mart-1-antigen and Vitiligo

Vitiligo is characterized by progressive skin depigmentation resulting from an autoimmune response targeting epidermal melanocytes. Melanocytes are particularly immunogenic by virtue of the contents of their melanosomes, generating the complex radical scavenging molecule melanin in a process that involves melanogenic enzymes and structural components, including tyrosinase, MART-1, gp100, TRP-2 and TRP-1. These molecules are also prime targets of the immune response in both vitiligo and melanoma. The immunogenicity of melanosomal proteins can partly be explained by the dual role of melanosomes, involved both in melanin synthesis and processing of exogenous antigens. Melanocytes are capable of presenting antigens in the context of MHC class II, providing HLA-DR+ melanocytes in perilesional vitiligo skin the option of presenting melanosomal antigens in response to trauma and local inflammation. Type I cytokine-mediated immunity to melanocytes in vitiligo involves T cells reactive with melanosomal antigens, similar to T cells observed in melanoma. In vitiligo, however, T cell tuning allows T cells with higher affinity for melanocyte differentiation antigens to enter the circulation after escaping clonal deletion in primary lymphoid organs. The resulting efficacious and progressive autoimmune response to melanocytes provides a roadmap for melanoma therapy.

Topics: Animals; Antigen Presentation; Antigens, Neoplasm; Autoantigens; Autoimmunity; Clonal Deletion; gp100 Melanoma Antigen; HLA-DR Antigens; Humans; Intramolecular Oxidoreductases; MART-1 Antigen; Melanins; Melanosomes; Membrane Glycoproteins; Neoplasm Proteins; Oxidoreductases; Pigmentation; T-Lymphocytes; Vitiligo

In this study, we developed an in vivo vitiligo induction model to explore the underlying mechanisms leading to Koebner's phenomenon and to evaluate the efficacy of therapeutic strategies. The model consisted of 12 pigmented test regions on the back of generalized vitiligo patients that were exposed to three Koebner induction methods: cryotherapy, 755 nm laser therapy, and epidermal abrasion. In addition, four cream treatments (pimecrolimus, tacrolimus, steroid and placebo) were randomly applied. Koebnerization was efficiently induced by all three induction methods. In general, cryotherapy was the best method of Koebner induction, followed by 755 nm laser therapy and epidermal abrasion. Reproducible results were obtained, which showed enhanced depigmented surface areas and higher amounts of T lymphocytes in placebo-treated test zones compared to active treated areas. Tacrolimus and local steroids were better inhibitors of Koebner's process (P < 0.05) compared to pimecrolimus. Our in vivo vitiligo induction model is very informative to investigate vitiligo induction and to determine the efficacy of topical treatments in vitiligo. This proof of concept confirms the efficient comparison of head-to-head therapeutic strategies intra-individually in a standardized, specific and better timed way.

Topics: Administration, Cutaneous; Adult; Cryotherapy; Dermabrasion; Double-Blind Method; Female; gp100 Melanoma Antigen; Humans; Immunosuppressive Agents; Langerhans Cells; Low-Level Light Therapy; Male; MART-1 Antigen; Middle Aged; Mometasone Furoate; Ointments; Pregnadienediols; Reproducibility of Results; T-Lymphocyte Subsets; Tacrolimus; Triamcinolone Acetonide; Vitiligo

A phase I/II trial was performed to evaluate the safety and immunogenicity of a novel melanoma vaccine comprising six melanoma-associated peptides defined as antigenic targets for melanoma-reactive helper T cells. Source proteins for these peptides include MAGE proteins, MART-1/MelanA, gp100, and tyrosinase.. Thirty-nine patients with stage IIIB to IV melanoma were vaccinated with this six-peptide mixture weekly at three dose levels, with a preceding phase I dose escalation and subsequent random assignment among the dose levels. Helper T-lymphocyte responses were assessed by in vitro proliferation assay and delayed-type hypersensitivity skin testing. Patients with measurable disease were evaluated for objective clinical response by Response Evaluation Criteria in Solid Tumors.. Vaccination with the helper peptide vaccine was well tolerated. Proliferation assays revealed induction of T-cell responses to the melanoma helper peptides in 81% of patients. Among 17 patients with measurable disease, objective clinical responses were observed in two patients (12%), with response durations of 1 and 3.9+ years. Durable stable disease was observed in two additional patients for periods of 1.8 and 4.6+ years.. Results of this study support the safety and immunogenicity of a vaccine comprised of six melanoma helper peptides. There is also early evidence of clinical activity.

