HLA-A2 Restricted, Melanocyte-Specific CD8+ T Lymphocytes Detected in Vitiligo Patients are Related to Disease Activity and are Predominantly Directed Against MelanA/MART1
2001; Elsevier BV; Volume: 116; Issue: 6 Linguagem: Inglês
10.1046/j.1523-1747.2001.01363.x
ISSN1523-1747
AutoresKarl S. Lang, Alexandra Muhm, Arnaud Moris, Stefan Stevanović, Hans-Georg Rammensee, Constanze Charlotte Caroli, Dorothee Wernet, Birgit Schittek, Evelyn Knauss-Scherwitz, Claus Garbe,
Tópico(s)Immunotherapy and Immune Responses
ResumoVitiligo is a skin and hair disorder characterized by circumscribed depigmented lesions due to lack of melanocytes in the respective areas. It has been suggested that vitiligo is caused by an autoimmune-mediated destruction of melanocytes. Recently, the presence of a high frequency of skin-homing melanocyte-specific cytotoxic T lymphocytes in the peripheral blood of patients with vitiligo was reported. Our study examines the frequency of melanocyte-specific cytotoxic T lymphocytes in vitiligo patients and its relationship to disease activity. Thirty-two patients with moderate to active vitiligo and 17 control subjects were included. Melanocyte specific reactive CD8+ T cells were identified by enzyme-linked immunospot assay after stimulation with five peptides from gp100, four peptides from MelanA/MART1, and two peptides from tyrosinase. In selected patients, intracellular interferon-γ staining for the detection of specific reactive CD8+ T cells was additionally performed. In seven of 10 patients (70%) with actively progressive disease CD8+ T cells directed against melanocyte epitopes were detected, whereas only in four of 22 patients (18%) with moderate disease activity such specific reactivity was found. MelanA/MART1 peptides were immunodominant in nine patients reacting against EAAGIGILTV and three patients reacting against ILTVILGVL. Intracellular interferon-γ staining confirmed the findings obtained by the enzyme-linked immunospot technique. The present study supports the hypothesis that vitiligo is a cytotoxic T lymphocyte-mediated autoimmune disease. The presence of melanocyte-specific reactive CD8+ T cells seems to be closely related to disease activity. Vitiligo is a skin and hair disorder characterized by circumscribed depigmented lesions due to lack of melanocytes in the respective areas. It has been suggested that vitiligo is caused by an autoimmune-mediated destruction of melanocytes. Recently, the presence of a high frequency of skin-homing melanocyte-specific cytotoxic T lymphocytes in the peripheral blood of patients with vitiligo was reported. Our study examines the frequency of melanocyte-specific cytotoxic T lymphocytes in vitiligo patients and its relationship to disease activity. Thirty-two patients with moderate to active vitiligo and 17 control subjects were included. Melanocyte specific reactive CD8+ T cells were identified by enzyme-linked immunospot assay after stimulation with five peptides from gp100, four peptides from MelanA/MART1, and two peptides from tyrosinase. In selected patients, intracellular interferon-γ staining for the detection of specific reactive CD8+ T cells was additionally performed. In seven of 10 patients (70%) with actively progressive disease CD8+ T cells directed against melanocyte epitopes were detected, whereas only in four of 22 patients (18%) with moderate disease activity such specific reactivity was found. MelanA/MART1 peptides were immunodominant in nine patients reacting against EAAGIGILTV and three patients reacting against ILTVILGVL. Intracellular interferon-γ staining confirmed the findings obtained by the enzyme-linked immunospot technique. The present study supports the hypothesis that vitiligo is a cytotoxic T lymphocyte-mediated autoimmune disease. The presence of melanocyte-specific reactive CD8+ T cells seems to be closely related to disease activity. cluster of differentiation cutaneous lymphocyte antigen cytomegalovirus cytotoxic T lymphocytes enzyme-linked immunospot peripheral blood mononuclear cells PBS with 0.05% Tween 20 Vitiligo is a skin and