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Comparison of T cell sites recognized by thymic and blood lymphocytes in Myasthenia Gravis patients
902
Abstracts
ISOLATION HUMAN
AND
MUSCLE
EXPRESSION OF cDNAS FOR NICOTINIC ACETYLCHOLINE
THE
RECEPTOR. Neurosciences Dr. D. Beeson, Molecular Medicine, University cliffe Hospital, Oxford. OX3 David Davis.
Beeson,
M.
Brydson,
Detailed study of the epitopes by the low level of AChR (< to overcome this problem subunits and their subsequent
Group, Institute of of Oxford, John Rad9DU Tel No 0865-752326 A. Vincent, J. NewsomOxford, England involved in myasthenia gravis is restricted Zpmol/g) found in human muscle. We aimed by cloning cDNAs for the human AChR expression.
A cDNA library has been constructed in AgtlO using poly(A+) RNA from human leg muscle. The library was initially screened with cDNA probes coding for chicken AChR subunits and, following isolation of positives, with cDNAs for the human AChR subunits. cDNAs containing the full coding sequence have been isolated for the o! and 7 -subunits, and in the case of x and r -subunits for the full sequence except for the initial 5’ coding region. The full coding region of the p-subunit has and has been shown previously known in man), acids including a 23 amino acid prepeptide sequence.
been determined to contain 501
(not amino
Restriction fragments of both Torpedo and human o( -subunit cDNAs, coding for almost all the mature protein, and for smaller polypeptide have been expressed in E. Coli strain RB791, using the segments, Subsequent modification of the vector has expression vector pKK 233-2. Milligram quantities increased expression to 5-lOmg/litre of cell culture. have polypeptide been purified using preparative of each gel In this way we have constructed an extensive electrophoresis. library of overlapping 6 -subunit polypeptides. This approach provides the first example of a recombinant human antigen prepared for the investigation of an autoimmune disease, and has already (see proved invaluable in defining T cell epitopes in MC patients Harcourt et al).
COMPARISON OF T CELL SITES RECOGNIZED BY THYMIC BLOOD LYMPHOCYTES IN MYASTHENIA GRAVIS PATIENTS
AND
S. BERRIH-AKNIN, CNRS URA-D1159, 133 av. de la Resistance, 92350 LE PLESSIS ROBINSON - FRANCE Tel. 46 30 21 33 p. 3565 S. BERRIH-AKNIN, 92350-LE PLESSIS
S. COHEN-KAMINSKY, ROBINSON, France,
D. NEUMANN, and S. FUCHS and REHOVOT 76100 - Israel.
The synthetic peptides are interesting tools for the definition of T cell antigenic sites since T cell were proved to recognize conformation insensitive epitopes. The recent availability of the entire acetylcholine receptor AChR sequence makes it possible to use a synthetic peptide approach for mapping functional AChR regions. Several reports have attempted to define T cell sites involved in Myasthenia Gravis. Our previous work has indicated that peptides 168-181 and 351-368 from the human a-subunit represent potentially interesting sites since several patients and not controls were responding to these peptides.
Abstracts In order to compare the T-ceil specificities of thymic and blood lymphocytes, we have used 10 different peptides from torpedo or human sequence in a proliferation assay. Our results indicate a positive proliferative response to 3 peptides in several blood lymphocyte preparations whereas the autologous thymic cells were unresponsive. We attempted to sensitize the assay by addition of recombinant IL-2 in order to expand the proliferating clones. The degree of proliferation was much higher in presence of IL-2 but the non responding patients remained negative. Our data confirm that synthetic peptides are useful to pinpoint the T cell sites. The addition of IL-2 leads to a general increased proliferation but does not recruite new responder patients. Differences in reactivity to peptides observed between thymic lymphocytes and PBL could be explained by a low number of antigen-presenting cells in the thymic cell suspensions. Alternatively, the AChFi sites recognized by thymic T cells might be different from those recognized by the peripheral T cells. This last hypothesis is consistent with data showing that the AChR-like molecule present in the thymus is probably antigenically different from that expressed in the muscle.
