Human retinal pigment epithelium-induced CD4+CD25+ regulatory T cells suppress activation of intraocular effector T cells.

Murine retinal pigment epithelial (RPE) cells suppress T-cell activation by releasing soluble inhibitory factors and promote the generation of regulatory T cells in vitro. These T cells exposed to RPE supernatants (RPE-induced Treg cells) can suppress the activation of bystander effector T cells via the production of transforming growth factor-beta (TGFbeta). In the present study, we showed that human RPE-induced Treg cells are also able to acquire regulatory function when human RPE cell lines were pretreated with recombinant TGF beta 2. These RPE-induced Treg cells produced TGF beta 1 and IL-10 but not IFN gamma, and they significantly suppressed the activation of target cell lines and intraocular T-cell clones established from patients with active uveitis. Moreover, CD4(+)CD25(+) RPE-induced Treg cells expressed CTLA-4 and Foxp3 molecules, and the CD25(+) Treg cells profoundly suppressed the T-cell activation. Thus, in vitro manipulated Treg cells acquire functions that participate in the establishment of immune tolerance in the eye.

Bilateral anterior granulomatous keratouveitis with sunset glow fundus in a patient with autoimmune polyglandular syndrome.

AIM: To report the case of a patient with bilateral anterior granulomatous keratouveitis and sunset glow fundus. METHOD: Review of case record. RESULTS: A 15-year-old patient had bilateral anterior granulomatous keratouveitis and sunset glow fundus similar to findings in Vogt-Koyanagi-Harada disease (VKH). However, the patient also suffered additional autoimmune disease against endocrine glands. In addition to anti-thyroid antibody and anti-glutamic acid decarboxylase antibody, anti-melanocyte autoantibody was detected in serum from this patient. The authors finally diagnosed autoimmune polyglandular syndrome. CONCLUSION: If resistance against treatment exists for VKH, particularly in pediatric cases, this disease should be considered and other endocrine disorders examined.

Human iris pigment epithelium suppresses activation of bystander T cells via TGFbeta-TGFbeta receptor interaction.

Iris pigment epithelial (IPE) cells from the anterior segment in the eye are able to suppress activation of bystander responder T cells in vitro. The cultured IPE cells fully suppress proliferation and cytokine production by responder T cells via direct cell-to-cell contact. We have now investigated whether primary cultured human iris pigment epithelial (h-IPE) cells that were established from fresh iris tissues can also inhibit the activation of T cells in vitro. We found that cultured h-IPE cells significantly inhibited T cell proliferation and the IFN-gamma production by the target T cells from both the allogeneic and autogeneic peripheral blood mononuclear cells (PBMCs). The h-IPE cells also inhibited the activation of CD4(+) T cells from patients with active uveitis. The suppression by h-IPE occurred in a completely contact-dependent manner. The h-IPE constitutively expressed transforming growth factor beta (TGFbeta) and the receptors, and the T cells exposed to h-IPE greatly expressed Smad transcripts. In addition, TGFbeta2-siRNA transfected h-IPE failed to inhibit activation of responder T cells. Similarly, h-IPE cells in the presence of anti-TGFbeta neutralizing antibodies or recombinant TGFbeta receptor blocking proteins failed to inhibit the T-cell activation. In conclusion, cultured human iris pigment epithelium fully inhibits T cell activation in vitro. Our data support the hypothesis that the ocular resident cells play a critical role in immunosuppression in the eye.

T-cell suppression by programmed cell death 1 ligand 1 on retinal pigment epithelium during inflammatory conditions.

