A Shift in the Phenotype of Melan-A-Specific
CTL Identifies Melanoma Patients with an
Active Tumor-Specific Immune Response
This information is current as
of June 17, 2017.
P. Rod Dunbar, Caroline L. Smith, David Chao, Mariolina
Salio, Dawn Shepherd, Fareed Mirza, Martin Lipp, Antonio
Lanzavecchia, Federica Sallusto, Alun Evans, Robin
Russell-Jones, Adrian L. Harris and Vincenzo Cerundolo
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The Journal of Immunology is published twice each month by
The American Association of Immunologists, Inc.,
1451 Rockville Pike, Suite 650, Rockville, MD 20852
Copyright © 2000 by The American Association of
Immunologists All rights reserved.
Print ISSN: 0022-1767 Online ISSN: 1550-6606.
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J Immunol 2000; 165:6644-6652; ;
doi: 10.4049/jimmunol.165.11.6644
http://www.jimmunol.org/content/165/11/6644
A Shift in the Phenotype of Melan-A-Specific CTL Identifies
Melanoma Patients with an Active Tumor-Specific Immune
Response1
P. Rod Dunbar,* Caroline L. Smith,* David Chao,‡ Mariolina Salio,* Dawn Shepherd,*
Fareed Mirza,* Martin Lipp,§ Antonio Lanzavecchia,¶ Federica Sallusto,¶ Alun Evans,储
Robin Russell-Jones,储 Adrian L. Harris,† and Vincenzo Cerundolo2*
T
he discovery that melanomas can be recognized and killed
by human CTL has allowed a number of CTL epitopes to
be defined (1). These epitopes are important targets for
immunotherapy, and since many of them are expressed in other
tumors, there are hopes that immunotherapeutic strategies initially
tested in melanoma may prove broadly applicable to other tumors.
Among the known melanoma Ags recognized by CTL, melan-A
or MART-1 is probably the best studied, and it has become an
extremely useful target for immunotherapy. Melan-A was originally defined in a patient with a favorable disease course after
vaccination with an autologous tumor cell line, by cloning from
her blood HLA-A2-restricted CTL capable of lysing this tumor
cell line (2). Expression libraries of tumor cDNAs showed that the
target of these CTL was melan-A, a lineage-specific molecule also
expressed on normal melanocytes (2, 3). The dominant HLA-A2restricted epitope of melan-A originally appeared to be melan-
*Molecular Immunology Group Nuffield Department of Medicine, and †Imperial
Cancer Research Foundation, Institute of Molecular Medicine, and ‡Nuffield Department of Surgery, University of Oxford, John Radcliffe Hospital, Oxford, United Kingdom; §Max-Delbrueck-Center for Molecular Medicine, Berlin-Buch, Germany; ¶Institute of Research in Biomedicine, Bellinzona, Switzerland; and 储Skin Tumor Unit,
St. John’s Institute of Dermatology, St. Thomas’ Hospital, Westminster, London,
United Kingdom
Received for publication April 13, 2000. Accepted for publication August 30, 2000.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
This work was supported by funding from the Cancer Research Campaign, the U.K.
Medical Research Council, the Cancer Research Institute, the Imperial Cancer Research Foundation, and the Axe Immunologie des Tumeurs de La Ligue Nationale
contre le Cancer.
2
Address correspondence and reprint requests to Dr. Vincenzo Cerundolo, Molecular
Immunology Group, Institute of Molecular Medicine, John Radcliffe Hospital, Oxford
OX3 9DS, U.K. E-mail address: [email protected]
Copyright © 2000 by The American Association of Immunologists
A27–35 (AAGIGILTV) (4), but subsequent experiments showed
melan-A26 –35 (EAAGIGILTV) is also recognized by CTL, and
binds better to HLA-A2 (5). Many laboratories have since found it
relatively easy to generate CTL specific for this epitope from
HLA-A2⫹ melanoma patients (6, 7), and it therefore seemed that
melan-A26/7–35 might be the most commonly recognized epitope
among all the known targets of melanoma Ag-specific CTL (6).
Many studies have also demonstrated that similar CTL could be
generated from healthy individuals (6), although this usually required repeated priming of PBL, and was possible in fewer healthy
individuals than melanoma patients (8). Nevertheless, the comparative ease with which melan-A26/7–35-specific CTL could be obtained in many healthy individuals suggested that precursor frequencies are elevated in a large percentage of the general
population. One explanation proposed was that melan-A26/7–35specific CTL may merely be cross-reactive with this epitope,
having been originally primed against a microbial epitope (9).
However, peptide stimulation experiments suggested that melanA26/7–35-specific CTL in healthy subjects did not derive from the
memory compartment, whereas they did in melanoma patients
(10). Unfortunately, the technical difficulties of characterizing
CTL activity ex vivo have imposed severe limitations on such
comparisons between patients and healthy controls. Similarly, any
correlation between CTL responses in different patients and their
clinical features has proven technically daunting.
