A GMCSF-neuroantigen fusion protein is a potent tolerogen in experimental autoimmune encephalomyelitis (EAE) that is associated with efficient targeting of neuroantigen to APC

The Department of Microbiology and Immunology, East Carolina University, Brody School of Medicine, Greenville, North Carolina, USA.
Journal of leukocyte biology (Impact Factor: 4.29). 12/2009; 87(3):509-21. DOI: 10.1189/jlb.0709520
Source: PubMed


Cytokine-NAg fusion proteins represent an emerging platform for specific targeting of self-antigen to particular APC subsets as a means to achieve antigen-specific immunological tolerance. This study focused on cytokine-NAg fusion proteins that targeted NAg to myeloid APC. Fusion proteins contained GM-CSF or the soluble extracellular domain of M-CSF as the N-terminal domain and the encephalitogenic 69-87 peptide of MBP as the C-terminal domain. GMCSF-NAg and MCSF-NAg fusion proteins were approximately 1000-fold and 32-fold more potent than NAg in stimulating antigenic proliferation of MBP-specific T cells, respectively. The potentiated antigenic responses required cytokine-NAg covalent linkage and receptor-mediated uptake. That is, the respective cytokines did not potentiate antigenic responses when cytokine and NAg were added as separate molecules, and the potentiated responses were inhibited specifically by the respective free cytokine. Cytokine-dependent targeting of NAg was specific for particular subsets of APC. GMCSF-NAg and MCSF-NAg targeted NAg to DC and macrophages; conversely, IL4-NAg and IL2-NAg fusion proteins, respectively, induced an 1000-fold enhancement in NAg reactivity in the presence of B cell and T cell APC. GMCSF-NAg significantly attenuated severity of EAE when treatment was completed before encephalitogenic challenge or alternatively, when treatment was initiated after onset of EAE. MCSF-NAg also had significant tolerogenic activity, but GMCSF-NAg was substantially more efficacious as a tolerogen. Covalent GMCSF-NAg linkage was required for prevention and treatment of EAE. In conclusion, GMCSF-NAg was highly effective for targeting NAg to myeloid APC and was a potent, antigen-specific tolerogen in EAE.

