Abrogation of anti-retinal autoimmunity in IL-10 transgenic mice due to reduced T cell priming and inhibition of disease effector mechanisms

Experimental autoimmune uveitis (EAU) induced by immunization of animals with retinal Ags is a model for human uveitis. The immunosuppressive cytokine IL-10 regulates EAU susceptibility and may be a factor in genetic resistance to EAU. To further elucidate the regulatory role of endogenous IL-10 in the mouse model of EAU, we examined transgenic (Tg) mice expressing IL-10 either in activated T cells (inducible) or in macrophages (constitutive). These IL-10-Tg mice and non-Tg wild-type controls were immunized with a uveitogenic regimen of the retinal Ag interphotoreceptor retinoid-binding protein. Constitutive expression of IL-10 in macrophages abrogated disease and reduced Ag-specific immunological responses. These mice had detectable levels of IL-10 in sera and in ocular extracts. In contrast, expression of IL-10 in activated T cells only partially protected from EAU and marginally reduced Ag-specific responses. All IL-10-Tg lines showed suppression of Ag-specific effector cytokines. APC from Tg mice constitutively expressing IL-10 in macrophages exhibited decreased ability to prime naive T cells, however, Ag presentation to already primed T cells was not compromised. Importantly, IL-10-Tg mice that received interphotoreceptor retinoid-binding protein-specific uveitogenic T cells from wild-type donors were protected from EAU. We suggest that constitutively produced endogenous IL-10 ameliorates the development of EAU by suppressing de novo priming of Ag-specific T cells and inhibiting the recruitment and/or function of inflammatory leukocytes, rather than by inhibiting local Ag presentation within the eye.     

Pregnancy ameliorates induction and expression of experimental autoimmune uveitis

Female patients suffering from autoimmune uveitis are reported to experience a temporary remission during pregnancy. Experimental autoimmune uveitis (EAU) is a model for human uveitis. Here we examine the effect of pregnancy on the development of EAU and its associated immunological responses. Susceptible C57BL/6 mice were immunized with interphotoreceptor retinoid-binding protein (IRBP). EAU scores and Ag-specific responses were evaluated 21 days later. Mice immunized during pregnancy developed significantly less EAU than nonpregnant controls. Their lymph node cells and splenocytes produced a distinct pattern of cytokines in response to IRBP: reduced IFN-gamma and IL-12 p40, but unchanged levels of TNF-alpha, IL-4, IL-5, and IL-10. Anti-IRBP Ab isotypes revealed an up-regulation of IgG1, indicating a possible Th2 bias at the humoral level. Ag-specific proliferation and delayed hypersensitivity, as well as mitogen-induced IFN-gamma production, remained undiminished, arguing against an overall immune deficit. Interestingly, pregnant mice that received an infusion of IRBP-primed lymphoid cells from nonpregnant donors also developed reduced EAU, suggesting that pregnancy suppresses not only the generation, but also the function of mature uveitogenic effector T cells. Pregnant mice at the time of immunization exhibited elevated levels of TGF-beta, but not of IL-10, in the serum. We suggest that protection from EAU during pregnancy is due primarily to a selective reduction of Ag-specific Th1 responses with only marginal enhancement of Th2 function, and that these effects may in part be secondary to elevated systemic levels of TGF-beta.     

Activation of invariant NKT cells ameliorates experimental ocular autoimmunity by a mechanism involving innate IFN-gamma production and dampening of the adaptive Th1 and Th17 responses

Invariant NKT cells (iNKT cells) have been reported to play a role not only in innate immunity but also to regulate several models of autoimmunity. Furthermore, iNKT cells are necessary for the generation of the prototypic eye-related immune regulatory phenomenon, anterior chamber associated immune deviation (ACAID). In this study, we explore the role of iNKT cells in regulation of autoimmunity to retina, using a model of experimental autoimmune uveitis (EAU) in mice immunized with a uveitogenic regimen of the retinal Ag, interphotoreceptor retinoid-binding protein. Natural strain-specific variation in iNKT number or induced genetic deficiencies in iNKT did not alter baseline susceptibility to EAU. However, iNKT function seemed to correlate with susceptibility and its pharmacological enhancement in vivo by treatment with iNKT TCR ligands at the time of uveitogenic immunization reproducibly ameliorated disease scores. Use of different iNKT TCR ligands revealed dependence on the elicited cytokine profile. Surprisingly, superior protection against EAU was achieved with alpha-C-GalCer, which induces a strong IFN-gamma but only a weak IL-4 production by iNKT cells, in contrast to the ligands alpha-GalCer (both IFN-gamma and IL-4) and OCH (primarily IL-4). The protective effect of alpha-C-GalCer was associated with a reduction of adaptive Ag-specific IFN-gamma and IL-17 production and was negated by systemic neutralization of IFN-gamma. These data suggest that pharmacological activation of iNKT cells protects from EAU at least in part by a mechanism involving innate production of IFN-gamma and a consequent dampening of the Th1 as well as the Th17 effector responses.     