PURPOSE: To determine whether retinal pigment epithelial (RPE) cells can inhibit in vitro T-cell activation during inflammatory conditions. METHODS: Primary cultured RPE cells were established from normal C57BL/6 mice. Target bystander T cells were established from normal splenic T cells with anti-CD3 antibodies. T-cell activation was assessed for proliferation by both examining [(3)H]-thymidine incorporation and the production of interferon (IFN)gamma or IL-17, as determined by ELISA. Expression of programed cell death 1 ligand 1 (PD-L1) on RPE or recombinant mouse IFNgamma-pretreated RPE cells was evaluated using oligonucleotide microarray, RT-PCR, immune staining, and flow cytometry. Expression of programed cell death 1 (PD-1)(+) on target T cells was evaluated by flow cytometry. Anti-mouse PD-L1 or PD-L2 neutralizing antibodies or target T cells from PD-1 knockout donors were used for the assay. RESULTS: IFNgamma-pretreated RPE greatly suppressed activation of bystander T cells, especially the IFNgamma production by the target T cells (Th1 cells, but not Th17 cells) via direct cell contact. By examining cell surface candidate molecules, IFNgamma-pretreated RPE expressed much higher levels of PD-L1 compared with the control nontreated RPE. Although primary RPE did not express the costimulatory molecule, expression of the molecule was induced on the surface of IFNgamma-pretreated RPE. PD-L1(+) RPE in the presence of IFNgamma selectively suppressed PD-1(+) T-cell activation. IFNgamma-pretreated RPE in the presence of anti-PD-L1 neutralizing antibodies, but not anti-PD-L2, failed to suppress T-cell production of IFNgamma. In addition, these RPE cells failed to suppress the production of IFNgamma by CD4(+) T cells from PD-1 null donors. CONCLUSIONS: Suppression of T-cell activation was obtained in cultures only when RPE expressed negative costimulators. Therefore, the authors propose that in vitro, Th1 cytokine-exposed ocular resident cells can express this molecule and it is this expression that causes the suppression of the bystander Th1-type cells.

Human corneal endothelial cells expressing programmed death-ligand 1 (PD-L1) suppress PD-1+ T helper 1 cells by a contact-dependent mechanism.

PURPOSE: This study was designed to determine whether human corneal endothelial (HCE) cells could regulate the activation of bystander T cells in vitro. METHODS: HCE cell lines were established from primary HCE cells. Target-activated T cells were used allogeneic T cells and Jurkat T-cell lines. As an additional target, T-cell clones from uveitis patients were established from aqueous humor via a limiting dilution. T-cell activation was assessed for proliferation by [(3)H]-thymidine incorporation, carboxyfluorescein succinimidyl ester incorporation, or IFNgamma production. Expression of co-stimulatory molecules on IFNgamma-treated corneal endothelial and non-treated cells was evaluated by flow cytometry, RT-PCR, or immunohistochemistry. Expression of co-stimulatory receptors on target T cells was evaluated by flow cytometry. Blocking antibodies was used to abolish the HCE-inhibitory function. RESULTS: HCE cells suppressed both in vitro proliferation and IFNgamma production by CD4(+) T cells via a cell contact-dependent mechanism. HCE constitutively expressed co-stimulatory molecules programmed death-ligand 1 (PD-L1) and PD-L2, and their expression was enhanced by IFNgamma. HCE efficiently inhibited the proliferation of Th1 cells that overexpressed PD-1 among various activated T-cell lines and clones established from patients with uveitis or corneal endotheliitis. A neutralizing mAb for PD-L1, but not PD-L2, blocked the suppressive effect of HCE on Th1 cells. CONCLUSIONS: HCE can impair the effector functions and activation of Th1 infiltrating CD4(+) T cells via the PD-1/PD-L1 interaction. The data support the hypothesis that corneal endothelium may contribute to maintenance of the privileged immune status of the anterior chamber of the eye by inducing peripheral immune tolerance.

Retinal pigment epithelium-derived CTLA-2alpha induces TGFbeta-producing T regulatory cells.

T cells that encounter ocular pigment epithelium in vitro are inhibited from undergoing TCR-triggered activation, and instead acquire the capacity to suppress the activation of bystander T cells. Because retinal pigment epithelial (RPE) cells suppress T cell activation by releasing soluble inhibitory factors, we studied whether soluble factors also promote the generation of T regulatory (Treg) cells. We found that RPE converted CD4(+) T cells into Treg cells by producing and secreting CTLA-2alpha, a cathepsin L (CathL) inhibitor. Mouse rCTLA-2alpha converted CD4(+) T cells into Treg cells in vitro, and CTLA-2alpha small interfering RNA-transfected RPE cells failed to induce the Treg generation. RPE CTLA-2alpha induced CD4(+)CD25(+)Foxp3(+) Treg cells that produced TGFbeta in vitro. Moreover, CTLA-2alpha produced by RPE cells inhibited CathL activity in the T cells, and losing CathL activity led to differentiation to Treg cells in some populations of CD4(+) T cells. In addition, T cells in the presence of CathL inhibitor increased the expression of Foxp3. The CTLA-2alpha effect on Treg cell induction occurred through TGFbeta signaling, because CTLA-2alpha promoted activation of TGFbeta in the eye. These results show that immunosuppressive factors derived from RPE cells participate in T cell suppression. The results are compatible with the hypothesis that the eye-derived Treg cells acquire functions that participate in the establishment of immune tolerance in the posterior segment of the eye.