hair disorder characterized by circumscribed depigmented lesions of variable size and shape, which tend to enlarge over time and in some cases cover the whole body. This disease was found at a prevalence of 0.4–1% of the world's population, regardless of sex, age, and color of the skin. In 30–40% there is a positive family history. The etiology of vitiligo is not yet clearly known (Chakraborty et al., 1996Chakraborty D.P. Roy S. Chakraborty A.K. Vitiligo, psoralen, and melanogenesis: some observations and understanding.Pigment Cell Res. 1996; 9: 107-116Crossref PubMed Scopus (42) Google Scholar;Maresca et al., 1997Maresca V. Roccella M. Roccella F. et al.Increased sensitivity to peroxidative agents as a possible pathogenic factor of melanocyte damage in vitiligo.J Invest Dermatol. 1997; 109: 310-313Crossref PubMed Scopus (231) Google Scholar), but it is believed by many authors that vitiligo is caused by an autoimmune-mediated destruction of melanocytes in the epidermis. It has been suggested that this destruction is caused by autoantibodies (Naughton et al., 1983Naughton G.K. Eisinger M. Bystryn J.C. Detection of antibodies to melanocytes in vitiligo by specific immunoprecipitation.J Invest Dermatol. 1983; 81: 540-542Crossref PubMed Scopus (119) Google Scholar;Norris et al., 1988Norris D.A. Kissinger R.M. Naughton G.M. Bystryn J.C. Evidence for immunologic mechanisms in human vitiligo: patients' sera induce damage to human melanocytes in vitro by complement-mediated damage and antibody-dependent cellular cytotoxicity.J Invest Dermatol. 1988; 90: 783-789Abstract Full Text PDF PubMed Google Scholar;Baharav et al., 1996Baharav E. Merimski O. Shoenfeld Y. et al.Tyrosinase as an autoantigen in patients with vitiligo.Clin Exp Immunol. 1996; 105: 84-88Crossref PubMed Scopus (96) Google Scholar;Hann et al., 1996Hann S.K. Koo S.W. Kim J.B. Park Y.K. Detection of antibodies to human melanoma cells in vitiligo and alopecia areata by Western blot analysis.J Dermatol. 1996; 23: 100-103PubMed Google Scholar;Fishman et al., 1997Fishman P. Merimski O. Baharav E. Shoenfeld Y. Autoantibodies to tyrosinase: the bridge between melanoma and vitiligo.Cancer. 1997; 79: 1461-1464https://doi.org/10.1002/(sici)1097-0142(19970415)79:8[#60]1461::aid-cncr3[#62]3.0.co]2-eCrossref PubMed Scopus (0) Google Scholar) or may be mediated by cellular immunity (Le et al., 1996Le P.I. van den Wijngaard R.M. Westerhof W. Das P.K. Presence of T cells and macrophages in inflammatory vitiligo skin parallels melanocyte disappearance.Am J Pathol. 1996; 148: 1219-1228PubMed Google Scholar;Mahmoud et al., 1998Mahmoud F. Abul H. al-Saleh Q. Haines D. Burleson J. Morgan G. Peripheral T-cell activation in non-segmental vitiligo.J Dermatol. 1998; 25: 637-640Crossref PubMed Scopus (20) Google Scholar). It is an interesting finding that vitiligo was found more frequently in melanoma patients (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 (258) Google Scholar;Duhra and Ilchyshyn, 1991Duhra P. Ilchyshyn A. Prolonged survival in metastatic malignant melanoma associated with vitiligo.Clin Exp Dermatol. 1991; 16: 303-305Crossref PubMed Scopus (57) 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 (82) 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). The prevalence of vitiligo in patients with metastatic melanoma is 2–4%, which is clearly higher than in the normal population. Furthermore, vitiligo was observed following successful immunotherapy of melanoma (Lacour et al., 1992Lacour J.P. Caldani C. Thyss A. Schneider M. Ortonne J.P. Vitiligo-like depigmentation and morpheas after specific intralymphatic immunotherapy for malignant melanoma.Dermatology. 