Topics: Adult; Aged; Aged, 80 and over; Antigens, Differentiation; Antigens, Neoplasm; Cancer Vaccines; Cell Proliferation; Female; gp100 Melanoma Antigen; Humans; Male; MART-1 Antigen; Melanocytes; Melanoma; Membrane Glycoproteins; Middle Aged; Monophenol Monooxygenase; Neoplasm Proteins; Neoplasm Staging; Skin Neoplasms; Skin Tests; T-Lymphocytes, Helper-Inducer; Vitiligo

Rhododendrol, 4-(4-hydroxyphenyl)-2-butanol, Rhododenol(®) (RD), a naturally occurring phenolic compound, was developed as a tyrosinase inhibitor for skin-lightening/whitening cosmetics. In 2013, skin depigmentation was reported in consumers using RD-containing skin-brightening cosmetics; this condition is called RD-induced leukoderma.. The etiology of RD-induced leukoderma is still largely unknown. Here, to assess the depigmentation potential of RD, we developed a new mouse model of leukoderma by topically applying RD.. Hairless hk14-SCF Tg mice with melanocytes distributed in the epidermis were used for this study. RD was applied on the dorsal skin of the mice daily for 28 days. Then, immunohistological, biochemical, and electron microscopic analyses were performed on biopsy samples taken from these mice.. The depigmentation in the RD-treated sites appeared on Day 14. Histological examination indicated a loss of epidermal melanocytes at Day 7. On the other hand, the melanocyte number did not decrease in the albino mice having the same background as the hairless hk14-SCF Tg, but without tyrosinase activity. Biochemical analyses showed that the eumelanin content decreased in the RD-treated sites and metabolites of RD-quinone, i.e., non-protein thiol adducts and protein-SH adducts, were produced. Electron microscopic analyses revealed double-membrane-walled structures containing electron-dense material, which might be typical for melanin-containing autophagosomes and a dilated endoplasmic reticulum (ER), which would indicate ER stress.. These data suggested that RD exerted tyrosinase-dependent melanocyte cytotoxicity and that tyrosinase-dependent accumulation of ER stress from activation of the autophagy pathway contributed to melanocyte cytotoxicity.

Topics: Administration, Topical; Animals; Asian People; Autophagy; Butanols; Cell Count; Disease Models, Animal; Endoplasmic Reticulum Chaperone BiP; Endoplasmic Reticulum Stress; Enzyme Inhibitors; Epidermal Cells; Epidermis; Heat-Shock Proteins; Humans; MART-1 Antigen; Melanins; Melanocytes; Mice; Mice, Hairless; Mice, Inbred C57BL; Mice, Transgenic; Models, Animal; Monophenol Monooxygenase; Skin Lightening Preparations; Skin Pigmentation; Vitiligo

Occurence of vitiligo lesions is caused by the destruction of melanocytes in a ected skin and therefore by the re- duction of pigment melanin content. Questions remain about the presence of residual melanocytes in the depigmented skin and optimal methods of their identi cation.. Skin biopsy samples from 16 patients with non-segmental vitiligo and from 10 healthy volunteers were investigated for Melan-A (A103 clone)+ melanocytes expression by immunohistochemical analysis and for melanin by histochemical studies with section staining by Fontana-Masson method.. For some patients including those with long-standing disease (up to 40 years) Melan-A+ cells and melanin granules were detected in depigmented skin as indication that the residual melanocytes are preserved in vitiligo lesions. More than three-fold decrease of Melan-A+ melanocytes amount was revealed in perilesional normally pigmented skin of vitiligo patients (P < 0.001) compared with the skin of healthy volunteers. Clinically intact skin involvement in the pathological process should be taken into consideration if local treatment methods are prescribed.. In some vitiligo patients the residual melanocytes are preserved in depigmented skin. Melan-A marker is useful for identi cation of melanocytes in vitiligo patients' skin.

Topics: Adult; Biomarkers; Case-Control Studies; Female; Humans; Male; MART-1 Antigen; Melanocytes; Middle Aged; Vitiligo; Young Adult

Patients with melanoma may develop skin depigmentation spontaneously or following therapy, referred to as melanoma-associated leucoderma (MAL). As clinical presentation of MAL may precede primary/metastatic melanoma detection, recognition of MAL is important to prevent its misdiagnosis as vitiligo and the subsequent application of immunosuppressive treatment. To reveal the immunity involved in MAL development, we investigated the presence of antibody and T-cell immune responses directed against the melanocyte-differentiation-antigens MART-1 (Melan-A), tyrosinase and gp100 in patients with MAL, as compared to patients with vitiligo. Autoantibodies to gp100 and tyrosinase were commonly found in both diseases. Interestingly, MART-1 antibodies were only present in patients with MAL. Melanocyte antigen-specific T cells were found in all patients, with relatively more specific T cells in patients with active vitiligo. Although MAL and vitiligo may appear clinically similar, our results indicate that the humoral immune responses against MART-1 differ between these diseases, which can help to differentiate MAL from vitiligo.

