Ocular Infiltrating CD4⁺ T Cells from Patients with Vogt-Koyanagi-Harada Disease Recognize Human Melanocyte Antigens

Abstract

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 (tyrosinase_450-462: 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 tyrosinase_450-462, 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-γ in response to the two peptides. The T cells were active memory Th1-type lymphocytes, and they recognized the tyrosinase peptide and produced IFN-γ in response to HLA-DRB1*0405⁺ melanoma cells. Cytomegalovirus envelope glycoprotein H (CMV-egH_290-302) had high amino acid homology with the tyrosinase peptide. In addition, some of the VKH-TCCs recognized CMV-egH_290-302 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.

Vogt-Koyanagi-Harada (VKH) disease is a systemic disorder that affects the eye, meninges, ear, skin, and hair. Although the pathogenic mechanism of the disease has still not been completely determined, there is strong clinical and experimental evidence indicating that VKH disease is an autoimmune disease that acts against melanocytes. All tissues affected by the disease contain melanocytes ¹ ; there is depigmentation of the affected tissues (e.g., poliosis, vitiligo, and the sunset-glow fundus of the eye in the late stage of the disease); the disease is more prevalent in pigmented ethnic groups, such as the Japanese, than in nonpigmented groups, such as whites ² ; and the peripheral blood mononuclear cells (PBMCs) react to melanocyte antigens. ^{3 4} In addition, the disease is characterized by a significant association with HLA-DR4, and the genomic type is DRB1*0405 or DRB1*0410. ^{2 5} Among the pigmented ethnic groups, the Japanese have a higher prevalence of VKH than do Africans who have darker skin; 30% to 40% of Japanese people have HLA-DR4. ^{5 6}  

Although the disease appears to be caused by autoimmune responses to melanocytes or melanocyte-associated antigens, it is still not known what antigens or antigen peptides among the melanocyte-associated antigens are responsible for the disease. It has been shown that several melanoma peptides are recognized by cytotoxic T lymphocytes (CTLs), and MART-1, ⁷ gp100 (Pmel-17), ⁸ gp75, ⁹ tyrosinase, ^{10 11} and melanocyte lineage-specific peptides are expressed not only on melanoma cells but also on human melanocytes. Many epitopes of these peptides are recognized by HLA-class I-restricted CTLs. For example, in our previous study, the CTLs from the eyes of patients with VKH recognized MART-1 peptide in an HLA-A2-restricted manner. ¹² However, VKH disease is known to be highly associated with HLA-DR4 (HLA-DRB1*0405). Therefore, the candidate peptides responsible for the immunopathogenic mechanism of the disease should be recognized by T cells in an HLA-DR4-restricted manner. 

Tyrosinase and gp100 are melanoma-associated antigen peptides that have been shown to be recognized by CD4⁺ T cells in an HLA-DR4-restricted manner. ^{13 14} The purpose of this study was to determine whether these melanocyte-associated peptides are responsible for the immunopathogenic mechanisms of VKH disease. In addition, we examined whether a viral infection could be a trigger mechanism. We first identified the melanocyte antigen peptides that could play a role in the development of the disease, and then used database screening to search for homology between the melanocyte peptides and antigen peptides of exogenous pathogens. 

Materials and Methods

Subjects

The procedures used in this research conformed to the tenets of Declaration of Helsinki, and informed consent was obtained from each participant. There were nine patients with VKH disease, five with other types of uveitis, and eight healthy donors. All the patients with VKH had typical ocular signs and symptoms and fluorescein angiographic findings of VKH disease. They also had systemic signs, such as pleocytosis of the cerebrospinal fluid (CSF) and abnormal audiograms. Their HLA serotypes and DNA genotypes, allele DRB1, DQA1, and DQB1, were determined by SRL Inc. (Tokyo, Japan). Samples of aqueous humor (AH) were obtained from some of the patients. 

Antigen Peptides

Two HLA-DRB1*0405 human binding melanocyte peptides were used: a 13-mer peptide (tyrosinase_450-462: SYLQDSDPDSFQD) and a 16-mer peptide (gp100_44-59: WNRQLYPEWTEAQRLD). The major histocompatibility complex (MHC) class II binding motif of these two peptides was HLA-DRB1*0405 binding peptide. ¹⁵ An influenza peptide HA_307-319 (PKYVKQNTLKLAT) was used as the negative control. These peptides are known to bind to HLA-DRB1*0401 with high affinity. ¹⁶ Some analogues of tyrosinase_450-462 were also used. We selected these analogues of tyrosinase_450-462 from the report of Topalian et al. ¹³ All peptides were synthesized by BioSynthesis Inc. (Lewisville, TX), and the purity of the peptides was >95%, as determined by HPLC analysis. 

