- Email: [email protected]
Hypoallergenic recombinant variants of Bet v 1, the major allergen of birch pollen: Tools for specific immunotherapy of birch pollen allergy?
Pathogenesis of autoimmunity
Tuesday, 24 June 1997 - Oral presentations
dismutase (SOD) and as part of the E. cc/i outer membrane protein PhoE. These constructs were introduced in Mycob&eriurn vaccae and S&none//a fyphimurium, respectively. Recombinant bactetia were used for immunisation studies in mice. Reeuftsz The 29 aa long Der pl peptide could be expressed at high levels in M. vaccae and S. typbirtwtium. The recombinant bacteria were shown to be potent inducers of T-cells specific for the Der pl peptide and these T-cells produced high amounts of IFNy. We are currently investigating whether Th2 responses against this peptide can be modulated or prevented by immunisation with the recombinants expressing the peptide. Conclusion: Peptides as long as long as 29 aa c%nbe expressed at high levels as part of the SOD protein in M.vaccae and as part of outer membrane protein PhoE in S. fyphimudum. lmmunisation with recombinant bacteria results in Thl responses against the allergenic peptide.
Th cells is principally possible. These properties make this molecule acandidate for specific Immunotherapy of birch pollen allergy.
16:36-l
0.514
0.5.14.1 0.5.13.7
Tkeatment with mu&e CTLAMgG inhibits alnvay eosinophilla, hyperresponslveness and attenuates IgE upregulatlon in a murine model of allergic asthma
Claudia L. Hofstra ’ , lngrid Van Ark ’ , Bob Shields 2, Betty Chan ‘, Paula M. Jardieu *, Frans P. Nijkamp ‘, Antoon J.M. van Oosterhoui ’ ‘Department of Pharrnac&gy and FWhcphysiobg)! Ufrecht UniverSity,Faculty of Pharmacy, TS Utrechf, The Netherlands, *Department of Immuncl~ Genentech, South San Franciisco,USA Patients with allergic asthma suffer from airway obstruction, inflammation, bronchial hyperresponsiveness and high serum IgE levels. This disease is suggested to be regulated by T cells. Activation of antigen-specific T cells requires two signals: a specific signal generated by the allergen-MHC complex and a second signal provided by costimulatory molecules on the antigen presenting cell. One of the most important pathways of costimulation is the interaction of CD28 on the T cell with 87-l/87-2 on antigen presenting calls. In the present study we used a murine model of allergic asthma to investigate the CD28-B7 pathway by in viw blocking of this interaction by mCTLA4-IgG. For these studies a chimeric fusion protein of murine CTLA4 and human IgG, was constructed. Mice were sensitized with ovalbumin and repeated challenged with ovalbumin or saline inhalations. During the challenge period mice were treated with mCTLA4-IgG or control antibody. 24 hrs after the last challenge ovalbumin-specific IgE levels in serum, cellular infiltration in the bronchoalveolar lavage and in viw airway hyperresponsiveness to methacholine were determined. In ovalbumin challenged mice treated with control antibody a significant increase in the number of eosinophils (13 f 4 x 103 eosinophils, P < 0.05) in the bronchoalveolar lavage, in viw ainvay hyperresponsiveness (49 f 1596,P < 0.05) and increased serum levels of ovalbumin-specific IgE (1133 f 260 EU/ml versus 220 f 63 EU/ml, P c 0.01) were observed compared to saline challenged mice. In ovalbumin challenged mice treated with mCTLA4_IgG, the number of eosinophils in the bronchoalveolar lavage and the development of airway hyperresponsiveness were completely inhibited. After mCTLA4-IgG treatment no uoreoulation of ovalbuminsoeclflc IaE levels in serum was observed in ovaldum& versus saline chaflen& mice (respectively 680 f 138 EU/ml and 139 f 26 EU/ml). In conclusion, these data indicate the therapeutic potential of blocking T cell costimulation by CTLACIgG as a possible immune-suppressive treatment for patients with asthma.
0513.6
Hypoallergenlc recombinant variants of Bet v 1, the major allergen of Mrch pollen: Tools for specific immunotheraov . of birch pollen allemv? s
.