[Detection of herpesvirus genome by multiplex polymerase chain reaction (PCR) and real-time PCR in ocular fluids of patients with acute retinal necrosis].

PURPOSE: The human herpesvirus (HHV) family consists of types 1 to 8 (HHV1-8). The purpose of this study was to investigate the detection of HHV DNA, especially HSV1 (herpes simplex virus 1, HHV1), HSV2 (herpes simplex virus 2, HHV2), and VZV (varicella-zoster virus, HHV3) in ocular fluids of patients with acute retinal necrosis(ARN). METHODS: The intraocular genome for HHV1-8 was determined in 19 ocular fluid samples (12 vitreous fluid and 7 aqueous humor samples) taken from ARN patients (n=14). The samples were tested for the presence of virus DNA by two systems of polymerase chain reaction (PCR): the multiplex PCR screening test and real-time quantitative PCR. RESULTS: Multiplex PCR demonstrated VZV (n=16, 84%), HSV1 (n = 1.5%) or HSV2 (n = 2.11%)genomic DNA in all the samples. In real-time PCR, a high copy number of virus DNA was detected. The virus DNA-positive samples contained Epstein-Barr virus (EBV, HHV4) DNA in 9 of 19 samples (47%). No HHV6-8 DNA was detected in the ocular samples, and no virus DNA was detected in the serum samples. CONCLUSIONS: The genome for HHV1-3 was detected in the patients with ARN. All cases contained a high copy number for the virus DNA that indicates viral replication. PCR systems are useful for determing whether virus infections are associated with uveitis.

[Murine ciliary body pigment epithelial cells inhibit activation of T cells].

PURPOSE: Pigment epithelial(PE) cells cultured from the eye possess the novel property of suppressing T cells in vitro. Although cultured iris PE (IPE) and retinal PE (RPE) achieve suppression, the function of ciliary body PE (CBPE) is still unknown. We investigated whether CBPE can suppress the activation of T cells in vitro. METHODS: CBPE, IPE and RPE cells(controls) were established from normal C57BL/6 mice. T-cell activation was assessed by proliferative response and cytokine production, by ELISA. The expression of candidate genes on ocular PE was evaluated with on oligonucleotide microarray, and PE proliferation and PE morphology were also evaluated. RESULTS: CBPE significantly suppressed T cell activation in vitro. Cell division of CBPE was much greater than in other PE, and the cells were confluent from on early phase. Microarray analysis showed that the expression of candidate genes in CBPE is similar to the expression in other PE cells. CONCLUSIONS: CBPE fully suppress T cell activation. Thus, ocular pigment epithelial cells display inhibitory function in the immune privileged site.

Transforming growth factor beta-producing Foxp3(+)CD8(+)CD25(+) T cells induced by iris pigment epithelial cells display regulatory phenotype and acquire regulatory functions.

The ocular pigment epithelial (PE) cells convert T cells into T regulators (Tregs) in vitro. The PE-induced Tregs fully suppress activation of bystander responder T cells. Iris PE (IPE) cells from anterior segment in the eye produce costimulatory molecules and transforming growth factor beta (TGFbeta) that is delivered to CD8(+) Tregs. We have now examined whether T cells exposed to cultured IPE express CD25 and Foxp3, and to determine if the CD25(+) IPE-exposed T cells display regulatory functions in vitro. We have found that cultured B7-2(+) IPE converted CTLA-4(+) T cells into CD25(+) Tregs that suppress the activation of bystander T cells. The CD8(+) IPE-induced Tregs constitutively expressed CD25. Through TGFbeta-TGFbeta receptor interactions, the IPE converted these T cells into CD25(+) Tregs that express Foxp3 transcripts. The CD8(+) IPE-induced Tregs produced immunoregulatory cytokines, e.g., interleukin-10 and TGFbeta. In addition, IPE-exposed T cells that downregulated Foxp3 mRNA failed to acquire the regulatory function. In conclusion, ocular pigment epithelial cells convert CD8(+) T cells into CD25(+) Tregs by inducing the transcription factor Foxp3. Thus, T cells that encounter ocular parenchymal cells participate in the T-cell suppression.

