Scand. J. Immunol. 36, 855 863, 1992
Oligoclonal T Cells in Rheumatoid Arthritis: Identification Strategy and Molecular Characterization of a Clonal T-Cell Receptor U. KORTHAUER. B. HENNERKES. H. MENNINGER*. H. W. MAGES, J. ZACHERt. A. J. POTOCNIK, F. EMMRICH &L R. A. KROCZEK Max-Planck-Society Research Unit for Rh(.'iim;itology..Immiinology at the Institute for Clinical Immunology of the University, Erlangen. and Departments of *Medicine and tSurgcry, BRK Center for Rheumatology. Bad Abbach, Germany
Korthiiuer U, Mennerkes B, Menninger H, Mages HW. Zacher J. Potocnik AJ. Emmrich F. Kroc7.ek RA. OligoelonalT Cells in Rheumaioid Arthritis: Identification Strategy and Molecular Characterization of a Clonal T-Cell Receptor. Scand J Immunol l992;36:855-63 Immunodominant antigens in rheumatoid arthritis (RA) should induce an expansion of T cells bearing a corresponding T-cell receptor (TCR). We therefore analysed the TCR repertoire at the site of inflammation using two fundamentally different strategies. The ;('/«/TCR repertoire was examined by generating 'representative' T-eell clone panels, which were subsequently tested for clonality by restriction mapping oftheTCR/i gene loctjs. No clonality was detected in large T-cell clone panels generated wilh cells from three patients. However, when we selectively analysed the TCR repertoire of in vivo pre-actiiated. interleukin-2 UL-2)-responsiveT cells, significant T-cell/ TCR elonality was found in 2 out o r 4 patients. The clonal T cells represented a minority of the total T-cell population wilh an estimated lYequency of 1 in 300 to 1 in 1000 cells. Molecular characterization of a clonal TCR and the use of a specific TCR V/J MoAb ruled out an overrepresentation ofT cells bearing the same V/( element in the total T-cell population, rendering the involvement of superantigens in the induction of T-cell clonality in this case unlikely. Dr R. .4. Kroczek. Max-Planck-Socieiy Research Unit for Rheumatology j Immunology. Sctiwabachanlage 10. 8520 Ertangen, Germany
Various viral and bacterial agents have been implicated in the pathogcnesis of rheumatoid arthritis (RA), without a definitive proof. In the search for a causative antigen we have therefore taketi an indirecl approach. If T cells were crucial in the pathogenesis of RA, then the TCR repertoire at the site of inflammation can be expected to reflect the antigen(s) involved since relevant antigens should expand T cells bearing the corresponding T-cell receptor (TCR). The isolation of such a clonal TCR would then potentially allow the identification of the causative antigen. As a first step we tested the following hypothesis: 'Oligoclonal T cells, induced by an immunodominant antigen X, represent a major proportion of T cells at the site of inflammation'. Two fundamentally different strategies were used to test this hypothesis. The first approach was directed at the analysis of the totat TCR repertoire, while in the second approach only the TCR repertoire of in
vivo pre-activated T cells was examined. In the selective approach T-cell clonality was detected in 2 out of 4 patients, and one of the clonal TCRs was molecularly characterized.
MATERIALS AND METHODS Isotalion of T cells from synovial tissue, synorial fluid and peripheral hlooti. Synovial tissue (ST) from knee joints was obtained at synovectomy from three patients (SU. WK, HJ) wilh classic RA [1] oriong duration ( > 5 years). All patients had clinical signs of inflammalion in the involved joint at the time of surgery. ST was digested with 2 mg.ml collagenase (Biochrom, Berlin) and 0.15 mg.ml DNAse (Paesel, Frankfurt) for 2 h at 37 C in Ca-^' /Mg-' -free Hanks' balanced salt solution (HBSS) and subsequently fraetionated by centrifugation on discontinuous gradients consisting of lO'lii. l&Vf. 5O'!^r. and 6O'Vli Percoll (Pharmacia, Uppsala, Sweden) in Ca-\'Mg-'--free HBSS. The fraetion enriched tor T cells was removed from ihe interface between the 507n and bO'Vit Percol! layers. Synovial fluid (SF) was 855
856
(/. Korthauer et at.
