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Cytotoxic T Lymphocyte Reactivity to gp100, MelanA/MART-1, and Tyrosinase, in HLA-A2-Positive Vitiligo Patients

2003; Elsevier BV; Volume: 121; Issue: 3 Linguagem: Inglês

10.1046/j.1523-1747.2003.12413.x

1523-1747

Rochelle L. Mandelcorn-Monson, Neil H. Shear, Eddy Yau, Suryaprakash Sambhara, Brian H. Barber, David Spaner, Mark DeBenedette,

Immunotherapy and Immune Responses

Vitiligo is a common depigmentation disorder thought to result from autoimmune destruction of melanocytes. Recent studies suggest a role for cell-mediated immune responses to melanocyte differentiation antigens, including gp100, MelanA/MART-1, and tyrosinase, in vitiligo pathogenesis. This study investigated T cell reactivity to MelanA/MART-1, tyrosinase, and gp100, in HLA-A2-positive patients with vitiligo. Melanocyte-specific T cell responses were measured ex vivo via enzyme-linked immunospot assay following stimulation with MelanA/MART-1, tyrosinase, and modified gp100 epitopes. Antigen-specific T lymphocyte reactivity to gp100 peptides was seen in 15 of 17 (88%) patients, with many demonstrating very high reactivity at levels comparable with those observed with common recall antigens. Reactivity to gp100 was noted to be associated with disease activity. Antigen-specific T lymphocyte reactivity to MelanA/MART-1 and tyrosinase peptides was not observed ex vivo in our patients, and only one patient demonstrated responses to MelanA/MART-1 and tyrosinase peptides following in vitro re-stimulation. Our findings implicate T cell reactivity to gp100 in patients with active disease and support the concept of an immunopathologic mechanism in vitiligo, in which cell-mediated responses to normal melanocyte antigens play a crucial part. Vitiligo is a common depigmentation disorder thought to result from autoimmune destruction of melanocytes. Recent studies suggest a role for cell-mediated immune responses to melanocyte differentiation antigens, including gp100, MelanA/MART-1, and tyrosinase, in vitiligo pathogenesis. This study investigated T cell reactivity to MelanA/MART-1, tyrosinase, and gp100, in HLA-A2-positive patients with vitiligo. Melanocyte-specific T cell responses were measured ex vivo via enzyme-linked immunospot assay following stimulation with MelanA/MART-1, tyrosinase, and modified gp100 epitopes. Antigen-specific T lymphocyte reactivity to gp100 peptides was seen in 15 of 17 (88%) patients, with many demonstrating very high reactivity at levels comparable with those observed with common recall antigens. Reactivity to gp100 was noted to be associated with disease activity. Antigen-specific T lymphocyte reactivity to MelanA/MART-1 and tyrosinase peptides was not observed ex vivo in our patients, and only one patient demonstrated responses to MelanA/MART-1 and tyrosinase peptides following in vitro re-stimulation. Our findings implicate T cell reactivity to gp100 in patients with active disease and support the concept of an immunopathologic mechanism in vitiligo, in which cell-mediated responses to normal melanocyte antigens play a crucial part. enzyme-linked immunospot peripheral blood mononuclear cells glycoprotein 100 cytotoxic T cell Vitiligo is a common acquired depigmentation disorder characterized by loss of epidermal melanocytes (Kovacs, 1998Kovacs S.O. Vitiligo.J Am Acad Dermatol. 1998; 38: 647-666Abstract Full Text Full Text PDF PubMed Scopus (331) Google Scholar). Although the precise etiology of vitiligo is unknown, an autoimmune mechanism has been proposed (Kemp et al., 2001Kemp E.H. Waterman E.A. Weetman A.P. Autoimmune aspects of vitiligo.Autoimmunity. 