1992; 184: 283-285Crossref PubMed Scopus (11) 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 (817) Google Scholar;Overwijk et al., 1999Overwijk W.W. Lee D.S. Surman D.R. et al.Vaccination with a recombinant vaccinia virus encoding a "self" antigen induces autoimmune vitiligo and tumor cell destruction in mice: requirement for CD4(+) T lymphocytes.Proc Natl Acad Sci USA. 1999; 96: 2982-2987https://doi.org/10.1073/pnas.96.6.2982Crossref PubMed Scopus (342) Google Scholar). Particularly after immunization of melanoma patients with peptide pulsed dendritic cells, triggering tumor peptide-specific cytotoxic T lymphocyte (CTL) response, and likewise, following high-dose interleukin-2 therapy, vitiligo-like depigmentations were documented (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 (291) 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 [see comments].Nat Med. 1998; 4: 328-332Crossref PubMed Scopus (2638) Google Scholar). Furthermore, the presence of clonally expanded T cells with identical BV regions has been observed in areas of melanocyte destruction of both vitiligo-like leukoderma and melanoma regression (Becker et al., 1999Becker J.C. Guldberg P. Zeuthen J. Brocker E.B. Straten P.T. Accumulation of identical T cells in melanoma and vitiligo-like leukoderma [see comments].J Invest Dermatol. 1999; 113: 1033-1038https://doi.org/10.1046/j.1523-1747.1999.00805.xCrossref PubMed Scopus (66) Google Scholar). Recently,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 (363) Google Scholar demonstrated the presence of a high frequency of skin-homing melanocyte-specific CTL in the peripheral blood of seven of nine patients with vitiligo. This study was planned in order to correlate the occurrence of such T cells with disease state. We established a sensitive enzyme-linked immunospot (ELISPOT) assay for detection of CD8+ T cells specific for 11 different peptides presented by melanocytes from MelanA/MART1, tyrosinase and gp100. Furthermore, we applied intracellular interferon (IFN) -γ staining as an additional marker for antigen specificity, and we examined the expression of the skin homing marker cutaneous lymphocyte antigen (CLA) on these cells. Thirty-two patients with active to moderately progressive vitiligo and 17 healthy asymptotic persons as control individuals were included in this study after obtaining informed consent. All of these study subjects were HLA-A*0201-positive. The disease classification of vitiligo was based on case history. Patients with 10% and higher increase of vitiligo areas during the last 3 mo were classified as actively progressive, patients with less increase but signs of progression for the last 6 mo were classified as moderately progressive, and patients without a clear increase of vitiligo areas during the last 6 mo were classified as having stable disease. Altogether, 137 patients with vitiligo were screened (76 with stable disease, 61 with active to moderately progressive disease; 32 of 61 were HLA-A*0201 positive). Ten of the 32 vitiligo patients included were classified as having actively progressive disease and 22 patients as having moderately progressive disease. Three of the 10 patients with actively progressive disease had a vitiligo associated autoimmune disease, two thyroid disease and one pernicious anemia. The 17 control individuals were not known to have familiar disposition of any autoimmune disorder. Peripheral blood mononuclear cells (PBMC) were isolated from 100 ml heparinized blood samples by density gradient centrifugation using Ficoll (FicoLite Linearis, Bettingen/Main, Germany). PBMC were washed four times with phosphate-buffered saline (PBS) in order to remove platelets. Thereafter, PBMC were resuspended in culture medium RPMI 1640 (GIBCO BRL, Karlsruhe, Germany) containing 24 mM NaHCO3 (Merck, Darmstadt, Germany), 10 mM HEPES (Roth, Karlsruhe, Germany), 1.6 mM glutamine (GIBCO BRL), 100 IU penicillin-streptomycin per ml (GIBCO BRL) and 10% fetal bovine serum (PAA Laboratories GmbH, 4020 Linz, Austria). Cells were used for the ELISPOT assay and CLA detection or frozen with culture medium containing 40% fetal bovine serum and 15% dimethylsulfoxide. PBMC were thawed at 37°C, and washed in culture medium if needed for further examinations. The following HLA-A2 restricted peptide epitopes from melanocyte proteins were used for stimulation in the ELISPOT assay: LLDGTATLRL (Kawakami et al., 1995Kawakami Y. Eliyahu S. Jennings C. et al.Recognition of multiple epitopes in the human melanoma antigen gp100 by tumor-infiltrating T lymphocytes associated with in vivo tumor regression.J Immunol. 