Clin. exp. Immunol. (1992) 90, 440-446

An altered repertoire of T cell receptor V gene expression by rheumatoid synovial fluid T lymphocytes C. LUNARDI, C. MARGUERIE & A. K. SO Rheumatology Unit, Royal Postgraduate Medical School, London, UK (Acceptedfor publication 14 September 1992)

SUMMARY The pattern of T cell receptor V gene expression by lymphocytes from rheumatoid synovial fluid and paired peripheral blood samples was compared using a polymerase chain reaction (PCR)-based assay. Eight rheumatoid arthritis (RA) patients who had varying durations of disease (from 2 to 20 years) were studied. In all patients there was evidence of a different pattern of V gene expression between the two compartments. Significantly increased expression of at least one Vot or V/ gene family by synovial fluid T cells was observed in all the patients studied. Three different Va (Va 10, 15 and 18) and three V/ (V/ 4, 5 and 13) families were commonly elevated. Sequencing of synovial V/I transcripts demonstrated that the basis of increased expression of selected V gene families in the synovial fluid was due to the presence of dominant clonotypes within those families, which constituted up to 53% of the sequences isolated from one particular synovial V gene family. There were considerable differences in the NDJ sequences found in synovial and peripheral blood T cell receptor (TCR) transcripts of the same V/I gene family. These data suggest that the TCR repertoire in the two compartments differs, and that antigen-driven expansion of particular synovial T cell populations is a component of rheumatoid synovitis, and is present in all stages of the disease.

Keywords rheumatoid arthritis T cell receptor V gene usage

(TCR) present within the rheumatoid joint is not known. INTRODUCTION have been chain reaction (PCR)-based methods nd/Ige . is achronicinflammatPolymerase usdtsuyTCexrsinadshwdhtV Rheumatoid arthritis (RA) IS a chronic inflammatory disease fies wr select e xpresi s edi Ras oviandmembrane affecting particularly synovial joints. The cause of the disease is andlsynovial fluid- a t serantigensvmal are unknown, but there is evidence that T cells play a key role in its pathogenesis. T cells isolated from rheumatoid synovium and in activation of selected TCRs. synovial fluid (SF) express activation antigens such as HLA the expression the In Va study, havefamilies characterized A in rheumatoid and V/wegene an y of recpt e f r, synovialoffluid class II molecules, the IL-2 r mleclete trnd thsVAiamilywof knownthis anpeihrlbodTclsuigaPC-sdasywih molecules on their surface [1-3]. RA is strongly associated with and peripheral blood T cells, using a PCR-based assay which two lass11 HA-D antgens DRIandDR4,whic hav in atigens the expression of an individual V gene family between compared anidsequence DR4,ence which tommca a highlA-Donserve tetocmatet.W aeaaye huaodsnva spanningththe common highly conserved amino acid We have analysed rheumatoid synovial the two compartments. ein[] hs idnssgetta fluid, as this represented a sample of all T cells present within the thirdallelichypervatiabenpregio [4].tThese tatin findings g T elst s joint compartment. Our results showed that there was selective expression of a number of Vot and V/I gene families within RA.chr mayhe aipertoirt po gncetic mechdatanismine a of rheumatoid synovial fluid when compared with paired periphsite cheroi todffTel froun tat Thel etionlirely eral blood, and the increased expression was secondary to the memory blood and there is evidence to show that T cells of of specific T cell clonotypes which were unique to the blooda expansion medmor teareis theatTell coprtet jon marker, are otepredominant bearing the CD45RO phenotype, within synovial fluid [5,6]. However, attempts to study the T cell repertoire in rheumatoid synovitis have been hampered by the PATIENTS AND METHODS lack of serological reagents against the majority of the V-gene repertoire. The presence of oligoclonal T cell populations within Patients and clinical material the rheumatoid synovium has been reported [7], but how this Paired samples of heparinized peripheral blood (PB) and SF correlates with changes in the repertoire of T cell receptors were obtained from eight patients (seven female and one male) with RA as defined by the American College of Rheumatology Correspondence: A. K. So, Rheumatology Unit, Royal Postgracriteria [1I3]. Synovial fluid was obtained by arthrocentesis of the duate Medical School, Du Cane Road, London W12 ONN, UK. 440 Rheumatoid arthritis

