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*
Division of Clinical Onco-Immunology, Ludwig Institute for Cancer Research, Lausanne Branch, University Hospital, and
Multidisciplinary Oncology Center, University Hospital, Lausanne, Switzerland;
Ludwig Institute for Cancer Research, Lausanne Branch, University of Lausanne, Epalinges, Switzerland;
§
Institute of Molecular Medicine, Nuffield Department of Medicine, John Radcliffe Hospital, Headington, Oxford, United Kingdom; and
¶
Laboratory of Tumor Immunology, Division of Oncology, University Hospital, Geneva, Switzerland
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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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T cell Ag recognition is based on the interaction of a clonotypic
TCR-
ß with antigenic peptides presented by a given HLA class I or
class II molecule. Thus, it is conceivable that the structural
constraints imposed by the recognition of immunodominant tumor Ags in
TIL(N) would select a limited number of TCR that exhibit at least some
common features. Previous attempts to demonstrate restricted TCR V gene
segment usage by tumor-reactive TIL have yielded contrasting results.
In some studies, limited TCR V gene segment usage was indeed found
(4, 5), while other reports suggested a more diverse TCR V
gene repertoire (6). The main difficulty in interpreting
the results from these studies was that even in those cases where
antitumor reactivity and HLA restriction of the analyzed populations
were demonstrated, no information was available concerning the Ag(s)
recognized. Thus, the search for correlations between TCR V gene
segment usage and tumor Ag-specific recognition has been limited to the
analysis of few available Ag-specific T cell clones
(7, 8, 9, 10, 11).
Overall, these studies have been greatly limited by the difficulty of
identifying tumor-reactive CD8+ T cells of
defined single Ag specificity within polyclonal polyspecific T cell
populations. We have recently shown that direct visualization of
CD8+ T cells specific for the immunodominant
epitope from the melanoma-associated Ag Melan-A can be achieved through
staining with fluorescent HLA-A*0201/Melan-A peptide tetramers
(A2/Melan-A tetramers thereafter) (12). Staining with
A2/Melan-A tetramers is highly sensitive, allowing the identification
of Melan-A-specific cells at frequencies as low as 0.2% in
CD8+ lymph node cells (12) and even
lower in CD8+ circulating lymphocytes (
0.04%)
(13). We have previously shown that the frequency of
A2/Melan-A tetramer+ cells detected in bulk
cultures directly correlates with peptide-specific cytotoxicity
(14). The specificity of
A2/tetramer+ staining was further validated by
assessment of the functional specificity of sorted A2/Melan-A
tetramer+ populations (12, 14). In
the present study we used A2/Melan-A tetramers in combination with
anti-CD8 and a panel of anti-BV mAb to directly and
quantitatively analyze the TCR BV region usage by Melan-A-specific CTL
in TILN and TIL as well as in peptide-stimulated PBMC cultures from
HLA-A2 melanoma patients.
The results of these analyses revealed that small proportions of A2/Melan-A tetramer+ CD8+ cells expressing several different BV can be found in a single sample, thus showing the existence of a large repertoire of Melan-A-specific T cells. However, enrichment of A2/Melan-A tetramer+ CD8+ T cells expressing certain BV was also found. A2/Melan-A tetramer+ CD8+ T cells expressing BV14 were over-represented in the majority of the samples analyzed, while over-representation of other BV appeared to be characteristic of single samples. These findings suggest that the repertoire of Melan-A-specific T cells available in different A2+ melanoma patients could be only partially overlapping.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Melanoma patients subjected to therapeutic surgical resection of lymph node (LN) or metastatic lesions were selected for this study on the basis of HLA-A2 Ag expression as assessed by HLA typing. Individual samples collected by surgical dissection were finely minced with needles in sterile RPMI 1640 supplemented with 10% FCS. Cell suspensions from each sample were placed in 24-well tissue culture plates (Costar, Cambridge, MA) in 2 ml of IMEM (Life Technologies, Basel, Switzerland) supplemented with 0.24 mM Asn, 0.55 mM Arg, 1.5 mM Gln, 10% pooled human A+ serum (CTL medium), 100 U/ml IL-2, and 10 ng/ml IL-7. Cells were cultured 2–3 wk before FACS analysis. For peptide stimulation experiments of PBMC, CD8+ lymphocytes were positively selected by magnetic cell sorting from PBMC of HLA-A*0201 melanoma patients using a MiniMACS device (Miltenyi Biotec, Sunnyvale, CA). Cells of the CD8- fraction were irradiated (30 Gy) and used as autologous APC. CD8+ highly enriched lymphocytes (1 x 106/condition) were stimulated with peptide (1 µM) and irradiated autologous APC in 2 ml of CTL medium (14) containing human rIL-2 (100 U/ml; Glaxo, Geneva, Switzerland; provided by Dr. M. Nabholz, Institut Suisse de Recherches Experimentales sur le Cancer, Epalinges, Switzerland) and human rIL-7 (10 ng/ml; R&D Systems Europe, Oxon, U.K.). Cells were cultured for 2 wk before A2/Melan-A tetramer-guided ß-chain variable (BV) chain usage analysis.