The development of tetrameric MHC class I/peptide complexes
(11) has greatly aided the analysis of tumor-specific CTL responses. In keeping with the relative ease with which melan-Aspecific CTL can be derived in vitro, tetramers based on this
epitope have to date proven more useful in this work than those
based on any other known melanoma epitope. After initial studies
confirmed that melan-A tetramer⫹ CD8⫹ cells represented true
0022-1767/00/$02.00
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In a significant proportion of melanoma patients, CTL specific for the melan-A26/7–35 epitope can be detected in peripheral blood
using HLA-A2/peptide tetramers. However, the functional capacity of these CTL has been controversial, since although they prove
to be effective killers after in vitro expansion, in some patients they have blunted activation responses ex vivo. We used phenotypic
markers to characterize melan-A tetramerⴙ cells in both normal individuals and melanoma patients, and correlated these markers
with ex vivo assays of CTL function. Melanoma patients with detectable melan-A tetramerⴙ cells in peripheral blood fell into two
groups. Seven of thirteen patients had a CCR7ⴙ CD45R0ⴚ CD45RAⴙ phenotype, the same as that found in some healthy controls,
and this phenotype was associated with a lack of response to melan-A peptide ex vivo. In the remaining six patients, melan-A
tetramerⴙ cells were shifted toward a CCR7ⴚ CD45R0ⴙ CD45RAⴚ phenotype, and responses to melan-A peptide could be readily
demonstrated ex vivo. When lymph nodes infiltrated by melan-A-expressing melanoma cells were examined, a similar dichotomy
emerged. These findings demonstrate that activation of melan-A-specific CTL occurs in only some patients with malignant melanoma, and that only patients with such active immune responses are capable of responding to Ag in ex vivo assays. The Journal
of Immunology, 2000, 165: 6644 – 6652.
The Journal of Immunology
tients, were HLA-A2⫹ by PCR. Clinical characteristics of each melanoma
patient appear in Table I. All PBL obtained were cryopreserved immediately after separation, and analyzed after thawing and culturing briefly in I5
to allow metabolic recovery. TILN were surgically resected, and were mechanically disrupted before cryopreservation as single cell suspensions. On
thawing, the numbers of tumor cells were counted as well as the number of
lymphocytes, to provide an index of the extent of tumor infiltration, expressed as a percentage of the total cell count accounted for by tumor cells.
Expression of melan-A was examined in TILN by RT-PCR (2), and protein
expression confirmed in one sample by Western blotting using the primary
Ab A103 (Novocastra, Newcastle, U.K.) (25). When tumor samples were
available from patients, these were examined by immunohistochemistry,
including staining for melan-A with the primary Ab A103 (Novocastra)
(25). A tumor cell line was established from patient M2 by mechanically
disrupting a surgically excised skin metastasis and culturing in D10 medium. This cell line was subjected to FACS analysis after incubation for
72 h with or without 100 U/ml IFN-␥ and 1 ng/ml TNF-␣.
Peptides and tetramers
Peptides synthesized by FMOC chemistry were: melan-A, ELAGIGILTV,
a variant of the 26 –35 epitope, which binds better to HLA-A2 than the
natural peptide due to an altered anchor residue, but which is recognized by
the same CTL as the natural peptide (26); influenza, GILGFVFTL, the
influenza matrix protein 58 – 66 epitope (27); EBV, GLCTLVAML, the
280 –288 epitope from the lytic protein BMLF1 of EBV (28, 29).
Tetramers were all made from HLA-A2.1 heavy chain, and the peptides
above, and are referred to in the text simply as melan-A, influenza, and
EBV tetramers. HLA-A2/peptide complexes were synthesized as previously described (11, 12, 30). Briefly, the HLA-A2.1 heavy chain cDNA
was modified by substitution of the trans-membrane and cytosolic regions
with a sequence encoding the BirA biotinylation enzyme recognition site.
This modified HLA-A2.1 and ␤2-microglobulin were synthesized in a prokaryotic expression system (pET; R&D Systems, Minneapolis, MN), purified from bacterial inclusion bodies, and allowed to refold with the relevant peptide by dilution. Refolded complexes were purified by FPLC and
biotinylated using BirA (Avidity, Denver, CO), then combined with PElabeled streptavidin (Sigma, St. Louis, MO) at a 4:1 molar ratio to form
tetramers. Tetramers were titrated against appropriate CTL clones to determine the dose that induced maximal staining (12, 14).
FACS staining and analysis
The HLA-A2⫹ lymphoblastoid cell line T2 was maintained in RPMI 1640
medium with 10% FCS. The melanoma cell line SK-mel-29 was maintained in Dulbecco’s modified medium with 10% FCS (D10). FACS buffer
was PBS with 1% FCS. Cell culture media for patient samples were
Iscove’s medium plus 5% human serum (I5) or I5 plus 100 U/ml human
rIL-2 (Chiron, Emeryville, CA).
Cells were stained with the appropriate PE-labeled tetramer at 37°C for 20
min before washing in FACS buffer at 37°C, and incubating for 30 min on
ice with Abs, including Tricolor anti-CD8␣ (Caltag, Burlingame, CA) or
peridinin chlorophyl protein anti-CD8␣ (Becton Dickinson, Mountain
View, CA), FITC anti-CD45RA (Dako, Glostrup, Denmark), and APC
anti-CD45R0 (Becton Dickinson). For CCR7 staining, the rat mAb 3D12
(22) was incubated for 30 min before two washes in FACS buffers and
incubation with FITC mouse anti-rat IgG1/2a (PharMingen, San Diego,
CA) along with the other Abs. After extensive washes in ice-cold FACS
buffer, cells were kept on ice without fixation and analyzed on a Becton
Dickinson FACScalibur using CellQuest software. For analysis, small lymphocytes were gated according to forward/side scatter profiles. Tetramer⫹
CD8⫹ were defined as CD8high cells with a PE fluorescence of at least 100
fluorescence units (while CD8⫹ cells in the same sample unstained with
tetramer had a maximum fluorescence of 10 fluorescence units). CD8low
cells were excluded from analysis because most CD8low cells stained with
anti-CD8␣ Abs were CD8␣⫹ CD8␤⫺ CD3⫺ CD45RA⫹ (data not shown).