1995; 154: 3961-3968PubMed Google Scholar) VLYRYGSFSV (Kawakami et al., 1995Kawakami Y. Eliyahu S. Jennings C. et al.Recognition of multiple epitopes in the human melanoma antigen gp100 by tumor-infiltrating T lymphocytes associated with in vivo tumor regression.J Immunol. 1995; 154: 3961-3968PubMed Google Scholar), ITDQVPFSV (Kawakami et al., 1995Kawakami Y. Eliyahu S. Jennings C. et al.Recognition of multiple epitopes in the human melanoma antigen gp100 by tumor-infiltrating T lymphocytes associated with in vivo tumor regression.J Immunol. 1995; 154: 3961-3968PubMed Google Scholar), and YLEPGPVTA (Cox et al., 1994Cox A.L. Skipper J. Chen Y. et al.Identification of a peptide recognized by five melanoma-specific human cytotoxic T cell lines.Science. 1994; 264: 716-719Crossref PubMed Scopus (798) Google Scholar) from gp100. From tyrosinase we investigated MLLAVLYCL (Wolfel et al., 1994Wolfel T. Van Pel A. Brichard V. Schneider J. Seliger B. Meyer zBKH, Boon T. Two tyrosinase nonapeptides recognized on HLA-A2 melanomas by autologous cytolytic T lymphocytes.Eur J Immunol. 1994; 24: 759-764Crossref PubMed Scopus (379) Google Scholar) and YMNGTMSQV (Wolfel et al., 1994Wolfel T. Van Pel A. Brichard V. Schneider J. Seliger B. Meyer zBKH, Boon T. Two tyrosinase nonapeptides recognized on HLA-A2 melanomas by autologous cytolytic T lymphocytes.Eur J Immunol. 1994; 24: 759-764Crossref PubMed Scopus (379) Google Scholar), which can post-translationally be modified in 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). AAGIGILTV (Coulie et al., 1994Coulie P.G. Van Brichard V. Pel A. et al.A new gene coding for a differentiation antigen recognized by autologous cytolytic T lymphocytes on HLA-A2 melanomas [see comments].J Exp Med. 1994; 180: 35-42Crossref PubMed Scopus (855) Google Scholar), ILTVILGVL (Castelli et al., 1995Castelli C. Storkus W.J. Maeurer M.J. et al.Mass spectrometric identification of a naturally processed melanoma peptide recognized by CD8+ cytotoxic T lymphocytes.J Exp Med. 1995; 181: 363-368Crossref PubMed Scopus (180) Google Scholar), EAAGIGILTV (Kawakami et al., 1994Kawakami Y. Eliyahu S. Sakaguchi K. et al.Identification of the immunodominant peptides of the MART-1 human melanoma antigen recognized by the majority of HLA-A2-restricted tumor infiltrating lymphocytes.J Exp Med. 1994; 180: 347-352Crossref PubMed Scopus (745) Google Scholar;Romero et al., 1997Romero P. Gervois N. Schneider J. et al.Cytolytic T lymphocyte recognition of the immunodominant HLA-A*0201-restricted Melan-A/MART-1 antigenic peptide in melanoma.J Immunol. 1997; 159: 2366-2374PubMed Google Scholar) and its modified form ELAGIGILTV (Valmori et al., 1998Valmori D. Fonteneau J.F. Lizana C.M. et al.Enhanced generation of specific tumor-reactive CTL in vitro by selected Melan-A/MART-1 immunodominant peptide analogues.J Immunol. 1998; 160: 1750-1758PubMed Google Scholar) from MelanA/MART1. A mixture of viral epitopes was used as a positive control: CLGGLLTMV (Lee et al., 1993Lee S.P. Thomas W.A. Murray R.J. et al.HLA A2.1-restricted cytotoxic T cells recognizing a range of Epstein-Barr virus isolates through a defined epitope in latent membrane protein LMP2.J Virol. 1993; 67: 7428-7435PubMed Google Scholar), LLDFVRFMGV (White et al., 1996White C.A. Cross S.M. Kurilla M.G. et al.Recruitment during infectious mononucleosis of CD3+CD4+CD8+ virus-specific cytotoxic T cells which recognise Epstein-Barr virus lytic antigen BHRF1.Virology. 1996; 219: 489-492https://doi.org/10.1006/viro.1996.0277Crossref PubMed Scopus (35) Google Scholar), and GLCTLVAML (Steven et al., 1997Steven N.M. Annels N.E. Kumar A. Leese A.M. Kurilla M.G. Rickinson A.B. Immediate early and early lytic cycle proteins are frequent targets of the Epstein-Barr virus-induced cytotoxic T cell response.J Exp Med. 1997; 185: 1605-1617Crossref PubMed Scopus (272) Google Scholar) from Epstein–Barr virus (EBV), GILGFVFTL (Bednarek et al., 1991Bednarek M.A. Sauma S.Y. Gammon M.C. Porter G. Tamhankar S. Williamson A.R. Zweerink H.J. The minimum peptide epitope from the influenza virus matrix protein. Extra and intracellular loading of HLA-A2.J Immunol. 