Enhanced Generation of Specific Tumor-Reactive CTL In Vitro by Selected Melan-A/MART-1 Immunodominant Peptide Analogues

Cell lines and clones

Tumor cell lines and EBV-transformed B cell lines were maintained in DMEM (Life Technologies, Gaithersburg, MD) supplemented with 10% FCS, 0.55 mM Arg, 0.24 mM Asn, and 1.5 mM Gln. Melanoma cell lines Me 256, Me 260, Me 275, and Me 290 were established at the Ludwig Institute for Cancer Research, Lausanne Branch, from surgically excised melanoma metastases from patients LAU84, LAU149, LAU50, and LAU203, respectively. CTL line 198NS, which is specific for peptide MAGE-3_271-279 presented by HLA-A2, was derived as previously described (20). Melan-A-specific CTL clones M77.80 (Vβ3) and M77.86 (Vβ14) were derived from TIL of melanoma patient M77 as described (21). Briefly, CTL clones were obtained from limiting dilution cultures in the presence of irradiated autologous tumor cells, EBV-transformed B lymphocytes, PHA, and rIL-2. Clones were derived from wells having a probability of clonality higher than 90% according to single hit Poisson distribution. They were subsequently expanded by plating 5 × 10³ cells, every 3 to 4 weeks, into microtiter plates together with irradiated feeder cells (5 × 10⁴ allogenic PBMC and 2 × 10⁴ EBV-transformed-B-LAZ cells). CTL clones 1.13 (Vβ14), 7.10 (Vβ17), and Mel 1.33 (Vβ9) were derived from PBMC of normal HLA-A2 donors (N. Gervois, S. Le Guiner, N. Labarriere, J.-F. Fonteneau, A. S. Beignon, E. Diez, and F. Jotereau, manuscript in preparation). All the clones used in this study efficiently recognized HLA-A*0201-positive Melan-A-expressing tumor cell lines.

Synthetic peptides

Peptides were synthesized by standard solid phase chemistry on a multiple peptide synthesizer (Applied Biosystems, Foster City, CA) by using standard F-moc for transient NH₂-terminal protection, and were analyzed by mass spectrometry. All peptides were >90% pure as indicated by analytical HPLC. Lyophilized peptides were diluted in DMSO and stored at −20°C.

HLA-A*0201 binding assay

The peptide binding capacity to HLA-A*0201 was assessed in a functional competition assay based on inhibition of recognition of the antigenic peptide MAGE-3_271-279 by the HLA-A*0201-restricted CTL line 198NS (20). Briefly, 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 MAGE-3_271-279 (50 μl) was then added together with specific CTL (5000 cells/well) (50 μl). Chromium release was measured after a 4-h incubation at 37°C. The concentration of each competitor peptide required to achieve 50% inhibition of target cell lysis was then determined and indicated as [nM] 50%. To facilitate comparison, the relative competitor activity of each peptide was calculated as the [nM] 50% of the unmodified Melan-A nonapeptide AAGIGILTV divided by the [nM] 50% of the competitor peptide.

Assessment of the stability of Melan-A-derived peptides/HLA-A*0201 complexes

The stability of peptide/HLA-A*0201 complexes was assayed using the TAP-deficient T2 cells. These cells were loaded with peptide by overnight incubation at room temperature with saturating concentrations (10 μM) of the different peptides and human β2 microglobulin (3 μg/ml) (Sigma, Buchs, Switzerland) in serum-free medium (X-VIVO 10; BioWhittaker, Walkersville, MD). After peptide removal and addition of emetine (10⁻⁴ M; Sigma) to block protein synthesis, cells were incubated at 37°C for the indicated time periods. For each time point, an aliquot of cells was stained with mAb BB7.2 (HLA-A2 specific) to measure HLA-A2 Ag expression. Influenza matrix_58-66 peptide, which has been shown to bind to HLA-A*0201 with high affinity and form stable peptide/HLA-A*0201 complexes (22), was used as an internal standard. Results are expressed as: Where background mean fluorescence represents the fluorescence value obtained on a sample of T2 cells treated in similar conditions except for the absence of exogenous peptide (23).