Frequencies of melan-A tetramer-positive cells were averaged from between two and six replicate stainings. Minimal melan-A tetramer binding
was observed on HLA-A2⫺ CD8⫹ PBL (⫽5 cells/105 CD8⫹ cells), but in
view of this slight background staining, HLA-A2⫹ samples containing less
than 20 melan-A tetramer⫹ cells per 105 CD8⫹ cells were considered negative for melan-A tetramer staining, and excluded from further analysis.
FACS analysis of MHC class I and HLA-A2 expression on cell lines
was performed using the mAbs W6/32 and BB7.2, respectively (American
Type Culture Collection, Manassas, VA).
Subjects and samples
Enzyme-linked immunospot (ELISPOT) analysis
Materials and Methods
Cell lines and reagents
Healthy subjects were blood donors, registered with the U.K. National
Blood Service. HLA-A2-negative healthy donors were used to define background levels of nonspecific binding by HLA-A2 tetramers. All other subjects discussed in the text, including healthy donors and melanoma pa3
Abbreviations used in this paper: TILN, tumor-infiltrated lymph node; ELISPOT,
enzyme-linked immunospot.
Where sufficient PBL were available, ELISPOT analysis for IFN-␥ secretion was performed according to the protocol provided by the manufacturer
(Mabtech, Stockholm, Sweden). PBL were thawed and cultured overnight
in I5 medium to ensure good viability before assay, and plated in duplicate
at up to 5 ⫻ 105 cells/well. In some experiments, CD8⫹ cells were enriched
from PBL using immunomagnetic separation (MACS; Miltenyi Biotec,
Bergsich Gladbach, Germany) before assay. Peptide was added at 10 ␮M
to appropriate wells, and cells incubated for 40 h before development.
Downloaded from http://www.jimmunol.org/ by guest on June 17, 2017
tumoricidal CTL (12), we showed that some lymph nodes infiltrated with melan-A-expressing melanoma cells contain high frequencies of melan-A tetramer⫹ cells, and that these cells have
phenotypic markers consistent with previous Ag exposure (13).
Small populations of melan-A tetramer⫹ CD8⫹ cells could also be
visualized in the peripheral blood of melanoma patients (13–15),
and in some patients these cells responded to peptide Ag in vitro
by proliferating, forming highly tumoricidal cell lines (12, 15).
Melan-A-specific CTL clones could also be generated by directly
sorting single melan-A tetramer⫹ CD8⫹ cells from melanoma patient peripheral blood (14). As expected from earlier work,
melan-A tetramer⫹ CD8⫹ cells could also be detected in the PBL
of some healthy individuals, and these cells were CD45RA⫹
CD45R0⫺ CD28⫹, a phenotype suggestive of a naive rather than
a memory status (16). Several melanoma patients also had tetramer⫹ CD8⫹ cells exclusively of this phenotype, while others
showed mixed populations, in which CD45RA expression had
been lost (13, 17), and CD45R0 had been gained on a proportion
of tetramer⫹ cells (16). These data suggested that only a minority
of circulating melan-A-specific CTL in a minority of melanoma
patients had a history of Ag exposure. However, CD45 isoforms
are not in themselves adequate markers of memory/naivety, since
memory cells can change expression patterns in vitro (18, 19), and
subpopulations of memory CTL specific for viral Ags can express
converse CD45 isoforms in vivo (20, 21).
Recently, expression of the chemokine receptor CCR7, which is
involved in homing to lymphoid tissue, has emerged as a useful
phenotypic marker in determining the memory status of T lymphocytes, particularly when used in conjunction with CD45 isoforms (22). CCR7 is expressed only by naive cells and central
memory cells, while effector memory cells and terminally differentiated effectors are CCR7⫺ (22). CCR7 and CD45 isoform
expression also seems to distinguish between the functional
capacity of different populations of T lymphocytes (22). The
functional capacity of circulating tumor-specific lymphocytes
has become an issue of some importance, since data published
recently suggested melan-A-specific CTL circulating in melanoma patients are anergic (23).
We analyzed CCR7 and CD45 isoform expression on melan-A
tetramer⫹ cells in the peripheral blood of melanoma patients and
normal controls, taking advantage of a modified tetramer-staining
protocol, which improves the specificity and intensity of tetramer
staining (24), and minimizes sample manipulation before assay.
The results of these phenotyping experiments were then compared
with assays of CTL function ex vivo, and further extended by
analysis of tumor-infiltrated lymph nodes (TILN)3 using similar
techniques.
