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Immunology |
Division of Clinical Onco-Immunology, Ludwig Institute for Cancer Research, Centre Hospitalier Universitaire Vaudois, 1011 Lausanne [V. D., V. R-G., D. L., D. S., P. B., J-C. C., P. R., D. V.]; Division of Oncology, Laboratory of Tumor Immunology, University Hospital, 1211 Geneva 14 [P-Y. D., A-L. Q., V. S.]; Ludwig Institute for Cancer Research, Lausanne Branch, University of Lausanne, 1066 Epalinges [D. R., P. G., J-C. C.]; and Multidisciplinary Oncology Center, University Hospital, 1011 Lausanne [D. L.], 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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The potency of antitumor CTL responses directed against peptide antigenic sequences derived from tumor Ags is influenced by parameters such as frequency and specificity of Ag expression in tumors, frequency of specific T cells, and avidity of specific CTLs for the tumor target (13 , 14) . Whereas CTL responses to most of the Ag groups listed above are relevant to a specific type of tumor, cancer testis Ags are ideal candidates for generic vaccination of cancers patients. We and others (15 , 16) have reported recently that natural CTL responses to the cancer testis Ag NY-ESO-1 can be detected in melanoma patients as well as in patients bearing other types of tumors. In the case of MAGE-encoded Ags, however, despite the identification of numerous CTL-defined antigenic peptides derived from individual MAGE gene products (2) , specific CTLs have been thus far detected only rarely in cancer patients. Thus, both the extent and the nature of MAGE Ag-specific CTL repertoire have remained unexplored.
MAGE-A10 is a nuclear protein (17) expressed by tumors of different histological types, with the highest frequencies (33–50%) observed in melanoma, bladder carcinoma, lung carcinoma, esophageal, and head and neck squamous carcinoma. Recently, an HLA-A2-restricted MAGE-A10 encoded epitope recognized by a CTL clone from a melanoma patient has been found to correspond to peptide 254–262 (18) . We have observed recently that, in contrast to previously analyzed CD8+ T cells, responses to other MAGE antigenic peptides, CD8+ T cells specific for peptide MAGE-A10254–262 are found frequently in peptide-stimulated PBMCs from A2-expressing melanoma patients and healthy donors (19) . Therefore, peptide MAGE-A10254–262 is currently an important candidate vaccine for immunotherapy of cancer. In this perspective, however, a deeper knowledge of the functional diversity of MAGE-A10254–262-specific T cells as well as the correlation of the latter with tumor recognition and, possibly, with TCR structural diversity, would be instrumental for evaluating the efficacy of vaccination trials aimed at eliciting MAGE-A10254–262-specific, tumor-reactive T cells. With this aim, in the present study we have characterized MAGE-A10254–262-specific T cells derived from six melanoma patients and two healthy donors in terms of avidity of Ag recognition and specific tumor lysis. On the basis of the analysis of specific clonal populations, we observed that MAGE-A10254–262-specific T cells can recognize peptide MAGE-A10254–262 within a relatively large range of avidity. Importantly, MAGE-A10-specific T cells able to recognize peptide MAGE-A10254–262 with high avidity (comparable with that of T cells specific for virally derived Ags) showed a superior capacity to lyse MAGE-A10-expressing tumors. In addition, we observed that, under defined experimental conditions, the brightness of specific T-cell staining with A2/MAGE-A10254–262 peptide tetramers (tetramers hereafter) correlate with avidity of Ag recognition thus that high and low avidity MAGE-A10254–262-specific T cells can be separated by FACS.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Ag Recognition Assay.
Ag recognition was assessed by chromium-release assay. Briefly, chromium-labeled target T2 cells (1000) were incubated in the presence of serial dilutions of parental peptide or analogues and effector cells at an effector:target cell ratio of 10:1. Chromium release was measured in the supernatant after 4 h of incubation at 37°C. The % specific lysis was calculated as:
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Tetramers, mAbs, and BV Repertoire Analysis.
