Eur. J. Immunol. 1991.22: 2749-2754
Gerd PluschkeO,
Gem Rickeno, Heiko Taubeo, Susanne Kroningero, Inga MelchersO, Hans Hartmut Peter+, Klaus Eichmann. and Ulrich Krawinkelo Kliniscbe Forscbergrruppe fiir Rbeumatologieo, Abteilnng Rbeumatologie Md Klinisehe Immunologic+ des UniversitatskWkums Fhiburg and Max-Plan&-Institut fiir Immunobiologie., Freiburg
V, gene repertoire
2749
Biased Tcell receptor Varegion repertoire in the SyoOvial fluid of rheumatoid arthritis patients* Synovial T lymphocytes seem to contribute to the pathogenesis of rheumatoid arthritis (RA). Since very little is known about the structural heterogeneity of theirTeel1 antigen receptors (RR), we analyzed TcR a chain mRNA of synovial fluid Tcells from two RA patients. X R a chain cDNA was amplified by the polymerase chain reaction with single-sided specificity for the a chain constant (Ca)gene segment, and the nuclwtide sequences of 51 functionally rearranged cDNA clones were determined. "benty different V, genes and 26 different J, gene segmentswere utilized in these cDNA clones.Three of theV, gene segments which are frequently (8%-17% total) expressed in synovial fluid Tcells have rarely been found in the TcR repertoire of peripheral blood T cells from healthy individuals.The T cell responses in the rheumatic synovia analyzed here are not oligoclonal, but the usage of TcR V, genes is biased.
1 Introduction
Analysis of the diversity of TcR chain mFWA by screening conventional cDNA libraries with probes for the C regions Rheumatoid arthritis (RA) is characterized by chronic mostly is not suitable for clinical samplesbecause it requires inflammation of synovial membranes in peripheral joints. large amounts of starting material. The polymerase chain Since the rheumatoid synovial tissue and fluid contain an reaction (PCR) is useful in the analysis of nucleotide elevated number of activated T cells [l-31, it is thought that sequences from minute quantities of starting material. T lymphocytes specific for synovial (auto)antigens are Conventional PCR employs two specific oligonucleotide involved in the pathogenesis of RA. This concept is primers that flank the sequence of interest [20]. However, supported by the observation that genes of the MHC play a as sequence information for only part of the V, gene role in the inheritanceof susceptibilityto RA: 80%-90% of segment germ-line repertoire is available, this technique is RA patients carry either the Dw4 or Dw14 subtype of the of limited use in the analysis of TcR a chain mRNA. Sottini DR4 serotype or the DR1 serotype [4]. Evidence for et al. [21] recently reported the analysis of the synovial V, oligoclonality or a limited TcR gene segment usage of repertoire of three RA patients employingthe conventional autoreactiveTcells in autoimmunediseases is accumulating PCR technique with a set of 22 V, primers. In order to [5-71, but in RA in attempts to identify dominant Vg survey the entire repertoire of Va regions used by T rearrangements by Southern blot analysis [8-141 so far have lymphocytes from two RA patients, we used in this study yielded conflicting results. PCR with single-sided specificity [19, 22-24] for C, sequences common to all TcR a chain mRNA. We find the Analysis of TcR V gene segment usage by human T frequent usage of threeV, genes which inTcR from PBL of lymphocytes is hampered by the fact that only a few healthy persons OCCUT rarely, if at all. A primer detecting V-specific mAb are available and that only part of the V the most frequent of theseV, genes was not included in the gene segment sequences are known. of these study of Sottini et al. [21]. Our data suggest that as yet sequences were obtained by analysis of libraries unknown antigens in rheumatic synovia select T lymphofrom human thymocytes and peripheral blood T cells of cytes which express in their antigen receptor a distinct set of healthy donors [15-191. These known Va gene segments V, genes. have been subgrouped into 29 families,which are defined as groups of V segments that share >75% nucleotide sequence similarity. Southern blot hybridization analysis 2 Materials and methods have shown that these families contain between one and nine members [16-191. Fourty-six different J, segments 2.1 RA patients have been listed by Wilson et al. [15], but the statistically estimated number of Ja gene segments is 100 or more Synovial cells from therapeutic arthrocenteses were obtained from two RG patients who fulfilled at least five of ~71. the American Rheumatism Association criteria for the classification of RA [25]. Patient 1carried DR4-Dw4 and DRwU-Dw19 MHC class 11molecules. Patient 2 was typed [I 97151 DR7-Dw7DRw8.1-Dwl (A. Becking, personal communication). * This work was supported by the Deutsche Forschungsgemeinschaft through grant Pe 151/10. Coriwpondence: Ulrich KrawWel, Fakultrrt fir Biologie-Immu- 2.2 Isolation of RNA and syntBesis of uled cDNA nologie, Postfach 55 60,D-7750 Konstanz, FRG AbbrewWow U meumatoid arthritis PCa: Polymerase chain reaction SF(L): Synovial fluid (mononuclear cells)
0 VCH Verlagsgesellschaft mbW, D-6940 Weinheim, 1991
Synovial fluid mononuclear cells (SFL) were isolated by Ficoll-Hypaque (Pharmacia, Freiburg, F'RG) density gradient centrifugationfollowed by two washesin WMI 1640. OO14-2980/91/1111-2749$3.~ + .25/0