Loss of STAT3 in CD4+ T cells prevents development of experimental autoimmune diseases

Th17 cells are implicated in CNS autoimmune diseases. We show that mice with targeted-deletion of Stat3 in CD4(+) T cells (CD4(Stat3)(-/-)) do not develop experimental autoimmune uveoretinitis (EAU) or experimental autoimmune encephalomyelitis. Defective Th17 differentiation noted in CD4(Stat3)(-/-) mice is compensated by exaggerated increases in Foxp3-, IL-10-, IL-4-, and IFN-gamma-expressing T cells, suggesting critical roles of STAT3 in shaping Ag-specific CD4(+) T cell repertoire. In mice with EAU, a high percentage of IL-17-expressing T cells in their peripheral lymphoid organs also secrete IFN-gamma while these double-expressors are absent in CD4(Stat3)(-/-) and wild-type mice without EAU, raising the intriguing possibility that uveitis maybe mediated by Th17 and IL-17-expressing Th1 cells. Resistance of Stat3-deficient mice to EAU derives in part from an inability of uveitogenic Th17 and Th1 cells to enter eyes or brain of the CD4(Stat3)(-/-) mouse because of the reduction in the expression of activated alpha4/beta1 integrins on CD4(Stat3)(-/-) T cells. Adoptive transfer of activated interphotoreceptor retinoid-binding protein-specific uveitogenic T cells induced in CD4(Stat3)(-/-) mice a severe EAU characterized by development of retinal folds, infiltration of inflammatory cells into the retina, and destruction of retinal architecture, underscoring our contention that the loss of STAT3 in CD4(+) T cells results in an intrinsic developmental defect that renders CD4(Stat3)(-/-) resistant to CNS inflammatory diseases. STAT3 requirement for IL-17 production by Th17, generation of double positive T cells expressing IL-17 and IFN-gamma, and for T cell trafficking into CNS tissues suggests that STAT3 may be a therapeutic target for modulating uveitis, sceritis, or multiple sclerosis.     

Suppression of experimental autoimmune uveitis in mice by induction of anterior chamber-associated immune deviation with interphotoreceptor retinoid-binding protein.

Immunization with bovine interphotoreceptor retinoid-binding protein induces autoimmune uveitis in B10.A mice. We have examined whether this soluble retina-specific Ag can induce anterior chamber-associated immune deviation when injected into the anterior chamber (AC) of the eye, and whether this deviant immune response has any effect on uveitis is susceptible mice. The results of these experiments indicate that interphotoreceptor retinoid-binding protein (IRBP) injected intracamerally altered the subsequent immune response of B10.A mice such that a) they were not able to develop IRBP-specific delayed hypersensitivity, nor (b) were they able to express significant autoimmune uveitis following a uveitogenic regimen. Moreover, spleen cells from mice that received IRBP in the AC suppressed uveitis when adoptively transferred into naive recipients. The splenic suppressor cells were able to prevent autoimmune uveitis in recipient mice when administered after the uveitogenic regimen. Most important, IRBP-specific splenic cells from mice treated with IRBP in the AC when injected into mice with established uveitis caused an abrupt cessation of the intraocular inflammation. The ability of intracamerally-injected soluble Ag to induce suppressor T cells that act on the efferent limb of the immune response suggests that the anterior-chamber-associated immune deviation phenomenon may have physiologic relevance in terms of preservation of the integrity of ocular tissue and renders this approach particularly suitable for treating already established experimental autoimmune diseases of this type. These results are discussed in terms of other methods that have been devised experimentally to suppress and prevent autoimmune uveitis and encephalomyelitis.     

