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Specificities of multiple sclerosis cerebrospinal fluid and serum antibodies against mimotopes
34
P o s t e r A b s t r a c t s / J o u r n a l o f N e u r o i m r n u n o l o g y 90 (1998) 1 3 - 1 0 5
173
176
Isolation and C h a r a c t e r i z a t i o n o f the Fraction o f Synthetic C o p o l y m e r 1 B o u n d to R e c o m b i n a n t H L A - D R Molecules M. Ffidkis-Hareli. J. Neveu, R.A. Robinson, W.S. Lane, Harvard University, USA, K.W. Wucherpfennig, HarvardMediL'alSchool, USA,J.L. Strominger, Harvard
From Specificity to Degeneracy to Molecular Mimicry:
University, USA
Copolymer 1 (Cop I ) is a random synthetic amino acid copolymer of L-alanine, L-glutamic acid, L-lysine and L-tyrosine effective both in suppression of experimental allergic encephalomyelitis (EAE) and in the treatment of relapsing forms of multiple sclerosis (MS). Cop 1 binds promiscuously, with high affinity and in an antigen-specific manner to purified human HLA-DRI, DR2 and DR4 molecules. To isolate the bound fraction of Cop 1 with no interference from endogenous peptides, recombinant "empty" HLA-DRI, DR2 and DR4 molecules produced in insect cells were employed in the present study. Bound Cop 1 was eluted by acid extraction and subjected to amino acid analysis, HPLC separation and pool sequencing. Amino acid composition, HPLC profiles and sequencing patterns of Cop I bound to DR I, DR2 or DR4 molecules were similar to those of the unseparated Cop I, These findings suggest that nearly the entire mixture of Cop I components binds and one or more of these maybe active component(s). Further characterization of class 11 MHC binding motifs of Cop 1 will form a basis for design and synthesis of efficient Cop 1-derived peptides for use in MS therapy. This work is part of a collaboration with Michael Sela, Ruth Arnon and Dvora Teitelbaum on characterization of the binding motifs of Cop 1.
A n t i g e n Recognition of H u m an Autoreactive and Pathogen-specific CD4+ T Cells !~. H e m ~ NINDS, NIH, USA,C. Pinilla, TPIMS, USA,B. Gran, H. McFarland, NINDS, NIH, USA,R. Houghten, MPS, USA,R. Marlin, NINDS, NIH, USA
CD4+ T cells recognize by their T-cell-raceptor (TCR) peptidesbound to MHC-class I1 molecules. Initially,this interactionwas considered highly specific. However, recently it became evidentthat a specific TCR can interact with many [igands. Here, we define recognition patterns of a set of myelin basic protein (MBP)- and influenzavirus-specific human T cell clones (TCCs) using a soluble syntheticpeptide combinatoriallibrary in the positionalscanningformat (PS-SCL). The use of PS-SCLsallows determination of important residues for each position of the antigenicpeptide and deduction of recognition motifs for each individualTCC. All MBP-specificTCCs had motifs that identifiedMBP as a rather suboptimalantigen. In additionbased on the recognition motifs, high potency ligandswere found for the MBP-speeificTCCs, some of these ligandsderived from viral antigens such as Human-Herpes-Virus 7. For one TCC, a crossreaetive peptide derived from myelin oligodendracyteglycoproteinwas identified as well as 5 amino acid long synthetic peptides that were more potent than the autoantigen used to establishthe TCC. These data demonstrate that antigen recognition by CD4+ T cells is highly degenerate allowingpositiveengagementby many ligands. Although initialactivationof these cells in vivo will most likelyrequire a high affinity interaction (such as a viral antigen), the activatedcells, after upregulationand activation of coreeeptors, have a high potential for cross-recognitionof self antigens. These results shed new light on the extent of cross-recognitionin the immunesystem and its importance for occurrence of autoimmunediseases.
