Clin. exp. Immunol. (1992) 90, 93-98
Antigen-specific T cell recognition of affinity-purified and recombinant thyroid peroxidase in autoimmune thyroid disease D. L. EWINS, P. S. BARNETT, S. RATANACHAIYAVONG, C. SHARROCK*, J. LANCHBURYt, A. M. McGREGOR & J. P. BANGA Department of Medicine, King's College School of Medicine and Dentistry, * Tissue Typing Laboratory, St. George's Hospital Medical School, and tDepartment of Molecular Immunogenetics, Guys Hospital, London, UK
(Acceptedfor publication 19 June 1992)
SUMMARY The T cell proliferative responses of peripheral blood lymphocytes from 20 patients with autoimmune thyroid disease (AITD) and 20 healthy controls were analysed to immunoaffinity-purified thyroid peroxidase (TPO) and recombinant antigen preparations generated in Escherichia coli as glutathiones-transferase fusion proteins. The epitope specificity of the T cell response was investigated using a selection of eight discrete recombinant fragments encompassing the whole of the extracellular region of the TPO molecule. Significant differences in the proliferative responses between patients and controls were observed to the full length, affinity-purified TPO molecule (P < 0 002) as well as to the recombinant fragments Rlc (residues 145-250) (P < 0-001) and R2b (residues 457-589) (P < 0-001) suggesting the presence of at least two distinct T cell determinants on this autoantigen. One of these T cell epitopes, localized within the region Rlc, has not previously been identified by studies with synthetic peptides.
Keywords thyroid peroxidase recombinant autoantigen T cell responses Hashimoto's thyroiditis Graves' disease
INTRODUCTION
lines generated from these animals are capable of adoptively transferring the disease to naive, syngeneic animals [3]. Whilst considerable progress has been made in understandinadmpigthauotbdyrsnetoTO[,6]
.hyroi and Graves' disease Hrgan-shecimot isoyroidis dissearescommon organ-specific disorders chandravtes'zedby characterized by immune responses Hashimoto's
s
are
common
muc les misnown abutogntion ofsponby th 'pa thogen T cels in.human dase.omeituies ha edemontrate
towards a number of thyroid proteins which include thyroid peroxidase (TPO) and the thyrotropin hormone receptor reautoantibodies to TPO are found in spectively. High levels of of
T cel
inhmndsae.oestde
aedmntae
evidence of sensitization to thyroid microsome preparations [7]. ThavibltyopufedrprtonofPO ndsm Theticapepti ha revaed onlyaatlow aand be e synthetic peptides has revealed only a low and a variable T cell response with no significant difference between patients and [8]. More recently, a limited number of lof synthetic peptides have bee usedto d in me tT toesio have been used to define some of the T cell epitopes on peptides TP inhmndsaeungitritahyoal[]r peripherlbo dilymphocytes [0.her describe teu of immunaffinitymp human IPO and a number ofedif immunoaffinity purified human TPO and a number of different colinas tsou otin o tud atin-scificTce
spectively Highlevels[1] and autoantbodieston Tor ofoof the both these conditions may be responsible forae some immunologically mediated destruction of the thyroid follicular cell in utoimunehypohyrodism[2].TPO as ben cells in autoimmune hypothyroidism [2]. TPO has been sown shown obe identicalto to the conventionally thyroid microy recognized g y somal antigen and is the essential enzyme in the biosynthesis of thehormones hyridhrmoes []. he conig ofTPOhas how tha the thyroid [1]. The cloning of TPO has shown that it comprises a 933-amino acid polypeptide chain, with a large, Nterminal extracellular region of 845 residues, a single membrane spanning region and a short cytoplasmic tail (reviewed in [1]) Experimental immunization of certain strains of mice with purified TPO [3] or TPO-specific synthetic peptides [4] in Freund's complete adjuvant induces severe thyroiditis characterized by an intense inflammatory infiltrate into the gland. One of the pathogenetic mechanisms in experimental thyroiditis is the activation of the TPO-specific T cells, since activated T cell in
~ ~ ~ ~ ~ ~controls
. autoimmune thyroid disease o
proliferative responses in human
(AITD). PATIENTS AND METHODS
Correspondence: Dr J.P. Banga, Department of Medicine, King's College School of Medicine and Dentistry, Bessemer Road, London SE5 9PJ, UK.
