T cell receptor variable region repertoire in lymphocytes from rheumatoid arthritis patients

Immunobiol., vol. 185, pp. 483-491 (1992)

Rapid Communication

Klinische Forschergruppe fur Rheumatologie, Universitatsklinikum Freiburg, Germany

T Cell Receptor Variable Region Repertoire in Lymphocytes from Rheumatoid Arthritis Patients ULRICH KRAWINKEL 1 and GERD PLUSCHKE 2 Received May 12, 1992 . Accepted May 18, 1992

Introduction Rheumatoid arthritis (RA) is a disease characterized by chronic inflammation affecting the synovial membranes of articulating joints. T lymphocytes, as regulators of immune responses, are thought to be involved in the initiation and perpetuation of RA (1). Supportive evidence for this idea comes from observations that activated T cells represent a fraction of 10-15 % of the synovial mononuclear cell infiltrate (2, 3), and that RA is strongly associated with the DR4 and the DRI alleles of the class II genes of the major histocompatibility complex (MHC) (4-8). Moreover, elimination or inhibition of T cells by various treatment sometimes leads to an amelioration of RA (9-11). In situ activation and clonal expansion of specific T cell populations caused and selected by as yet unknown synovial antigen(s) is suggested by the observation of dominant T cell receptor (TCR) rearrangement patterns in T lymphocytes isolated from synovial tissue and cultured in vitro (12-14). Direct analyses of the synovial TCRrepertoire utilizing the polymerase chain reaction (PCR) did not confirm the existence of few dominant T cell clones in the synovial fluid. However, the preferential usage of TCR variable region gene families which are rarely used in blood T lymphocytes suggested a role for superantigen in the pathogenesis of RA (15-17). Here we review our studies of the synovial and peripheral TCR-repertoire in RA-patients and discuss them in the context of related data from other groups in the field. Experimental Approaches Commonly used techniques to analyze the TCR V -region repertoire of RA -patients are outlined in Table 1. We studied the synovial TCR -Va Present address: 1 Fakultat fur Biologie, Universitat Konstanz, Postfach 5560, 7750 Konstanz, Germany, and 2 Ciba-Geigy AG, CH-4002 Basel, Switzerland

484 . U. KRAWINKEL and G. PLUSCHKE Table 1. Experimental strategies to analyze the TCR a-chain (a) and fJ-chain (b, c) repertoire of RA-patients

A. Analysis of the synovia! TCR Va-region repertoire - mRNA is isolated from SFL and cDNA is synthesized with reverse transcriptase. - A dG-tail is added to the 3 end of the eDNA utilizing terminal transferase. - PCR-amplification of tailed cDNA employing a Ca-specific primer and oligo-dC as a nonspecific primer. - Amplification-products of 100-500 bp length are purified on a gel. - Isolated fragments are re-amplified using a nested Ca-primer and oligo-de. - Products of correct size are isolated after gel-purification. - Ligation of amplification-products into a plasmid vector, transformation of bacteria, screening of colonies with another nested Ca-oligonucleotide. 50 % of the colonies react positively. - The VJ-junctional nucleotide sequence of cloned TCR Va-regions is determined.

B. Analysis of the expressed TCR Vf3-region repertoire - mRNA is isolated from SFL and PBL, eDNA-synthesis with reverse transcriptase - PCR-amplification of eDNA employing a radioactively labeled CfJ-specific primer and 22 family-specific VfJ-primers. As an internal standard Ca-regions are amplified utilizing radioactively labeled Ca-primers. - VfJ- and Ca-amplification products are separated on a gel, bands are isolated and the VfJ!Ca ratio is determined by counting the incorporated radioactivity. C. Analysis of transcripts carrying Vf32-gene segments - cDNA from SFL is amplified by PCR utilizing a CfJ-specific and a VfJ2-specific primer. Both primers carry restriction sites suitable for cloning the resulting PCR -products into plasmid vectors. - Gel-purification of PCR-products. - VfJ2-carrying DNA-fragments are cloned in bacteria and the VfJ2DJ-junction nucleotide sequences are determined.

repertoire by employing the anchor PCR technique (Table 1a). The TCRrepertoire usually is analyzed according to the protocol of CHOI et al. (1S), with the PCR-primers described therein (Table 1b). The V~D~J~­ junctional sequences are determined on DNA-fragments obtained by PCRamplification of mRNA from synovial fluid (SFL) lymphocytes (Table 1c).