174
177
Fine Specifieities o f T Cell and B Cell Responses to Myelin]Oligodendrocyte Glycoprotein in C o m m o n M a r m o s e t s C.E Genairt, N. Belmar, P. Diaz-Villoslada, S.L. Hauser, Universityof Califorma,
B Lymphocytes P r o d u c i n g D e m y e l i n a t i n g A u t o a n t i b o d i e s : D e v e l o p m e n t and Function in Gene-targcted Transgenic Mice T. Litzenbnrger, R. Fassler, Max-Planck-InstitutfurNeurobiologie,Germany, J. Bauer, Max-Planek-Institutfi~rBtochemie,Germany,H. Las sm ann, C. Linhlgton, Instituteof Neurology, Universityof Vienna,Austria, H. Wekerle, A. I gles ias, Max-Planck-lnstitut
USA
fur Neurobiologie,Germany Objective: To define the encephalitogenic B cell and T cell epitopes of myelin/olidedendrocyte glycoprotein (MOG) in MS-like marmoset EAE.
Methods: In C. jacchus marmosets with demyelinating EAE induced with recombinant rat MOG (rMOG: extracellular domain aa 1-125), the fine specificities of T cell reactivity (proliferative responses in PBMC) and B cell reactivity (serum antibody) to MOG were serially studied using overlapping 15met PIN-pepddes (offsets of I and 3), corresponding to amino-acid sequences of both rat and human MOG (Chiron Mimotopes, San Diego, CA). Results: All animals studied (n=6) had a prominent and sustained T cell response restricted to aa 28-36, a sequence totally conserved across species. A single marmoset responded to a second T cell epitope located within aa 63-72. Serum antibody responses (n= 10) mapped to 4 different regions of MOG including 2 major epitopes, aa 13-21 and aa 67-73 (100% and 60% of animals, respectively). No epitope spreading was observed either for T cells or antibodies in animals with relapsing EAE that were monitored for up to 93 days. Conclusions: Encephalitogenic responses to MOG in MS-like, marmoset EAE appear restricted to a limited number of B cell and T cell epitopes. These findings may provide a rationale for specific immunotherapy in human MS.
The cellular basis of self-tolerance of B cells specific for brain autoantigens was studied in transgenic mice engineered to produce high liters of autoantibodies against the myelin oligodendrocyte glycoprotein (MOG), a surface component of central nervous system (CNS) myelin. We generated "knock-in" mice by replacing the germline JH locus with the rearranged lg H chain variable (V) gene of a pathogenic MOG-spocificmonoclonal antibody. In transgeniemice B cells reach normal numbers in bone marrow and periphery and express exclusivelytransgenic H chains, resulting in high titers of MOG-specific serum lgs. Additionally, about one third of transgenic B cells bind MOG, thus demonstrating the absence of active tolerisation. Upon immunization with MOG the mature transgenic B cell population undergo normal differentiation to plasma cells secreting MOG-specific IgG antibodies, during which both lg isotype switching and somatic mutation occur. Naive transgenic mice fail to develop either spontaneous neurological disease or pathological evidence of demyelination. However,the presence of the transgene both accelerates and exacerbates experimental autoimmune encephalitis, irrespective of the identityof the initial autoimmuneinsult.
175
178
In Vitro Analysis of A n t i g e n Presentation by G l i a l Cells O. Gresser, S. Riese, A. Regnier-Vigouroux, Universnyof Heidelberg, Germany
Speeificities o f Multiple Sclerosis C e r e b r o s p i n a l F l u i d and S e r u m Antibodies Against Mimotopes C. Jglivet-Revnaud, H. Perron, L. Generenez, U/v/R103 CNRS-BioMerieux,Lyon, France, l~ Dalbon, Bio~[erieux,Marcy l 'EtoiIe,France, B. Montrant, UMRI 03 C'N/CS-
BioMerieur,Lyon, France
Aslrocytex and micro~lia have the capacities to function as antigen presenting cells (APe) in the brain. Our goal is to analyse the mechanisms by which these cells process :rod pruseut antigens to T lympocytes ill comparison with a protessiolml APe such as B lymphocytes. Onr apprmlch to study tile hmuunc conlpetencc of Ibesc cells is based on a ftmctional analysis using primary cultures of nlurine aStl'ouytes mid mlcroglia and a model antigen. We use apomin, a neul'otoxin id the bee venom, and a panel of npanlin-speclfic MIle ll-rcstricted "f cell lines. Afiur actlw~tion by cytokines, aslrocytes and nticroglia tire c o c t d t t l r e d with T cells in Ihe presence or the absence of apumin. The T ceil response to the antigenic sdmulatiun is measured by Ihe quantific:aion of IL-2 production (CTL-L assay). In addition we use ELISA on live cells to cbaracterise the cell sure'ace expression of imnmne molecules xuch as B7 or ICAMI ilwolved in antigen presenlation and T cell activation. Their functional inlportance it also being studied by using antibodies ill the antigen presentation assay. We will discuss the first results of our nlodel system charactcrisatioll Ibm display a complex profile of expression and functional irapormnce of ilnmune molecules. We further want to investigate the kinetics of antigen processing and presentation, the generation of a peptidic repertoire and the enzymes involved in the processing.
