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Immunology |
Torrey Pines Institute for Molecular Studies and Mixture Sciences, Inc., San Diego, California 92121 [C. P., R. A. H.]; Division of Clinical Onco-Immunology, Ludwig Institute for Cancer Research, University Hospital, 1011 Lausanne, Switzerland [V. R-G., V. D., J-C. C., P. R., D. V.]; Multidisciplinary Oncology Center, University Hospital, 1005 Lausanne, Switzerland [P. G.]; and Molecular Statistics and Bioinformatic Section, Biometric Research Branch, National Cancer Institute, NIH, Bethesda, Maryland 20892 [R. S., Y. Z.]
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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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Several approaches have been designed to this end. One approach consists in narrowing down the gene segment encoding the antigenic peptide by using target cells transfected with gene fragments. The deduced amino acid sequence of this segment is used to produce synthetic peptides that are then tested in a target-sensitization assay for recognition by tumor-reactive T-cell clones. An alternative approach is based on the acid elution and high-performance liquid chromatography fractionation of peptides present at the surface of in vitro cultured tumor cell lines (11) , which are then tested for recognition by tumor-reactive T-cell clones. By using this approach, the naturally presented peptide tyrosinase368–376 was shown to differ from the tyrosinase gene-deduced sequence (12) as a result of a posttranslational modification (13) . However, this approach is technically challenging, and its success relies on the relative abundance of few tumor-derived peptides at the surface of the few tumor cell lines that are able to grow in vitro to the high numbers required for peptide acid elution. Finally, a third strategy is based on the synthesis of putative antigenic peptides, corresponding to linear amino acid sequences of a known tumor antigen-associated protein and selected on the basis of the ability to bind to a particular MHC-class I molecule. The synthetic peptides are used to stimulate CD8+ T cells from either cancer patients or normal individuals. If peptide-specific CTL clones are obtained, they are used to assess whether the antigenic peptide is presented at the surface of tumor cells. However, a major drawback of this approach is that CTLs obtained in this way often fail to recognize tumor cells endogenously expressing the protein because the selected peptide is not generated efficiently by the processing machinery of the cell (14) .
Overall, these procedures are time consuming, labor intensive and not free from pitfalls. A novel approach for the direct identification of tumor-associated peptide epitopes is based on the use of combinatorial peptide libraries in positional scanning format. Typically, such libraries are composed of trillions of peptides systematically arranged in mixtures with defined amino acids at each position. The PS-SCL5 approach has been successfully applied in both the identification of biologically active peptides (15, 16, 17) and the study of B- and T-cell specificity (18) .
As a first step toward the use of the PS-SCL approach for the identification of CTL-defined tumor antigens, we analyzed in detail the recognition of PS-SCLs by tumor-reactive CD8+ T-cell clones that are specific for the cell-lineage-differentiation antigen Melan-A and that differ in terms of avidity and fine specificity. Our results show that, at least for some clones, most of the amino acids present in the native sequence could be identified among the most active peptide mixtures. Interestingly, through this approach, we could identify Melan-A peptide analogues with increased activity as well as agonist peptides containing multiple amino acid substitutions. In addition, biometrical analysis of the data generated by the PS-SCL screening allowed the identification of the native ligand in public databases. These data demonstrate that PS-SCLs can be used for the identification and optimization of tumor-associated CTL epitopes.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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T-Cell Clones and Lines.
Melan-A-specific CTL clones were derived from tumor-infiltrated lymph node cells of patient LAU 203 by limiting dilution cultures in the presence of irradiated allogeneic PBMCs, phytohemagglutinin, and hrIL-2 as described previously (20)
. They were subsequently expanded by periodical (3–4-week) restimulation into microtiter plates together with irradiated feeder cells in the presence of PHA and hrIL2. For peptide stimulation of PBMC, CD8+ lymphocytes were positively selected by magnetic cell sorting from PBMCs of HLA-A*0201 melanoma patients using a miniMACS device (Miltenyi Biotec GmBH, Sunnyvale, CA). Cells of the CD8- fraction were irradiated (3,000 rads) and used as autologous APCs. CD8+ highly enriched lymphocytes (1 x 106/condition) were stimulated with peptide (1 mM) and irradiated autologous APCs in 2 ml of medium containing hrIL-2 (100 units/ml; Glaxo, Geneva, Switzerland; kindly provided by Dr. M. Nabholz, ISREC, Epalinges, Switzerland) and hrIL-7 (10 ng/ml; R&D Systems, Europe, Oxon, United Kingdom). Cells underwent two additional cycles of weekly restimulation with peptide-pulsed APCs prior to A2/Melan-A tetramer analysis. Where indicated, CD8+ tetramer+ and CD8+ tetramer- cells were isolated by fluorescence activated cell sorting.
