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Immunology |
Division of Clinical Onco-Immunology, Ludwig Institute for Cancer Research, Lausanne Branch [D. V., V. D., D. L., D. R., M. J. P., D. S., J-C. C., P. R.], and Multidisciplinary Oncology Center [D. L., F. L.], University Hospital, 1011 Lausanne, Switzerland; Laboratoire d’Immunologie, Institut de Recherches Cliniques de Montréal, HZW IR7 Québec, Canada [P. C., K. E.]; Department of Medicine, Division of Experimental Medicine, McGill University, Montréal, Québec H3A 1A3, Canada [P. C.]; and Department of Internal Medicine I, Saarland University Medical School, D-66421 Homburg, Germany [U. S.]
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ABSTRACT |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Six CT antigen families have been identified thus far, either by using tumor-reactive CTL clones from melanoma patients (1) or by immunoscreening of cDNA expression libraries from tumors [serological analysis of recombinant cDNA expression libraries (SEREX)] with autologous sera from cancer patients (2, 3, 4) . Whereas CD8+ T cells directed against melanocyte differentiation antigens are readily detectable in both peripheral blood lymphocytes and TILs from melanoma patients, T-cell responses to "MAGE-related" CT antigens have been detected only rarely in cancer patients, even after vaccination (5) . T-cell reactivity to "SEREX-defined antigens" has only recently been addressed (6 , 7) . In particular, three peptides corresponding to overlapping sequences spanning the 155–167 region of the CT antigen NY-ESO-1 have been shown to be recognized by a tumor-reactive CTL line from a melanoma patient in an HLA-A2-restricted fashion (6) .
Here, we report a detailed study of CD8+ T-cell responses to NY-ESO-1 in HLA-A2 melanoma patients, which includes identification of an optimal antigenic peptide sequence(s), assessment of tumor reactivity of specific CTLs, generation of HLA-A2/NY-ESO-1 peptide tetramers, and-tetramer based identification of NY-ESO-1-specific CD8+ T cells in peripheral blood and melanoma lesions.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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) were selected for this study. Melanoma cell lines Me 275 and Me 242
were established in our laboratory from melanoma metastases surgically
excised from patients LAU 50 and LAU 92, respectively. The melanoma
cell line NA8-MEL was kindly provided by Dr. F. Jotereau (U211;
Institut National de la Santé et de la Recherche Médicale,
Nantes, France). NY-ESO-1-specific serum antibody titers were
determined by ELISA, as described previously, with minor modifications
(8)
. Two patients showed detectable levels of
NY-ESO-1-specific antibodies: LAU 50 (antibody titer 1:1'400) and LAU
156 (antibody titer > 1:10'000). No detectable levels
of NY-ESO-1-specific antibodies were observed in patients LAU 42, LAU
97, LAU 165, LAU 198, LAU 212, LAU 233, and LAU 253. Patient LAU 203
was not tested.
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For peptide stimulation experiments, CD8+ lymphocytes were positively selected by magnetic cell sorting from PBMCs of HLA-A2 melanoma patients using a miniMACS device (Miltenyi Biotec GmBH, Sunnyvale, CA). Cells from the CD8- fraction were irradiated (3.000 rads) and used as autologous APCs. CD8+ highly enriched lymphocytes (1 x 106/well) were stimulated with peptide (1 µM) and irradiated autologous APCs in 2 ml of CTL medium (10) containing hrIL-2 [10 units/ml; Glaxo, Geneva, Switzerland; kindly provided by Dr. M. Nabholz (Institut Suisse de Recherches Experimentales sur le Cancer, Epalinges, Switzerland)] and hrIL-7 (10 ng/ml; R&D System Europe, Oxon, United Kingdom). Cells were cultured for the indicated period of time before ELISPOT or A2/NY-ESO-1 tetramer analysis.
HLA-A2 Binding and Antigen Recognition Assays.
Peptide binding to HLA-A2 was assessed in a functional competition
assay based on inhibition of recognition of the antigenic peptide
Tyrosinase 368-376 (YMDGTMSQV) by the HLA-A2-restricted CTL clone
LAU132/2 (10)
. Briefly, T2 cells were
51Cr-labeled in the presence of the anticlass I
mAb W6/32. Various concentrations of competitor peptides ranging from
1–104
nM (50 µl) were incubated
with 51Cr-labeled T2 cells (50 µl; 1000
cells/well) for 15 min at room temperature. A suboptimal dose (1
nM) of the antigenic peptide (50 µl) was then added
together with specific CTL (5000 cells/well; 50 µl). Chromium release
was measured after a 4-h incubation at 37°C. The concentration of
each competitor peptide required to achieve 50% inhibition of target
cell lysis was then determined as [nM] 50%. To
facilitate comparison, the relative competitor activity of each peptide
was calculated as the [nM] 50% of the reference
influenza A matrix peptide 58-66 divided by the [nM] 50%
of the competitor peptide. Antigen recognition was assessed using
standard 51Cr release assays. Target cells were
labeled with 51Cr for 1 h at 37°C and
washed two times. Labeled target cells (1000 cells in 50 µl) were
incubated in the presence of various concentrations of peptide (50
µl) for 15 min at room temperature before the addition of effector
cells (50 µl). In the case of TILNs, the effector cells were
preincubated for at least 20 min at 37°C with unlabeled K562 cells
(50,000/well) to eliminate nonspecific lysis due to natural
killer-like effectors. Chromium release was measured in
supernatant (100 µl) harvested after a 4-h incubation at 37°C. The
percentage of specific lysis was calculated as: 100 x
[(experimental - spontaneous
release)/(total - spontaneous release)].
The relative antigenic activity of each peptide was calculated as the [nM] 50% of the parental NY-ESO-1 nonapeptide 157–165 SLLMWITQC divided by the [nM] 50% of the competitor peptide.
