T-Cell Receptor/3 Usage by 35 Different Antigen-Specific T-Cell Clones Restricted by HLA-Dw4 or-Dw14.1 Torbj0rn Hansen, Erik Qvigstad, Knut E. A. Lundin, and Erik Thorsby

ABSTRACT: We studied whether antigen-specific T cells being restricted by the very similar HLA-Dw4 and/ or -Dw 14.1 molecules might demonstrate homogeneities in parts of their TCR. TCCs were generated from three individuals who were all HLA-Dw4/Dw14.1 heterozygous. Thirty-five TCCs specific for PPD or TT and restricted by HLA-Dw4 and/or -Dw14.1 were selected for

TCR/3 gene sequencing. We found that 19 different V 3 genes from 13 V/~ families were expressed by these TCCs. Thus, it seems that many different TCRV/3 genes may be used by TCCs restricted by these HLA molecules. For PPD-specific TCCs, a possible biased usage of V/38, as well as possible preferential usage of a CDR3 motif, were found. Human Immunology 35, 149-156 (1992~

ABBREVIATIONS

APC CDR mAb PBMC PCR PPD

antigen-presenting cell complementarity determining region monoclonal antibody peripheral blood mononuclear cell polymerase chain reaction purified protein derivative

RA RACE TCC TCR TT

rheumatoid arthritis rapid amplification of cDNA ends T-cell clone T-cell receptor tetanus toxoid

INTRODUCTION The T-cell receptor (TCR) is a membrane-spanning heteroduplex composed of an ~ chain covalently linked to a/3 chain. The ligand for the T C R is a peptide generated by intracellular proteolysis and bound by a given HLA (MHC) molecule (reviewed in Marrack and Kappler [ 1] and Davis [2]). The three-dimensional structure of the TCR is unknown. However, based on the amino acid sequence homology to the Fab fragment of immunoglobulin molecules, models have been proposed, according to which the complementarity determining regions (CDRs) 1 and 2 of the ~ and/3 chains interact with the s-helices of HLA, while CDR3 interacts with the peptide bound in the groove o f the HLA molecule [ 3 - 5 ] . The CDR1 and 2 are encoded by the Vo~ and V/3 genes, while the CDR3 is encoded by the V(D)J junction. From the Institute of Transplantation Immunology, The National Hospital, Oslo, Norway. Address reprint requests to Dr. T. Hansen, Institute of Transplantation Immunology, The National Hospital, N-0027 Oslo, Norway. Received April 21, 1992," accepted September 14, 1992. Human Immunology 35, 149-156 (1992) © American Society for Histocompatibility and Immunogenetics, 1992

If the CDR1 and 2 bind to HLA, one might assume that the expressed Vgene products o f T cells recognizing peptides bound by the same HLA molecule might demonstrate structural similarities, especially in the CDR1 and 2. We decided to study antigen-specific T cells restricted by the HLA-DR4 subtypes Dw4 or Dw14.1. These molecules have been recognized as risk factors for rheumatoid arthritis (RA) [ 6 - 9 ] . If antigen-specific Dw4- or Dw 14-restricted T cells demonstrate common structural features in their TCRs irrespective of peptide specificity, autoimmune T cells involved in the pathogenesis of RA may also possess these structural characteristics, and it might become possible to prevent or treat RA by the use of appropriate monoclonal antibodies (mAbs) [10, 11]. We therefore generated T-cell clones (TCCs) specific for purified protein derivative (PPD) or tetanus toxoid (TT) and selected those being restricted by Dw4 and/or Dw14.1. The only differences between Dw4 and Dw14.1 involve aa residue 71, where Dw4 has lysine 149 0198-8859/92/$5.00

150

and Dw14.1 arginine, and residue 86, where Dw4 has glycine and Dw14.1 valine. We then sequenced the TCRfl genes of these TCCs and looked for possible correlations between TCR structure and specificity.