_-
C. Ebner ’ , F. Ferreira *. ’ Institute of General and Experimental Pathology; University of Vienna, Austria, * lnsMute of Biology and Genetics, University of Salzburg, Austria Introduction: Bet v 1, the major allergen of birch pollen consists of multiple closely related isoforms. These variants are highly similar but possess distinct immundooical orooerties. Mat&~ andkhods: Birch pollen-allergic subjects were tested in skin tests and by bronchial provocation for reactivity with natural Bet v 1 and Bet v 1-isoform d. Allergen-specific T lymphocytes (l cell lines, peptide-mapped T cell clones) were reacted with natural Bet v 1, Bet v 1d and Bet v 1a, an isoform with high IgE-binding capacity. Resuf& In all skin tests. Be1 v Id revealed a significantly lower activity to induce typical tieal and flare reactions than natural Bet v 1 or Bet v la,. Bronchial challenge revealed a higher PC20FEVl for Bet v Id. On the other hand, Bet v 1d displayed excellent T cell reactivity. Peptkfe-mapped TCC proved the presence of all important T ceil epitopes. Concluelon:Betv Id displays reduced allergenicity in viw. As it is recognized by T cells, the capacity to modulate the immune response at the level of
219
6:30
Room L
Pathogenesis of autoimmunity
TNF-triggered CNS inflammation, demyelination and T cell autoreactivlty in tranegenlc mice: A role for mature lymphocytes In disease modulation but not disease inltlation
G. Kassiotis, K. Akassoglou, G. Kollias, L. Probert. Department of Molecular Genetics, Hellenic Pasteur Institute, Amens, Hellas Introduction: TNF has been associated with the aetiology and pathogenesis of inflammatory and autoimmune diseases including those affecting the central nervous system (CNS). We have shown that the local overexpression of TNF transgenes in the CNS of mice is sufficient to bigger CNS inflammation and demyelination, and the recruitment of T lymphocytes and macrophages (PNAS: 1995,92,11294-E; J Immunol: 1997,158,43t?-45). Methods: To determine the functional conttfbution of CNS-infiltrating lymphocytes to disease development, Tg6074 mice which show CNS-specific expression of a muTNFglobin transgene, were backcrossed into a CD4 or RAG-1 deficient background. T cell proliferation responses of naive spleen cells taken from immunocompetent mice, to known autoantigens including MBP, PLP, MOG and collagen, were also measured. Results: The initiation of CNS inflammatoty and demyelinatfng disaase was unaltered in Tg6074 x RAG-l-‘- and Tg6074 x CD4-‘- mice when compared to immunocompetent Tg6074 littermates. Interestingly, disease severity and mortality rate were significantly increased in both Tg6074 backcmsses. T cell proliferation assays revealed the presence of a substantial CNS-directed autoimmune component with naive spleen cells taken from sick, but noi healthy mice, proliferating specifically in response to MOG and PLP peptides. Concfuslons: Local expression of TNF is sufficient to trigger CNS inflammatory demyelinating disease in the absence of mature T and B lymphocytes. Mature T and B lymphocytes, and to a lesser extent CD4+ T lymphocytes, contribute an overall disease suppressive effect. Following disease onset, a substantial CNS-specific autoreactive T lymphocyte response of so far undefined function is elicited. These results have important implications for our understanding of the pathogenesis of inflammatory and autoimmune diseases affecting the CNS, and transgenic mice which overexpress TNF in their CNS represent an important animal model system for the furlher study and management of related human diseases such as multiple sclerosis.
0.5.14.2
Gene targeting shows TNF to be an essential mediator of EAE in C57BL16 mice
S. Riminton, H. Khmer. D. Strickland. F. Lemckert, J. Sedgwick. lmmunocatholoav GIVUD. Centenarv Institute for Cancer Medicine and Cell Siolog~‘Sydrn+~NSW: kustralia _
Introduction: Tumor necrosis factor (TNF) has numerous known functions that may contribute to the pathology of T cell-dependent autoimmune diseases, either as a mediator of inflammation. or as a direct effector of tissue darnaoe. To investigate this we have used TNF gene-deleted mice. The position of the TNF genomic locus within the murine MHC prevents intercrossing of gene-deleted 129 mice to a more susceptible strain. We have therefore deleted TNF directly in C57BU6 mice, a strain highly susceptible to the model of myelin oligodendrocyte glycoprotein (MOG)-induced experimental autoimmune encephalomyelitis (EAE). Matarlalsand Methods:To induce EAE, mice were immunised with pMOG_5 (50 pg, sc) in complete Freunds adjuvant, and petussis toxin (200 ng iv, day 0 and day 2). The TNF genomic locus was disrupted in C57BU6 embryonic stem cells, using gene targeting by homologous recombination, and homozygous TNF-‘- mice were produced (Nucleic Acids Research 1997, 25, 917-918). Wild-type (WT.TNF*/+) C57BU6 mice were used as controfs. Resuftsz lmmunisation of WT C57BU6 mice with pMOG_ resulted in a severe ascending paralysis and wasting illness peaking around day 15, and spontaneously improving to leave a chronic, non-relapsing disability. The neuropathology was characterized by florrid CNS cellular inflammation and demyelination. Disease in TNF-‘- mice was markedly delayed in onset and of a reduced overall duration, but the peak severity of clinical manifestations was unaltered. Significantly, VCAM-1 expression on vascular endothelium and T cell
Tuesday, 24 June 1997 - Oral presentations
dismutase (SOD) and as part of the E. cc/i outer membrane protein PhoE. These constructs were introduced in Mycob&eriurn vaccae and S&none//a fyphimurium, respectively. Recombinant bactetia were used for immunisation studies in mice. Reeuftsz The 29 aa long Der pl peptide could be expressed at high levels in M. vaccae and S. typbirtwtium. The recombinant bacteria were shown to be potent inducers of T-cells specific for the Der pl peptide and these T-cells produced high amounts of IFNy. We are currently investigating whether Th2 responses against this peptide can be modulated or prevented by immunisation with the recombinants expressing the peptide. Conclusion: Peptides as long as long as 29 aa c%nbe expressed at high levels as part of the SOD protein in M.vaccae and as part of outer membrane protein PhoE in S. fyphimudum. lmmunisation with recombinant bacteria results in Thl responses against the allergenic peptide.
