Activation of OX40 Prolongs and Exacerbates Autoimmune Experimental Uveitis

Department of Pediatrics, Oregon Health & Science University, Portland, OR 97239, USA.
Investigative ophthalmology & visual science (Impact Factor: 3.4). 09/2011; 52(11):8520-6. DOI: 10.1167/iovs.11-7664
Source: PubMed


T cells are essential for the development of autoimmune uveitis. Although the costimulatory molecule OX40 promotes T-cell function and expansion, it is unclear whether OX40 is implicated in ocular inflammation. The purpose of this study was to examine the role of OX40 in uveitis.
Experimental autoimmune uveitis (EAU) was induced in B10.RIII mice by subcutaneous injection of interphotoreceptor retinoid-binding protein peptide 161-180 (IRBP(161-180)). Some mice received an intravenous administration of OX40-activating antibody on days 0 and 4 after IRBP(161-180) sensitization or on days 10 and 14 of uveitis onset. The severity of EAU was evaluated by histology at different time points. In addition, ocular inflammatory cytokine expression was determined by real time-PCR, and peripheral activated CD4(+)CD44(+)CD62L(-) T cells and IL-7Rα expression were analyzed by flow cytometry. The activated CD4(+)CD44(+) lymphocytes were rechallenged with IRBP(161-180) in vitro to assess their antigen recall response.
The authors demonstrated a marked OX40 expression by infiltrating lymphocytes in enucleated human eyes with end-stage inflammation. In addition, the administration of OX40-activating antibody prolonged and exacerbated the disease course of EAU. Moreover, activation of OX40 not only increased CD4(+)CD44(+)CD62L(-) lymphocyte number, it upregulated IL-7Rα expression in the activated T-cell population. Lastly, these cells exhibited a stronger interferon-γ response to IRBP(161-180) restimulation in vitro.
The results reveal a pathogenic role of OX40 in uveitis. Furthermore, the upregulation of IL-7R in CD4(+)CD44(+) lymphocytes suggests that the activation of OX40 promotes the generation or expansion of uveitogenic memory T cells.

