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Experimental Therapeutics, Preclinical Pharmacology |
Institut de Biologie, INSERM U463, 44093 Nantes Cédex 1 [N. G., N. L., S. L., J-F. F., Y. G., E. D., F. J.]; Unité de thérapie cellulaire et génique, CHRU de Nantes, 44093 Nantes [M-C. P.]; and Departement de dermatologie du CHRU de Nantes, 44093 Nantes [B. D.], France
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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We then describe a relatively simple and efficient procedure that allowed us to obtain systematically high amounts (in the range of billion) of high avidity Melan-A/MART-1-specific T cells from the PBLs of HLA-A2 melanoma patients and healthy donors in 3 months. Because this antigen is expressed by most melanoma tumors, this procedure should be useful for checking the efficiency of adoptive immunotherapy of melanoma tumors and using functionally well-defined Melan-A/MART-1-specific CTLs in a large group of patients.
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Immunotherapeutic trials using ex vivo-expanded TILs have reported relative success (10) . However, we have shown recently that a majority of melanoma TILs are either not tumor-specific or not avid enough to secrete cytokines in response to melanoma cells (data not shown). The development of strategies allowing to generate high amounts of high avidity tumor-reactive CTLs is therefore now a critical issue for the development of melanoma-specific adoptive therapies.
The discovery of antigenic tumor epitopes has offered a strategy to obtain antigen-specific CTLs. This relies on performing repeated in vitro stimulations of PBLs by peptide-pulsed cells to obtain specific lymphocytes. Although most melanoma antigenic peptides stimulate the growth of peptide-specific PBLs in vitro (11, 12, 13, 14, 15) , a large fraction of these, as well as of in vivo-detectable tumor antigen-specific T cells, were of low avidity and exhibited limited responses to tumor cells endogenously expressing the antigen (16) . It was therefore necessary to derive methods allowing the expansion of high avidity T cells. Recently, peptide-MHC tetramers have been used to sort specific T cells, and by this approach, two groups already reported the isolation of high avidity tumor-reactive clones from either TILs or PBLs (17 , 18) . However, FACS sorting is not yet applicable at a clinical grade to perform immunotherapies.
We describe here an alternative procedure to isolate Melan-A/MART-1 (thereafter Melan-A)-specific CTL clones of high reactivity toward melanoma cells endogenously expressing this antigen. To design this procedure, we have analyzed how conditions of ex vivo peptide presentation, i.e., the peptide presenting cell type, the nature of the peptide, its concentration, and the different cytokines added during PBL stimulation, could affect the growth of low and high avidity-specific CTLs. It has been shown before that cytokine secretion by T cells requires higher avidity interactions with target cells than with lysis (19 , 20) . Therefore, specific CTLs that developed only lysis in response to peptide were considered to be of low avidity, and those also producing cytokines were considered to be of high avidity. We show that, with selected conditions of peptide presentation, high avidity tumor-reactive CTL clones could be generated from the blood of all HLA-A*0201 donors, including melanoma patients.
This procedure opens the possibility of Melan-A-targeted CTL-based therapy for a large group of melanoma patients.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Synthetic Peptides.
The Melan-A peptides27–35 AAGIGILTV and
26–35EAAGIGILTV and the decamer analogue
ELAGIGILTV were purchased from Genosys (Cambridge, United Kingdom).
Purity (>70%) was controlled by reversed-phase high-performance
liquid chromatography. Peptides were lyophilized, dissolved in DMSO at
10 mg/ml, and stored at -80°C.
mAbs.
Phycoerythrin-conjugated MQ1–17H12 (anti-IL-2) and FITC-conjugated
4S.B3 (anti-IFN-
) purified antibodies were obtained from PharMingen
SA (San Diego, CA). A103 (anti-Melan-A) antibody was obtained from DAKO
(Trappes, France).
Immunolabeling of Melanoma Cells with Monoclonal Anti-Melan-A
Antibody.
Cells (105 cells/well) were incubated with
A103 mAb at appropriate an concentration in PBS containing 0.1% BSA
and 0.1% Saponin for 30 min at 4°C. After two washes with the same
buffer, cells were incubated for 30 min with the secondary FITC-labeled
antibody. After washing, fluorescence analysis was immediately
performed on a FACScan flow cytometer using CellQuest software (Becton
Dickinson, Grenoble, France). Five thousand cells were gated with
forward scatter/side scatter parameters and analyzed.