2750
Eur. J. Immunol. 1991.21: 2749-2754
G. Pluschke, G. Ricken, H. Taube et al.
Total RNA was prepared from about lo7 cells by acid guanidinium thiocyanate-phenol-chloroform extraction [26]. The first cDNA strand was synthesized in a 50-pl reaction mixture containing total RNA equivalent to 5 x 106 mononuclear cells, 50 nm Tris-HC1 (pH 8.3), 6 m M MgC12,40 nm KC1,l mM DTT, each dNTP at 1m, 10 U placental ribonuclease inhibitor (BRL, Gaithersburg, MD), 4 pg (dT)~.~-primer, 100 U of avian myeloblastoid virus reverse transriptase (Boehringer Mannheim, Mannheim, FRG) and 20 pCi = 740 kBq [32P]dCTP(Amersham Buchler, Braunschweig, FRG).RNA was heated at 65 "C for 3 min and chilled on ice. The reaction mixture was incubated for 1h at 42"C, heated to 70°C for 5 min and subsequently loaded onto a Bio-Gel A-5m (Bio-Rad, Richmond, CA) column equilibratedwith 0.5 mM Tris-HC1 and 0.05 m~ EDTA (pH 7.9). One-drop fractions were collected and the first peak of radioactivity was pooled and concentrated by ethanol precipitation. An oligo(dG)-tail was added to the 3' end of the cDNA utilizing terminal deoxynucleotide transferase (BRL, Gaithersburg, MD) . Reaction mixtures of 25 pl containing 100 nm sodium cacodylate, 1m~ dGTP, 2 m~ CoCl2 and 50 enzyme units were incubated for 20 min, at 37°C. The reaction was stopped by heating for 15min at 65 "C and the tailed cDNA was precipitated in ethanol.
plasmids, kinasing of Klenow-treated PCR products, ligation of vectors to cDNA fragments, transformation of bacterial hosts and selection of plasmid-containingcolonies by hybridization to oligonucleotideFR16 was done according to standard methods [27]. Bacteria from_suchcolonies superinfectedwith M13 helper phages a d dot blots of containing single-stranded phagemids were screened with the complementary C, oligonucleotides FRl6 and FR17. Sizing of plasmid inserts was done by agarose gel electrophoresisof Eco RI-digested plasmids. Inserts longer than 150 bp were sequenced by the Sequenase method
[%I* 2.5 Genomic V, genes Genomic V, genes were isolated from a partial Sau 3 A library of human placenta DNA in vector lambda FIX (Stratagene). As a probe we used cDNA clone M29 (see Fig. 3). Library screening, Southernhybridization analysis, subcloning of V,-carrying DNA fragments into the Bluescript vector and sequencing of inserts was performed utilizing standard procedures [27]. 2.7 Processing of DNA sequences
2 3 Amplification of tailed cDNA Reaction mixtures (100 pl) containing tailed cDNA, Ca primer FR8 and oligo (dC) primer FR5 (Table 1) at 1p ~ 50 m~ KC1,lO m~ Tris-HC1 (pH 8.4), 1.5 m~ MgCl2 and each dNTP at 200 p~ were heated to 95 "C for 5 min, 2 units of Thermophilus aquaticus (Taq) DNA polymerase (BRL) were added and the mixture was overlaid with mineral oil (Sigma, St. Louis, MO). Amplification conditions were 3 min at 72 "C, 1min at 92 "C and 2 min at 50 "C per cycle in a Hybaid MB 101 heating block (Hybaid, Teddington, GB). After 25 cycles the amplificationproduct was electrophoresed through 2% agarose. DNA of the appropriate size (350-600 bp) was isolated and reamplified in 25 cycles with the oligo (dC) primer FR5 and the nested C, primer FR4. PCR products separated again in 2% agarose gels were transfered to Gene Screen Plus hybridization membranes (NEN, Boston, MA) and were probed with the nested 32P-labeledC, oligonucleotide (FR16).
,
Sequence data were processed utilizing the UWGCG programs [29]. New sequences are available from the EMBL Data Library (accession numbers X58157X58168).
3 Results 3.1 PCR with single-sided speci5dty PCR with single-sided specificity was used to analyze the TcR a chain repertoire of synovialfluid T lymphocytesfrom two patients with R4,Mononuclear cells were isolated from synovial fluids of therapeutic arthrocenteses and total RNA was extracted without prior culturing of the cells. mRNA was transcribed into cDNA and a poly(dG) tail was added. The product then was amplified with C, anti-sense Table 1. Sequence of oligonucleotides used as PCR primers or probesa)
2.4 PCR primers
All PCR primers used in this study are listed in Table 1 (Sect. 3.1).
2.5 Cloning of PCR products Appropriate size fractions of the products of the second set of amplification were isolated from 2% agarose gels and cloned into the Eco FU site of pT7T3 18 U (Pharmacia) or a) The C, oligonucleotide FR8 was used as primer in the first PCR, the C, oligonucleotideFR4 was used as internal primer in Bluescript I1 SK (Stratagene, La Jolla, CA). For the the second reaction. The complementary oligonucleotides generation of cohesive ends [24], PCR DNA was treated FR16 and FR17 were used as probes. FR16 corresponds to the with 20 U of Klenow enzyme (Boehringer Mannheim) for coding strand, FR17 to the inverted complement of the coding 40 min at 37°C in a buffer containing 25 mh4 Tris-HC1 strand. The oligo(dC) primer FR5 and the C, primer FR4 have (PH 7.3), 20 n w NaCl, 5 nm MgCl2, 1mM DTT, 20 WM Eco RI sites introduced to facilitate clonine " of PCR uroducts. dCTP and dGTP. Digestion an2 dephosphorylation of '
Eur. J. Immunol. 1991. 21: 2749-2754
V, gene repertoire
primers in combination with the oligo(dC) primer FR5 (Table 1). For cloning of the amplified cDNA into the Eco RI site of suitable plasmids, a procedure was chosen that does not require restriction enzyme digestion and thus leaves internal Eco RI sites uncleaved. Both primers used for the final amplification start with partial Eco RI restriction sites (SAATTC3'). The 3' 4 5' exonuclease activity of the Klenow enzyme therefore can be exploited to produce cohesive 5'AATI'3' ends in the presence of dGTP and dCTP Klenow-treated and kinased cDNAwere ligated into Eco RI-digested, dephosphorylated vectors. More than 50% of the cloned inserts finally hybridized to C, oligonucleotides.We sequenced all cDNA of a size between 150 and 550 bp. This length is sufficient to allow the unambiguous identification of the used J and V gene segments.