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    • "To validate the effect of roscovitine on a self-antigen-mediated posterior uveitis, we used IRBP-induced EAU model. Compared to the control group, as we have previously reported [30], further activation of uveitogenic T cells by anti-OX40 activating antibody exacerbated ocular inflammation as evidenced by more severe EAU score (Figure 7B). Some OX40 activating antibody-treated B10.RIII mice also intraperitoneally received 10 μg roscovitine every other day from days 0 to 21 after IRBP immunization. "
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    ABSTRACT: T cells are essential for the development of uveitis and other autoimmune diseases. After initial activation, CD4+ lymphocytes express the co-stimulatory molecule OX40 that plays an important role in T cell proliferation. Cyclin dependent kinase 2 (CdK2) plays a pivotal role in the cell cycle transition from G1 to S phase. In addition, recent research has implicated CdK2 in T cell activation. Thus, we sought to test the immunosuppressive effect of roscovitine, a potent CdK2 inhibitor, on CD4+ T cell activation, proliferation, and function. Mouse CD4+ T cells were activated by anti-CD3 and anti-CD28 antibodies. The expression of OX40, CD44, and CdK2 were analyzed by flow cytometry. In addition, cell cycle progression and apoptosis of control and roscovitine-treated T lymphocytes were measured by BrdU incorporation and annexin V assay, respectively. Furthermore, the immunoregulatory effect of roscovitine was evaluated in both ovalbumin-induced uveitis and experimental autoimmune uveitis (EAU) models. In this study, we found that T cell activation induced OX40 expression. Cell cycle analysis showed that more CD4+OX40+ cells entered S phase than OX40- T cells. Concurrently, CD4+OX40+ cells had a higher level of CdK2 expression. Roscovitine treatment blocked activated CD4+ cells from entering S phase. In addition, roscovitine not only reduced the viability of CD4+ lymphocytes but also suppressed T cell activation and cytokine production. Finally, roscovitine significantly attenuated the severity of T cell-dependent, OX40-enhanced uveitis. These results implicate CdK2 in OX40-augmented T cell response and expansion. Furthermore, this study suggests that roscovitine is a novel, promising, therapeutic agent for treating T cell-mediated diseases such as uveitis.
    Full-text · Article · Dec 2013 · PLoS ONE
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    • "Moreover, flow cytometry showed that Th17 cells expressed OX40 ligand, and interleukin-17 neutralizing antibody was successful in the attenuation of OX40-mediated uveitis [22]. In another study of experimental autoimmune uveitis, OX40 activation was found to promote the expansion uveitogenic memory T cells, as evidenced by the upregulation of IL-7R in CD4+CD44+ lymphocytes [23]. "
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    ABSTRACT: This study aims to investigate the role of OX40 ligand (OX40L) in ocular inflammation via abrogation of retinal pigment epithelium (RPE)-mediated immunosuppression using an in vitro expression approach. OX40L cDNA was polymerase chain reaction-amplified and cloned into an eYFP fusion vector. Cultured retinal pigment epithelial cells (ARPE-19) were transfected with the vector. Total RNA from unstimulated or inflammatory cytokine-stimulated ARPE cells were isolated and analyzed for OX40L expression by reverse transcription-polymerase chain reaction. Peripheral blood mononuclear cells (PBMCs) were isolated from healthy human donors. Human ARPE cells (±OX40L ± GITR ligand (GITRL) expression) and PBMCs were co-cultured for in vitro proliferation studies. Polymerase chain reaction confirmed the insertion of the OX40L gene into the fusion vector. Flow cytometry and fluorescence microscopy further confirmed surface expression of OX40L on ARPE cells after transfection. OX40L expression was induced in the RPE cells stimulated with pro-inflammatory cytokines. In the co-culture studies, there was a significant reversal (20% to 30%) of the RPE-induced suppression of activated PBMCs when the ARPE cells were transfected with OX40L. When both OX40L and GITRL were concomitantly transfected into ARPE cells, there was an additive reversal of RPE-mediated T cell suppression, when compared to the reversal caused by RPE cells expressing either OX40L alone or GITRL alone. Using an in vitro approach, we found that OX40L causes an abrogation of the RPE-mediated immunosuppression. OX40L appears to be regulated in the ARPE-19 cell line and may play an important role in the pathogenesis of various ocular inflammatory conditions.
    Full-text · Article · Jan 2013 · Journal of Ophthalmic Inflammation and Infection
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    • "Previously, we reported that EAU subsided by day 21 [24]. Here, we found that low dose rapamycin prolonged the course of uveitis. "
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    ABSTRACT: Rapamycin, a potent immune modulator, is used to treat transplant rejection and some autoimmune diseases. Uveitis is a potentially severe inflammatory eye disease, and 2 clinical trials of treating uveitis with rapamycin are under way. Unexpectedly, recent research has demonstrated that low dose rapamycin enhances the memory T cell population and function. However, it is unclear how low dose rapamycin influences the immune response in the setting of uveitis. B10.RIII mice were immunized to induce experimental autoimmune uveitis (EAU). Ocular inflammation of control and rapamycin-treated mice was compared based on histological change. ELISPOT and T cell proliferation assays were performed to assess splenocyte response to ocular antigen. In addition, we examined the effect of rapamycin on activation-induced cell death (AICD) using the MitoCapture assay and Annexin V staining. Administration of low dose rapamycin exacerbated EAU, whereas treating mice with high dose rapamycin attenuated ocular inflammation. The progression of EAU by low dose rapamycin coincided with the increased frequency of antigen-reactive lymphocytes. Lastly, fewer rapamycin-treated T cells underwent AICD, which might contribute to exaggerated ocular inflammation and the uveitogenic immune response. These data reveal a paradoxical role for rapamycin in uveitis in a dose-dependent manner. This study has a potentially important clinical implication as rapamycin might cause unwanted consequences dependent on dosing and pharmacokinetics. Thus, more research is needed to further define the mechanism by which low dose rapamycin augments the immune response.
    Full-text · Article · May 2012 · PLoS ONE
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