PBL Stimulation.
Stimulated cells were total PBLs from HLA-A2 donors, either healthy or
melanoma patients. Peptide-pulsed irradiated HLA-A2 melanoma lines or
PBLs were used as stimulator cells. Loading with Melan-A peptides was
done by incubating stimulator cells with the peptide (50
µM for the natural peptides or 1 µM for the
peptide analogue) at 37°C in serum-free medium for 2 h.
Stimulator cells were washed twice to eliminate unbound peptide.
Stimulations were performed either in 24-well culture plates by mixing
5.105 peptide-pulsed stimulator cells and
1.106 responder PBLs or in 96-well culture plates
by mixing peptide-pulsed stimulator cells, either
2.104 melanoma cells or 105
PBLs, with 105 responder PBLs in RPMI medium
containing 8% human serum and cytokines. Irradiated stimulator cells
were added again twice at 7-day intervals. Three cytokine combinations
were used: either 5 ng/ml IL-6 (Sigma, St. Louis, MO) and 5 ng/ml IL-12
(Sigma) for the first week and 10 units/ml IL-2 and 5 ng/ml IL-7
thereafter, or IL-6 for the first week and IL-2 (50 U/ml) thereafter or
no cytokine for the first week and IL-2 (50 units/ml) thereafter.
51Chromium Microcytotoxicity Assay.
Cytotoxic activity was measured in a standard 4-h assay against
51Cr-labeled cells. Briefly, target cells
(peptide-pulsed T2 or melanoma cells) were incubated with 100 µCi
Na251CrO4
(Oris Industrie, Gif-sur-Yvette, France) at 37°C for 1 h.
Various amounts of effector cells were then added to
103 target cells in a final volume of 100 µl.
For peptide recognition assays, T2 cells were preincubated with
Melan-A27–35 peptide (10 µM) for
30 min at room temperature and then washed before the addition of
effector cells. After 4 h, 25 µl of supernatant were mixed with
100 µl of scintillation cocktail (Optiphase Supermix, Wallak, United
Kingdom) for measurement of radioactive content. A culture was
considered positive when the percentage of lysis of peptide-pulsed T2
cells exceeded the percentage of specific lysis of T2 cells by at least
15%.
Flow Cytometric Analysis of Intracellular Cytokines.
Antigen-specific lymphocytes were quantified among stimulated PBLs by
intracellular cytokine labeling using a method described by Jung
et al. (22)
. PBLs were stimulated by
peptide-pulsed or unpulsed T2 cells (or by antigen expressing melanoma
cells) at the ratio of 1:2 in RPMI 10% FCS in the presence of
Brefeldin A (10 µg/ml, Sigma, St. Louis, MO). Peptide-pulsed T2 cells
were prepared by a 1-h incubation at 37°C with 50
µM of Melan-A27–35
peptide in RPMI 1640 medium without FCS, followed by two washings.
After 6 h, cells were fixed for 10 min at room temperature in a
solution of PBS 4% paraformaldehyde. T cells
(5.104) were stained
with anticytokine mAbs at a concentration of 5 µg/ml for 30 min at
room temperature. Reagent dilutions and washes were done with PBS
containing 0.1% BSA (A-9647, Sigma) and 0.1% saponin (S-2149, Sigma).
After staining, cells were resuspended in PBS, and 5000 events were
analyzed on a FACScan (Becton Dickinson).
Cloning and Selection of Melan-A-specific CTLs.
PBLs from microcultures containing a significant fraction of Melan-A
peptide-reactive cells were cloned in U-bottomed 96-well plates
(Falcon) by limiting dilution. Growth was induced using PHA (1 µg/ml)
and irradiated allogeneic feeder cells: EBV-transformed LAZ cells
(2.104/well) and allogeneic PBLs
(105/well). Microcultures showing >95%
probability of monoclonality, according to the single-hit Poisson law,
were transferred into new plates with freshly irradiated feeder and
PHA. Melan-A-specific CTL clones were selected on the basis of their
capacity to lyse Melan-A peptide-pulsed T2 cells but not T2 cells
alone.