Hydrodynamic vaccination with DNA encoding an immunologically privileged retinal antigen protects from autoimmunity through induction of regulatory T cells

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 microg 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-beta. In contrast, depletion of CD25(+) cells after vaccination and before challenge markedly abrogated protection. IRBP-specific CD4(+)CD25(high) 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.     

Pertussis toxin enhances Th1 responses by stimulation of dendritic cells

Pertussis toxin (PTX) has been widely used as an adjuvant to induce Th1-mediated organ-specific autoimmune diseases in animal models. However, the cellular and molecular mechanisms remain to be defined. In this study, we showed that dendritic cells (DC) stimulated with PTX (PTX-DC) were able to substitute for PTX to promote experimental autoimmune uveitis (EAU). EAU induced by PTX-DC revealed a typical Th1 response, characterized by high uveitogenic retinal Ag interphotoreceptor retinoid-binding protein (IRBP)-specific IFN-gamma and IL-12 production in the draining lymph nodes, as well as increased levels of anti-IRBP IgG2a and decreased levels of anti-IRBP IgG1 in the serum of IRBP-immunized mice. Furthermore, PTX-DC preferentially induced T cells to produce the Th1 cytokine, IFN-gamma. After being stimulated with PTX, DC exhibited up-regulation of MHC class II, CD80, CD86, CD40, and DEC205. PTX-DC had also increased allostimulatory capacity and IL-12 and TNF-alpha production. Serum IL-12 was increased in naive mice that received PTX-DC i.p. In addition, PTX activated extracellular signal-regulated kinase in DC. Following the inhibition of extracellular signal-regulated kinase signaling, the maturation of PTX-DC was reduced. Subsequently, the ability of PTX-DC to promote IFN-gamma production by T cells in vitro and to induce EAU in vivo was blocked. The results suggest that PTX might exert an adjuvant effect on DC to promote their maturation and the production of proinflammatory cytokines, thereby eliciting a Th1 response.     

IL-22-induced regulatory CD11b+ APCs suppress experimental autoimmune uveitis

We have previously reported that IL-17(+) interphotoreceptor retinoid-binding protein (IRBP) 161-180-specific T cells have a strong pathogenic effect in experimental autoimmune uveitis (EAU) induced in B10RIII mice; however, this pathogenic activity is not solely attributable to the major cytokine, IL-17, produced by these cells. To determine whether other cytokines produced by Th17 cells show a stronger association with their pathogenic activity, we studied the role of IL-22 in EAU. IL-22 is one of the major cytokines produced by these cells. Our results showed that administration of small doses of IL-22 to EAU-susceptible mice significantly reduced the severity of EAU. In addition, mice treated with IL-22 generated decreased numbers of IFN-γ(+) and IL-17(+) uveitogenic T cells, but increased numbers of Foxp3(+) regulatory T cells. Mechanistic studies showed that the effect of the injected IL-22 was on CD11b(+) APCs, which expressed increased levels of IL-22R during induction of disease following immunization with uveitogenic Ag. In vitro IL-22 treatment of CD11b(+) APCs collected from Ag-primed mice resulted in increased expression of programmed death ligand-1 and the production of increased amounts of IL-10 and TGF-β. Moreover, IL-22-treated CD11b(+) APCs caused IRBP161-180-specific T cells to lose their uveitogenic activity and acquire immunosuppressive activity, which suppressed the induction of EAU by additional pathogenic IRBP161-180-specific effector T cells.     

Essential role of the MyD88 pathway, but nonessential roles of TLRs 2, 4, and 9, in the adjuvant effect promoting Th1-mediated autoimmunity

Induction of tissue-specific experimental autoimmune diseases involves an obligatory adjuvant effect to trigger an innate response of a type that will drive a Th1-biased adaptive response. This is achieved by use of CFA containing mycobacteria (Mycobacterium tuberculosis), whose recognition by cells of the innate immune system depends on TLRs that signal through the adaptor molecule MyD88. We examined the role of selected components of the MyD88 pathway in promoting experimental autoimmune uveitis (EAU). Mice deficient in MyD88, TLR2, TLR4, or TLR9 were immunized with the retinal Ag interphotoreceptor retinoid-binding protein in CFA, and their EAU scores and associated immunological responses were examined. MyD88-/- mice were completely resistant to EAU and had a profound defect in Th1, but not Th2, responses to autoantigen challenge. Surprisingly, TLR2-/-, TLR4-/-, and TLR9-/- mice were fully susceptible to EAU and had unaltered adaptive responses to interphotoreceptor retinoid-binding protein. Examination of IL-1R family members, which share the common adaptor MyD88 with the TLR family, revealed that IL-1R-deficient mice, but not IL-18-deficient mice, are resistant to EAU and have profoundly reduced Th1 and Th2 responses. These data are compatible with the interpretation that TLR9, TLR4, and TLR2 signaling is either not needed, or, more likely, redundant in the adjuvant effect needed to induce EAU. In contrast, signaling through the IL-1R plays a necessary and nonredundant role in EAU and can by itself account for the lack of EAU development in MyD88 mice.     

