Cellular and molecular mechanisms involved in T cell-mediated autoimmunity against immunologically privileged retinal antigens (Ags) are being studied. The questions are aimed at elucidating the natural development and maintenance of self-tolerance to retinal Ags, and defining the processes that lead to their pathological breakdown. The goal is to use this knowledge for designing novel and rational strategies for immunotherapy. The experimental approaches utilize the model of experimental autoimmune uveoretinitis (EAU), which resembles immune-mediated uveitic diseases in humans that can lead to blindness. EAU is induced in mice and rats by immunization with retinal Ags such as IRBP, Arrestin (S-Ag), or their component peptide epitopes, or by infusion of cultured lymphocytes that recognize these Ags. The mechanisms controlling disease susceptibility and pathogenesis are being defined at the genetic, developmental, and immunological levels using cellular and molecular approaches. Novel approaches to disease regulation are devised based on these findings. ? ? As an attempt to strengthen what we believe to be deficient peripheral tolerance to retinal Ags, we are studying therapeutic induction of tolerance in adult mice by hydrodynamic IV vaccination with a 10 ?g of naked DNA plasmid encoding an IRBP fragment. It is highly effective in preventing disease, but only moderately effective in reversing it. We are currently studying the cellular mechanisms leading to the protection. Another approach to induce peripheral tolerance is infusion of autologous B cells retrovirally transduced with a uveitogenic antigen, which leads to long term protection from EAU. This can be achieved in HLA Tg mice with an epitope of S-Ag recognized by human uveitis patients, suggesting clinical relevance of this approach (4). ? ? Innate immune responses to microbes are thought to play a role as environmental triggers of autoimmunity. Because EAU induction is dependent on mycobacterial adjuvant, which provides strong innate stimuli, we studied the dependence of EAU induction on signaling through the MyD88 as well as TLR2, TLR 4 and TLR9 receptors. Unexpectedly, although MyD88 KO mice were highly resistant, TLR2, 4 and 9 single KOs and double KOs 2/4, 4/9 and 2/9 were fully susceptible and had normal responses to IRBP. In contrast, mice deficient in IL-1R (but not in IL-18), cytokines which also signal through MyD88, were resistant. Thus, absence of IL-1 signaling can by itself account for resistance of MyD88 KO mice to eau, and points to IL-1R as a potential therapeutic target for uveitis (2). ? ? We have previously shown that the threshold of susceptibility to EAU is in controlled in part by thymic derived """"""""natural"""""""" CD4+CD25+ regulatory T cells (T-regs). We investigated the requirement for expression of the cognate Ag (IRBP) in the thymus to generate these cells. While thymic expression of IRBP was needed to generate IRBP-specific T-regs, EAU-relevant T-regs that limit disease did not need to be IRBP specific. Polyclonal T-regs found in IRBP KO mice could be activated by microbial (including innate TLR-mediated) stimuli to protect from EAU. This study also demonstrated that generation of natural T-regs to a native auto-Ag in a mouse with a full T cell repertoire requires a cognate interaction (3).? ? Dendritic cells (DC) are important Ag presenting cells (APC) in induction of immune and autoimmune responses. We developed a new model EAU model where disease is induced with in vitro-matured, Ag pulsed DC (which can be obtained in large numbers from mice injected hydrodynamically with Flt-3L). When matured (by culture with LPS and anti-CD40), pulsed with a uveitogenic IRBP peptide and injected into naive syngeneic mice, these DC elicit ocular inflammation. Interestingly, DC-induced EAU differs from """"""""traditional"""""""" EAU induced by immunization in terms of clinical manifestations, the type of effector response elicited and the nature of the inflammatory infiltrate in the eye (granulocytic vs. monocytic). This shows (i) that distinct forms of EAU follow exposure to the same Ag presented to the immune system in a different context, (ii) provide an alternative EAU model representing types of uveitis not well represented by the traditional EAU model, and (iii) help explain the heterogeneity of clinical uveitis in the face of recognition directed predominantly at the same Ag (retinal arrestin) (manuscript submitted). ? ? In collaboration with the Oppenheim group at NCI we continue to study the ability of auto-Ags to act as chemoattractants, a finding which may be connected to pathogenicity. Previously we demonstrated that lymphocytes and immature dendritic cells exhibit chemotactic responses to the retinal Ags S-Ag and IRBP. The chemokine receptors CXCR5 and CXCR3 mediated the chemotactic effect of IRBP, while only CXCR3 was required for the S-Ag signal. We now found that auto-Ag bind to a different region of CXCR3 than does its cognate ligand CXCL11 (manuscript submitted). ? ? We are dissecting the respective roles in EAU of IL-12 and IL-23, related heterodimeric cytokines which share the p40 chain, using mice deficient in the non-shared receptors (p35 for IL-12 and p19 for IL-23), and the respective neutralizing monoclonal antibodies. Data indicate that IL-23, not IL-12, is an obligatory cytokine for induction of EAU. This may in part be due to its ability to promote the IL-17 response, which has recently been identified as an important proinflammatory cytokine in autoimmune and inflammatory disease. Unlike the IL-12-driven IFN-?, the IL-23 driven IL-17 is an effector cytokine in EAU and its neutralization during the efferent phase of disease is therapeutic. However, the IL-17 effector T cell can be dispensable, as severe EAU follows infusion of polarized Th1 IRBP specific T cells that produce only IFN-?, whose phenotype is stable. In contrast the requirement for IL-23 is a nonredunant and may therefore transcend the known property of IL-23 to promote the IL-17 response (manuscript submitted).? ? Ocular immune privilege, composed in part of inhibition of inflammatory processes in the eye, plays an important role in uveitis. We have previously shown that retinal glial Muller cells inhibit proliferation of T cells in an Ag-nonspecific fashion by a contact dependent mechanism. Using primary cultures of murine Muller cells, we have examined a large number of candidate molecules that might be involved in this phenomenon. Preliminary data identify thrombospondin-1, a molecule with many functions including activation of TGF-?, as partly responsible part of the suppressive effect of Muller cells. TGF-? may constitute a 2nd pathway, in part independent of TSP-1. T cells that have interacted with Muller cells become regulatory and are able to themselves inhibit activation and proliferation of fresh T cells. ? ? Within the scope of our studies on genetic susceptibility to uveitis, we have defined a number of genes that may be involved in susceptibility, using combined approaches of (i) classical genetics aided by SSLP analysis in an F2 cross of susceptible and resistant rat strains, (ii) microarray analysis of genes expressed by these strains validated by real-time PCR and gene knockout mouse strains, (iii) and in silico analysis of inbred mouse strains of varying EAU susceptibility using a SNP database. In addition, using closely spaced SSLP markers we have identified several new genetic regions associated with susceptibility, that are shared with regions reported for other autoimmune and non-autoimmune diseases, among them experimental autoimmune encephalomyelitis, arthritis and type 2 diabetes. This indicates that uveitis uses similar pathways as other autoimmune and inflammatory diseases, which may suggest use of common therapeutic approaches (manuscript submitted).
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