Scand. J. Immunol. 36, 681-688, 1992

T-Cell Receptor V-Gene Usage in Synovial Fluidand Synovial Tissue from RA Patients

S. GUDMUNDSSON, J R O N N E L I D , A. KARLSSON-PARRA,J. LYSHOLM*, B. GUDBJORNSSONt. B. WIDENFALKJ,C. H. JANSON§ & L. KLARESKOGDepartment of Clinical Immunology. Uppsala Utiiversity Hospital, Sweden, 'Department ofRheumatology. Falu Hospital, Falun. Sweden, tDepartment of Rheumatology, Uppsala UniversityHospital, t Department of Hand Surgery, Uppsala University Hospital, and ^Department ofImmunology, Karolinska lnstitutet, Stockholm, Sweden

Gudmundsson S. Ronnelid J, Karlsson-Parra A. Lysholm J, Gudbjornsson B, Widenfalk B.Janson CH. Klareskog L. T-Cell Receptor V-Gene Usage in Synovial Fluid and Synovial Tissuefrom RA Patients. Scand J Immunol 1992:36:681-8

The question of whether Ihcre is a preferential use of certain V genes in T cells entering aninflamed joint has hitherto been studied mainly using unfractionated eells from synovial fluid andtissue respectively, and no clear answer to the queslion has yet been provided. Concomitantly.evidence has been provided that the use of V genes may dilVer considerably between CD4 ' andCD8*^ T cells, and consequently thai detection of biased V-gene expression within aninflammatory lesion may require separate analysis of the Iwo T-cell subsets.

In this paper we have therefore studied T-cell receplor V-gene expression in rheumatoidarlhritis by means of double stainings of synovial fluid and blood for available anti-TCRmonoclonal antibodies and antibodies to CD4 and CD8. respectively. Double stainings were alsoperformed with anti-TCR antibodies and antibodies to activation markers HLA-DR and 1L-2R.A certain bias towards the preferential use of certain V genes was seen particularly in the synovialfluid samples within both the CD4 ' and CDS * T-cell populations, but no uniform pattern wasevident among the 35 patients investigated.

Sveinn Gudtnundsson MD. Deparinieiil of Clinical Immunology, Uppsala Unirer.sitv Ho.spiial. S-751 85 Upp.sala. Sweden

Activated T lymphocytes are thought to beinvolved in the development ofsynovitis as well asin other manifestations of rheumatoid arthritis(for a review see Ref. 1). It is likely that at leastsome of these cells are activated loeally in thesynovial tissue, in contact with synovial MHCclass ll-expressing cells and antigen [2]. It is alsolikely that T cells of particular importance for thepathogenesis of the disease are restricted by aparticular MHC class II HLA-DR4 moleculewhich is characterized by a detined sequence in itsji chain [3]. Although these features suggest thatthe T cells driving the disease may be activated bydefined antigen(s) (see discussion in Ref. 3). it hasso far been difficult to define such an antigen.

Another way to utilize the knowledge of theMHC class II restriction of this T-cell-dependentdisease is by taking advantage of the fact that Tcells that escape elimination in the thymus. but

are still capable of mediating an autoimmuneresponse, tend to make use of a restricted numberof variable T-cell receptor (TCR) genes [4, 5], If Tcells restricted by the particular HLA-DR4 fichain that confers susceptibility to rheumatoidarthritis (RA) would also tend to make use of oneor a few variable TCR ji genes, this would openpossibilities both for a further analysis of thespecificity of T cells driving the disease and forspecific therapy where a subset of potentiallyarthritogenic T cells can be affected (see Refs 6-8).