obtained from four patients (WM. HT, SA. RK) wilh classic RA, high erythrocyle sedimentation rate ( >60 mm/h) and an acute efTu.sion inio a joint. All seven patients were on non-steroidal anti-inflammatory drugs: palienl WM in addition received prednisolone. Mononuclear cells were .separated from heparinized SF or peripheral blood (PB) on a Ficol! metrizoitte (Nycomed, Oslo, Norway) density gradient. T-lymplunyie cliinin^. In the •representative" approach, T cells from ST were seeded inio roundbottomed microlitre plales at a density of 0.6cellS''well and grown under standard conditions in the presence of 5x10'' irradiated (5000 rad) autologous peripheral blood (PB) mononuclear cells feeders. 1:200 phytohaemagglutlnin (PHA)(Gibco/BRL, Paisley, UK), 100 U/ml recombinanl IL-2 (rIL-2: Sandoz. Basel. Switzerland) and 2iy'Ai Lymphocult-T (Biotest, Dreieich, Germany) in complete RPMM64() medium supplemented with 10% autologous serum. In the '.selective' approach mononuclear cells were seeded al a density of 30 3Q0 cells/well (SF) or 5 .10 cells,, well (PB) m the pre.sence of 100 U.ml rIL-2 and 10";, fetal calf serutn (Boehringer Mannheim, Germany). Cells were expanded from positive wells on plates which, by Poisson dislribulion of positive: negative wells, indicated a clonal outgrowth of TceSls |2]. In both approaches T-cell clones were further expanded, using for stimulation i:600 PHA, supernatant from MLA-144 cells containing IL-2 [S], and heterologous PB mononuclear cells as feeders. TCR analysis hy Southern hybridization. For the analysis of TCR/( and TCRy gene rearrangements, 5 /ig of genomic DNA, extracted from cells by a standard method [4], were digested with an appropriale restriction enzyme, separated on a d.l"/., agarose gel and transferred onto a nylon membrane. A 686-bp C/f| probe (MI3IBI0BBI, [5]) was used to detecl TCR rearrangements involving C/i| and C/^. The TCRy probe pH60 was the 70()-bp fragment of clone MI y\ 160 containing Jyi [6|. Prehybridization and hybridization were conducted at 42 C using 50"/,. formamide aceording to standard protocols. Subsequent washing of the blots included a high stringency step (0.1 x SSC. 1",. SDS at 50 C tor 30 min). Molecular cloning of TCRi and ji chains. Oligo (dT) primed double-stranded cDNA was synthesized from 20 ;(g of total RNA prepared by standard methods [7, 8] using RAV reverse transcriptase (Amersham. Bucks. UK), RNaseH. F-. loh DNA polymerase I and E. coli DNA ligase. followed by incubation wilh T4 DNA polymerase for blunt-end formation. Afler addition of EcoK\ linkers, the cDNA was ligaled to lambda gtlO arms (Slratagenc. La Jolla, CA. USA). The lambda gl 10 cDNA library was screened for TcR^ and TcR/i transcripts using ihe Cx probe pYI4 [9] and the C/(| probe M131 BIOBBL Fuil-length clones were subcloned into pBluescHpt II SK/KS (Stratagene) and manually sequenced using Ihe Sequenase'" system (Uniled Stales Biochemical. Cleveland. OH, USA). Polymerase chain reaction i PCRl. Total RNA from 10'* cells was prepared according to Chomczynski & Sacchi [10] with 15 /(g IRNA (Boehringer Mannheim) as carrier. First strand cDNA was synthesized using MMLV reverse transcriptase (Gibco BRL) and Ca(5'GGTGAATAGGCAGACAGACTTG-3 ) or C/J ( 5 GTGGGAGATCTCTGCTTCTG-3) primers. To one-third of ihe reaction products the appropriale \y.
(5-CACTGCTCAGCCATGC-3 ) or V/f ( 5 TGCTGCTTTGTCTCCTGG-3 ) primers were added and the cDNA amplified with Taq polymerase (Perkin Flmer Cctus, Fmeryville, CA, USA) in .15 cycles (94 C for 40 s. 57 C for 60 s, 72 C for 7(1 s). The amplified material was .separaicd on a 2"';. agarose gel and analysed by Southern hybridization using the cloned \i and V^ regions indicated in Fig. 4 as '-P-labelled probes. Flow cyionielry. Phenotypic analysis was performed as described previously [I!]. In brief, 2 x U)'' cells were stained with a V/y5.3-specitic MoAb (clone ICl; IgGI), a V/((i-specific MoAb (clone OT145; IgGI: both T-Cell Sciences, Cambridge. MA. USA). TCRa /i-panspecific MoAb BMA 031 (fgG2b. Behringwerke, Marburg. Germany) or the isotype control MoAb 534 (IgG I, kind gift of Dr O. Majdic), and analysed on an EPICS 753 (Coulter Electronics. Hialeah. FL, USA) using propidium iodide gating for exclusion of dead eells.