Cytokines

Human rIL-2 (Glaxo, Geneva, Switzerland) was kindly provided by Dr. M. Nabholz (ISREC, Epalinges, Switzerland) and human rIL-7 was donated by Dr. N. Vita of Sanofi Recherche (Labège, France). One unit of IL-2 is defined as the concentration that gives 50% maximal proliferation of CTLL-2.

Generation of TILN and Melan-A-specific CTL

TILN were generated from tumor-infiltrated lymph nodes of HLA-A*0201⁺ melanoma patients obtained by surgery at the Centre Pluridisciplinaire d’Oncologie, CHUV, Lausanne, Switzerland. Tumoral lymph node fragments were minced to single cell suspensions and cultured in 24-well tissue culture plates (Costar corporation, Cambridge, MA) in 2 ml of Iscove’s Dulbecco medium supplemented with Asn, Arg, and Gln and 10% pooled human A⁺ serum in the presence of IL-2 and IL-7 (100 U/ml and 10 ng/ml, respectively). After 2 to 3 wk of cell culture, the TILNs were tested for cytolytic activity and their cell surface phenotype was determined by flow cytometry. The TILN chosen for this study were >90% CD3⁺CD8⁺ cells that had cytolytic activity against autologous or HLA-A*0201⁺ tumor cell lines and recognized the Melan-A_(26)27-32 peptides presented by HLA-A*0201. Peptide-specific CTL were generated as previously described (20) with minor modifications. Briefly, PBMC from HLA-A*0201⁺ melanoma patients were isolated by centrifugation in Ficoll-Paque (Pharmacia, Uppsala, Sweden). After enrichment for CD3⁺ lymphocytes by treatment with Lympho-Kwik T (One Lambda, Canoga Park, CA), CD8⁺ T lymphocytes were isolated using a miniMACS device (Miltenyi Biotec GmBH, Sunnyvale, CA). The resulting populations routinely contained >75% CD8⁺ T cells and were used as responder cell populations. Purified CD8⁺ T cells were plated at 1 to 2 × 10⁶ cells/well together with 2 × 10⁶ stimulator cells/well in 24-well plates in a total volume of 2 ml of Iscove’s medium supplemented with 10% human serum, Asn, Arg, and Gln (complete medium) in the presence of IL-7 (10 ng/ml) and IL-2 (10 U/ml). Stimulator cells were prepared as follows: 2 × 10⁶ autologous PBMC were incubated for 2 h at 37°C in serum-free medium (X-VIVO 10; BioWhittaker) with a Melan-A-derived peptide (20 μg/ml) and human β2 microglobulin (3 μg/ml). Peptide-pulsed PBMC were then washed, irradiated (3000 rad), and adjusted to the appropriate volume before addition to the CD8⁺-enriched responder cell populations. On day 7, cells were restimulated with peptide-pulsed autologous PBMC in complete medium supplemented with IL-2 (10 U/ml). Subsequent restimulations were performed weekly with peptide-pulsed and irradiated autologous PBMC. CTL activity was first tested at the end of the second in vitro restimulation.

Assessment of Ag recognition by TILN, Melan-A-specific CTL clones, and peptide-induced CTL

Ag recognition was assessed using chromium-release assays. Target cells were labeled with ⁵¹Cr for 1 h at 37°C and washed two times. Labeled target cells (1,000 cells in 50 μl) were then added to varying numbers of effector cells (50 μl) in V-bottom microwells in the presence or absence of 1 μg/ml of the antigenic peptide (50 μl). In the peptide titration experiments, target cells (1,000 cells in 50 μl) were incubated in the presence of various concentrations of peptide (50 μl) for 15 min at room temperature before the addition of effector cells (50 μl). In each case the effector cells were preincubated for at least 20 min at 37°C with unlabeled K562 cells (50,000/well) to eliminate nonspecific lysis due to NK-like effectors present in stimulated T cell populations. Chromium release was measured in supernatant (100 μl) harvested after 4 h of incubation at 37°C. The percent specific lysis was calculated as:

Analysis of mRNA expression

The analysis of mRNA expression was performed as described (24). Briefly, total cellular RNA was extracted by the guanidine-isothiocyanate/cesium chloride procedure. cDNA synthesis from 2 μg of RNA was accomplished by priming with oligo(dT) and aliquots corresponding to 100 ng of RNA were amplified by 30 cycles of PCR using oligonucleotide primers specific for the gene Melan-A (24). A 10-μl aliquot from each reaction was run on a 2% agarose gel and visualized by ethidium bromide fluorescence. To verify RNA integrity, a 21-cycle PCR assay with primers specific for β-actin was conducted in each case.

Identification of the Melan-A-derived peptide(s) recognized by TILN from HLA-A*0201 melanoma patients

Several overlapping antigenic peptide sequences have been mapped in the Melan-A protein region containing the immunodominant HLA-A2-restricted CTL epitope (11). To determine which of these peptides were recognized by TILN populations derived from two HLA-A*0201 melanoma patients, we tested the peptides listed in Table I⇓. In an attempt to minimize Ag-specific selection in vitro, we used TILN that were cultured for a short time period in the presence of IL-2 and IL-7 alone. Two melanoma lines (Me 290 and Me 260) were used as a source of Melan-A-expressing target cells. As shown in Figure 1⇓, both TILN populations were able to lyse the HLA-A*0201-positive melanoma line Me 290 equally well in the absence or presence of exogenously added peptide Melan-A_26-35. In contrast, the HLA-A*0201-negative melanoma line Me 260 was not recognized by the two TILN populations, either in the absence or in the presence of exogenously added peptide. T2 target cells, which express HLA-A*0201 but not Melan-A, were lysed only in the presence of exogenously added peptide. These results show that the two TILN populations used in this study recognized Melan-A-derived epitopes expressed in HLA-A*0201-positive melanoma cells.

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FIGURE 1.

Ag specificity and HLA-A2 restriction of TILN LAU 132 and LAU203. Lysis of Me 290 (□, ▪), Me260 (▵, ▴), and T2 (○, •) by TILN from HLA-A*0201 melanoma patients was measured in a 4-h ⁵¹Cr release assay in the absence (open symbols) or presence (closed symbols) of exogenously added peptide Melan-A_26-35 (M10, 1 μM).

Peptide Melan-A_26-35 (EAAGIGILTV) was recognized at least one order of magnitude more efficiently than peptide Melan-A_27-35 (AAGIGILTV) by both TILN (Table I⇑). In contrast, peptide Melan-A_27-36 (AAGIGITLVI) was poorly recognized while the partially overlapping peptide Melan-A_32-40 (ILTVILGVL) was not recognized at all. Similar results have been reported with TILN populations from three other HLA-A*0201 melanoma patients (16). It thus appears that peptide Melan-A_26-35 is recognized most efficiently by TILN populations from HLA-A*0201 melanoma patients compared with other overlapping peptides. Based on these results, we selected peptides Melan-A_26-35 and Melan-A_27-35 for further studies.

Binding of Melan-A peptide analogues to HLA-A*0201

In an attempt to identify Melan-A peptides with enhanced binding activity to HLA-A*0201, we synthesized the peptide analogues listed in Table II⇓. Peptide analogues Melan-A_27-35 A28L and A28 M contain single amino acid substitutions corresponding to the HLA-A*0201 dominant anchor residues at position 2 of the nonapeptide. The same substitutions were introduced at position 2 of the decapeptide (peptide analogues Melan-A_26-35 A27L and Melan-A_26-35 A27M). For comparison, we also synthesized the analogues Melan-A_27-35 A27L and A27 M as well as Melan-A_26-35 A28L and A28M. Two additional peptide analogues were synthesized (peptides Melan-A_26-35 E26F and E26Y) in which E at position 1 of the decapeptide (described as being associated with less efficient peptide-HLA-A2 binding) was replaced by an aromatic amino acid (Y or F) known to favor decapeptide binding to HLA-A2 (19).