MATERIALS A N D METHODS

T-cell donors. The T-cell donors RT and IW were female RA patients fulfilling the 1987 revised criteria of the American Rheumatism Association. They are rheumatoid factor positive and have erosive disease. Their HLA types are RT: HLA-A1,2; B8, 15; Dw4,Dw14.1 (DRBI*0401,0404); DQw8 (DQAI*030I, DQB l *0302 ); DPw4 (DPA I *OI O1, DPB I *0401,0402 ); and IW: HLA-A 11,19; B27; Dw4,Dw14.1 (DRB l*0401,0404); DQw7,DQw8 (DQAI*0301,0302, DQBl*0301,0302); and DPw2, -w4 (DPAI*OIO1,DPBI*0201,0401). The third donor, AKG, is a healthy HLA identical sister of IW. T-cell cloning. This was done essentially as described [12]. Briefly, peripheral blood mononuclear cells (PBMCs) were isolated by flotation on Lymphoprep. The PBMCs were cultured in bulk for 6 days at 37°C, 5% CO 2 in RPMI 1640 supplemented with penicillin, streptomycin, 10% pooled human serum and antigen (PPD, 2.5 /a,g/ml [The Veterinary Institute, University of Oslo, Norway]; or TT, 0.1 >g/ml [The National Institute of Public Health, Oslo, Norway]). T-cell blasts were separated on Percoll density gradients (IW and AKG) or by anti-Tac Ab and Dynabeads (RT) [13], and seeded at 0.3 cells/well with antigen and 30 Gy irradiated autologous PBMCs as feeders. Growing TCCs were expanded and frozen in aliquotes.

Determination of TCC specificity. A total of 2 × 104 TCCs and 5 × 104 irradiated PBMCs as antigen-presenting cells (APCs) were cocultivated with or without antigen (PPD or TT) for 64-72 hours in RPMI 1640 substituted with antibiotics and 10% normal human serum. For the last 16 hours, [~H]thymidine was added to the well. The median [3H]thymidine incorporation of triplicate cultures was used as a measure of proliferation. To establish whether DR molecules were presenting the antigen, the HLA-DRc~-chain-specific mAb L243 [14] was added at 10 /~g/ml to some of the cultures. We defined a positive response as one fulfilling all of the following three criteria: (a) counts per minute (cpm) higher than 1000, (b) cpm higher than three times the background without added antigen, and (c) cpm being above one-third of the maximal response seen with APCs from any of the donors tested. TCR sequencing. T-cell blasts were isolated by Percoll

T. Hansen et al.

gradients, and cytoplasmatic RNA was extracted with phenol [15]. TCRB mRNA was reverse transcribed using 20 pmol of the C/3 primer I: 5 'CACTGTGCACCTCCTTC-3' (Genosys, Houston, TX, USA) (C/3 sequences from Tunnacliffe et al. [16]) and avian myeloblastosis virus or Moloney murine leukemia virus reverse transcriptase at 42°C in 20 ~l for 1 hour, followed by boiling for 2 minutes. From this stage, two alternative strategies were used. The first strategy was to make a polymerase chain reaction (PCR) using the C/3 primer II: 5'-GGAGAgCTCaaGCTTCTGATGGCTCA-3'(this primer is modified compared to the C~1,2 sequence upstream of the C/3 primer I to contain restriction sites for SacI and HindIII) and primers corresponding to relatively conserved regions of the leader segments [17]. Leader primer I was 5 '-CCTGCCATGGGATCCAGGCTCCTCTGCT-3'. Leader primer II was 5'-CCAGGATCCATGCTGAAGCTTCTGCTGCTT-3'. Leader primer Ill was 5'-CCAGTCGACTGTGTCAAGTGGGGCTTTTACTTTCT-3 r. If none of the leader primers worked, the second strategy was to perform rapid amplificaton of cDNA ends (RACE) [18] by a slightly modified procedure. First-strand TCRB cDNA was synthesized as above and boiled for 2 minutes. The volume was adjusted to 80/.d by adding STE (0.15 M NaC1, 10 mM Tris-HCl, pH 8.0, and 1 mM EDTA). The cDNA was gel filtered through an STE-equilibrated Sephacryl S-300 cDNA spun column (Pharmacia) to remove excess primer and deoxynucleotides. The filtered cDNA was precipitated in 0.3 M Na-acetate, pH 5.0, and 70% ethanol, washed in 70% ethanol, and solved in 7 ~l H20. To make a poly-A tail at the 3' end, we added 4/~l 5 x tailing buffer (Boehringer Mannheim, Mannheim, Germany), 6/.~l 5 mM CoC12, 2/a,l 2 mM dATP, and 1/.d terminal deoxy nucleotidyle transferase (Boehringer). Tailing was performed for 10 minutes at 37°C and was stopped by heating to 95°C. The tailed cDNA was precipitated and solved in H20 as above. RACE amplification was performed in 100/A volume using 25 pmol of CB primer III: 5'-CCATTCACCCACCGCTCAGCTCCA-3', 10 pmol of adapter + T primer: 5 '-CCACTGCAGTCGACTCTAGATTTTTTTTTTTTTTTTT-3' (includes restriction sites for PstI, SalI, and XbaI), and 25 pmol of adapter-T primer: 5'CCACTGCAGTCGACTCTAGA-3'. The cycling conditions were one initial cycle with 95°C for 2 minutes, 55°C for 5 minutes, and 72°C for 40 min, followed by 40 cycles of 95°C for 40 seconds, 55°C for 1 minute 30 seconds, and 72°C for 2 minutes, followed by a final extension for 10 minutes at 72°C. The PCR products were size selected on agarose gel, purified by Geneclean (Bio 101), digested with restriction enzymes (for RACE products, we used the natural BglII site upstream of Cfl primer III), cloned in M13, and sequenced.