mRNA Expression of Melanocyte Antigens on Ocular Tissues

To confirm that tyrosinase and gp100 were present in human ocular tissues, especially in patients with VKH, the expression of the mRNA of tyrosinase and gp100 in human iris was determined by RT-PCR. The samples of iris and trabecular meshwork were obtained from a patient with VKH disease during glaucoma surgery. A melanoma cell line, MMAc, was used as the positive control. Total cellular RNA was extracted from the tissues (TRIzol Reagent; Invitrogen, Gaithersburg, MD), and the melanoma cell line was prepared. Reverse transcription was performed (SuperScriptTM II; Invitrogen). The cDNA obtained was tested for the presence of defined gene sequences by PCR (Ex-Taq kit; Takara Shuzo Co., Ltd., Shiga, Japan) on 25 μL using specific primer pairs. The following primers were used for the PCR reaction: β-actin sense, 5′-CTT CGC GGG CGA CGA TGA-3′, and anti-sense, 5′-CGT ACA TGG CTG GGG TGT TG-3′, yielding an amplification product of 340-bp; tyrosinase sense, 5′-AAG AAA TCC AGA AGC TGA CAG GAG ATG-3′ and anti-sense, 5′-TGC TTT GAG AGG CAT CCG CTA TC-3′, amplifying a 423-bp fragment; gp100 sense, 5′-CTG TGC CAG CCT GTC TAC-3′, and anti-sense, 5′-CAC CAA TGG GAC AAG AGC AG-3′, amplifying a 334-bp fragment. Amplification of tyrosinase, gp100, and β-actin as positive control transcripts required 40 cycles, including a 30-second annealing step at 55°C and a 30-second extension at 72°C. 

The RT-PCR products were run on 1.5% agarose gels in the presence of ethidium bromide, and the gels were then photographed under ultraviolet transillumination. 

TCCs and T-Cell Lines

An aliquot of AH was obtained from patients with different types of uveitis whose eyes were inflamed. PBMCs were obtained from the patients before and during systemic corticosteroid therapy. TCCs were established from these samples by the limited-dilution method. ^{12 17 18} There were 16 TCCs from the AH of four patients with VKH (P2–5, Table 1 , Fig. 4 ) and 2 TCCs from the PBMCs of a patient (P1) with VKH disease. The TCCs established from other types of uveitis were used: three clones from the AH and four clones from the vitreous fluid of three patients with Behçet’s disease, and four clones from the AH of a patient with sarcoidosis. The TCCs established from PBMCs of healthy donors (HD1 and -2 in Table 1 ) were also used. The cloning efficiency was approximately 15% to 50%. 

T-cell lines (TCLs) were also established from the PBMCs of six patients with VKH disease (P4–9, Tables 2 and 3 ), two patients with Behçet’s disease (P14, P15), a patient with sarcoidosis (P13), and eight healthy donors (HD1–8). The blood samples were obtained before systemic corticosteroid therapy, and the TCLs were established from freshly isolated blood samples by coculturing the cells with feeder cells. These cells were used for assays. 

To establish these T cells (TCCs and TCLs), we used T cell growth factors such as x-irradiated (50 Gy) allogeneic PBMCs as the feeder cells, human recombinant IL-2 (rIL-2), and PHA-P (Difco Laboratory, Inc., Detroit, MI). The medium used for the culture was RPMI 1640 + 10% fetal bovine serum (FBS). The feeder cells were added to each well along with rIL-2 every 7 days until an outgrowth of cells was observed. For an in vitro assay, we used 1 × 10⁶ TCCs or TCLs. The culture period for the T cells was ∼4 weeks. The experiments were performed at least twice with similar results. 

Assays for Cytokine Production and Phenotype of TCC

Cell-free supernatants were obtained from each TCC by centrifuging and collecting the supernatant. An ELISA kit (R&D Systems, Minneapolis, MN) was used to detect and quantify the cytokines and chemokines. The ability of T cells to produce cytokines and chemokines was determined by using the TCCs of patients with VKH. The cytokines and chemokines measured were IL-1α, IL-2, IL-4, IL-6, IL-10, TNF-α, IFN-γ, GM-CSF, IL-8 (CXC chemokine ligand 8, CXCL-8), MIP-1α (CC chemokine ligand 3, CCL-3) and -β (CCL-4), and RANTES (CCL-5). 