Analysis of TCR usage among synovial fluideells, and to some extent synovial tissue eells, hasso far been carried out by a number of groups.They have used either a limited number ofmonoclonal antibodies for analysis of the totalnumber of T cells in the synovial fluid [9], or V^specific oligonucleotide probes to quantitate

681

682 S. Gudmundsson el al.

mRNA correspotiding to various V/( genes citherin the unfractionated synovial fluid or tissue bymeans of polymerase chain reaction (PCR)amplification [10 12], or in in vitro expanded Teells by means of direct blotting techniques [13-16]. Quite variable results have been obtained,particularly from the analyses on in vitroexpanded cell populations, which may be due toselection biases introduced by the various cultur-ing techniques (for results and discussions, secRefs 14 and 16). The few analyses published usingPCR amplifieation techniques have also yieldeduneonclusive results so far. In one study a certainrelative aeeumulation of V/fM TCR among RAsynovial fluid cells was reported [11], which wasnot eonfirmed in another study [10]; the interpre-tation ofthesc studies is. so far, also litnitcd by thedifficulties encountered in tnaking the actualPCR-based techniques sufficiently quantitative.Thus, it appears that monoclonal antibodies thatpermit a direct quantitative analysis of synovialcell V-gcne expression remain important tools inthe search for restricted V-gene usage amonginflammatory cells, something that Is furtherctnphasized by the demonstration of different V-gene usage among CD4-and CD8-positiveT cellsrespectively by means of double itnmtinofluores-cence labelling techniques [17].

Against this baekground, we have in the pres-ent sludy analysed both synovial fluid and syno-vial tissue T cells in a number ol" synovialspecimens from RA patients using a wider panelofTCR-speeific monoclonal antibodies than pre-viously published. By means of separate analysisboth for CD4- and CD8-positive T-celt subsets,as well as T-cell subsets carrying dilTerent acti-vation markers, we also wanted to see whetherthere was a bias regarding TCR rearrangementwithin relevant T-cell subsets.

MATERIALS AND METHODS

Patients, biopsies and .synorial fluid specimens. Syno-vial tissue biopsies from patients sufTering from activeRA were obtained in conjunction with routine surgeryat the Department of Hand Surgery, Uppsala Univer-sity Hospital. Synovial fluids (SF) from knee joints ofactive RA patients as well ;is heparinized peripheralblood from these patients were provided from theDepartment of Rheumatology. Falu Lasarctt, Swedenand from the Department of Rheumatology. UppsalaUniversity Hospital. Peripheral blood (PBL) fromnonnal healthy controls was obtained from blooddonors. Biopsies from normal human lymph nodes andnonnal spleen were obtained in conjunction with

routine surgery and the biopsies kindly provided by DrClaes Juhlin, Department of Surgery, Uppsala Univer-sity Hospital,

The synovial biopsies were snap-frozen as earlierdescribed in detail [9] and kept frozen at —70 C untilcryosectioncd. Mononucloar cells from the synoviallluid and peripheral blood samples were obtained byFicoll Hypaque centrifugation.

Antihtiilics. Monoclonal antibodies reactive with thefollowing TCR V-gene segments were obtained from T-Cell Sciences Inc. (Cambridge. MA. USA): V/y5.1 (LC4.Ref. IS); V/i5.2-1-5.3(ICI,Ref. 19); V/y5.3 (WI 12, Ref.20); V/(6.7 (OTI45, Ref. 21); V/(8 (idGK. Ref. 20) andV^12 (S511, Ref. 22). The monoelonai antibody(MoAb) Fl (Va2.3) was produced in our laboratory[2.1]. Notably, the TCR MoAb listed above may alsohave reactivities with V-gene segments other than theindieated ones. Other antibodies used were antibodiesto gamma-delta and to the ^(/(TCR (T-Cell Sciences),Leu4 (anti-CD.l), Leu3a (anti-CD4). Leu2 (anti-CD8),anti-HLA-DR. and anti-I L-2 receptor (anti-CD2.^) (allfrom Bctton-Dickinson. Sunnyvale. CA, USA).

Imniuni'hisUnhcmiiol stdiuin^s. Immiinohistoehemi-cal stainings were done on frozen sections of 9 biopsiesfrom inflamed synovial tissue from RA patients andfrom normal lymph nodes and one normal spleen.Stainings were made as previously described [24] utiliz-ing a goat anti-mouse secondary reagent and a mono-elonai peroxidase-anti-peroxidase (PAP) reagenl (Dak-opatts, Giostrup. Denmark) in tbe third step. In theestimation of the fraetion of T eells stained with therespective anti-TCR MoAbs. the Leu4-positive cellswithin a given area of the biopsy were first counted.Thereafter, cells stained with the respective anti-TCRMoAb.s were counted on serial sections encompassingthe same area.