As illustrated in Figure 2⇓, binding of the various peptides to HLA-A*0201 was evaluated in a functional competition assay based on inhibition of specific recognition of peptide MAGE-3_271-279 by the HLA-A*0201-restricted CTL line 198NS (20). The nonsubstituted nonapeptide was a relatively weak competitor (50% inhibition at 60 μM) compared with the internal positive control peptide (Influenza A matrix _58-66 peptide, 50% inhibition at 1 μM) (Fig. 2⇓, Table II⇑). As expected, the HLA-A1-restricted MAGE-3_168-176 peptide did not show competitor activity even at the highest concentration tested. Substitution of A at position 2 of the nonapeptide with either L or M (Melan-A_27-35 A28L, A28M) resulted in a 30- to 40-fold increase in competitor activity. In contrast, substitution of A at position 1 of the nonapeptide with L or M (Melan-A_27-35 A27L, A27M) had no apparent effect. Peptide Melan-A_26-35 was a fourfold better competitor than the peptide Melan-A_27-35. Substitution of A at position 2 of the decapeptide with L (Melan-A_26-35 A27L) resulted in an additional twofold increase in competitor activity, while substitution with M (Melan-A_26-35 A27M) had no further effect. Substitution of L or M for A at position 3 of the decapeptide (Melan-A_26-35 A28L, A28M) reduced its competitor activity. In contrast, substitution of Y or F for E at position 1 (Melan-A E26Y, E26F) significantly enhanced the competitor activity of the decapeptide (Table II⇑).

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FIGURE 2.

Binding of Melan-A Ag peptide analogues to HLA-A*0201. Binding was measured in a functional competition assay by using the MAGE-3_271-279-specific, HLA-A*0201-restricted CTL line 198NS. Test peptides were incubated at various concentrations (1 μM to 100 μM) with ⁵¹Cr-labeled T2 cells (1000/well) for 15 min at room temperature. A suboptimal dose of the antigenic peptide (MAGE-3 _271-279, 1 nM) was then added together with the effector cells at 5:1 E:T. The assay was then carried on as a conventional chromiun release assay. For details of peptide origin and amino acid sequence, see Table II⇑.

Assessment of the dissociation rate of Melan-A peptide analogues from HLA-A*0201

The stability of complexes formed between HLA-A*0201 and the different Melan-A peptides was assayed on T2 cells, which lack TAP function and consequently are defective in properly loading class I molecules with antigenic peptides generated in the cytosol. The association of exogenously added peptides with thermolabile, empty HLA-A2 molecules stabilizes them and results in an increase in the level of surface HLA-A2 recognizable by conformation-dependent α1/α2-specific mAb such as W6/32 or BB7.2 (25). Indeed, overnight incubation of T2 cells with saturating amounts of HLA-A*0201 binding peptides and human β2 microglobulin resulted in increased surface expression of HLA-A*0201 molecules. After peptide removal and addition of emetine to inhibit protein synthesis, T2 cells were incubated at 37°C and the amount of HLA-A*0201 molecules remaining at the cell surface was determined after various incubation times (as illustrated in Figure 3⇓A for some peptides). The stability of each peptide/HLA-A*0201 complex was then normalized relative to that observed for the Influenza A matrix _58-66 peptide/HLA-A*0201 complex (Fig. 3⇓, B–D). HLA-A*0201 complexes formed with peptides Melan-A_27-35 and Melan-A_26-35 were unstable, reaching background levels in less than 1 h of incubation at 37°C (Fig. 3⇓, A–C). Similarly, complexes formed with peptides Melan-A_27-35 A27L and A27M dissociated rapidly (Fig. 3⇓B). In contrast, peptides Melan-A_26-35 A27L, A27M (Fig. 3⇓C), E26Y, and E26F (Fig. 3⇓D) formed complexes that were relatively stable over a 6-h period. Complexes of intermediate stability were observed with the remaining peptide analogues tested (Fig. 3⇓, B and C).