Tetramers
Complexes were synthesized as previously described (12, 15). Briefly, purified recombinant HLA heavy chain and ß2-microglobulin were obtained by means of a prokaryotic expression system (pET, R&D Systems, Minneapolis, MN). The heavy chain was modified by deletion of the transmembrane-cytosolic tail and COOH-terminal addition of a peptide sequence containing the BirA enzymatic biotinylation site. Heavy chain, ß2-microglobulin, and peptide were refolded by dilution. The 45-kDa refolded product was isolated by fast protein liquid chromatography and then biotinylated by recombinant BirA (Avidity, Denver, CO) in the presence of biotin, ATP, and Mg 2+ (all from Sigma, St. Louis, MO). Tetramers were prepared by addition of PE-streptavidin and then concentrated to 1 mg/ml. As the antigenic peptide, the Melan-A26–35 E26A/A27L analogue (ALAGIGILTV) was used, which has a higher binding stability to HLA-A*0201, and T cell antigenicity and immunogenicity higher than those of the natural Melan-A decapeptide EAAGIGILTV or the nonapeptide AAGIGILTV (16).
Monoclonal Abs and flow cytometric analysis
A panel of 21 anti-BV Abs was used in this study (Immunotech, Beckman-Coulter, Marseilles, France). Anti-BV1, -2, -3, -5.1, -5.2, -7, -8, -11, -12, -13.1, -13.6, -14, -16, -17, -20, -21.3, and -22 mAbs were FITC conjugated, whereas anti-BV5.3, -9, -18, and -23 were used as purified mAbs. Anti-CD8 PercP was purchased from Becton Dickinson (San Jose, CA). Staining and washing were performed in PBS, 5% FCS, and 0.2% sodium azide. For indirect fluorescence labeling, cells were incubated 1) with tetramers (0.2 µg/sample in 20 µl) for 20 min at room temperature and washed, 2) with purified anti-BV mAbs for 30 min at 4°C and washed, 3) with sheep anti-mouse FITC-labeled Ab for 30 min at 4°C and washed, 4) with IgG1 and IgG2a Abs for 10 min at 4°C, and 5) with anti-CD8 PercP-labeled mAb for 30 min at 4°C. Staining with directly labeled anti-BV mAbs was similarly performed for steps 1, 2, and 5. After completing the staining scheme, the cells were washed once and analyzed immediately in a FACSCalibur (Becton Dickinson). Data analysis was performed using CellQuest software.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Melan-A has been previously shown to be one of the most prevalent
melanoma-associated Ags, as it is recognized by the large majority of
TIL cultures (17). In a previous report we observed that
short term cultured TILN from A2 melanoma patients often contain high
percentages of A2/Melan-A tetramer+ cells that
may allow TCR repertoire analysis by flow cytometry (12).