The phenotype of a TCC was determined by the double-color immunofluorescence technique with flow cytometry. T cells were washed with PBS and were incubated at 4°C for 30 minutes with the following antibodies: FITC-conjugated mAb NU-Ts/c (CD8; Nichirei, Tokyo, Japan); anti-HLA-DR (BD Biosciences, Mountain View, CA); CD45RA (BD PharMingen, SanDiego, CA); CXCR3 (Genzyme, Cambridge, MA); TCR-α/β (BD PharMingen); a PE-conjugated mAb, NU-Th/i (CD4; Nichirei); CD45RO (BD PharMingen); CXCR1 (Genzyme); and TCR-γ/δ (BD PharMingen). Anti-Tac mAb (CD25) was used as the primary reagent, with FITC-conjugated goat IgG against mouse IgG (Tago, Inc., Burlingame, CA) used as the secondary reagent. The biotinylated NOK-1 (CD95 ligand) was used as the primary reagent and FITC-conjugated avidin (BD PharMingen) was the secondary reagent. 

Database Screening to Melanocyte Antigens

Database screening (GenBank database screening) was performed to determine whether tyrosinase_450-462 and gp100_44-59 had homologous amino acid sequences to exogenous antigens. The human cytomegalovirus envelope glycoprotein H peptide (CMV-egH_290-302) was found to have a high amino acid homology to tyrosinase_450-462 peptide (Table 4) . The MHC class II binding motif of the CMV-egH_290-302 was also DRB1*0405. ¹⁵  

Assay for T-Cell Responses to Peptides

To examine whether the T cells respond to tyrosinase and gp100, the T cells, TCCs, and TCLs, from patients with VKH disease were cocultured with antigen-presenting cells (APCs) in the presence of tyrosinase peptide, gp100 peptide, or a control peptide, as described later. The APCs used were LDR4 transfectant cells with HLA-DRB1*0405 ¹⁹ and melanoma cell lines. The APCs were irradiated (50 Gy) and incubated overnight (5 × 10⁴ cells/well). Then, one of the peptides at doses of 1 to 500 μM and the T cells were added to the wells and cocultured for 72 hours. 

In the screening assay, the peptides were used at a concentration of 50 μM because titration of the CD4⁺ T cell response to the peptides showed that a peptide concentration of more than 50 μM was needed to evoke significant reactivity. ¹³ After the incubation, cell-free culture supernatants were harvested and used to measure RANTES or IFN-γ. To determine the specificity of the response, the APCs (MMAc) were pretreated with mAbs that included anti-HLA-DR (IgG_2b); anti-HLA-A, B, and C (IgG₁); and anti-CD4 (IgG₁; all BD PharMingen). As an isotype control, purified mouse IgG_2a,κ (BD PharMingen) was added to the appropriate cultures. To examine whether the T cells respond to CMV antigens, the TCCs from patients with VKH disease were cocultured with LDR4 cells in the presence of CMV-egH_290-302. The specific response was evaluated by RANTES production assay. 

Statistical Evaluation of Results

Each experiment was repeated at least twice with similar results. All statistical analyses were conducted with Student’s t-test. Differences were considered statistically significant at P < 0.05. 

Results

HLA Genotypes in Patients with VKH

All nine patients with VKH disease were HLA-DR4⁺. The HLA genotypes were DRB1*0405 in eight patients and DRB1*0410 in one patient. Four HLA-DR4⁺ patients with Behçet’s disease were DRB1*0405⁺, and one patient with sarcoidosis was DRB1*0410⁺. Of the eight HLA-DR4⁺ healthy donors, seven were DRB1*0405⁺, and one was DRB1*0406⁺. All patients with VKH were DQB1*0401⁺ and DQA1*0302⁺. 

Detection of Tyrosinase and gp100 Gene Transcriptions in Human Iris

To determine whether tyrosinase and gp100 are present in ocular tissues, the expression of their genes was determined by RT-PCR using ocular tissues of a patient with VKH disease (Fig. 1) . A strong expression of the mRNA of tyrosinase was detected in iris tissues but not in trabecular meshwork tissues (Fig. 1A) . The expression of the mRNA of gp100 was also detected in iris tissues and in melanoma cell lines as the positive control (Fig. 1B) . β-Actin was detected in all samples (data not shown). 

Recognition of Tyrosinase and Gp100 by T Lymphocytes

First, we evaluated the cytokine profiles in VKH-TCCs. The TCCs developed from ocular-infiltrating cells of four patients with VKH constitutively produced significant amounts of different cytokines and chemokines, and the mean values were (in pg/mL): IL-6, 46; IL-8/CXCL-8, 1031; TNF-α, 37; IFN-γ, 64; GM-CSF, 24; MIP-1α/CCL-3, 370; MIP-1β/CCL-4, 278; and RANTES/CCL-5, 581. In preliminary study using these cytokines and chemokines, the VKH TCCs indicated large response for IFN-γ and RANTES in the presence of human melanocyte antigens, but not IL-6, IL-8, GM-CSF, and TNF-α. The RANTES and IFN-γ production assays were used in the following experiments because they were produced in high amounts by the ocular-infiltrating T cells compared with the T cells from the PBMCs of patients with VKH and the ocular fluid from other types of uveitis. 