Cylofluarinii'lric analysis. Cytofluorimetric analysiswas made on suspended cells from 35 synovial fluidsamples and 13 parallel peripheral hlood samples andfrom 15 normal individuals. Stainings were made withthe various anti-TCR antibodies or anli-Leu4 as theprimary reagents followed by an FITC-labelled goatanti-mouse IgG antibody, Kor double labellings phyco-erythrin-conjugated anti-HLA-DR. anti-CD4. arti-CDS and anti-CD25 antibodies (Beeton-Dickinson)were added after blocking the secondary anti-mouseantibody with l"'(i normal mouse serum. Labelledsamples were analysed in a FACScan cytofluorimeter(Beeton-Diekinson).

RESULTS

Itwnutwhtstochemical stainings oti biopsies of

ilieumatoid synovial tissue

Stainings with the V/i5,2-F5.3, V/J5.3,V/i6.7 and anti-gamma-delta TCR were distinctlypositive for lymphocyte subpopulations in bothlymph nodes and synovial specimens, as was theanti-Lcu4 antibody. No stainings on the frozenseetions were, however, seen with the anti-a^TCR antibody, and only weak stainings, difficult

•fa)

TCR I-Gene U.sage in RA Paiienis 683

f. A ^

V%S

X:..

•A^-^^

:V«»" -̂>'• #•?

• V

• -

- • • ^ ^ *

riy^"-^^'«.-;--> \*-"^- *!

F I G . I. Results of immunohisUichcniiciil slaiiiings oTseri;!! sections Irom a rheumaloid synovial tissue with a[Ui-I.,eu4(anti-CD.1) antibodies (a) and ami V/y5.2 4-5.3 antibodies Ib).

684 S. Gudmundsson et al.

TABLE I, Numbers of cells stained with certain anti-TCR antibodies infrozen sections from inflamed synovial tis.sucs*

Synovialtissues

ABCDEFGHI

\{fS.2 + i

223327343

V^5.3

1121

< 1222I

Antibody

\fi$_\

222424234

used

V^6.7

254244333

CD3

100100100100100100100100100

2< [

31

< 14

< 132

* Percentages given represent the number of cells within a given areaof the section that were stained with the respective anti-TCR antibodyas a fraction of the number of cells within the same area that werestained with the Leu4 (anti-CD3) antibody.

to evaluate, were seen with the anti-V^8.1.V^12.1 and V^6.7, Interestingly, cells that werepositive for either one of the well-staining anti-TCR antibodies tended to be accumulated incertain areas of the biopsies as small clusters (Fig.la and tb). No obvious bias towards the stainingfor any of the presently used antibodies was seen,however, when taking the results from the entiresections into account (Table I).

TABLE II. Percentage of CD3+ lymphocytes reactivewith TCR MoAbs in peripheral blood lymphocytes ofRA patients

MedianPercentiles(10 907o)

Exceedingupper limit*

Vp5.2-1-5.3V/?5.3VfS6.7VjiSV^12Va2.3V^5.1

6.03.16.057.05.14.35.9

1.4-20,10-13.2

1.63-15,32.8-17.280.8-14.8

1.78-12.881.92-11.68

4/111/114/115/126/124/123/12

* Percentages represent the numbers of cells bindingthe respective anti-TCR MoAb as a fraction of numbersof cells binding the Leu4 antibody, applying the samegating procedures for side and forward scattering. Thelast column indicates the number of patients where thefraction of T cells binding the respective antibodiestwice exceeds the upper limit, defined by the 10-90%percentile of a normal material (for previous use andreference of this criterion see Ref. 17). This approachalso applies to Table III.