Antigen Recognition Assay.
Antigen recognition was assessed using a chromium-release assay as described previously (20)
. Briefly, target cells were labeled with 51Cr for 1 h at 37°C and washed twice. Labeled target cells (1000 cells/well) were incubated in the presence of libraries (100 µg/ml final), or, in peptide titration experiment, with various concentrations of peptide, for 15 min at room temperature before the addition of effector cells at a L:T ratio of 10:1. Chromium release was measured in supernatants harvested after 4-h incubation at 37°C. The percentage specific lysis was calculated as:
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Scoring Matrix and Database Searches.
A scoring matrix for each individual experiment was generated by transforming the percentage specific lysis of each mixture defined with 1 of the 20 L-amino acids in each of the 10 positions of the decamer libraries into numerical values that account for the SDs of replicates.6
The final scoring matrix for database search was derived by taking the average of the score matrices of multiple experimental data. On the basis of the assumption of independent and additive contribution of each position of a peptide, the score for an individual peptide was then calculated by adding the individual stimulatory values of the 10 amino acids in a decamer. A program was designed to use the matrix to score all of the overlapping 10-mers contained in the GenPept database and thus identify sequences with the predicted highest stimulatory values.
HLA-A*0201 Binding Assay.
The peptide-binding capacity to HLA-A*0201 was assessed in a functional competition assay based on the inhibition of recognition of the antigenic peptide tyrosinase368–376 (YMDGTMSQV) (12)
by HLA-A*0201-restricted CTL clone LAU 156 34 as described previously (20)
. Briefly, T2 cells were 51Cr labeled in the presence of anti-class I mAb W6/32. Various concentrations of competitor peptides (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 CTLs (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 and is indicated as [nM] 50%. To facilitate comparison, the relative competitor activity of each peptide was calculated as the IC50 of the native Melan-A decapeptide divided by the IC50 of the competitor peptide.
Tetramers, mAbs, and Flow Cytometry Immunofluorescence Analysis.
Tetramers were prepared as described previously (21)
. As the antigenic peptide, the Melan-A26–35 A27L analogue (ELAGIGILTV) was used; it has a higher binding affinity and stability than the native Melan-A decapeptide (EAAGIGILTV) or the nonapeptide (AAGIGILTV; Ref. 22
). Interchangeability of native Melan-A decapeptide and the A27L analogue in terms of staining specificity has been assessed previously (21)
. Peptide-stimulated CD8+-highly enriched T cells were stained with PE-labeled tetramers (10 µg/ml in 20 µl of mixture) during 1 h at room temperature; then 20 µl of anti-CD8FITC mAb (1:25 dilution; Becton Dickinson, San Jose, CA) were added and incubated for an additional 30 min at 4°C. Cells were washed once in the same buffer and analyzed by flow cytometry. Data analysis was performed using Cell Quest software.
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RESULTS |
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, the clones efficiently lysed the Melan-A-expressing autologous melanoma line Me 290, whereas they failed to lyse the Melan-A-expressing but HLA-A2- melanoma line Me 242. However, they largely differed in the recognition of antigenic synthetic peptides Melan-A26–35 (EAAGIGILTV) and Melan-A27–35 (AAGIGILTV) in terms of both avidity and fine specificity (Fig. 1B)
. Clone LAU 203 1.3 recognized peptide Melan-A27–35 better than it did Melan-A26–35; clone LAU 203 1.5 recognized peptide Melan-A26–35 better than it did Melan-A27–35; and clone LAU 203 17 recognized both peptides with similar efficiency. Fig. 1B
also shows that antigenic peptides containing an amidated carboxyl terminus were less efficiently recognized than peptides containing a free COOH terminus. This result was explained by a decreased HLA-A2 binding capacity of amidated nona- and deca- Melan-A peptide as compared with the corresponding carboxyl-free peptides as determined by functional competition assay (Ref. 20
; and data not shown). However, the difference in COOH termini did not modify the hierarchy of peptide recognition. The selected clones were used to test the recognition of a nonapeptide PS-SCL and a decapeptide PS-SCL by standard chromium-release assay.