The lymphocytic responses to the HLA-DRB1*0405-restricted, melanocyte-associated peptides tyrosinase_450-462 and gp100_44-59 were tested by the RANTES-production assay (Table 1) . One CD4⁺ TCC (VKH1-2) established from the PBMCs of patient 1, one CD4⁺TCC (VKH2-1) from the AH of patient 2 and two CD4⁺TCCs (VKH3-1 and -4) from the AH of patient 3 responded more strongly to tyrosinase_450-462 than to HA_307-319, a control peptide. One CD4⁺TCC (VKH3-3) from the AH of patient 3 and two CD4⁺ TCCs (VKH4-1 and -2) from the AH2 of patient 4 responded more strongly to gp100_44-59 than to HA_307-319. In contrast, the TCCs from HLA-DR4⁺ patients with Behçet’s disease and sarcoidosis did not respond to tyrosinase_450-462 or to gp100_44-59 (Table 1) . The TCCs from PBMCs of HLA-DR4⁺ healthy donors also did not respond to all peptides (Table 1) . 

All TCLs (TCL1-6) from the PBMCs of six patients with VKH (P4-9) responded more strongly to tyrosinase_450-462 and gp100_44-59 peptides than to HA_307-319. In contrast, the TCLs from a patient with Behçet’s disease and with sarcoidosis did not respond to the two peptides (Table 2) . The TCLs from eight healthy donors who had no history of uveitis and were aged-matched to the patients with VKH did not respond to the two peptides (Table 2) . In addition, all TCLs from patients with VKH produced IFN-γ in the presence of these melanocyte peptides much greater than control TCLs (Table 3) . 

A dose-response study was performed to confirm that the T-cell response to tyrosinase_450-462 peptide and gp100_44-59 peptide was significant. The production of RANTES by VKH3-1 CD4⁺ TCC from the AH of a patient with VKH disease was upregulated by tyrosinase_450-462 stimulation in a dose-dependent manner (Fig. 2A) . Similarly, the VKH4-1 CD4⁺ TCC responded strongly and in a dose-dependent manner to gp100_44-59 (Fig. 2B) . A change of one amino acid at the anchor position of tyrosinase_450-462 abolished the RANTES production by the TCL (TCL-2) in response to the peptide (Fig. 2C) . In addition, the production of IFN-γ by VKH3-1 or VKH4-1 CD4⁺ TCCs was upregulated by the melanocyte-peptide stimulation in a dose-dependent manner (Fig. 2D) . 

The phenotypes of the TCCs were analyzed by flow cytometry. The VKH3-1 which produced RANTES in response to tyrosinase_450-462 were CD4⁺, CD25⁺, CD45RO⁺, CD95⁺, HLA-DR⁺, CXCR3⁺, and TCR-α/β⁺ cells (Fig. 3A) . The cells were negative for CD8, CD45RA, CD95L, CXCR1, and TCR-γ/δ. Other TCCs (VKH2-1, VKH3-4, VKH3-10, and VKH5-2) established from ocular-infiltrating cells in patients with VKH indicated similar phenotypes (data not shown). 

To verify that VKH3-1 TCCs truly recognized tyrosinase peptide and responded to the peptide, a melanoma cell line (MMAc) was cocultured with the VKH3-1 TCC in the presence of anti-HLA-class I mAb, anti-HLA-class II (DR) mAb, or anti-CD4 mAb. The MMAc cell line is known to express melanocyte antigens on HLA-DRB1*0405. The VKH3-1 TCC produced a significant amount of IFN-γ in response to MMAc (Fig. 3B) , and IFN-γ production was significantly reduced by anti-DR mAb and anti-CD4 mAb, but not by anti-class I mAb (Fig. 3B)or isotype control. Similarly, the VKH2-1 TCC produced a significant amount of IFN-γ in response to MMAc, and the production was significantly reduced by anti-DR mAbs (data not shown). These results indicate that VKH3-1 TCCs are the Th1-type T cells that recognize melanocyte antigens in an HLA-DRB1*0405-restricted manner and produce significant amounts of IFN-γ. 