3.2 Heterogeneity of TcR a chain cDNA Fifty-fiveV,J,C, sequences were obtained from SFL of two patients; 51 of these were in-frame and therefore should
represent functional transcripts. Fig. 1 shows the V,J, junctions of all cDNA clones obtained, and Fig. 2 shows the complete sequences of yet undescribed gene segments used in these clones. Clones C11 and F72, KSR2 and M57 and M43, M78 and A172 differ in length and the number of 3'-terminal G residues, but are otherwise identical. However, this is the only hint at oligoclonality in the synovial T cell response. All other cDNA represent mRNA with unique V,J, combinations, indicating a largely polyclonal response and, in addition, an unbiased amplification procedure. The V, gene usage listed in Fig. 1is summarizedin Table 2. V,FRl, also tentativelydesignated V,w23 [19], is frequently used in SFL clones (clones K42,L113, K64, K50,J33 and L97 from patient 1, KSR2 and M57 from patient 2) but rarely in libraries from PBL [16-191. It has been described as segment of the functional TcR a chain gene of the autoreactive T cell clone UA-S2 from human rheumatic synovia 1301. Sequences of all V,FRl-using clones are compared in Fig. 2. Differences in theV, sequences could result from single-base exchanges during the amplification procedure and/or may represent allelic variation. Jn-segmnt
clone vn-farily
**
Ll6 B11 L32 P106 Clll P721
~ 3 9 L127 K65 J43 P86 C13 c2 Ll15 x49 L58 L9
c29 F4 L123 K32
J66 E4 818 K2 560
1 2 2 2 2 2
a 3 3
4 6 8 8
10 11 11 11 11 14 14 14 14 15 15 15 19
L ~ O J104 x12 L113 K64 K5O
aa
533
FR1 FR1
L97 PlOO PlOl P31 P24 P96
22
8x1 FR1 FR1 PR1 PR3
FR3 FR4 FR5
FR6
2751
GTG GGG ACC ACG GGA GGA GGA AAC AAA CTC ACC TTT TAC TTC TGT GCT ATG AGC TCT TCT GOT TCT GCA AGG CM CTG ACC TTT TAC CTC TOT GCA GTC C M CGT CCC GGC ACT GCC AGT AM CTC ACC TTT TAC CTC TOT GCA GGT AAC ACC GAC AAG CTC ATC TlT TAC CTC TGT GCC GTG AAC ATC GCA GAC GAC TAC AAG CTC AGC TTT TAC CTC TGT GTG GTG AAC ATC GCA GAC GAC TAC AAG CTC A& TTT TAC CTC TGT GTG G W CCA CGA TCA GGA GGA W TAC ATA CCT ACA TAC CTC TGT GTG EGG AGC CAT AAC ACC GAC AAG CTC ATC TTT TAC TTC TOT GCT TGG AGC CCT TAC AAT AAC AAT GAC ATG Coc TTT TAC TTC TGT GCT TAC TAT TGC ACC GTC AGA TCC TLT GG6 M T OCT GGT GOT ACT AGC TAT GGA AAG CM A(3L ATG AGC CCA TCG AAC TAC GAC AAG GTG ATA l T T TAC TTC TGT GCA GCA AGT AGG GAC AGA GAT GAC AAG ATC ATC l T T TAC TTC TGT GCA GAG AAG GAC AAT GCA GGC AAC AT0 cfc ACC TTT TAC T l T TOT GCA GGA GGG GGT TTC CGT TCT AAC GAC TAC AAG CTC M C TIT TAC CTC TGT GCA GTG GAA GGC CAG GCA GGA ACT GCT FK: ATC l l T TAC TAC TGT GCT GTG GAC AGA GAT m C M G ATC ATC T" TAC TAC TGT GCT QTG CAT TCA GGA GGA W T GCT GAC GUA CK: ACC TTT TAC TAC TGT GCT QTG QAG GAT CTA GAA GCT GCA GGC AAC AAG CTA ACT TTT TAC TAC TGC GCT TCG TCA GGA TAC AOC ACC CSC'ACC TTT TAT TTC TGT WT TAP A.C.. A. GAC ACG GGC AGG AGA IXA Cl" ACT Tll' -..-. -~~~ ...~ TAT TTC TGT GCT TAT AQC3 AGC TCA M T TAT GGA GGA Aoc CM GGA M T eK: ATC TAT TTC TGT GCT TAT AGG AGT ATT CAG GGA GCC CAO M G m GTA TAT TTC TGT GCT ATG AGA TCT TTC AAC CAG GCA GGA ACT GCTCTG ATC TAC TTC TGT GCA GAG AGT TCA TCA GGA GGA AGC TAC ATA CCT *CA TAC TTC TGT GCA GAG GAC GCA GGC M C ATG CTC ACC TAC TTC TGT GCA wrc ccc TCG 660 GAA ACC AGT GGC TCT Mow ACC TAC ATC TGT GCT CTG ncT 6CC TCT TCT GGT TCT GCA AGG C M CM ACC lTl' TAC TTC TGT GCT FK: AGl' GAT TCA GGA TAC AGC ACC 2Tc ACC TTT TAC TTC TGT GCT GTC ClV CCG ATG GAA GCT GCA GGC AAC AAG CTA ACT TTT TAC CTC TGT GCT lXT A m GAT AGC AGC TAT ..