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:
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Introduction |
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.
| |
Materials and Methods |
HLA-Peptide Tetrameric Complexes and Flow Cytometry.
HLA-
peptide tetrameric complexes were synthesized as previously
described (17). In brief, purified HLA heavy chain and 
2 microglobulin were synthesized using a prokaryotic expression system
(pET; Novagen, Milwaukee, WI). The heavy chain was modified by deletion of the transmembrane/cytosolic tail and COOH-terminal addition of a sequence containing the BirA enzymatic biotinylation site. Heavy chain, 2 microglobulin, and peptide
were refolded by dilution. A*0201-binding peptides were tyrosinase 369-377 YMDGTMSQV (18) and MelanA 26-35 ELAGIGILTV (7, 8, 21). The 45-kD refolded product was isolated
using fast protein liquid chromatography (FPLC), biotinylated by
BirA (Avidity, Denver, CO) in the presence of biotin (Sigma
Chemical Co., St. Louis, MO), ATP (Sigma Chemical Co.) and
Mg2+ (Sigma Chemical Co.). The biotinylated product was separated from free biotin by gel filtration and ion exchange using
FPLC. Streptavidin-PE conjugate (Sigma Chemical Co.) was added
in a 1:4 molar ratio and the tetrameric product was concentrated
to 1 mg/ml. Analysis of cells for the expression of cell surface markers was performed using a FACScan® (Becton Dickinson & Co., Mountain View, CA) and CellQuest software (Becton Dickinson & Co.). Frozen PBMC were thawed and incubated for 24-48 h
in RPMI 1640 supplemented with 10% FCS to allow recovery of cell viability. 106 cells were centrifuged at 300 g for 5 min and
resuspended in 50 µl cold PBS. Anti-CLA antibody (rat IgM;
PharMingen, San Diego, CA) was added and the samples incubated on ice for 20 min. After two washes with PBS, anti-rat
IgM-FITC (PharMingen) was added and the cells left for a further incubation on ice for 20 min. After two washes with PBS,
the tetramer and anti-CD8-Tricolor (Caltag Laboratories, Burlingame, CA) were added and incubated for another 20 min. The
samples were washed two more times with PBS before formaldehyde fixation. Triple-color analysis was performed with tetramer-PE, anti-CD8-tricolor, and anti-CLA. Controls for the tetramers
included staining A*0201-negative individuals and the use of an
irrelevant A*0201-tetramer (SLYNTVATL p17Gag 77-85; reference 22).
Subjects.
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 counts 
media control) × 100/(detergent counts media control).
Media controls were between 10 and 15% of detergent controls.
Cell 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 |
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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Fig. 1.
Uncultured CD8+ T cells stained with A2-MelanA tetramer
and anti-CLA antigen from: (A) an A*0201-positive patient with vitiligo in
whom the majority of MelanA-specific CTLs are positive for CLA (percentages given in each quadrant); (B) an A*0201-positive normal control
individual in whom there were no detectable MelanA-specific CTLs ex
vivo (pattern observed in five of six normal controls); (C) the A*0201-
positive normal control individual in whom there were detectable MelanA-specific CTLs ex vivo, but which were all negative for CLA (pattern observed in one of six normal controls); (D) an A*0201-negative patient with
vitiligo in whom there were no detectable A2-MelanA tetramer+ CTLs
(observed in all A*0201-negative vitiligo patients). E shows the percentage
of CD8+ T cells staining with A2-MelanA tetramer in the A*0201-positive
normal controls and A*0201-positive vitiligo patients. F confirms a significant difference between the percentage of CLA-positive MelanA-specific
CTLs in the PBMC of A*0201-positive patients and controls (P < 0.05).
|
|
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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Fig. 2.
Optimized peptide-stimulated IL-7-based cultures showing
the CD8+ T cells stained with A2-MelanA tetramer and anti-CLA from:
(A) an A*0201-positive patient with vitiligo in whom the majority of cultured MelanA-specific CTLs are positive for CLA (percentages given in
each quadrant); (B) an A*0201-positive normal control individual in
whom there was a low frequency of cultured MelanA-specific CTLs that
were predominantly negative for CLA (pattern observed in five of six
normal controls); (C) the A*0201-positive normal control individual in
whom there were detectable MelanA-specific CTLs ex vivo, and in
whom it was possible to culture high levels of MelanA-specific CTLs that
were all persistently negative for CLA (pattern observed in one of six normal controls); (D) an A*0201-negative patient with vitiligo in whom
there were no detectable A2-MelanA tetramer+ CTLs (observed in all
A*0201-negative vitiligo patients) after a period of stimulation in vitro. E
shows the percentage of CD8+ T cells from the cultures staining with
A2-MelanA tetramer in the A*0201-positive normal controls and
A*0201-positive vitiligo patients. F shows a significant difference between the percentage of CLA-positive MelanA-specific CTLs in the cultures from A*0201-positive patients and controls (P < 0.05).
|
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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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Fig. 3.
(A-C) Cytolytic activity of MelanA/A*0201-specific CTL lines derived from an
A*0201-positive vitiligo patient
either unsorted (A), enriched for
CD8+ A2-MelanA tetramer+
cells (B) or enriched for CD8+
A2-MelanA tetramer cells (C).
The unsorted and CD8+-tetramer+ cells were both able to
lyse peptide-pulsed targets, but
only the CD8+ A2-MelanA
tetramer+ population showed
significant lysis of A*0201-matched melanoma cells (ME
275 and SK-mel-29). (D-E) The
corresponding percentages of
CD8+ T cells staining with the
A2-MelanA tetramer are shown:
the unsorted population contained 17% antigen-specific cells
(D); the CD8+ A2-MelanA
tetramer+ population contained
72% antigen-specific cells (E);
and the CD8+ A2-MelanA
tetramer population contained
1.5% antigen-specific cells.
|
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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.
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
This work was supported by the Medical Research Council, UK, and the Cancer Research Campaign.
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