Expansion of T-Cell Clones.
Melan-A-specific clones were expanded using a polyclonal T-cell
stimulation protocol described previously (23)
. Briefly,
2000 clone cells/well were distributed in 96-well plates with 200 µl
of culture medium (RPMI with 8% human serum and IL-2 150 units/ml) and
irradiated feeder cells: LAZ EBV-B cells
(2.104/well) and allogeneic PBLs
(105/well). PHA was added (1 µg/ml). Culture
medium was changed 2 days later. Clone T cells were split when their
number exceeded 2.105/well.
Sorting of Melan-A-specific Cells Using HLA-A2/ELA Tetramers.
HLA-A0201/ELAGIGILTV tetramer was a generous gift from F. Romanier
(Immunotech, Marseilles, France). T cells
(1.106) were incubated for 1 h at
room temperature in 200 µl of PBS/0.1% BSA containing diluted
HLA/peptide tetramers (25 µg/ml). After two washes with 200 µl of
PBS/0.1% BSA, labeled T cells were sorted using a FACS (Vantage,
Becton Dickinson, Mountain View, CA).
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. As shown
in Fig. 1a,
microcultures
stimulated by PBLs did not contain a significant fraction of
IFN--labeled cells. In contrast, significant fractions of
IFN--labeled cells (ranging from 1.1 to 37%) were found in
microcultures stimulated by melanoma cells (Fig. 1b).
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, three cell
lines (M17, M119, and M113) efficiently induced lysis and IFN-
secretion in all of the microcultures containing
106 stimulated PBLs. Nonetheless, this high
stimulation capacity was dependent upon peptide addition, although two
of these cell lines (M113 and M119) expressed the Melan-A antigen
endogenously, as shown by Melan-A protein labeling (data not shown) and
by reverse transcription-PCR (24)
. Two melanoma lines, M44
and IGR1/54, were inefficient in stimulating the growth of
Melan-A-specific CTLs.
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, the fraction of IFN--positive cells
increased proportionally with the peptide concentration and passed
beyond 10% in some wells only at the highest peptide concentration (50
µM).
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shows that 1 µM of the
peptide analogue induced higher fractions of high avidity CTLs than 50
µM of the Melan-A peptide 27–35. These fractions induced
by the peptide analogue and by peptide 27–35 ranged respectively from
3.8 to 39% and from 1.3 to 9.2% for donor H4 and from 1.1 to 5.4%
and from 0.4 to 2.7% for donor H5. Therefore, the peptide analogue
induced a higher expansion of high avidity Melan-A-specific CTLs per
microculture. However, it did not improve the percentage of positive
microcultures.
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-producing lymphocytes were measured after three stimulations.
Results confirmed that autologous PBLs were poorly efficient (Fig. 4a),
as shown before with PBLs
from healthy donors. These results also show that autologous melanoma
cells were as efficient as allogeneic melanoma lines (Fig. 4b).
Therefore, the efficiency of melanoma cells in
stimulating highly reactive Melan-A-specific lymphocytes is not due to
an allorestricted effect.
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, results obtained using Il-6 and IL-2
were similar to those obtained with the four cytokines. Therefore, IL-7
and IL-12 did not appear to be essential for the generation of highly
reactive Melan-A-specific T cells from in vitro-stimulated
PBLs. In contrast, IL-2 alone was clearly less efficient to induce a
high growth of IFN--producing T cells.
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show that these
PBL-derived clones lysed melanoma lines at similar levels to those of
melanoma lines lysed by TIL-derived clones. However, all of them
secreted significantly higher amounts of GM-CSF and IFN- than did
TIL clones, and two of the four clones shown on Fig. 7
also secreted
higher amounts of IL-2 than did TIL clones.
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Highly Tumor-reactive T-Cell Populations Can Be Sorted by Specific
Tetramer from Stimulated PBL.