Topics: Adolescent; Adult; Aged; Antibody Formation; Antibody Specificity; CD8-Positive T-Lymphocytes; Demography; Female; Humans; Hypopigmentation; Male; MART-1 Antigen; Melanocytes; Melanoma; Middle Aged; Vitiligo; Young Adult

Regressing nevi are considered an example of an efficient early antitumoral response preventing the development of neoplasia. The underlying mechanism has not been elucidated, although an immune-based destruction of melanocytes is supposed. The aim of this study was to provide evidence of an effective immunosurveillance of pigment lesions in a patient at high risk of melanoma.. A patient with the dysplastic nevus syndrome and a history of melanoma was included in this study. Since 2003, a marked regression of almost all nevi was observed. Immunohistochemistry was performed and the antigen specificity of T-cells was analyzed on T-cells isolated from a regressing nevus by flow cytometry using HLA-A2-peptide tetramers containing Mart-1(26-35), gp100(280-288), gp100(209-217) and tyrosinase(369-377). Immunohistochemistry of the regressing nevi showed a strong infiltrate of CD4 + and CD8 + T-cells. Flow cytometric analyses demonstrated the presence of a CD8 + T-cell response against gp100(280-288) and Mart-1(26-35) both in peripheral blood and in a regressing nevus.. These findings indicate that an immune reaction against melanocyte differentiation antigens can target specifically nevi without signs of vitiligo and suggests that boosting the anti-melanocyte immune response in patients at high risk for melanoma may prevent tumor development at an early stage.

Topics: Blotting, Western; CD4-Positive T-Lymphocytes; CD8-Positive T-Lymphocytes; Dysplastic Nevus Syndrome; Flow Cytometry; gp100 Melanoma Antigen; HLA-A2 Antigen; Humans; Immunoenzyme Techniques; Male; MART-1 Antigen; Melanocytes; Melanoma; Middle Aged; Monophenol Monooxygenase; Skin Neoplasms; T-Lymphocytes, Cytotoxic; Vitiligo

We investigated the possibility of introducing exogenous T cell receptor (TCR) genes into T cells by lentiviral transduction, without prior stimulation of endogenous TCR with anti-CD3. TCR transfer is used to impose tumor antigen specificity on recipient T cells, but sustained activation required for retroviral transduction may affect the clinical efficacy of engineered T cells. Cytokine stimulation makes T cells susceptible to lentiviral transduction in the absence of TCR triggering, but this advantage has never been exploited for TCR transfer. Autoimmune diseases are a source of high-affinity TCRs specific for self/tumor antigens. We selected, from a patient with vitiligo, a Mart1-specific TCR based on intrinsic interchain pairing properties and functional avidity. After lentiviral transduction of human peripheral blood mononuclear cells, preferential pairing of exogenous alpha and beta chains was observed, together with effective recognition of Mart1(+) melanoma cells. We tested transduction efficiency on various T cell subsets prestimulated with interleukin (IL)-2, IL-7, IL-15, and IL-21 (alone or in combination). Both naive and unfractionated CD8(+) T cells could be transduced without requiring endogenous TCR triggering. IL-7 plus IL-15 was the most powerful combination, allowing high levels of transgene expression without inducing T cell differentiation (34 +/- 5% Mart1-TCR(+) cells in naive CD8(+) and 16 +/- 6% in unfractionated CD8(+)). Cytokine-prestimulated, Mart1-redirected naive and unfractionated CD8(+) cells expanded better than CD3-CD28-prestimulated counterparts in response to both peptide-pulsed antigen-presenting cells and Mart1(+) melanoma cells. This strategy allows the generation of tumor-specific T cells encompassing truly naive T cells, endowed with an intact proliferative potential and a preserved differentiation stage.