Lymphocytic Response of Patients with VKH to CMV Antigen and Melanocyte Antigen

Because tyrosinase_450-462 is highly homologous to CMV-egH_290-302 (Table 4) , we also examined its lymphocytic response to CMV-egH_290-302. Six CD4⁺ TCCs established from the AH of patients with VKH responded significantly to CMV-egH_290-302 (Fig. 4) . Some of the CD4⁺ T cells also responded significantly to tyrosinase_450-462 but not to the peptide of control AH. Thus, TCCs established from patients with VKH without antigen stimulation contain CMV-egH_290-302 peptide–specific T cells. 

Discussion

The T lymphocytes infiltrating the eye of patients with VKH were reactive to the peptides derived from human melanocyte antigens (e.g., tyrosinase_450-462 and gp100_44-59). The lymphocytic response to tyrosinase_450-462 was dose dependent, structure dependent, and disease specific. A TCC that recognized tyrosinase_450-462 was assayed for the phenotype, and it was an active memory Th1-type lymphocyte. Some of T cells directly recognized the peptides and produced RANTES when cocultured with HLA-DRB1*0405 transfected LDR-4 cells as APCs. In addition, PBMCs from the patients with VKH responded when incubated with the tyrosinase_450-462 and gp100_44-59. Taken together, these findings indicate that VKH disease is an autoimmune disease against the melanocyte antigens, and tyrosinase is one of the responsible antigens in the immunopathogenic mechanism of VKH disease. 

Tyrosinase is a type I transmembrane protein consisting of 529 amino acids and regulates melanin production, ²⁰ whereas gp100 is a membrane glycoprotein consisting of 661 amino acids and is present in melanosomes where melanin is synthesized. ²¹ Tyrosinase and gp100 are expressed on both melanoma and melanocytes. Kawakami and Rosenberg ²² reported that in patients with malignant melanoma who had immunotherapy against melanoma-specific antigens including tyrosinase and gp100 antigens, vitiligo developed as an adverse side effect of the therapy. They also demonstrated that IgG specific for melanoma protein preferentially expressed on melanoma and melanocytes was detected in the sera of 7 of 11 patients with VKH disease and some patients with melanoma. ²³ These tumor antigens were isolated from a patient with vitiligo. These observations suggest that the recognition of the melanoma-differentiation antigen can be associated with autoimmune depigmentation, vitiligo, which is one of the characteristic clinical symptoms of VKH disease. Therefore, it was hypothesized that the melanoma and melanocyte-specific antigens which recognized infiltrating T cells could be potential candidates for self-antigens of autoimmune disease against melanocytes. 

Our previous results have demonstrated that MART-1-specific cytotoxic T cells isolated from the eye of patients with VKH disease lysed melanocytes in an HLA-A2-restricted manner. ¹² However, MART-1 is not an appropriate candidate for the pathogenic antigen of VKH disease because VKH is known to be associated with HLA-DR4 and not with HLA-A2. The pathogenic antigen should be HLA-DR4-restricted or an HLA-DRB*0405-restricted antigen. 

Earlier, Yamaki et al. ^{24 25} showed that the PBMCs of patients with VKH disease exhibited low but significant proliferation to tyrosinase peptides, and immunization of experimental animals with tyrosinase induced ocular inflammation. Recently, Damico et al. ²⁶ also reported that T cells established from PBMCs of patients with VKH responded to some peptides of tyrosinase, TRP-1, TRP-2, and Pmel-17 (gp100). Therefore, we focused on the role of ocular-infiltrating lymphocytes in VKH disease. For this purpose, we used TCCs established from cells infiltrating the eye of patients with VKH by the limited-dilution method. ^{12 17 18} Our results showed that the TCCs obtained from the eyes of patients with VKH intrinsically produced large amounts of IFN-γ, and RANTES. RANTES has a strong chemotactic effect on neutrophils and T lymphocytes and are produced by a variety of cells including T lymphocytes, particularly helper memory T cells. ²⁷ IFN-γ is a major Th1 cytokine and has been reported to be involved in the pathogenesis of VKH disease. ²⁸ It is assumed that these cytokines can best represent the recognition of T cells in the reappearance of antigens. Our results showed that the production of RANTES by the TCCs obtained from patients with VKH was significantly upregulated when cultured with tyrosinase_450-462 or gp100_44-59, but not with control peptides. The TCCs obtained from eyes with other uveitic diseases, such as Behçet’s disease and sarcoidosis, did not respond significantly to the two peptides, indicating that the lymphocytic response to the peptides was disease specific. 

The lymphocytic response was blocked by anti-DR and anti-CD4 antibodies indicating that the response was MHC class II–restricted. The phenotype of the TCCs was active memory Th1 cells. The TCLs established from the PBMCs of patients with VKH disease, but not those with Behçet’s disease or sarcoidosis or healthy control subjects, also induced a significant increase of RANTES production in response to the two melanocyte-associated peptides. These results indicated that active memory T lymphocytes are present in the eye and the peripheral circulation of patients with VKH disease, and they play a significant role in recognizing the melanocyte-associated peptides to induce immunologic response to the peptides. 