Thus, samples from a total of six TILN and six TIL were first analyzed
by staining with an anti-CD8 mAb together with A2/Melan-A
tetramers. We have previously established that staining of LN cell
suspensions with A2/Melan-A tetramers allows the identification of
Melan-A-specific cells at frequencies as low as 0.2% of the
CD8+ subpopulation of LN cells. This detection
limit was calculated from background staining observed in a series of
seven HLA-A2- LNs (12). Based on
these data, percentages of CD8+ A2/Melan-A
tetramer+ cells largely over the tetramer
detection limit were found in four of six TILN and four of six TIL
(Table I
). These samples were selected
for repertoire analysis.
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for TILN LAU 233. Reanalysis of the
CD8+ A2/Melan-A tetramer+
and CD8+ tetramer-
populations showed a high degree of purity (>96%). After overnight
culture, the sorted populations were assayed for Ag-specific cytolytic
activity. As shown in Fig. 1B, sorted
CD8+ A2/Melan-A tetramer+ T
cells specifically lysed peptide-loaded T2 cells even at a low E:T cell
ratio, whereas no significant specific lysis was observed for the
CD8+ Melan-A tetramer-
population. The former population displayed a potent specific lytic
activity in the presence of parental Melan-A peptide concentrations as
low as 100 pM, suggesting that they expressed relatively high avidity
TCRs (Fig. 1C). In addition, CD8+
A2/Melan-A tetramer+ cells specifically lysed
autologous tumor cells (Fig. 1D). Similar results were
obtained for other TIL(N) populations (LAU 203 and LAU 156; data not
shown). Finally, lysis of nonautologous melanoma tumor cell lines by
CD8+ A2/Melan-A tetramer+
cells was strictly dependent on coexpression of both HLA-A2 and Melan-A
(Fig. 1D).
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The TCR BV usage of A2/Melan-A tetramer+
cells present within TIL(N) samples was analyzed using a panel of 21
mAbs directed against the variable region of the TCR ß-chain together
with anti-CD8 mAb and A2/Melan-A tetramers. An illustration of the
results obtained is shown in Fig. 1
for patient LAU 233 (BV14, Fig. 1E; BV17, Fig. 1F). It is of note that in some
cases (Fig. 1F) the intensity of the tetramer signal
detected in the tetramer+
BV+ fraction was lower than that of the
tetramer+ BV-
signal, most likely as the result of steric
hindrance between tetramers and some anti-BV Abs competing for
neighboring binding sites. Indeed, inversion in the order of addition
of reagents (i.e., staining with the tetramer before staining with the
anti-BV mAb) resulted in the reciprocal pattern; that is, a lower
intensity of BV signal in the tetramer+
BV+ fraction, but a comparable intensity of the
tetramer signal in both tetramer+
BV+ and tetramer+
BV- fractions (not shown). This phenomenon was
observed for certain anti-BV mAbs, but not for others (Fig. 1E), probably dependent on the topology of their TCR binding
sites.
A compilation of the results of BV usage analysis for TILN from four
melanoma patients is illustrated in Fig. 2 for CD8+
A2/Melan-A tetramer+ T cells (Fig. 2A)
and CD8+ A2/Melan-A
tetramer- T cells (Fig. 2B). The
fraction of BV repertoire covered within CD8+
A2/Melan-A tetramer+ TILN populations by our
panel of anti-BV mAb was similar in different TILN (70% for LAU
253, 74% for LAU 50, and 78% for LAU 233), with the exception of TILN
LAU 203, where only 41% of the available BV repertoire was covered.