6645
6646
FUNCTIONAL ACTIVITY OF MELAN-A-SPECIFIC CTL IN MELANOMA PATIENTS
Table I. Melanoma patient characteristics
Sex
Age
M1
M2
M3
M4
M5
M6
M7
M8
M9
M10
M11
M12
M13
M14
M15
M16
M17
M18
M19
M20
M21
M22
M23
M24
M25
M26
M27
M28
M29
M30
F
F
F
M
F
M
M
F
M
F
F
M
M
M
F
M
F
M
F
F
F
M
M
M
M
M
F
M
M
F
64
65
47
68
58
58
26
59
43
67
59
49
55
78
83
48
59
53
54
56
65
40
46
29
51
49
47
51
55
43
5
1
0.5
4
5
0.7
0
0
7
0
0
1.5
1.7
0.5
6
14
15
3
3
10
0.3
0.7
0
0
0
0
0.5
0
0.6
1.6
Stageb
IV
IV
IV
IV
IV
III
IIA
IA
III
IIA
IIB
III
IV
IIB
III
IV
IV
IV
III
IV
IV
III
IB
IB
IB
III
III
III
III
III
Sites of Metastasis
Lymph
Lymph
Lymph
Lymph
Lymph
Lymph
–
–
Lymph
–
–
Lymph
Lymph
–
Lymph
Lymph
Lymph
Lymph
Lymph
Lymph
Liver
Lymph
–
–
–
Lymph
Lymph
Lymph
Lymph
Lymph
nodes,
nodes,
nodes,
nodes,
nodes,
nodes
skin
adrenal
skin, liver, lungs, bone
adrenal
skin, lungs
nodes
nodes
nodes, lungs
nodes,
nodes,
nodes,
nodes,
nodes
nodes,
Prior Therapy
local
lungs
skin
chest wall
skin
node
node
nodes
nodes
nodes
nodes
Surgery,
Surgery,
Surgery,
Surgery,
Surgery,
Surgery,
–
–
Surgery,
–
–
Surgery,
Surgery,
Surgery
Surgery,
Surgery,
Surgery,
Surgery,
Surgery,
Surgery,
Surgery
Surgery
–
–
–
–
Surgery,
Surgery
Surgery
Surgery
Prior
Immunotherapy
chemotherapy
chemotherapy
chemotherapy
chemotherapy
chemotherapy
chemotherapy
chemotherapy
chemotherapy
chemotherapy
chemotherapy, laser therapy
chemotherapy
chemotherapy, radiotherapy
chemotherapy
chemotherapy
chemotherapy, radiotherapy
chemotherapy*
–
–
–
IFN-␥
IFN-␥
–
–
–
–
–
–
–
–
–
IFN-␥
–
IFN-␣
–
–
–
–
IFN-␣
–
–
–
–
–
–
–
–
a
Years elapsed between excision of primary lesion and sample studied.
American Joint Committee on Cancer staging system.
ⴱ, Two TILN were obtained from M27, one pre- and one post-chemotherapy.
b
Results are presented normalized for CD8 counts in each PBL sample
(determined by FACS), and represent mean values for each peptide after
background (no peptide) has been subtracted. ELISPOT analysis of TILN
was conducted in a similar fashion, except that CD8⫹ cells were separated
away from tumor cells by immunomagnetic selection (MACS). CD8⫹ cells
were cultured in I5 medium for 48 h before assay, then 4000 CD8⫹ cells
were plated in duplicate ELISPOT wells containing T2 cells pulsed or not
pulsed with 10 ␮M melan-A peptide.
Analysis of CTL expansion
A total of 106 PBL from healthy donors and melanoma patients with detectable melan-A tetramer⫹ CD8⫹ cells was cultured at 37°C in I5 plus 100
U/ml human rIL-2 for 7 days after addition of 10 ␮M melan-A peptide or
no peptide, before harvesting and staining with tetramer and Abs, as described above.
Results
Normal donor PBL: phenotype and functional characteristics of
melan-A tetramer⫹ CD8⫹ cells
Melan-A tetramer⫹ cells could be detected in the peripheral blood
of 5 of 10 healthy blood donors (Fig. 1). These cells were CCR7⫹
CD45R0⫺ (Fig. 2, a and d). In contrast, influenza and EBV tetramer⫹ (memory) cells in healthy blood donors were never
CCR7⫹ CD45R0⫺ (data not shown). Melan-A tetramer⫹
CD45R0⫺ cells were also CD45RA⫹ (Fig. 2g), so that the full
phenotype of these cells was CCR7⫹ CD45RA⫹ CD45R0⫺, consistent with the naive phenotype proposed in recent work (22).
ELISPOT analysis revealed no detectable secretion of IFN-␥ from
the PBL of these healthy donors in response to melan-A peptide
(Fig. 3), even when 5 ⫻ 105 enriched CD8⫹ cells were plated per
well (Fig. 4), and viral recall responses could readily be visualized
(Fig. 4). Hence, the CCR7⫹ CD45R0⫺ phenotype in melan-A tetramer⫹ cells in normal donors is associated with a lack of recall
response to peptide Ag.
Melanoma patient PBL: phenotype and functional
characteristics of melan-A tetramer⫹ CD8⫹ cells
Peripheral blood from 26 patients with malignant melanoma
(whose clinical characteristics are provided in Table I) was examined with melan-A tetramers. Melan-A tetramer⫹ cells were repeatably detected on ex vivo staining in 13 of the 26 patients (Fig.