Melan-A-reactive CTL stimulated either by the natural peptide 27–35 or
by the ELA peptide were sorted by FACS using HLA-A2/ELAGIGILTV peptide
tetramers and were tested for their capacity to produce cytokines upon
stimulation by HLA-A2+/Melan-A+ cell lines. Fractions of
tetramer-labeled cells found among the PBLs stimulated by peptide
27–35 and by the ELA analogue were respectively of 2 and 39% before
sorting and of 94 and 99% after (data not shown). We measured the
production of IFN- and IL-2 of these two sorted populations in
response to melanoma cells. These responses were identical in terms of
percentage of labeled cells and mean of fluorescence (Fig. 8)
. This shows an equal capacity of
individual lymphocytes stimulated by one of these two peptides to
secrete IFN- and IL-2.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Here we show that most melanoma cells, once loaded with Melan-A peptides, systematically induced the growth of high avidity Melan-A-specific and tumor reactive CTLs from only 106 PBLs. After three peptide stimulations, the percentage of high avidity-specific CTLs detected in microcultures ranged from 0.5 to 37%. Such an efficient generation of Melan-A-specific CTLs can be related to the high frequency of Melan-A-specific effectors in the blood of many donors, as recently shown using HLA-A2-peptide tetramers (27) As reported before (14 , 28) , stimulation by peptide-pulsed PBLs also induced Melan-A-specific CTLs, as shown by the specific lysis that developed in most of these microcultures. Nonetheless, we show that CTLs generated by this way were of too low avidity to secrete cytokines in response to natural Melan-A peptide.
We also showed that a density of MHC-peptide complexes, higher than the
naturally occurring density, was required for efficient CTL generation
by melanoma cells. Indeed, in the absence of a peptide addition,
Melan-A-expressing melanoma lines were inefficient. In addition,
although the lowest concentration of the natural peptide (0.5
µM) induced high avidity CTLs in some microcultures, 50
µM induced a much higher fraction of such CTLs in all of
the microcultures. A minimal density of MHC peptides on melanoma cells
is therefore required for efficient stimulation and expansion of high
avidity Melan-A-specific PBLs in vitro, and there was a
clear-cut dose response relationship from 0.5 to 50
µM for the generation of high avidity CTLs.
This may explain why a melanoma line, M44, with relatively low HLA-A2
expression (24)
was inefficient in inducing high avidity
CTLs (Table 1)
. It has been previously reported that high
concentrations of viral peptides preferentially induced low avidity T
cells by inducing apoptosis of high avidity T cells (29)
.
This apparent discrepancy with our results may be explained by the low
affinity of natural Melan-A peptide for the HLA-A*0201 molecule. This
likely limits the density of HLA-A2/peptide complexes expressed by
melanoma cells even in presence of a high concentration of this peptide
(50 µM). Therefore, high avidity CTLs generated
in the present study have been probably induced, as in the study of
Alexander-Miller et al. (29)
, by melanoma cells
bearing relatively low MHC peptide densities. In support of this
interpretation, we recently obtained results similar to those of
Alexander-Miller et al. (29)
using the Melan-A
peptide analogue ELA of high avidity for HLA-A2, i.e., more
efficient induction of high avidity Melan-A-specific CTLs with 1
µM than with 10 or 50
µM (data not shown). This suggests that high
ELA peptide concentrations, like viral peptides used by
Alexander-Miller et al. (29)
, induced apoptosis
of high avidity CTLs.
In addition to the density of the MHC peptide expression, the density of the ICAM-1 molecule on melanoma cells also seemed critical, as suggested by the complete inefficiency of a melanoma line (IGR1/54) lacking ICAM-1 to stimulate specific CTLs. This is in accordance with our previous results showing that melanoma cells lacking ICAM-1 induced a suboptimal activation of Melan-A- and NA17-A-specific CTL clones (24) . Although not shown in this study, the same was true for the LFA-3/CD2 interaction as far as a melanoma line that expressed LFA-3 at a low density was found inefficient in activating CTL clones. Furthermore, we had shown before that increasing LFA-3 density on melanoma cells by transfection always resulted in higher cytokine responses of melanoma-associated antigen-specific CTL clones (30) . A possible explanation for the inefficiency of PBLs in generating high avidity-specific CTLs may be the low expression of ICAM-1 and LFA-3 by most of these cells compared with melanoma cells (data not shown). In contrast, the classical costimulatory interaction between B7 molecules and CD28, often considered critical for T-cell activation, was not required in the present stimulations as far as melanoma cells lack B7 molecules. Nonetheless, T-cell stimulations independent of the CD28-CD80 interaction have already been reported especially for memory CD8 and even for naive T cells in stimulations with high MHC-peptide densities (31, 32, 33) .