Tetramers, mAbs, and Flow Cytometry Immunofluorescence Analysis.
HLA-A2/peptide tetramers were synthesized as described (11
, 12) . Antigenic peptides included influenza A matrix peptide
58-66 (GILGFVFTL), HIV-derived peptide pol 476-484 (ILKEPVHGV), and
NY-ESO-1 parental peptide 157-165 (SLLMWITQC) or peptide analogue C165A
(SLLMWITQA). The cells were first incubated with tetramers in 20 µl
of PBS containing 2% BSA and 0.2% azide during 1 h at room
temperature, then 20 µl of anti-CD8FITC or of a mixture
containing anti-CD8FITC and
anti-CD45-RACyc or
anti-CD8PerCP and
anti-CD62LFITC were added, and the incubation was
continued for another 20 min at 4°C. All of the mAbs used were
purchased from Becton Dickinson (Basel, Switzerland). Cells were washed
once in the same buffer and analyzed by flow cytometry. Data analysis
was performed using Cell Quest software.
IFN-
ELISPOT Assay.
IFN- ELISPOT assay (13)
was performed in
nitrocellulose-lined 96-well microplates (Millipore MAHA S45;
Millipore, Bedford, MA) using a IFN- ELISPOT kit (Mabtech,
Stockholm, Sweden) according to the manufacturer’s instructions, with
minor modifications. Plates were coated overnight with antibody to
human IFN- and washed six times. T2 cells (5 x 104
/well) were then added together with the
indicated number of responder T cells and peptide (1 µM
where indicated). After incubation for 20 h at 37°C, cells were
removed and plates were developed with a second (biotinylated)
antibody to human IFN- and streptavidin-alkaline phosphatase. Spots
were counted using a stereomicroscope with a magnification of x15.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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production by ELISPOT (13)
. To
this end, we used three different peptides corresponding to overlapping
sequences spanning the NY-ESO-1 155-167 region (peptide 157-167
SLLMWITQCFL, peptide 157-165 SLLMWITQC, and peptide 155-163 QLSLLMWIT)
that have been previously shown to be recognized by a NY-ESO-1-reactive
CTL line (6)
. As shown in Fig. 1
, high frequencies of IFN- producing cells were readily observed with
any of the three NY-ESO-1 peptides. From the ELISPOT data we estimated
that about 20% of TILs were NY-ESO-1 specific.
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-producing cells were also detected in the same TIL population
after stimulation with two other HLA-A2-restricted CTL antigenic
peptides, Melan-A peptide 26-35 (EAAGIGILTV) and Tyrosinase peptide
368-376 (YMDGTMSQV; data not shown). No reactivity was detected against
other known HLA-A2-restricted CTL antigenic peptides, including gp100
peptides 154-162 (KTWGQYWQV), 209-217 (ITDQVPFSV), 280–288
(YLEPGPVTA), 457–466 (LLDGTATLRL), and 476-485 (VLYRYGSFSV; Ref.
14
); 619-628 (RLMKQDFSV) and 639-647 (RLPRIFCSC; Ref.
15
); Tyrosinase peptide 1-9 (MLLAVLYCL; Ref.
16
); and the recently defined CAMEL peptide 1-11
(MLMAQEALAFL; Ref. 17
; data not shown).
Monoclonal NY-ESO-1-specific CD8+ T-cell
populations were generated by limiting dilution cloning of the TIL in
the presence of PHA and allogeneic feeder cells, as described
previously (9)
. In agreement with the ELISPOT data, 5 of
24 (21%) CTL clones were found to specifically recognize
NY-ESO-1-derived peptides presented in association with HLA-A2. Fig. 2
shows the cytolytic activity of a representative clone (ESO 5) that was
able to specifically lyse melanoma line Na8-MEL
(A2+, NY-ESO-1-) in the
presence, but not in the absence, of exogenously added NY-ESO-1 157-165
nonapeptide. In addition, this CTL clone efficiently lysed melanoma
line Me 275 (A2+,
NY-ESO-1+) in the absence of added peptide. In
contrast, no lysis of melanoma line Me 242 (A2-,
NY-ESO-1+) was observed, even in the presence of
added peptide. Furthermore, other A2+, but not
A2-, NY-ESO-1-expressing melanoma lines
generated in our laboratory were efficiently lysed by CTL clone ESO 5
(data not shown).
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, NY-ESO-1 nonapeptide 155-163 was 100-fold less efficient
than Flu-MA 58-66. NY-ESO-1 nonapeptide 157-165 was even less active
(data not shown). Surprisingly, NY-ESO-1 peptide 157-167 displayed a
competitor activity higher than that of either nonapeptide (only
25-fold less efficient than Flu-MA 58-66; data not shown). These
results led us to speculate that the poor binding of NY-ESO-1
nonapeptide 157-165 could be due to the presence of a cysteine (C)
residue at the carboxyl terminus of the peptide. To test this
hypothesis, we produced single amino acid-substituted nonapeptides
containing either A or L at position 165 (NY-ESO-1 C165A and NY-ESO-1
C165 L). As shown in Fig. 3A
, both substitutions
dramatically enhanced peptide binding to HLA-A2 (100-fold as compared
with the parental sequence). Recognition of both parental and analogue
peptides by CTL clone ESO 5 is shown in Fig. 3B
. Each of the
three parental peptides was specifically recognized at high peptide
concentrations. However, the nonapeptide 157-165 was recognized more
efficiently (50% maximal lysis at 0.6 nM) than
the other parental peptides (50% maximal lysis at 50
nM). In addition, both NY-ESO-1 C165 L and
NY-ESO-1 C165A nonapeptide analogues were recognized more efficiently
than the parental nonapeptide 157-165 (50% maximal lysis at 10
pM and 0.5 pM,
respectively).
