By
From the * Institute of Molecular Medicine, Nuffield Department of Clinical Medicine, Oxford, OX3
9DS, United Kingdom; and the Ludwig Institute for Cancer Research, Lausanne Branch, University
of Lausanne, Epalinges, 1011 Switzerland
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Abstract |
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Vitiligo is an autoimmune condition characterized by loss of epidermal melanocytes. Using tetrameric complexes of human histocompatibility leukocyte antigen (HLA) class I to identify antigen-specific T cells ex vivo, we observed high frequencies of circulating MelanA-specific, A*0201-restricted cytotoxic T lymphocytes (A2-MelanA tetramer+ CTLs) in seven of nine HLA-A*0201-positive individuals with vitiligo. Isolated A2-MelanA tetramer+ CTLs were able to lyse A*0201-matched melanoma cells in vitro and their frequency ex vivo correlated with extent of disease. In contrast, no A2-MelanA tetramer+ CTL could be identified ex vivo in all four A*0201-negative vitiligo patients or five of six A*0201-positive asymptomatic controls. Finally, we observed that the A2-MelanA tetramer+ CTLs isolated from vitiligo patients expressed high levels of the skin homing receptor, cutaneous lymphocyte-associated antigen, which was absent from the CTLs seen in the single A*0201-positive normal control. These data are consistent with a role of skin-homing autoreactive melanocyte-specific CTLs in causing the destruction of melanocytes seen in autoimmune vitiligo. Lack of homing receptors on the surface of autoreactive CTLs could be a mechanism to control peripheral tolerance in vivo.
Key words: human; skin; cytotoxic T lymphocytes; fluorescence-activated cell sorter; autoimmunity ![]() |
Introduction |
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itiligo is a common progressive depigmentary condition that is believed to be due to the autoimmune-mediated destruction of epidermal melanocytes. Melanocyte-specific autoantibodies have been described in vitiligo patients, but their pathogenic role remains uncertain (1). Titers of such autoantibodies in individuals with melanoma-associated hypopigmentation are similar to those in normal controls, suggesting that, in these patients, other mechanisms may lead to melanocyte loss (4). The observation that melanocyte proteins are targets for antimelanoma CTLs (6) raises the possibility that destruction of epidermal melanocytes in vitiligo patients could be due to a melanocyte-specific CTL response. Indeed, the spontaneous appearance of vitiligo has been associated with an improved prognosis in individuals with metastatic melanoma (9). Histology of the advancing margins of vitiligo reveals a lymphocytic infiltrate predominantly composed of CD8+ T cells expressing the skin homing receptor, cutaneous lymphocyte-associated antigen (CLA),1 which is a modified form of P selectin-binding glycoprotein 1 (11). Recently, dendritic cell-based vaccination strategies for treatment of melanoma have been accompanied by the acquisition of tumor-specific CTL responses and cutaneous depigmentation, suggesting that melanocyte-specific CTLs can play a role in melanocytic destruction (15). Animal models of vitiligo are also consistent with a role for autoreactive CD8+ T cells (16), but it has remained unclear whether antigen-specific CD8+ T cells are involved in vitiligo or indeed any other human autoimmune condition. Here, we demonstrate the presence of high frequencies of skin-homing melanocyte-specific CTLs in the peripheral blood of patients with autoimmune vitiligo compared with healthy volunteers.
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Materials and Methods |
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HLA-Peptide Tetrameric Complexes and Flow Cytometry.
HLA- peptide tetrameric complexes were synthesized as previously described (17). In brief, purified HLA heavy chain andSubjects.
13 individuals with vitiligo were recruited through the Vitiligo Society (London, UK) and nine were found to be HLA-A*0201 positive when screened using allele-specific PCR. Six patients had associated autoimmune conditions (four were HLA-A*0201 positive) including thyroid disease and pernicious anaemia, but the remaining seven (five were HLA-A*0201 positive) had no other clinical disorders. None of the patients were on immunosuppressive therapy. The negative control individuals were healthy asymptomatics with no history of autoimmune disease.Cytotoxicity Assays.
A*0201-positive EBV-transformed B cell lines, T2 cells, or melanoma cells (provided by Dr. P. van der Bruggen, Ludwig Institute for Cancer Research, Bruxelles, Belgium) were incubated at 37°C for 1 h in the presence of Na2 51CrO4 (Amersham Pharmacia Biotech, Inc., Piscataway, NJ) at 2 µCi/µl. The cells were washed once in medium containing 10% FCS and incubated either with or without peptide at 1 µM for 1 h. The targets were washed a further two times before plating into 96-well round-bottomed plates at 2,500 cells per well. 100 µl of medium containing cells at documented E/T ratios were added to each well after counting in the presence of trypan blue. The plates were incubated at 37°C for 4 h before harvesting 20 µl of supernatant. Percentage lysis was estimated from (experimental countsCell Culture.