Blockade of costimulation through B7/CD28 inhibits experimental autoimmune uveoretinitis, but does not induce long-term tolerance

It has been reported that costimulation blockade can result in T cell anergy. We investigated the effects of blocking costimulatory molecules in vivo on the development of experimental autoimmune uveoretinitis (EAU), a model for autoimmune uveitis in humans that is induced in mice by immunization with the retinal Ag interphotoreceptor retinoid binding protein. B10.A mice immunized with a uveitogenic regimen of interphotoreceptor retinoid-binding protein were treated with Abs to B7.1 and B7.2 for 2 wk. Evaluation of EAU and immunological responses 1 wk later showed that disease had been abrogated, and cellular responses were suppressed. To determine whether the costimulation blockade resulted in tolerance, adult-thymectomized mice immunized for uveitis and treated with anti-B7 or anti-CD28 were rechallenged for uveitis induction 5 wk after the initial immunization. Although confirmed to be disease free after the initial immunization, both anti-B7- and anti-CD28-treated mice developed severe EAU and elevated cellular responses after the rechallenge, equivalent to those of control mice. We conclude that in this model costimulatory blockade in vivo prevents the development of autoimmune disease, but does not result in long-term tolerance. The data are compatible with the interpretation that B7/CD28 blockade prevents generation of effector, but not of memory, T cells.     

Retinal self-antigen induces a predominantly Th1 effector response in Axl and Mertk double-knockout mice

The TAM family of receptors (Tyro3, Axl, and Mertk) plays an important role in the negative regulation of response of dendritic cells (DCs) and macrophages to pathogenic stimuli, and mice lacking this receptor family develop spontaneous lupus-like systemic autoimmunity against a variety of tissues, including retina. To study the molecular mechanism underlying the TAM regulation of APC functions and subsequent effects on the induction of an autoimmune response against the eye, we examined CD4 T cell differentiation following retinal self-antigen immunization. CD4 T cells prepared from naive or interphotoreceptor retinoid-binding protein (IRBP)1-20-immunized Axl and Mertk double-knockout (dko) mice reacted to activation using anti-CD3 and anti-CD28 Abs or to bolster by self-antigen in vitro with a predominantly Th1 effector response, as characterized by increased IFN-γ production and higher frequency of IFN-γ-positive CD4 T cells. The Th17 effector response to IRBP immunization was similar in dko mice to that in wild-type controls, as shown by ELISA measurement of IL-17A in the culture medium and flow cytometric analysis of IL-17A-secreting CD4 T cells. Interestingly, APCs or DCs isolated from IRBP-immunized dko mice exhibited a greater ability to drive the Th1 response. The production of two driving cytokines for Th1 differentiation, IL-12 and IL-18, was dramatically increased in dko DCs and macrophages, and LPS stimulation bolstered their production. The preferential development into the Th1 subset in dko mice suggests that the cytokine milieu produced by the mutant mice in vivo or by mutant APCs in vitro selectively creates a differentiation environment favoring the Th1 effector response.     

Role of CD25+ dendritic cells in the generation of Th17 autoreactive T cells in autoimmune experimental uveitis

In the current study, we showed that in vivo administration of an anti-CD25 Ab (PC61) decreased the Th17 response in C57BL/6 mice immunized with the uveitogenic peptide interphotoreceptor retinoid-binding protein, while enhancing the autoreactive Th1 response. The depressed Th17 response was closely associated with decreased numbers of a splenic dendritic cell (DC) subset expressing CD11c(+)CD3(-)CD25(+) and decreased expansion of γδ T cells. We demonstrated that ablation of the CD25(+) DC subset accounted for the decreased activation and the expansion of γδ T cells, leading to decreased activation of IL-17(+) interphotoreceptor retinoid-binding protein-specific T cells. Our results show that an enhanced Th17 response in an autoimmune disease is associated with the appearance of a DC subset expressing CD25 and that treatment of mice with anti-CD25 Ab causes functional alterations in a number of immune cell types, namely DCs and γδ T cells, in addition to CD25(+)αβTCR(+) regulatory T cells.     