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. At day 7,
high proportions of NY-ESO-1-specific IFN--producing cells were
detected in CD8+ T-cell cultures from patient LAU
156 stimulated with either of the two peptides. Similar results were
obtained with cultures from patient LAU 50, albeit only after
stimulation with NY-ESO-1 peptide C165A. Analysis of cultures at day 14
after stimulation confirmed the results obtained at day 7. In addition,
specific IFN--producing cells were now detected among
CD8+ T cells from patient LAU 50 stimulated with
the parental nonapeptide. Remarkably, patients LAU 156 and LAU 50 were
the only two patients among the nine tested (patient LAU 203 was not
tested) with both detectable serum levels of anti NY-ESO-1 antibodies
and NY-ESO-1-expressing tumors as assessed by RT-PCR and Western blot
analysis (data not shown). Specific IFN--producing cells were
detected at day 14 in CD8+ T-cell cultures from
an additional patient, LAU 198, after stimulation with peptide NY-ESO-1
C165A.
Enumeration of NY-ESO-1-specific CD8+ T Cells in
Peptide-stimulated Blood Lymphocytes from Melanoma Patients.
To directly enumerate NY-ESO-1-specific CD8+ T
cells in the cultures described above, we prepared fluorescent
A2/NY-ESO-1 peptide tetramers (11)
. Because high-affinity
peptide binding to HLA-A2 greatly facilitates the generation of stable
tetramer preparations, the peptide analogue NY-ESO-1 C165A was chosen
for tetramer assembly. As shown in Fig. 4
, A2/NY-ESO-1 C165A tetramers (A2/NY-ESO-1 tetramers thereafter)
specifically stained CTL clone ESO 5. No detectable staining over
background values was observed for the Flu-MA 58–66-specific CTL clone
FLU 17 (Fig. 4A
). Conversely, A2/Flu-MA 58–66 tetramers
stained specifically the latter but not CTL clone ESO 5 (Fig. 4B
). Remarkably, the efficacy (both in terms of mean
fluorescence intensity and tetramer dose response) of A2/NY-ESO-1
tetramer staining of clone ESO 5 was comparable with that of A2/Flu-MA
58–66 tetramer staining of clone FLU 17 (Fig. 4C
). Specific
staining of CTL clone ESO 5 was also obtained with tetramers containing
the parental peptide NY-ESO-1 157-165, although with lower staining
efficiency compared with tetramers containing the peptide analogue. In
addition, both NY-ESO-1 tetramers detected identical proportions of
CD8+ T cells in polyclonal PBMC cultures
stimulated with NY-ESO-1 peptide 157-165 (data not shown).
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was performed at day 14 after
stimulation (Fig. 5
). In agreement with the ELISPOT results (Table 1)
, relatively high
proportions of A2/NY-ESO-1 tetramer+ lymphocytes
were detected in cultured CD8+ T cells from
patients LAU 50 and LAU 156. In the case of patient LAU 50, the
majority of tetramer+ lymphocytes displayed a
bright fluorescence signal, whereas a tetramer+
dull population was also clearly detectable in the cultures from
patient LAU 156. Moreover, high proportions of A2/NY-ESO-1
tetramer+ lymphocytes with intermediate
fluorescence intensity were detected in cultures from patient LAU 198.
Interestingly, significant numbers of A2/NY-ESO-1
tetramer+ cells were also detected in cultures
from patients LAU 97 and LAU 42, although these cultures scored
negatively in the ELISPOT assay (Table 1)
. The positive staining of
these cultures with tetramers was confirmed after a second round of
in vitro stimulation. No A2/NY-ESO-1
tetramer+ lymphocytes were detected in cultures
from the remaining patients. Altogether, these results indicated that 5
of the 10 patients tested exhibited significant numbers of circulating
NY-ESO-1 peptide-reactive CD8+ T cells that could
be expanded in vitro.
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) were isolated by tetramer-guided cell sorting and
immediately tested for IFN- production. In addition,
antigen-specific cytolytic activities of both subpopulations were
assessed after mitogen-driven expansion. As shown in Fig. 6
, specific IFN--producing cells were detected only in
CD8+ tetramer+, but not in
CD8+ tetramer-
subpopulations derived from cultures stimulated with either NY-ESO-1
peptide 157-165 (Fig. 6A
) or NY-ESO-1 peptide analogue C165A
(Fig. 6B
). Moreover, CD8+
tetramer+, but not CD8+
tetramer-, subpopulations were able to kill
peptide-pulsed target cells as well as autologous tumor cells (Fig. 6C
).
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, high numbers of NY-ESO-1 tetramer+
CD8+ T cells were found in TILNs from patient LAU
50 and, in agreement with the ELISPOT and cloning data mentioned above,
in TILs from patient LAU 156. However, no NY-ESO-1
tetramer+ CD8+ T cells were
detectable in fresh or short-term cultured TIL or TILN samples from the
eight other patients analyzed.