1). In contrast, tetramers made from other melanoma epitopes only
rarely detected tumor-specific CTL in peripheral blood without Ag
stimulation (data not shown). The frequency of melan-A tetramer⫹
cells in melanoma patients was often similar to those found in
normal subjects (Fig. 1), with only four patients showing more
frequent melan-A-specific CTL than the normal controls. When
the phenotype of these cells was studied, the patients fell into two
distinct groups. In one group (group B), comprising 7 of the 13
patients with detectable CTL, melan-A tetramer⫹ cells were
CCR7⫹ CD45R0⫺ (Fig. 2, b and e), the same phenotype as in the
healthy controls. In contrast, influenza and EBV tetramer⫹ cells,
where detected in these patients, were never CCR7⫹ CD45R0⫺
(data not shown). In the other group (group A), comprising 6 of 13
patients with detectable CTL, a substantial proportion of melan-A
tetramer⫹ cells was CCR7⫺ CD45R0⫹ (Fig. 2, c and f). Tetramer⫹
CD45R0⫹ cells in group A patients were also CD45RA⫺ (Fig. 2h),
so that these patients had substantial populations of CCR7⫺
CD45RA⫺ CD45R0⫹ cells, the converse phenotype of those
found in group B patients and healthy controls. However, in all
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Patient
Years
Since
Primarya
The Journal of Immunology
6647
group A patients, some melan-A tetramer⫹ cells still displayed the
phenotype typical of group B patients and healthy controls (Fig. 2,
c, f, and h), while CCR7⫹ CD45R0⫹ cells were rare. Hence, the
distinguishing characteristic of group A is the appearance of
CCR7⫺ CD45RA⫺ CD45R0⫹ cells, among the melan-A tetramer⫹ population. The percentage of CCR7⫺ CD45R0⫹ cells
among the melan-A tetramer⫹ cells in group A patients ranged
from 24 to 89%, with the highest phenotypic shifts seen in those
with the highest frequencies of melan-A tetramer⫹ cells.
FIGURE 2. Phenotypes of melan-A tetramer⫹
CD8⫹ cells in peripheral blood. PBL from melanoma patients and healthy controls were quadruple stained with melan-A tetramer and anti-CD8,
along with either anti-CD45R0 and anti-CCR7
(d–f) or anti-CD45R0 and anti-CD45RA (g and h).
Three representative samples are shown, gated on
lymphocytes to show the size of the tetramer⫹
CD8⫹ population (a– c), or gated on tetramer⫹
CD8⫹ cells to show the phenotype of these cells
only (d– h).
In ELISPOT assay, only patients from group A were capable of
producing IFN-␥ in response to melan-A peptide (Fig. 3). CD8
enrichment allowed substantial numbers of melan-A-reactive cells
to be visualized in patients from group A, but did not result in any
melan-A-specific signal from subjects with melan-A tetramer⫹
cells bearing an exclusively CCR7⫹ CD45RA⫹ CD45R0⫺ phenotype (Fig. 4). Comparison of the frequencies of ELISPOT-reactive cells with melan-A tetramer⫹ cells in group A revealed that on
average, one-third of tetramer⫹ cells were detected in ELISPOT
Downloaded from http://www.jimmunol.org/ by guest on June 17, 2017
FIGURE 1. Frequencies of melan-A tetramer⫹ CD8⫹ cells in peripheral blood. PBL from melanoma patients (f and o) and healthy controls (䡺) were
stained directly with melan-A tetramer and anti-CD8, and frequencies of tetramer-high cells calculated as a proportion of total CD8 cells. Subsequent
phenotyping of melan-A tetramer⫹ cells showed the melanoma patients could be divided into two groups: group B (o) had the same phenotype as healthy
controls (CCR7⫹ CD45R0⫺), while group A (f) had circulating melan-A tetramer⫹ cells that were CCR7⫺ CD45R0⫹.
6648
FUNCTIONAL ACTIVITY OF MELAN-A-SPECIFIC CTL IN MELANOMA PATIENTS
FIGURE 3. Frequencies of IFN-␥-secreting CD8⫹ cells compared with tetramer⫹
CD8⫹ cells. PBL from melanoma patients
(filled symbols) and healthy controls (E) were
stained with melan-A tetramer and anti-CD8,
and frequencies of tetramer⫹ cells as a proportion of total CD8 cells were calculated (x-axis).
The same PBL were tested in ELISPOT assay
for secretion of IFN-␥ in response to melan-A
peptide, and spot-forming cells calculated as a
proportion of total CD8 cells (y-axis). a, PBL
from group A melanoma patients (f) showed a
correlation between spot-forming cells and tetramer⫹ cells. b, PBL from healthy controls (E)
and group B melanoma patients (Œ) showed no
spot-forming cells, despite detectable melan-A
tetramer⫹ cells. Two group A melanoma patients (also shown on the larger scale in a) are
shown for comparison.
Melanoma-infiltrated lymph nodes: phenotype and function of
melan-A tetramer⫹ CD8⫹ cells
TILN from four patients (described in Table I) were examined,
including two TILN from the same patient. The TILN had variable
percentages of infiltrating tumor cells, as shown in Fig. 6 (M30
was 9%). Melan-A expression was confirmed in all TILN samples
by RT-PCR, and in TILN from patient M29 by Western blot.