In vitro activation of CD8 interphotoreceptor retinoid-binding protein-specific T cells requires not only antigenic stimulation but also exogenous growth factors

In a previous study, we demonstrated that immunization with the uveitogenic peptide interphotoreceptor retinoid-binding protein (IRBP) 1-20 induces both CD4 and CD8 uveitogenic T cells in the B6 mouse. In the current study, we determined the role of the CD8 IRBP-specific T cells in the pathogenesis of experimental autoimmune uveitis. We also determined the conditions that facilitated the activation of CD8 autoreactive T cells. Our results showed that the beta2-microglobulin(-/-) mouse had a greatly decreased susceptibility to induction of experimental autoimmune uveitis by adoptive transfer of IRBP-specific T cells from B6 mice. We also showed that unlike CD4 autoreactive T cells, activated CD8 autoreactive T cells produced only a limited number and amounts of growth factors. As a result, in the absence of exogenously supplied growth factor(s), CD8 T cell activation and expansion were aborted. However, the growth and expansion of triggered CD8 autoreactive T cells could be supported by various cytokines. In addition to factors produced by activated CD4 autoreactive T cells, factors produced by nonlymphoid cells, such as IL-7 and IL-15, and unidentified factors in the culture supernatants of astrocytes and retinal pigment epithelial cells support the CD8 autoreactive T cells as well. Finally, we showed that, although several cytokines augmented the CD8 T cell response in vitro, different cytokines appeared to act on different CD8 subsets or on different activation/differentiation phases of CD8 autoreactive T cells. As a result, cytokines, such as IL-7, supported the proliferation and survival of CD8 IRBP-specific T cells, while others had only a growth-promoting effect.     

Exogenous nitric oxide inhibits experimental autoimmune uveoretinitis development in Lewis rats by modulation of the Th1-dependent immune response.

Experimental autoimmune uveoretinitis (EAU) is a T cell-mediated autoimmune disease of posterior uvea that closely resembles a human disease. The uveitogenic effector T cell has a Th1-like phenotype [high interferon-gamma (IFN-gamma), low interleukin-4 (IL-4)], and genetic susceptibility to EAU that is associated with an elevated Th1 response. Suppression of CD4+ Th1 cells for the treatment of autoimmune disease is an attractive potential therapeutic approach. Nitric oxide (NO) has been implicated in the pathogenesis of several inflammatory and autoimmune diseases. In this study, we investigated the potential role of NO as an immunoregulator to alter Th1/Th2 cytokine production, as well as to inhibit the interphotoreceptor retinoid binding protein (IRBP)-induced EAU, a CD4+ Th1 cell-mediated autoimmune disease. Injection of IRBP (100 microg) into two footpads resulted in severe EAU. The beginning peak of the disease was days 12 to 15 after immunization. Oral treatment with molsidomine (MSDM), a NO donor, began 24 h before IRBP immunization to the end of the experiments, which resulted in a significant inhibition of the disease by clinical and histopathological criteria. When MSDM was administered until day 21, a complete reduction of incidence and severity of EAU was observed. To investigate the cytokine alterations from Th1 to Th2 cytokines by MSDM, the cytokines were assayed in a culture medium of IRBP-stimulated inguinal lymphocytes. IRBP-immunized rats secreted a high concentration of IFN-gamma and a low concentration of IL-10. In contrast, MSDM treatment enhanced IL-10 secretion and tended to decrease IFN-gamma secretion. In conclusion, we show that the administration of NO suppresses EAU by altering the Th1/Th2 balance of inflammatory immune responses. We suggest that NO may be useful in the therapeutic control of autoimmune uveitis.     