Topics: Antigens, Neoplasm; CD8-Positive T-Lymphocytes; Cell Line, Tumor; Genetic Therapy; Genetic Vectors; Humans; Immunologic Memory; Lentivirus; MART-1 Antigen; Melanoma; Neoplasm Proteins; Receptors, Antigen, T-Cell; Transduction, Genetic; Vitiligo

Unlike in common melanocytic naevi, an acquired leukoderma (halo) surrounding a congenital melanocytic naevus is a rare phenomenon. A 6-year-old boy developed a depigmentation around a congenital melanocytic naevus on the right thigh. Simultaneously, segmental vitiligo appeared on the thigh, lower abdomen and buttock of the same side with sharp midline demarcation. Examination for associated autoimmune diseases proved negative. The simultaneous occurrence of a halo phenomenon around a congenital melanocytic naevus and segmental vitiligo, as well as identical histological and immunohistological findings in both pigmented lesions, suggest shared immunological mechanisms.

Topics: Antigens, Neoplasm; Child; Dermis; Humans; Immunohistochemistry; Male; MART-1 Antigen; Melanocytes; Neoplasm Proteins; Nevus, Pigmented; Skin Neoplasms; Skin Pigmentation; T-Lymphocytes, Cytotoxic; Vitiligo

To detect the serum levels of melanocyte antibodies and explore the relation between melanoma antigen recognized by T-cells (MART-1) and vitiligo in children.. The serum samples were collected from children with vitiligo to test the autoantibodies, and divided into low- and high-titer group according to the test results. Melanocytes were incubated with the serum samples, and the changes of melanocyte surface antigen were evaluated using specific MART-1 antibody.. The serum melanocyte antibody levels in children with vitiligo were significantly higher than those in normal subjects. The expression level of melanocyte surface antigen MART-1 increased obviously after incubation of the melanocyte with high antibody titer serum samples, and MART-1 was found to specifically bind to specific MART-1 antibody.. Melanocytes MART-1 may correlate to the autoimmune mechanism in children with vitiligo.

Topics: Adolescent; Autoantibodies; Child; Child, Preschool; Enzyme-Linked Immunosorbent Assay; Female; Humans; Male; MART-1 Antigen; Melanocytes; T-Lymphocytes; Vitiligo

Vitiligo, a skin disorder characterized by the spontaneous destruction of melanocytes, is believed to be of autoimmune origin. We investigated the presence and functionality of CD8(+) T-cells specific for the melanocyte-associated antigens Melan-A, gp100, tyrosinase, and TRP-2 in the blood of HLA-A2(+) vitiligo patients. We enumerated antigen-specific CD8(+) T cells by major histocompatibility complex multimer staining directly ex vivo, as well as after 9 days of in vitro stimulation and assessed IFN-gamma secretion by enzyme-linked immunospot (Elispot) assay. Tyrosinase-, gp100-, or TRP-2-specific CD8(+) T cells could not be identified in the peripheral blood of individuals with vitiligo. Although Melan-A-specific T cells were detectable at levels comparable to Flu-MP-specific T cells by multimer staining, these lymphocytes did not express the skin-homing receptor cutaneous lymphocyte antigen, were phenotypically naïve (CD45RA(+)), and were unresponsive in the IFN-gamma Elispot assay, suggesting that they are unlikely to be involved in the etiopathogenesis of vitiligo.

Topics: Antigens, Neoplasm; Case-Control Studies; CD8-Positive T-Lymphocytes; gp100 Melanoma Antigen; HLA-A2 Antigen; Humans; Interferon-gamma; MART-1 Antigen; Membrane Glycoproteins; Membrane Proteins; Monophenol Monooxygenase; Neoplasm Proteins; Peptide Fragments; Vitiligo

Hypopigmented mycosis fungoides is an uncommon clinical variant of cutaneous T-cell lymphoma. We hypothesized that hypomelanosis in hypopigmented mycosis fungoides may have a similar mechanism as in vitiligo, a condition in which it is believed that alterations in expression of CD117 (stem cell factor receptor/KIT protein) on epidermal melanocytes and abnormal interactions between melanocytes and surrounding keratinocytes may play a pathogenic role. To test the hypothesis that similar mechanisms might also explain hypopigmentation in hypopigmented mycosis fungoides, skin specimens from five cases each of hypopigmented mycosis fungoides and vitiligo were studied immunohistochemically for immunophenotype of the infiltrating cells, CD117 (expressed by epidermal melanocytes), and pan melanoma cocktail of antigens (gp100, tyrosinase, and MART-1) expression; cases of conventional mycosis fungoides and normal skin were studied in parallel as controls. Our findings confirm a predominance of CD8+ neoplastic T cells in hypopigmented mycosis fungoides. Similarly, the epidermal lymphocytic infiltrate in vitiligo was also composed of CD8+ cytotoxic T cells, in contrast to an epidermal infiltrate composed of CD4+ T cells in conventional mycosis fungoides. The average number of epidermal CD117 expressing cells followed the same pattern of decreased expression in hypopigmented mycosis fungoides as in vitiligo, whereas the levels in conventional mycosis fungoides were higher, and similar to that observed in normal skin. Furthermore, a decreased number of melanocytes per high-power field of the length of the biopsy was present in hypopigmented mycosis fungoides and vitiligo, as compared with either conventional mycosis fungoides or normal skin, suggesting a correlation between decreased expression of CD117 and decreased number of melanocytes. We propose that decreased expression of CD117 and its downstream events in melanocytes may be initiated by cytotoxic effects of melanosomal-antigen-specific CD8+ neoplastic T lymphocytes, resulting in destabilization of CD117 and leading to dysfunction and/or loss of melanocytes in the epidermis of hypopigmented mycosis fungoides.