1991; 147: 4047-4053PubMed Google Scholar) from influenza and 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 pp 65: frequency, specificity, and T-cell receptor usage of pp 65-specific CTL.J Virol. 1996; 70: 7569-7579PubMed Google Scholar) from cytomegalovirus (CMV). Reaction against the peptide YLLPAIVHI (Hunt et al., 1992Hunt D.F. Henderson R.A. Shabanowitz J. et al.Characterization of peptides bound to the class I MHC molecule HLA-A2.1 by mass spectrometry [see comments].Science. 1992; 255: 1261-1263Crossref PubMed Scopus (1008) Google Scholar) from p72 helicase was assessed as unspecific stimulation. Peptides and their origin are listed in Table I.Table IPeptides used for the detection of specifically reactive cytotoxic T cellsPeptide sequenceProteinPositionOriginKTWGQYWQVgp100154–162HumanITDQVPFSVgp100209–217HumanYLEPGPVTAgp100280–288HumanLLDGTATLRLgp100457–466HumanVLYRYGSFSVgp100476–485HumanMLLAVLYCLTyrosinase1–9HumanYMNGTMSQVTyrosinase369–377HumanAAGIGILTVMelanA27–35HumanEAAGIGILTVMelanA26–35HumanELAGIGILTVMelanA (modified)26–35HumanILTVILGVLMelanA32–40HumanYLLPAIVHIp72 RNA helicase146–154HumanGLCTLVAMLBMLF-1259–267EBVLLDFVRFMGVEBNA-6284–293EBVCLGGLLTMVlmp-2426–434EBVGILGFVFTLMatrix protein58–66Influenza ANLVPMVATVpp65495–503CMV Open table in a new tab Peptides were synthesized in an automated peptide synthesizer 432A (Applied Biosystems, Weiterstadt, Germany) following the Fmoc/tBu strategy. Synthesis products were analyzed by high-performance liquid chromatography (System gold, Beckman Instruments, Munich, Germany) and MALDI-TOF mass spectrometry (G2025A, Hewlett-Packard, Waldbronn, Germany). Peptides of less than 80% purity were purified by preparative high-performance liquid chromatography. Peptides were dissolved in dimethylsulfoxide in a concentration of 10 mg per ml and further diluted to a final concentration of 1 mg per ml peptide with H2O twice distilled. For generation of stimulator cells, TAP-deficient HLA-A2-positive T2 (Salcedo et al., 1994Salcedo M. Momburg F. Hammerling G.J. Ljunggren H.G. Resistance to natural killer cell lysis conferred by TAP1/2 genes in human antigen-processing mutant cells.J Immunol. 1994; 152: 1702-1708PubMed Google Scholar) were incubated with 50 µg peptide per ml for 8 h in fetal bovine serum free culture medium. At the end of peptide incubation T2 cells were irradiated with 170 Gy (Gammacell 1000 Elite, MDS Nordion, Kanata, Ontario, Canada) and unbound peptide was removed by washing with RPMI 1640 containing 10% fetal bovine serum. Nitrocellulose 96 well plates (MAHA 45, Millipore, Bedford, MA) were coated with 50 µl per well human IFN-γ-specific antibody (20 µg per ml; Biosource, Camarillo, CA) diluted in coating buffer (35 mM sodium bicarbonate, 15 mM sodium carbonate and 3 mM sodium acid) for 3 h at 37°C. Unbound antibody was removed by four washing steps with PBS. Remaining protein binding sites of the nitrocellulose plate were blocked with culture medium for 1 h at 37°C. PBMC (50,000 per well) and peptide-loaded T2 cells (70,000 per well) were co-cultured in triplicates in coated nitrocellulose 96 well plates. After 40 h of incubation at 37°C, 7% CO2 cells were removed by washing seven times with PBS containing 0.05% Tween 20 (PBS/T). 50 µl of biotinylated human IFN-γ antibody (Biosource), diluted to 2 µg per ml in PBS containing 0.5% bovine serum albumin and 0.02% sodium azide, was used for the detection of bound IFN-γ. After 3 h unbound antibodies were removed by six washes with PBS/T and 50 µl of avidin peroxidase complex (ABC Vectastain-Elite Kit, Vector Laboratories, Burlingame, CA) was added. Two hours after addition of avidin peroxidase the plate was washed three times with PBS/T and three times with PBS. In the last washing step the complete plate was submerged in PBS. Subsequently, the reaction was developed with 3-amino 9-ethyl carbazole (Sigma, St Louis, MO). After 5 min the color reaction was