__ _.-.-. . - AAA . - TTC ~ ATC TJX TAC CTC TGT GCT GTG AGG CCO GAA TAT GGA GGA AGC CAA GGA AAT CTC ATC TTT TAC CTC TGT GCT GTG ACT CTC TAT AAC CAG GGA GGA AAG CTT ATC TTC TAC CTC TGT GCT GTC CCC AAC CAG GCA GGA ACT GCT CTG ATC TTT TAC CTC TGT GCT GCG GTT AGC AGC TAT AAA TT(i ATC TTC TAC CTC TOT GCT GTG TGG CAT AAC CAG GGA GGA AAG CTT ATC TAC TTC TGT GCC GTC AAC M T GAC ATG CGC TlT TAC TTC TGT GCC TAC TTC TGT GCA GCC TCT GGG GAA GCA GEC AAA TCA ACC TTT TAT AGG GAC ACC GGT M E CAG TTC TAT TTl' TAT TTC TGT GCT TTG GAT TCA GGA AAC ACA CCT CTT GTC TTT TAT TTC TGT GCT ~~~~~
___
____
A021 C AA17
I N
N T f FB.3 J
FR6
P AC17 N
S P W
AC25 AD17 ABl9 FB.1 U S
T AC17
FR5 C AD17 ACZS
B FR1 H S
B H FR3
FR4 0
F
EathKtd
A152
PRZ
TAC TTC TOT GTT TAC CTC TGT GTG TAC CTC TCT GTG TAC TTC TGT GCA TAC TTC TGT GCA TAC TTC TGT GCA TAT TTC TOT GCT TAC TTC TGT GCA TAC TTC TGT GCA TAC CTC TGT GCT TAC CTC TOT GC3T TAC TN: TGT GCA
X60
6 10 18 19
TAC TTC 'IUT OCA TAC CTC TGT GCA TAC TTC TGT dCh TAC ATC TGT OCT
U36 U143
ua
1 2
2.m
A1724 8 n43# 8 U70X U33
8
14 15 15 mR21 FRl n5711 FR1
All1 A60
FB3
A179 F71
GTG GG% GAT CGA GTG AAC A M GNN GCA ACT M C TCC GCA W M C TCC GCA MIT M C TCC HAT A00 AOA GGA ATC TAT TCA GAG GAG ACT ATA AAG GGT GTG GTT GTT GGG CGG TCA
AT GAG AGA Boc G GAG QTC 6 ACA CCC CCT G TCT
Coc GGC NNN AAC AAC AAC GGA M A GGA
Doc GAC TTC AAC AM TTT TAC lTT
AGA GGT GCT GAC GGA CTC NNG GGU NGC NNN NCA NTT Cno GCA GGA ACT GCT CTG CAG GCA GGA ACT GCT CTG CAG GCA GGA ACT GCT CTG GGA GGT GCT GAC GGA CTC TGG GAT AAC M T GAC AT0 GGT AGC AAC TAT AM CTG TCC GAT TCC GGG TAT GCA CTC TCC GAT TCC GGG TAT GCA CTC GGA GGA GGT GCT GAC GGA CTC
TTC ACG GGA abu GGA AAC CCA OQA GGA G@I'GCT GAC
ACC 'I" ACN ATC TTT ATC m ATC TTT ACC TTT CGC TTT ACA TTT AAC TTC M C ACC TTT
AM CTC ACC TTT
-4
w
0 S S
S W
FR3 1[ pB1
W
AGZl CTC AcC TTT W ACT AAT AAT Ocn 6GC AAC ATG CTC ACC TTT AC17 TCA GGA GGA GGT GCT GAC GGA CTC ACC TTT W GUA
Figure 1. V-J junctional sequences of in-frame TcR a chain transcripts from SF Tcells.V, gene segment families are designated according to Wilson et al. [W]. J, gene segments are designated according to Kimura et al. [16] and Klein et al. [17]. Clones labeled are identical in sequencebut differ in size and tail length.
*
Eur.J. Immunol. 1991. 21: 2749-2754
G. Pluschke, G. Ricken, H. 'Igube et al.
2752
Thirteen independent cDNA clones obtained from patient 'Rble 2. V, usage in SFL of RA patients 1 and 5 clones obtained from patient 2 have sequences which correspond to the previously published V, sequence SFL sn PBL in cDNA clone H A W 0 [16]. This sequence defines the Patient1 Patient2 Healthypersons Va14 family [15]. In Southern hybridization analyses of (%I 6) (%I8) V,,family fibroblast DNA usha clone HAVT20 as probe two bands were detected [16]. We screened a genohc library made 16.7(2112) lS.l(lW6) 15.4(6/39) 2 from human placental DNA with V,14 cDNA M29 as a 25 (3/12) 9.1 (6'66) 5.1(2/39) 8 probe and obtained nine independent clones representing < S(W2) 10.6 (7/66) 11 12.8(5/39) two germ-line V, genes, V,14.1 and V,14.2 (Fig. 3). The < l.S(W66) 10.3(4/39)b) 8.4(l/12)b) 14 V,14 family thus appears to comprise only two members.