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. Only very low levels of specific lysis were obtained with clone LAU 203 1.3 with either the nona- or the decapeptide PS-SCL (less than 10%; not shown). Higher levels of specific lysis were obtained with clones LAU 203 1.5 and LAU 203 17, mainly with the decapeptide PS-SCL. Fig. 2
shows, as an example, the results obtained for clone LAU 203 1.5 and the decapeptide PS-SCL. At position 1, the highest activity was detected for the mixture having Arg (R) at this position. The mixtures containing at position 2 those amino acids known to function as strong anchors for binding to HLA-A*0201, i.e., Val (V), Ile (I), Leu (L), and Met (M), were among the most active in the lytic assay. In agreement with these data, we have previously shown that substitution of A27 of Melan-A26–35 with Leu results in improved recognition by Melan-A-specific CD8+ T cells (23)
. At position 3, the two most active mixtures were defined with Thr (T) and Ala (A), the latter being the residue present at this position in the native decapeptide sequence. Similarly, at positions 4–7, the most active mixtures were defined with amino acids present at the corresponding positions in the native sequence. At position 8, several mixtures were active including, for example, those defined with His (H) and Phe (F) and, to a lesser extent, the mixture defined with Leu, which is the amino acid present at this position in Melan-A26–35. The mixture defined with Pro (P) at position 9 was well recognized followed by Thr, which is the residue present in the native peptide. Finally, at position 10, several mixtures including the ones defined with the native Val were identified. Thus, remarkably, for 8 of the 10 positions, mixtures containing amino acids corresponding to the native amino acid present at the selected position were among the most antigenic.
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, mixtures containing the amino acid corresponding to the native one for 7 of the 10 positions were among the most active. Interestingly, the mixture defined with Ala was identified among the most active at position 1 in line with our previous study that demonstrated that substitution of E26 with Ala resulted in improved recognition by most Melan-A-specific CD8+ T-cell clones (20)
. We have also observed that this substitution synergizes with the substitution of A27 with Ala, generating a potent analogue (ALAGIGILTV; Ref. 24
). Again, anchor amino acids for binding to HLA-A*0201 were identified at position 2. At position 3, mixtures containing Thr and Trp in addition to the native Ala were the most active. With the exception of position 7, at which a modest level of activity was detected for mixtures defined with Trp or Phe, either a Trp or the native amino acid were identified at the remaining positions. A relatively low level of specific lysis was obtained for clone LAU 203 1.5 with the nonapeptide PS-SCL in agreement with the preference of this clone for the native decapeptide. In contrast, clone LAU 203 17, which recognized native Melan-A nona- and decapeptides with similar efficiency, also efficiently recognized the nonapeptide PS-SCL. However, in this case, the most active mixtures did not correspond to the ones defined with the native amino acids. All together, these results illustrate that, although no conclusive data could be obtained with the nonapeptide PS-SCL, for two of three Melan-A clones analyzed, most of the amino acids composing the native antigenic peptide could be identified with the decapeptide PS-SCL.
A biometrical data analysis (Ref. 25
and Zhao et al.6
) that allows the comparison of the results of the PS-SCL screening with protein databases was used to assess whether the information derived from the decapeptide PS-SCL scan would have been adequate for the identification of the native ligand in case it had been unknown. The percentage specific killing values obtained for each mixture of the decapeptide PS-SCL were used to generate a score matrix. The score matrix was then used to calculate a predicted stimulatory score for each decapeptide present in proteins of a public database. Retrieved peptides were ranked according to their predicted stimulatory score. Table 2
shows the ranking for Melan-A26–35 obtained with the data generated with clones LAU 203 1.5 and LAU 203 17. The results show that the known native ligand ranked 4th and 22nd for clones LAU 203 17 and LAU 203 1.5, respectively, from a total of 
13 million decapeptides.
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and 4
for clones LAU 203 1.5 and 17, respectively.
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. In contrast, peptides that contained single or multiple substitutions but not Val at position 2 were poorly recognized. These results can be explained, at least partially, by the improved binding to HLA-A*0201 consequent to the introduction of an anchor residue at position 2. Indeed, peptide 65 (containing Val as a single substitution at position 2) was significantly better recognized than the native decapeptide (66-fold) and showed an increased binding (3-fold; Fig. 5A
) to HLA-A*0201.