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). This value was clearly above background values obtained with the
unrelated A2/HIV tetramer in eight HLA-A2 normal donors (<0.01%; data
not shown). This patient presented a primary skin melanoma of the lower
limb in October 1992. A few months later, numerous cutaneous metastases
appeared in the same limb. He was subjected to isolated limb perfusion
with melphalan and adjuvant IFN-
therapy. A complete response
ensued, lasting until 1995 when an inguinal lymph node
dissection revealed that 1 of 14 nodes was infiltrated by melanoma
cells. Since then, the patient remained tumor free until his last
control in April 1999. A2/NY-ESO-1 tetramer staining of blood samples
collected at different time points of his clinical history is shown in
Fig. 8
. A2/NY-ESO-1 tetramer+
CD8+ T cells were clearly detectable in three
consecutive blood samples collected between 1997 and 1999 but were
below the detection limit at an earlier time point. Of note,
A2/NY-ESO-1 tetramer+ cells displayed a typical
memory phenotype at all time points analyzed
(CD45RA-, CD28+; Fig. 8
and data not shown). Moreover, staining with anti-CD62L mAb subdivided
NY-ESO-1-specific CD8+ T cells in two
subpopulations of comparable size. Thus, although the frequency of
circulating NY-ESO-1-specific CD8+ T cells is
usually below the limit of detection by tetramer staining, the data
obtained with patient LAU 50 clearly indicate that
CD8+ T-cell memory to NY-ESO-1 can persist for
several years in clinically tumor-free melanoma patients.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The CT antigens include presently the products of at least six gene families: MAGE, GAGE, BAGE (1) , SSX-2, SCP1 (2 , 3) , and NY-ESO-1 (4) . Because of their tumor specificity, CT antigens are good candidates for the design of cancer vaccines aimed at eliciting tumor-reactive CTL responses. Indeed, one of these antigens, the MAGE-3.A1 peptide, has been already used in a Phase I-II clinical trial (5) . Although a relatively high tumor response rate was observed among a group of 25 HLA-A1 patients, there was no evidence of specific CTL induction after peptide immunization. Moreover, naturally occurring CD8+ T-cell responses to CT antigens have been reported only occasionally possibly due to the very low frequency of specific precursors (19) . Together, these observations raise questions on the immunogenicity of CT antigens and, hence, on the induction of specific CTL responses by appropriate peptide immunization.
The recently identified CT antigen NY-ESO-1 has been shown to be involved in both cellular and humoral immune responses. High titers of IgG antibodies were first demonstrated in a patient with a squamous cell carcinoma of the esophagus (4) . Circulating antibodies to NY-ESO-1 have been found in about 10% of melanoma patients and in lower proportions of patients with other tumor types, such as ovarian, lung, and breast adenocarcinomas (8) . In this study, we focused on the assessment of naturally occurring CD8+ T-cell responses to the HLA-A2-restricted NY-ESO-1 antigenic peptides previously described by Jäger et al. (6) . Using a NY-ESO-1-specific CTL clone isolated from a TIL population, we confirmed previous observations made with a polyclonal CTL line indicating that three overlapping NY-ESO-1 peptides could be efficiently recognized at saturating peptide concentrations. Our study established that among these three peptides NY-ESO-1 peptide 157-165 (SLLMWITQC) was the most efficiently recognized in antigenic peptide titration assays.
The cysteine residue at the carboxyl terminus of NY-ESO-1 peptide 157-165 is not only suboptimal for binding to HLA-A2 (20) but may also be disadvantageous due to its propensity to form cystine dimers on oxidation. As expected, we found that NY-ESO-1 peptide analogues carrying major HLA-A2 anchor residues (leucine or valine) at the COOH terminus exhibited enhanced binding to HLA-A2. Surprisingly, the peptide analogue containing a COOH-terminal alanine, an amino acid that has not been identified as a major anchor residue, also bound to HLA-A2 more efficiently than the parental nonapeptide. Moreover, this peptide analogue was more efficiently recognized by cloned CTL. Importantly, the CTL generated after in vitro stimulation with this peptide analogue were able to efficiently cross-recognize the naturally processed NY-ESO-1 peptide. As reported recently for another CTL-defined antigenic peptide analogue (21) , additional studies using HLA-A2-transgenic mice should establish whether the NY-ESO-1 analogue described here displays enhanced immunogenicity in vivo.
The availability of a potent NY-ESO-1 peptide analogue provided the opportunity to generate stable fluorescent tetramers for the direct visualization of NY-ESO-1-specific CD8+ T cells. We have previously shown that an analogue of the HLA-A2-restricted Melan-A/MART-1 antigenic peptide was superior to the parental peptide in terms of tetramer staining efficiency (12) . We made similar observations in this study when comparing HLA-A2 tetramers assembled with either the NY-ESO-1 parental peptide or its analogue. It is noteworthy, however, that the two tetramers exhibited virtually identical staining properties when used at saturating concentrations.
Fluorescent tetramers were used to monitor naturally occurring CD8+ T-cell responses to NY-ESO-1 in HLA-A2 melanoma patients. Five of the 10 patients analyzed had circulating NY-ESO-1-specific CD8+ T cells that were readily detectable with tetramers after a single round of in vitro peptide stimulation. Although we could not directly determine the frequency of circulating NY-ESO-1-specific CD8+ T cells in the majority of the patients, our results suggest that these cells are much more frequent than CD8+ T cells directed against the CT MAGE-3-derived antigenic peptide EVDPIGHLY (19) . NY-ESO-1 seropositive patients exhibited relatively strong NY-ESO-1 CD8+ T-cell responses in vitro. The melanoma lesions of these patients were found to express NY-ESO-1 by RT-PCR as well as by Western blot. Surprisingly, low but significant NY-ESO-1 CD8+ T-cell responses were also detected in three seronegative patients. It is noteworthy that the melanoma lesions from these latter patients did not express NY-ESO-1 mRNA.4 It is conceivable that these patients may have been primed by NY-ESO-1 antigen expressed in melanoma lesions that were not accessible for RT-PCR analysis. If so, the NY-ESO-1-specific CD8+ T cells should express an antigen-experienced phenotype. Moreover, their existence would implicate that CD8+ T-cell responses to this antigen may take place in the absence of specific humoral responses. Alternatively, the circulating NY-ESO-1-specific lymphocytes may express a naive phenotype. Indeed, we have recently reported that both melanoma patients and normal individuals display high numbers of circulating Melan-A-specific CD8+ T cells displaying a naive phenotype (22) . Additional analyses of the naive/memory phenotype of circulating NY-ESO-1-specific CD8+ T cells in NY-ESO-1 seronegative patients should clarify these issues.