HLA-A2/MAGE-A10254–262 peptide tetramers were synthesized as described (20
, 21)
using peptide MAGE-A10254–262. Anti-CD8FITC and anti CD8PerCP were purchased from Becton Dickinson (San Jose, CA). To enumerate MAGE-A10-specific T cells in the cultures, samples were stained with tetramersPE (0.1 µg/sample in 20 µl) for 1 h at room temperature and then with anti-CD8FITC for an additional 30 min at 4°C. For BV repertoire analysis, a panel of 21 anti-BV mAbs (purchased from Immunotech, Beckman-Coulter, Marseilles, France) was used. Anti-BV 5.3, -9, -18 and -23 were used as purified mAbs, whereas the remaining BV mAbs were FITC conjugated. Staining and washing were performed in PBS, 0.2% BSA, and 0.02% sodium azide. Cells were: (a) stained with tetramers-PE (5 µg/ml) for 1 h at room temperature and washed; (b) with purified anti-BV mAbs for 20 min at 4°C and washed; (c) with goat antimouse FITC-labeled mAb for 30 min at 4°C and washed; (d) with IgG1 and IgG2a mAbs for 10 min at 4°C; and (e) with anti-CD8-PerCP 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, cells were washed once with the same buffer and analyzed in a FACSCalibur, FACScan, or FACSVantage (Becton Dickinson). Data analysis was performed using the Cell Quest software. For tetramer-guided cell sorting, the same protocol was used except for the fact that the buffer contained no sodium azide. For PCR analysis, total RNA was prepared from MAGE-A10256–262-specific clones using TRIzol (Life Technologies, Inc., Paisley, United Kingdom) and converted to cDNA by standard methods using reverse transcriptase and an oligo(dT) primer. cDNA was then amplified using a panel of validated 5' sense primers specific for the 24 BV subfamilies and 1 3' antisense primer specific for the BC gene segment (22)
. TCR BV-BC PCR products were cloned into pBS-SK+ vector (Stratagene, La Jolla, CA). Competent XL-1 blue Escherichia coli (Stratagene) were transformed and plated for blue/white color selection on medium containing 5-bromo-4-chloro-3-indolyl-ß-D-galactopyranoside. Plasmid DNA was extracted from white colonies using the Qiagen Plasmid Mini kit (Qiagen, Hilden, Germany) and sequenced using the Dye Terminator cycle Sequencing kit (ABI PRISM; Perkin-Elmer, Foster City, CA) according to the manufacturer’s instructions.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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or under limiting dilution conditions to isolate T-cell clones. A total number of six polyclonal lines (from patients LAU 50, LAU 155, LAU 119, LAU 42, and healthy donors HD 591 and HD 795) were generated. Specific T-cell clones were obtained from LAU 50, LAU 155, LAU 203, LAU 169, LAU 42, and from HD 795.