Gerd PluschkeO,
Gem Rickeno, Heiko Taubeo, Susanne Kroningero, Inga MelchersO, Hans Hartmut Peter+, Klaus Eichmann. and Ulrich Krawinkelo Kliniscbe Forscbergrruppe fiir Rbeumatologieo, Abteilnng Rbeumatologie Md Klinisehe Immunologic+ des UniversitatskWkums Fhiburg and Max-Plan&-Institut fiir Immunobiologie., Freiburg
V, gene repertoire
2749
Biased Tcell receptor Varegion repertoire in the SyoOvial fluid of rheumatoid arthritis patients* Synovial T lymphocytes seem to contribute to the pathogenesis of rheumatoid arthritis (RA). Since very little is known about the structural heterogeneity of theirTeel1 antigen receptors (RR), we analyzed TcR a chain mRNA of synovial fluid Tcells from two RA patients. X R a chain cDNA was amplified by the polymerase chain reaction with single-sided specificity for the a chain constant (Ca)gene segment, and the nuclwtide sequences of 51 functionally rearranged cDNA clones were determined. "benty different V, genes and 26 different J, gene segmentswere utilized in these cDNA clones.Three of theV, gene segments which are frequently (8%-17% total) expressed in synovial fluid Tcells have rarely been found in the TcR repertoire of peripheral blood T cells from healthy individuals.The T cell responses in the rheumatic synovia analyzed here are not oligoclonal, but the usage of TcR V, genes is biased.
1 Introduction
Analysis of the diversity of TcR chain mFWA by screening conventional cDNA libraries with probes for the C regions Rheumatoid arthritis (RA) is characterized by chronic mostly is not suitable for clinical samplesbecause it requires inflammation of synovial membranes in peripheral joints. large amounts of starting material. The polymerase chain Since the rheumatoid synovial tissue and fluid contain an reaction (PCR) is useful in the analysis of nucleotide elevated number of activated T cells [l-31, it is thought that sequences from minute quantities of starting material. T lymphocytes specific for synovial (auto)antigens are Conventional PCR employs two specific oligonucleotide involved in the pathogenesis of RA. This concept is primers that flank the sequence of interest [20]. However, supported by the observation that genes of the MHC play a as sequence information for only part of the V, gene role in the inheritanceof susceptibilityto RA: 80%-90% of segment germ-line repertoire is available, this technique is RA patients carry either the Dw4 or Dw14 subtype of the of limited use in the analysis of TcR a chain mRNA. Sottini DR4 serotype or the DR1 serotype [4]. Evidence for et al. [21] recently reported the analysis of the synovial V, oligoclonality or a limited TcR gene segment usage of repertoire of three RA patients employingthe conventional autoreactiveTcells in autoimmunediseases is accumulating PCR technique with a set of 22 V, primers. In order to [5-71, but in RA in attempts to identify dominant Vg survey the entire repertoire of Va regions used by T rearrangements by Southern blot analysis [8-141 so far have lymphocytes from two RA patients, we used in this study yielded conflicting results. PCR with single-sided specificity [19, 22-24] for C, sequences common to all TcR a chain mRNA. We find the Analysis of TcR V gene segment usage by human T frequent usage of threeV, genes which inTcR from PBL of lymphocytes is hampered by the fact that only a few healthy persons OCCUT rarely, if at all. A primer detecting V-specific mAb are available and that only part of the V the most frequent of theseV, genes was not included in the gene segment sequences are known. of these study of Sottini et al. [21]. Our data suggest that as yet sequences were obtained by analysis of libraries unknown antigens in rheumatic synovia select T lymphofrom human thymocytes and peripheral blood T cells of cytes which express in their antigen receptor a distinct set of healthy donors [15-191. These known Va gene segments V, genes. have been subgrouped into 29 families,which are defined as groups of V segments that share >75% nucleotide sequence similarity. Southern blot hybridization analysis 2 Materials and methods have shown that these families contain between one and nine members [16-191. Fourty-six different J, segments 2.1 RA patients have been listed by Wilson et al. [15], but the statistically estimated number of Ja gene segments is 100 or more Synovial cells from therapeutic arthrocenteses were obtained from two RG patients who fulfilled at least five of ~71. the American Rheumatism Association criteria for the classification of RA [25]. Patient 1carried DR4-Dw4 and DRwU-Dw19 MHC class 11molecules. Patient 2 was typed [I 97151 DR7-Dw7DRw8.1-Dwl (A. Becking, personal communication). * This work was supported by the Deutsche Forschungsgemeinschaft through grant Pe 151/10. Coriwpondence: Ulrich KrawWel, Fakultrrt fir Biologie-Immu- 2.2 Isolation of RNA and syntBesis of uled cDNA nologie, Postfach 55 60,D-7750 Konstanz, FRG AbbrewWow U meumatoid arthritis PCa: Polymerase chain reaction SF(L): Synovial fluid (mononuclear cells)
0 VCH Verlagsgesellschaft mbW, D-6940 Weinheim, 1991
Synovial fluid mononuclear cells (SFL) were isolated by Ficoll-Hypaque (Pharmacia, Freiburg, F'RG) density gradient centrifugationfollowed by two washesin WMI 1640. OO14-2980/91/1111-2749$3.~ + .25/0