Topics: Antigens, Neoplasm; Biomarkers; CD4-Positive T-Lymphocytes; Cell Count; gp100 Melanoma Antigen; Immunoenzyme Techniques; Immunophenotyping; MART-1 Antigen; Melanocytes; Membrane Glycoproteins; Monophenol Monooxygenase; Mycosis Fungoides; Neoplasm Proteins; Proto-Oncogene Proteins c-kit; Retrospective Studies; Skin; Skin Neoplasms; T-Lymphocytes, Cytotoxic; Vitiligo

Vitiligo is a skin disorder characterized by depigmented macules secondary to melanocyte loss. An unusual facet is its relation to melanoma: cytotoxic T lymphocytes directed to melanocyte antigens are found in both conditions and imply a breakdown of tolerance, yet the resulting immune reaction is the opposite. The mechanisms at the basis of these opposite effects are not known. Here, we performed a direct comparison of whole melanocyte-specific T cell populations in the two diseases. We demonstrate that neither precursor frequencies of Melan-A/MART-1-specific T lymphocytes nor their status of activation differ significantly. However, by using a tetramer-based T cell receptor down-regulation assay, we documented a higher affinity of vitiligo T cells. We calculated that the peptide concentration required for 50% of maximal receptor down-regulation differed by 6.5-fold between the two diseases. Moreover, only vitiligo T cells were capable of efficient receptor down-regulation and IFN-gamma production in response to HLA-matched melanoma cells, suggesting that this difference in receptor affinity is physiologically relevant. The differences in receptor affinity and tumor reactivity were confirmed by analyzing Melan-A/MART-1-specific clones established from the two diseases. Our results suggest that the quality, and not the quantity, of the melanocyte-specific cytotoxic responses differs between the two pathologies.

Topics: Antigens, Neoplasm; Cell Line; Clone Cells; Down-Regulation; Female; Humans; Male; MART-1 Antigen; Melanocytes; Melanoma; Neoplasm Proteins; Receptors, Antigen, T-Cell; T-Lymphocytes, Cytotoxic; Vitiligo

Vitiligo patients possess high frequencies of circulating CD8+ T lymphocytes specific for the melanocyte differentiation antigen Melan-A/MART-1. These self-specific T cells exhibit intact functional properties and their T cell receptors are selected for a narrow range of high affinities of antigen recognition, suggesting their important role in the pathogenesis of vitiligo. In order to understand the molecular base for this unexpected, optimal T cell receptor recognition of a self-antigen, a tetramer-guided ex vivo analysis of the T cell receptor repertoire specific for the Melan-A antigen in a patient affected by vitiligo is reported. All T cell receptors sequenced corresponded to different clonotypes, excluding extensive clonal expansions and revealing a large repertoire of circulating Melan-A-specific T lymphocytes. A certain degree of T cell receptor structural conservation was noticed, however, as a single AV segment contributed to the alpha chain rearrangement in 100% of clones and a conserved amino acid sequence was found in the beta chain complementarity determining region 3 of various high affinity cells. We suggest that the conserved alpha chain confers self-antigen recognition, necessary for intrathymic selection and peripheral homeostasis, to many synonymous T cell receptors, whereas the beta chain fine tunes the T cell receptor affinity of the specific cells. In addition, we demonstrate that many high avidity T cell clones from this patient were capable of specifically lysing normal, HLA-matched melanocytes. These autoreactive clones persisted for more than 3 y in the patient's peripheral blood. These data, together with the skin-homing potential of the clones, directly point to the in vivo pathogenic role of melanocyte-specific cytotoxic T lymphocytes in vitiligo.