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. Anti-BV mAbs were also used to assess the BV segment usage of isolated clonal populations. Tables 1
and 2
shows the results of this analysis for polyclonal cultures and representative clones. For 4 of the polyclonal cultures analyzed (LAU 50, LAU 155, LAU 119, and LAU 42), BV segment usage of >90% of tetramer+ cells was determined by using this method. However, for cultures from healthy donors HD 591 and HD 795, BV usage of only 
50% of tetramer+ cells was determined. Most of tetramer+ cells in the polyclonal culture from LAU 50 expressed BV 22, whereas a small but significant proportion expressed BV 21.3 (Fig. 1B)
. Both BV 21.3 (clone LAU 50/15)- and BV 22-expressing clones (clone LAU 50/22) were isolated from this culture. Tetramer+ cells in the polyclonal culture from patient LAU 155 expressed exclusively BV 21.3. Accordingly, a BV 21.3-expressing clone (LAU 155/6D1) was isolated from this culture. Tetramer+ cells in the polyclonal culture from patient LAU 119 exclusively expressed BV 22. BV gene segment usage of a T-cell clone derived from patient LAU 203 (clone LAU 203/1B8) could not be determined by staining with anti BV antibodies and was determined by PCR analysis (BV 13; Table 2
). A BV 22-expressing clone was isolated from peptide-stimulated PBMCs of patient LAU 169 (LAU 169/4E8); no polyclonal culture was obtained from this patient. Among tetramer+ cells in the polyclonal culture of HD 591, 22% expressed BV 21.3, whereas 32% expressed BV 22. Among tetramer+ cells in the polyclonal culture of HD 795, 43% expressed BV 17 and 9% expressed BV 22. Two clones were derived from this culture; one of them (HD 795/4D11) expressed BV 17, whereas BV gene segment usage of the other one (HD 795/2B6) could not be determined by staining with anti-BV antibodies but was determined by PCR (BV 13; Table 2
). Finally, tetramer+ cells in the polyclonal culture from patient LAU 42 expressed BV 1 (63%) and BV 13.1 (30%) segments. Interestingly, this last patient expressed the HLA-A*0206 allele instead of the HLA-A*0201 allele expressed by the large majority of Caucasians.
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. This analysis revealed CDR3 regions of variable size (6–10 amino acids) and sequence among clones suggesting the lack of a high degree of restriction in the repertoire selection of MAGE-A10254–262-specific CD8+ T cells. Strikingly, however, clones LAU 203/1B8 and HD 795/2B6, both using BV 13 but different BJ segments, exhibited identical CDR3 sequences (6 amino acids long), with the exception of a single amino acid substitution (in bold in Table 2
). Thus, despite the apparent lack of restriction, conserved structural features were also observed among MAGE-A10254–262-specific TCRs.
Efficiency and Fine Specificity of T-Cell Recognition of MAGE-A10254–262-specific CD8+ T Cells.
To assess the efficiency of antigen recognition by MAGE-A10-specific T cells, peptide titration assays were performed for 10 independent T-cell populations in a standard 4-h chromium release assay using the T2 cell line as target cells. An example of the results obtained with this analysis is shown in Fig. 2
for two cell clones. Data obtained for 10 independent populations are summarized in Table 3
. Although all MAGE-A10254–262-specific T cells were able to efficiently lyse target cells in the presence of saturating concentrations of MAGE-A10254–262 peptide, the peptide concentration required to obtain 50% of maximal lysis was highly variable among the different populations ranging from 0.05 nM to >100 nM. Polyclonal cultures and clones isolated from patients LAU 50, LAU 155, LAU 119, and LAU 203 and from donor HD 591 recognized the MAGE-A10 peptide with relatively high efficiency (50% maximal lysis between 0.05 and 0.4 nM) and are thereafter functionally defined as high-avidity T cells. In contrast, clones isolated from donor HD 795 and patients LAU 42 and LAU 169 recognized the Ag with relatively low efficiency (50% maximal lysis at 100 nM) and are thereafter functionally defined as low-avidity T cells. Both high- and low-avidity-specific T cells were isolated from melanoma patients expressing MAGE-A10 in their tumor cells (LAU 50, LAU 119, and LAU 169; Ref. 19
). Similarly, both high- and low-avidity-specific T cells were isolated from patients bearing MAGE-A10-negative tumors as well as from healthy donors (LAU 155, LAU 203, LAU 42, HD 591, and HD 795). Thus, we found no clear correlation between MAGE-A10 expression in their tumor cells and isolation of high versus low avidity MAGE-A10-specific T cells.
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Correlation between Avidity of Peptide Recognition, Specific Tumor Lysis, and Tetramer Staining.