In line with our previous findings with Tyrosinase- and Melan-A-tetramer+ lymphocytes (10 , 23) , NY-ESO-1-tetramer+ CD8+ T cells could be isolated by flow cytometry sorting and expanded in vitro in cultures containing allogeneic PBMCs, PHA, and hrIL-2. Moreover, the cultured tetramer+ cells were able to kill melanoma cells either sensitized with NY-ESO-1 peptide or expressing the naturally processed antigenic peptide. It, thus, seems that NY-ESO-1-specific CD8+ T cells are not necessarily in an anergic state, in contrast to the recently reported tyrosinase-specific CD8+ T cells derived from a melanoma patient (24) . Although there is no direct evidence that the NY-ESO-1-specific CD8+ T cells identified in this study by tetramer staining can exert lytic functions in vivo, their ability to proliferate and display potent effector functions in vitro suggest that appropriate stimulation and/or addition of growth factors may easily overcome any putative anergy in vivo. NY-ESO-1-specific CD8+ T cells among TILs from patients LAU 50 and LAU 156 reveal the existence of tumoricidal NY-ESO-1-specific responses in vivo at tumor sites.
Direct ex vivo enumeration of NY-ESO-1-specific
CD8+ T cells in the five patients with detectable
in vitro responses indicated that the frequencies of these
cells in blood were below the tetramer detection limit (0.01%), with
one exception. Indeed, the frequency of NY-ESO-1
tetramer+ cells in patient LAU 50 was 
1/2'500–5'000 circulating CD8+ lymphocytes.
Remarkably, this high frequency was stable over a prolonged period of
time. The NY-ESO-1-specific CD8+ T cells
exhibited an activated/memory phenotype and could be divided into two
distinct subpopulations by staining with anti-CD62L mAb. These
subpopulations may correspond, respectively, to the resting memory
(CD62L+) and effector memory
(CD62L-) CD8+ T cells that
have been described recently (25
, 26)
. It is unclear
whether the coexistence of these two phenotypically distinct memory
cells in the circulating lymphocyte pool may reflect an ongoing
CD8+ T-cell response that is sustained over time.
Conceivably, resting memory cells could become activated by tumor
antigen-presenting dendritic cells after homing into lymph nodes via
CD62L. Following activation (and CD62L down-regulation), these cells
could recirculate for some time before migrating into tumor lesions. In
a previous study, we showed selective accumulation of Melan-A-specific
CD8+ T cells with an antigen-experienced
phenotype in metastatic lymph nodes (12)
. In contrast, the
Melan-A-specific CD8+ T-cell populations found in
the blood were a mixture of naive and memory lymphocytes at least in
some patients (22)
. Unfortunately, the relatively large
numbers of cells required for flow cytometry analysis has thus far
hampered an extensive ex vivo assessment of the phenotype of
antigen-specific CD8+ T cells present at tumor
sites. Future studies using tetramers for in situ detection
of antigen-specific CD8+ T cells and laser
scanning confocal microscopy might provide information on the
functional state of tumor antigen-specific T cells that are located in
tumor lesions and in tumor-invaded lymph nodes.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported in part by the Leenaards Foundation,
Swiss Cancer League Grant KFS 633-2 1998 (to M. J. P.), and the
Medical Research Council of Canada Doctoral Award (to P. C.). 
2 To whom requests for reprints should be
addressed, at Division of Clinical Onco-Immunology, Ludwig Institute
for Cancer Research, CHUV-BH 19-602, 1011 Lausanne, Switzerland. Phone:
41-21-314-0176; Fax: 41-21-314-7477; E-mail: Danila.Valmori{at}inst.hospvd.ch
3 The abbreviations used are: Melan-A,
Melan-A/MART-1; CT, cancer testis; TIL, tumor-infiltrating lymphocyte;
TILN, tumor-infiltrated lymph node; hrIL, human recombinant
interleukin; PBMC, peripheral blood mononuclear cell; PHA,
phytohemagglutinin; mAb, monoclonal antibody; RT-PCR, reverse
transcription-PCR; APC, antigen-presenting cell; ELISPOT, Enzyme-linked
immunospot assay.
Received 12/20/99. Accepted 6/20/00.
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REFERENCES |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-interferon-secreting cells. J. Immunol. Methods, 110: 29-36, 1988.[Medline]
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 L. Derre, M. Bruyninx, P. Baumgaertner, M. Ferber, D. Schmid, A. Leimgruber, V. Zoete, P. Romero, O. Michielin, D. E. Speiser, et al. Distinct sets of {alpha}{beta} TCRs confer similar recognition of tumor antigen NY-ESO-1157-165 by interacting with its central Met/Trp residues PNAS, September 30, 2008; 105(39): 15010 - 15015. [Abstract] [Full Text] [PDF]
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L. Derre, M. Bruyninx, P. Baumgaertner, E. Devevre, P. Corthesy, C. Touvrey, Y. D. Mahnke, H. Pircher, V. Voelter, P. Romero, et al. In Vivo Persistence of Codominant Human CD8+ T Cell Clonotypes Is Not Limited by Replicative Senescence or Functional Alteration J. Immunol., August 15, 2007; 179(4): 2368 - 2379. [Abstract] [Full Text] [PDF]