Melan-A tetramer⫹ CD8⫹ cells were also detectable in all samples. Two TILN, from patients M29 and M30, showed frequencies
of melan-A tetramer⫹ cells similar to those found in healthy controls and group B patient PBL, less than 100 cells per 105 CD8⫹
cells (Fig. 6d, and data not shown). Melan-A tetramer⫹ CD8⫹
cells in these TILN had a CCR7⫹ CD45R0⫺ phenotype (Fig. 6h),
identical with PBL from healthy controls and group B melanoma
patients. Patients M27 and M28 had melan-A tetramer⫹ cells that
were not of this phenotype. Patient M28 had a melan-A tetramer⫹
population of 0.27% of CD8⫹ cells (Fig. 6c), which were largely
CCR7⫺ CD45R0⫹ (Fig. 6g), although some CCR7⫹ CD45R0⫹
cells were also present (Fig. 6g). Patient M27, in the first TILN
sample, had a massively expanded population of melan-A tetramer⫹ CD8⫹ cells compared with all other patients, equivalent to
13% of the CD8⫹ cells (Fig. 6a), and the vast majority of these
cells were CCR7⫺ CD45R0⫹ (Fig. 6e), although 6% were CCR7⫹
CD45R0⫹ (Fig. 6e). A second TILN, obtained from patient M27 8
mo later, had a similar expansion of melan-A tetramer⫹ CD8⫹
cells (Fig. 6b), although these cells were split evenly between the
CCR7⫺ CD45R0⫹ and CCR7⫹ CD45R0⫹ phenotypic patterns
(Fig. 6f). Perhaps most strikingly, in neither of these TILN, in
which expansion of melan-A-specific CTL had clearly been stimulated, did significant numbers of these CTL bear the CCR7⫹
CD45R0⫺ phenotype seen in healthy PBL (Fig. 6, e and f). Hence,
in the TILN, as in the PBL, melan-A tetramer⫹ cells had strikingly
different phenotypes in different patients, with some patients showing exclusively a CCR7⫹ CD45R0⫺ phenotype, and some patients
showing a shift to the converse phenotype. This shift in phenotype
was associated with a slightly greater level of tumor infiltration in
the small number of samples investigated in this study (Fig. 6).
ELISPOT assay was performed on TILN1 from patient M27 to
test whether the expanded population of melan-A tetramer⫹ cells
(13% of total CD8⫹ cells) was able to release IFN-␥ in response
FIGURE 4. ELISPOT analysis of IFN-␥ secretion by enriched CD8⫹ cells. PBL from a melanoma patient (M1) and a healthy control with detectable
melan-A tetramer⫹ CD8⫹ cells (N1) were enriched for CD8⫹ cells by immunomagnetic sorting. A total of 5 ⫻ 105 CD8⫹ cells/well was then plated in
duplicate into an ELISPOT assay for secretion of IFN-␥, with or without melan-A peptide or influenza matrix peptide at 10 ␮M. The assay was developed
after a 40-h incubation.
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(Fig. 3). This discrepancy is very similar to that reported for influenza-specific CTL (16). To confirm that melan-A tetramer⫹
cells in group A patients were capable of functional responses to
Ag, PBL were cultured in the presence of IL-2 for 7 days after
addition of melan-A peptide. Melan-A tetramer⫹ cells from group
A patients expanded rapidly under these experimental conditions
(Fig. 5).
The Journal of Immunology
6649
FIGURE 5. Tetramer analysis of in vitro
expansion in response to melan-A peptide.
PBL from a melanoma patient (M2) were
plated at 1 ⫻ 106 cells/well, with or without
10 ␮M melan-A peptide, then cultured in the
presence of IL-2 for 7 days. Cells were then
harvested and stained with melan-A tetramer
and anti-CD8, and frequencies of tetramer⫹
CD8⫹ cells in each culture were calculated as
a percentage of the total CD8⫹ cells.
Tumor phenotype in patients with active melan-A-specific CTL
Patient M1, who had the highest level of melan-A-specific CTL
measured by both tetramer staining and ELISPOT, had tissue
available from a sternal metastasis for examination by immunohistochemistry. Although the tumor cells expressed melan-A
strongly, and a CD8⫹ lymphocytic infiltrate was present, MHC
class I expression was not detected on tumor cells (data not
shown). From patient M2, who had the second strongest melanA-specific CTL response, a resected skin metastasis was cultured
in vitro, and a tumor cell line was established. This cell line proved
to have a selective loss of HLA-A2 (Fig. 8). Interestingly, this cell
line exhibited substantial expression of total MHC class I, indicating that at least one allele other than HLA-A2 was still expressed (Fig. 8). Treatment with IFN-␥ up-regulated total expression of MHC class I slightly, but did not restore HLA-A2
expression (Fig. 8).
Discussion
Quantification of melan-A-specific CTL in peripheral blood
In this study, we used optimized tetramer-staining techniques to
study melan-A-specific CTL directly ex vivo, to correlate phenotypic markers with functional parameters.
Circulating CTL specific for melan-A were detectable ex vivo in
13 of 26 patients with malignant melanoma and 5 of 10 healthy
controls. These percentages of tetramer positivity in PBL are far
higher than for other tumor epitopes studied to date with tetramers
(31–33), in which only rare patients have circulating tumor-specific CTL detectable ex vivo. Given that so many normal subjects
also had melan-A tetramer⫹ cells, the high percentage of positivity
in melanoma patients is not surprising, and clearly may relate to a
higher precursor frequency in many HLA-A2⫹ individuals rather
than a response to the melanoma-associated Ag. Four patients with
melanoma (M1– 4) had a higher frequency of melan-A tetramer⫹
cells than any normal control, suggesting they might have some
degree of Ag-specific CTL expansion. However, it is important to
note that even the highest tetramer⫹ frequency seen in this patient
series (0.3% of CD8⫹ cells) is relatively low compared with those
seen in acute viral infections (21, 30).