Novel function of IFN-gamma: negative regulation of dendritic cell migration and T cell priming

IFN-gamma is considered to be a Th1 cytokine with immunomodulatory effects on a variety of immune cells. In this study, we determined whether dendritic cell (DC) function was aberrant in IFN-gamma knockout (GKO) mice. The results demonstrated that IFN-gamma deficiency did not interfere with bone marrow-derived DC development and maturation in vitro. However, functional analysis showed that bone marrow-derived DC from GKO mice had altered cytokine secretion, allostimulatory and Ag presentation capacity, chemokine receptor expression, and in vitro chemotaxis. LPS induced the recruitment of DC from different organs into the spleen; epicutaneously sensitized DC with hapten (FITC) accumulated in the draining lymph nodes and CD11c(+) DC levels in the draining lymph nodes from autoantigen (interphotoreceptor retinoid-binding protein) immunized mice were enhanced in GKO mice as compared with wild-type mice. After treatment of GKO mice with i.p. IFN-gamma injection restored IFN-gamma levels in vivo, DC migration decreased in response to LPS or FITC. IFN-gamma altered the adaptive immune responses in vivo, since T cell priming and IL-2 production were increased in interphotoreceptor retinoid-binding protein-immunized GKO mice. Furthermore, in IFN-gamma-treated GKO mice, experimental autoimmune uveitis score enhancement and T cell activation were eliminated. Taken together, IFN-gamma appears to play a negative regulatory role on in vivo DC function, resulting in suppression of Ag-specific T cell priming.     

IL-4 and IL-10 are both required for the induction of oral tolerance

Protection from the development of experimental autoimmune uveitis (EAU) can be induced by feeding mice interphotoreceptor retinoid binding protein before uveitogenic challenge with the same protein. Two different regimens are equally effective in inducing protective tolerance, although they seem to do so through different mechanisms: one involving regulatory cytokines (IL-4, IL-10, and TGF-beta), and the other with minimal involvement of cytokines. Here we studied the importance of IL-4 and IL-10 for the development of oral tolerance using mice genetically engineered to lack either one or both of these cytokines. In these animals we were able to protect against EAU only through the regimen inducing cytokine-independent tolerance. When these animals were fed a regimen that in the wild-type animal is thought to predominantly induce regulatory cells and is associated with cytokine secretion, they were not protected from EAU. Interestingly, both regimens were associated with reduced IL-2 production and proliferation in response to interphotoreceptor retinoid binding protein. These findings indicate that both IL-4 and IL-10 are required for induction of protective oral tolerance dependent on regulatory cytokines, and that one cytokine cannot substitute for the other in this process. These data also underscore the fact that oral tolerance, manifested as suppression of proliferation and IL-2 production, is not synonymous with protection from disease.     

Characterization of rat CD8+ uveitogenic T cells specific for interphotoreceptor retinal-binding protein 1177-1191

The uveitogenic T cells that mediate experimental autoimmune uveitis are commonly assumed to be exclusively CD4(+). In the present study, we showed that, although a panel of long-term cultured rat uveitogenic T cell lines specific for the interphotoreceptor retinal-binding protein peptide, R16, all expressed CD4, approximately 40% of the R16-specific uveitogenic T cells freshly prepared from Ag-immunized rats were CD8(+)alphabetaTCR(+), as demonstrated by CFSE staining. We showed that the expansion of these CD8(+)alphabetaTCR(+) T cells was Ag-specific and that highly purified CD8(+) R16-specific T cells were able to induce uveitis on transfusion into naive rats. Moreover, CD8(+) uveitogenic T cells more readily switched phenotype from, and to, TCR(-)CD8(-)CD4(-) during in vivo or in vitro activation compared with their CD4(+) counterparts. In a previous study, we showed that highly purified CD8(+) myelin oligodendrocyte glycoprotein-specific T cells induced more severe autoimmune encephalomyelitis than the corresponding CD4(+) T cells. In this study, we show that an interphotoreceptor retinal-binding protein peptide consistently activated a high proportion of CD8(+)alphabetaTCR(+) T cells, which were uveitogenic in Lewis rats.     