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M. Condomines, D. Hose, P. Raynaud, M. Hundemer, J. De Vos, M. Baudard, T. Moehler, V. Pantesco, M. Moos, J.-F. Schved, et al. Cancer/Testis Genes in Multiple Myeloma: Expression Patterns and Prognosis Value Determined by Microarray Analysis J. Immunol., March 1, 2007; 178(5): 3307 - 3315. [Abstract] [Full Text] [PDF]
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 F. F. Costa, K. Le Blanc, and B. Brodin Concise Review: Cancer/Testis Antigens, Stem Cells, and Cancer Stem Cells, March 1, 2007; 25(3): 707 - 711. [Abstract] [Full Text] [PDF]
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S. M. Walton, M. Gerlinger, O. de la Rosa, N. Nuber, A. Knights, A. Gati, M. Laumer, L. Strauss, C. Exner, N. Schafer, et al. Spontaneous CD8 T Cell Responses against the Melanocyte Differentiation Antigen RAB38/NY-MEL-1 in Melanoma Patients J. Immunol., December 1, 2006; 177(11): 8212 - 8218. [Abstract] [Full Text] [PDF]
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G. Zeng, M. E. Aldridge, X. Tian, D. Seiler, X. Zhang, Y. Jin, J. Rao, W. Li, D. Chen, M. P. Langford, et al. Dendritic Cell Surface Calreticulin Is a Receptor for NY-ESO-1: Direct Interactions between Tumor-Associated Antigen and the Innate Immune System J. Immunol., September 15, 2006; 177(6): 3582 - 3589. [Abstract] [Full Text] [PDF]
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B. Janjic, P. Andrade, X.-F. Wang, J. Fourcade, C. Almunia, P. Kudela, A. Brufsky, S. Jacobs, D. Friedland, R. Stoller, et al. Spontaneous CD4+ T Cell Responses against TRAG-3 in Patients with Melanoma and Breast Cancers J. Immunol., August 15, 2006; 177(4): 2717 - 2727. [Abstract] [Full Text] [PDF]
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M. A. Purbhoo, D. H. Sutton, J. E. Brewer, R. E. Mullings, M. E. Hill, T. M. Mahon, J. Karbach, E. Jager, B. J. Cameron, N. Lissin, et al. Quantifying and Imaging NY-ESO-1/LAGE-1-Derived Epitopes on Tumor Cells Using High Affinity T Cell Receptors. J. Immunol., June 15, 2006; 176(12): 7308 - 7316. [Abstract] [Full Text] [PDF]
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 P. Baumgaertner, N. Rufer, E. Devevre, L. Derre, D. Rimoldi, C. Geldhof, V. Voelter, D. Lienard, P. Romero, and D. E. Speiser Ex vivo Detectable Human CD8 T-Cell Responses to Cancer-Testis Antigens Cancer Res., February 15, 2006; 66(4): 1912 - 1916. [Abstract] [Full Text] [PDF]
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V. Vignard, B. Lemercier, A. Lim, M.-C. Pandolfino, Y. Guilloux, A. Khammari, C. Rabu, K. Echasserieau, F. Lang, M.-L. Gougeon, et al. Adoptive Transfer of Tumor-Reactive Melan-A-Specific CTL Clones in Melanoma Patients Is Followed by Increased Frequencies of Additional Melan-A-Specific T Cells J. Immunol., October 1, 2005; 175(7): 4797 - 4805. [Abstract] [Full Text] [PDF]
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M. Gannage, M. Abel, A.-S. Michallet, S. Delluc, M. Lambert, S. Giraudier, R. Kratzer, G. Niedermann, L. Saveanu, F. Guilhot, et al. Ex Vivo Characterization of Multiepitopic Tumor-Specific CD8 T Cells in Patients with Chronic Myeloid Leukemia: Implications for Vaccine Development and Adoptive Cellular Immunotherapy J. Immunol., June 15, 2005; 174(12): 8210 - 8218. [Abstract] [Full Text] [PDF]
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 F. van Rhee, S. M. Szmania, F. Zhan, S. K. Gupta, M. Pomtree, P. Lin, R. B. Batchu, A. Moreno, G. Spagnoli, J. Shaughnessy, et al. NY-ESO-1 is highly expressed in poor-prognosis multiple myeloma and induces spontaneous humoral and cellular immune responses Blood, May 15, 2005; 105(10): 3939 - 3944. [Abstract] [Full Text] [PDF]
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 N. Naka, S. Joyama, Y. Tsukamoto, K. Yoshioka, N. Hashimoto, T. Ujiiye, T. Hayashi, M. Kawase, M. Mano, S. Ishiguro, et al. Quantification of SSX mRNA Expression in Human Bone and Soft Tissue Tumors Using Nucleic Acid Sequence-Based Amplification J. Mol. Diagn., May 1, 2005; 7(2): 187 - 197. [Abstract] [Full Text] [PDF]
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M. Schnurr, Q. Chen, A. Shin, W. Chen, T. Toy, C. Jenderek, S. Green, L. Miloradovic, D. Drane, I. D. Davis, et al. Tumor antigen processing and presentation depend critically on dendritic cell type and the mode of antigen delivery Blood, March 15, 2005; 105(6): 2465 - 2472. [Abstract] [Full Text] [PDF]
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G. Bricard, H. Bouzourene, O. Martinet, D. Rimoldi, N. Halkic, M. Gillet, P. Chaubert, H. R. MacDonald, P. Romero, J.-C. Cerottini, et al. Naturally Acquired MAGE-A10- and SSX-2-Specific CD8+ T Cell Responses in Patients with Hepatocellular Carcinoma J. Immunol., February 1, 2005; 174(3): 1709 - 1716. [Abstract] [Full Text] [PDF]
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X.-Y. Shang, H.-S. Chen, H.-G. Zhang, X.-W. Pang, H. Qiao, J.-R. Peng, L.-L. Qin, R. Fei, M.-H. Mei, X.-S. Leng, et al. The Spontaneous CD8+ T-Cell Response to HLA-A2-Restricted NY-ESO-1b Peptide in Hepatocellular Carcinoma Patients Clin. Cancer Res., October 15, 2004; 10(20): 6946 - 6955. [Abstract] [Full Text] [PDF]
