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

Abstract

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.

GM-CSF–Neuroantigen Fusion Proteins Reverse Experimental Autoimmune Encephalomyelitis and Mediate Tolerogenic Activity in Adjuvant-Primed Environments: Association with Inflammation-Dependent, Inhibitory Antigen Presentation

“…Previous studies discounted the possibility that administration of GMCSF-MOG in saline elicited anti-GM-CSF antibodies(14). Also, previous studies (14–16) showed that treatment with GM-CSF alone or with noncovalently linked NAg lacked tolerogenic activity but nonetheless had the same theoretical potential as GMCSF-NAg to generate anti-GM-CSF antibodies. The use of B cell-deficient mice provided a definitive assessment of this issue.…”

“…GMCSF-NAg fusion proteins comprised of GM-CSF and dominant myelin epitopes of MBP, MOG, and PLP were potent NAg-specific tolerogens in rat and mouse models of EAE(14-16). GMCSF-NAg proteins were tolerogenic vaccines when administration was completed before immunization and were effective interventions when GMCSF-NAg treatment was initiated after onset of EAE.…”

“…Fusion proteins comprised of GM-CSF as the N-terminal domain and a myelin epitope as the C-terminal domain represent highly effective vaccine tolerogens. A rat GMCSF-NAg fusion protein that included the 69–87 epitope of myelin basic protein was tested in the Lewis rat model of EAE(16). The GM-CSF domain of the fusion protein targeted the NAg domain to myeloid APC to engender an approximate 1000-fold enhancement of antigenic potency by a mechanism that was blocked by free GM-CSF and that depended upon physicallinkage of the cytokine and NAg domains.…”

The Immune Basis of Allergic Lung Disease

“…Consistent with these mucosal sources, GM-CSF mRNA is increased in the airways of human asthmatics (Chung and Barnes, 1999) where it promotes maturation, activation, and growth of monocytes and DCs, resulting in increased antigen presentation and activation of antigen-specific B and T cells (Chung and Barnes, 1999). GM-CSF also contributes to T H 17-induced inflammatory responses (Ganesh et al, 2009;Blanchfield and Mannie, 2010;Hamilton and Anderson, 2004;Ritz et al, 2002).…”

GM-CSF–Based Fusion Cytokines as Ligands for Immune Modulation

“…It is unclear how this could induce Ag-specific tolerance, but the investigators speculated that APCs may be involved, because the IL-16/MBP fusion was effective at having APCs present MBP through MHC class II to Rsl.11 T cells (11). Blanchfield and Mannie (14) further explored how cytokines could be fused to more effectively deliver MBP to APCs by combining it with GM-CSF, finding that this fusion was also capable of significantly reducing the severity of EAE in rats. The investigators believed that the GM-CSF moiety acts as a delivery vehicle for MBP, because the two need to be linked for the fusion to suppress EAE.…”