,.lli third~~~~~~~~~~~~ ,yevral

HLa-DR-restnioriteantipathogenpresicmentatio toisynovil

TCR V gene expression by rheumatoid SF T lymphocytes knee in each patient. All patients had clinically active disease at the time of study. In addition to heparin, 3000 U of hyaluronidase (CP Pharmaceuticals, Wrexham, UK) were added to the SF samples. Mononuclear cells were obtained by centrifugation through Ficoll (Lymphoprep, Nycomed, Oslo, Norway) and used immediately for RNA preparation.

Preparation of RNA and cDNA Total RNA was prepared using the guanidinium isothiocyanate/caesium chloride method. TCRa and # chain-specific first strand cDNA was synthesized in two separate reactions using 5 pig of total RNA and the appropriate constant region specific oligonucleotide primer (Cacl 3' and C#I 3'-the sequences are given in Table 1).

PCR amplification of TCR V gene segments PCR amplification of the V gene segments present in cDNA was performed using 18 TCR Va, 24 TCR VP family-specific oligonucleotides as 5' primers and TCRa and ,B constant region specific oligonucleotides (Ca 3' and C# 3') as 3' primers which were internal to those used for cDNA synthesis. The sequences of the V family-specific and C primers used are listed in Table 1, and the relative positions of the primers shown in Fig. 1. The Table 1. Sequences of oligonucleotide primers used for polymerase chain raction(PCR)

441

amount of TCR x and fl chain cDNA was assessed by a separate PCR of the constant region using a pair of C region-specific primers (Ca 5' with Cal 3' and Cf3 5' with Cf3I 3', see Fig. I and Table 1 for primer positions and sequences). PCR was performed for 30 cycles of 1 min at 940C, 1 min at 550C and 1 min at 72-C. PB and SF pairs were always amplified using the same PCR reaction buffer and the reactions were performed at the same time. Negative controls (with no added cDNA) were included in each PCR experiment.

Quantification of PCR product A 5 pl aliquot of each PCR sample was transferred to nylon membrane (Hybond-N +, Amersham) using a slot-blot apparatus (HYBRI-SLOT manifold, BRL, Gaithersburg, MD). Hybridization was carried out with radiolabelled Ca or C# probes which overlapped with both the constant and the variable region PCR products. The TCRa probe was a 363-bp Taq I fragment of Jurkat TCRa chain cDNA, spanning nucleotide 533-896 [14], and the TCRB probe was a 518-bp fragment obtained by PCR, spanning nucleotide 445-963 of the Jurkat TCRB cDNA [15]. The sequences of the PCR primers used (C/4 and CflI 3') are listed in Table 1. Filters were then exposed for autoradiography. The hybridization signal was assessed using scanning densitometry (Chromoscan III, JoyceLoebl) and direct counting cut filter in aThe /3coer. Thebybackground h of iztio w fragments racted. The background hybridization was subtracted. The correlation of expression indexes obtained using the two methods was high (correlation coefficient >0 90), and the /3counter method was employed in all except the first three patients. To compare the expression of a particular TCR V family in the SF with PB, an expression index was calculated based on the densitometry and/or # counts

aalysiscounter.

TCRa COI3' (cDNA) Ca5' Ca3'

5'-+3' CAAAGCTTTTCTCGACCAGC GTGACAAGTCTGTCTGCCTA CTCTCAGCTGGTACACGGC AGCCCAGTCTGTGAGCCAGC GAAGGTTTACAGCACAGCTC

Val1

Va2

Vsf/Csf

.

i

Vpb/Cpb=expression index TCRB

C#13' (cDNA)

CTCTTGACCATGGCCATAAC GATACTGCCTGAGCAGCCGC

C#5'

C/B' Cf4

TTCTGATGGCTCAAACAC GGACCGGAACAAGGTGTTCCCA

V#24

GCTGCTGTTCCACTACTATGA

The C primers were used as described in Patients and Methods. The Va3 to Vaxl8 and the VP/I to VB20 primers have been published [19,20].