RF.SULTS Failure to detect ctonat T celts in 'representative' T-cetl panels T cells were enriched from ST specimetis and randomly cioned by immediate limiting dilution using the polyclonal mitogen PHA. In three experiments giving sufficient numbers of T-cell clones, single T-cell clones were further expanded (Fig. I. 'representative' approach). Genomic DNA from single T-cell clones was then e.xtracted and subjected to a multistep restriction mapping analysis (Table I). Only T-cell clones giving identical restriction patterns with all enzymes and hybridization probes used, when run on the same gel. were regarded as in vivo expanded multiples of an originally single T cell. As a result, in three large "representative' T-cell clone panels generated from ST resident T cells of different patients, no T-ceil clonality could be detected. The data, including the cloning efficiencies, are summarized in Table II.
Identification of ctonat T cells in 'selective' T-cell panels T cells were immediately cloned by limiting dilution with rIL-2 as the sole stimulant; autoiogous feeders were used to prevent the outgrowth of alloreactive T cells. This protocol yielded low numbers of T-cell clones from ST, and further experiments were therefore carried out with T cells obtained from SF of acute knee joint effusions (Fig. I, 'selective' approach). As a result, we could identify live multiples within 28 examined T-cell clones {WM, Fig. 2A, B, C. D,
A Clonal TCR in Rheumatoid Arthritis
'SELECTIVE' APPROACH
•REPRESENTATIVE' APPROACH
SYNOVIAL TISSUE SYNOVIAL FLUID DIGEST WITH COLLAGENASE/DNAse
PERCOLL GRADIENT
METRIZOATE / FICOLL GRADIENT
T-CELL FRACTION
MONONUCLEAR CELLS
PHA + IL-2 + COND. MEDIUM
IL-2
AUTOLOGOUS FEEDERS
AUTOLOGOUS FEEDERS
—1
857
-
-
RESTRICTION MAPPING OF GENOMIC DNA FiCi. I. Schematic representation of the strategies used to identify clonal T cells in T-cell populations al the site of inHamnitttion.
858
U, Korthauer et al. TABLE I.
Sequence of restriction mapping
analysis
Step 1 Step 2 Step 3 Step 4 Step 5 Step 6
Digest
Hybridization probe
£(
Oligoclonal T Cells in Rheumatoid Arthritis: Identification Strategy and Molecular Characterization of a Clonal T-Cell Receptor U. KORTHAUER. B. HENNERKES. H. MENNINGER*. H. W. MAGES, J. ZACHERt. A. J. POTOCNIK, F. EMMRICH &L R. A. KROCZEK Max-Planck-Society Research Unit for Rh(.'iim;itology..Immiinology at the Institute for Clinical Immunology of the University, Erlangen. and Departments of *Medicine and tSurgcry, BRK Center for Rheumatology. Bad Abbach, Germany
Korthiiuer U, Mennerkes B, Menninger H, Mages HW. Zacher J. Potocnik AJ. Emmrich F. Kroc7.ek RA. OligoelonalT Cells in Rheumaioid Arthritis: Identification Strategy and Molecular Characterization of a Clonal T-Cell Receptor. Scand J Immunol l992;36:855-63 Immunodominant antigens in rheumatoid arthritis (RA) should induce an expansion of T cells bearing a corresponding T-cell receptor (TCR). We therefore analysed the TCR repertoire at the site of inflammation using two fundamentally different strategies. The ;('/«/TCR repertoire was examined by generating 'representative' T-eell clone panels, which were subsequently tested for clonality by restriction mapping oftheTCR/i gene loctjs. No clonality was detected in large T-cell clone panels generated wilh cells from three patients. However, when we selectively analysed the TCR repertoire of in vivo pre-actiiated. interleukin-2 UL-2)-responsiveT cells, significant T-cell/ TCR elonality was found in 2 out o r 4 patients. The clonal T cells represented a minority of the total T-cell population wilh an estimated lYequency of 1 in 300 to 1 in 1000 cells. Molecular characterization of a clonal TCR and the use of a specific TCR V/J MoAb ruled out an overrepresentation ofT cells bearing the same V/( element in the total T-cell population, rendering the involvement of superantigens in the induction of T-cell clonality in this case unlikely. Dr R. .4. Kroczek. Max-Planck-Socieiy Research Unit for Rheumatology j Immunology. Sctiwabachanlage 10. 8520 Ertangen, Germany
Various viral and bacterial agents have been implicated in the pathogcnesis of rheumatoid arthritis (RA), without a definitive proof. In the search for a causative antigen we have therefore taketi an indirecl approach. If T cells were crucial in the pathogenesis of RA, then the TCR repertoire at the site of inflammation can be expected to reflect the antigen(s) involved since relevant antigens should expand T cells bearing the corresponding T-cell receptor (TCR). The isolation of such a clonal TCR would then potentially allow the identification of the causative antigen. As a first step we tested the following hypothesis: 'Oligoclonal T cells, induced by an immunodominant antigen X, represent a major proportion of T cells at the site of inflammation'. Two fundamentally different strategies were used to test this hypothesis. The first approach was directed at the analysis of the totat TCR repertoire, while in the second approach only the TCR repertoire of in
vivo pre-activated T cells was examined. In the selective approach T-cell clonality was detected in 2 out of 4 patients, and one of the clonal TCRs was molecularly characterized.