2750
Eur. J. Immunol. 1991.21: 2749-2754
G. Pluschke, G. Ricken, H. Taube et al.
Total RNA was prepared from about lo7 cells by acid guanidinium thiocyanate-phenol-chloroform extraction [26]. The first cDNA strand was synthesized in a 50-pl reaction mixture containing total RNA equivalent to 5 x 106 mononuclear cells, 50 nm Tris-HC1 (pH 8.3), 6 m M MgC12,40 nm KC1,l mM DTT, each dNTP at 1m, 10 U placental ribonuclease inhibitor (BRL, Gaithersburg, MD), 4 pg (dT)~.~-primer, 100 U of avian myeloblastoid virus reverse transriptase (Boehringer Mannheim, Mannheim, FRG) and 20 pCi = 740 kBq [32P]dCTP(Amersham Buchler, Braunschweig, FRG).RNA was heated at 65 "C for 3 min and chilled on ice. The reaction mixture was incubated for 1h at 42"C, heated to 70°C for 5 min and subsequently loaded onto a Bio-Gel A-5m (Bio-Rad, Richmond, CA) column equilibratedwith 0.5 mM Tris-HC1 and 0.05 m~ EDTA (pH 7.9). One-drop fractions were collected and the first peak of radioactivity was pooled and concentrated by ethanol precipitation. An oligo(dG)-tail was added to the 3' end of the cDNA utilizing terminal deoxynucleotide transferase (BRL, Gaithersburg, MD) . Reaction mixtures of 25 pl containing 100 nm sodium cacodylate, 1m~ dGTP, 2 m~ CoCl2 and 50 enzyme units were incubated for 20 min, at 37°C. The reaction was stopped by heating for 15min at 65 "C and the tailed cDNA was precipitated in ethanol.
plasmids, kinasing of Klenow-treated PCR products, ligation of vectors to cDNA fragments, transformation of bacterial hosts and selection of plasmid-containingcolonies by hybridization to oligonucleotideFR16 was done according to standard methods [27]. Bacteria from_suchcolonies superinfectedwith M13 helper phages a d dot blots of containing single-stranded phagemids were screened with the complementary C, oligonucleotides FRl6 and FR17. Sizing of plasmid inserts was done by agarose gel electrophoresisof Eco RI-digested plasmids. Inserts longer than 150 bp were sequenced by the Sequenase method
[%I* 2.5 Genomic V, genes Genomic V, genes were isolated from a partial Sau 3 A library of human placenta DNA in vector lambda FIX (Stratagene). As a probe we used cDNA clone M29 (see Fig. 3). Library screening, Southernhybridization analysis, subcloning of V,-carrying DNA fragments into the Bluescript vector and sequencing of inserts was performed utilizing standard procedures [27]. 2.7 Processing of DNA sequences
2 3 Amplification of tailed cDNA Reaction mixtures (100 pl) containing tailed cDNA, Ca primer FR8 and oligo (dC) primer FR5 (Table 1) at 1p ~ 50 m~ KC1,lO m~ Tris-HC1 (pH 8.4), 1.5 m~ MgCl2 and each dNTP at 200 p~ were heated to 95 "C for 5 min, 2 units of Thermophilus aquaticus (Taq) DNA polymerase (BRL) were added and the mixture was overlaid with mineral oil (Sigma, St. Louis, MO). Amplification conditions were 3 min at 72 "C, 1min at 92 "C and 2 min at 50 "C per cycle in a Hybaid MB 101 heating block (Hybaid, Teddington, GB). After 25 cycles the amplificationproduct was electrophoresed through 2% agarose. DNA of the appropriate size (350-600 bp) was isolated and reamplified in 25 cycles with the oligo (dC) primer FR5 and the nested C, primer FR4. PCR products separated again in 2% agarose gels were transfered to Gene Screen Plus hybridization membranes (NEN, Boston, MA) and were probed with the nested 32P-labeledC, oligonucleotide (FR16).
,
Sequence data were processed utilizing the UWGCG programs [29]. New sequences are available from the EMBL Data Library (accession numbers X58157X58168).
3 Results 3.1 PCR with single-sided speci5dty PCR with single-sided specificity was used to analyze the TcR a chain repertoire of synovialfluid T lymphocytesfrom two patients with R4,Mononuclear cells were isolated from synovial fluids of therapeutic arthrocenteses and total RNA was extracted without prior culturing of the cells. mRNA was transcribed into cDNA and a poly(dG) tail was added. The product then was amplified with C, anti-sense Table 1. Sequence of oligonucleotides used as PCR primers or probesa)
2.4 PCR primers
All PCR primers used in this study are listed in Table 1 (Sect. 3.1).
2.5 Cloning of PCR products Appropriate size fractions of the products of the second set of amplification were isolated from 2% agarose gels and cloned into the Eco FU site of pT7T3 18 U (Pharmacia) or a) The C, oligonucleotide FR8 was used as primer in the first PCR, the C, oligonucleotideFR4 was used as internal primer in Bluescript I1 SK (Stratagene, La Jolla, CA). For the the second reaction. The complementary oligonucleotides generation of cohesive ends [24], PCR DNA was treated FR16 and FR17 were used as probes. FR16 corresponds to the with 20 U of Klenow enzyme (Boehringer Mannheim) for coding strand, FR17 to the inverted complement of the coding 40 min at 37°C in a buffer containing 25 mh4 Tris-HC1 strand. The oligo(dC) primer FR5 and the C, primer FR4 have (PH 7.3), 20 n w NaCl, 5 nm MgCl2, 1mM DTT, 20 WM Eco RI sites introduced to facilitate clonine " of PCR uroducts. dCTP and dGTP. Digestion an2 dephosphorylation of '
Eur. J. Immunol. 1991. 21: 2749-2754
V, gene repertoire
primers in combination with the oligo(dC) primer FR5 (Table 1). For cloning of the amplified cDNA into the Eco RI site of suitable plasmids, a procedure was chosen that does not require restriction enzyme digestion and thus leaves internal Eco RI sites uncleaved. Both primers used for the final amplification start with partial Eco RI restriction sites (SAATTC3'). The 3' 4 5' exonuclease activity of the Klenow enzyme therefore can be exploited to produce cohesive 5'AATI'3' ends in the presence of dGTP and dCTP Klenow-treated and kinased cDNAwere ligated into Eco RI-digested, dephosphorylated vectors. More than 50% of the cloned inserts finally hybridized to C, oligonucleotides.We sequenced all cDNA of a size between 150 and 550 bp. This length is sufficient to allow the unambiguous identification of the used J and V gene segments.