Topics: Antigens, Neoplasm; Autoantigens; CD8-Positive T-Lymphocytes; Clone Cells; Female; HLA-A Antigens; HLA-A2 Antigen; Humans; MART-1 Antigen; Melanocytes; Neoplasm Proteins; Receptors, Antigen, T-Cell; Vitiligo

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.

Topics: Antigens, Neoplasm; Autoantigens; CD8-Positive T-Lymphocytes; Cohort Studies; Enzyme-Linked Immunosorbent Assay; Epitopes; Female; gp100 Melanoma Antigen; HLA-A2 Antigen; Humans; Male; MART-1 Antigen; Melanocytes; Membrane Glycoproteins; Monophenol Monooxygenase; Neoplasm Proteins; T-Lymphocytes, Cytotoxic; Vitiligo

Recent studies have demonstrated the presence of circulating MelanA (MART1)-specific cytotoxic T lymphocytes in a significant number of vitiligo patients when compared to control subjects. High levels of the skin-homing receptor cutaneous lymphocyte-associated antigen were expressed on the T cells and their frequency correlated with the extent of depigmentation and disease activity in the vitiligo patients. The present study was designed to examine vitiligo patient sera for the presence of autoantibodies to MelanA. The incidence of autoantibodies to MelanA in patients with vitiligo (n = 51) and in healthy individuals (n = 20) was examined using a radiobinding assay with 35S]-labelled MelanA and using Western blot analysis with a glutathione S-transferase (GST)-MelanA fusion protein. Autoantibodies to MelanA could not be detected in any of the vitiligo patient sera or control sera analysed using either of these detection systems. It is therefore possible that MelanA only induces cellular rather than humoral autoreactivity in vitiligo.

Topics: Adolescent; Adult; Aged; Antibody Specificity; Antigens, Neoplasm; Autoantibodies; Blotting, Western; Cell Differentiation; Female; Glutathione Transferase; Humans; Male; MART-1 Antigen; Melanocytes; Middle Aged; Neoplasm Proteins; Protein Biosynthesis; Radioligand Assay; Recombinant Fusion Proteins; Transcription, Genetic; Vitiligo

Selection and activation of T cells is tightly regulated by both antigen-specific receptors and co-receptors to ensure that responses to self antigens are largely avoided. By T cell receptor clonotypic mapping and staining with tetrameric HLA-peptide complexes, we demonstrate the presence of melanocyte differentiation antigen MART-1 specific T cells in the areas of destruction of both neoplastic and normal melanocytic cells in a case of a primary melanoma and its associated hypopigmentation. These self reactive T cells expressed CD94/NKG2 major histocompatibility complex class I specific C-type lectin-like receptors. This family of receptors includes both activating and inhibitory isoforms. Thus, we performed a detailed analysis that revealed the exclusive presence of inhibitory NKG2-A/B receptors in the vitiligo-like leukoderma, whereas both the inhibitory receptors and the activating NKG2-C/E isoforms were present within the tumor. Our data suggest the differential expression of killer inhibitory receptors as a possible mechanism to regulate T cell responses to self antigens.

Topics: Antigens, CD; Antigens, Neoplasm; Autoimmunity; Clone Cells; Gene Expression Regulation, Neoplastic; Humans; Immune Tolerance; Immunohistochemistry; Lectins, C-Type; MART-1 Antigen; Melanoma; Membrane Glycoproteins; Neoplasm Proteins; NK Cell Lectin-Like Receptor Subfamily C; NK Cell Lectin-Like Receptor Subfamily D; Receptors, Antigen, T-Cell, alpha-beta; Receptors, Immunologic; Receptors, Natural Killer Cell; Skin Neoplasms; T-Lymphocytes; Vitiligo

Lichen sclerosus (LS) shares with vitiligo a milky-white appearance. By biopsy, pathognomonic dermal sclerosis readily distinguishes LS from vitiligo and other causes of leukoderma. To determine what the mechanism of hypopigmentation is in LS, we examined samples from LS cases for alterations in melanin content (Fontana-Masson stain) and melanocyte number (HMB-45 [PMEL-17/gp100], Mel-5 [TRP-1], Mart-1 [Melan A]) and compared these findings with those in controls of normal skin, acute scars, vitiligo, and lichen planus (LP; a common inflammatory cause of hyperpigmentation). The degree and extent of melanization found in LS overlapped with that in acute scars showing predominantly hypomelanized keratinocytes, with that in LP containing regions with numerous melanophages, and with that in vitiligo exhibiting focal regions of keratinocytes devoid of melanin pigment. By hematoxylin-eosin staining and immunocytochemistry for Mel-5 and Mart-1, LS had a lower mean count of melanocytes than acute scars, LP, and normal skin per 200 basal keratinocytes. In addition, a few LS cases had a significant loss of melanocytes comparable to that of vitiligo. Surprisingly, Mart-1 identified rare melanocytes in 67% of vitiligo cases and a significantly larger pool of melanocytes in LS and controls other than those labeled by Mel-5. Furthermore, LP and evolving lesions of LS contained the highest Mart-1 counts. HMB-45-immunoreactive melanocytes were found in the majority of acute scars and in LP and late-stage LS lesions at significantly lower levels than Mel-5- and Mart-1- labeled melanocytes, but they were not found in vitiligo or normal skin. We propose that several mechanisms may play a role in the production of leucoderma in LS: 1) decreased melanin production; 2) block in transfer of melanosomes to keratinocytes; and 3) melanocyte loss. The latter finding may be the pathogenic connection (lichenoid dermatitis of LS triggering an autoimmune reaction to melanocytes) that underlies the documented association of LS with vitiligo.