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M. Matsuo, Y. Nagata, E. Sato, D. Atanackovic, D. Valmori, Y.-T. Chen, G. Ritter, I. Mellman, L. J. Old, and S. Gnjatic IFN-{gamma} enables cross-presentation of exogenous protein antigen in human Langerhans cells by potentiating maturation PNAS, October 5, 2004; 101(40): 14467 - 14472. [Abstract] [Full Text] [PDF]
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C. Lonchay, P. van der Bruggen, T. Connerotte, T. Hanagiri, P. Coulie, D. Colau, S. Lucas, A. Van Pel, K. Thielemans, N. van Baren, et al. Correlation between tumor regression and T cell responses in melanoma patients vaccinated with a MAGE antigen PNAS, October 5, 2004; 101(suppl_2): 14631 - 14638. [Abstract] [Full Text] [PDF]
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M. van Oijen, A. Bins, S. Elias, J. Sein, P. Weder, G. de Gast, H. Mallo, M. Gallee, H. van Tinteren, T. Schumacher, et al. On the Role of Melanoma-Specific CD8+ T-Cell Immunity in Disease Progression of Advanced-Stage Melanoma Patients Clin. Cancer Res., July 15, 2004; 10(14): 4754 - 4760. [Abstract] [Full Text] [PDF]
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M. Ayyoub, C. S. Hesdorffer, G. Metthez, S. Stevanovic, G. Ritter, Y.-T. Chen, L. J. Old, D. Speiser, J.-C. Cerottini, and D. Valmori Identification of an SSX-2 Epitope Presented by Dendritic Cells to Circulating Autologous CD4+ T Cells J. Immunol., June 1, 2004; 172(11): 7206 - 7211. [Abstract] [Full Text] [PDF]
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 A. I. Webb, M. A. Dunstone, W. Chen, M.-I. Aguilar, Q. Chen, H. Jackson, L. Chang, L. Kjer-Nielsen, T. Beddoe, J. McCluskey, et al. Functional and Structural Characteristics of NY-ESO-1-related HLA A2-restricted Epitopes and the Design of a Novel Immunogenic Analogue J. Biol. Chem., May 28, 2004; 279(22): 23438 - 23446. [Abstract] [Full Text] [PDF]
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E. Maraskovsky, S. Sjolander, D. P. Drane, M. Schnurr, T. T. T. Le, L. Mateo, T. Luft, K.-A. Masterman, T.-Y. Tai, Q. Chen, et al. NY-ESO-1 Protein Formulated in ISCOMATRIX Adjuvant Is a Potent Anticancer Vaccine Inducing Both Humoral and CD8+ T-Cell-Mediated Immunity and Protection against NY-ESO-1+ Tumors Clin. Cancer Res., April 15, 2004; 10(8): 2879 - 2890. [Abstract] [Full Text] [PDF]
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H. Gyobu, T. Tsuji, Y. Suzuki, T. Ohkuri, K. Chamoto, M. Kuroki, H. Miyoshi, Y. Kawarada, H. Katoh, T. Takeshima, et al. Generation and Targeting of Human Tumor-Specific Tc1 and Th1 Cells Transduced with a Lentivirus Containing a Chimeric Immunoglobulin T-Cell Receptor Cancer Res., February 15, 2004; 64(4): 1490 - 1495. [Abstract] [Full Text] [PDF]
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M. J. Palmowski, L. Lopes, Y. Ikeda, M. Salio, V. Cerundolo, and M. K. Collins Intravenous Injection of a Lentiviral Vector Encoding NY-ESO-1 Induces an Effective CTL Response J. Immunol., February 1, 2004; 172(3): 1582 - 1587. [Abstract] [Full Text] [PDF]
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M. Rodolfo, R. Luksch, E. Stockert, Y.-T. Chen, P. Collini, T. Ranzani, C. Lombardo, P. Dalerba, L. Rivoltini, F. Arienti, et al. Antigen-Specific Immunity in Neuroblastoma Patients: Antibody and T-Cell Recognition of NY-ESO-1 Tumor Antigen Cancer Res., October 15, 2003; 63(20): 6948 - 6955. [Abstract] [Full Text] [PDF]
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D. Nagorsen, C. Scheibenbogen, F. M. Marincola, A. Letsch, and U. Keilholz Natural T Cell Immunity against Cancer Clin. Cancer Res., October 1, 2003; 9(12): 4296 - 4303. [Abstract] [Full Text] [PDF]
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M. Ayyoub, D. Rimoldi, P. Guillaume, P. Romero, J.-C. Cerottini, D. Valmori, and D. Speiser Tumor-reactive, SSX-2-specific CD8+ T Cells Are Selectively Expanded during Immune Responses to Antigen-expressing Tumors in Melanoma Patients Cancer Res., September 1, 2003; 63(17): 5601 - 5606. [Abstract] [Full Text] [PDF]
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 I. D. Davis, M. Jefford, P. Parente, and J. Cebon Rational approaches to human cancer immunotherapy J. Leukoc. Biol., January 1, 2003; 73(1): 3 - 29. [Abstract] [Full Text] [PDF]
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E.-F. Solomayer, M. Feuerer, L. Bai, V. Umansky, P. Beckhove, G. C. Meyberg, G. Bastert, V. Schirrmacher, and I. J. Diel Influence of Adjuvant Hormone Therapy and Chemotherapy on the Immune System Analysed in the Bone Marrow of Patients with Breast Cancer Clin. Cancer Res., January 1, 2003; 9(1): 174 - 180. [Abstract] [Full Text] [PDF]
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X. Zheng, J.-X. Gao, H. Zhang, T. L. Geiger, Y. Liu, and P. Zheng Clonal Deletion of Simian Virus 40 Large T Antigen-Specific T Cells in the Transgenic Adenocarcinoma of Mouse Prostate Mice: An Important Role for Clonal Deletion in Shaping the Repertoire of T Cells Specific for Antigens Overexpressed in Solid Tumors J. Immunol., November 1, 2002; 169(9): 4761 - 4769. [Abstract] [Full Text] [PDF]