Relatively small proportions of CD8+ A2/Melan-A
tetramer+ T cells were detected that express many
of the BV tested. However, high proportions of
CD8+ A2/Melan-A tetramer+ T
cells were found to express only certain BV. In particular,
CD8+ A2/Melan-A tetramer+ T
cells expressing BV14 were present in a relatively high percentage
(from 9.4–33.7%) in all the samples tested. In contrast,
over-representation of CD8+ A2/Melan-A
tetramer+ T cells expressing some other BV was
characteristic of single samples (i.e., BV2 and BV13.1 for patient LAU
50, BV17 for patient LAU 233, BV20 for patient LAU 203). The increased
proportion of CD8+ A2/Melan-A
tetramer+ T cells expressing a certain BV could
not simply be explained by selective growth of cells expressing that
particular BV in the culture, because a parallel expansion was often
not detected in the corresponding CD8+ A2/Melan-A
tetramer- T cell fraction (i.e., for patient LAU
253, BV14 represent 34% of CD8+ A2/Melan-A
tetramer+ T cells but only 3.7% of
CD8+ A2/Melan-A tetramer-
T cells). Over-representation of T cells expressing certain BV up to
21.1% (BV1 patient LAU 253) was also detected among
CD8+ A2/Melan-A tetramer-
cells, possibly reflecting T cell responses driven by Melan-A-unrelated
tumor Ags. This hypothesis may be supported by the fact that BV usage
analysis of short term cultured normal lymph node cells from two
A2+ melanoma patients failed to reveal expansion
of CD8+ T cells expressing a certain BV at levels
>6% (data not shown).
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A similar analysis of BV usage by CD8+
A2/Melan-A tetramer+ cells was performed on four
TIL from three melanoma patients (Fig. 3). In general, the BV usage by
CD8+ A2/Melan-A tetramer+
cells present in the TIL appeared to be much more restricted than that
in TILN. Remarkably, BV14+ cells represented 93
and 87% of CD8+ A2/Melan-A
tetramer+ cells in TIL LAU 156A and LAU 156B,
respectively. Because these two TIL were obtained from two independent
resections of the same metastatic lesion at an 8-mo interval, this
could indicate the presence (and prevalence) of a unique Melan-A
specific clonotype within the lesion. For TIL 343, two BVs (BV3, 17%;
BV16, 63%) were over-represented. From this patient the corresponding
TILN (obtained in the course of the same surgical intervention) were
also available, although in very limited numbers. TILN LAU 343
contained 90% of CD8+ cells, but only 0.25% of
those were A2/Melan-A tetramer+. However, and
interestingly, 71% of the CD8+ A2/Melan-A
tetramer+ cells were BV3 positive (not shown).
Thus, the tetramer-guided analysis of BV usage in TIL allowed us to
determine the BV segment used by the large majority of Melan-A-specific
T cells. In striking contrast, for TIL LAU 212, we failed to detect any
significant proportion of CD8+ A2/Melan-A
tetramer+ cells expressing any of the anti-BV
mAb included in our panel, thus suggesting over-representation of
alternative BV segments. Over-representation of T cells expressing
certain BV were also detected among CD8+
A2/Melan-A tetramer- cells (i.e., BV8 for TIL
LAU 156B, BV22 for TIL LAU 343), possibly in relation to T cell
responses to other tumor Ags.
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Several attempts to analyze the TCR repertoire selected in vitro
by stimulation with Melan-A natural peptides or analogues have recently
been reported (9, 18). To verify the feasibility of this
approach and to directly compare the BV usage by Melan-A-specific CTL
present in TILN and in peptide-stimulated PBMC from the same patient,
we performed the experiment illustrated in Fig. 4. CD8+ highly
enriched PBMC from melanoma patient LAU 203 were stimulated with either
the nonapeptide Melan-A27–35 or the decapeptide
Melan-A26–35 in the presence of autologous APC.
Two parallel cultures were set for each peptide used. The analysis of
BV repertoire usage of CD8+ A2/Melan-A
tetramer+ cells detected in the cultures at the
end of a 2-wk stimulation period revealed significant expansion of
CD8+ A2/Melan-A tetramer+
cells expressing many different BV, including the ones previously
identified in the autologous TILN (BV13.1, BV14, and BV20; Fig. 2).