It is of interest to note that vitiligo develops in patients with malignant melanoma as an adverse side effect of immunotherapy targeting melanoma-associated antigens, including tyrosinase and gp100. ²² This clinical observation suggests that the immunologic recognition of the melanoma-associated antigen can be associated with the autoimmune depigmentation vitiligo, which is one of the most characteristic clinical features of VKH disease. 

An important finding in our study was that the CD4⁺ T lymphocytes, that recognize the tyrosinase peptide, responded significantly to an antigen that had homologous amino acids to tyrosinase peptide. This antigen was the common cytomegalovirus of humans. The clinical onset of VKH is characterized by a sudden appearance of bilateral ocular inflammation with prodromal symptoms such as a common cold. This suggests that some viral infection may act as a trigger mechanism. We are now conducting experiments on whether T cells established from ocular fluids or PBMCs of patients with VKH cross-react with tyrosinase_450-462 and CMV-egH_290-302; whether VKH-PBMCs under antigen stimulation respond to the antigens; whether seropositivity to CMV is higher in patients with VKH disease; and whether the CMV infection in patients with VKH is latent. 

In conclusions, patients with VKH disease have immune responses specific to the melanocyte antigens, tyrosinase_450-462 and gp100_44-59. The melanocyte antigen-specific T cells could be associated with the cause and pathologic course of VKH disease. However, the mechanisms by which Th1 lymphocytes are sensitized by self-antigens, tyrosinase, and gp100 and cause the immune responses to the antigens are still unknown. We are now investigating whether melanocyte antigens from patients with VKH disease cross-react with exogenous antigens such as those of viruses. 

 Supported by Scientific Research Grant (B) 1437055 of the Ministry of Education, Culture, Sports, Science and Technology, Japan.

 Submitted for publication December 5, 2005; revised January 10 and 29, 2006; accepted March, 22, 2006.

 Disclosure: S. Sugita, None; H. Takase, None; C. Taguchi, None; Y. Imai, None; K. Kamoi, None; T. Kawaguchi, None; Y. Sugamoto, None; Y. Futagami, None; K. Itoh, None; M. Mochizuki, None

 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked “advertisement” in accordance with 18 U.S.C. §1734 solely to indicate this fact.

 

Table 1.

 

RANTES Production by CD4⁺TCCs of VKH Patients in Response to Tyrosinase & Gp100 Peptides

Disease	Patient No. (DRB1*)	TCCs					RANTES Production by TCCs (pg/mL)
				Source	Clone No.	Phenotype	TCCs Alone	Peptide (−)	HA307–319	Tyrosinase450–462	Gp10044–59
VKH	P1 (0405)	PBMC	VKH1-1	CD4	846	1104	1108	1040	986
			VKH1-2	CD4	238	1056	1157	2746	NT
	P2 (0405)	AH	VKH2-1	CD4	1084	1002	1002	1648	NT
	P3 (0405)	AH	VKH3-1	CD4	<15	<15	<15	619	NT
			VKH3-2	CD4	1616	2502	2521	2487	2512
			VKH3-3	CD4	450	494	751	860	1657
			VKH3-4	CD4	2223	2657	2643	3358	NT
			VKH3-5	CD4	1103	1794	1971	1799	1698
			VKH3-6	CD4	1594	1799	1794	2055	1704
	P4 (0410)	AH	VKH4-1	CD4	264	414	899	768	1398
			VKH4-2	CD4	324	576	607	658	1661
			VKH4-3	CD4	<15	<15	<15	<15	<15
			VKH4-4	CD4	571	1449	1655	1552	1737
Behçet’s	P10 (0405)	AH	B1-1	CD4	<15	<15	<15	<15	<15
			B1-2	CD4	726	1060	1140	878	1006
			B1-3	CD4	<15	<15	<15	<15	<15
	P11 (0405)	Vitreous	B2-1	CD4	<15	<15	<15	<15	<15
	P11 (0405)	Vitreous	B3-2	CD4	<15	<15	<15	<15	<15
			B3-3	CD4	<15	<15	<15	<15	<15
			B3-4	CD4	760	1123	1174	1146	1174
SAR	P13 (0410)	AH	S1-1	CD4	<15	<15	<15	<15	<15
			S1-2	CD4	34	120	128	98	132
			S1-3	CD4	<15	<15	<15	<15	<15
			S1-4	CD4	30	80	92	22	84
Healthy donor	HD1 (0405)	PBMC	H1-1	CD4	<15	<15	<15	<15	<15
	HD2 (0406)	PBMC	H2-1	CD4	<15	<15	<15	<15	<15
			H2-2	CD4	<15	431	513	391	409
			H2-3	CD4	387	236	320	329	409