TCR3 Usage by DR4-Restricted TCC

TCRa was amplified using RACE as above except that the cDNA was generated using the Ca primer I: 5'GCCACTTTCAGGAGGAG-3';the PCR was performed using the Ca primer II: 5'-TCGGAACCCAATCACTGACAG3', and adapter primers. For cloning of the RACE products, the natural HindIII site upstream of Ca primer II was exploited. The Ca sequence can be found in Yoshikai et al. [19]. At least three M13 clones were sequenced for each TCC, and consensus sequences constructed in case the three clones were not 100% identical. HLA typing. HLA typing was performed by serology (class I and some class II specificities) and DNA typing (class II alleles) using hybridization with oligonucleotide probes to PCR-amplified DNA [7]. RESULTS

HLA restriction of TCCs studied. Initially, 47 PPD- or TT-specific TCCs from the three donors (RT, IW, or AKG) were analyzed. TCR sequencing demonstrated that five of the TCCs were double clones or duplicates of other TCCs (data not shown). They were therefore excluded from further analysis. The remaining 42 TCCs were tested using the corresponding antigen and autologous APCs in the presence or absence of the DRa-chainspecific mAb L243. Five TCCs were not significantly inhibited by this mAb and were not studied further (data not shown). The remaining 37 TCCs were then tested with APCs from a panel of donors. Since two of these TCCs in preliminary tests recognized antigen both when presented by DR4- and DR7-expressing APCs, and therefore might be restricted by DRw53, they were excluded from further analysis. Representative results of repeated tests of the remaining 35 TCCs using the corresponding antigen (PPD or TT) and autologous or allogeneic APCs expressing Dw4 or Dw14.1, as well as the percent inhibition in the presence of the DR-specific mAb L243, are shown in Table 1. Fourteen TCCs were restricted by Dw4, 20 by Dw14.1, and one (RT3) apparently recognized antigen both when presented by Dw4 or Dw14.1. All TCCs (except RT3) were also tested with antigen and APCs from a Dwl0 +, Dw4-, Dw14.1- donor. No antigenspecific responses (32-256 cpm) were seen using APCs from this donor, showing that these TCCs could not recognize antigen when presented by this subtype of DR4. TCR sequences. The TCR~ genes of most TCCs were successfully amplified using leader primer 1, and that of AKG7 by using leader primer 3. In the latter case, it was necessary to reamplify size-selected PCR product to get sufficient material for cloning. The TCR~ genes of five

151

TCCs (RT9, -13, -15, AKG22, and -26) were amplified by the RACE technique. In several cases, we found that the TCCs in addition had a transcript of a nonproductively rearranged TCR~ gene (not shown). Figure 1A shows the deduced amino acid sequences of the TCR/~ chains. Nineteen different V/3 genes from 13 families were used by the TCCs studied, and nine of 13 J/3 gene elements were expressed. The putative CDR1 and CDR2, defined as aa 26-31 and 47-57, respectively, are indicated using the model of Chothia et al. [4]. From Fig. 1A, it can be seen that AKG6 and AKG8 had identical TCR 3 aa sequences. Therefore, we decided to sequence the a genes as well. The TCRa sequences of these two TCCs are shown in Fig. lB. The TCCs both used Val.4 and Ja HAVT27, but their sequences differed for 2 aa in the CDR3a. Figure 2 shows the nucleotide (nt) sequences in the CDR3a and CDR3/3. AKG6 and AKG8 had different nt sequences in CDR33 although the deduced aa sequences were identical. In Table 2, the V/3 and J/3 expressed by the TCCs are listed in the same order as in Table 1. We also grouped the V/3 according to Chothia et al. [4]. Members of the same group have at least 50% aa identity.