Patients and controls Peripheral blood mononuclear cells (PBMC) were prepared from 20 patients with AITD by gradient centrifugation using
93
D. L. Ewins et al.
94
Lymphoprep (Nycomed, Birmingham, UK). They included 18 patients with Hashimoto's thyroiditis who were clinically and biochemically euthyroid on thyroxine replacement therapy, and two patients with untreated active Graves' disease. All patients had high titre anti-microsomal autoantibody levels ( > 1:10 000) by ELISA [I1]. Control PBMC were obtained from 20 autoantibody-negative healthy hospital staff and from four patients with non-thyroid autoimmune disease, two with primary biliary cirrhosis (PBC) and two with systemic lupus erythematosus
(SLE).
(TPO-A)
NH2.
456 457
Rlo +Rlb 160
R c
(TPO-BI) 841 COOH________
COOH 84, 871 C 933
5
841
457 (TPO-B2)
,, 145
250
R2a
320
456R2b 457 589 577
R3
8
45
Fig.
A utoantigen preparations
(APTPo):. wafpurifinity-puromedthyroidperoxidase oTP from waspurifiedb deoxnitychae(oC)asog threviodly microsomes by MoAb affinity chromatography as previously Huma
ubilized
described [12]. Recombinant thyroid peroxidase. Eight separate recombinant proteins representing the entire extracellular region of TPO were produced as glutathione-s-transferase (GST) fusion proteins in E. coli using the plasmid vector pGEX-2T [6] (Fig. 1). Two large, non-overlapping, fusion proteins encompassed the entire extracellular region of the molecule corresponding to residues 1-456 (TPO-A) and 457-841 (TPO-B1). One further large fusion protein from the alternatively spliced TPO gene (TPO-2), and thus lacking amino acid residues 533-590, was also generated and termed TPO-B2. Smaller, overlapping recombinant fragments of TPO have also been produced as previously described [6] (Fig. 1). The recombinant preparations were purified from other bacterial proteins by 3 M urea extraction [13] followed by preparative gel electrophoresis employing an electroendosmotic preparative gel (ELFE, Genofit, Heidelberg, Germany) [14,151. Before cell culture work SDS was removed by detergent exchange with 0 03% DOC. GST was prepared from nonrecombinant pGEX-2T transfected E. coli and subjected to purification as above. The protein concentration of all antigen preparations was determined by a commercial protein assay (BCA, Pierce, UK). Due to the large variation in molecular weight of the antigens used (ranging from 33 kD for recombinant fragment Rlc to 105 kD for APTPO) the molarity of each preparation was determined and equimolar concentrations produced by dilution in PBS containing 003% DOC. All antigen preparations were irradiated (30 gy) before cell culture using a cell irradiator (Gammacell 1000, Vinten Instruments,
Ontario, Canada). Culture conditions Two hundred microlitre aliquots of PBMC in culture medium (RPMI 1640 containing 2 mm glutamine, 100 U/ml penicillin, 100 yg/ml streptomycin and 0 25 pg/ml amphotericin (GIBCO, Paisley, UK)), supplemented with 10% heat inactivated autologous serum were added to 96-well flat-bottomed microtitre plates (Linbro, Flow Laboratories, Rickmansworth, UK) at 105 cells/well. Antigen preparations were added to the PBMC cultures in triplicate at final concentrations of 0-5, 2, 10 and 50 nm (corresponding to approximately 10-4000 ng/ml). Phytohaemagglutinin (PHA) (Sigma, Poole, Dorset) and tetanus toxoid (Wellcome, Beckenham, UK) were added as positive controls to ensure that individual PBMC were capable of proliferation. The cells were cultured for 5 days at 370C in the presence of 5% CO2 in air. Tritiated (3H)-thymidine (Amersham
1. Schematic representation of the thyroid peroxidase (TPO) molecule demonstrating localization of overlapping recombinant TPO fragments encompassing the extracellular region of the molecule. The amino acid residue numbers are indicated. TM, Transmembrane region; C, cytoplasmic domain. The hatched area represents the residues that
are absent in the alternately spliced form of TPO (TPO-2).