 Two melanocyte peptides (tyrosinase and gp100, 50 μM) pulsed in LDR-4 cell (HLA-DRB1*0405 transfectant) were assessed for recognition by TCCs established from active uveitis with VKH disease. Behçet’s disease, sarcoidosis, or healthy donors (no uveitis). HA peptide which bind HLA-DRB1*0401 was used as a negative control peptide. The LDR-4 cells as antigen-presenting cells were irradiated at 50 Gy and added to TCCs at a ratio of 1:10. After 72 hours the active response of these T lymphocytes was evaluated by RANTES-ELISA. The RANTES secretion by LDR-4 cells without T cells in the absence of peptide has been not detected (<15 pg/mL). The certain responded data are indicated by Italic type. Peptide (−), TCCs + LDR4 TCCs, T cell clones; SAR, sarcoidosis; PBMC, peripheral blood mononuclear cells; AH, aqueous humor; NT, not tested.

Figure 4.

 

Capacity of CD4⁺ VKH TCCs to produce RANTES in response to CMV antigen. The six TCCs established from aqueous humor in three patients with VKH (P2, P3, P5) were assayed in the presence of the peptides, CMV-egH_290-302 (CMV), tyrosinase_450-462 (TY), or HA control peptide (HA; each 50 μM). The LDR-4 cell as APCs were irradiated at 50 Gy and added to TCCs at a ratio of 1:20. (□) TCCs alone (no APCs). After 72 hours, cell-free culture supernatants were harvested and assessed for RANTES. Data are the mean ± SD of triplicate ELISA determinations. *P < 0.05, **P < 0.005, ***P < 0.0005, compared with TCC alone.

Table 2.

 

RANTES Production by T-Cell Lines of VKH Patients in Response to Tyrosinase and Gp100 Peptides

Disease	Patient No. (DRB1*)	TCLs			RANTES Production by TCLs (pg/mL)
				Source	Cell Line No.	TCLs Alone	Peptide(−)	HA307–319	Tyrosinase450–462	Gp10044–59
VKH	P4 (0410)	PBMC	TCL-1	1367	1372	1323	1717	2357
	P5 (0405)	PBMC	TCL-2	<15	<15	42	481	404
	P6 (0405)	PBMC	TCL-3	1439	1448	1627	2953	2787
	P7 (0405)	PBMC	TCL-4	928	1368	1373	1629	2248
	P8 (0405)	PBMC	TCL-5	1112	1251	1245	2151	3020
	P9 (0405)	PBMC	TCL-6	3662	3016	3509	9682	5551
Behçet’s	P14 (0405)	PBMC	TCL-7	769	392	428	260	443
	P15 (0405)	PBMC	TCL-8	365	354	306	355	380
SAR	P13 (0410)	PBMC	TCL-9	565	655	602	608	619
Healthy donor	HD1 (0405)	PBMC	TCL-10	230	321	235	270	301
	HD2 (0406)	PBMC	TCL-11	<15	<15	<15	<15	<15
	HD3 (0405)	PBMC	TCL-12	<15	<15	<15	<15	<15
	HD4 (0405)	PBMC	TCL-13	102	154	143	149	155
	HD5 (0405)	PBMC	TCL-14	<15	<15	<15	<15	<15
	HD6 (0405)	PBMC	TCL-15	<15	<15	<15	<15	<15
	HD7 (0405)	PBMC	TCL-16	122	104	155	151	166
	HD8 (0405)	PBMC	TCL-17	223	243	253	249	231

 Two melanosyte peptides (tyrosinase and gp100, 50 μM) pulsed in LDR-4 cell were assessed for recognition by TCLs established from PBMC with VI disease and Behçet’s disease. The TCLs of control were also established from PBMC in two HLA-DR4⁺ Behçet’s disease, one sarcoidosis, and eight HLA-DR4-healthy donor. The assay was performed as described in Table 1 . TCLs, T cell lines.

Table 3.