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Y. Nagata, S. Ono, M. Matsuo, S. Gnjatic, D. Valmori, G. Ritter, W. Garrett, L. J. Old, and I. Mellman Differential presentation of a soluble exogenous tumor antigen, NY-ESO-1, by distinct human dendritic cell populations PNAS, August 6, 2002; 99(16): 10629 - 10634. [Abstract] [Full Text] [PDF]
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G. Zeng, Y. Li, M. El-Gamil, J. Sidney, A. Sette, R.-f. Wang, S. A. Rosenberg, and P. F. Robbins Generation of NY-ESO-1-specific CD4+ and CD8+ T Cells by a Single Peptide with Dual MHC Class I and Class II Specificities: A New Strategy for Vaccine Design Cancer Res., July 1, 2002; 62(13): 3630 - 3635. [Abstract] [Full Text] [PDF]
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D. Valmori, C. Scheibenbogen, V. Dutoit, D. Nagorsen, A. M. Asemissen, V. Rubio-Godoy, D. Rimoldi, P. Guillaume, P. Romero, D. Schadendorf, et al. Circulating Tumor-reactive CD8+ T Cells in Melanoma Patients Contain a CD45RA+CCR7- Effector Subset Exerting ex Vivo Tumor-specific Cytolytic Activity Cancer Res., March 1, 2002; 62(6): 1743 - 1750. [Abstract] [Full Text] [PDF]
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M. Ayyoub, S. Stevanovic, U. Sahin, P. Guillaume, C. Servis, D. Rimoldi, D. Valmori, P. Romero, J.-C. Cerottini, H.-G. Rammensee, et al. Proteasome-Assisted Identification of a SSX-2-Derived Epitope Recognized by Tumor-Reactive CTL Infiltrating Metastatic Melanoma J. Immunol., February 15, 2002; 168(4): 1717 - 1722. [Abstract] [Full Text] [PDF]
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V. Dutoit, V. Rubio-Godoy, M.-A. Doucey, P. Batard, D. Lienard, D. Rimoldi, D. Speiser, P. Guillaume, J.-C. Cerottini, P. Romero, et al. Functional Avidity of Tumor Antigen-Specific CTL Recognition Directly Correlates with the Stability of MHC/Peptide Multimer Binding to TCR J. Immunol., February 1, 2002; 168(3): 1167 - 1171. [Abstract] [Full Text] [PDF]
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V. Rubio-Godoy, V. Dutoit, D. Rimoldi, D. Lienard, F. Lejeune, D. Speiser, P. Guillaume, J.-C. Cerottini, P. Romero, and D. Valmori Discrepancy between ELISPOT IFN-gamma secretion and binding of A2/peptide multimers to TCR reveals interclonal dissociation of CTL effector function from TCR-peptide/MHC complexes half-life PNAS, August 17, 2001; (2001) 181348898. [Abstract] [Full Text] [PDF]
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V. Dutoit, V. Rubio-Godoy, P.-Y. Dietrich, A.-L. Quiqueres, V. Schnuriger, D. Rimoldi, D. Lienard, D. Speiser, P. Guillaume, P. Batard, et al. Heterogeneous T-Cell Response to MAGE-A10254-262: High Avidity-specific Cytolytic T Lymphocytes Show Superior Antitumor Activity Cancer Res., August 1, 2001; 61(15): 5850 - 5856. [Abstract] [Full Text] [PDF]
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T. Ono, T. Kurashige, N. Harada, Y. Noguchi, T. Saika, N. Niikawa, M. Aoe, S. Nakamura, T. Higashi, A. Hiraki, et al. Identification of proacrosin binding protein sp32 precursor as a human cancer/testis antigen PNAS, March 13, 2001; 98(6): 3282 - 3287. [Abstract] [Full Text] [PDF]
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P.-Y. Dietrich, P. R. Walker, A.-L. Quiquerez, G. Perrin, V. Dutoit, D. Liénard, P. Guillaume, J.-C. Cerottini, P. Romero, and D. Valmori Melanoma Patients Respond to a Cytotoxic T Lymphocyte-defined Self-Peptide with Diverse and Nonoverlapping T-Cell Receptor Repertoires Cancer Res., March 1, 2001; 61(5): 2047 - 2054. [Abstract] [Full Text]
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D. Valmori, V. Dutoit, V. Rubio-Godoy, C. Chambaz, D. Liénard, P. Guillaume, P. Romero, J.-C. Cerottini, and D. Rimoldi Frequent Cytolytic T-Cell Responses to Peptide MAGE-A10254-262 in Melanoma Cancer Res., January 1, 2001; 61(2): 509 - 512. [Abstract] [Full Text]
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D. Rimoldi, V. Rubio-Godoy, V. Dutoit, D. Lienard, S. Salvi, P. Guillaume, D. Speiser, E. Stockert, G. Spagnoli, C. Servis, et al. Efficient Simultaneous Presentation of NY-ESO-1/LAGE-1 Primary and Nonprimary Open Reading Frame-Derived CTL Epitopes in Melanoma J. Immunol., December 15, 2000; 165(12): 7253 - 7261. [Abstract] [Full Text] [PDF]
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V. Rubio-Godoy, V. Dutoit, D. Rimoldi, D. Lienard, F. Lejeune, D. Speiser, P. Guillaume, J.-C. Cerottini, P. Romero, and D. Valmori Discrepancy between ELISPOT IFN-gamma secretion and binding of A2/peptide multimers to TCR reveals interclonal dissociation of CTL effector function from TCR-peptide/MHC complexes half-life PNAS, August 28, 2001; 98(18): 10302 - 10307. [Abstract] [Full Text] [PDF]
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, influenza A matrix peptide 58-66; •, NY-ESO-1
parental peptide 157-165 SLLMWITQC; 
, NY-ESO-1 peptide analogue
SLLMWITQL; 
, NY-ESO-1 peptide analogue SLLMWITQA. B,
NY-ESO-1 parental or analogue peptides were tested for antigen
recognition by CTL clone ESO 5 in a 4-h chromium release assay as
detailed in "Materials and Methods." 
, SLLMWITQCFL; 
,
QLSLLMWIT; •, SLLMWITQC; 