PLC, Amersham, UK) was then added at 0 25 yCi/well and the cells harvested 18 h later onto scintillant-coated filters (Beckman, CA) using an automated cell harvester (Skatron AS, Flow) and the activity counted on a scintillation counter (Rackbeta, LKB). Results were expressed as a stimulation index (SI). For the recombinant preparations this was defined as the mean activity in the presence of the fusion protein divided by the activity achieved with recombinant GST, whereas for APTPO the denominator was culture medium alone. The SI was calculated for each of the four separate antigen concentrations utilized and the peak value used for subsequent analysis. Results were arbitrarily regarded as positive if the SI was >3. T cell proliferation experiments were repeated in 10 of the patients using different preparations of affinity-purified and recombinant TPO. Statistical analysis Positive and negative responses were analysed by the X2-test and the actual proliferative values were compared by the Wilcoxon rank sum test. Tissue typing PBMC from patients and controls were tissue typed by serological analysis in Terasaki plates using 120 class I and 60 class II antisera in the two-colour fluorescence test [16]. Additional DR and DQ typing was performed in the majority of samples by restriction fragment length polymorphism (RFLP) analysis [17], with DR4 subtyping by DR4.B1 selective DNA amplification followed by specific oligonucleotide hybridization [18]. The DPBI typing was performed following in vitro DNA amplification and hybridization with sequence-specific oligonucleotide probes using a modification of the described [19] method. RESULTS
Preparations of TPO
Affinity chromatography has previously been shown
to
yield
TPO of very high purity with a single polypeptide band being observed at 105 kD following SDS-PAGE [12]. For the preparation of the various rTPO fragments in E. coli, extraction of bacterial inclusion bodies by 3 Molar urea was found to enrich considerably the recombinant protein in the insoluble
95
T cell responses to thyroid peroxidase
;b~ on sewingl§|
~4~
~
~
~
~
~
l _ ii! __ | I I __ |
~ ~ ~ ~~ L ~ ~.~~ ..~~~...
. . . . .. . . . . . . . . .
..n
M
.'I.'2
31 4
5
6
7
a
2
lo
4'"' 51
6
eoe .Critical!~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~wih3Mlrue
Fig. 2. (a) SDS-PAGE analysis demonstrating urea extraction of bactenial inclusion bodies for recombinant thyroid peroxidase-glutathione-stransferase (TPO GST) fusion proteins Ri a +Rib (lanes 1-4) and R2a (lanes 5-8). The arrow shows the position of the recombinant proteins comigrating at approximately 43 kD. Lanes 1 and 5, total inclusion body preparation of induced bacteria expressing the fusion polypeptides; lanes 2 and 6, and 3 and 7, two consecutive 3 Molar urea extractions of inclusion body preparations showing virtual absense of recombinant fusion proteins; lanes 4 and 8, insoluble pellets after 3 Molar urea extraction showing the enrichment of recombinant fusion proteins. M refers to mol. wt markers at (from top to bottom) 66, 45, 36, 29,24, 20 and 14 kD respectively. (b) Protein staining of SDS-PAGE gel demonstrating purified recombinant fusion fragments of TPO. Lane 1, recombinant GST, lanes 2, 3,4, 5 and 6, recombinant fusion proteins R 1la + b, R 1lc, R2a, R2b and R3 respectively. M, mol. wt markers (from top to bottom) 66, 45, 36, 29, 20 and 14 kD.