Role of thrombospondin-1 in T cell response to ocular pigment epithelial cells.

Ocular pigment epithelium (PE) cells promote the generation of T regulators (PE-induced Treg cells). Moreover, T cells exposed to PE acquire the capacity to suppress the activation of bystander T cells via TGFbeta. Membrane-bound TGFbeta on iris PE cells interacts with TGFbeta receptors on T cells, leading to the conversion of T cells to CD8(+) Treg cells via a cell contact-dependent mechanism. Conversely, soluble forms of TGFbeta produced by retinal PE cells can convert CD4(+) T cells into Treg cells in a manner that is independent of cell contact. In this study, we looked at the expression of immunoregulatory factors (TGFbeta, thrombospondins, CD59, IL-1 receptor antagonist, etc.) in PE cells as identified via an oligonucleotide microarray. Several thrombospondin-binding molecules were detected, and thus we focused subsequent analyses on thrombospondins. Via the conversion of latent TGFbeta to an active form that appears to be mediated by thrombospondin 1 (TSP-1), cultured iris PE and retinal PE cells induce a PE-induced Treg cell fate. After conversion, both ocular PE and PE-induced Treg cells express TSP-1. Regulatory T cell generation was amplified when the T cells also expressed TSP-1. In addition, PE-induced Treg cells significantly suppressed activation of bystander T cells via TSP-1. These results strongly suggest that the ability of ocular PE and PE-induced Treg cells to suppress bystander T cells depends on their capacity to produce TSP-1. Thus, intraocular TSP-1 produced by both ocular parenchymal cells and regulatory T cells is essential for immune regulation in the eye.

B7+ iris pigment epithelial cells convert T cells into CTLA-4+, B7-expressing CD8+ regulatory T cells.

PURPOSE: To determine whether iris PE (IPE) promotes the generation of regulatory T-cells (Tregs) with cell contact via B7-2/CTLA-4 interactions. METHODS: T cells were cocultured with IPE cells obtained from eyes of normal and B7-deficient mice, x-irradiated, and used as regulators. IPE T regulator cells (IPE Tregs) of normal and CD28- or CTLA-4-deficient mice were established. Target bystander T cells were established from normal splenic T cells with anti-CD3 antibodies. T-cell activation was assessed for proliferation by [3H]-thymidine incorporation. Neutralizing anti-B7-1 and/or B7-2 antibodies, anti-CTLA-4 antibodies, CTLA-4-Ig fusion proteins were used to abolish regulatory function. IPE-exposed CD8+ T cells were evaluated for expression of B7, CTLA-4, and Foxp3 by using RT-PCR and flow cytometry. CD8+ IPE Tregs were depleted of B7-2+ and CTLA-4+ T cells and assayed for suppressive activity by adding them to bystander T cells. RESULTS: T cells acquired T regulatory activity when exposed to cultured IPE. Ciliary body PE cells did not promote conversion of T cells into Tregs. IPE converted CD8+, but not CD4+, T cells into Tregs by direct cell contact. In the conversion, IPE and responding T cells must both express endogenously synthesized B7-1 and B7-2, and the T cells must also express CTLA-4. Expression of CD28 molecules was not necessary for Treg generation. In addition, the CD8+ Tregs that fully suppress activation of bystander T cells expressed Foxp3. CONCLUSIONS: IPE cells promote conversion of T cells into Tregs solely through a contact-dependent mechanism. T cells exposed to IPE cells acquire full regulatory capacity.

Retinal and ciliary body pigment epithelium suppress activation of T lymphocytes via transforming growth factor beta.