2001; 34: 65-77Crossref PubMed Scopus (105) Google Scholar). This stems from the fact that many vitiligo patients also exhibit other autoimmune disorders, such as autoimmune thyroiditis, Addison's disease, alopecia areata, pernicious anemia, and type I diabetes (Kemp et al., 2001Kemp E.H. Waterman E.A. Weetman A.P. Autoimmune aspects of vitiligo.Autoimmunity. 2001; 34: 65-77Crossref PubMed Scopus (105) Google Scholar). Further support for this hypothesis is provided by the finding that many vitiligo patients have antibodies to melanocytes (Naughton et al., 1983Naughton G.K. Eisinger M. Bystryn J.C. Antibodies to normal human melanocytes in vitiligo.J Exp Med. 1983; 158: 246-251Crossref PubMed Scopus (191) Google Scholar;Bystryn and Naughton, 1985Bystryn J.C. Naughton G.K. The significance of vitiligo antibodies.J Dermatol. 1985; 12: 1-9Crossref PubMed Scopus (50) Google Scholar;Song et al., 1994Song Y.H. Connor E. Li Y. Zorovich B. Balducci P. Maclaren N. The role of tyrosinase in autoimmune vitiligo.Lancet. 1994; 344: 1049-1052Abstract PubMed Scopus (161) Google Scholar;Kemp et al., 1998Kemp E.H. Gawkrodger D.J. Watson P.F. Weetman A.P. Autoantibodies to human melanocyte-specific protein pmel17 in the sera of vitiligo patients: A sensitive and quantitative radioimmunoassay (RIA).Clin Exp Immunol. 1998; 114: 333-338Crossref PubMed Scopus (59) Google Scholar), and the extent of depigmentation is correlated with the incidence and level of anti-melanocyte antibodies (Naughton et al., 1983Naughton G.K. Eisinger M. Bystryn J.C. Antibodies to normal human melanocytes in vitiligo.J Exp Med. 1983; 158: 246-251Crossref PubMed Scopus (191) Google Scholar). Moreover, patients may respond to immunosuppressive treatments, such as psoralen with ultra-violet A radiation (Parrish et al., 1976Parrish J.A. Fitzpatrick T.B. Shea C. Pathak M.A. Photochemotherapy of vitiligo. Use of orally administered psoralens and a high-intensity long-wave ultraviolet light system.Arch Dermatol. 1976; 112: 1531-1534Crossref PubMed Scopus (194) Google Scholar), topical corticosteroids (Kumari, 1984Kumari J. Vitiligo treated with topical clobetasol propionate.Arch Dermatol. 1984; 120: 631-635Crossref PubMed Scopus (93) Google Scholar), and cytotoxic drugs (Tsuji and Hamada, 1983Tsuji T. Hamada T. Topically administered fluorouracil in vitiligo.Arch Dermatol. 1983; 119: 722-727Crossref PubMed Scopus (83) Google Scholar). Recent evidence has emerged for a role for cell-mediated immunity in vitiligo pathogenesis. Infiltrating activated T lymphocytes have been observed at the periphery of vitiligo lesions (Badri et al., 1993Badri A.M. Todd P.M. Garioch J.J. Gudgeon J.E. Stewart D.G. Goudie R.B. An immunohistological study of cutaneous lymphocytes in vitiligo.J Pathol. 1993; 0: 149-155Crossref Scopus (131) Google Scholar). A recent immunopathologic study of lesional skin of vitiligo patients noted a high frequency of cutaneous lymphocyte antigen-positive activated cytotoxic T cells clustered in perilesional skin in the vicinity of disappearing melanocytes (van den Wijngaard et al., 2000van den Wijngaard R. Wankowicz-Kalinska A. Le Poole C. Tigges B. Westerhof W. Das P. Local immune response in skin of generalized vitiligo patients. Destruction of melanocytes is associated with the prominent presence of CLA+ T cells at the perilesional site.Lab Invest. 2000; 80: 1299-1309Crossref PubMed Scopus (215) Google Scholar). Melanocytes in close proximity to activated lymphocytes focally expressed HLA-DR and intercellular adhesion molecule-1, suggesting a major role for skin-homing T cells in melanocyte death (van den Wijngaard et al., 2000van den Wijngaard R. Wankowicz-Kalinska A. Le Poole C. Tigges B. Westerhof W. Das P. Local immune response in skin of generalized vitiligo patients. Destruction of melanocytes is associated with the prominent presence of CLA+ T cells at the perilesional site.Lab Invest. 