PBMCs were incubated with 100 µM peptide for 1 h before dilution at 2 × 106 cells/ml in RPMI 1640 supplemented with 5% human serum and 25 ng/ml IL-7 (23). Lymphocult-T (Biotest, Dreieich, Germany) was added at day 4 to a final concentration of 10%. The cells were harvested at day 12 for flow cytometry analysis or cytotoxicity assays. ![]() |
Results and Discussion |
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To test the hypothesis that high frequencies of melanocyte-specific CTLs correlate with vitiligo, we quantified
the frequency of ex vivo melanocyte-specific CTLs by
constructing HLA-A*0201-peptide tetrameric complexes
(17, 24) based on two A*0201-binding peptides derived
from melanocyte proteins MelanA (MelanA 26-35) and tyrosinase (tyrosinase 369-377) (7, 8, 18). A modified MelanA 26-35 peptide (alanine to leucine at position 2) has
previously been shown to increase the binding affinity for
A*0201 without affecting CTL recognition (21). HLA
heavy chain was expressed in Escherichia coli with an engineered COOH-terminal signal sequence containing a biotinylation site for the enzyme BirA. After the refolding of
heavy chain, 2 microglobulin, and peptide, the complex was biotinylated and tetramer formation induced by the addition of streptavidin. By using fluorescently labeled streptavidin, the tetramer was used to stain and sort antigen-specific cells by flow cytometry. HLA-peptide tetrameric
complexes bind to antigen-specific CTLs with high specificity such that CTL clones and lines directed to different
epitope peptides bound to the same HLA molecule do not
stain. Tetramer binding is known to correlate well with both peptide-specific cytolytic activity and IFN-
production
(24), and even down to low frequencies of antigen-specific T cells (1 in 5,000 CD8+ T cells or less) it is possible
to directly isolate tetramer-positive cells by FACS® (26).
By staining of PBMC directly ex vivo, without any antigen-specific stimulation, we observed high frequencies of MelanA-specific CTLs in seven of nine A*0201-positive vitiligo patients, but in only one of six A*0201-positive asymptomatic controls (Fig. 1, A-D). Frequencies were similar to those previously obtained for A*0201-restricted influenza A matrix protein in asymptomatic patients (26). The identification of A*0201-restricted MelanA-specific CTLs was dependent on the presence of HLA-A*0201, as none of four A*0201-negative vitiligo controls had detectable CTLs ex vivo. No tyrosinase (369-377)-specific CTLs were observed directly ex vivo in any patient group. It is certainly possible that melanocyte-specific CTLs exist to epitopes other than those tested with the HLA-tetrameric complexes. The one A*0201-positive asymptomatic control with a frequency of MelanA-specific CTLs similar to that observed in the vitiligo patients had no personal or family history suggestive of autoimmune disease. To explain such a high frequency of MelanA-specific CTLs in the absence of disease, we examined the skin-homing capacity of CTLs from the cohort. The majority of MelanA-specific CTLs from the vitiligo patients expressed the skin-homing receptor CLA, in contrast to those from the asymptomatic control who had detectable CTLs, in whom the CTLs were all negative for CLA (Fig. 1, A-F). There was a statistically significant difference between the number of CLA-positive A2-MelanA tetramer+ CTLs between patients and controls (P < 0.05). These data confirmed that high frequencies of autoreactive CTLs could be detected directly ex vivo in patients with vitiligo, but were only associated with disease if they were able to home to the skin.