FIGURE 6. Phenotypes of melan-A tetramer⫹ CD8⫹ cells in TILN. TILN from three melanoma patients (including two different nodes from patient
M27) were quadruple stained with melan-A tetramer, anti-CD8, anti-CD45R0, and anti-CCR7. a– d, Scatter plots of tetramer and CD8 stains (gated on small
lymphocytes by forward/side scatter profile) show the size of the tetramer⫹ CD8⫹ population, as indicated. e– h, Scatter plots gated on tetramer⫹ CD8⫹
cells show the phenotype of these cells with respect to expression of CCR7 and CD45R0. The proportion of the cells in each sample accounted for by tumor
cells is shown above each pair of plots, as an index of tumor infiltration.
Downloaded from http://www.jimmunol.org/ by guest on June 17, 2017
to peptide Ag. To minimize contamination of the assay with
melan-A-expressing tumor cells, CD8⫹ cells were separated from
tumor cells, and rested in tissue culture for 2 days. In subsequent
ELISPOT assay, secretion of IFN-␥ in response to melan-A peptide was detected in 4.5% of the CD8⫹ cells plated (Fig. 7), a
similar proportion of tetramer⫹ cells to that detected in PBL.
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FUNCTIONAL ACTIVITY OF MELAN-A-SPECIFIC CTL IN MELANOMA PATIENTS
FIGURE 7. ELISPOT analysis of IFN-␥ secretion by enriched CD8⫹
cells from melanoma-infiltrated lymph nodes. CD8⫹ lymphocytes from a
melanoma-infiltrated lymph node (TILN1 from patient M27) were enriched by immunomagnetic sorting, and rested for 48 h in tissue culture.
Four thousand cells/well were then plated in duplicate into an ELISPOT
assay for secretion of IFN-␥, with T2 cells pulsed or not pulsed with 10 ␮M
melan-A peptide. The assay was developed after 40-h incubation.
During such a response, the phenotype of melan-A tetramer⫹
cells in the PBL typically shifted to CCR7⫺ CD45RA⫺ CD45R0⫹.
Cells with this phenotype have been termed effector memory cells
(22) and are expected to have effector function, including the ability to secrete IFN-␥ (22), as detected in our assays. Populations of
melan-A-specific CTL that fit the proposed central memory population (CCR7⫹ CD45RA⫺ CD45R0⫹) were only rarely seen in
peripheral blood (Fig. 2, and data not shown). However, in TILN,
CCR7⫹ CD45R0⫹ cells with a putative central memory phenotype
were detected (Fig. 6), albeit as smaller populations than the effector memory cells (CCR7⫺ CD45R0⫹). Interestingly, of two
TILN taken from the same patient 8 mo apart, it was at the later
time point that the most central memory cells were found among
the melan-A-specific CTL.
Lack of effector function correlates with a naive phenotype
Phenotype of melan-A-specific CTL in peripheral blood
and TILN
FIGURE 8. Expression of HLA-A2 and total
MHC class I on melanoma lines. The melanoma
cell line M2, derived from patient M2, was
stained for surface expression of HLA-A2 (mAb
BB7.2) and total MHC class I (mAb W6/32) and
analyzed by FACS, with and without prior treatment with IFN-␥ and TNF-␣. The HLA-A2⫹
melanoma cell line SK-mel-29 served as a positive control.
Priming of melan-A-specific CTL precursors is often weak in
melanoma
In some patients with malignant melanoma (group B), melan-A
tetramer⫹ cells were readily detected, but there was no phenotypic
evidence that these cells had ever responded to Ag. This phenomenon was observed even in TILN, in which melan-A-expressing
tumor cells were effectively adjacent to melan-A CTL precursors
(patients M29 and M30). It therefore seems likely that these patients are in a state analogous to the peripheral ignorance proposed
from some animal tumor models (35, 36), in which tumor-specific
CTL are present both in the circulation and the lymphoid tissue,
but have not been primed by tumor Ag. As noted above, unprimed
CTL were seen even in the circulation of patients who had managed to prime at least a proportion of their melan-A-specific CTL.
Hence, even in the circulation of melanoma patients with active
immunity against melan-A, priming of CTL may not be complete.
In fact, only in those TILN in which expansion of melan-A-specific CTL had occurred, had all melan-A tetramer⫹ cells converted
away from the naive CCR7⫹ CD45R0⫺ phenotype (Fig. 6). All
these data suggest priming of melan-A-specific CTL by malignant
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We used expression of the chemokine receptor CCR7 alongside
CD45 isoforms to analyze the phenotype of melan-A tetramer⫹
CD8⫹ cells, to resolve questions about the nature of these cells that
could not be answered using CD45 isoform expression pattern
alone. The CD45R0⫺ CD45RA⫹ phenotype classically ascribed to
naive cells suffers from the ability of memory cells such as EBVspecific memory and effector CTL to revert to this phenotype (20,
21). Hence, new markers of memory status are needed, and CCR7
is clearly one such candidate (22). In control experiments, we
found no EBV- or influenza-specific CTL with a CCR7⫹
CD45R0⫺ phenotype, suggesting that memory CTL do not bear
this combination of markers. This CCR7⫹ CD45R0⫺ phenotype
was found on melan-A tetramer⫹ cells from both healthy controls
and some melanoma patients (group B), confirming that the melanA-specific CTL precursors detected in these individuals are unlikely to be memory CTL.