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. The peptides exhibited an enhanced binding to HLA-A*0201 (Fig. 5A)
that could, at least partially, explain their improved recognition. In agreement with this interpretation is the observation that enhanced recognition was not clone specific, because peptides 65, 81, and 89 were recognized much more efficiently than was the native decapeptide and were recognized also by polyclonal Melan-A-specific cultures derived from two additional melanoma patients (Fig. 5B)
. Peptides containing Trp at positions 3–8, either as a single or a multiple substitution, were, in general, poorly recognized by clone LAU 203 17 with few exceptions (peptides 96 and 100). Indeed and remarkably, peptide 96 (ALTGWWWLTV), which contains six substituted positions, was recognized at least as efficiently as the native peptide was by clone LAU 203 17, as well as by polyclonal cultures (Fig. 5B)
. Efficient recognition of peptide 96 by Melan-A-specific T cells could not be solely explained by its improved binding to HLA-A*0201 as compared with the native decapeptide sequence. Indeed, peptide 93, which also exhibited highly improved binding, was not recognized. In addition, the C-terminal carboxyl analogue of peptide 96 was not recognized by Melan-A-specific CTLs, although it was able to bind to HLA-A*0201 with higher affinity than did its C-terminal amide form (data not shown).
As shown in Fig. 6A
, peptide 96 was immunogenic and able to stimulate the expansion of Melan-A specific T-cell precursors. Remarkably, in vitro stimulation of CD8+ T cells from patient LAU 203 with this peptide resulted in a significant expansion of Melan-A-specific T cells, as assessed by staining with A2/Melan-A tetramers (21)
, more efficiently than stimulation with the native decapeptide, although less efficiently as compared with an enhanced analogue (peptide 81). To assess the extent of cross-reactivity between CTLs elicited by stimulation with the peptide analogue and the native decapeptide, CD8+ tetramer+ and CD8+ tetramer- fractions were isolated by tetramer-guided cell sorting, and their lytic activity was assayed on T2 or tumor cell targets (Fig. 6B)
. Comparable levels of specific lysis on T2 cells were detected for the CD8+ tetramer+ cell fraction in the presence of peptides 63 and 96. The two peptides were recognized with comparable efficiency by CD8+ tetramer+ T cells as determined by peptide titration assay (Fig. 6C)
For the CD8+ tetramer- cell fraction, however, a lower but significant level of specific lysis was detected on T2 cells in the presence of peptide 96 but not in the presence of peptide 63, thus suggesting the presence, within this cell fraction, of a subpopulation non-cross-reactive with the native peptide (Fig. 6B)
. In addition, CD8+ tetramer+ but not CD8+ tetramer- cells were able to efficiently lyse Melan-A-expressing tumor cells (Fig. 6B)
. All together, these results show that peptides with little sequence homology but that are fully cross-reactive with the native peptide can be identified through the screening and deconvolution of PS-SCLs.
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DISCUSSION |
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It is not immediately clear why the native sequence was not identified with the nonapeptide PS-SCLs. This is particularly intriguing in the case of clone LAU 203 17, which recognizes nona- and decapeptides with comparable avidity. However, a similar analysis of the nonapeptide PS-SCL [specific for peptide tyrosinase368–376 (12) ] with a CTL clone resulted in the identification of most of the amino acids present at the corresponding position in the native tyrosinase peptide sequence among the most active peptide mixtures (data not shown) which argues against a general lack of recognition of the nonapeptide PS-SCL by CTL clones. Thus, in the case of CTL clones of unknown specificity, it would be advantageous to test PS-SCLs of different lengths to maximize the chances of obtaining positive results. Also, the reasons why some tumor-reactive clones (e.g., clone LAU 203 1.3) fail to efficiently recognize any of the analyzed peptide libraries are not clear and remain to be further investigated.
The peptide libraries used in the present study are made of synthetic peptides having a C-terminal amide. Although the C-terminal amide Melan-A26 (27)-35 peptides bind HLA-A*0201 with a lower affinity than do their C-terminal carboxyl forms and are recognized less efficiently by Melan-A-specific CTLs, the overall effect of C-termini amidation on the recognition of PS-SCLs is difficult to evaluate. Indeed, it has been recently shown that C-terminal amidation of Melan-A26–35 protects the peptide from enzymatic degradation and significantly increases the peptide half-life in serum (26) . Protection from enzymatic degradation could be critical for the recognition of single peptides present at subfemtomolar concentration in the PS-SCLs. Future experiments will directly address these issues by comparing the recognition of amidated and carboxyl-free PS-SCLs.