The ability of MAGE-A10-specific CD8+ T cells to specifically lyse the MAGE-A10-expressing tumor cell line Me 275 was assessed by standard chromium release assay (Fig. 3A
and data not shown). All populations were able to lyse Me 275 in the presence of MAGE-A10254–262 peptide. However, only some of them efficiently lysed Me 275 in absence of peptide. Specific tumor lysis tightly correlated with avidity of peptide recognition of MAGE-A10254–262-specific populations. As shown in Fig. 3B
, MAGE-A10254–262-specific cells requiring <1 nM of antigenic peptide to achieve 50% maximal lysis on T2 cells were also able to lyse MAGE-A10-expressing tumor cells. In marked contrast, polyclonal cultures or clones that required higher peptide concentrations to achieve 50% maximal lysis failed to do so.
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ß, CD8, LFA, and CD28 were used to stain representative CTL clones of different avidity. There were no detectable differences in the levels or TCR expression as well as in the expression of accessory molecules (data not shown). This suggested that the TCR itself was the main determinant imparting the differential avidity of the CTL clones. Hence, a correlation between avidity of CTL clones and the efficacy of tetramer staining could be expected. To address this question, CTL clones of different avidity (Fig. 4A)
were stained with tetramers during 1 h at room temperature. Under these conditions, two of three high-avidity clones (LAU 50/22 and LAU 155/6D1) displayed a brighter tetramer staining as compared with the three low-avidity clones analyzed (HD 795/2H11, HD 795/4D11, and LAU 168/4E8), whereas one did not (LAU 50/15; Fig. 4A
). However, interestingly, upon prolonged staining (4 h), high-avidity clones were stained significantly more efficiently than low-avidity clones. Similar results were obtained upon staining of the polyclonal populations from which the clones were derived (Fig. 4A)
. To analyze whether this phenomenon could be exploited to separate high and low tumor-reactive MAGE-A10254–262-specific populations, T cells present among polyclonal populations, we performed the experiment illustrated in Fig. 2B
. Polyclonal populations from HD 795, LAU 50, and LAU 155 (Fig. 4A)
were mixed in approximately equivalent proportions. The resulting population, stained with tetramers during 4 h at room temperature, showed a broad range of tetramer staining so that it could be arbitrarily separated into tetramer bright and tetramer low populations by FACS (Fig. 4B)
. The functional characteristics of tetramerhigh and tetramerlow populations were then analyzed in a chromium release assay in the presence of graded peptide concentrations. Tetramerhigh T cells exerted 50% maximal lysis at a 100-fold lower peptide dose as compared with tetramerlow T cells. In agreement with these data, tetramerbright T cells displayed a considerably higher tumor-specific lysis than tetramerlow T cells (Fig. 4C)
. It is of note that the total population displayed a functional activity comparable with that of tetramerhigh T cells demonstrating that the functional heterogeneity of the unseparated population would have passed unnoticed in a standard analysis.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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T-cell Ag recognition is based on the interaction of clonotypic TCR-ß with antigenic peptides presented by a given HLA molecule. It is likely that, because of the structural constrains imposed by Ag recognition, a limited number of TCRs exhibiting at least some common features will be selected upon stimulation with Ag, thus defining a limited Ag-specific TCR repertoire. Numerous studies have focused on the complexity of TCR usage by T cells of defined Ag specificity (25)
. The human TCR repertoire expressed by CTLs specific for MHC class-I restricted viral peptides has been shown to be highly restricted and very similar in different individuals sharing the presenting HLA allele (26, 27, 28, 29, 30)
. Interestingly, overexpression of a restricted set of TCR BV gene families was found in metastatic lesions of melanoma patients treated with an autologous Dinitrophenyl-modified tumor cell vaccine (31)
