACCGACAAANuVD5.2 GCCTCGGGGTATAT GTGGCA ACCCCCCT ATATCGGAGGGAT ACCGACAAANuVD5.3 GCCTCGGGGTATA GGCAT TCC GAGGGATACGAGC CCGACAAANuVD5.4 GCCTCGGGGT GGCATAT TG5GTT TCGGAGGGATACG ACCGACAAANuVD5.5 GCCTCGGG CC TGGCAT TCCCCCT ATATCGGAGGGATACGAG CTACCGACAAANuVD5.6 GCCTCGGG TACA ACGTGGCATAT ATCGGAGGGAT TC TGTCGGTAGCTACCGACAAA

____________________________________________________________________________________________________

V66 N1 D61 N2 D62 N3 J61____________________________________________________________________________________________________

germline TCTCTGGGAGCT GTGGCATATCA ATCGGAGGGATACGAG CTACCGACAAANuVD6.1 TCTCTGGGAGC GTGGCA CTACCGACAAANuVD6.2 TC CGG CCGACAAANuVD6.3 TCTCTGGGAGCT GGGA GGATACG GC GCTACCGACAAA

________.___________________________________________________________________________________________-

Va4 N1 D61 N2 D62 N3 J61____________________________________________________________________________________________________

germline TGTGCTCTGGGG GTGGCATATCA ATCGGAGGGATACGAG CTACCGACAAANuVA4.1 TGTGCTCTGGGG GCCGGG ATCGGAGGGAT TCAT TACCGACAAA

____________________________________________________________________________________________________

Figure 2. Nucleotide sequences of nude bone marrow-derived TcR 5-chain cDNA clones. * D62 tandem duplication; P nucleotidesunderlined.

equal frequency, e.g. each translation of the three functional spliced from a potential splice donor site in the 3' coding regionV65 rearrangements shown in Fig. 2 used a different D62 of the V gene segment to the splice acceptor site in the first C6reading frame. These data indicate that the majority of the exon. An alternative splicing reaction appears to have combinedsequenced TcR 6-chain transcripts can potentially contribute to a potential splice donor sequence located eight codons from thethe repertoire of y6 T cells in nude BM. No restriction in the 3' end of the V6 1 gene segment (CTGGTACGT; underlined indiversity of the TcR yb repertoire was obvious. Fig. 4), homologous to the consensus splice donor sequence

CAGGTAAGT,39 with the splice acceptor site ATATTTTCA-Unusual TcR 6-chain transcripts in nude BMC GAA at the 5' end of the C6 gene. In the second clone, a V66

gene segment was directly combined in a different manner to C6.Two of the sequenced TcR 6-chain transcripts were truncated Thirteen codons are lacking from the 3' end of the V66 geneforms lacking D and J gene segments (Fig. 4). In the first clone, segment and the first six codons ofCb were deleted. No potentialan almost full-length fragment of the V61 gene segment was splice donor site was detectable in the 3' coding region of V66,

256 S. Spieji et al.

V61 D62 J61_____________________________________________________________________

germline G S D Y R R D T T D KNuVD1.1 G S D M E Y R R D T G L A T D K

_____________________________________________________________________

V62 D61 D62 J61_____________________________________________________________________

germline G G K W H I I G G I R T D KG I S S E G Y E

NuVD2.1 G G K D G A T D KNuVD2.2 G G P L H I G G T T D KNuVD2.3 G G K G A G G I R T T D KNuVD2.4 G G L I G G I R A D KNuVD2.5 G G K G G A I G G I R A T D KNuVD2.6 G G K A L G A T D KNuVD2.7 G G K P G S S E G Y E G D K

_____________________________________________________________________

V63 D62 J61_____________________________________________________________________

germline I L R Y R R D T T D KNuVD3.1 I L R V P Y R R D T D KNuVD3.2 out of frame

_____________________________________________________________________

V64 D61 D62 J61_____________________________________________________________________

germline L M E W H I S E G Y E T D KG I S I G G I R

NuVD4.1 L M E R V H I G G I R A A KNuVD4.2 L M E I S E G Y A R S KNuVD4.3 out of frameNuVD4.4 L M E S I R V L P P I R A P D KNuVD4.5 L M E R E G Y G S L T D K