VB21 and VB24 sequences have recently been described [21].

Cloning and sequencing of NDJ region DNA obtained from PCR amplification of V gene families was subcloned into M 13 mp 1 8 DNA, and transformed into Escherichia coli TG2 strain. Sequencing was performed using T7

modified DNA polymerase (Sequenase, US Biochem, Cleveland, OH).

RESULTS

Fig. 1. Strategy of polymerase chain reaction (PCR) amplification of

Experimental protocol and expression index Assay reproducibility. Employing the procedures described, we were able to detect amplified DNA for each Va and V/3 family studied, in both the PB and SF compartments following 30 cycles of PCR. In control experiments using varying numbers f PCR cycles, we found that lower cycle numbers (15-25 cycles) may fail to amplify V gene families which were expressed at low levels (data not shown).Thirty cycles was therefore chosen as the reaction condition for studying TCR gene expression in the patient samples. The relative expression of a particular or

T cell receptor (TCR) Va and VB gene subfamilies. See Patients and Methods and Table 1 for details of PCR and primers employed. Solid boxes indicate the relative positions of the primers used for the V-family

gene family in the synovial compartment compared with paired PB was calculated as a ratio of hybridization signal intensity of

PCR, and thehatched boxes the primers used fortheC-region PCR. The products were then hybridized with a constant region fragment which overlapped with both C and V region amplifications. Their lengths and relative positions are shown. Relative expression indices were calculated from the signal intensities.

the individual V gene-amplified products in the two compartments, after correcting for the amount of TCR mRNA as detected by the C region amplification, and was expressed as a relative expression index. The reproducibility of this method of analysis was assessed by comparing the expression indices for all

' 518 bp TCR

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C. Lunardi, C. Marguerie & A. K. So

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The experiments were performed twice from two independent cDNA syntheses, and the expression indices calculated (J/B, experiments 1 and 2). The P value of the correlation coefficient was 0 0001.

(Jurkat: Val,VJJ8. 1 and HPB-ALL: Va 12, Vfl5.3). A comparison of V gene expression was undertaken between two samples which differed in the numbers of T cells bearing a given receptor, with the total number of T cells kept constant (2 x 107). For development of an expression index for Val and V/38.l, V region and C region polymerase chain

the Va families of one pair of synovial and PB RNA samples (patient 2) analysed in two independent sets of experiments. The results showed that there was a strong correlation between results obtained from the two experiments (correlation coefficient = 0 78, P < 0 0001 ) (Fig. 2). Similar reproducibility was obtained for the Vfl primers (data not shown).

reactions (PCRs) were performed using a sample containing 2 x 107 Jurkat cells as reference, and samples which contained 2 x 104, 2 x 105 and 2 x 106 Jurkat cells for comparison. The T cell population was made up to 2 x 107~ by HPB-ALL cells, which does not express the relevant V gene families. Similar experiments were performed for the Val12 family using HPB-ALL cell line, and expression indices were calculated for differing ratios of HPB-ALL cells as above. Jurkat cells were used in

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expression index changed with variations in the amount of V gene RNA in paired samples. This was performed using two T cell lines, Jurkat and HPB-ALL, which expressed Vfl8.1 and Vacl, and V/35.3 and Val2 respectively. The expression index was calculated between pairs of samples. In one of the pair were 2 x 107 Jurkat cells. In the other sample was a variable amount ofJurkat cells, with the total cell number kept constant at 2 x 107 by addition of HPB-ALL cells (as they express different TCRs). The numbers of Jurkat cells in the different samples were 2 x 1 04, 2 x 105, and 2 x 106, therefore giving cell ratios of 1000: 1, 100: 1, and 10:1l respectively. Expression indices were calculated from the PCR-amplified products using V/38 and Vas 1 specific primers. A similar set of experiments was performed on HPBALL cell mixtures of differing ratios, using the Va 12 primer. V/35. 1 primer was used as a negative control in this experiment, to demonstrate that the primers are specific (as HPB-ALL expresses Vfl5.3). The results showed that the change in expression index with differing ratios of cells was non-linear (Fig. 3). The expression index increased to > 2 when the cell ratios were between 100 and 1000. Duplicate experiments were performed from the same RNA sample at each dilution, with the same results (data not shown). We have taken an expression index of 2 as an indicator of significantly increased expression of a particular V gene family in one compartment over another. Increased expression of Va and Vfi genes in RA synovialfluid Eight paired SF and PBL samples derived from different RA patients were studied. Their clinical characteristics are summarized in Table 2. In each case, increased expression of one or more Va or Vfi families in the SF was detected. In the majority,