Th cells is principally possible. These properties make this molecule acandidate for specific Immunotherapy of birch pollen allergy.
16:36-l
0.514
0.5.14.1 0.5.13.7
Tkeatment with mu&e CTLAMgG inhibits alnvay eosinophilla, hyperresponslveness and attenuates IgE upregulatlon in a murine model of allergic asthma
Claudia L. Hofstra ’ , lngrid Van Ark ’ , Bob Shields 2, Betty Chan ‘, Paula M. Jardieu *, Frans P. Nijkamp ‘, Antoon J.M. van Oosterhoui ’ ‘Department of Pharrnac&gy and FWhcphysiobg)! Ufrecht UniverSity,Faculty of Pharmacy, TS Utrechf, The Netherlands, *Department of Immuncl~ Genentech, South San Franciisco,USA Patients with allergic asthma suffer from airway obstruction, inflammation, bronchial hyperresponsiveness and high serum IgE levels. This disease is suggested to be regulated by T cells. Activation of antigen-specific T cells requires two signals: a specific signal generated by the allergen-MHC complex and a second signal provided by costimulatory molecules on the antigen presenting cell. One of the most important pathways of costimulation is the interaction of CD28 on the T cell with 87-l/87-2 on antigen presenting calls. In the present study we used a murine model of allergic asthma to investigate the CD28-B7 pathway by in viw blocking of this interaction by mCTLA4-IgG. For these studies a chimeric fusion protein of murine CTLA4 and human IgG, was constructed. Mice were sensitized with ovalbumin and repeated challenged with ovalbumin or saline inhalations. During the challenge period mice were treated with mCTLA4-IgG or control antibody. 24 hrs after the last challenge ovalbumin-specific IgE levels in serum, cellular infiltration in the bronchoalveolar lavage and in viw airway hyperresponsiveness to methacholine were determined. In ovalbumin challenged mice treated with control antibody a significant increase in the number of eosinophils (13 f 4 x 103 eosinophils, P < 0.05) in the bronchoalveolar lavage, in viw ainvay hyperresponsiveness (49 f 1596,P < 0.05) and increased serum levels of ovalbumin-specific IgE (1133 f 260 EU/ml versus 220 f 63 EU/ml, P c 0.01) were observed compared to saline challenged mice. In ovalbumin challenged mice treated with mCTLA4_IgG, the number of eosinophils in the bronchoalveolar lavage and the development of airway hyperresponsiveness were completely inhibited. After mCTLA4-IgG treatment no uoreoulation of ovalbuminsoeclflc IaE levels in serum was observed in ovaldum& versus saline chaflen& mice (respectively 680 f 138 EU/ml and 139 f 26 EU/ml). In conclusion, these data indicate the therapeutic potential of blocking T cell costimulation by CTLACIgG as a possible immune-suppressive treatment for patients with asthma.
0513.6
Hypoallergenlc recombinant variants of Bet v 1, the major allergen of Mrch pollen: Tools for specific immunotheraov . of birch pollen allemv? s
.