The antibody specificities of cerebrospinal fluids (CSF) and sera from patients with multiple sclerosis (MS) have been analyzed using a random pentadecapeptide library displayed on phage. With sera, the selected peptides appeared to be immunoreactive but were not significantly detected as disease-specific epitopes. In contrast, with MS CSF, the screening of the phage library using different protocols of biopannings with different MS CSF. evidenced several motifs (mimotopes) selected with high frequency. These peptide sequences were totally different from the sequences of peptides selected with MS sere. Five selected mimotopes, reproduced as biotinylated synthetized peptides, were specifically recognized in ELISA by 8 to 11 out of 15 MS CSF and by none of CSF from patients with other neurological disease (ND). Moreover, the combination of only 3 immunnreaetive peptldes allowed the detection of specific antibodies in 13 out of 15 MS CSF. Homology search with the MS CSF selected peptides indicated that homologies were found with the env C terminal region of MSRV, a new MS-associated retrovirus which has been recently reported. In data bases, homologies were also found with the Feline Sarcoma Virus p30 and "Fax protein of HTLV.
P o s t e r A b s t r a c t s / J o u r n a l o f N e u r o i m r n u n o l o g y 90 (1998) 1 3 - 1 0 5
173
176
Isolation and C h a r a c t e r i z a t i o n o f the Fraction o f Synthetic C o p o l y m e r 1 B o u n d to R e c o m b i n a n t H L A - D R Molecules M. Ffidkis-Hareli. J. Neveu, R.A. Robinson, W.S. Lane, Harvard University, USA, K.W. Wucherpfennig, HarvardMediL'alSchool, USA,J.L. Strominger, Harvard
From Specificity to Degeneracy to Molecular Mimicry:
University, USA
Copolymer 1 (Cop I ) is a random synthetic amino acid copolymer of L-alanine, L-glutamic acid, L-lysine and L-tyrosine effective both in suppression of experimental allergic encephalomyelitis (EAE) and in the treatment of relapsing forms of multiple sclerosis (MS). Cop 1 binds promiscuously, with high affinity and in an antigen-specific manner to purified human HLA-DRI, DR2 and DR4 molecules. To isolate the bound fraction of Cop 1 with no interference from endogenous peptides, recombinant "empty" HLA-DRI, DR2 and DR4 molecules produced in insect cells were employed in the present study. Bound Cop 1 was eluted by acid extraction and subjected to amino acid analysis, HPLC separation and pool sequencing. Amino acid composition, HPLC profiles and sequencing patterns of Cop I bound to DR I, DR2 or DR4 molecules were similar to those of the unseparated Cop I, These findings suggest that nearly the entire mixture of Cop I components binds and one or more of these maybe active component(s). Further characterization of class 11 MHC binding motifs of Cop 1 will form a basis for design and synthesis of efficient Cop 1-derived peptides for use in MS therapy. This work is part of a collaboration with Michael Sela, Ruth Arnon and Dvora Teitelbaum on characterization of the binding motifs of Cop 1.