Strikingly, in TILN, in which expansions of melan-A-specific
CTL had clearly taken place (Fig. 6), this CCR7⫹ CD45R0⫺ phenotype was entirely absent from melan-A tetramer⫹ cells. Hence,
conversion away from this phenotype is associated with strong in
vivo proliferative CTL responses against the tumor, and Ag-specific effector function that is measurable ex vivo. In the PBL of six
patients, melan-A tetramer⫹ cells that no longer expressed the
CCR7⫹ CD45RA⫹ CD45R0⫺ phenotype could be found, suggesting an active immune response against melan-A had occurred in
these patients. Indeed, in functional assays of recall responses,
these were the patients who responded to melan-A peptide, demonstrating that these CTL were not anergic. Therefore, in both PBL
and TILN, loss of the CCR7⫹ CD45RA⫹ CD45R0⫺ phenotype
was associated with a functional immune response against
melan-A.
Our data suggest that many melan-A-specific CTL that appear anergic ex vivo may in fact be naive or unprimed, because they are
phenotypically identical with those found in many normal subjects
(CCR7⫹ CD45RA⫹ CD45R0⫺). Functional assays that depend on
rapid response to peptide Ag do not detect these cells, because they
probably require prolonged stimulation to become activated (34).
Hence, we failed to detect secretion of IFN-␥ in response to peptide when samples contained such naive phenotype CTL precursors, while this assay readily detected the presence of Ag-experienced (CCR7⫺ CD45RA⫺ CD45R0⫹) CTL. Our data suggest that
brisk effector function can be readily detected ex vivo provided
there is evidence of CTL priming.
The Journal of Immunology
melanoma is weak. This should perhaps not be unduly surprising,
because tumor cells usually lack costimulatory molecules (37, 38),
and tumor Ags have no direct route into the MHC class I pathway
of professional APCs.
How is active immunity against melan-A generated?
Clinical consequences of active immunity against melan-A
When clinical parameters in group A and group B patients were
compared, no striking correlation with disease progression was
evident. Indeed, further study will be necessary to determine
whether the character of the melan-A-specific CTL response has
any impact on clinical course, because we did not follow patients
longitudinally or study patients at equivalent disease stages. Nevertheless, the clinical features of group A patients offer important
clues as to the possible impact of an active immune response
against melan-A.
Most importantly, the two patients from group A with the strongest melan-A-specific CTL responses both died within 1 yr of
developing metastatic disease. Hence, active immunity to
melan-A, comprising activated Ag-experienced melan-A-specific
CTL circulating at numbers of up to 400 cells/ml of blood, did not
protect these patients from succumbing to their tumors. If priming
of melanoma Ag-specific CTL is a relatively late event, then it is
possible that these patients simply generated a melan-A-specific
CTL response too late to show substantial clinical benefit. The loss
of HLA-A2 expression observed in some melanoma cells from
these patients would have rendered these cells impervious to the
effects of the HLA-A2-restricted melan-A-specific CTL, even in
the presence of IFN-␥ and TNF-␣ (in the case of patient M2). The
maintenance of total MHC class I expression in some of these cells
(Fig. 8), presumably due to continued expression of at least one
other MHC class I allele, may also have allowed these cells to
escape attack by NK cells. Although similar antigenic loss variants
have previously been reported in melanoma (41, 42), in this study
we confirm that this phenomenon can occur in patients with functional Ag-specific CTL detectable ex vivo. It is possible that the
pressure of melan-A CTL attack in patient M2 may have played a
role in selecting the tumor variant we propagated in tissue culture.
However, it is important to note that MHC class I loss variants are
also likely to be present in patients without an active immune
response against melan-A, so our data do not necessarily support
the concept of CTL pressure selecting tumor variants in vivo.
Implications for immunotherapy of melanoma
These findings have important implications for immunotherapy of
melanoma. If many melanoma patients are failing to prime their
melan-A-specific CTL, then clearly immunogens that are capable
of priming these CTL may be clinically helpful. The fact that CTL
precursors for the melan-A26/7–35 epitope seem to be present at
relatively high frequencies in such a large proportion of HLA-A2⫹
individuals is encouraging, because it may prove easier to generate
a sizeable CTL response against this epitope than for epitopes in
which precursor CTL are rare. In contrast, CTL responses against
melan-A26/7–35 are clearly not protective in late disease, at least in
part due to the development of tumor escape loss variants. Even so,
it is clear that a sound immunotherapeutic strategy in melanoma
may be to prime melan-A26/7–35-specific CTL early in disease,
when tumor burden and genetic variation are at a minimum. Using
polyvalent agents to induce CTL against other melanoma Ags
would also seem advisable, to minimize the risk of Ag loss variants
emerging. Powerful immunogens capable of reliably priming tumor-specific CTL are needed, and developing agents that are also
simple to store and administer will greatly assist their wider use in
early disease.
Acknowledgments
We thank Pru Bahl for expert technical assistance.
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