2000; 80: 1299-1309Crossref PubMed Scopus (215) Google Scholar). An increased level of soluble interleukin (IL)-2 receptor (Caixia et al., 1999Caixia T. Hongwen F. Xiran L. Levels of soluble interleukin-2 receptor in the sera and skin tissue fluids of patients with vitiligo.J Dermatol Sci. 1999; 21: 59-62Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar;Yeo et al., 1999Yeo U.C. Yang Y.S. Park K.B. Sung H.T. Jung S.Y. Lee E.S. Shin M.H. Serum concentration of the soluble interleukin-2 receptor in vitiligo patients.J Dermatol Sci. 1999; 19: 182-188Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar), IL-6, and IL-8 (Yu et al., 1997Yu H.S. Chang K.L. Li H.F. Wu M.T. Wu C.S. Wu C.S. Alterations in IL-6, IL-8, GM-CSF, TNF-alpha, and IFN-gamma release by peripheral mononuclear cells in patients with active vitiligo.J Invest Dermatol. 1997; 108: 527-529Abstract Full Text PDF PubMed Scopus (85) Google Scholar), has been observed in vitiligo patients, which also suggests that T cell activation may be a component in vitiligo pathogenesis. Further evidence for a part played by cytotoxic T cells (CTL) in vitiligo stems from studies of melanoma patients. Vitiligo is found more frequently in patients with metastatic melanoma (Nordlund et al., 1983Nordlund J.J. Kirkwood J.M. Forget B.M. Milton G. Albert D.M. Lerner A.B. Vitiligo in patients with metastatic melanoma: A good prognostic sign.J Am Acad Dermatol. 1983; 9: 689-696Abstract Full Text PDF PubMed Scopus (259) Google Scholar;Bystryn et al., 1987Bystryn J.C. Rigel D. Friedman R.J. Kopf A. Prognostic significance of hypopigmentation in malignant melanoma.Arch Dermatol. 1987; 123: 1053-1055Crossref PubMed Scopus (184) Google Scholar;Cui and Bystryn, 1995Cui J. Bystryn J.C. Melanoma and vitiligo are associated with antibody responses to similar antigens on pigment cells.Arch Dermatol. 1995; 131: 314-318Crossref PubMed Scopus (86) Google Scholar;Cavallari et al., 1996Cavallari V. Cannavo S.P. Ussia A.F. Moretti G. Albanese A. Vitiligo associated with metastatic malignant melanoma.Int J Dermatol. 1996; 35: 738-740Crossref PubMed Scopus (16) Google Scholar) and is associated with an improved prognosis (Nordlund et al., 1983Nordlund J.J. Kirkwood J.M. Forget B.M. Milton G. Albert D.M. Lerner A.B. Vitiligo in patients with metastatic melanoma: A good prognostic sign.J Am Acad Dermatol. 1983; 9: 689-696Abstract Full Text PDF PubMed Scopus (259) Google Scholar;Bystryn et al., 1987Bystryn J.C. Rigel D. Friedman R.J. Kopf A. Prognostic significance of hypopigmentation in malignant melanoma.Arch Dermatol. 1987; 123: 1053-1055Crossref PubMed Scopus (184) Google Scholar). Vitiligo-like depigmentation has been observed following successful immunotherapy of melanoma (Rosenberg and White, 1996Rosenberg S.A. White D.E. Vitiligo in patients with melanoma: Normal tissue antigens can be targets for cancer immunotherapy.J Immunother Emphasis Tumor Immunol. 1996; 19: 81-84Crossref PubMed Scopus (292) Google Scholar;Nestle et al., 1998Nestle F.O. Alijagic S. Gilliet M. et al.Vaccination of melanoma patients with peptide- or tumor lysate-pulsed dendritic cells.Nat Med. 1998; 4: 328-332Crossref PubMed Scopus (2640) Google Scholar;van Elsas et al., 1999van Elsas A. Hurwitz A.A. Allison J.P. Combination immunotherapy of B16 melanoma using anti-cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) and granulocyte/macrophage colony-stimulating factor (GM-CSF)-producing vaccines induces rejection of subcutaneous and metastatic tumors accompanied by autoimmune depigmentation.J Exp Med. 1999; 190: 355-366Crossref PubMed Scopus (820) Google Scholar;Yee et al., 2000Yee C. Thompson J.A. Roche P. et al.Melanocyte destruction after antigen-specific immunotherapy of melanoma: Direct evidence of T cell-mediated vitiligo.J Exp Med. 2000; 192: 1637-1644Crossref PubMed Scopus (368) Google Scholar), including high-dose IL-2 therapy (Rosenberg and White, 1996Rosenberg S.A. White D.E. Vitiligo in patients with melanoma: Normal tissue antigens can be targets for cancer immunotherapy.J Immunother Emphasis Tumor Immunol. 