Hydrodynamic Vaccination with DNA Encoding an Immunologically Privileged Retinal Antigen Protects from Autoimmunity through Induction of Regulatory T Cells

Abstract

The eye is an immunologically privileged organ whose Ags serve as targets for experimental autoimmune uveitis (EAU), a model for human uveitis. We used a hydrodynamic i.v. injection of naked DNA to express the uveitogenic retinal Ag interphotoreceptor retinoid-binding protein (IRBP) in the periphery, thus revoking its immune-privileged status. IRBP was expressed in the liver within hours of administration of as little as 10 μg of IRBP-DNA. Vaccinated mice were highly protected from EAU induced by immunization with IRBP for at least 10 wk after vaccination. Protection was partial in a reversal protocol. Mechanistic studies revealed specific hyporesponsiveness to IRBP without immune deviation, no evidence for apoptosis either by the Fas- or Bcl-2-regulated (mitochondrial) pathway and apparent lack of dependence on CD8+ cells, IL-10, or TGF-β. In contrast, depletion of CD25+ cells after vaccination and before challenge markedly abrogated protection. IRBP-specific CD4+CD25high T cells could be cultured from vaccinated mice and transferred protection to unvaccinated, EAU-challenged recipients. In vitro characterization of these cells revealed that they are Ag specific, anergic, express FoxP3, CTLA-4, and glucocorticoid-induced TNFR, and suppress by contact. Thus, expression of IRBP in the periphery by DNA vaccination results in tolerance that acts at least in part through induction of IRBP-specific, FoxP3+CD4+CD25+ regulatory T cells. DNA vaccination may offer a new approach to Ag-specific therapy of uveitis.

Understanding Autoimmune Uveitis through Animal Models The Friedenwald Lecture

“…An efficient way to induce Tregs is by hydrodynamic injection of naked DNA encoding a large fragment of IRBP, which is followed by expression of IRBP in the liver and induction of tolerance manifested as resistance to EAU induction. 25 It turns out that this tolerance is mediated at least in part by induction of Treg cells, which can be isolated from protected mice and expanded in culture by stimulation with immature DCϩIRBP 161-180 . When these expanded iTregs are infused into recipient mice that are subsequently immunized for EAU, they protect from induction of disease.…”

“…Within hours after injection of the DNA, IRBP protein is expressed in the liver. 25 Alternatively, we have transduced autologous B lymphocytes ex vivo with an IRBP construct using retrovirus, and infused them back into the mouse. 26 Both procedures resulted in a long-lived tolerance that prevented induction of EAU by a subsequent immunization with IRBP.…”

A look at autoimmunity and inflammation in the eye

“…The utility of antigen-specific therapy in humans is suggested by encouraging results in a clinical trial mentioned above using oral tolerance to arrestin (27). Peripheral de novo tolerogenic expression of retinal antigens can be achieved in the adult by naked DNA vaccination with an IRBP expression plasmid or by infusion of autologous B cells engineered by retroviral transduction to express a uveitogenic fragment of IRBP (22,23). Importantly, this tolerogenic B cell-based therapy has been shown to control EAU in HLA-DR3 transgenic mice (93) and could have significant clinical potential.…”

“…Thus, circulating retinal antigen-specific T cells are likely to be "ignorant" of their cognate antigen rather than tolerant and can be activated by a chance encounter with antigen, possibly in the form of a microbial component that structurally mimics their cognate tissue antigen (19). Indeed, it has been demonstrated that forced expression of a retinal antigen in the periphery (as a transgene, by retroviral delivery, or by vaccination with naked DNA) results in tolerance and in resistance to the subsequent induction of autoimmunity (20)(21)(22)(23). Therefore, by sequestering retinal antigens within the eye and hindering peripheral tolerance, immune privilege may actually predispose to ocular autoimmunity (24).…”

Autoimmunity in the immune privileged eye: pathogenic and regulatory T cells

“…IRBP could be detected in the liver by Western blotting within 8 h of vaccination. Vaccinated mice were highly protected from EAU induced by immunization with IRBP and were significantly protected also in a reversal protocol, indicating the utility of this approach in ongoing disease [35]. Interestingly, although detectable IRBP signal in the liver was gone by 50 h after vaccination, the vaccinated animals remained highly protected from a uveitogenic challenge for at least 10 weeks.…”