stopped by rinsing with water. Computer-assisted video image analysis was used to count spots (Carl Zeiss Vision GmbH, Hallbergmoos, Germany). Pictures of wells were digitized by image software. The difference of contrast was checked for several parameters such as area, saturation, shape, slope, contrast, and color. Only spots corresponding to the parameters determined were counted. The number of spots for the negative control peptide (YLLPAIVHI) was subtracted from the number of spots for peptides. The result is taken as the number of specifically reactive spots. If more spots were found in the control sample than in the test peptide samples negative values resulted. Values of the control collective consisting of 17 healthy persons were taken as reference values. The cut-off level was defined as the mean value of the control collective plus 2 SD. This means that 97.7% of the control persons were defined as the normal range of values and that 2.3% of control persons were expected to show elevated values. Cut-off level was calculated separately for each peptide tested. Higher values than defined by the cut-off level were regarded as representing specific PBMC reactivity. The Cytofix/Cytoperm kit (PharMingen, San Diego, CA) was used for intracellular IFN-γ staining. Thawed cells were incubated for 3 h in a 24 well culture plate at 37°C. This leads to adherence of monocytes. The monocyte-free supernatant was used for further studies. Clusters of dead cells were filtered through a cell strainer (40 µm; Nylon, Falcon, Becton Dickinson, New York, NY). Peripheral blood lymphocytes (106 per ml) were incubated in 4.5 ml polystyrene tubes (Greiner GmbH, Frickenhausen, Germany) with 106 peptide loaded T2 cells (2 h, 37°C). To stop IFN-γ secretion Golgi Stop (Cytofix/Cytoperm Kit, PharMingen) was added (0.7 µl per ml). After another 10 h, cells were washed with PBS and stained in fluorescence-activated cell sorter-buffer with 10 µg/100 µl CD8 tricolor antibody (CALTAG Laboratories, Burlingame, CA) and 20 µl fluorescein isothiocyanate labeled CLA antibody (rat IgM, PharMingen) for 30 min at 4°C. After removing unbound antibodies cells were fixed and permeabilized with 300 µl Cytofix/Cytoperm solution (Cytofix/Cytoperm Kit, PharMingen) for 20 min at 4°C. Cells were washed twice with 1 ml of Perm/Wash solution (Cytofix/Cytoperm Kit, PharMingen) and stained for 30 min in 100 μl Perm/wash solution with phycoerythrin-labeled mouse IFN-γ antibody (PharMingen) diluted to 1.5 µg per ml. Antibody was removed by two washes and cells were resuspended in PBS. Cells were analyzed on a FACSCalibur (Becton Dickinson, San Jose, CA) with logarithmic amplification through a viable cell gate determined by forward and side scatter. Fresh PBL (3 × 106) were incubated for 2 h in serum-free culture medium with 50 µg peptide per ml followed by co-culture with 106 autologous PBMC in a 24 well plate (Greiner GmbH, Frickenhausen, Germany). On day 4 culture medium was supplemented with 5% Lymphocult (Biotest, Germany) containing interleukin-2 and other unspecified T cell growth factors. T cell cultures were restimulated weekly with 3 × 106 peptide loaded autologous PBMC per well (31 Gy irradiated) and supplemented with Lymphocult. ELISPOT analysis was performed 7 d after restimulation. The Wilcoxon Sum-of-Ranks (Mann–Whitney) test for comparing two unmatched samples was used for testing differences in the ELISPOT values between different subgroups of study subjects. p < 0.05 was statistically significant. Of the 32 patients tested 10 had actively progressive disease and in 22 the disease progression was moderate. In the PBMC of 11 patients (34%) T cells against the tested epitopes were detected. Seven of them had active disease. Four patients who reacted against the peptides had moderate disease progression (Table II).Table IIPatient's characteristics, disease activity, and specific CTL reactivityaReactions with more spots than the 97.7 percentile (mean ± 2 SD) of controls was assessed as low response (+), reactions higher than mean ± 3 SD was assessed as moderate (++), and