The role of cytokines in inducing the growth of specific T cells during
peptide stimulation is thus far unclear. Various cytokine combinations
have been used previously with success, but neither their requirement
nor their relative efficiency was established. Van der Bruggen et
al. (12)
used IL-6 and IL-12 for the first week of
stimulation, then IL-7 and IL-2. Others used IL-7 and IL-2 (11
, 34)
. IL-1ß, IL-4, and IL-10 were also used for human CTL
induction (15
, 35
, 36)
. In side-by-side experiments, we
found no significant difference between the efficiency of the four
cytokine combination (IL-6/IL-12 then IL-7/IL-2) and that of the two
cytokine combination (IL-6 then IL-2) in generating high avidity CTLs.
In contrast, the use of IL-2 alone dramatically reduced the frequency
of these cells. Furthermore, cytokines secreted by melanoma cells could
contributed to the differentiation and activation of Melan-A-specific
PBLs. Indeed, we have observed differences between melanoma lines in
cytokine transcription that could also account for their different
efficiency to stimulate CTLs. Among cytokine RNAs, those differentially
expressed by melanoma lines were IL-1, tumor necrosis factor 
and
ß, IL-6, IL-7, IL-10, and
1L-15.5
The efficiency of the M17 melanoma line in stimulating specific CTLs in
the absence of exogenous Il-7 could be due to the endogenous expression
of this cytokine by M17 cells (detected by reverse transcription-PCR).
To improve the generation of high avidity Melan-A-specific CTLs, we also tested the efficiency of a recently described Melan-A peptide analogue ELAGIGILTV, which was designed by Cerottini’s group as one of the most interesting variants of Melan-A 27–35 and 26–35 natural epitopes (25) . We show that, when loaded on M17 melanoma cells, this peptide induced a higher expansion of high avidity CTLs than the natural nonamer, although it did not increase the frequency of positive cultures. Furthermore, this peptide analogue was as efficient as the natural peptide at a concentration 50 times lower.
With the perspective to derive CTL for use in immunotherapy, it was
particularly critical to control if Melan-A peptide reactive T cells
derived with allogeneic melanoma cells and with this peptide analogue
were not alloreactive and of sufficient avidity to recognize tumor
cells endogenously expressing the natural epitopes. To this end,
Melan-A peptide reactive lymphocytes were sorted from the cultures,
either by cloning or by using HLA-A2/ELA tetramers. We observed that
these lymphocytes exclusively recognized melanoma cells expressing both
HLA-A2 and Melan-A and were of high avidity toward these cells as shown
by the secretion of not only IFN- but also IL-2. Using Vß
labeling, we found that several clones (between one and five) were
derived from 105 or 106
stimulated PBLs. All these clones had a similar lysis but significantly
higher capacity to secrete IFN- and GM-CSF than TIL-derived clones,
and for a significant fraction, a higher capacity to secrete IL-2 in
response to melanoma cells. Regarding the recent demonstration that
IFN- and GM-CSF secretion by tumor-specific CTLs plays a critical
role in inducing tumor-specific responses (7)
, these
PBL-derived clones represent interesting effectors to be used for
antigen adoptive immunotherapy. Although not shown, Il-2 secretion
could be a critical function for adoptive therapy, especially for the
survival of transferred CTLs in vivo. Finally, we could
systematically expand all melanoma-specific clones by a factor of 1000
within 1 month.
In conclusion, stimulation conditions designed here were found
efficient to derive high amounts of high avidity and highly
tumor-reactive CTL clones from all melanoma donors. Another procedure
to obtain high avidity tumor-reactive CTL clones was reported recently
by Yee et al. (18)
using autologous peptide
pulsed dendritic cells to stimulate PBLs and HLA-A2-Melan-A peptide
tetramers to sort high avidity CTLs that were then cloned. Another
group also succeeded to derive such clones from patient TIL or
stimulated PBL using FACS sorting with peptide MHC tetramers and then
cloning (17)
. However, clinical grade MHC class I peptide
tetramers are not yet available. In the procedure described here,
IFN- labeling was an alternative way to select populations
sufficiently enriched in high avidity T cells to allow successful
cloning. Another interest of the present procedure is the use of a
single established melanoma line to stimulate PBLs of all donors, which
obviates the need to grow high amounts of autologous dendritic cells as
done in other recently described protocols (17
, 18)
.