1996; 19: 81-84Crossref PubMed Scopus (292) Google Scholar) and infusions of peptide-pulsed dendritic cells (Nestle et al., 1998Nestle F.O. Alijagic S. Gilliet M. et al.Vaccination of melanoma patients with peptide- or tumor lysate-pulsed dendritic cells.Nat Med. 1998; 4: 328-332Crossref PubMed Scopus (2640) Google Scholar) and MelanA/MART-1-specific CTL clones (Yee et al., 2000Yee C. Thompson J.A. Roche P. et al.Melanocyte destruction after antigen-specific immunotherapy of melanoma: Direct evidence of T cell-mediated vitiligo.J Exp Med. 2000; 192: 1637-1644Crossref PubMed Scopus (368) Google Scholar). Furthermore, cytotoxic T cells generated from melanoma tissue also recognize differentiation antigens expressed by normal melanocytes (Kawakami et al., 2000Kawakami Y. Dang N. Wang X. et al.Recognition of shared melanoma antigens in association with major HLA-A alleles by tumor infiltrating T lymphocytes from 123 patients with melanoma.J Immunother. 2000; 23: 17-27Crossref PubMed Scopus (85) Google Scholar;Yee et al., 2000Yee C. Thompson J.A. Roche P. et al.Melanocyte destruction after antigen-specific immunotherapy of melanoma: Direct evidence of T cell-mediated vitiligo.J Exp Med. 2000; 192: 1637-1644Crossref PubMed Scopus (368) Google Scholar), suggesting a link between autoimmunity and tumor immunity. The possibility that melanocyte differentiation antigens represent the molecular targets of a cell-mediated autoimmune response in vitiligo has recently been investigated. These antigens include melanosomal proteins, such as MelanA/MART-1, tyrosinase, and gp100, involved in biosynthesis of melanin (Norlund et al, 1983). Recent studies have noted a specific cellular immune response predominantly directed against the melanosomal protein Melan-A/MART-1 in HLA-A2-positive vitiligo patients (Ogg et al., 1998Ogg G.S. Rod D.P. Romero P. Chen J.L. Cerundolo V. High frequency of skin-homing melanocyte-specific cytotoxic T lymphocytes in autoimmune vitiligo.J Exp Med. 1998; 188: 1203-1208Crossref PubMed Scopus (365) Google Scholar;Lang et al., 2001Lang K.S. Caroli C.C. Muhm A. et al.HLA-A2 restricted, melanocyte-specific CD8(+) T lymphocytes detected in vitiligo patients are related to disease activity and are predominantly directed against MelanA/MART1.J Invest Dermatol. 2001; 116: 891-897Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar;Palermo et al., 2001Palermo B. Campanelli R. Garbelli S. et al.Specific cytotoxic T lymphocyte responses against Melan-A/MART1, tyrosinase and gp100 in vitiligo by the use of major histocompatibility complex/peptide tetramers: The role of cellular immunity in the etiopathogenesis of vitiligo.J Invest Dermatol. 2001; 117: 326-332Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar), where CD8+ T cells displaying MelanA/MART-1-specific reactivity ex vivo were demonstrated in the peripheral blood of these patients. In this study we investigated T cell reactivity to MelanA/MART-1, tyrosinase, and gp100, in vitiligo patients, in order to elucidate the role of cell-mediated immunity in disease pathogenesis. Herein we show that T cell reactivity to the modified forms of gp100 epitopes can be readily detected ex vivo in vitiligo patients with active disease, suggesting that a component of this cell-mediated autoimmune disease is directed to the melanocyte gp100 protein. We also show that there is an association of anti-gp100 reactivity with the active disease state of individual patients in our cohort of