10 (a)
|P
Antigen-specific T cell recognition of affinity-purified and recombinant thyroid peroxidase in autoimmune thyroid disease D. L. EWINS, P. S. BARNETT, S. RATANACHAIYAVONG, C. SHARROCK*, J. LANCHBURYt, A. M. McGREGOR & J. P. BANGA Department of Medicine, King's College School of Medicine and Dentistry, * Tissue Typing Laboratory, St. George's Hospital Medical School, and tDepartment of Molecular Immunogenetics, Guys Hospital, London, UK
(Acceptedfor publication 19 June 1992)
SUMMARY The T cell proliferative responses of peripheral blood lymphocytes from 20 patients with autoimmune thyroid disease (AITD) and 20 healthy controls were analysed to immunoaffinity-purified thyroid peroxidase (TPO) and recombinant antigen preparations generated in Escherichia coli as glutathiones-transferase fusion proteins. The epitope specificity of the T cell response was investigated using a selection of eight discrete recombinant fragments encompassing the whole of the extracellular region of the TPO molecule. Significant differences in the proliferative responses between patients and controls were observed to the full length, affinity-purified TPO molecule (P < 0 002) as well as to the recombinant fragments Rlc (residues 145-250) (P < 0-001) and R2b (residues 457-589) (P < 0-001) suggesting the presence of at least two distinct T cell determinants on this autoantigen. One of these T cell epitopes, localized within the region Rlc, has not previously been identified by studies with synthetic peptides.
Keywords thyroid peroxidase recombinant autoantigen T cell responses Hashimoto's thyroiditis Graves' disease
INTRODUCTION
lines generated from these animals are capable of adoptively transferring the disease to naive, syngeneic animals [3]. Whilst considerable progress has been made in understandinadmpigthauotbdyrsnetoTO[,6]
.hyroi and Graves' disease Hrgan-shecimot isoyroidis dissearescommon organ-specific disorders chandravtes'zedby characterized by immune responses Hashimoto's
s
are
common
muc les misnown abutogntion ofsponby th 'pa thogen T cels in.human dase.omeituies ha edemontrate
towards a number of thyroid proteins which include thyroid peroxidase (TPO) and the thyrotropin hormone receptor reautoantibodies to TPO are found in spectively. High levels of of
T cel
inhmndsae.oestde
aedmntae
evidence of sensitization to thyroid microsome preparations [7]. ThavibltyopufedrprtonofPO ndsm Theticapepti ha revaed onlyaatlow aand be e synthetic peptides has revealed only a low and a variable T cell response with no significant difference between patients and [8]. More recently, a limited number of lof synthetic peptides have bee usedto d in me tT toesio have been used to define some of the T cell epitopes on peptides TP inhmndsaeungitritahyoal[]r peripherlbo dilymphocytes [0.her describe teu of immunaffinitymp human IPO and a number ofedif immunoaffinity purified human TPO and a number of different colinas tsou otin o tud atin-scificTce
spectively Highlevels[1] and autoantbodieston Tor ofoof the both these conditions may be responsible forae some immunologically mediated destruction of the thyroid follicular cell in utoimunehypohyrodism[2].TPO as ben cells in autoimmune hypothyroidism [2]. TPO has been sown shown obe identicalto to the conventionally thyroid microy recognized g y somal antigen and is the essential enzyme in the biosynthesis of thehormones hyridhrmoes []. he conig ofTPOhas how tha the thyroid [1]. The cloning of TPO has shown that it comprises a 933-amino acid polypeptide chain, with a large, Nterminal extracellular region of 845 residues, a single membrane spanning region and a short cytoplasmic tail (reviewed in [1]) Experimental immunization of certain strains of mice with purified TPO [3] or TPO-specific synthetic peptides [4] in Freund's complete adjuvant induces severe thyroiditis characterized by an intense inflammatory infiltrate into the gland. One of the pathogenetic mechanisms in experimental thyroiditis is the activation of the TPO-specific T cells, since activated T cell in
~ ~ ~ ~ ~ ~controls
. autoimmune thyroid disease o
proliferative responses in human
(AITD). PATIENTS AND METHODS
Correspondence: Dr J.P. Banga, Department of Medicine, King's College School of Medicine and Dentistry, Bessemer Road, London SE5 9PJ, UK.