_____________________________________________________________________

V65 D61 D62 J61_____________________________________________________________________

germline A S G Y W H I I G G I R T D KS E G Y EY R R D T

NuVD5.1 A S G Y C G G I K A T D KNuVD5.2 out of frameNuVD5.3 A S G Y R H S E G Y E P D KNuVD5.4 A S G W H I G F R R D T T D KNuVD5.5 out of frameNuVD5.6 out of frame

_____________________________________________________________________

V66 D61 D62 J61

germline A L W E G I S S E G Y E T D KNuVD6.1 A L W E R G T T D KNuVD6.2 A P A D KNuVD6.3 A L W E L G G Y G A T D K

Va4 J61

germline C A L G T D KNuVA4.1 out of frame.____________________________________________________________________

Figure 3. Predicted amino acid sequences of nude BM-derived V3-chain transcripts.

and no alternative splice acceptor site was present downstream adult nude mice contain 30-50% of TcRyb+ cells which arein the 5' coding region of Cb. It is uncertain how this transcript either 'double-negative' or CD8+. TcR y-chain expression iswas processed. It is possible that the deletion of the second clone high in spleen cells (SC) of young nude mice40 while TcR a andoccurred during amplification. In both clones, the open reading TcR f-chain expression is only detectable in SC from old nudeframe of the V6 gene segment continues into the Cb exon, mice.4' SC from 8-week-old nude mice contain in-frame TcR 6-suggesting that they may encode functional peptides. chain transcripts composed of V65-D16l-D62-J61-C6 genes.42

A large fraction of T cells from nude liver are TcRyb + and

DISCUSSION preferentially express a V66/Vyl or Vy2 TcR.'9 During foetalontogeny of normal mice, there is an ordered expression ofdifferent TcRyb in the thymus. By day 14 of gestation, thymic y6cells express Vbl/Vy3 receptors on the surface.43 Some days

T-cell populations from BM, spleen and lymph node (LN) of later, a new wave of T cells is generated which expresses V6l/

Diversity of TcR b-chain transcripts

v61 CS

germline AAAGACGCTGGTACGTACTACTGTGGGTCAGATATK D A G T Y Y C G S D

NuVD1.2 AAAGACGCTGK D A E

TATTTTCAGAAAGCCAGCCTCCGGCCAAACCATCTGTS Q P P A K P S V

AAAGCCAGCCTCCGGCCAAACCATCTGTS Q P P A K P S V

____________________________________________________________________________________________________

V66 CS____________________________________________________________________________________________________

germline TTACAACCAGACGATTCGGGAAAGTATTTCTGTGCTCTCTGGGAGCT TATTTTCAGAAAGCCAGCCTCCGGCCAAACCATCTGTL Q P D D F G K Y F C A L W E S Q P P A K P S V

NuVD6.4 TTACAA CCATCTGTL Q P S V

Figure 4. Unusual nude BM-derived TcR 3-chain transcripts. Splice donor/acceptor sites (NuVD 1.2) and potential recombination sites(NuVD6.4) are underlined.

Vy4 receptors."4 Despite the potential for extensive junctionaldiversification, both early thymic y6 T-cell subsets selectivelyexpress a canonical sequence at the V3 1-D32-J32 junction. y6 Tcells in the adult thymus express different V3 gene segments(with a predominance of V35/Vy2) and display extensivejunctional diversity which results in a large repertoire.45 FoetalV3l/Vy3-expressing thymocytes are precursors of dendriticepidermal cells.46 Invariant Vb1/Vy4 receptors are exclusivelyexpressed by cells in the epithelium of the female reproductivetract.47 These y6 T-cell subsets are not detectable in epidermisand vaginal epithelium of nude mice. Unlike these epithelial y6cells, intestinal IEL employ diverse V3 segments paired withVy2, and exhibit extensive junctional diversity that include N-regions and use ofboth D3 elements characteristic of rearrange-ments occurring in the adult.48 Intestinal intraepithelial lympho-cytes (TEL) are found in nude mice22,25'35-37,49 and arise followingBM reconstitution of irradiated, thymectomized mice.50,5' Thisindicates that intestinal IEL can arise extrathymically. Likeintestinal IEL, y6 cells in the lung have a potentially largeantigenic repertoire. Different V3 genes are detectable and thejunctional sequences are highly diverse, with extensive N-regionnucleotide addition and the use of both D elements. In lung IELof BALB/c mice, however, a V35-D32-J 1 rearrangement withan invariant junctional sequence is dominant. This invariantsequence is also dominant in lung-derived y3 T cells from nudeBALB/c mice, but is not found in thymus of BALB/c mice. Thissuggests that lung IEL with this invariant sequence are selectedextrathymically.52