MATERIALS AND METHODS Isotalion of T cells from synovial tissue, synorial fluid and peripheral hlooti. Synovial tissue (ST) from knee joints was obtained at synovectomy from three patients (SU. WK, HJ) wilh classic RA [1] oriong duration ( > 5 years). All patients had clinical signs of inflammalion in the involved joint at the time of surgery. ST was digested with 2 mg.ml collagenase (Biochrom, Berlin) and 0.15 mg.ml DNAse (Paesel, Frankfurt) for 2 h at 37 C in Ca-^' /Mg-' -free Hanks' balanced salt solution (HBSS) and subsequently fraetionated by centrifugation on discontinuous gradients consisting of lO'lii. l&Vf. 5O'!^r. and 6O'Vli Percoll (Pharmacia, Uppsala, Sweden) in Ca-\'Mg-'--free HBSS. The fraetion enriched tor T cells was removed from ihe interface between the 507n and bO'Vit Percol! layers. Synovial fluid (SF) was 855
856
(/. Korthauer et at.
obtained from four patients (WM. HT, SA. RK) wilh classic RA, high erythrocyle sedimentation rate ( >60 mm/h) and an acute efTu.sion inio a joint. All seven patients were on non-steroidal anti-inflammatory drugs: palienl WM in addition received prednisolone. Mononuclear cells were .separated from heparinized SF or peripheral blood (PB) on a Ficol! metrizoitte (Nycomed, Oslo, Norway) density gradient. T-lymplunyie cliinin^. In the •representative" approach, T cells from ST were seeded inio roundbottomed microlitre plales at a density of 0.6cellS''well and grown under standard conditions in the presence of 5x10'' irradiated (5000 rad) autologous peripheral blood (PB) mononuclear cells feeders. 1:200 phytohaemagglutlnin (PHA)(Gibco/BRL, Paisley, UK), 100 U/ml recombinanl IL-2 (rIL-2: Sandoz. Basel. Switzerland) and 2iy'Ai Lymphocult-T (Biotest, Dreieich, Germany) in complete RPMM64() medium supplemented with 10% autologous serum. In the '.selective' approach mononuclear cells were seeded al a density of 30 3Q0 cells/well (SF) or 5 .10 cells,, well (PB) m the pre.sence of 100 U.ml rIL-2 and 10";, fetal calf serutn (Boehringer Mannheim, Germany). Cells were expanded from positive wells on plates which, by Poisson dislribulion of positive: negative wells, indicated a clonal outgrowth of TceSls |2]. In both approaches T-cell clones were further expanded, using for stimulation i:600 PHA, supernatant from MLA-144 cells containing IL-2 [S], and heterologous PB mononuclear cells as feeders. TCR analysis hy Southern hybridization. For the analysis of TCR/( and TCRy gene rearrangements, 5 /ig of genomic DNA, extracted from cells by a standard method [4], were digested with an appropriale restriction enzyme, separated on a d.l"/., agarose gel and transferred onto a nylon membrane. A 686-bp C/f| probe (MI3IBI0BBI, [5]) was used to detecl TCR rearrangements involving C/i| and C/^. The TCRy probe pH60 was the 70()-bp fragment of clone MI y\ 160 containing Jyi [6|. Prehybridization and hybridization were conducted at 42 C using 50"/,. formamide aceording to standard protocols. Subsequent washing of the blots included a high stringency step (0.1 x SSC. 1",. SDS at 50 C tor 30 min). Molecular cloning of TCRi and ji chains. Oligo (dT) primed double-stranded cDNA was synthesized from 20 ;(g of total RNA prepared by standard methods [7, 8] using RAV reverse transcriptase (Amersham. Bucks. UK), RNaseH. F-. loh DNA polymerase I and E. coli DNA ligase. followed by incubation wilh T4 DNA polymerase for blunt-end formation. Afler addition of EcoK\ linkers, the cDNA was ligaled to lambda gtlO arms (Slratagenc. La Jolla, CA. USA). The lambda gl 10 cDNA library was screened for TcR^ and TcR/i transcripts using ihe Cx probe pYI4 [9] and the C/(| probe M131 BIOBBL Fuil-length clones were subcloned into pBluescHpt II SK/KS (Stratagene) and manually sequenced using Ihe Sequenase'" system (Uniled Stales Biochemical. Cleveland. OH, USA). Polymerase chain reaction i PCRl. Total RNA from 10'* cells was prepared according to Chomczynski & Sacchi [10] with 15 /(g IRNA (Boehringer Mannheim) as carrier. First strand cDNA was synthesized using MMLV reverse transcriptase (Gibco BRL) and Ca(5'GGTGAATAGGCAGACAGACTTG-3 ) or C/J ( 5 GTGGGAGATCTCTGCTTCTG-3) primers. To one-third of ihe reaction products the appropriale \y.