Possible correlations between TCR structure and HLA specificity. As appears from Table 2, all three TCCs expressing V/35.7 were restricted by Dw14.1. Expression of V33.1, -8.1, or - 17.1 did not seem to be associated with a strong preference for Dw4 instead of Dw14.1 or vice versa. In Fig. 1A, the CDR1 and 2 are indicated. No obvious correlation of CDRs and restricting HLA molecule was detected. Table 2 also shows that six TCCs belonged to Chothia's group 2. Five of them were restricted by Dw4, while the last (RT3) could recognize antigen both when presented by Dw4 or Dw14.1. Thus, group 2 appears to be correlated to Dw4 restriction. Not counting RT3, this correlation has an uncorrected p value of 0.0072 by Fischer's exact test. Groups 1 and 4 did not correlate with either of these HLA molecules. Five of the TCCs (RT3, -13, -17, -32, and -48) had the motif DRG in CDR3. The sequence DRG is contained in one of the reading frames of D/31.1, and the TCCs with this motif expressed D/31.1. This is shown in Fig. 3. Four of the TCCs with the motif DRG in CDR3 were restricted by Dw14.1, while the fifth (RT3) could use both Dw4 and Dw14.1. Possible correlations to antigen specificity. The results given in Table 2 also suggest a correlation between expression of V38 and specificity for a PPD-derived peptide, and between expression of V/312 and specificity for a TTderived peptide. Most striking, six of 15 PPD-specific TCCs were V/38 + compared with one of 20 TT-specific

152

T. H a n s e n et al.

TABLE 1

Specificity of TCCs Autologous

Antigen-presenting ceils Dw4

Dw14.1

Probable restricting HLA molecule

Ag ÷

Ag-

Ag*

Ag-

Ag +

lnhib. a-DR mAb ~

82 a 134 70 118 26

21207 17759 20888 5937 15729

96 674 526 260 214

25328 32118 31815 13131 16521

98 90 156 68 190

3239 2911 4698 2076 1001

98 92 99 99 99

Dw4

1 9 13 15 17 31 32 47 48

142 58 44 46 54 64 46 74 46

4852 15013 6190 11871 4002 3632 4960 4936 6633

118 156 140 486 92 116 128 190 72

1503 2528 3490 4920 544 3153 2953 936 504

144 72 180 384 96 264 196 202 132

8824 40172 26047 27344 14731 37779 16626 11854 7723

98 98 92 96 98 99 99 96 96

Dw14.1

3

92

2492

184

5257

280

3824

87

Dw4/Dw14.1

848 854 1048 204 164

19191 14543 13815 2949 2809

1095 526 420 116 720

18110 13053 13962 5427 4093

410 210 142 104 154

356 420 186 346 900

98 77 93 94 91

Dw4

12 15 18 24 28

264 672 84 178 262

3401 10620 1735 8137 4014

66 304 98 570 94

150 1515 208 1898 220

188 410 72 344 472

4291 20198 2785 7433 5014

98 91 99 94 83

Dw14.1

AKG 1 4 5 10

404 56 390 206

29294 2034 91523 3538

1125 92 378 438

33296 2110 52342 3532

128 54 130 72

446 114 1365 176

86 80 95 80

Dw4

3 6 7 8 22 26

330 124 746 132 94 120

18115 8306 22848 3902 1759 3948

150 178 152 104 68 120

144 2292 234 396 80 208

828 128 812 130 86 64

18034 11067 17952 3221 1331 4495

99 82 97 99 97 97

Dwl4.1

TCC

Antigen

Ag-

RT 5 11 18 25 40

PPD

IW 4 8 14 17 23

TT

a % Inhibition = (1 - median cpm with L243/median cpm without L243) x 100c~. bThe numbers represent median cpm of triplicate cultures from representative experiments.

ones. The Vfl8 ÷ TT-specific TCC (AKG10) expressed VB8.3, which is quite different from VB8.1 and V/38.2 (Fig. 1A). All of the TCCs with the motif DRG in CDR3 were PPD specific, and a correlation between this motif and specificity for a PPD-derived peptide therefore seems likely.