However, although the percentages of CD8+
A2/Melan-A tetramer+ cells present in the
different cultures were roughly constant (3.5 and 4.6% for the
AAGIGILTV-stimulated cell cultures, and 4 and 4.4% for the
EAAGIGILTV-stimulated cell cultures), the expansion of
CD8+ A2/Melan-A tetramer+
cells expressing a certain BV varied considerably between parallel
cultures. Similar results were obtained with PBMC from another melanoma
patient (not shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Using A2/Melan-A tetramers we have recently shown that ex vivo stained
metastatic LN of A2 melanoma patients can contain significant numbers
of Ag-experienced Melan-A-specific T cells (12). We have
also observed that the proportion of such T cells can significantly
increase during the first weeks of in vitro culture in the presence of
exogenously added cytokines only. In this study we found that 8 of 12
short term cultured TIL and TIL(N) samples from A2 melanoma patients
contained increased proportions (from 4 to 24% of total
CD8+ cells; see Table I) of Melan-A-specific T
cells. These results further underline the immunodominant character of
anti-Melan-A-specific responses in the context of HLA-A*0201.
However, it is not clear why enrichment of Melan-A-specific T cells is
not always found. Interestingly, in the case of patient LAU 198 (for
whom no significant amount of CD8+ A2/Melan-A
tetramer+ T cells was observed), a melanoma cell
line derived from the analyzed lesion showed loss of HLA-A2 surface
expression, as assessed by staining with HLA-A2-specific Ab BB7.1 (data
not shown). In addition, whether the presence of increased numbers of
A2/Melan-A tetramer+ cells in ex vivo or short
term cultured TIL(N) samples of A2 melanoma patients may represent an
independent prognosis indicator for overall survival remains to be
established.
In the present study we have used A2/Melan-A tetramers containing the Melan-A26–35-enhanced analogue E26A/A27L (ALAGIGILTV). In a previous study we showed the interchangeability of A2/Melan-A tetramers containing both the parental Melan-A26–35 (EAAGIGILTV) peptide or the A27L (ELAGIGILTV) modified analogue for the staining of CTL-specific monoclonal populations (12). We have also extensively shown that A2/Melan-A A27L tetramer+ sorted populations from both TILN and TIL and peptide-stimulated PBL were functionally extremely active in killing both peptide-pulsed cells and autologous tumor cells. In contrast, no specific lysis was detected for A2/Melan-A A27L tetramer- populations (12, 13, 14). Similar results were obtained for the E26A/A27L peptide analogue (not shown). In addition, in our experience both polyclonal and monoclonal Melan-A-specific populations obtained from TILN or TIL or from PBL stimulated with parental Melan-A26–35 peptide, cross-recognized the A27L analogue with a similar or high avidity compared with the parental sequence (16). Overall, these data support the idea that the large majority of Melan-A-specific CD8+ T cells efficiently cross-recognize peptide A27L and E26A/A27L and are efficiently stained by A2/Melan-A A27L or E26A/A27L tetramers. However, the possibility that a very minor population of Melan-A-specific cells that do not cross-react with the analogue or exhibit very low avidity recognition could be missed by this approach cannot be formally excluded.
Numerous studies have focused on the complexity of TCR usage by T cells
of defined Ag specificity (26). Responses to T cell Ags
were sometimes rather diverse or, in other cases, strongly constrained
(27). The human TCR repertoire expressed by CTL specific
for MHC class I-restricted viral peptides has been shown to be
very limited. Indeed, similar or even identical TCR /ß primary
structures were found in different individuals sharing the presenting
HLA allele (19, 20, 21, 22, 28). In contrast to these
observations, the analysis of CTL clones derived from a single melanoma
patient and specific for an HLA-A1-restricted epitope derived from
MAGE-1 protein indicated that the TCR repertoire directed against this
tumor Ag was rather diverse (10). The presence of multiple
shared tumor-associated Ags has led to conflicting studies of T cell
variable usage in TIL cultures (6, 7, 24, 25). Overall,
the knowledge concerning the repertoire of tumor-specific T cells
remains very limited.