these latter experiments as the irrelevant cell type to make the total cell nube 2 x 0.AV3Ipie a se sangtv otot

demonstrate primer specificity. Duplicate experiments were performed with similar results. Primers used: 0, Val; 0, VfJ8 (Jurkat mixtures); *, Val2; *, Vf35.l (HPB-ALL mixtures).

increased expression of more than one V family was observed. The maximum number of Va and Vfl families found to be elevated were six and seven respectively. No correlation was seen between duration of disease, rheumatoid factor status, HLA-DR type and the V gene families which were elevated in the SF. We did not find a consistent increase in expression index of a particular V gene family which was common to all patients. Some families were increased more commonly in the patients' SF. Four of eight patients demonstrated increased expression of Val 5, while three of eight patients had increased expression of ValO, Vocl8, Vf34, V/35 and Vf313 families. Some V gene families were not increased in the SF in these patients: Val, Va 13, Vfll1, Vf315, Vfll6, Vfpl7, Vfll9, VfJ2l and Vfl24. Figure 4 summarizes the frequency of increased expression of the different V gene families in the group. Sequencing of the TCRJI NDJ region In order to determine if the increased expression of selected V gene families observed in the PCR assays was due to polyclonal activation of T cells, or was due to clonal activation of T cells within a specific V gene family, we analysed the NDJ region sequences of three Vfl gene families which showed elevated expression (expression index >2) in the synovial compartment (V/313.1 from patient 2, Vfl4 from patient 4, and Vf#l4 from

TCR V gene expression by rheumatoid SF T lymphocytes

443

Table 2. Selective T cell receptor (TCR) V gene expression and clinical features of rheumatoid arthritis (RA) patients studied

Patient

Sex

Disease duration (years)

Latex

Erosions

HLA-DR

1 2 3 4

F M F F

4 5 2 10

+ +

+ + + +

ND 3/4

F

5 6 7 8

-

5 2 9 20

F F F

+ + +

ND

1/7 4/4

+

3/4 1/4 1/4

+ + +

Va

VP

10 6 2, 15, 18 4,7,9, 10,

4,7,9,12,13.1,13.2 13.1

17

5.1, 5.2, 13.2, 18

14,15,16,17

3,4,5.1,5.2,6,8,10

1,2,3,4

18 6

2, 8, 15 3, 10, 11, 12,

5.2, 14, 18, 20

15, 18 HLA-DR typing was performed by restriction fragment length polymorphism (RFLP) [22]. Va and VPl families which demonstrated an expression index > 2 are shown. Latex test + indicated a rheumatoid factor titre of > 1/80 on more than two occasions.

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*

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10II I1121 13114915'16'171 19120'21'24' Fig. 4. Frequency of increased expression of Va (a) and V/I (b) families in 3

As negative controls we performed the same analysis on two Vfe families which were not preferentially expressed in the joint (expression index 2 were taken as an indicator of significantly increased expression in the synovial compartment relative to peripheral blood, and the numbers of patients indicated. For this analysis, V/I5. 1 and 5.2 and V/I13. 1 and 13.2 families were combined.

patient 7). In patient 7, a unique NDJ sequence constituted 16 of the 30 V/I14 clones that were sequenced from the SF. In the PB of this patient, this sequence was not observed at all (Table 3). A predominance of a particular NDJ sequence among the SF sequences compared with PB sequences was also found on sequencing the V#4 family from patient 4, and the V/I1 3.1 family from patient 2. These results indicated the presence of dominant clonotypic sequences within particular V gene families. The results are summarized in Table 4. To check that these results were not due to preferential priming during the PCR reaction, the VIl13. 1 family was amplified and sequenced from two independent cDNA syntheses from joint fluid and PB RNA of patient 2. Identical dominant clonotypic sequences were found from these two reactions.