. However, the knowledge concerning the repertoire of tumor-specific T cells of defined antigen specificity remains limited. Initial studies on the repertoire of T cells specific for the immunodominant HLA-A2-restricted peptide from the tumor Ag Melan-A26 (27, 28, 29, 30, 31, 32, 33, 34, 35)
gave conflicting results, some reporting restricted (32)
and others diverse (21
, 33
, 34)
BV gene segment usage. By using A2/Melan-A peptide tetramers, we have observed recently that Melan-A26 (27, 28, 29, 30, 31, 32, 33, 34, 35)
specific T cells present in both tumor-infiltrated lymph nodes and tumor-infiltrating lymphocytes of melanoma patients exhibit a large and diverse TCR repertoire. However, dominant clonal populations were also found in individual patients (24
, 35)
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Because of the rare CTL response to the initially defined MAGE-derived antigenic peptides, the repertoire of T cells specific for MAGE Ag has remained, until the present time, largely unexplored with one exception (36)
. In that study the analysis of CTL clones from a single melanoma patient and specific for an HLA-A1-restricted epitope derived from MAGE-1 protein suggested that the TCR repertoire directed against this Ag was rather diverse. In the present study, we observed that MAGE-A10254–262-specific CD8+ T cells can use at least five different BV segments. Whereas tetramer+ cells frequently used BV 22, BV 22+ tetramer+ T cells could recognize the antigenic peptide with either high or low avidity. The same was observed in the case of BV 13+ tetramer+ T cells. Thus, no strict correlation between BV segment usage and avidity of Ag recognition could be observed. Interestingly, 2 BV 13+ clones (LAU 203/1B8 and HD 795/2B6) recognized the antigenic peptide with high and low avidity, respectively, despite a high degree of similarity in their CDR3 regions. Further studies should unravel an eventual correlation with other structural features of the TCR, such as chain usage and preferential ß TCR chain pairing. In addition, analysis of a larger sample of responding HLA-A*0201 individuals is needed to substantiate the preferential BV 22 usage by MAGE-A10254–262-specific T cells. If so, the propensity of MAGE-A10254–262-specific T cells to preferentially use a limited number of BV segments could be exploited for increasing the rate of MAGE-A10254–262-specific T-cell detection based on the use of BV-specific, anti-TCR Abs to select and/or preferentially expand these T cells.
It is noteworthy that MAGE-A10254–262-specific T cells could be detected and isolated among peptide-stimulated PBMCs from a HLA-A*0206-expressing melanoma patient with HLA-A*0201/peptide tetramers. Interestingly, for the same patient, CD8+ T-cell responses to at least three additional CTL epitopes [from influenza matrix58–66, tyrosinase368–376, Melan-A26 (27, 28, 29, 30, 31, 32, 33, 34, 35)
, not shown] could also be visualized with HLA-A*0201/peptide tetramers. HLA-A*0201 and HLA-A*0206 differ at a single position (amino acid 9) of the -1 domain of the molecule, where Phe is present in HLA-A*0201 and Tyr in HLA-A*0201. It is likely that this difference results in only moderate conformational effects of the corresponding HLA/peptide complexes, thus allowing wide cross-recognition by specific T cells. Nevertheless, MAGE-A10254–262-specific T cells from patient LAU 42 were able to recognize the peptide MAGE-A10254–262 10-fold more efficiently when presented by HLA-A*0206 than by HLA-A*0201 (data not shown). The overlapping of CD8+ T cell Ag-specific repertoires restricted to different A2 subtypes is difficult to predict and would be most likely inversely related to the degree of structural conservation among different subtypes. To what extent HLA-A*0201/peptide tetramers can be used to analyze peptide-specific CD8+ T-cell responses in individuals expressing other HLA-A2 subtypes is presently unknown. Remarkably, >60% of tetramer+ cells in the polyclonal culture of patient LAU 42 but none in the culture from the seven HLA-A*0201-expressing individuals used BV1. Analysis of peptide-stimulated PBMCs from additional HLA-A*0206-expressing individuals would be required to determine whether the usage of BV 1 by tetramer+ cells of LAU 42 is indeed related to this particular HLA-A2 subtype.