Topics: Antigens, Neoplasm; Cell Count; Cicatrix; Glycoproteins; Humans; Hypopigmentation; Immunohistochemistry; Lichen Sclerosus et Atrophicus; MART-1 Antigen; Melanins; Melanocytes; Melanoma-Specific Antigens; Monophenol Monooxygenase; Neoplasm Proteins; Skin; Vitiligo

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-gamma 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-gamma 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.

Topics: Adult; Aged; Antigens, Differentiation, T-Lymphocyte; Antigens, Neoplasm; CD8-Positive T-Lymphocytes; Female; HLA-A2 Antigen; Humans; Interferon-gamma; Male; MART-1 Antigen; Melanocytes; Membrane Glycoproteins; Middle Aged; Neoplasm Proteins; Vitiligo

Vitiligo is a common skin disease characterized by the presence of well circumscribed, depigmented, milky white macules devoid of identifiable melanocytes. Although the detection of circulating anti-melanocytic antibodies and of infiltrating lymphocytes at the margin of lesions supports the view that vitiligo is an autoimmune disorder, its etiology remains unknown. In particular, it is still a matter of debate whether the primary pathogenic role is exerted by humoral or cellular abnormal immune responses. In this study, the presence of specific cytotoxic T lymphocyte responses against the melanocyte differentiation antigens Melan-A/MART1, tyrosinase, and gp100 in vitiligo patients have been investigated by the use of major histocompatibility complex/peptide tetramers. High frequencies of circulating melanocyte-specific CD8+ T cells were found in all vitiligo patients analyzed. These cells exerted anti-melanocytic cytotoxic activity in vitro and expressed skin-homing capacity. In one patient melanocyte-specific cells were characterized by an exceptionally high avidity for their peptide/major histocompatibility complex ligand. These findings strongly suggest a role for cellular immunity in the pathogenesis of vitiligo and impact on the common mechanisms of self tolerance.

Topics: Antigens, Neoplasm; CD8-Positive T-Lymphocytes; Cell Differentiation; Cell Line; Female; Flow Cytometry; gp100 Melanoma Antigen; HLA-A2 Antigen; Humans; Immunity, Cellular; Male; MART-1 Antigen; Melanocytes; Membrane Glycoproteins; Monophenol Monooxygenase; Neoplasm Proteins; Peptide Fragments; T-Lymphocytes, Cytotoxic; Vitiligo

Peptides derived from human tumor antigens have been used in a number of clinical trials to induce specific immune responses against autologous tumors in cancer patients. Although favorable clinical results were observed in single patients, immune responses correlating with tumor regression were either not detected or in case of responses, the T-cell specificity was difficult to demonstrate. In this study, we analyzed antigen-specific T-cell responses induced in the skin and in peripheral blood lymphocytes (PBL) in an HLA-A2-positive melanoma patient. The patient showed major regression of metastatic melanoma under continued immunization with peptides derived from the melanocyte differentiation antigens Melan A/MART-1, tyrosinase and gp100/Pmel17. Based on the identification of different T-cell receptor (TCR) families reactive with Melan A/MART-1, we have demonstrated that i.d. immunization with peptides alone leads to oligoclonal expansion of Melan A/MART-1-specific cytotoxic T lymphocytes (CTL), detectable in local delayed-type hypersensitivity (DTH) reactions and PBL. A monoclonal expansion of a Melan A/MART-1-specific TCR VB 16 CTL was reproducibly observed after in vitro stimulation with Melan A/MART-1 peptides. The same TCR VB 16 CTL clone was detected in skin biopsies taken from vitiligo areas. Our findings provide strong evidence for the effective induction of specific T-cell responses to Melan A/MART-1 by i.d. immunization with peptide alone, which accounts for dermal depigmentation, specific cytotoxicity against Melan A/MART-1-expressing melanoma cells and clinical tumor regression.