both Of the five families analysed, we Of found compartments. that four PB sequences were present in both compartments. sequences, 22 2% (patient 2, V/i13.1 family) had the same clonotype, which was found in 6- 1 % of the synovial sequences. The other three sequences were present in approximately equal frequency in both compartments. None of them represented compartment dominanc oty . d

DISCUSSION We have compared the expression of TCR V genes by rheumatoid SF T cells with paired PB T cells at the level of individual V gene families, and also by comparing the NDJ region sequences of a number of V/I gene families. Because we were concerned that in vitro expansion of the T cell populations (such as with IL2 or anti-CD3 MoAb) may change the relative expression of the V gene repertoire, we did not stimulate the lymphocytes isolated from either compartment, but used them directly for RNA extraction and PCR. In contrast to a previous report [8], we found that all the known Va and V/I families were expressed in both compartments, and showed that the TCR repertoire which was present within the joint fluid was extremely diverse. Our PCR assay compared the level of expression of each V gene family between the two compartments, rather than to attempt to quantify the amount of an individual V gene family RNA among the total poo1 of T cell RNA. This is because variations in PCR efficiencies of the different primers used would require individual calibration for each family analysed.

C. Lunardi, C. Marguerie & A. K. So

444

Table 3. Analysis ofV 14 NDJ sequences from 30 synovial fluid and 25 peripheral blood T cell clones of patient 7 Peripheral blood

Synovial fluid ND

V * CASS

* * * * *

* * * * *

* *

* *

*

CASS CASS CASS CASS CASS CASS CASS CASS CASS CASS CASS CASS CASS CASS CASS CASS CASS CAS CASS CASS CASS CASS CAS CASS CASS CASS CASS CASS CASS

PGQAVF LTS LTWR PGQAVF PGQAVF PGQAVF PGQAVF PQGAVF LSLAG YGGGFR LTF PQGAVF PQGAVF PQGAVF PQGAVF PQGAVF KDTA

PDIKR QASSGGP LSGTYGD SST PQGAVF PQGAVF MGGAGG LSLAG PQGAVF PQGAVF FVGGE LSKG PQGAVF

J

V

YGY 1.2 SYE 2.7 GAN 2.6 YGY 1.2 YGY 1.2 YGY 1.2 YGY 1.2 YGY 1.2 DTQ 2.3 ETQ 2.5 NTG 2.2 YGY 1.2 YGY 1.2 YGY 1.2 YGY 1.2 YGY 1.2 YNE 2.1 GYT 1.2 ETQ 2.5 EQF 2.1 GNT 1.3 YGY 1.2 YGY 1.2 YEQ 2.7 DTQ 2.3 YGY 1.2 YGY 1.2 TQY 2.3 NTE 1.1 YGY 1.2

CASS CASS CAS CASS CASS CASS CASS CAS CASS CASS CASS CASS CAS CASS CAS CASS CASS CASS CASS CAS CAS CASS CASS CASS CASS

ND

GWDGGA RGTSGE IG RITPSQIV LHNIRDA TGD LFQGEVN RQGN

STGTGI LLGQE SG SQGRP RPGEG LGRGD WTSGR LLGPGRTA LPGVG SRPLV LSGTWG RRQSI RKANRGRSI LAGGFS SDVTG STPHHS LTDRVT

J TGE 2.2 TQY 2.3 NTE 1.1 TEA 1.1 NTG 2.2 TEA 1.1 EQY 2.7 QET 2.7 EKL 1.4 NYG 1.2 YGY 1.2 EKL 1.4 YGY 1.2 IQY 2.4 GEL 2.2 TDT 2.3 NSP 1.6 EKL 1.4 EKL 1.4 NTE 1.1 YGY 1.2 GYT 1.2 EKL 1.4 ANV 2.6 QPQ 1.5

Amino acid sequence is shown in single-letter code. The last three to four amino acids of the V# 14 gene segment are shown, followed by the ND region sequence and the first three amino acids of the J region. The J region usage is shown. Sixteen (*) of the 30 clones sequenced from the synovial fluid were identical.