MAGE-A10254–262-specific T cells are functionally heterogeneous both in terms avidity of antigen recognition and of tumor-specific lytic activity. Only MAGE-A10254–262-specific CD8+ T cells able to recognize the Ag with high avidity were able to efficiently lyse MAGE-A10-expressing tumors. Similar findings have been reported in the case of CTLs specific for other tumor Ag (13) . High avidity of recognition of the tumor target has been shown to be important for antitumor efficacy in vivo (14) and could be critical in the case of suboptimal Ag expression (37) . One approach for cancer therapy consists in the isolation of tumor-specific T cells from cultures stimulated with peptides corresponding to immunogenic sequences for adoptive transfer into patients. Whereas the isolation and expansion of tumor Ag-specific CTLs can be highly accelerated by tetramer guided cell sorting followed by mitogen driven proliferation (23) , it is crucial to identify means of separating high- and low-avidity-specific CTLs from polyclonal populations. In this study, we found a good correlation between intensity of tetramer staining and avidity of T-cell recognition after prolonged incubation with tetramers (4 h) at room temperature. Under these experimental conditions, high and low tumor-reactive MAGE-A10254–262-specific T cells could be efficiently separated by FACS. Tetramer guided isolation of high and low tumor-reactive CTLs specific for other tumor Ags from polyclonal cultures has also been reported recently (13) . However, the molecular bases of this phenomenon are unclear. Indeed, the intensity of tetramer staining does not appear to be related to differences in the level of expression of the TCR, CD8, or accessory molecules, and it could be the consequence of differences in the association and/or dissociation kinetics of tetramers for individual TCRs. Whereas more rigorous studies would be required to verify these hypotheses, this phenomenon should be taken in consideration in the monitoring of both natural and induced tumor Ag-specific responses.
Additional strategies could be explored to select high avidity MAGE-A10254–262-specific T cells. It is noteworthy that in this study, PBMCs were stimulated in vitro with a relatively high dose of peptide (1 µM). In previous studies using splenocytes of mice immunized with recombinant viral Ag (38 , 39) , the dose of antigen appeared to be critical for the optimal elicitation of specific CD8+ T cells and was the function of both T cell avidity and activation state; indeed, if a high dose of Ag induced apoptosis of effector CTLs and optimally activate low avidity CTLs, stimulation with lower doses promoted the expansion of high-avidity CTLs. Whether such approach would be effective for the isolation of high avidity tumor-reactive CTLs from the PBMCs of patients with malignancies remains to be established.
We isolated both high- and low-avidity MAGE-A10254–262-specific T cells from PBMCs of healthy donors. In addition, both high- and low-avidity-specific T cells were also isolated from melanoma patients irrespective of MAGE-A10 expression on their melanoma lesions [this study and Valmori et al. (19) ]. Although these findings suggest the presence of an heterogeneous MAGE-A10254–262-specific T-cell repertoire in each individual, they do not exclude the presence, in patients bearing MAGE-A10-expressing lesions, of MAGE-A10254–262-specific T cells which would have been spontaneously primed in vivo by the tumor. Indeed, a high level of tumor reactivity could be expected by such cells. Unfortunately, the functional phenotype of MAGE-A10254–262 tetramer+ T-cell precursors in unstimulated PMBCs cannot be determined because of their low frequency (close or below tetramer detection limits, 1/10,000 CD8+ T cells; Ref. 19 ). Future studies on peptide-stimulated, purified CD45RAbright/CD45ROlow and CD45RAlow/CD45RObright populations (alone or in combination with other markers) will help assess a potential correlation between phenotype and efficiency of tumor recognition by MAGE-A10254–262-specific T cells in this patient population, which is eligible for MAGE-A10-based vaccination trials.