In the case of clones LAU 203 17 and 1.5, several amino acids that are different from the amino acid present at the corresponding position of the native sequence were also identified. In some cases, the replacement of native amino acids with these amino acids, either as single (peptide 65) or multiple substitutions (peptides 81 and 89), resulted in an improved recognition by CTLs as well as in highly improved immunogenicity. This effect was not clone specific and could be at least partially explained by an improved binding to HLA-A2. In several other cases, substitution of native amino acids with defined amino acids present in the active mixtures as single, and often as multiple substitutions, resulted in peptides of reduced antigenicity. Sometimes, however, as illustrated by our findings with peptide 96, multiple substitution of native amino acids with defined amino acids in the active mixtures may allow the identification of fully cross-reactive analogues without obvious sequence homology to the native sequence. The identification of these analogues depends strictly on the PS-SCL used, as suggested by the observation that the carboxyl-free analogue of peptide 96 was not recognized by Melan-A-specific CTLs, although it was able to bind to HLA-A*0201 with higher affinity than was its amidated form. It is of note that, taking into account that PS-SCLs were tested at 100 µg/ml final, the concentration of each individual peptide is of 5.5 x 10-15 M and 4.4 x 10-16 M in each mixture, for the nonapeptide and decapeptide PS-SCL, respectively. Because the recognition signal in our chromium-release assay is completely extinguished at peptide concentrations lower than 10-12 M, it is clear that multiple peptides in the mixtures are simultaneously recognized by the T-cell clone. Overall, these data are in agreement with previous observations on TCR recognition. Indeed, although TCR recognition was initially believed to be highly specific, recent studies have shown that it is, at the same time, highly degenerate (27) . Many different MHC-peptide complexes can bind to a single TCR with different affinities, which results in a variety of functions going from full T-cell activation to antagonism. Besides high-affinity ligands being able to fully activate mature T cells under physiological conditions, MHC-peptide complexes that bind to the TCR with low affinities play an important role during thymic selection (28) and in the survival of mature T cells in the periphery (29) .
These phenomena are particularly relevant when analyzing autoreactive T-cell clones. However, because of their complexity, the molecular basis as well as the extent of the degeneracy of T-cell recognition of antigen-specific clonal T-cell populations is difficult to evaluate. Degeneracy of antigen recognition by Melan-A-specific T cells has been previously addressed (30) by searching a protein database using the program findpatterns with an input pattern generated based on the recognition data of a total of 46 analogues that are singly substituted at positions P1-P9 of Melan-A27–35. Among the candidate mimicry peptides retrieved in that study, peptide gC448–488 from a herpes virus was cross-recognized by a Melan-A-specific CTL. However, this peptide was not recognized by several other monoclonal and polyclonal Melan-A-specific T-cell populations derived in our laboratory, including the ones used in the present study (data not shown). An alternative and more comprehensive method for the identification of epitope mimics from either human autoantigens or pathogens consists in screening protein databases with scoring matrices generated from PS-SCL screening data by transforming the stimulatory potency of each of the 20 amino acids in each position of the libraries into numerical values as described recently (Ref. 25 and Zhao et al.6 ). By applying this method, we were able to identify the native Melan-A peptide sequence among decapeptides present in proteins of public databases. Recognition of additional sequences identified with this analysis is currently being assessed in our laboratory. In conclusion, in the present study we have focused on the analysis of the T-cell recognition of PS-SCLs by using a 51Cr release assay that measures the lytic activity of in vitro cultured CD8+ T-cell populations. This provides a valid strategy to rapidly obtain information about sequences recognized by tumor-reactive CTL clones of clinical relevance. At the same time, it will be instrumental to analyze the extent of degeneracy in TCR recognition of tumors antigens. In addition, by simultaneously measuring other cell functions such as proliferation and cytokine secretion, more complete information could be obtained. These issues will be the focus of future studies.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 V. R-G. was supported by the Swiss Cancer League Grant SKL 782-2-1999. 
2 C. P. and V. R-G. contributed equally to this work.
3 To whom correspondence about the use of combinatorial peptide libraries should be addressed, at Torrey Pines Institute for Molecular Studies and Mixture Sciences, Inc., 3550 General Atomics Court, San Diego, CA, 92121. E-mail: cpinilla{at}tpims.org
4 To whom requests for reprints should be addressed, at Division of Clinical Onco-Immunology, 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
5 The abbreviations used are: PS-SCL, positional scanning synthetic combinatorial peptide library; PBMC, peripheral blood-derived mononuclear cell; hrIL, human recombinant interleukin; APC, antigen-presenting cell; mAb, monoclonal antibody; PE, phycoerythrin; TCR, T-cell receptor for antigen; L:T, lymphocyte:target cell (ratio).
6 Y. Zhao, B. Gran, C. Pinilla, S. Markovic-Plese, B. Hemmer, A. Tzou, L. Whithney, W. E. Biddison, R. Martine, and R Simon. Combinatorial peptide libraries and biometric score matrices permit the quantitative analysis of specific and degenerate interactions between clonotypic T-cell receptors and MHC-peptide ligands, in press.
Received 1/26/01. Accepted 4/26/01.
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, amino acid corresponding to Melan-A26–35 native peptide sequence.