reactions with more spots than mean ± 4 SD as strong (+++) response.Patient no.Sex/ageDuration of disease (mo)Disease activity% involved skinELAGIG ILTV (MART1)EAAGI GILTV (MART1)ILTVIL GVL (MART1)AAGIGI LTV (MART1)LLDGT ATLRL (gp100)VLYRY GSFSV (gp100)KTWGQ YWQV (gp100)YLEPG PVTA (gp100)ITDQV PFSV (gp100)YMNGT MSQV (tyr.)MLLAV LYCL (tyr.)4F/3046Active40++++++–––––––––5F/2954Active10–––––––––––9F/36120Active20++++++––++++–––––15F/56246Active10–+–––––––––18F/4754Active20–––––––––––19F/46174Active3––+++––––––+–22F/52114Active20–+–––––––––27F/2148Active10–––––––––––29F/4078Active80––+++–––––+–32M/4554Active15+++++++++++++++++––+++–1F/35174Moderate40–––––––––––2M/3854Moderate15–––––––––––3M/37210Moderate10–––––––––––6M/39120Moderate15–––––––––––7F/55120Moderate20–––––––––––8F/35354Moderate15+++++–––––+–+++10F/48294Moderate15–––––––––––11F/21174Moderate10–––––––––––12F/65272Moderate20–––––––––––13F/41282Moderate10–––––––––––14M/52294Moderate15–+–––––––––16F/64294Moderate90–+–––––––––17F/33306Moderate20–+–––––––––20F/34186Moderate25–––––––––––21M/69156Moderate10–––––––––––23F/5754Moderate90–––––––––––24F/1442Moderate15–––––––––––25M/4354Moderate3–––––––––––26M/36210Moderate15–––––––––––28F/51174Moderate5–––––––––––30F/53402Moderate75–––––––––––31F/4990Moderate8–––––––––––a Reactions with more spots than the 97.7 percentile (mean ± 2 SD) of controls was assessed as low response (+), reactions higher than mean ± 3 SD was assessed as moderate (++), and reactions with more spots than mean ± 4 SD as strong (+++) response. Open table in a new tab The most frequently encountered immunodominant epitopes were the MelanA/MART1 peptide EAAGIGILTV and its modified form ELAGIGILTV. PBMC of nine patients reacted against one or both of these peptides, reaching 70–150 spots per 105 cells. In none of the healthy individuals spot frequencies of this magnitude were observed (Figure 1). Patients reacted significantly stronger against the epitope EAAGILGITV than healthy controls (Wilcoxon, p = 0.0003). There was also a significant difference between patients with active disease and patients with moderate disease progression (Wilcoxon, p = 0.02). In two patients the peptide ILTVILGVL from MelanA/MART1 led to stimulation of T cells resulting in about 160 spots per 105 PBMC (Figure 1). This corresponded to 1–2% of the CD8+ cells in these patients (Figure 3). Another patient also reacted against this epitope, albeit at a lower frequency. Three patients reacted against peptides derived from the protein gp100. All of them also showed strong reactions against the MelanA/MART1 peptides EAAGIGILTV and its modified form ELAGIGILTV. Four patients were found reactive against tyrosinase peptides, they also reacted against the MelanA/MART1 peptides EAAGIGILTV or ILTVILGVL. The patients showing high numbers of positive spots against one peptide, usually had specific reactivity against several other peptides. In patient 32, reactivity against all four peptides deriving from MelanA/MART1 and against three gp100 peptides and one tyrosinase peptide was found. Patient 8 showed reactivity against five peptides and patient 9 against four peptides. Altogether, six of 11 patients reacted against several peptides. No reactivity in vitiligo patients was found against the gp100 peptide ITDQVPFSV. Weak reactivity in only one patient was observed against the gp100 peptide YLEPGPVTA and the tyrosinase peptide MLLAVLYCL. Only one of the control persons studied was found to show weak reactivity against the peptides YMNGTMSQV (tyrosinase) and ITDQVPFSV (gp100). Although the reactivity against the peptide VLYRYGSFSV was weak, we found a significant difference between patients and healthy donors (Wilcoxon, p = 0.004). Interestingly, 11 of the 32 patients showed a strong reaction against the viral epitope mixture (EBV, influenza A, CMV) compared with the normal individuals (Figure 1, Wilcoxon, p = 0.003). In further experiments we stimulated the strongest reacting three control persons and three patients with CMV peptide in the same EL
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