Patients and controls Peripheral blood mononuclear cells (PBMC) were prepared from 20 patients with AITD by gradient centrifugation using
93
D. L. Ewins et al.
94
Lymphoprep (Nycomed, Birmingham, UK). They included 18 patients with Hashimoto's thyroiditis who were clinically and biochemically euthyroid on thyroxine replacement therapy, and two patients with untreated active Graves' disease. All patients had high titre anti-microsomal autoantibody levels ( > 1:10 000) by ELISA [I1]. Control PBMC were obtained from 20 autoantibody-negative healthy hospital staff and from four patients with non-thyroid autoimmune disease, two with primary biliary cirrhosis (PBC) and two with systemic lupus erythematosus
(SLE).
(TPO-A)
NH2.
456 457
Rlo +Rlb 160
R c
(TPO-BI) 841 COOH________
COOH 84, 871 C 933
5
841
457 (TPO-B2)
,, 145
250
R2a
320
456R2b 457 589 577
R3
8
45
Fig.
A utoantigen preparations
(APTPo):. wafpurifinity-puromedthyroidperoxidase oTP from waspurifiedb deoxnitychae(oC)asog threviodly microsomes by MoAb affinity chromatography as previously Huma
ubilized
described [12]. Recombinant thyroid peroxidase. Eight separate recombinant proteins representing the entire extracellular region of TPO were produced as glutathione-s-transferase (GST) fusion proteins in E. coli using the plasmid vector pGEX-2T [6] (Fig. 1). Two large, non-overlapping, fusion proteins encompassed the entire extracellular region of the molecule corresponding to residues 1-456 (TPO-A) and 457-841 (TPO-B1). One further large fusion protein from the alternatively spliced TPO gene (TPO-2), and thus lacking amino acid residues 533-590, was also generated and termed TPO-B2. Smaller, overlapping recombinant fragments of TPO have also been produced as previously described [6] (Fig. 1). The recombinant preparations were purified from other bacterial proteins by 3 M urea extraction [13] followed by preparative gel electrophoresis employing an electroendosmotic preparative gel (ELFE, Genofit, Heidelberg, Germany) [14,151. Before cell culture work SDS was removed by detergent exchange with 0 03% DOC. GST was prepared from nonrecombinant pGEX-2T transfected E. coli and subjected to purification as above. The protein concentration of all antigen preparations was determined by a commercial protein assay (BCA, Pierce, UK). Due to the large variation in molecular weight of the antigens used (ranging from 33 kD for recombinant fragment Rlc to 105 kD for APTPO) the molarity of each preparation was determined and equimolar concentrations produced by dilution in PBS containing 003% DOC. All antigen preparations were irradiated (30 gy) before cell culture using a cell irradiator (Gammacell 1000, Vinten Instruments,
Ontario, Canada). Culture conditions Two hundred microlitre aliquots of PBMC in culture medium (RPMI 1640 containing 2 mm glutamine, 100 U/ml penicillin, 100 yg/ml streptomycin and 0 25 pg/ml amphotericin (GIBCO, Paisley, UK)), supplemented with 10% heat inactivated autologous serum were added to 96-well flat-bottomed microtitre plates (Linbro, Flow Laboratories, Rickmansworth, UK) at 105 cells/well. Antigen preparations were added to the PBMC cultures in triplicate at final concentrations of 0-5, 2, 10 and 50 nm (corresponding to approximately 10-4000 ng/ml). Phytohaemagglutinin (PHA) (Sigma, Poole, Dorset) and tetanus toxoid (Wellcome, Beckenham, UK) were added as positive controls to ensure that individual PBMC were capable of proliferation. The cells were cultured for 5 days at 370C in the presence of 5% CO2 in air. Tritiated (3H)-thymidine (Amersham
1. Schematic representation of the thyroid peroxidase (TPO) molecule demonstrating localization of overlapping recombinant TPO fragments encompassing the extracellular region of the molecule. The amino acid residue numbers are indicated. TM, Transmembrane region; C, cytoplasmic domain. The hatched area represents the residues that
are absent in the alternately spliced form of TPO (TPO-2).
