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*
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
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Skin Tumor Unit, St. John’s Institute of Dermatology, St. Thomas’ Hospital, Westminster, London, United Kingdom
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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-A2-restricted epitope of melan-A originally appeared to be melan- 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-A26/7–35-specific CTL may merely be cross-reactive with this epitope, having been originally primed against a microbial epitope (9). However, peptide stimulation experiments suggested that melan-A26/7–35-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-A-specific 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 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, 14, 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.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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).
Subjects and samples
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 patients, 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-
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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 PE-labeled 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
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).
Enzyme-linked immunospot (ELISPOT) analysis
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 x 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 µM to appropriate wells, and cells incubated for 40 h before
development. 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 µM 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 µM melan-A peptide or no peptide, before harvesting and staining with tetramer and Abs, as described above.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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 x
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.
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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 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.
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 (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).
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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).
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in response 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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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 melan-A-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).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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, 32, 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).
Phenotype of melan-A-specific CTL in peripheral blood and TILN
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 EBV-specific 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 melan-A-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.
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
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.
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 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?
The fact that only some patients primed their melan-A CTL precursors begs the question as to whether any factors can be distinguished that might correlate with CTL priming. It is tempting to suggest that priming of melanoma Ag-specific CTL is a late phenomenon, because only stage III or stage IV patients (patients M1-6, M27-8) had the group A phenotype indicating an active immune response. Conversely, four patients with no clinical evidence of metastatic disease (M7, M8, M10, M11) had the group B unprimed phenotype, suggesting substantial metastatic disease may be required for CTL priming. As noted above, however, many patients with metastases also showed no evidence of priming melan-A-specific CTL, so while metastatic disease may be required for priming CTL, it may not in itself be sufficient. Indeed, the current data suggest that the cross-presentation events postulated to explain the priming of tumor-specific CTL in humans (39, 40) may be quite rare.
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.
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Acknowledgments |
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Footnotes |
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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.
3 Abbreviations used in this paper: TILN, tumor-infiltrated lymph node; ELISPOT, enzyme-linked immunospot.
Received for publication April 13, 2000. Accepted for publication August 30, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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2-Microglobulin mutations, HLA class I antigen loss, and tumor progression in melanoma. J. Clin. Invest. 101:2720.[Medline]This article has been cited by other articles:
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V. Voelter, N. Rufer, S. Reynard, G. Greub, R. Brookes, P. Guillaume, F. Grosjean, T. Fagerberg, O. Michelin, S. Rowland-Jones, et al. Characterization of Melan-A reactive memory CD8+ T cells in a healthy donor Int. Immunol., August 1, 2008; 20(8): 1087 - 1096. [Abstract] [Full Text] [PDF]
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K. M. Hargadon, C. C. Brinkman, S. L. Sheasley-O'Neill, L. A. Nichols, T. N. J. Bullock, and V. H. Engelhard Incomplete Differentiation of Antigen-Specific CD8 T Cells in Tumor-Draining Lymph Nodes J. Immunol., November 1, 2006; 177(9): 6081 - 6090. [Abstract] [Full Text] [PDF]
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C. L. Smith, F. Mirza, V. Pasquetto, D. C. Tscharke, M. J. Palmowski, P. R. Dunbar, A. Sette, A. L. Harris, and V. Cerundolo Immunodominance of Poxviral-Specific CTL in a Human Trial of Recombinant-Modified Vaccinia Ankara J. Immunol., December 15, 2005; 175(12): 8431 - 8437. [Abstract] [Full Text] [PDF]
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E. Mallard, F. Vernel-Pauillac, T. Velu, F. Lehmann, J.-P. Abastado, M. Salcedo, and N. Bercovici IL-2 Production by Virus- and Tumor-Specific Human CD8 T Cells Is Determined by Their Fine Specificity J. Immunol., March 15, 2004; 172(6): 3963 - 3970. [Abstract] [Full Text] [PDF]