T-CELL EPITOPES IN PATIENTS WITH MYASTHENIA GRAVIS DEFINED BY THE USE OF RECOMBINANT OF THE HUMANACETYLCHOLINE
POLYPEPTIDES
RECEPTOR Dr. G. Ha-court,
Neuro-
sciences Group Institute of Molecular Medicine, Univ of Oxford. OX3 9DU Tel. n. 0865-752320 Gillian Harcourt, D. Beeson, N. Willcox, A. Vincent and J. Newsom-Davis. Oxford ENGLAND T lymphocytes which specifically recognise the human acetylcholine receptor (AChR) alpha subunit have been ieolated from patients with mya&henia grawic (MC) using an almost full-length alpha subunit recombinant polypeptide Ir37-437) which has been expressed in E.coli and purified by gel electraphoresis. The T-cell recognition sites ofo patients have been furthur defined using shorter recombinant fragments and synthetic peptides. Three T cell clones and one line isolated from Patient CD (HLA DR 1,7) exhibited a. similar response, recognising all polypeptides that contain the amino acid sequence 37-87 and this response was restricted by DR 1 in all three clones. However, eight clones and two lines derived from patient MP (HLA DR 1,3) showed qreater heteroaeneitv. Two of the clones showed a slmifar pattern to that of patient CD, qecogn&ing all fragments containing AA 37-07, again restricted by DR 1. In contrast, six other clones and two lines from patient MP seemed to recognise an epitope nearer to the carboxy terminus of the alpha subunit, between AA 346-437. and aft were restricted by DR 3, Furthur studies are in progress using shorter peptides to’ define more precisely and to investigate the heterogeneity of responses patients. The study of furthur patients may also reveal whether geneity is related to the HLA antigens restricting the recognition epitopes.
MICE IMMUNISED WITH RECOMBINANT SUBUNIT OF THE HUMAN ACETYLCHOLINE RECEPTOR (AChR) DEVELOP ANTI-AChR ANTIBODIES BUT NOT EXPERIMENTAL AUTOIMMUNE MYASTHENIA DAVIS (EAMG) Dr. A. Jenny, Neurosciences group, Institute of Molecular Medicine, University of Oxford. OX3 9DU Tel No 0865 752327 Andrew Jermy, D. Beeson, A. Vincent, N. Willeox and J. Newsom-Davis. Oxford, England.
the epitopes in other this heteroof these
903
Abstracts
ISOLATION HUMAN
AND
MUSCLE
EXPRESSION OF cDNAS FOR NICOTINIC ACETYLCHOLINE
THE
RECEPTOR. Neurosciences Dr. D. Beeson, Molecular Medicine, University cliffe Hospital, Oxford. OX3 David Davis.
Beeson,
M.
Brydson,
Detailed study of the epitopes by the low level of AChR (< to overcome this problem subunits and their subsequent
Group, Institute of of Oxford, John Rad9DU Tel No 0865-752326 A. Vincent, J. NewsomOxford, England involved in myasthenia gravis is restricted Zpmol/g) found in human muscle. We aimed by cloning cDNAs for the human AChR expression.
A cDNA library has been constructed in AgtlO using poly(A+) RNA from human leg muscle. The library was initially screened with cDNA probes coding for chicken AChR subunits and, following isolation of positives, with cDNAs for the human AChR subunits. cDNAs containing the full coding sequence have been isolated for the o! and 7 -subunits, and in the case of x and r -subunits for the full sequence except for the initial 5’ coding region. The full coding region of the p-subunit has and has been shown previously known in man), acids including a 23 amino acid prepeptide sequence.
been determined to contain 501
(not amino
Restriction fragments of both Torpedo and human o( -subunit cDNAs, coding for almost all the mature protein, and for smaller polypeptide have been expressed in E. Coli strain RB791, using the segments, Subsequent modification of the vector has expression vector pKK 233-2. Milligram quantities increased expression to 5-lOmg/litre of cell culture. have polypeptide been purified using preparative of each gel In this way we have constructed an extensive electrophoresis. library of overlapping 6 -subunit polypeptides. This approach provides the first example of a recombinant human antigen prepared for the investigation of an autoimmune disease, and has already (see proved invaluable in defining T cell epitopes in MC patients Harcourt et al).