HLA-A2-positive individuals. Study subjects consisted of 27 patients with nonsegmental vitiligo each of whom had been seen at the Cosmetic Clinic at the Sunnybrook and Women's College Health Sciences Center Dermatology Unit. Following informed consent, historical data were obtained via direct interview, questionnaire, and chart review. Patients were questioned regarding the progression of their vitiligo as well as their personal and family history of vitiligo and associated autoimmune diseases, including thyroiditis, alopecia areata, pernicious anemia, myasthenia gravis, and type I diabetes. Patients were classified as having progressive disease if they had developed new lesions within the previous 6 mo. A family history was regarded as being positive if there was one or more first, second, or third degree relatives affected. Patients and blood samples were assigned a number to ensure blinding of laboratory investigators to the subjects' clinical characteristics. Immunologic analysis by patient number was completed prior to amalgamation with historical data. This study was approved by the Institutional Review board of Sunnybrook and Women's College Health Sciences Center (SWCHSC), and all patients signed informed consents before giving blood. HLA-A2-positive patients were identified by flow cytometry using fluorescein isothiocyanate conjugated allele-specific antibody BB7.2 (Parham and Brodsky, 1981Parham P. Brodsky F.M. Partial purification and some properties of BB7.2. A cytotoxic monoclonal antibody with specificity for HLA-A2 and a variant of HLA-A28.Hum Immunol. 1981; 3: 277-299Crossref PubMed Scopus (282) Google Scholar). Of the total of 27 patients screened for the study, 18 were found to be HLA-A2 positive and this group constitutes the study population. Eight healthy asymptomatic HLA-A2-positive individuals were also included as a control group. Peripheral blood mononuclear cells (PBMC) were isolated from 40 to 50 mL heparinized blood samples by density gradient centrifugation over Ficoll-Hypaque 1.077 (Amersham Pharmacia, Sweden). PBMC were washed with Hank's balanced salt solution (Gibco-BRL Burlington, Ontario, Canada) and frozen in RPMI-1640 medium (Gibco-BRL) containing 20% AB serum (Sigma Oakville, Ontario, Canada) and 10% dimethyl sulfoxide and stored in liquid nitrogen until analysis. The following HLA-A2-restricted peptides were used for stimulation in the ELISPOT assay. For gp100 protein: the modified epitopes 209.2m (IMDQVPSFV) and 280.9v (YLEPGPVTV) (Parkhurst et al., 1996Parkhurst M.R. Salgaller M.L. Southwood S. Robbins P.F. Sette A. Rosenberg S.A. Kawakami Y. Improved induction of melanoma-reactive CTL with peptides from the melanoma antigen gp100 modified at HLA-A*0201-binding residues.J Immunol. 1996; 157: 2539-2548PubMed Google Scholar); for MelanA/MART-1: AAGIGILTV (Coulie et al., 1994Coulie P.G. Brichard V. Van Pel A. et al.A new gene coding for a differentiation antigen recognized by autologous cytolytic T lymphocytes on HLA-A2 melanomas.J Exp Med. 1994; 180: 35-42Crossref PubMed Scopus (857) Google Scholar); and for tyrosinase: YMDGTMSQV (Skipper et al., 1996Skipper J.C. Hendrickson R.C. Gulden P.H. et al.An HLA-A2-restricted tyrosinase antigen on melanoma cells results from posttranslational modification and suggests a novel pathway for processing of membrane proteins.J Exp Med. 1996; 183: 527-534Crossref PubMed Scopus (368) Google Scholar). Also included were GILGFVFTL (Gotch et al., 1987Gotch F. Rothbard J. Howland K. Townsend A. McMichael A. Cytotoxic T lymphocytes recognize a fragment of influenza virus matrix protein in association with HLA-A2.Nature. 