Topics: Antigens, Neoplasm; Female; Humans; Hypersensitivity, Delayed; Immunization; MART-1 Antigen; Melanoma; Melanoma-Specific Antigens; Middle Aged; Neoplasm Proteins; T-Lymphocytes, Cytotoxic; Vitiligo

There is now considerable evidence that human tumors often express antigens that render them susceptible to lysis by cytotoxic T lymphocytes (CTLs). These findings have raised hope for the development of cancer vaccines to trigger a tumor-specific immune response in cancer patients. To optimize the immunogenicity of cancer vaccines, it is important to improve the monitoring of the immune response. The use of tetrameric soluble major histocompatibility complex (MHC) class I/peptide complexes ("tetramers") to identify tumor-specific CTLs has shown that these novel reagents allow rapid and accurate analysis of human CTL responses in cancer patients. We have used fluorescence-driven cell sorting to clone tumor-specific CTLs after staining with tetrameric MHC class I/peptide complexes. Analysis of melanoma-infiltrated lymph nodes revealed that strong CTL responses often occur in vivo, and that the reactive CTLs have substantial proliferative and tumoricidal potential.

Topics: Antigens, Neoplasm; beta 2-Microglobulin; Epitopes, T-Lymphocyte; Flow Cytometry; Histocompatibility Antigens Class I; HLA Antigens; HLA-A2 Antigen; Humans; Lymph Nodes; MART-1 Antigen; Melanocytes; Melanoma; Monitoring, Immunologic; Neoplasm Proteins; T-Lymphocytes, Cytotoxic; Vitiligo

Current strategies for the immunotherapy of melanoma include augmentation of the immune response to tumor antigens represented by melanosomal proteins such as tyrosinase, gp100, and MART-1. The possibility that intentional targeting of tumor antigens representing normal proteins can result in autoimmune toxicity has been postulated but never demonstrated previously in humans. In this study, we describe a patient with metastatic melanoma who developed inflammatory lesions circumscribing pigmented areas of skin after an infusion of MART-1-specific CD8(+) T cell clones. Analysis of the infiltrating lymphocytes in skin and tumor biopsies using T cell-specific peptide-major histocompatibility complex tetramers demonstrated a localized predominance of MART-1-specific CD8(+) T cells (>28% of all CD8 T cells) that was identical to the infused clones (as confirmed by sequencing of the complementarity-determining region 3). In contrast to skin biopsies obtained from the patient before T cell infusion, postinfusion biopsies demonstrated loss of MART-1 expression, evidence of melanocyte damage, and the complete absence of melanocytes in affected regions of the skin. This study provides, for the first time, direct evidence in humans that antigen-specific immunotherapy can target not only antigen-positive tumor cells in vivo but also normal tissues expressing the shared tumor antigen.

Topics: Antigens, Neoplasm; Female; Humans; Immunotherapy, Adoptive; MART-1 Antigen; Melanocytes; Melanoma; Middle Aged; Neoplasm Proteins; Skin; Skin Neoplasms; T-Lymphocytes, Cytotoxic; Vitiligo

The cloning of genes encoding melanoma antigens has opened new possibilities for the treatment of patients with cancer; however, most tumor rejection antigens recognized by tumor infiltrating lymphocytes are the products of genes that are also expressed by normal melanocytes. Hence, a large set of antigenic determinants of the self have not induced self-tolerance and these peptide determinants furnish target structures for immune responses directed against tumors. The notion that the immunotherapeutic targets involved in cancer regression comprise normal differentiation antigens is stressed by the association between vitiligo-like leukoderma, due to destruction of normal melanocytes, and melanoma regression, due to destruction of cancer cells. Nevertheless, this is the first report to demonstrate by means of a new technique based on reverse transcription polymerase chain reaction and denaturing gradient gel electrophoresis, the presence of clonally expanded T cells with identical BV regions in areas of destruction of both normal and neoplastic cells.

Topics: Amino Acid Sequence; Antigens, Neoplasm; Base Sequence; Humans; Lymphocytes, Tumor-Infiltrating; Male; MART-1 Antigen; Melanoma; Middle Aged; Molecular Sequence Data; Neoplasm Proteins; Receptors, Antigen, T-Cell, alpha-beta; Reverse Transcriptase Polymerase Chain Reaction; T-Lymphocytes; Vitiligo