Several attempts to analyze the TCR repertoire specific for Melan-A have been made. Some studies initially reported restricted TCR V gene usage (AV2, BV7, and BV14) by Melan-A-specific T cell clones (7). However, subsequent studies failed to confirm this trend (6, 8, 9, 11). The results of the present study show that a large repertoire of Melan-A-specific CD8+ T cells is available for each patient. In agreement with these structural data we have recently reported a large functional heterogeneity of Ag recognition by Melan-A-specific CTL clones (16). Whether a correlation can be found between Melan-A-specific T cell fine specificity and BV usage remains to be established. Indeed, it would be of great interest to use the approach described here to assess the functional properties and, in particular, the tumoricidal potential of Melan-A-specific populations expressing single BV and isolated by tetramer-guided cell sorting. However, the feasibility of these studies is limited by the large number of cells required for the assessment of Ag-specific cytolytic activity. The development of new techniques based on flow cytometric analysis of single-cell cytolytic functions may allow new means of addressing this issue in the near future.
The BV repertoire was larger in PBMC and TILN compared with TIL. This could be explained if, despite the relatively high frequencies at which A2/Melan-A tetramer-positive precursors can be detected in both normal LN and PBMC of A2 melanoma patients (12, 14), only few specific circulating precursors can infiltrate tumor metastases. A2/Melan-A tetramer-positive cells expressing BV14 were over-represented in most samples analyzed, whereas over-representation of other BV was characteristic of individual samples. These results may at least partially explain the BV14 restriction previously reported for Melan-A-specific CTL (7) and may reflect a relatively high frequency of BV14-bearing cells in circulating Melan-A-specific CTL precursors. Alternatively, it may be related to the high affinity/avidity of Ag recognition by BV14-bearing CTL. These issues are currently under investigation.
In the present study the analysis of the T cell repertoire of Melan-A-specific CTL was based on the use of a panel of anti-BV Abs. This approach constitutes only a first level of analysis of the complexity of Ag-specific T cell responses represented by the differential use of the BV, and does not reveal whether the BV-restricted response analyzed is, for example, of monoclonal or polyclonal nature or whether Ag-specific clonotypes present in the population exhibit any other common features. To analyze this second level of complexity we are currently performing molecular TCR analysis (26) of Melan-A-specific T cells isolated by tetramer-guided cell sorting. The combination of these powerful techniques will be instrumental to dissect changes in the repertoire of tumor-specific CTL responses during the course of the disease as well as in vaccinated patients.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Danila Valmori, Division of Clinical Onco-Immunology, Ludwig Institute for Cancer Research, Hôpital Orthopédique, Niv. 5, avenue Pierre-Decker 4, 1005 Lausanne, Switzerland.
3 Abbreviations used in this paper: TIL, tumor-infiltrating lymphocytes; TILN, tumor-infiltrated lymph nodes; LN, lymph node; BV, ß-chain variable.
Received for publication December 27, 1999. Accepted for publication April 12, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and ß chains of the human T-cell antigen receptor recognizing HLA-A2 and influenza A matrix peptide. Proc. Natl. Acad. Sci. USA 88:8987.1-w29/Vß1-w24) for the study of human T cell receptor variable V gene segment usage by polymerase chain reaction. Eur. J. Immunol. 22:1261.[Medline]
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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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V. Rubio-Godoy, V. Dutoit, Y. Zhao, R. Simon, P. Guillaume, R. Houghten, P. Romero, J.-C. Cerottini, C. Pinilla, and D. Valmori Positional Scanning-Synthetic Peptide Library-Based Analysis of Self- and Pathogen-Derived Peptide Cross-Reactivity with Tumor-Reactive Melan-A-Specific CTL J. Immunol., November 15, 2002; 169(10): 5696 - 5707. [Abstract] [Full Text] [PDF]