Disruption of CD40/CD40-ligand interactions in a retinal autoimmunity model results in protection without tolerance

We examined the role of CD40/CD40L interactions on the development of experimental autoimmune uveoretinitis (EAU), a cell-mediated, Th1-driven autoimmune disease that serves as a model for autoimmune uveitis in humans. EAU-susceptible B10.RIII mice immunized with the retinal autoantigen interphotoreceptor retinoid binding protein in CFA and treated with anti-CD40L Ab (MR1) had reduced incidence and severity of disease. Real-time PCR analysis revealed that the innate and adaptive responses of protected mice were reduced, without an obvious shift toward a Th2 cytokine profile. In contrast to some other reports, no evidence was found for regulatory cells in adoptive transfer experiments. To determine whether CD40L blockade resulted in long-term tolerance, mice protected by treatment with MR1 Ab were rechallenged for uveitis after circulating MR1 Ab levels dropped below the detection limit of ELISA. MR1-treated mice developed severe EAU and strong cellular responses to interphotoreceptor retinoid binding protein, comparable to those of control mice. These responses were higher than in mice that had not received the primary immunization concurrently with anti-CD40L treatment. We conclude that 1) CD40/CD40L interaction is required for EAU and its disruption prevents disease development; 2) CD40L blockade inhibits the innate response to immunization and reduces priming, but does not result in immune deviation; and 3) protection is dependent on persistence of anti-CD40L Abs, and long-term tolerance is not induced. Furthermore, immunological memory develops under cover of CD40L blockade causing enhanced responses upon rechallenge. Taken together, our data suggest that ongoing CD40/CD40L blockade might be required to maintain a therapeutic effect against uveitis.     

A new model of autoimmune disease. Experimental autoimmune uveoretinitis induced in mice with two different retinal antigens

Experimental autoimmune uveoretinitis (EAU) is an organ-specific, T lymphocyte-mediated autoimmune disease, which serves as a model for several human ocular inflammations of an apparently autoimmune nature. EAU pathology in some rodents and in monkeys can readily be induced by immunization with several different retinal proteins; however, advancing research into the cellular mechanisms of this disease has raised the need for an EAU model in an immunologically and genetically well defined species. We report here the induction of EAU in the mouse, which has hitherto been considered a species refractory to EAU, with two retinal Ag, the retinal soluble Ag and the interphotoreceptor retinoid-binding protein. Although all the mouse strains tested exhibited lymphocyte responses and antibody titers to both retinal Ag, EAU was inducible in only some of the strains, and the uveitogenic responses to retinal soluble Ag and interphotoreceptor retinoid-binding protein appeared to be mutually exclusive. The EAU model in mice was found to differ in several respects from the EAU model in other rodent species. Induction of the disease was achieved with a relatively high dose of Ag and an intensified immunization protocol, and the onset of disease was later, the duration was longer, and the course was less acute. Anterior segment involvement was slight or nonexistent, and damage to the retina and uvea was of a focal rather than of a diffuse nature. Murine EAU appeared to approximate some types of human uveitis more closely than the EAU models described in other rodent species with respect to its pathologic manifestations as well as its more chronic course. The relatively longer duration of the active stage of disease in murine EAU should facilitate therapeutic intervention in established disease, which was not feasible in the more acute models of EAU. The extensive knowledge of the immunologic parameters of the mouse and the availability of genetically defined strains should be of great value in the study of cellular mechanisms and immunogenetics of ocular autoimmune disease.     

Galectin-1 suppresses autoimmune retinal disease by promoting concomitant Th2- and T regulatory-mediated anti-inflammatory responses

Intraocular inflammatory diseases are a common cause of severe visual impairment and blindness. In this study, we investigated the immunoregulatory role of galectin-1 (Gal-1), an endogenous lectin found at sites of T cell activation and immune privilege, in experimental autoimmune uveitis (EAU), a Th1-mediated model of retinal disease. Treatment with rGal-1 either early or late during the course of interphotoreceptor retinoid-binding protein-induced EAU was sufficient to suppress ocular pathology, inhibit leukocyte infiltration, and counteract pathogenic Th1 cells. Administration of rGal-1 at the early or late phases of EAU ameliorated disease by skewing the uveitogenic response toward nonpathogenic Th2 or T regulatory-mediated anti-inflammatory responses. Consistently, adoptive transfer of CD4(+) regulatory T cells obtained from rGal-1-treated mice prevented the development of active EAU in syngeneic recipients. In addition, increased levels of apoptosis were detected in lymph nodes from mice treated with rGal-1 during the efferent phase of the disease. Our results underscore the ability of Gal-1 to counteract Th1-mediated responses through different, but potentially overlapping anti-inflammatory mechanisms and suggest a possible therapeutic use of this protein for the treatment of human uveitic diseases of autoimmune etiology.