3.2 Heterogeneity of TcR a chain cDNA Fifty-fiveV,J,C, sequences were obtained from SFL of two patients; 51 of these were in-frame and therefore should
represent functional transcripts. Fig. 1 shows the V,J, junctions of all cDNA clones obtained, and Fig. 2 shows the complete sequences of yet undescribed gene segments used in these clones. Clones C11 and F72, KSR2 and M57 and M43, M78 and A172 differ in length and the number of 3'-terminal G residues, but are otherwise identical. However, this is the only hint at oligoclonality in the synovial T cell response. All other cDNA represent mRNA with unique V,J, combinations, indicating a largely polyclonal response and, in addition, an unbiased amplification procedure. The V, gene usage listed in Fig. 1is summarizedin Table 2. V,FRl, also tentativelydesignated V,w23 [19], is frequently used in SFL clones (clones K42,L113, K64, K50,J33 and L97 from patient 1, KSR2 and M57 from patient 2) but rarely in libraries from PBL [16-191. It has been described as segment of the functional TcR a chain gene of the autoreactive T cell clone UA-S2 from human rheumatic synovia 1301. Sequences of all V,FRl-using clones are compared in Fig. 2. Differences in theV, sequences could result from single-base exchanges during the amplification procedure and/or may represent allelic variation. Jn-segmnt
clone vn-farily
**
Ll6 B11 L32 P106 Clll P721
~ 3 9 L127 K65 J43 P86 C13 c2 Ll15 x49 L58 L9
c29 F4 L123 K32
J66 E4 818 K2 560
1 2 2 2 2 2
a 3 3
4 6 8 8
10 11 11 11 11 14 14 14 14 15 15 15 19
L ~ O J104 x12 L113 K64 K5O
aa
533
FR1 FR1
L97 PlOO PlOl P31 P24 P96
22
8x1 FR1 FR1 PR1 PR3
FR3 FR4 FR5
FR6
2751
GTG GGG ACC ACG GGA GGA GGA AAC AAA CTC ACC TTT TAC TTC TGT GCT ATG AGC TCT TCT GOT TCT GCA AGG CM CTG ACC TTT TAC CTC TOT GCA GTC C M CGT CCC GGC ACT GCC AGT AM CTC ACC TTT TAC CTC TOT GCA GGT AAC ACC GAC AAG CTC ATC TlT TAC CTC TGT GCC GTG AAC ATC GCA GAC GAC TAC AAG CTC AGC TTT TAC CTC TGT GTG GTG AAC ATC GCA GAC GAC TAC AAG CTC A& TTT TAC CTC TGT GTG G W CCA CGA TCA GGA GGA W TAC ATA CCT ACA TAC CTC TGT GTG EGG AGC CAT AAC ACC GAC AAG CTC ATC TTT TAC TTC TOT GCT TGG AGC CCT TAC AAT AAC AAT GAC ATG Coc TTT TAC TTC TGT GCT TAC TAT TGC ACC GTC AGA TCC TLT GG6 M T OCT GGT GOT ACT AGC TAT GGA AAG CM A(3L ATG AGC CCA TCG AAC TAC GAC AAG GTG ATA l T T TAC TTC TGT GCA GCA AGT AGG GAC AGA GAT GAC AAG ATC ATC l T T TAC TTC TGT GCA GAG AAG GAC AAT GCA GGC AAC AT0 cfc ACC TTT TAC T l T TOT GCA GGA GGG GGT TTC CGT TCT AAC GAC TAC AAG CTC M C TIT TAC CTC TGT GCA GTG GAA GGC CAG GCA GGA ACT GCT FK: ATC l l T TAC TAC TGT GCT GTG GAC AGA GAT m C M G ATC ATC T" TAC TAC TGT GCT QTG CAT TCA GGA GGA W T GCT GAC GUA CK: ACC TTT TAC TAC TGT GCT QTG QAG GAT CTA GAA GCT GCA GGC AAC AAG CTA ACT TTT TAC TAC TGC GCT TCG TCA GGA TAC AOC ACC CSC'ACC TTT TAT TTC TGT WT TAP A.C.. A. GAC ACG GGC AGG AGA IXA Cl" ACT Tll' -..-. -~~~ ...~ TAT TTC TGT GCT TAT AQC3 AGC TCA M T TAT GGA GGA Aoc CM GGA M T eK: ATC TAT TTC TGT GCT TAT AGG AGT ATT CAG GGA GCC CAO M G m GTA TAT TTC TGT GCT ATG AGA TCT TTC AAC CAG GCA GGA ACT GCTCTG ATC TAC TTC TGT GCA GAG AGT TCA TCA GGA GGA AGC TAC ATA CCT *CA TAC TTC TGT GCA GAG GAC GCA GGC M C ATG CTC ACC TAC TTC TGT GCA wrc ccc TCG 660 GAA ACC AGT GGC TCT Mow ACC TAC ATC TGT GCT CTG ncT 6CC TCT TCT GGT TCT GCA AGG C M CM ACC lTl' TAC TTC TGT GCT FK: AGl' GAT TCA GGA TAC AGC ACC 2Tc ACC TTT TAC TTC TGT GCT GTC ClV CCG ATG GAA GCT GCA GGC AAC AAG CTA ACT TTT TAC CTC TGT GCT lXT A m GAT AGC AGC TAT ..