V~

Patients

SFL for our studies were obtained from therapeutic arthrocenteses of four RA-patients who fulfilled at least five of the American Rheumatism Association criteria for RA classification (19). Patients ALN, AKR, PTW carry the HLA-DR4 subtypes which predispose to RA, whereas patient KSR was typed DR7-DW7/DRWS.1-DW1 (20). TCR Va-region Repertoire

In our analysis, 55 Va-Ja-Ca transcripts from synovial lymphocytes of RA-patients PTW and KSR were characterized as eDNA-clones (16). 51

TCR-Repertoire of RA-Patients

485

Table 2. Frequency and Vn-Jr; junctional sequences of transcripts using Vcd4, Vcd5 and VexFRI-segments in Sl~L from two RA-patients. Frequencies for PBL from healthy individuals are calculated from data in references 21-24. Amino acid sequences are gi\'en in the one-Iettercode SFL of patient PTW Vu14 JC( (4/39) - nCA SSGYSTL U-C« -YFCA YRSIQGAQKLAF-Cu -YrCA YTDTGRRALTF-Cu - YfCA--YRSSNYGGSQGNLlF-Cu Vrd5 J« (3/39) -YFCA EDAGNMLTF-Cc; - YFCA ESSSGGSYIPTF-C« - YFCA---MRSFNQAGTALlJ'-C(( VuFRI--Jex (6/39) - YLCA AVSSYKLIF-Cu - YLCA----VPNQAGTALll:-Cc; -YLCA VTLYNQGGKLll:-Cc; -YLCA---VRPEYGSSQGNLlF-Cu - YLCA--VLPMFAAG N KL TF-Cn -YLCA VMDSSYKLII:-C(( SFL of patient KSR Vc;14 In(I/12) - YFCA-YRRGMYSGGGADG LU-C« Y((15 JCt (2/12) -YFCA ERWDNNDMRF-Cu - YrCA---ESIKGGGSNYKL U-Cu

VClFRI--Ju (2/12) -YLCA VVSDSGYALNF-Cc( - YLCA VVSDSG YALNF-Cu PBL of healthy persons'" Vcd4ju (0/66) Vn15Jn (0/66) VnFRIJn (8/238)

clones were in-frame and thus should represent functional TCR-transcripts. Sequence analysis showed that the synovial T cell population of both patients was essentially polyclonal although two pairs and one triplet of identical Va-Ja-junctional sequences indicated the clonal expansion of lymphocytes using Vn-segments from families Va2, VuFRl and Va8. In both patients high expression of Vn-segments belonging to families Vu2, Va8, Va14, Vo.IS and VctFRl was observed. T lymphocytes using Vn2and Vn8-related V-segments are frequent in PBL of healthy individuals but expression of segments from families Vnl4, VatS and VaFRl is rarely found (21-24). The latter families comprise 4 genes in the germ line, i.e. they represent < S '}';) of the entire Va-gene repertoire. In the expressed reper-

PTW

ALN

not detectable 1.5-0.4

2.2 (10.3 °;;,/4.6 %) 2.8 (6.3 %12.2 %) 1.8-0.2

Vj36 Vr:\8 all others

Vj39 all others

2.2 (13.9 %/6.6 %)

Vj32 VPTSVNTEAFFGGTRL TVV -Cj3 Vj32--APRRYTDTQYFGPGTRLTVL-Cj3 Vj32--ADRRYSGNTIYFGEGTWLTVV-Cj3

1.3-0.3

all others

Vj32

Vj32--AHRQQDPQHFGDGTRLSIL-C[:I Vj32-VLERGRPQNIQYFGAGTRLSVL-Cj3 Vj32--RDRRDGTYEQYFGPGTRL TVT -Cj3 Vj32 RQYSGNTIYFGEGTWLTVV -Cj3

2.6 (26.9 %/10.4 %)

Vj32

AKR

VDJ-junction

Vj3-expression ratio SFLlPBL

Vj3-family

Patient

3x Ix

4x

2x Ix

4x

32x

Frequency

Table 3. Comparison of Vj3-gene expression in SFL and PBL from RA-patients. The amino acid sequences of 47 Vj32-Dj3-Jj3 junctions are shown in the oneletter-code

~

'"

is :r:~

c:::

r

>-;;j

o

"'0::l

r

'"

Z

>"'1

'"

;:>::

~

0'

00

.,.

TCR-Repertoire of RA-Patients . 487

toire of synovial lymphocytes from our RA-patients, however, 30-35 % of TCR a-chain transcripts carry Va14-, Va16 and VaFRl-related sequences (Table 2). Most interestingly, the respective Va-Ja-junctional sequences in Va14-, Va15- and VaFRl-transcripts from both patients share structural motives, and the VaFRI-response of patient KSR appears to be oligoclonal (T able 2 and 4). These findings support the idea that the synovial T cell response of RA-patients is selected by as yet unknown antigen(s). Note, that only patient PTW carries the HLA-DR4 subtype which is genetically associated with RA.