1987; 326: 881-882Crossref PubMed Scopus (332) Google Scholar), the dominant epitope from influenza A (Flu) matrix protein (MP), NLVPMVATV (Wills et al., 1996Wills M.R. Carmichael A.J. Mynard K. Jin X. Weekes M.P. Plachter B. Sissons J.G. The human cytotoxic T-lymphocyte (CTL) response to cytomegalovirus is dominated by structural protein pp65. Frequency, specificity, and T-cell receptor usage of pp65-specific CTL.J Virol. 1996; 70: 7569-7579PubMed Google Scholar) from cytomegalovirus (CMV) p65, and the HIV p17Gag protein derived peptide SLYNTVATL (Parker et al., 1992Parker K.C. Bednarek M.A. Hull L.K. et al.Sequence motifs important for peptide binding to the human MHC class I molecule, HLA-A2.J Immunol. 1992; 149: 3580-3587PubMed Google Scholar). All peptides were synthesized at Aventis Pasteur (Toronto, Canada), in an automated multiple peptide synthesizer (MPS-396, Advanced ChemTech, Louisville, KY), using standard Fmoc chemistry. The cleavage was performed on-line, using the reagent K TFA/water/thioanisole/phenol/ethanedithiol, 82.5:5:5:5:2.5). The crude peptides were precipitated with diethylethyl ether, washed (3×diethyl ether), dissolved in 20% acetonitrile and lypholized. The identities of the products were confirmed by ion spray mass spectroscopy. Nitrocellulose 96 well plates (MAHA 45, Millipore, Bedford, Massachusetts) were coated with 100 μL per well of human interferon (IFN)-γ specific antibody 5 μg per mL (clone 1-D1K, MABTECH, Stockholm, Sweden) in coating buffer (0.1 M Na2HPO4, pH 9.0) overnight at 4°C. Unbound antibody was removed by washing three times with phosphate-buffered saline. Nonspecific sites on the plates were blocked with 1% bovine serum albumin in phosphate-buffered saline for 1 h at room temperature. Plates were washed three times with phosphate-buffered saline and the appropriate number of responder PBMC were added. Plates were incubated overnight at 37°C, 5% CO2. Cells were discarded and plates were washed three times with deionized water, and five times with phosphate-buffered saline/Tween. Biotinylated anti-IFN-γ antibody (clone 7-B6-1, MABTECH) was added in 100 μL per well (1 μg per mL). Plates were then incubated for 3.5 h at room temperature. This was followed by the addition of a 1:500 dilution of extra avidin–alkaline phosphatase (Sigma) in 100 μL blocking buffer/Tween added per well and plates were incubated for 1 h at room temperature. The plates were developed using NBT/BCIP phosphatase substrate solution (Sigma) and blue spots were counted using an AID Inc. ELISPOT Image Analyzer and software (Cell Technology Inc. Jessup, MD). PBMC were thawed, washed, and incubated overnight in AIM-V medium (Gibco-BRL) containing 50 μM 2-mercaptoethanol (Sigma) at 37°C in 5% CO2. For the direct detection of IFN-γ secretion, 1×105 PBMC were added directly to the ELISPOT plate with the appropriate peptides listed in the figure legends. Plates were developed 18 h later. To attempt to allow peptide specific T cells to increase in numbers, PBMC were plated in complete AIM-V medium (10% AB serum, Valley Biomedical, [Winchester, VA] 50 μm 2-mercaptoethanol), at 1–2×106 cells per well in a 48 well plate. The next day, 10 μg per mL of peptides were added plus 10 ng per mL recombinant IL-7 (R&D Systems, Minneapolis, Minnesota). Recombinant IL-2 (Chiron, Emeryville, California) was added on days 2 and 6 at 20 IU per mL. Cells were harvested 9 d after stimulation and added to the ELISPOT plate at 1×105 cells per well. For each peptide-stimulated group, the same melanocyte-derived peptide was added to wells in triplicate. To control for nonspecific cell culture responses, a known HLA-A2 binding peptide from the HIV GAG protein was added to samples from each group. This served as a control from nonspecific responses. All peptides were added at a final concentration of 10 μg per mL. The Wilcoxon signed-rank test was used to compare responses between paired