Therefore, it is feasible to grow high amounts of high avidity CTL clones specific for Melan-A antigen from the PBLs of all melanoma patients. Reinfusion of such cells to melanoma patients should allow adequate determination of the efficiency of adoptive immunotherapy against this melanoma antigen.
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FOOTNOTES |
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1 Supported by contract grant sponsors (Institut
National de la Santé et de la Recherche Médicale; Grant
6494 from the Association pour la Recherche contre le Cancer) and funds
from the Ligue Nationale contre le Cancer "axe immunologie des
tumeurs" and the Ligue Départementale de Loire Atlantique. 
2 The two first authors contributed equally to
this work.
3 To whom requests for reprints should be
addressed, at Institut National de la Santé et de la Recherche
Médicale U463, 9 Quai Moncousu, 44093 Nantes Cédex 1,
France. Phone: 33-2-40-08-47-20; Fax: 33-2-40-35-66-97; E-mail: jotereau{at}nantes.inserm.fr
4 The abbreviations used are: IL,
interleukin; TIL, tumor-infiltrating lymphocyte; PBL, peripheral blood
lymphocyte; FACS, fluorescence-activated cell sorting; mAb, monoclonal
antibody; PHA, phytohemagglutinin; GM-CSF, granulocyte macrophage
colony-stimulating factor.
5 P. Lemarre, S. Raher, N. Labarriere, B. Dreno,
F. Jotereau, and A. Godard, unpublished data.
Received 10/20/99; revised 12/15/99; accepted 12/16/99.
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REFERENCES |
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and granulocyte/macrophage colony-stimulating factor) producing donor tumor-infiltrating lymphocytes. J. Immunol., 160: 334-344, 1998. receptor II-mediated apoptosis of cytotoxic T lymphocytes. J. Exp. Med., 188: 1391-1399, 1998.This article has been cited by other articles:
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  I. M. Belyakov, S. Kozlowski, M. Mage, J. D. Ahlers, L. F. Boyd, D. H. Margulies, and J. A. Berzofsky Role of {alpha}3 domain of class I MHC molecules in the activation of high- and low-avidity CD8+ CTLs Int. Immunol., December 1, 2007; 19(12): 1413 - 1420. [Abstract] [Full Text] [PDF]
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 L. Derre, M. Corvaisier, B. Charreau, A. Moreau, E. Godefroy, A. Moreau-Aubry, F. Jotereau, and N. Gervois Expression and Release of HLA-E by Melanoma Cells and Melanocytes: Potential Impact on the Response of Cytotoxic Effector Cells. J. Immunol., September 1, 2006; 177(5): 3100 - 3107. [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. Y. Mapara and M. Sykes Tolerance and Cancer: Mechanisms of Tumor Evasion and Strategies for Breaking Tolerance J. Clin. Oncol., March 15, 2004; 22(6): 1136 - 1151. [Abstract] [Full Text] [PDF]
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T. Woodberry, J. Gardner, S. L. Elliott, S. Leyrer, D. M. Purdie, P. Chaplin, and A. Suhrbier Prime Boost Vaccination Strategies: CD8 T Cell Numbers, Protection, and Th1 Bias J. Immunol., March 1, 2003; 170(5): 2599 - 2604. [Abstract] [Full Text] [PDF]
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L. Derre, M. Corvaisier, M.-C. Pandolfino, E. Diez, F. Jotereau, and N. Gervois Expression of CD94/NKG2-A on Human T Lymphocytes Is Induced by IL-12: Implications for Adoptive Immunotherapy J. Immunol., May 15, 2002; 168(10): 4864 - 4870. [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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) or not
preincubated (
) for 1 h at 37°C with 50 µM
Melan-A 
) against two
Melan-A-expressing cell lines, M77 and M113. For the cytotoxicity
assays, the target cells were 51Cr-labeled and incubated
for 4 h with CTLs at an effector/target ratio of 3:1. For cytokine
production, clones were stimulated by Melan-A-expressing cell lines M77
and M113 for 6 h. The supernatants were collected, and cytokines
were measured by ELISA assay.