DISCUSSION In this study, we have analyzed 35 antigen-specific TCCs restricted by the RA-associated Dw4 or Dw14.1 molecules. We looked for possible correlations between TCRfl gene usage and HLA or antigen specificity. We found that the VB genes used by the TCCs were quite heterogeneous. The Dw4 and Dw14.1 molecules

B

DAGVIQSPRHEVTEMGQEWTI~CKP ISGHNS LFWYRQTMMRGLELL IYFNNNVPIDD SGMPEDRFSAKMPNASFSTLKIQPSEPRDSAVYFCASS .............................................................................................. ............................................................................................... ............................................................................................... .............................................................................................. ............................. DY ................................................................ --R-T-T---K ......... M--Q-L---T V ........ Q ..... A--R-RA-L ...... K ..... E--D-TLA ...................

DTAVSQTPKYLVTQMGNDKSIKCEQ NLGHDT MYWYKQDSKKFLKIM FSYNNKELIIN ETVPNRFSPKSPDKAHLNLHINSLELGDSAVYFCAS ............................................................................................

DTKVTQRPRLLAKASEQKAKMDCVP

DAGITQSPItHKVTETGTPVTLRCHQ --E ........ I .... RQ---A --E ........ I .... RQ---A

NAGVTQTPKFQVLKTGQSMTLQCAQ DMNHNS MYWYRQDPGMGLRLI YYSASEGTTDK GEVPNGYNVSRLNKREFSLRLESAAPSQTSVYFCAS .............................................................................................

EAQVTQNPRYLITVTGKKLTVTCSQ

DGGITQSPKYL RKEGQNVTLSCEQ NLNHDA MYWYRQDPGQGLRLI YYSQIVNDFQK GDIAEGYSVSREKKESFPLTVTSAQKNPTAFYLCAS ............................................................................................. .............................................................................................

NAGVMQNPRHLVRRRGQEARLRCSP

VCt

QSVTQPDIHITVSEGASLELRCNY

.............................................................................................

RT5 RTI3 RT17 RT32 RT48 RTI5 AKGI0

RTI8 AKG1

AKG7

AKG4 AKG2 2 AKG26

RT31 IWI8

AKG3

RT9 IW4 IW14

IWI2

TCC

AKG6

AKG8

MKGHSH

NMNHEY

TENHRY W--NN W--NN

NFKEQTE NFKEQTE

H-F .... T .............. H-F .... T ..............

AEIINERFLAQCSKNSSCTLEIQSTESGDTALYFCASS

N .............. N ..............

SRLLSDRFSAERPKGSFSTLEIQRTEQGDSAMYLCAST

LLKYFSGDT

VYLQKENIIDE

YYSMNVEVTDK

LVQGIKGFEAEFKRSQSSFNLItKPSVHWSDAAEYFCA

SGMPKERFSAEFPKEGPSILRIQQVVRGDSAAYFCASS

GDVPEGYKVSRKEKRNFPLILESPSPNQTSLYFCASS

AA

VGS

-N-

S

S

SS-

PATGGR

RGQD SRGEI TPGQGS

LKIRTPAG

RSERD VSGGR

ESMGQGRG AH AQE

KEQGRRRSE

RQVGTGG ELVEAD

VGH MPDRGR KDRGQ VDRGR TKDRGV FSRD GSRDRD

QDRGSL RAIFAGAGG QDNSG QVERL

LAGDFA

LYEGPGTA KPGAFA RDRV RQGP LAPQQG

LKLAGG

LGGA LSG

HGLVIG

-N-D-N-

~

E

E

V

...................

I

I

Cot

1.4

1.4

Vct

EQYFGPGTRLTVT

QPQHFGDGTRLSIL TEAF--Q .... TVV YGYT--S .... TVV

TDTQYFGPGTRLTVL

SYEQYFGPGTRLTVT QET ......... L-L

TEAFFGQGTRLTVV SNQPQH--D .... SIL N ..............

ELFFGEGSRLTVL

NSPLHFGNGTRLTVT YGYT--S ......