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M. J. Dobrzanski, J. B. Reome, J. A. Hollenbaugh, and R. W. Dutton Tc1 and Tc2 Effector Cell Therapy Elicit Long-Term Tumor Immunity by Contrasting Mechanisms That Result in Complementary Endogenous Type 1 Antitumor Responses J. Immunol., February 1, 2004; 172(3): 1380 - 1390. [Abstract] [Full Text] [PDF]
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M. J. Dobrzanski, J. B. Reome, J. A. Hollenbaugh, J. C. Hylind, and R. W. Dutton Effector Cell-Derived Lymphotoxin {alpha} and Fas Ligand, but not Perforin, Promote Tc1 and Tc2 Effector Cell-Mediated Tumor Therapy in Established Pulmonary Metastases Cancer Res., January 1, 2004; 64(1): 406 - 414. [Abstract] [Full Text] [PDF]
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L. H. Butterfield, A. Ribas, W. S. Meng, V. B. Dissette, S. Amarnani, H. T. Vu, E. Seja, K. Todd, J. A. Glaspy, W. H. McBride, et al. T-Cell Responses to HLA-A*0201 Immunodominant Peptides Derived from {alpha}-Fetoprotein in Patients with Hepatocellular Cancer Clin. Cancer Res., December 1, 2003; 9(16): 5902 - 5908. [Abstract] [Full Text] [PDF]
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M. Salio, N. Dulphy, J. Renneson, M. Herbert, A. McMichael, A. Marchant, and V. Cerundolo Efficient priming of antigen-specific cytotoxic T lymphocytes by human cord blood dendritic cells Int. Immunol., October 1, 2003; 15(10): 1265 - 1273. [Abstract] [Full Text] [PDF]
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M. W. J. Schreurs, K. B. J. Scholten, E. W. M. Kueter, J. J. Ruizendaal, C. J. L. M. Meijer, and E. Hooijberg In Vitro Generation and Life Span Extension of Human Papillomavirus Type 16-Specific, Healthy Donor-Derived CTL Clones J. Immunol., September 15, 2003; 171(6): 2912 - 2921. [Abstract] [Full Text] [PDF]
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M. J. Pittet, V. Rubio-Godoy, G. Bioley, P. Guillaume, P. Batard, D. Speiser, I. Luescher, J.-C. Cerottini, P. Romero, and A. Zippelius {alpha}3 Domain Mutants of Peptide/MHC Class I Multimers Allow the Selective Isolation of High Avidity Tumor-Reactive CD8 T Cells J. Immunol., August 15, 2003; 171(4): 1844 - 1849. [Abstract] [Full Text] [PDF]
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M. Ayyoub, A. Zippelius, M. J. Pittet, D. Rimoldi, D. Valmori, J.-C. Cerottini, P. Romero, F. Lejeune, D. Lienard, and D. E. Speiser Activation of Human Melanoma Reactive CD8+ T Cells by Vaccination with an Immunogenic Peptide Analog Derived from Melan-A/Melanoma Antigen Recognized by T Cells-1 Clin. Cancer Res., February 1, 2003; 9(2): 669 - 677. [Abstract] [Full Text] [PDF]
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S. Mantovani, B. Palermo, S. Garbelli, R. Campanelli, G. Robustelli della Cuna, R. Gennari, F. Benvenuto, E. Lantelme, and C. Giachino Dominant TCR-{alpha} Requirements for a Self Antigen Recognition in Humans J. Immunol., December 1, 2002; 169(11): 6253 - 6260. [Abstract] [Full Text] [PDF]
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L. Peng, J. Kjaergaard, G. E. Plautz, M. Awad, J. A. Drazba, S. Shu, and P. A. Cohen Tumor-Induced L-Selectinhigh Suppressor T Cells Mediate Potent Effector T Cell Blockade and Cause Failure of Otherwise Curative Adoptive Immunotherapy J. Immunol., November 1, 2002; 169(9): 4811 - 4821. [Abstract] [Full Text] [PDF]
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M. J. Palmowski, E. M.-L. Choi, I. F. Hermans, S. C. Gilbert, J.-L. Chen, U. Gileadi, M. Salio, A. Van Pel, S. Man, E. Bonin, et al. Competition Between CTL Narrows the Immune Response Induced by Prime-Boost Vaccination Protocols J. Immunol., May 1, 2002; 168(9): 4391 - 4398. [Abstract] [Full Text] [PDF]
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A. Zippelius, M. J. Pittet, P. Batard, N. Rufer, M. de Smedt, P. Guillaume, K. Ellefsen, D. Valmori, D. Lienard, J. Plum, et al. Thymic Selection Generates a Large T Cell Pool Recognizing a Self-Peptide in Humans J. Exp. Med., February 19, 2002; 195(4): 485 - 494. [Abstract] [Full Text] [PDF]
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A. Perez-Diez, P. J. Spiess, N. P. Restifo, P. Matzinger, and F. M. Marincola Intensity of the Vaccine-Elicited Immune Response Determines Tumor Clearance J. Immunol., January 1, 2002; 168(1): 338 - 347. [Abstract] [Full Text] [PDF]
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D. E. Speiser, M. Colonna, M. Ayyoub, M. Cella, M. J. Pittet, P. Batard, D. Valmori, P. Guillaume, D. Lienard, J.-C. Cerottini, et al. The Activatory Receptor 2B4 Is Expressed In Vivo by Human CD8+ Effector {alpha}{beta} T Cells J. Immunol., December 1, 2001; 167(11): 6165 - 6170. [Abstract] [Full Text] [PDF]
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J.-S. Blanchet, D. Valmori, I. Dufau, M. Ayyoub, C. Nguyen, P. Guillaume, B. Monsarrat, J.-C. Cerottini, P. Romero, and J. E. Gairin A New Generation of Melan-A/MART-1 Peptides That Fulfill Both Increased Immunogenicity and High Resistance to Biodegradation: Implication for Molecular Anti-Melanoma Immunotherapy J. Immunol., November 15, 2001; 167(10): 5852 - 5861. [Abstract] [Full Text] [PDF]
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M. Salio, D. Shepherd, P. R. Dunbar, M. Palmowski, K. Murphy, L. Wu, and V. Cerundolo Mature Dendritic Cells Prime Functionally Superior Melan-A-Specific CD8+ Lymphocytes as Compared with Nonprofessional APC J. Immunol., August 1, 2001; 167(3): 1188 - 1197. [Abstract] [Full Text] [PDF]
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M. J. Pittet, A. Zippelius, D. E. Speiser, M. Assenmacher, P. Guillaume, D. Valmori, D. Lienard, F. Lejeune, J.-C. Cerottini, and P. Romero Ex Vivo IFN-{{gamma}} Secretion by Circulating CD8 T Lymphocytes: Implications of a Novel Approach for T Cell Monitoring in Infectious and Malignant Diseases J. Immunol., June 15, 2001; 166(12): 7634 - 7640. [Abstract] [Full Text] [PDF]
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and 
) and healthy
controls (
) 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 (
) 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-
) 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.