Mice with severe combined immunodeficiency (SCID)53recombine TcR3 V, D, J gene segments at low frequency, andmost of the attempted recombinations result in large deletionsfrom the ends of the coding elements.5 P nucleotide additions ofexcessive length have been observed in SCID mice. Usually,mono- or dinucleotides forming palindromes (P) with thetermini of the adjoining coding segments are frequently addedto coding joints." Occasionally, P nucleotide additions of up tofour nucleotides have been found in normal mice. In SCID mice,up to 15 P nucleotides were inserted during y-chain rearrange-

ments.55 One clone (NuVD5.6) with nine P nucleotides added inthe D32-J31 junctional region was observed. This is unprece-

dented in 3-chain rearrangements. Only mono- or dinucleotideswere added in all other clones containing P nucleotides, with theexception of the addition of three P nucleotides in clone

NuVD 1. 1. There is thus no evidence that P nucleotide additionis deregulated in BMC of nude mice.

The truncated cDNA clone NuVD1.2 probably representsan alternative splice that resulted in deletion of the D and Jsegments. Similar aberrant splice events of TcR chains wereobserved in thymocytes from normal mice (B. Arden, unpub-lished observation) and in TcR 3-chain transcripts from SCIDmice (S. SpieB and J. Reimann, unpublished observation). Theconsensus splice donor site identified within the V61 genesegment was not found in the 3' regions of other V6 genes. Thesecond transcript with a similar deletion (NuVD6.4) did notdisplay cryptic splice sites near the deletion breakpoints.Instead, a short stretch of five identical nucleotides (AACCA)was observed on either side which may have caused deletionduring amplification of cDNA. As the predicted proteinsequence of the V6 segment is in-frame with that of C3, it istempting to speculate that a truncated protein lacking the thirdcomplementarity determining region could be expressed. How-ever, its tertiary structure would be severely affected, becauseone cysteine of the V segment, as well as important invariantresidues of the J segment, are missing.

An extensive diversity was detected in TcR 3-chain tran-scripts expressed by nude BMC and 80% of the sequenced TcRb-chain transcripts were found to be in-frame. Some V3 genesegments (e.g. V32, V34, V35, V36) seemed to be used morefrequently than others (e.g. V31, V33). This was apparent fromthe intensity of the signal detectable in Southern analyses ofDNA amplified by PCR. The diversity detectable in DNAcloned from these PCR products supported this interpretation:with small yields of amplified DNA, many of the sequencedclones were identical (see, e.g. V31/C3 amplification data). Incontrast, with the abundant DNA obtained, e.g. with V32-,V35- or V36-specific PCR primers, all sequenced clones weredifferent. Technical factors such as PCR amplification ofunequal efficiency with different primer pairs may have distortedthe picture, but would underestimate the existing TcR3 diver-sity. The described data indicate that the potential repertoire ofTcRyb-expressing T cells in the nude BM is extensive. Thediversity generated by usage of Vb gene segments from all sixfamilies was further increased by extensive junctional diversityin all sequenced 3-chain transcripts. Db1 and D32 were used inmost clones and extensive template-independent N-regiondiversity was evident. TcR rearrangement in adult nude mice

257

258 S. Spiefi3 et al.

therefore does not resemble TcR /3- or TcR 6-chain rearrange-ment in foetal ontogeny of euthymic mice in which very limitedjunctional diversity is detectable.55 The nude mouse TcRrepertoire is thus different from the foetal TcR repertoire ofeuthymic mice and is more reminiscent of the thymic repertoireof adult euthymic mice.

ACKNOWLEDGMENTS

We gratefully acknowledge the help of Professor Dr D. W. Knochel insequencing PCR-amplified DNA. This work was supported by grantsfrom the Deutsche Forschungsgemeinschaft (SFB322/B12) and theFRITZ-THYSSEN-STIFTUNG to J.R.

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