The functional heterogeneity of MAGE-A10254–262-specific T cells should also be taken in account when considering therapeutic approaches based on the use of this antigenic peptide for immunization of cancer patients. It can be anticipated that in vivo recruitment of high or low tumor-reactive MAGE-A10254–262-specific precursors may be dependent on the immunization protocol used. If immunization with MAGE-A10254–262 peptide in adjuvant could be expected to recruit only high-avidity T cells because of the low peptide dose, immunization strategies aimed at inducing a high antigen load, such as, e.g., immunization with serum protease-resistant peptides, could also result in the recruitment of low-avidity CTLs. On the other hand, the in vivo expansion potential of the two CTL subsets could also be different, because low avidity T cells could be only suboptimally activated by Ag stimulation and therefore be more dependent from help than high-avidity CTLs. Answering these questions may require the establishment of appropriate animal models as well as the accurate monitoring of MAGE-A10 vaccination trials in cancer patients.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported in part by Grant SKL 782-2-1999 from the Swiss Cancer League (to V. R-G.). 
2 To whom requests for reprints should be addressed, at Division of Clinical Onco-Immunology, Ludwig Institute, Hôpital Orthopédique, Avenue Pierre-Decker, 4, 1005 Lausanne, Switzerland. Phone: 41-21-314-01-76; Fax: 41-21-314-74-77; E-mail: Danila.Valmori{at}inst.hospvd.ch
3 The abbreviations used are: Ag, antigen; PBMC, peripheral blood mononuclear cell; TCR, T-cell receptor; mAb, monoclonal antibody; FACS, fluorescence-activated cell sorting.
Received 2/13/01. Accepted 5/31/01.
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J. Lin, L. Lin, D. G. Thomas, J. K. Greenson, T. J. Giordano, G. S. Robinson, R. A. Barve, F. A. Weishaar, J. M. G. Taylor, M. B. Orringer, et al. Melanoma-Associated Antigens in Esophageal Adenocarcinoma: Identification of Novel MAGE-A10 Splice Variants Clin. Cancer Res., September 1, 2004; 10(17): 5708 - 5716. [Abstract] [Full Text] [PDF]
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S. Yang and F. G. Haluska Treatment of Melanoma with 5-Fluorouracil or Dacarbazine In Vitro Sensitizes Cells to Antigen-Specific CTL Lysis through Perforin/Granzyme- and Fas-Mediated Pathways J. Immunol., April 1, 2004; 172(7): 4599 - 4608. [Abstract] [Full Text] [PDF]
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E. M.-L. Choi, J.-L. Chen, L. Wooldridge, M. Salio, A. Lissina, N. Lissin, I. F. Hermans, J. D. Silk, F. Mirza, M. J. Palmowski, et al. High Avidity Antigen-Specific CTL Identified by CD8-Independent Tetramer Staining J. Immunol., November 15, 2003; 171(10): 5116 - 5123. [Abstract] [Full Text] [PDF]
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V. Dutoit, P. Guillaume, M. Ayyoub, C. S. Hesdorffer, I. F. Luescher, and D. Valmori Decreased Binding of Peptides-MHC Class I (pMHC) Multimeric Complexes to CD8 Affects Their Binding Avidity for the TCR But Does Not Significantly Impact on pMHC/TCR Dissociation Rate J. Immunol., May 15, 2003; 170(10): 5110 - 5117. [Abstract] [Full Text] [PDF]
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M. P. Rubinstein, A. N. Kadima, M. L. Salem, C. L. Nguyen, W. E. Gillanders, M. I. Nishimura, and D. J. Cole Transfer of TCR Genes into Mature T Cells Is Accompanied by the Maintenance of Parental T Cell Avidity J. Immunol., February 1, 2003; 170(3): 1209 - 1217. [Abstract] [Full Text] [PDF]
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D. Schrama, E. Fuchs, E.-B. Brocker, P. thor Straten, and J. C. Becker Identical T-cell Receptor Transcripts in Multiple Melanoma Metastases Cancer Res., October 15, 2002; 62(20): 5664 - 5667. [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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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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) or in the presence (
, •) of exogenously added peptide MAGE-A10254–262 (1 µM). B, for each population, the concentration of peptide MAGE-A10254–262 required to obtain 50% maximal was plotted against the % specific lysis of MAGE-A10-expressing tumor cell line Me 275.