The ocular microenvironment is immunosuppressive and anti-inflammatory. Pigment epithelial (PE) cells isolated from the eye possess a new property of suppressing T cell receptor-dependent activation of T cells in vitro. This property depends on their capacity to produce cell-surface and soluble inhibitory molecules. The iris pigment epithelia (IPE) do so through direct cell-to-cell contact with naïve T cells, and this suppressive contact is mediated by interactions between B7 and membrane-bound TGFbeta that are expressed constitutively on IPE. We have now examined whether other ocular PE cells, e.g., retinal pigment epithelia (RPE) and ciliary body pigment epithelia (CBPE), have a similar suppressive property by a similar process. We have found that RPE and CBPE significantly suppress the activation of bystander T cells via soluble inhibitory factors. RPE and CBPE secrete different soluble inhibitory factors including TGFbeta1 and TGFbeta2. Although IPE cells suppress the activation of bystander T cells by membrane-bound TGFbeta, the RPE and CBPE do so by soluble forms of active TGFbeta through mechanisms independent of cell contact. These ocular PE cells are capable modifying T cell function by enhancing production of regulatory cytokines including TGFbeta. We propose that this mechanism of suppression via TGFbeta ensures that soluble active TGFbeta is released into the ocular microenvironment in order to create the immune privilege of the posterior segment of the eye.

Ocular infiltrating CD4+ T cells from patients with Vogt-Koyanagi-Harada disease recognize human melanocyte antigens.

PURPOSE: To determine whether patients with Vogt-Koyanagi-Harada (VKH) disease have immune responses specific to the melanocyte antigens tyrosinase and gp100. METHODS: T-cell clones (TCCs) were established from cells infiltrating the aqueous humor and from peripheral blood mononuclear cells (PBMCs) of patients with VKH. The target cells were LDR4-transfected cells (HLA-DRB1*0405). The TCCs were cocultured with LDR4 in the presence of tyrosinase (tyrosinase450-462: SYLQDSDPDSFQD), gp100 (gp100(44-59): WNRQLYPEWTEAQRLD), or a control peptide. The immune response was evaluated by cytokine production. The responding melanocyte peptide-specific VKH-TCCs were characterized by an immunofluorescence method with flow cytometry. A search was made for molecular mimicry among tyrosinase450-462, gp100(44-59), and exogenous antigens, such as viruses, by database screening. RESULTS: Cells infiltrating the eye and PBMCs in HLA-DR4+ (HLA-DRB1*0405, 0410) patients with VKH contained a population of CD4+ T lymphocytes that recognized tyrosinase and gp100 peptides and produced RANTES and IFN-gamma in response to the two peptides. The T cells were active memory Th1-type lymphocytes, and they recognized the tyrosinase peptide and produced IFN-gamma in response to HLA-DRB1*0405+ melanoma cells. Cytomegalovirus envelope glycoprotein H (CMV-egH290-302) had high amino acid homology with the tyrosinase peptide. In addition, some of the VKH-TCCs recognized CMV-egH290-302 peptide, as well as the tyrosinase peptides. CONCLUSIONS: In VKH there are tyrosinase and gp100 peptide-specific T cells that can mediate an inflammatory response. Such melanocyte antigen-specific T cells could be associated with the cause and pathology of VKH disease.

Cytokine profile in aqueous humor and sera of patients with infectious or noninfectious uveitis.

PURPOSE: To determine the cytokine expression profile at the protein level in aqueous humor (AqH) and sera of patients with uveitis. METHODS: Patients with various clinical entities of strictly diagnosed infectious or noninfectious uveitis were tested. AqH and sera were collected from patients with uveitis. AqH was also collected during surgery from patients with cataract, as control specimens. Interferon (IFN)-gamma, tumor necrosis factor (TNF)-alpha, and interleukin (IL)-2, -4, -5, and -10 were measured from nondiluted samples simultaneously, with microparticle-based flow cytometric analysis. RESULTS: In AqH IFN-gamma was the most abundant cytokine in both infectious (mean, 3240.5 pg/mL) and noninfectious (mean, 115.6 pg/mL) uveitis, and IL-10 was the second (mean, 402.1 pg/mL, infectious uveitis; 7.5 pg/mL, noninfectious uveitis). The expression level of other cytokines in AqH was generally higher in infectious uveitis than in noninfectious uveitis, but the levels were lower than that of IL-10. There was no remarkable difference, however, in the cytokine expression pattern in AqH of the different clinical entities of uveitis. Sera from patients with noninfectious uveitis contained IFN-gamma (mean, 45.0 pg/mL), but the other serum cytokines in both types of uveitis were low or under the detectable level. CONCLUSIONS: IFN-gamma is the most abundant cytokine in infectious and noninfectious uveitis, with a remarkable difference between the two groups. The data suggest that cytokines in AqH of infectious uveitis are locally produced, whereas in noninfectious uveitis, IFN-gamma is produced both in the eye and the peripheral blood.