COMPARISON OF T CELL SITES RECOGNIZED BY THYMIC BLOOD LYMPHOCYTES IN MYASTHENIA GRAVIS PATIENTS
AND
S. BERRIH-AKNIN, CNRS URA-D1159, 133 av. de la Resistance, 92350 LE PLESSIS ROBINSON - FRANCE Tel. 46 30 21 33 p. 3565 S. BERRIH-AKNIN, 92350-LE PLESSIS
S. COHEN-KAMINSKY, ROBINSON, France,
D. NEUMANN, and S. FUCHS and REHOVOT 76100 - Israel.
The synthetic peptides are interesting tools for the definition of T cell antigenic sites since T cell were proved to recognize conformation insensitive epitopes. The recent availability of the entire acetylcholine receptor AChR sequence makes it possible to use a synthetic peptide approach for mapping functional AChR regions. Several reports have attempted to define T cell sites involved in Myasthenia Gravis. Our previous work has indicated that peptides 168-181 and 351-368 from the human a-subunit represent potentially interesting sites since several patients and not controls were responding to these peptides.
Abstracts In order to compare the T-ceil specificities of thymic and blood lymphocytes, we have used 10 different peptides from torpedo or human sequence in a proliferation assay. Our results indicate a positive proliferative response to 3 peptides in several blood lymphocyte preparations whereas the autologous thymic cells were unresponsive. We attempted to sensitize the assay by addition of recombinant IL-2 in order to expand the proliferating clones. The degree of proliferation was much higher in presence of IL-2 but the non responding patients remained negative. Our data confirm that synthetic peptides are useful to pinpoint the T cell sites. The addition of IL-2 leads to a general increased proliferation but does not recruite new responder patients. Differences in reactivity to peptides observed between thymic lymphocytes and PBL could be explained by a low number of antigen-presenting cells in the thymic cell suspensions. Alternatively, the AChFi sites recognized by thymic T cells might be different from those recognized by the peripheral T cells. This last hypothesis is consistent with data showing that the AChR-like molecule present in the thymus is probably antigenically different from that expressed in the muscle.
T-CELL EPITOPES IN PATIENTS WITH MYASTHENIA GRAVIS DEFINED BY THE USE OF RECOMBINANT OF THE HUMANACETYLCHOLINE
POLYPEPTIDES
RECEPTOR Dr. G. Ha-court,
Neuro-
sciences Group Institute of Molecular Medicine, Univ of Oxford. OX3 9DU Tel. n. 0865-752320 Gillian Harcourt, D. Beeson, N. Willcox, A. Vincent and J. Newsom-Davis. Oxford ENGLAND T lymphocytes which specifically recognise the human acetylcholine receptor (AChR) alpha subunit have been ieolated from patients with mya&henia grawic (MC) using an almost full-length alpha subunit recombinant polypeptide Ir37-437) which has been expressed in E.coli and purified by gel electraphoresis. The T-cell recognition sites ofo patients have been furthur defined using shorter recombinant fragments and synthetic peptides. Three T cell clones and one line isolated from Patient CD (HLA DR 1,7) exhibited a. similar response, recognising all polypeptides that contain the amino acid sequence 37-87 and this response was restricted by DR 1 in all three clones. However, eight clones and two lines derived from patient MP (HLA DR 1,3) showed qreater heteroaeneitv. Two of the clones showed a slmifar pattern to that of patient CD, qecogn&ing all fragments containing AA 37-07, again restricted by DR 1. In contrast, six other clones and two lines from patient MP seemed to recognise an epitope nearer to the carboxy terminus of the alpha subunit, between AA 346-437. and aft were restricted by DR 3, Furthur studies are in progress using shorter peptides to’ define more precisely and to investigate the heterogeneity of responses patients. The study of furthur patients may also reveal whether geneity is related to the HLA antigens restricting the recognition epitopes.
MICE IMMUNISED WITH RECOMBINANT SUBUNIT OF THE HUMAN ACETYLCHOLINE RECEPTOR (AChR) DEVELOP ANTI-AChR ANTIBODIES BUT NOT EXPERIMENTAL AUTOIMMUNE MYASTHENIA DAVIS (EAMG) Dr. A. Jenny, Neurosciences group, Institute of Molecular Medicine, University of Oxford. OX3 9DU Tel No 0865 752327 Andrew Jermy, D. Beeson, A. Vincent, N. Willeox and J. Newsom-Davis. Oxford, England.
the epitopes in other this heteroof these
903