TCR VB-region Repertoire The relative expression of VB-families was assessed in SFL and PBL of three RA-patients (Table 3). For comparison, we also analyzed the relative VB-family expression in PBL of two healthy individuals. This did not differ . from the one of PBL in patients AKR and ALN (GINTER et al., submitted). The SFL of both patients, however, showed a relative overexpression of the VB2-family. In patient ALN, we also found a synovial overexpression of families VB6 and VB8. Patient PTW did not overexpress a particular VBfamily in his synovial TCR VB-chain response. On the contrary, we could not detect VB9-transcripts in SFL and PBL. The latter finding cannot be explained by a deletion of structural genes since amplification products of correct size were obtained in the PCR-analysis of PTW -DNA employing primers complementary to 3 different regions of the VB-9 gene. A deletion of VB9-carrying T lymphocytes in some RA-patients also has been found by PALIARD et al. (15). In order to assess the c10nality of the synovial VB2-response of patients AKR and ALN we cloned VB2-carrying mRNAs and sequenced 47 VDJjunctions. In both patients the VB2-responsc is clearly oligoclonal. Moreover, the VDJ-junctions of distinct clones are Structurally similar (Tables 3 and 4). The properties of the VB2-response observed in SFL from RA-patients support the conclusion derived from our analysis of the Table 4. Similarity of amino acid sequence motives within the respective V(D)J-junctions of TCR-transcripts carrying Va14-, Va15-, VaFRl and V~2-gene segments Similarity of V(D)J-junctional sequences Va14-J Va15-J YSTL YTDT YRST YRSS YRRG

ESS EST ERW MRS EDA

VaFRI-J VTLY VSSY VPN.

VVS. VLP. VRP. VMD.

RDRR .. APRRYT ADRRYS .. RQYS VP .... AHRQ .. LER ...

488 . U.

KI(AWINKFI.

and

G. PI USCHt..F

synovial Va-region repertOIre: there IS evidence for an antigen-driven Immune response.

Conclusions Independent quantitative analyses (summarized in Table 5) of the TCR Vregion repertoire in SFL of RA-patients provide evidence that the synovial immune response is selected by as yet unknown antigen(s). Some qualities of this response, namely polyclonality in combination with preferential usage or underrepresentation of distinct V-region families, and an apparent lack of conventional MHC-restriction lead some authors to the speculation that bacterial or viral superantigen(s) are involved (15, 17). However, as individual patients often differ from each other in their patterns of V -region expression in their synovial T lymphocytes (15-17, 25-27) it is obvious that such responses are influenced by multiple parameters. Two of our three patients analyzed for their VB-region repertoire expressed enhanced synovial levels of VB2-carrying transcripts which seem Table 5. Synopsis of quantitative analyses of synovial niB TCR responses in RA-patients Synovial T cell responses in RA B-chains

n-chains

KSR (ref. 16)

not anal yzed

high frequency ofVn14,15 FR1transcripts, Vn14, 15-responses polyclonal, but similar VJ-junctions, oligoclonal VnFRI-response

PTW (ref. 16, and':')

no VB9-transcripts

high frequency of VnI4,15, FR1transcripts, responses poly clonal, but similar VJ -junctions

ALN

high frequency of VB2-transcripts, oligoclonal VB2-response

not analyzed

AKR

high frequency of VB2,6,8-transcripts, oligoclonal VB2-response

not

high frequency of VfI14-transcripts, no Vf,9-transcripts, oligoclonal V[iI4-response

not analyzed

high frequency ofVB14,17-transcripts coming from few T cell clones

not analyzed

high frequency of V[B-transcripts coming from few T cell clones

not analyzed

RA-patient(s)

C) C)

4 of 7 patients (ref. IS)

4 of 5 patients (ref. 17) 3 of 5 patients (ref. 17)

':. GINnR, KRAWINKEL, PLUSCHKF.

SFL. Submitted.

analyzed

Analysis ofTCR Vf3-region expression in rheumatoid

TCR-Repertoire of RA-Patients . 489

to stem from few T lymphocyte clones. A V~2-response can be elicited by M proteins of group A streptococci (28). One may thus speculate that such bacteria are involved in the pathogenesis of RA. Our patients analyzed for their synovial TCR Va-region repertoire differed in their HLA-DR subtypes but both showed high expression of families Va14, ValS and VaFRl which are rarely used in peripheral blood lymphocytes. This may indicate the involvement of superantigen(s), although it has not been reported as yet that superantigens directly select the expression of Va-region families. The possibility exists that Va-segments influence the response to superantigen through preferential pairing of superantigen-selected TCR V~-segments with specific Va-segments. However, we wish to state, that the term «superantigen» need not necessarily be evoked to explain the properties of synovial T cell responses in RApatients. Selection by conventional antigen is a sufficient albeit not an exclusive explanation. A role of genes from families Va14 and VaFRl in autoimmune responses also is suggested by the finding that TCR from auto reactive T cell lines carry Va-segments belonging to these families. The autoreactive clone UAS2 (29) from the synovium of a patient with reactive arthritis expresses a VaFR1-carrying TCR a-chain (30), and two clones reactive against small nuclear riboproteins (autoantigens in mixed connective tissue disease) use Va14-segments in their TCRs (M. SCHLESIER, pers. comm.). In summary, although RA is a disease with a complex pathogenesis, there clearly seems to be a role for (auto )antigen-activated T lymphocytes in the sequence of reactions that finally lead to the destruction of joint tissue. Acknowledgements We thank H. H. PETER, 1. MELCHERS and M. SCHLESIER for discussion, and C. HOFFMANN for preparing the manuscript. The work was supported by the Deutsche Forschungsgemeinschaft through grant PelS1/l.

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