A n t i g e n Recognition of H u m an Autoreactive and Pathogen-specific CD4+ T Cells !~. H e m ~ NINDS, NIH, USA,C. Pinilla, TPIMS, USA,B. Gran, H. McFarland, NINDS, NIH, USA,R. Houghten, MPS, USA,R. Marlin, NINDS, NIH, USA
CD4+ T cells recognize by their T-cell-raceptor (TCR) peptidesbound to MHC-class I1 molecules. Initially,this interactionwas considered highly specific. However, recently it became evidentthat a specific TCR can interact with many [igands. Here, we define recognition patterns of a set of myelin basic protein (MBP)- and influenzavirus-specific human T cell clones (TCCs) using a soluble syntheticpeptide combinatoriallibrary in the positionalscanningformat (PS-SCL). The use of PS-SCLsallows determination of important residues for each position of the antigenicpeptide and deduction of recognition motifs for each individualTCC. All MBP-specificTCCs had motifs that identifiedMBP as a rather suboptimalantigen. In additionbased on the recognition motifs, high potency ligandswere found for the MBP-speeificTCCs, some of these ligandsderived from viral antigens such as Human-Herpes-Virus 7. For one TCC, a crossreaetive peptide derived from myelin oligodendracyteglycoproteinwas identified as well as 5 amino acid long synthetic peptides that were more potent than the autoantigen used to establishthe TCC. These data demonstrate that antigen recognition by CD4+ T cells is highly degenerate allowingpositiveengagementby many ligands. Although initialactivationof these cells in vivo will most likelyrequire a high affinity interaction (such as a viral antigen), the activatedcells, after upregulationand activation of coreeeptors, have a high potential for cross-recognitionof self antigens. These results shed new light on the extent of cross-recognitionin the immunesystem and its importance for occurrence of autoimmunediseases.
174
177
Fine Specifieities o f T Cell and B Cell Responses to Myelin]Oligodendrocyte Glycoprotein in C o m m o n M a r m o s e t s C.E Genairt, N. Belmar, P. Diaz-Villoslada, S.L. Hauser, Universityof Califorma,
B Lymphocytes P r o d u c i n g D e m y e l i n a t i n g A u t o a n t i b o d i e s : D e v e l o p m e n t and Function in Gene-targcted Transgenic Mice T. Litzenbnrger, R. Fassler, Max-Planck-InstitutfurNeurobiologie,Germany, J. Bauer, Max-Planek-Institutfi~rBtochemie,Germany,H. Las sm ann, C. Linhlgton, Instituteof Neurology, Universityof Vienna,Austria, H. Wekerle, A. I gles ias, Max-Planck-lnstitut
USA
fur Neurobiologie,Germany Objective: To define the encephalitogenic B cell and T cell epitopes of myelin/olidedendrocyte glycoprotein (MOG) in MS-like marmoset EAE.
Methods: In C. jacchus marmosets with demyelinating EAE induced with recombinant rat MOG (rMOG: extracellular domain aa 1-125), the fine specificities of T cell reactivity (proliferative responses in PBMC) and B cell reactivity (serum antibody) to MOG were serially studied using overlapping 15met PIN-pepddes (offsets of I and 3), corresponding to amino-acid sequences of both rat and human MOG (Chiron Mimotopes, San Diego, CA). Results: All animals studied (n=6) had a prominent and sustained T cell response restricted to aa 28-36, a sequence totally conserved across species. A single marmoset responded to a second T cell epitope located within aa 63-72. Serum antibody responses (n= 10) mapped to 4 different regions of MOG including 2 major epitopes, aa 13-21 and aa 67-73 (100% and 60% of animals, respectively). No epitope spreading was observed either for T cells or antibodies in animals with relapsing EAE that were monitored for up to 93 days. Conclusions: Encephalitogenic responses to MOG in MS-like, marmoset EAE appear restricted to a limited number of B cell and T cell epitopes. These findings may provide a rationale for specific immunotherapy in human MS.