Cytoftuorimetric analysis of tnononuclear cellsfrom SF and PBL of RA patients

Two types of experiments were performed.Initially only single stainings with the anti-TCRmonoclonal antibodies on appropriately Ficoll-separated and gated mononuclear synovial fluid-derived cells were carried out; subsequently alsovarious double staining experiments were per-formed in parallel with continued stainings on theentire cell population. A summary of the resultsfrom single staining experiments on 35 patients isshown in Tables II and III. For comparison, theresults of similar analysis of PBL of normals aregiven in Table IV. No consistent over- or undcr-rcpresentation of any particular V gene wasapparent, even though both increases and de-creases in relative numbers of cells stained with

TABLE III. Percentage of CD3^ lymphocytes reactivewith TCR MoAbs in synovial fluid lymphocytes of RApatients

MedianPercentiles(10-90%)

Exceedingupper limit*

V/?6.7

Va2.3

6.03.755.27.43.15.554.2

1.19-13.00-8.99

0.9-11.02.0 15.9

0-6.821.94-9.121.35-7.28

6/351/352/34

12/356/356/341/33

* See footnote to Table II.

TCR V-Gene Usage in RA Patients 685

TABLF IV. Percentage of CD3^ lymphocytesreactive with TCR MoAbs in peripheral bloodlymphocyles of normals

V/f5.2 + 5.;V/(5,3\li6.1vpsVf}]2V:x2.3V/!5.l

Median

i 2.92.73.84.42.22.73.6

Pcrcentiies(10-90%)

2.0 5.10.8-7.52.5 6.03.0 4.91.4 4.21.9-3.92.5 4.8

Upperlimit*

10.115.011.99.78.47.89,6

* Upper limit is defined us twice the upper valueof 10 90 percentile.

the various anti-TCR antibodies were seen insingle patients; the reference values against whieheomparisons were made were derived after analy-sis of peripheral blood cells of normal blooddonors, and these results were in good agreement

with previous results frotn healthy individuals[17]. In a limited number of the patients, cellsfrom peripheral blood were analysed in parallelwith the synovial fluid cells; in these eases nonotable differences were found either betweenpatterns in peripheral blood and synovial fluid ofRA patients as compared with controls (seeTables II. Ill and IV), or between synovial fluidcells and peripheral blood eells of the samepatients {data not shown).

The cells were subsequently stained in two-colour fluorescence analysis using on the onehand the anti-TCR antibodies and on the otherantibodies against CD4, CD8 or the activationmarkers HLA-DR or IL-2R. The data frotn theseparate analysis of CD4- and CD8-positivesynovial fluid cells are summarized in Figs 2 and3. As seen from these figures, there were increasesin numbers of cells stained with eertain TCR V-gene segment antibodies, within both the CD4-

20

18

16

14

12

10

8

6

4

2

OE+0

5.2 *- 5.3 V/3 5.3

•

; Ii :

6

4

2

OE + 0

SF PBL Normals

20

SF PBL Normals

V/3 12

SF PBL Normals

VQ2.3

SF PBL Normals

t

t

SF PBL Normals SF PBL Normals SF PBL Normals

FiG- 2- Percentage of CD4 ^ lymphocytes from synovial fluid or blood samples reactive with TCR MoAhs; the medianvalue, the IO-90'I ,̂ percenliles and twice (he upper limit are indicated.

686 S. Gudtnundsson et al.

V/3 5,120

18

16

14

12

8

6

4

2

OE-l-0

V/3 5.2 + 5.3 V)3 6.7

I

I

'• . i 1 : I:' • T

SF PBL Normals

V/3 8

SF PBL Normals

12

SF PBL Normals

Vo2.3

SF PBL Normals

SF PBL Normals SF PBL Normals SF PBL Normals

FIG. 3. Percentage of CDK ' lymphocytes from synovijil iluid or blood samples reactive with TCR MoAbs: the medianvalue, the 10 90"/,, percentiles and twice the upper limit are indicated.

positive and the CD8-positive T-ccll subsets.There was. however, no obvious preference (orany of the TCR \ji families when looking at thegroup of patients analysed by means of doublestainings tbrCD4and CD8. The slight increase inmean level of stainings for several of the TCRantibodies on synovial fluid derived eells is so farunexplained.