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FIGURE 3.

Assessment of the dissociation rate of Melan-A peptide analogues from HLA-A*0201. After overnight incubation with saturating amounts of peptide, T2 cells were treated with emetine (to inhibit protein synthesis) and incubated at 37°C. At the indicated time points, cells were stained for HLA-A2 Ag expression. The mean fluorescence intensity was determined for each histogram (A), where NC refers to the HLA-A2 Ag expressed by T2 cells treated in the same conditions but in the absence of exogenous peptide. The stability of each peptide/HLA-A*0201 complex was normalized relative to that observed for the Influenza A matrix _58-66 peptide/HLA-A*0201 complex (B-D).

Recognition of Melan-A peptide analogues by Melan-A-specific TILN and CTL clones

The relative antigenic activity of the peptide analogues was quantitated in a standard CTL assay using Melan-A-specific TILN and CTL clones. A dose-response curve was generated for each peptide analogue (Fig. 4⇓). The antigenic activity of each peptide analogue was then calculated relative to that of the Melan-A_27-35 peptide (which was 60 and 30 nM for 50% maximal lysis by TILN LAU 203 and LAU 132, respectively, Table III⇓). The two Melan-A-specific TILN populations recognized the reference nonapeptide with a similar efficiency (Table III⇓). Substitution of A at position 1 of the nonapeptide with L or M resulted in enhanced peptide recognition of about 10-fold. Surprisingly, substitution of A at position 2 of the nonapeptide with either L or M, which strongly increased peptide binding to HLA-A*0201, resulted in a >50-fold reduction of their antigenic activity.

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FIGURE 4.

Recognition of Melan-A peptide analogues by Melan-A-specific TILN. The relative antigenic activity of Melan-A peptides was measured by assessing lysis of T2 cells incubated with increasing concentrations of the Melan-A peptide analogues by TILN LAU 132 and LAU 203 in a 4-h ⁵¹Cr release cytotoxicity assay at a lymphocyte to target cell ratio of 30:1.

In agreement with the results mentioned previously, peptide Melan-A_26-35 was better recognized than peptide Melan-A_27-35 by both TILN populations. Remarkably, substitution of A at position 2 of the decapeptide with L or M further improved the efficiency of peptide recognition. However, L or M substitution for A at position 3 of the decapeptide resulted in strongly reduced efficiency of recognition, as expected from the relatively low HLA-A*0201 binding activity displayed by these two peptide analogues. Finally, substitution of E at position 1 with Y and, particularly, with F, in accordance with the effect on peptide binding, also resulted in highly improved peptide recognition (Table III⇑).

To further document these findings, we tested the different peptides and their analogues for recognition by five independent, HLA-A*0201-restricted Melan-A-specific CTL clones known to lyse appropriate melanoma target cells. As shown in Table IV⇓, the CTL clones recognized the peptide Melan-A_27-35 with variable efficiency, as reflected by the concentration of peptide required to achieve 50% of maximal target cell lysis (between 15 and 4000 nM). Substitution of A at position 1 of the nonapeptide with L enhanced peptide recognition by four of the five clones, whereas a similar substitution at position 2 resulted in a general loss of antigenic activity. Three of the five CTL clones recognized peptide Melan-A_26-35 more efficiently than peptide Melan-A_27-35. Nevertheless, all five CTL clones recognized very efficiently the decapeptide analogue containing L at position 2. In contrast, substitution of A at this position with M decreased efficiency of peptide recognition to variable degrees. Moreover, substitution of L for A at position 3 of the decapeptide resulted in greatly reduced efficiency of recognition by four of the five clones. Surprisingly, substitution of A at position 3 with M, in contrast with the findings obtained with TILN, improved peptide recognition by four of the five clones. Finally, substitutions at position 1 of Melan-A_26-35 resulted in reduced recognition by several of the clones tested.