The cellular basis of self-tolerance of B cells specific for brain autoantigens was studied in transgenic mice engineered to produce high liters of autoantibodies against the myelin oligodendrocyte glycoprotein (MOG), a surface component of central nervous system (CNS) myelin. We generated "knock-in" mice by replacing the germline JH locus with the rearranged lg H chain variable (V) gene of a pathogenic MOG-spocificmonoclonal antibody. In transgeniemice B cells reach normal numbers in bone marrow and periphery and express exclusivelytransgenic H chains, resulting in high titers of MOG-specific serum lgs. Additionally, about one third of transgenic B cells bind MOG, thus demonstrating the absence of active tolerisation. Upon immunization with MOG the mature transgenic B cell population undergo normal differentiation to plasma cells secreting MOG-specific IgG antibodies, during which both lg isotype switching and somatic mutation occur. Naive transgenic mice fail to develop either spontaneous neurological disease or pathological evidence of demyelination. However,the presence of the transgene both accelerates and exacerbates experimental autoimmune encephalitis, irrespective of the identityof the initial autoimmuneinsult.
175
178
In Vitro Analysis of A n t i g e n Presentation by G l i a l Cells O. Gresser, S. Riese, A. Regnier-Vigouroux, Universnyof Heidelberg, Germany
Speeificities o f Multiple Sclerosis C e r e b r o s p i n a l F l u i d and S e r u m Antibodies Against Mimotopes C. Jglivet-Revnaud, H. Perron, L. Generenez, U/v/R103 CNRS-BioMerieux,Lyon, France, l~ Dalbon, Bio~[erieux,Marcy l 'EtoiIe,France, B. Montrant, UMRI 03 C'N/CS-
BioMerieur,Lyon, France
Aslrocytex and micro~lia have the capacities to function as antigen presenting cells (APe) in the brain. Our goal is to analyse the mechanisms by which these cells process :rod pruseut antigens to T lympocytes ill comparison with a protessiolml APe such as B lymphocytes. Onr apprmlch to study tile hmuunc conlpetencc of Ibesc cells is based on a ftmctional analysis using primary cultures of nlurine aStl'ouytes mid mlcroglia and a model antigen. We use apomin, a neul'otoxin id the bee venom, and a panel of npanlin-speclfic MIle ll-rcstricted "f cell lines. Afiur actlw~tion by cytokines, aslrocytes and nticroglia tire c o c t d t t l r e d with T cells in Ihe presence or the absence of apumin. The T ceil response to the antigenic sdmulatiun is measured by Ihe quantific:aion of IL-2 production (CTL-L assay). In addition we use ELISA on live cells to cbaracterise the cell sure'ace expression of imnmne molecules xuch as B7 or ICAMI ilwolved in antigen presenlation and T cell activation. Their functional inlportance it also being studied by using antibodies ill the antigen presentation assay. We will discuss the first results of our nlodel system charactcrisatioll Ibm display a complex profile of expression and functional irapormnce of ilnmune molecules. We further want to investigate the kinetics of antigen processing and presentation, the generation of a peptidic repertoire and the enzymes involved in the processing.
The antibody specificities of cerebrospinal fluids (CSF) and sera from patients with multiple sclerosis (MS) have been analyzed using a random pentadecapeptide library displayed on phage. With sera, the selected peptides appeared to be immunoreactive but were not significantly detected as disease-specific epitopes. In contrast, with MS CSF, the screening of the phage library using different protocols of biopannings with different MS CSF. evidenced several motifs (mimotopes) selected with high frequency. These peptide sequences were totally different from the sequences of peptides selected with MS sere. Five selected mimotopes, reproduced as biotinylated synthetized peptides, were specifically recognized in ELISA by 8 to 11 out of 15 MS CSF and by none of CSF from patients with other neurological disease (ND). Moreover, the combination of only 3 immunnreaetive peptldes allowed the detection of specific antibodies in 13 out of 15 MS CSF. Homology search with the MS CSF selected peptides indicated that homologies were found with the env C terminal region of MSRV, a new MS-associated retrovirus which has been recently reported. In data bases, homologies were also found with the Feline Sarcoma Virus p30 and "Fax protein of HTLV.