PLC, Amersham, UK) was then added at 0 25 yCi/well and the cells harvested 18 h later onto scintillant-coated filters (Beckman, CA) using an automated cell harvester (Skatron AS, Flow) and the activity counted on a scintillation counter (Rackbeta, LKB). Results were expressed as a stimulation index (SI). For the recombinant preparations this was defined as the mean activity in the presence of the fusion protein divided by the activity achieved with recombinant GST, whereas for APTPO the denominator was culture medium alone. The SI was calculated for each of the four separate antigen concentrations utilized and the peak value used for subsequent analysis. Results were arbitrarily regarded as positive if the SI was >3. T cell proliferation experiments were repeated in 10 of the patients using different preparations of affinity-purified and recombinant TPO. Statistical analysis Positive and negative responses were analysed by the X2-test and the actual proliferative values were compared by the Wilcoxon rank sum test. Tissue typing PBMC from patients and controls were tissue typed by serological analysis in Terasaki plates using 120 class I and 60 class II antisera in the two-colour fluorescence test [16]. Additional DR and DQ typing was performed in the majority of samples by restriction fragment length polymorphism (RFLP) analysis [17], with DR4 subtyping by DR4.B1 selective DNA amplification followed by specific oligonucleotide hybridization [18]. The DPBI typing was performed following in vitro DNA amplification and hybridization with sequence-specific oligonucleotide probes using a modification of the described [19] method. RESULTS
Preparations of TPO
Affinity chromatography has previously been shown
to
yield
TPO of very high purity with a single polypeptide band being observed at 105 kD following SDS-PAGE [12]. For the preparation of the various rTPO fragments in E. coli, extraction of bacterial inclusion bodies by 3 Molar urea was found to enrich considerably the recombinant protein in the insoluble
95
T cell responses to thyroid peroxidase
;b~ on sewingl§|
~4~
~
~
~
~
~
l _ ii! __ | I I __ |
~ ~ ~ ~~ L ~ ~.~~ ..~~~...
. . . . .. . . . . . . . . .
..n
M
.'I.'2
31 4
5
6
7
a
2
lo
4'"' 51
6
eoe .Critical!~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~wih3Mlrue
Fig. 2. (a) SDS-PAGE analysis demonstrating urea extraction of bactenial inclusion bodies for recombinant thyroid peroxidase-glutathione-stransferase (TPO GST) fusion proteins Ri a +Rib (lanes 1-4) and R2a (lanes 5-8). The arrow shows the position of the recombinant proteins comigrating at approximately 43 kD. Lanes 1 and 5, total inclusion body preparation of induced bacteria expressing the fusion polypeptides; lanes 2 and 6, and 3 and 7, two consecutive 3 Molar urea extractions of inclusion body preparations showing virtual absense of recombinant fusion proteins; lanes 4 and 8, insoluble pellets after 3 Molar urea extraction showing the enrichment of recombinant fusion proteins. M refers to mol. wt markers at (from top to bottom) 66, 45, 36, 29,24, 20 and 14 kD respectively. (b) Protein staining of SDS-PAGE gel demonstrating purified recombinant fusion fragments of TPO. Lane 1, recombinant GST, lanes 2, 3,4, 5 and 6, recombinant fusion proteins R 1la + b, R 1lc, R2a, R2b and R3 respectively. M, mol. wt markers (from top to bottom) 66, 45, 36, 29, 20 and 14 kD.
10 (a)
|P