(5-CACTGCTCAGCCATGC-3 ) or V/f ( 5 TGCTGCTTTGTCTCCTGG-3 ) primers were added and the cDNA amplified with Taq polymerase (Perkin Flmer Cctus, Fmeryville, CA, USA) in .15 cycles (94 C for 40 s. 57 C for 60 s, 72 C for 7(1 s). The amplified material was .separaicd on a 2"';. agarose gel and analysed by Southern hybridization using the cloned \i and V^ regions indicated in Fig. 4 as '-P-labelled probes. Flow cyionielry. Phenotypic analysis was performed as described previously [I!]. In brief, 2 x U)'' cells were stained with a V/y5.3-specitic MoAb (clone ICl; IgGI), a V/((i-specific MoAb (clone OT145; IgGI: both T-Cell Sciences, Cambridge. MA. USA). TCRa /i-panspecific MoAb BMA 031 (fgG2b. Behringwerke, Marburg. Germany) or the isotype control MoAb 534 (IgG I, kind gift of Dr O. Majdic), and analysed on an EPICS 753 (Coulter Electronics. Hialeah. FL, USA) using propidium iodide gating for exclusion of dead eells.
RF.SULTS Failure to detect ctonat T celts in 'representative' T-cetl panels T cells were enriched from ST specimetis and randomly cioned by immediate limiting dilution using the polyclonal mitogen PHA. In three experiments giving sufficient numbers of T-cell clones, single T-cell clones were further expanded (Fig. I. 'representative' approach). Genomic DNA from single T-cell clones was then e.xtracted and subjected to a multistep restriction mapping analysis (Table I). Only T-cell clones giving identical restriction patterns with all enzymes and hybridization probes used, when run on the same gel. were regarded as in vivo expanded multiples of an originally single T cell. As a result, in three large "representative' T-cell clone panels generated from ST resident T cells of different patients, no T-ceil clonality could be detected. The data, including the cloning efficiencies, are summarized in Table II.
Identification of ctonat T cells in 'selective' T-cell panels T cells were immediately cloned by limiting dilution with rIL-2 as the sole stimulant; autoiogous feeders were used to prevent the outgrowth of alloreactive T cells. This protocol yielded low numbers of T-cell clones from ST, and further experiments were therefore carried out with T cells obtained from SF of acute knee joint effusions (Fig. I, 'selective' approach). As a result, we could identify live multiples within 28 examined T-cell clones {WM, Fig. 2A, B, C. D,
A Clonal TCR in Rheumatoid Arthritis
'SELECTIVE' APPROACH
•REPRESENTATIVE' APPROACH
SYNOVIAL TISSUE SYNOVIAL FLUID DIGEST WITH COLLAGENASE/DNAse
PERCOLL GRADIENT
METRIZOATE / FICOLL GRADIENT
T-CELL FRACTION
MONONUCLEAR CELLS
PHA + IL-2 + COND. MEDIUM
IL-2
AUTOLOGOUS FEEDERS
AUTOLOGOUS FEEDERS
—1
857
-
-
RESTRICTION MAPPING OF GENOMIC DNA FiCi. I. Schematic representation of the strategies used to identify clonal T cells in T-cell populations al the site of inHamnitttion.
858
U, Korthauer et al. TABLE I.
Sequence of restriction mapping
analysis
Step 1 Step 2 Step 3 Step 4 Step 5 Step 6
Digest
Hybridization probe
£(