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Through the use of repeated antigen-specific stimulation in vitro, it has previously been possible to generate tyrosinase- and MelanA-specific CTLs, from normal controls, that have the capacity to lyse melanoma target cells (27, 28). We examined whether it would be possible to use the HLA-peptide tetrameric complexes to compare the frequency of melanocyte-specific CTLs generated by peptide-specific stimulation from our cohort of vitiligo patients and asymptomatic controls. Using an optimized peptide-specific stimulation protocol in the presence of IL-7 (23), we screened peptide-stimulated cultures at 2 wk for staining with the A2-MelanA and A2/tyrosinase tetramers. High frequencies of melanocyte-specific CTLs were observed in cultures from the A*0201-positive vitiligo patients, but only low frequencies were observed in five of the six asymptomatic controls (Fig. 2, A-D). The asymptomatic control with detectable MelanA-specific CTLs ex vivo had a similar frequency of CTLs generated by peptide stimulation as the vitiligo patients. No A2-MelanA tetramer+ CTLs were observed in cultures from A*0201-negative vitiligo patients. We were also able to generate tyrosinase-specific CTLs from A*0201-positive vitiligo patients and normal controls, but these were consistently at least an order of magnitude lower than the MelanA-specific CTL frequencies (data not shown). The rank order of frequencies of MelanA-specific CTLs generated by peptide stimulation was identical to the order obtained directly ex vivo, indicating that all individuals had cells with a relatively consistent ability to proliferate and survive in culture for 2 wk. We looked at whether CLA was expressed by the peptide-stimulated melanocyte-specific CTLs (Fig. 2 F) and observed a significant difference between patients and controls (P < 0.05), namely high expression on CTLs generated from vitiligo patients and low or absent staining from controls. Although the cultures from vitiligo patients maintained CLA expression at levels above those observed in normal controls, there was evidence of a reduction in CLA staining compared with uncultured MelanA-specific CTLs consistent with previous observations (13). These data showed that it was possible through optimized stimulation protocols to generate MelanA- and tyrosinase-specific CTLs from A*0201-positive vitiligo patients and normal controls, but the frequencies of such CTLs are significantly higher in the patients and, in addition, the cells are able to home to the skin. Therefore, it was possible to isolate autoreactive CTLs from asymptomatic controls, but these were maintained at a low frequency (in five of six asymptomatic controls) and lacked the expression of CLA, a marker associated with the ability to home to skin. Lack of tissue homing receptors on the surface of potentially autoreactive CTLs may be a mechanism to prevent autoreactivity in vivo and to control peripheral tolerance.
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We examined whether the melanocyte-specific CTLs
were capable of causing melanocytic destruction by sorting
tetramer-positive cells from polyclonal lines generated by
multiple rounds of peptide stimulation from one vitiligo
patient, using FACS®, and then assessed the ability of the
cells to lyse both peptide-pulsed target cells and A*0201-positive melanoma cells (Fig. 3, A-F). CD8+ A2-MelanA
tetramer+ cells were able to lyse both peptide-pulsed targets and melanoma cells (Fig. 3, B and E), in contrast to
CD8+ A2-MelanA tetramer cells, which had no detectable cytolytic activity (Fig. 3, C and F). These data confirmed that tetramer-binding cells were able to recognize
and lyse melanoma cells that present the epitope peptide
through endogenous processing pathway.
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If melanocyte-specific CTLs are important in causing the tissue damage observed in vitiligo, there may be an association between the frequency of CTLs and the extent of disease. We measured the area involved by vitiligo in all patients and, in those with <20% of skin surface involved by vitiligo, there were significantly less MelanA-specific CTLs identified directly ex vivo (P < 0.05). Furthermore, in the two vitiligo patients with the lowest frequencies of MelanA-specific CTLs, the vitiligo had been stable for many years and was not progressing.
Using HLA-peptide tetrameric complexes, we have shown an association between high frequencies of skin-homing melanocyte-specific CTLs and autoimmune vitiligo. Furthermore, the frequency of such CTLs correlates with the extent of disease. Therefore, in addition to the previously documented melanocyte-specific autoantibodies (1), these data are consistent with a role for skin-homing melanocyte-specific CTLs in vitiligo. The ability of such cells to home to sites of potential tissue damage may be a means to control peripheral tolerance in vivo. This raises the possibility that CD8-dependent cell-mediated immunity may be an important generalized effector mechanism in autoimmune conditions and therefore a potential target for therapeutic intervention.
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Footnotes |
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Address correspondence to Vincenzo Cerundolo, Institute of Molecular Medicine, Nuffield Department of Clinical Medicine, Oxford, OX3 9DS, UK. Phone: 44-1865-222-412; Fax: 44-1865-222-502; E-mail: vcerundo{at}worf.molbiol.ox.ac.uk
Received for publication 2 June 1998 and in revised form 30 June 1998.
We are grateful to the patients of the Vitiligo Society for blood samples.
This work was supported by the Medical Research Council, UK, and the Cancer Research Campaign.
Abbreviation used in this paper CLA, cutaneous lymphocyte-associated antigen.
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