Taken collectively, the results of this analysis indicated that two Melan-A peptide analogues, namely Melan-A_26-35 A27L and, to a lesser extent, Melan-A_27-35 A27L, were better recognized than the corresponding natural peptides by the various CTL lines and clones tested, whereas much more variability was observed with the other peptide analogues tested.

Induction of Melan-A-specific, tumor-reactive CTL by in vitro stimulation with Melan-A peptide analogues

Based on the above-mentioned data, the peptide analogue Melan-A_26-35 A27L was selected for in vitro CTL induction studies. The ability of this peptide to induce melanoma-reactive CTL specific for Melan-A was evaluated by stimulating CD8⁺-enriched T lymphocytes derived from PBMC of melanoma patients with autologous irradiated PBMC pulsed with peptide. Stimulation with Melan-A_27-35 and Melan-A_26-35 peptides was also performed in each experiment to allow an appropriate comparison between the CTL responses generated with the natural and the modified peptides. The results obtained on day 7 after the second restimulation of CD8⁺-enriched T cells from melanoma patient LAU 203 are shown in Figure 5⇓. Peptide-specific CTL activity was barely detectable in the cultures stimulated with the natural peptides, whereas the culture stimulated with the Melan-A_26-35 A27L peptide analogue exhibited strong CTL activity. This activity was directed not only against the peptide analogue used for stimulation but was also cross-reactive with the nonsubstituted Melan-A decapeptide. More importantly, the CTLs induced by stimulation with the Melan-A_26-35 A27L peptide analogue were able to lyse the Melan-A-expressing autologous melanoma cell line Me 290 (Fig. 5⇓). After three additional rounds of stimulation, peptide-specific activity as well as tumor reactivity was also clearly detectable in those cultures stimulated with the natural peptides (data not shown). Similar results were obtained after stimulation of PBMC CD8⁺ T cells from patient LAU 132 (data not shown).

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FIGURE 5.

Induction of Mela-A-specific, tumor-reactive CTL by in vitro stimulation of PBMC with Melan-A peptide analogues. CD8⁺-enriched T cells from melanoma patient LAU 203 were restimulated twice with the indicated peptide-pulsed and irradiated autologous PBMC. CTL activity was measured 7 days after the second in vitro restimulation. Lysis on autologous tumor cells, Me 290 (□), or T2 was measured in a 4-h ⁵¹Cr release assay in the absence (○) or presence of exogenously added parental peptide Melan-A_26-35 (▪) or peptide analogue Melan-A_26-35 A27L (•).

These results were extended in an experiment including CD8⁺-enriched T cells from eight HLA-A*0201 melanoma patients. The T cells were stimulated in parallel with peptides Melan-A_27-35, Melan-A_26-35, and Melan-A_26-35 A27L. CTL activity of individual cultures was assayed on day 7 after the third in vitro restimulation using as a source of target cells T2 cells with or without exogenously added peptide as well as Melan-A-expressing melanoma cells described above. As shown in Table V⇓, HLA-A*0201-restricted Melan-A-specific tumor-reactive CTL were detected in PBMC cultures from five of eight patients after stimulation with the Melan-A_26-35 A27L peptide analogue. In contrast, none of the PBMC cultures stimulated with the nonsubstituted Melan-A nona- or decapeptides exhibited specific cytolytic activity. Together, these data indicate that the Melan-A_26-35 A27L peptide analogue displays greater immunogenicity than the two parental peptides with regard to induction in vitro of Melan-A-specific and tumor-reactive CTL.