Differences in the level of expression of V gene families by SF and PB T cells were observed. Taking an expression index of > 2 as an indicator of significantly increased expression, each patient studied had increased expression of at least one V gene family in their SF, and frequently multiple V gene families. Among the eight patients studied, we found that three Va and three VP families were increased in the SF of three or more patients, though in individual patients there was no correlation between increased V gene expression and clinical variables or HLA-DR status. In contrast to an earlier report [9], we did not find consistently increased expression of the VPB14 gene family in RA SF. This may be due to methodological differences in quantifying the level of expression, and whether the SF T cells were pretreated with IL-2 before analysis. What is the basis of increased TCR RNA expression of selected V gene families in the synovial compartment? In control experiments using T cell lines, we showed that an expression index of >2 indicated greater than 100-fold differences in cell numbers. Differences in expression may be due to an increase in

the numbers of T cells or to increased RNA expression, or a combination of the two. A previous study of TCR V gene usage in RA using a limited panel of MoAbs showed up to a 10-fold increase in the frequency of cells expressing VBS in the synovium compared to paired PB [16]. T cell activation will also lead to increased expression of TCR RNA of > 10-fold [17]. We therefore favour a combination of both the above mechanisms as the explanation for the observed increases in SF V gene expression. In order to determine whether increased expression of particular V gene families in the synovial compartment was due to polyclonal activation, or to expansion of a clonal subset bearing a particular V gene, we proceeded to sequence the NDJ regions of VP genes expressed in the PB and the synovial compartments, as these sequences define individual clonotypic TCRs. When the expression index was >2, sequences from the SF showed the presence of dominant T cell clonotypes. These dominant clonotypes comprised between 30% and 53% of the V/I sequences from the joint, and were not present in the paired

TCR V gene expression by rheumatoid SF T lymphocytes

445

Table 4. Sequencing of the NDJ region of VPi families expressed in synovial fluid (SF) and peripheral blood (PB) No.

Synovial

Peripheral

sequenced

fluid

blood

Patient

VP3

SF

PB

2

13.1*

33

36

4

7

4

23

22

NDJ sequence

n

%

n

CAS RFAGA TDT-Jfl2.3 CAS RVAGS NEQ-Jfl2.1 CASS SGA YEQ--Jfi2.7 CSV DLAGG TDT-Jfi2.3 CSV SQK NEQ---Jf2.1

15 2 2 2 2

45-4 61 6-1

8-7

0 8 0 0 2

16

53 3

0

0

8 3 2 2

29-6 11 1 7-4 74

0 1 0 1

0 31 0 3-1

14*

30

25

CASS PQGAVF YGY-J/31.2

13.1

26

28

All different

4*

27

32

CSV GEGLG YEQ--Jfl.5 CSVE EEGTA GAN-Jf32.6 CSV GQVG QPQ---J#1.5 CSV AGTV NEQ---JfJ2.1

87

%

0 22 2 0

0 9.1

* VB families which have an expression index > 2 in the SF compared with PB. The amino acid sequence of the NDJ region is shown in the single-letter code, and the joining segments utilized shown. Only sequences which were found in both compartments or which were present more than once in either compartment are shown. The sequences derived from the VB13. 1 family from patient 7 were all different from one another, in both joint and peripheral blood.

PB sample. The absence of the same dominant clonotypic sequences in the PB compartment suggested that expression of the clonotype was restricted to synovial T cells. In contrast, sequencing of two V# families which had an expression index of < 2 did not show dominant clonotypes in the SF. This provided further supportive evidence that an expression index of >2 reflected biologically significant differences in TCR expression. Some clonotypic sequences were present in both PB and synovial compartments, but this was not influenced by the expression index of the V gene family. When sequences were common to both compartments, their frequency in the synovial compartment was low (