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 C. M. Constantin, E. E. Bonney, J. D. Altman, and O. L. Strickland Major Histocompatibility Complex (MHC) Tetramer Technology: An Evaluation Biol Res Nurs, October 1, 2002; 4(2): 115 - 127. [Abstract] [PDF]
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S. Mandruzzato, E. Rossi, F. Bernardi, V. Tosello, B. Macino, G. Basso, V. Chiarion-Sileni, C. R. Rossi, C. Montesco, and P. Zanovello Large and Dissimilar Repertoire of Melan-A/MART-1-Specific CTL in Metastatic Lesions and Blood of a Melanoma Patient J. Immunol., October 1, 2002; 169(7): 4017 - 4024. [Abstract] [Full Text] [PDF]
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 M. V. Dhodapkar, J. Krasovsky, and K. Olson T cells from the tumor microenvironment of patients with progressive myeloma can generate strong, tumor-specific cytolytic responses to autologous, tumor-loaded dendritic cells PNAS, October 1, 2002; 99(20): 13009 - 13013. [Abstract] [Full Text] [PDF]
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H. Echchakir, G. Dorothee, I. Vergnon, J. Menez, S. Chouaib, and F. Mami-Chouaib Cytotoxic T lymphocytes directed against a tumor-specific mutated antigen display similar HLA tetramer binding but distinct functional avidity and tissue distribution PNAS, July 9, 2002; 99(14): 9358 - 9363. [Abstract] [Full Text] [PDF]
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D. Valmori, V. Dutoit, V. Schnuriger, A.-L. Quiquerez, M. J. Pittet, P. Guillaume, V. Rubio-Godoy, P. R. Walker, D. Rimoldi, D. Lienard, et al. Vaccination with a Melan-A Peptide Selects an Oligoclonal T Cell Population with Increased Functional Avidity and Tumor Reactivity J. Immunol., April 15, 2002; 168(8): 4231 - 4240. [Abstract] [Full Text] [PDF]
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 H. Pilch, H. Hohn, K. Freitag, C. Neukirch, A. Necker, P. Haddad, B. Tanner, P. G. Knapstein, and M. J. Maeurer Improved Assessment of T-Cell Receptor (TCR) VB Repertoire in Clinical Specimens: Combination of TCR-CDR3 Spectratyping with Flow Cytometry-Based TCR VB Frequency Analysis Clin. Vaccine Immunol., March 1, 2002; 9(2): 257 - 266. [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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 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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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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 S. Szmania, A. Galloway, M. Bruorton, P. Musk, G. Aubert, A. Arthur, H. Pyle, N. Hensel, N. Ta, L. Lamb Jr, et al. Isolation and expansion of cytomegalovirus-specific cytotoxic T lymphocytes to clinical scale from a single blood draw using dendritic cells and HLA-tetramers Blood, August 1, 2001; 98(3): 505 - 512. [Abstract] [Full Text] [PDF]
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V. Karanikas, D. Colau, J.-F. Baurain, R. Chiari, J. Thonnard, I. Gutierrez-Roelens, C. Goffinet, E. V. Schaftingen, P. Weynants, T. Boon, et al. High Frequency of Cytolytic T Lymphocytes Directed against a Tumor-specific Mutated Antigen Detectable with HLA Tetramers in the Blood of a Lung Carcinoma Patient with Long Survival Cancer Res., May 1, 2001; 61(9): 3718 - 3724. [Abstract] [Full Text]
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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. Schrama, M. H. Andersen, P. Terheyden, L. Schrøder, L. O. Pedersen, P. t. Straten, and J. C. Becker Oligoclonal T-Cell Receptor Usage of Melanocyte Differentiation Antigen-reactive T Cells in Stage IV Melanoma Patients Cancer Res., January 1, 2001; 61(2): 493 - 496. [Abstract] [Full Text]
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P. G. Coulie, V. Karanikas, D. Colau, C. Lurquin, C. Landry, M. Marchand, T. Dorval, V. Brichard, and T. Boon A monoclonal cytolytic T-lymphocyte response observed in a melanoma patient vaccinated with a tumor-specific antigenic peptide encoded by gene MAGE-3 PNAS, August 28, 2001; 98(18): 10290 - 10295. [Abstract] [Full Text] [PDF]
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;
Melan-A26–35; EAAGIGILTV) or to an irrelevant
antigenic peptide (
; NY-ESO-1157–165;
SLLMWITQC) (
), the
HLA-A2+ Melan-A-negative melanoma cell line NA-8
MEL (
), or the HLA-A2- melanoma cell lines,
Me 242 B1 (
) and Me 329 M2 (
), in the absence (open symbols) or
the presence (filled symbols) of the antigenic peptide
Melan-A26–35 (1 µM).