data. The Wilcoxon sum-of-ranks test for comparing two unmatched samples was used for testing differences in ELISPOT ratios between different subgroups of study subjects. p<0.05 was considered statistically significant. The ex vivo recall response to melanocyte peptide epitopes was measured in vitiligo patient PBMC by stimulating cultures with the HLA-A2-restricted peptides from MelanA/MART-1, tyrosinase, and gp100. To optimize the detection of cell-mediated anti-melanocyte activity to gp100, an admixture of both gp100 modified epitopes were added to the same culture wells. Modification of the gp100 epitopes 209 to 217 and 280 to 288, by substituting amino acids at the primary anchor residues for peptide/major histocompatibility complex binding, has been shown to increase major histocompatibility complex binding and improve CD8 T cell specific responses (Parkhurst et al., 1996Parkhurst M.R. Salgaller M.L. Southwood S. Robbins P.F. Sette A. Rosenberg S.A. Kawakami Y. Improved induction of melanoma-reactive CTL with peptides from the melanoma antigen gp100 modified at HLA-A*0201-binding residues.J Immunol. 1996; 157: 2539-2548PubMed Google Scholar). Previous reports have shown that the use of the modified gp100 epitopes can reveal CD8 T cell responses in the peripheral blood of patients with vitiligo (Palermo et al., 2001Palermo B. Campanelli R. Garbelli S. et al.Specific cytotoxic T lymphocyte responses against Melan-A/MART1, tyrosinase and gp100 in vitiligo by the use of major histocompatibility complex/peptide tetramers: The role of cellular immunity in the etiopathogenesis of vitiligo.J Invest Dermatol. 2001; 117: 326-332Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar) and melanoma (Nielsen et al., 2000Nielsen M.B. Monsurro V. Migueles S.A. et al.Status of activation of circulating vaccine-elicited CD8+ T cells.J Immunol. 2000; 165: 2287-2296Crossref PubMed Scopus (102) Google Scholar). We chose to assay for melanocyte-specific immunity using a short-term ex vivo assay designed to optimize the detection of cell-mediated anti-melanocyte activity without the need of an in vitro re-stimulation step. In vitro re-stimulation of PBMC cultures can result in the introduction of nonspecific T cell activation, which can mask peptide specific responses. In addition, culturing PBMC with peptides may drive primary responses instead of measuring true recall responses to previously seen antigen. Measurement of peptide-specific CD8+ T cell activity immediately ex vivo may more realistically reflect the activation state of peptide-specific CTL present in the peripheral blood. As a measure of antigen-specific T cell effector function, secretion of IFN-γ was monitored to determine the frequency of responding CD8+ T cells using the ELISPOT assay. To provide a reference point for the magnitude of the T cell response to melanocyte peptides, patient samples were also screened for peptide-specific responses to a mixture of CMV and Flu peptides representing the immunodominant HLA-A2 epitopes. The admixture of both Flu and CMV epitopes was done to gauge the overall capacity of T cells in the periphery to produce IFN-γ upon peptide stimulation immediately ex vivo. We reasoned that as viral infection with either Flu or CMV generates strong recall responses, their dominant epitopes would be good markers to measure IFN-γ produced by antigen-experienced CTL. To delineate reactivity of antigen-specific CD8+ T cells to melanocyte antigens in vitiligo patients, the number of spots generated by ELISPOT assay to test peptides was compared with the number of spots generated to control peptide for each patient. The control peptide was tested as a measure of overall background reactivity. To quantify reactivity to the melanocyte peptides, a ratio was