Concerning ihe possibility of a bias in TCRusage among T cells carrying certain activationmarkers, no notable differences were found instaining with the anti-TCR antibodies eitherbetween HLA-DR* and HLA-DR T cells orbetweenIL-2R' andIL-2R Tcellsamong the 12synovial fluids investigated with this method-ology (data not shown).

DISCUSSION

The present study represents an attempt lo use an

extended panel of monoclonal antibodies tofamilies of TCRs on T cells from synovial tissueand synovial fluid of RA patients in order todetermine whether biased usage of any particularvariable TCR genes can be seen among the T cellsinfiltrating the rheumatoid joint. The resultspoint in two directions. Kirst. it appears that noneofthc presently analysed variable TCR structuresare subject to a general over- or under-represen-lation in inflamed synovial tissue of RA patients.This is in line wilh previous studies using a morelimited number of anti-TCR monoclonal anti-bodies [9]. but the observations are now extendedalso to the subpopulations of T cells which aredefined by the expression of CD4/CD8 and of theactivation markers HLA-DR and CD25. respect-ively. Second, the results show that a certainoverusagc of one or more variable TCR genesmay occur in single synovial fluids, and evenwithin certain areas of the synovial tissue. This

TCR V-Genc U.sage in RA Patients 687

finding indicates a non-random recruitment of Tcells to the joint,, although the TCR structure onthe recruited T cells can differ between indi-viduals. In particular, there are examples ofstriking differences in TCR usage between theCD4-positivc and CDS-positive cells respectively.

The results presented should—as shouid alldata obtained using the current technology beinterpreted with caution, due to the fact that onlya limited part of the TCR structures can beanalysed with the antibodies presently availabie.We must also be aware of the difficulties ininterpreting the cytofluorimetric results in cellpopulations derived from inflamed tissues: back-ground stainings due to autofluorescencc mayblur the results particulariy when single aniibodystainings arc used (for further discussion, seeGudmundsson et al.. submillcd for publication),Stainings using the current gating and doublestaining protocols will in this context be optimallyreliable. Nevertheless, the observations of varia-tions in TCR usage between differeni areas o\'synovial tissue, and differences between TCRusage in synovial fluid and peripheral blood,indicate slrongiy ihat a non-random migrationand;or aclivalion o\' T lymphocytes takes placewithin the inflamed synovial joint in RA. To whatextent this non-random migration/activationmirrors specific immune reactions associated wilhthe pathogenesis o\' RA, or represents ct)nimonfeatures of arthritis or intlammalion in generalremains, however, to be seen.

Thus continued investigations along ihc routenow described should be carried (Uil usinya largerpatieni material suffering also from other types ofarthrilides using both newly available antibodiesand recently developed quantitative techniquesibr determination o\' mRNAs corresponding toihe various variable TCR genes [12], That suchexperiments may turn oul lo be fruitful is showni>om oiher kinds vii inflammatory diseases suchas sarcoidosis [25] and multiple sclerosis [26], Insarcoidosis there were, similarly lo the presentcase, substantial differences in TCR usagebetween C D4- and CD8-posilive cells from thesame inflammatory compartment.

In conclusion, the present analysis failed todetect a common pattern of T-cell receptor usageamong synovial eells in RA. The findings alsoindicate, however, that the apparent non-randomappearance of T-cell subpopulations expressingdifferent TCR V genes be used lo gain furtherknowledge of which factors within ihc joint

attract and activate disease-inducing T lympho-cytes.

ACKNOWLEDGMENTS

Siv Rogberg and Annika Andreasson providedexcellcnl technical assistance and Roger Festingave vaiuable advice during the cytofluorometryanalysis. The work was supported by funds fromihe Swedish Medicai Research Council, fromKingGustafV's 80-year foundation and from theSwedish Association Against Rheumatism,

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Received 6 March 1992Accepled 1 June 1992