_-
C. Ebner ’ , F. Ferreira *. ’ Institute of General and Experimental Pathology; University of Vienna, Austria, * lnsMute of Biology and Genetics, University of Salzburg, Austria Introduction: Bet v 1, the major allergen of birch pollen consists of multiple closely related isoforms. These variants are highly similar but possess distinct immundooical orooerties. Mat&~ andkhods: Birch pollen-allergic subjects were tested in skin tests and by bronchial provocation for reactivity with natural Bet v 1 and Bet v 1-isoform d. Allergen-specific T lymphocytes (l cell lines, peptide-mapped T cell clones) were reacted with natural Bet v 1, Bet v 1d and Bet v 1a, an isoform with high IgE-binding capacity. Resuf& In all skin tests. Be1 v Id revealed a significantly lower activity to induce typical tieal and flare reactions than natural Bet v 1 or Bet v la,. Bronchial challenge revealed a higher PC20FEVl for Bet v Id. On the other hand, Bet v 1d displayed excellent T cell reactivity. Peptkfe-mapped TCC proved the presence of all important T ceil epitopes. Concluelon:Betv Id displays reduced allergenicity in viw. As it is recognized by T cells, the capacity to modulate the immune response at the level of
219
6:30
Room L
Pathogenesis of autoimmunity
TNF-triggered CNS inflammation, demyelination and T cell autoreactivlty in tranegenlc mice: A role for mature lymphocytes In disease modulation but not disease inltlation
G. Kassiotis, K. Akassoglou, G. Kollias, L. Probert. Department of Molecular Genetics, Hellenic Pasteur Institute, Amens, Hellas Introduction: TNF has been associated with the aetiology and pathogenesis of inflammatory and autoimmune diseases including those affecting the central nervous system (CNS). We have shown that the local overexpression of TNF transgenes in the CNS of mice is sufficient to bigger CNS inflammation and demyelination, and the recruitment of T lymphocytes and macrophages (PNAS: 1995,92,11294-E; J Immunol: 1997,158,43t?-45). Methods: To determine the functional conttfbution of CNS-infiltrating lymphocytes to disease development, Tg6074 mice which show CNS-specific expression of a muTNFglobin transgene, were backcrossed into a CD4 or RAG-1 deficient background. T cell proliferation responses of naive spleen cells taken from immunocompetent mice, to known autoantigens including MBP, PLP, MOG and collagen, were also measured. Results: The initiation of CNS inflammatoty and demyelinatfng disaase was unaltered in Tg6074 x RAG-l-‘- and Tg6074 x CD4-‘- mice when compared to immunocompetent Tg6074 littermates. Interestingly, disease severity and mortality rate were significantly increased in both Tg6074 backcmsses. T cell proliferation assays revealed the presence of a substantial CNS-directed autoimmune component with naive spleen cells taken from sick, but noi healthy mice, proliferating specifically in response to MOG and PLP peptides. Concfuslons: Local expression of TNF is sufficient to trigger CNS inflammatory demyelinating disease in the absence of mature T and B lymphocytes. Mature T and B lymphocytes, and to a lesser extent CD4+ T lymphocytes, contribute an overall disease suppressive effect. Following disease onset, a substantial CNS-specific autoreactive T lymphocyte response of so far undefined function is elicited. These results have important implications for our understanding of the pathogenesis of inflammatory and autoimmune diseases affecting the CNS, and transgenic mice which overexpress TNF in their CNS represent an important animal model system for the furlher study and management of related human diseases such as multiple sclerosis.
0.5.14.2
Gene targeting shows TNF to be an essential mediator of EAE in C57BL16 mice
S. Riminton, H. Khmer. D. Strickland. F. Lemckert, J. Sedgwick. lmmunocatholoav GIVUD. Centenarv Institute for Cancer Medicine and Cell Siolog~‘Sydrn+~NSW: kustralia _
Introduction: Tumor necrosis factor (TNF) has numerous known functions that may contribute to the pathology of T cell-dependent autoimmune diseases, either as a mediator of inflammation. or as a direct effector of tissue darnaoe. To investigate this we have used TNF gene-deleted mice. The position of the TNF genomic locus within the murine MHC prevents intercrossing of gene-deleted 129 mice to a more susceptible strain. We have therefore deleted TNF directly in C57BU6 mice, a strain highly susceptible to the model of myelin oligodendrocyte glycoprotein (MOG)-induced experimental autoimmune encephalomyelitis (EAE). Matarlalsand Methods:To induce EAE, mice were immunised with pMOG_5 (50 pg, sc) in complete Freunds adjuvant, and petussis toxin (200 ng iv, day 0 and day 2). The TNF genomic locus was disrupted in C57BU6 embryonic stem cells, using gene targeting by homologous recombination, and homozygous TNF-‘- mice were produced (Nucleic Acids Research 1997, 25, 917-918). Wild-type (WT.TNF*/+) C57BU6 mice were used as controfs. Resuftsz lmmunisation of WT C57BU6 mice with pMOG_ resulted in a severe ascending paralysis and wasting illness peaking around day 15, and spontaneously improving to leave a chronic, non-relapsing disability. The neuropathology was characterized by florrid CNS cellular inflammation and demyelination. Disease in TNF-‘- mice was markedly delayed in onset and of a reduced overall duration, but the peak severity of clinical manifestations was unaltered. Significantly, VCAM-1 expression on vascular endothelium and T cell