__ _.-.-. . - AAA . - TTC ~ ATC TJX TAC CTC TGT GCT GTG AGG CCO GAA TAT GGA GGA AGC CAA GGA AAT CTC ATC TTT TAC CTC TGT GCT GTG ACT CTC TAT AAC CAG GGA GGA AAG CTT ATC TTC TAC CTC TGT GCT GTC CCC AAC CAG GCA GGA ACT GCT CTG ATC TTT TAC CTC TGT GCT GCG GTT AGC AGC TAT AAA TT(i ATC TTC TAC CTC TOT GCT GTG TGG CAT AAC CAG GGA GGA AAG CTT ATC TAC TTC TGT GCC GTC AAC M T GAC ATG CGC TlT TAC TTC TGT GCC TAC TTC TGT GCA GCC TCT GGG GAA GCA GEC AAA TCA ACC TTT TAT AGG GAC ACC GGT M E CAG TTC TAT TTl' TAT TTC TGT GCT TTG GAT TCA GGA AAC ACA CCT CTT GTC TTT TAT TTC TGT GCT ~~~~~
___
____
A021 C AA17
I N
N T f FB.3 J
FR6
P AC17 N
S P W
AC25 AD17 ABl9 FB.1 U S
T AC17
FR5 C AD17 ACZS
B FR1 H S
B H FR3
FR4 0
F
EathKtd
A152
PRZ
TAC TTC TOT GTT TAC CTC TGT GTG TAC CTC TCT GTG TAC TTC TGT GCA TAC TTC TGT GCA TAC TTC TGT GCA TAT TTC TOT GCT TAC TTC TGT GCA TAC TTC TGT GCA TAC CTC TGT GCT TAC CTC TOT GC3T TAC TN: TGT GCA
X60
6 10 18 19
TAC TTC 'IUT OCA TAC CTC TGT GCA TAC TTC TGT dCh TAC ATC TGT OCT
U36 U143
ua
1 2
2.m
A1724 8 n43# 8 U70X U33
8
14 15 15 mR21 FRl n5711 FR1
All1 A60
FB3
A179 F71
GTG GG% GAT CGA GTG AAC A M GNN GCA ACT M C TCC GCA W M C TCC GCA MIT M C TCC HAT A00 AOA GGA ATC TAT TCA GAG GAG ACT ATA AAG GGT GTG GTT GTT GGG CGG TCA
AT GAG AGA Boc G GAG QTC 6 ACA CCC CCT G TCT
Coc GGC NNN AAC AAC AAC GGA M A GGA
Doc GAC TTC AAC AM TTT TAC lTT
AGA GGT GCT GAC GGA CTC NNG GGU NGC NNN NCA NTT Cno GCA GGA ACT GCT CTG CAG GCA GGA ACT GCT CTG CAG GCA GGA ACT GCT CTG GGA GGT GCT GAC GGA CTC TGG GAT AAC M T GAC AT0 GGT AGC AAC TAT AM CTG TCC GAT TCC GGG TAT GCA CTC TCC GAT TCC GGG TAT GCA CTC GGA GGA GGT GCT GAC GGA CTC
TTC ACG GGA abu GGA AAC CCA OQA GGA G@I'GCT GAC
ACC 'I" ACN ATC TTT ATC m ATC TTT ACC TTT CGC TTT ACA TTT AAC TTC M C ACC TTT
AM CTC ACC TTT
-4
w
0 S S
S W
FR3 1[ pB1
W
AGZl CTC AcC TTT W ACT AAT AAT Ocn 6GC AAC ATG CTC ACC TTT AC17 TCA GGA GGA GGT GCT GAC GGA CTC ACC TTT W GUA
Figure 1. V-J junctional sequences of in-frame TcR a chain transcripts from SF Tcells.V, gene segment families are designated according to Wilson et al. [W]. J, gene segments are designated according to Kimura et al. [16] and Klein et al. [17]. Clones labeled are identical in sequencebut differ in size and tail length.
*
Eur.J. Immunol. 1991. 21: 2749-2754
G. Pluschke, G. Ricken, H. 'Igube et al.
2752
Thirteen independent cDNA clones obtained from patient 'Rble 2. V, usage in SFL of RA patients 1 and 5 clones obtained from patient 2 have sequences which correspond to the previously published V, sequence SFL sn PBL in cDNA clone H A W 0 [16]. This sequence defines the Patient1 Patient2 Healthypersons Va14 family [15]. In Southern hybridization analyses of (%I 6) (%I8) V,,family fibroblast DNA usha clone HAVT20 as probe two bands were detected [16]. We screened a genohc library made 16.7(2112) lS.l(lW6) 15.4(6/39) 2 from human placental DNA with V,14 cDNA M29 as a 25 (3/12) 9.1 (6'66) 5.1(2/39) 8 probe and obtained nine independent clones representing < S(W2) 10.6 (7/66) 11 12.8(5/39) two germ-line V, genes, V,14.1 and V,14.2 (Fig. 3). The < l.S(W66) 10.3(4/39)b) 8.4(l/12)b) 14 V,14 family thus appears to comprise only two members.