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    • "The finding that GM-CSF was a more effective tolerogenic fusion partner than M- CSF provided suggestive evidence that active induction of myeloid APC was more important than " quiescent maintenance " for induction of tolerance (Fleetwood et al., 2007; Hamilton, 2008). This outcome was expected because GM-CSF facilitates MHCII expression on myeloid APC whereas M-CSF confers viability to the macrophage lineage but does not maintain MHCII expression (Blanchfield and Mannie, 2010). GM-CSF promotes differentiation of MHCII + dendritic cells (DC) whereas M-CSF appears to be a maintenance factor for quiescent or non-inflammatory MHCII − macrophages. "
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    ABSTRACT: Myelin-specific induction of tolerance represents a promising means to modify the course of autoimmune inflammatory demyelinating diseases such as multiple sclerosis (MS). Our laboratory has focused on a novel preclinical strategy for the induction of tolerance to the major encephalitogenic epitopes of myelin that cause experimental autoimmune encephalomyelitis (EAE) in rats and mice. This novel approach is based on the use of cytokine-NAg (neuroantigen) fusion proteins comprised of the native cytokine fused either with or without a linker to a NAg domain. Several single-chain cytokine-NAg fusion proteins were tested including GMCSF-NAg, IFNbeta-NAg, NAgIL16, and IL2-NAg. These cytokine-NAg vaccines were tolerogenic, therapeutic vaccines that had tolerogenic activity when given as pre-treatments before encephalitogenic immunization and also were effective as therapeutic interventions during the effector phase of EAE. The rank order of inhibitory activity was as follows: GMCSF-NAg, IFNbeta-NAg > NAgIL16 > IL2-NAg > MCSF-NAg, IL4-NAg, IL-13-NAg, IL1RA-NAg, and NAg. Several cytokine-NAg fusion proteins exhibited antigen-targeting activity. High affinity binding of the cytokine domain to specific cytokine receptors on particular subsets of APC resulted in the concentrated uptake of the NAg domain by those APC which in turn facilitated the enhanced processing and presentation of the NAg domain on cell surface MHC class II glycoproteins. For most cytokine-NAg vaccines, the covalent linkage of the cytokine domain and NAg domain was required for inhibition of EAE, thereby indicating that antigenic targeting of the NAg domain to APC was also required in vivo for tolerogenic activity. Overall, these studies introduced a new concept of cytokine-NAg fusion proteins as a means to induce tolerance and to inhibit the effector phase of autoimmune disease. The approach has broad application for suppressive vaccination as a therapy for autoimmune diseases such as MS.
    Preview · Article · Aug 2012 · Frontiers in Immunology
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    • "Purity was assessed by SDS-PAGE. GMCSF-GP(69-87), referred to as GMCSF-NAg in [15], was a fusion of rat GM-CSF with the 69-87 encephalitogenic sequence of guinea pig (GP) myelin basic protein "Y-G-S-L-P-Q-K-S-Q-R-S-Q-D-E-N-P-V-V-H". The sequence of the GP69-88 peptide was: Y-G-S-L-P-Q-K-S-Q-R-S-Q-D-E-N-P-V-V-H-F. "
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    ABSTRACT: Vaccination strategies that elicit antigen-specific tolerance are needed as therapies for autoimmune disease. This study focused on whether cytokine-neuroantigen (NAg) fusion proteins could inhibit disease in chronic murine models of experimental autoimmune encephalomyelitis (EAE) and thus serve as potential therapeutic modalities for multiple sclerosis. A fusion protein comprised of murine GM-CSF as the N-terminal domain and the encephalitogenic MOG35-55 peptide as the C-terminal domain was tested as a tolerogenic, therapeutic vaccine (TTV) in the C57BL/6 model of EAE. Administration of GMCSF-MOG before active induction of EAE, or alternatively, at the onset of EAE blocked the development and progression of EAE. Covalent linkage of the GM-CSF and MOG35-55 domains was required for tolerogenic activity. Likewise, a TTV comprised of GM-CSF and PLP139-151 was a tolerogen in the SJL model of EAE. These data indicated that fusion proteins containing GM-CSF coupled to myelin auto-antigens elicit tolerance rather than immunity.
    Full-text · Article · Dec 2011 · BMC Immunology
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    ABSTRACT: Multiple sclerosis (MS) is considered to be a T cell-mediated autoimmune disease directed against myelinated nerves within the central nervous system. Current therapies available to MS patients have low efficacy and are immunosuppressive. Novel therapies that negatively regulate or delete autoreactive T cells, i.e., induce antigen-specific T cell tolerance, are key for the development of more efficacious and perhaps curative therapies. Our laboratory has developed a vaccine platform comprised of cytokine-antigen fusion proteins to promote T cell tolerance. In this study, GM-CSF and M-CSF cytokines were tested as domains in cytokine-neuroantigen (NAg) fusion proteins to assess targeting of NAg to different antigen presenting cell (APC) subsets. Fusion proteins were designed with a cytokine N-terminal domain and the encephalitogenic peptide 69-88 of guinea pig myelin basic protein (GP69-88) as the C-terminal domain. Studies measuring T cell activation in vitro , prevention of experimental autoimmune encephalomyelitis (EAE), and treatment of EAE showed that GMCSF-NAg was the most potent fusion protein, with the following rank order of activity (GMCSF-NAg > MCSF-NAg > GP69-88). GMCSF-NAg was 1000-fold more potent than GP69-88 in stimulating myelin basic protein (MBP)-specific T cell proliferation. The mechanism by which GMCSF-NAg promoted T cell activation involved cytokine receptor-mediated uptake of NAg by APC, since free GM-CSF inhibited the GMCSF-NAg potentiated response. GMCSF-NAg potently targeted NAg to dendritic cells and macrophages in vitro , but not to B or T cell APC. Covalent linkage between GM-CSF and NAg was required for enhanced potency of GMCSF-NAg in vitro and for the prevention and treatment of EAE in vivo . In conclusion, GMCSF-NAg potently targeted self-antigen to myeloid APC subsets and caused profound antigen-specific tolerance in EAE. In the future, cytokine-NAg fusion proteins may provide a novel tool to develop antigen-specific, tolerogenic vaccines for the treatment of MS. Ph.D.
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