TDTQYFGPGTRLTVL ............... NTEAF--Q ...... V NQPRSF-D .... SINYE ........... T GYT--S ...... V YGYT--S ...... V

ETQYFGPGTRLLVL TGELF--E-S--T-SYE ......... T-T D .......... T--

EQYFGPGTRLTVT

G-L---E-S ..... Y--Y ......... T Y .............. NT-A---Q ...... V

E

E E E

E

E E

E E E

E

E E

E E E E E E E

E E E E

E

E E E E

~QFrGPG~rL ~ .............. v Y--Y ......... T E

TDT-Y ..........

SYNEQFFGPGTRLTVL

TQYFGPGTRLTVL

GGGADGLTFGKGTHLIIQPY

-Jet

(A) Deduced T C R B aa sequences. (B) Deduced TCRc~ aa sequences.

LFWYVQSPGQGLQL

VYWYRQLPEEGLKFM

MSWYRQDPGLGLRQI

MYWYRQDPGHGLRLI HYSYGVKDTDK GEVSDGYSVSRSKTEDFLLTLESATSSQTSVYFCAIS -F ..... L ............. H--N ............. N---LP ...... A .......... -F ..... L ............. H--N ............. N---LP ...... A ..........

VYFQNEELIQK

S .... L ...... S .... L ......

VYWYRKKI~EELKFL

FIGURE 1

SYGATPY

.... ....

IKAHSY

L--N ....... L--N .......

-

ETG ..... R ................. ETG ..... R .................

RT25 IW8

TYFQNEAQLEK

............ ............ ............ .......... ..........

DTEVTQTPKHLVMGMTNKKSLKCEQ HMGHRAMYWYKQKAKKPPELM FVYSYEKLSIN ESVPSRFSPECPNSSLLNLHLHALQPEDSALYLCASS ..............................................................................................

LYWYRQTLGQGPEFL

,---,---,-,,--,--,,-, D---A---P-YS--L--NA-L ,---,---,-,,--,--,,-, P .... L--P-YS--L--NA---D P .... L--P-YS--L--NA---D

RT3 IWI7

ISEHNR

-Q-YEKEE-GR -Q-YEKEE-GR -Q-YEKEE-GR -Q-YR-EENGR -Q-YR-EENGR

.... .... .... .... ....

DTGVSQDPRHKITKRGQNVTFRCDP

R ....... K ....... R ....... K ....... R ....... K ....... R--S Q---NT ...... R--S Q---NT ......

VL---P--I VL---P--I VL---P--I AL---P--I AL---P--I

IW28

........... ........... ........... ........... ...........

D ..... D ..... D ..... ET .... ET ....

RT1 IWI5 IW24 IW23 AKG5

S-TH S-TH S-TH S-TH S-TH

KAGVTQTPR~LIKTRGQQVTLSCSP ISGHRS VSWYQQTPGQGLQFL FEYFSETQRNK GNFPGR~SGRQFSNSRSEt4~VSTLELGDSALYLCASS ....................................................................................................

GEVPDGYNVSRI/qKQNFLLGLESAAPSQTSVYFCASS

AKG6 ~a.KG8

HYSVGEGTTAK

DVKVTQSSRYLVKRTGEKVFLECVQ DMDHEN MFWYRQDPGLGLRLI ¥FSYDKVKMEE KGDIPEGYSVSREKKERFSLILESASTNQTSMYLCASS ...............................................................................................

MYWYRQDPGMGLRLI

RT40 RT47

DMNHEY

CDR2

VNAVSLRPKFRVLKTGQSMTLLCAQ

-

RTI1

-CDRI-

V~

TCC

5.7

18 • 1

17 . 1 17 • 1 17 . 1

14.1

13 •3 13.3

12.2 12.5 12.5

i0 . 1

9 •1 9 .1

8 •1 8.1 8.1 8.1 8.1 8.2 8 •3

7.1 7.1 7.3 7.3

6 .9

5.7 5.7 5.8 5.8

HAVT27

HAVT27

J~

2.3

2.1

2.3

J~

2 •7

1 •5 1 .1 1 •2

2 •3

2 •7 2.5

1.1 1.5 1.1

2 .2

1 •6 1 .2

2 •3 2.3 1.1 1.5 2.7 1.2 1 •2

2.5 2.2 2.7 2.3

2 .7

2 •2 2.7 2.1 1.1

2.7

S. 1 2 . 1 s.1 2.1

3.1

3.1

I. 3

v~

C~

4

C

154

T. Hansen et al.

TABLE 2

TCC

T C R Vfl and J/3 e x p r e s s e d by the T C C s

Vfl

V/~ group

J,8

Probable restricting HLA molecule

Antigen specificity

A

aagl A V G S G G G AKG6: GCT GTG GGT TCA GGA GGA GGT AKG8:

GCT GTG GGT GCA GCA GGA GGT A V G A A G G

RT 5 11 18 25 40

8.1 1.3 9.1 7.3b 3.1

1 1

2 2 4

2,3 2,3 1,6 2,7 2,1

Dw4

Dwl4.1 "

9 13 15 17 31 32 47 48

1 4 1 4 1 4 1

2.4 1.5 2.3 1.2 1.1 2.7 1.5 2.3 2.7

3

7.1

2

2.5

Dw4/Dwl4

4 8 14 17 23

17.1 7.3b 17.1 7.1 5.8

4 2 4 2

1.1 2.3 1.2 2.2 2.1

Dw4

12 15 18 24 28

18.1 5.7 13.3 5.7 6.9

1

2.7 2.2 2.5 2.7 2.7

Dwl4.1

AKG 1 4 5 10

9.1 12,2 5.8 8,3

2 4 1 1

1.2 1.1 1.1 1.2

Dw4 " " "

14,1 5.1 10.1 5.1 12.5 12.5

4 1 1 1 4 4

2.3 2.l 2.2 2.l 1.5 1.1

Dw14.1 "

1 L

B

aa93 S S L K L A G G N AKG6: AGC AGC CTA AAA CTA GCG GGG GGG AAT

" " "

5,7 17,1 8,1 8,2 8.1 13.3 8.1 3.1 8.1

1

PPD

AKG8:

F I G U R E 2 Nucleotide and deduced aa sequences in AKG6 and - 8 CDR3: (A) CDR3c~ and (B) CDRfl.

IW

3 6 7 8

22 26

1

l

4 1

1

AGC AGC TTG AAA CTA GCG GGG GGC AAT S S L K L A G G N

TT

"

a Grouped according to Chothia et al. [4]. Members of the same group have at least 50% identity of aa residues.

are v e r y similar, differing for o n l y 2 aa in the m e m b r a n e distal d o m a i n . Y e t 19 d i f f e r e n t Vfls f r o m 13 families w e r e e x p r e s s e d b y the 35 T C C s . T h e s t r o n g e s t sign o f p o s s i b l e n o n r a n d o m V/~ usage was that all six T C C s e x p r e s s i n g Vfi chains b e l o n g i n g to C h o t h i a et al.'s [4] s u b g r o u p 2 w e r e r e s t r i c t e d by D w 4 , t h e only e x c e p t i o n b e i n g R T 3 , which a p p a r e n t l y c o u l d i n t e r a c t with b o t h D w 4 and D w 1 4 . 1 . A l t h o u g h this c o r r e l a t i o n may be statistically significant, i n s p e c t i o n o f C D R 1 and 2 and

any o t h e r parts o f t h e s e V 3 chains d o e s n o t reveal any o b v i o u s e x p l a n a t i o n for the p o s s i b l e p r e f e r e n t i a l restriction by o n e o f two very similar H L A molecules. One possible structure-specificity correlation noted was that b e t w e e n V 3 8 and specificity for a P P D - d e r i v e d p e p t i d e . M y c o b a c t e r i a l antigens have b e e n i m p l i c a t e d in the p a t h o g e n e s i s o f R A . A w e l l - k n o w n animal m o d e l o f R A is a d j u v a n t arthritis in rats that is caused by Mycobacterium tuberculosis-specific C D 4 + T cells [20]. T h e r e f o r e , it has b e e n s p e c u l a t e d that Mycobacterium-specific T cells may be i n v o l v e d in R A in humans, Sottini and c o w o r k e r s r e p o r t e d that Vfl8 was a m o n g the p r e f e r e n t i a l l y transcribed Vfl g e n e s in the T cells f r o m synovial fluid o f two o f t h r e e R A patients [21]. T a k e n t o g e t h e r , these data may suggest that t h e r e may be an increase o f Vfl8 +, Mycobacterium antigen-specific, D R 4 - r e s t r i c t e d T cells in

F I G U R E 3 Nucleotide-anddeduced ~ s e q u e n c e s o f D B l . 1 and ~ o m CD3 o f t h e TCC w i t h t h e motif DRG. D~I.I: Reading Reading Reading

frame frame frame

i: 2: 3:

GGGACAGGGGGC G T G G G Q G A D R G

RT3:

AGC S

CAA GAC AGA GGT TCA Q D R G S

RTI3:

ATG CCG GAC AGG GGG CGA M P D R G R

RTI7:

AGC AAG GAC AGG GGA CAA S K D R G Q

RT32:

AGC GTC GAC AGG GGA AGA S V D R G R

RT48:

ACA AAG GAC AGG GGA GTC T K D R G V

TCR/3 Usage by DR4-Restricted TCC

RA patients. Obviously, more experiments are necessary to determine whether this is the case. For instance, the frequency of V/38 + T cells among the PBMCs of donor RT is not known. Another possible correlation found was between expression of the D R G motif, encoded by the third reading frame of D/31.1, and specificity for PPD-derived peptides presented by Dw14.1. I f a peptide is preferentially recognized by T C R with the D R G motif in the CDR3/3 region, there will probably be a relatively high precursor frequency of T cells specific for this peptide because of the particular ease by which nucleotide sequences coding for this motif can be generated during TCR gene rearrangement. This may cause that peptide to be immunodominant. Four of the DRG-motif-positive TCCs were also V¢38 +, possibly indicating some influence of V/3 on peptide recognition. Two of the TCCs (AKG6 and AKG8) had remarkably similar deduced aa sequences, the only difference being 2 aa in CDR3a. Probably, they recognized the same TTderived peptide in the groove of Dw14.1. Two of the T-cell donors are HLA-identical sisters. One donor (IW) suffers from RA while the other (AKG) is healthy. An interesting possibility is that differences in the expressed T-cell repertoire of the two sisters is part of the reason that they are discordant for the disease. Table 2 shows that V/35.7 and V/317.1 were both used by two IW TCCs, but by no A K G TCCs. Conversely, V/35.1 and V/312.5 were used twice by A K G TCCs, but not by any IW TCCs. Also, J¢32.7 was used by three IW TCCs and by no A K G TCCs. It is tempting to take these differences as an indication that the sisters have different T C R repertoires for T T peptides presented by Dw4 or Dw14.1, but the number of TCCs sequenced is too small for a firm conclusion. Several reports on T C R structure-specificity correlations have been published. In some studies [ 2 2 - 3 3 ] similarities in antigen or M H C restriction specificity were reflected in similarities in T C R structure, while in other studies [ 3 4 - 3 6 ] no or only quite weak specificity-structure correlations were discernible. Thus, the data appear to be conflicting. It may be that for some a n t i g e n - M H C combinations the TCRs used by different TCCs are similar, but that for some other combinations they are very diverse. One possibility suggested by Taylor et al. [34] is that homogeneous TCRs will mainly be found in responses to antigens that are similar to self-structures, because many potentially antigen-reactive T cells will have been negatively selected in the thymus, leaving just a few highly selected clones that can be activated by challenge within the antigen in question. It might also be that the TCRs used in primary immunoresponses are heterogeneous, but that after repeated immunizations a selection for TCCs with high-

155

affinity TCRs showing more homogeneity will occur. (The same difference might apply for in vitro compared with in vivo priming). The reason that TCRs restricted by the same HLA molecule can be so diverse may be that different CDR3~/CDR3/3/peptide configurations may cause different orientations of the TCR toward the HLA molecule and/or that the same p e p t i d e - H L A complex may be seen in more than one orientation [3-5]. Also, the nature of the peptide bound in the groove of the HLA molecule may influence the TCR repertoire for that p e p t i d e - H L A combination; for instance, the side chains of different amino acids may have different flexibility [37]. In conclusion, the TCR/3s used by HLA-Dw4- or -Dw14.1-restricted TCCs appear to be quite heterogeneous. One aim of the study was to see whether T cells restricted by Dw4 and/or Dw14.1, irrespective of which peptide they recognized, had common structural features in their TCR/3 chains that could be exploited for immunotherapy of RA. We found little evidence that this may be a likely possibility. ACKNOWLEDGMENTS

The technical assistance of Anne Brit Thoresen, Guri Jcsrum, Kari Lislerud, and Kari Lise Forset is appreciated. This work was supported by the Norwegian Research Council for Science and the Humanities and by Pronova.

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