determined by dividing the number of spots generated to test peptides on ELISPOT assay by the number of spots generated to control peptide. A ratio of more than 1.0 was indicative of reactivity to melanocyte peptides, whereas a ratio of 1.0 or less indicated no peptide reactivity (Table I). Significant reactivity to gp100 peptides relative to control peptide was demonstrated in HLA-A2-positive vitiligo patients (median ratio 3.33, percentile25–75 (1.5, 20.0), 95% confidence interval (2.3812, 9.1057); p=0.0001, one-sided signed-rank test (Table I). Fifteen of 17 patients (88%) demonstrated reactivity to gp100, with ratios greater than 1.0. Eight of 17 patients had at least a 4-fold greater reactivity above background where the magnitude of the anti-gp100 response approached or surpassed the level seen with the Flu/CMV combination. Figure 1 depicts melanocyte peptide reactivity in a sample of patients and is representative of the type of results generated in our assay. Reactivity to gp100 was not demonstrated among eight healthy HLA-A2-positive control donor PBMC (Figure 2).Table IClinical characteristics and melanocyte peptide-specific CTL reactivity ex vivo in HLA-A2+ vitiligo patientsPatient characteristicsPeptide-specific CTL reactivityaShown is the number of spots seen on ELISPOT assay/1×105 PBMC after ex vivo stimulation with melanocyte, HIV, and CMV/Flu peptide antigens.PatientSexActive disease?Other autoimmune disease?bIncluding thyroiditis, alopecia areata, pernicious anemia, myasthenia gravis, and type I diabetes.Family history?HIV control peptideGp100 peptides 209.2m/280.9vRatiocPeptide ratios were derived by dividing the number of spots generated to test peptides on ELISPOT assay by the number of spots generated to HIV control peptide. Note that 15 of 17 patients (88%) had gp100 reactivity ratios of greater than 1.0, and eight of 17 patients (47%) displayed reactivity to gp100 of at least 4-fold above control at levels comparable with those seen with the Flu/CMV combination, corresponding to a high level of CTL reactivity to gp100 in HLA-A2+ vitiligo patients (p<0.0001). In contrast, reactivity ratios of 1.0 to 2.0 to MART-1 and tyrosinase peptides were seen in only five of 18 patients (28%), and no patients displayed ratios of greater than 2.0, corresponding to an absence of CTL reactivity to MART-1 and tyrosinase antigens in our patient population. for Gp100Tyrosinase PeptideRatiocPeptide ratios were derived by dividing the number of spots generated to test peptides on ELISPOT assay by the number of spots generated to HIV control peptide. Note that 15 of 17 patients (88%) had gp100 reactivity ratios of greater than 1.0, and eight of 17 patients (47%) displayed reactivity to gp100 of at least 4-fold above control at levels comparable with those seen with the Flu/CMV combination, corresponding to a high level of CTL reactivity to gp100 in HLA-A2+ vitiligo patients (p<0.0001). In contrast, reactivity ratios of 1.0 to 2.0 to MART-1 and tyrosinase peptides were seen in only five of 18 patients (28%), and no patients displayed ratios of greater than 2.0, corresponding to an absence of CTL reactivity to MART-1 and tyrosinase antigens in our patient population. for tyrosinaseMART-1 peptideRatiocPeptide ratios were derived by dividing the number of spots generated to test peptides on ELISPOT assay by the number of spots generated to HIV control peptide. Note that 15 of 17 patients (88%) had gp100 reactivity ratios of greater than 1.0, and eight of 17 patients (47%) displayed reactivity to gp100 of at least 4-fold above control at levels comparable with those seen with the Flu/CMV combination, corresponding to a high level of CTL reactivity to gp100 in HLA-A2+ vitiligo patients (p<0.0001). In contrast, reactivity ratios of 1.0 to