 

IFN-γ Production by T-Cell Lines of VKH Patients in Response to Tyrosinase and Gp100 Peptides

Disease	Patient No. (DRB1*)	TCLs			IFN-γ Production by TCLs (pg/mL)
				Source	Cell Line No.	TCLs Alone	Peptide(−)	HA307–319	Tyrosinase450–462	Gp10044–59
VKH	P4 (0410)	PBMC	TCL-1	567	483	528	777	845
	P5 (0405)	PBMC	TCL-2	<5	28	16	186	51
	P6 (0405)	PBMC	TCL-3	255	199	185	423	342
	P7 (0405)	PBMC	TCL-4	394	350	373	452	528
	P8 (0405)	PBMC	TCL-5	929	882	822	1121	1160
	P9 (0405)	PBMC	TCL-6	268	236	286	452	311
Behçet’s	P14 (0405)	PBMC	TCL-7	179	142	125	172	166
	P15 (0405)	PBMC	TCL-8	993	847	892	801	790
SAR	P13 (0410)	PBMC	TCL-9	32	34	30	25	31
Healthy donor	HD1 (0405)	PBMC	TCL-10	68	62	65	54	42
	HD2 (0406)	PBMC	TCL-11	59	70	70	62	70
	HD3 (0405)	PBMC	TCL-12	11	<5	15	<5	<5
	HD4 (0405)	PBMC	TCL-13	<5	<5	<5	<5	<5
	HD5 (0405)	PBMC	TCL-14	19	21	22	<5	21
	HD6 (0405)	PBMC	TCL-15	<5	<5	<5	<5	<5
	HD7 (0405)	PBMC	TCL-16	<5	<5	<5	<5	<5
	HD8 (0405)	PBMC	TCL-17	23	24	33	49	21

 The assay was performed as described in Table 2 . TCLs, T cell lines.

Table 4.

 

Homology in Amino Acid Sequence of Tyrosinase_450–462 and CMV-egH_290–302

Peptide	Amino Acid Sequence	Binding HLA-DRB1*0405
Tyrosinase450–462	SYL q DSD pdsfqd	(+)
CMV-egH290–302	SYL k DSD fldaal	(+)

Figure 1.

 

Expression of the mRNA of the melanocyte antigens tyrosinase (A) and gp100 (B) in human iris and trabecular meshwork tissues. Iris (IR) and trabecular meshwork (TM) tissues were obtained from a patient with VKH disease, and MMAc melanoma cell lines were the positive control (PC). M, molecular size marker.

Figure 2.

 

Peptide dose–response and truncated and mutated peptide study. (A) Tyrosinase peptide assay using VKH3-1 CD4⁺ TCCs. (B) Gp100 peptide assay using VKH4-1 CD4⁺ TCCs. The ability of VKH TCCs to produce RANTES in response to the peptides was confirmed by the dose–response study. The TCCs were assayed in the presence of the peptide at the five concentrations shown. The LDR-4 cells as APCs were irradiated at 50 Gy and added to TCCs at a ratio of 1:20. (C) To identify the tyrosinase peptide-responding T cells, we first identified the primary HLA-binding anchors in tyrosinase_450-462. The VKH-TCL (TCL-2) established from PBMCs of a VKH patient was cultured and then pulsed with tyrosinase_450-462 or truncated and mutated analogues of tyrosinase_450-462. After 72 hours, cell-free culture supernatants were harvested and assessed for RANTES. (D) IFN-γ production by the melanocyte peptide-responding CD4⁺ TCCs. The ability of VKH TCCs (VKH3-1, left; VKH4-1, right) to produce IFN-γ in response to the peptides was confirmed by the dose–response study. The TCCs were assayed in the presence of the peptide at the concentrations shown. The LDR-4 cells as APCs were irradiated at 50 Gy and added to TCCs at a ratio of 1:20. Data are the mean ± SD of three ELISA determinations.

Figure 3.

 

Characterization of the CD4⁺ TCC, VKH3-1. (A) The phenotype of VKH3-1. VKH3-1-induced production of significant amounts of RANTES was determined by immunofluorescence flow cytometry. The surface markers of VKH3-1 were analyzed by the following antibodies: CD4, CD8, CD25, CD45RA, CD45RO, CD95 (Fas), CD95L (FasL), HLA-DR, CXCR1, CXCR3, TCR-α/β, and TCR-γ/δ. The T cells were gated for expression of two molecules (e.g., CD4 and CD8) in total live cells. (B) Reactivity of VKH3-1 is MHC class II restricted. T cells were co-incubated with target melanoma cells in the presence of anti-HLA-A, B, C; anti-DR; and anti-CD4 mAbs. As target cells, MMAc melanoma cell line (HLA-DRB1*0405/1502) were used. T cells and two targets were cocultured for 24 hours and supernatants were measured for IFN-γ secretion by ELISA. Data are the mean ± SD of triplicate ELISA determinations. *P < 0.05, compared with Abs (−).

The authors thank Nobuhiro Kamikawaji (Department of Genetics, Medical Institute of Bioregulation, Kyushu University, Fukuoka, Japan) for providing cell lines (LDR4 transfected cells), Tomoko Yoshida for technical assistance, and Duco Hamasaki for critical reading of the manuscript.