Dominant TCR-α Requirements for a Self Antigen Recognition in Humans

Stefania Mantovani, Belinda Palermo, Silvia Garbelli, Rita Campanelli, Gioacchino Robustelli della Cuna, Roberto Gennari, Federica Benvenuto, Erica Lantelme and Claudia Giachino
J Immunol December 1, 2002, 169 (11) 6253-6260; DOI: https://doi.org/10.4049/jimmunol.169.11.6253

Abstract

TCR-α and -β chains are composed of somatically rearranged V, D, and J germline-encoded gene segments that confer Ag specificity. Recent crystallographic analyses revealed that TCR-α has more contacts with peptide than TCR-β, suggesting the possibility that peptide recognition predominantly relies on TCR-α. T cells specific for the self Ag Melan-A/MART-1 possess an exceptionally high precursor frequency in human histocompatibility leukocyte Ag-A2 individuals. This provided a unique situation for assessment of the structural relationship between TCR and peptide/MHC ligand at both the pre- and postimmune levels. Molecular and phenotypic analysis of many different Melan-A-specific T cell populations revealed that a structural constraint is imposed on the TCR for engagement with Melan-A peptides presented by HLA-A2, namely the highly preferential use of a particular TCRAV segment, AV2. Examination of CD8 single-positive thymocytes indicated that this preferential use in forming the Melan-A-specific TCR is mainly imposed by intrathymic positive selection. Our data demonstrate a dominant function of TCRAV2 segment in forming the TCR repertoire specific for the human self Ag Melan-A/MART-1 and support the view that Ag recognition is mediated predominantly by TCR-α.

Antigen-specific immune responses are both initiated and maintained by TCR recognition of Ag peptide assembled within the MHC groove. Selection of the TCR repertoire during thymic development begins with positive selection of T cells expressing receptors with low affinity for self-MHC, and negative selection of the high-affinity cells (1). Persistence in the peripheral repertoire requires homeostatic division, and this also relies on TCR recognition of self-epitopes (1). TCR-α and -β chains are composed of somatically rearranged V, D, and J germline-encoded gene segments (2), and contain three highly variable complementarity-determining regions (CDRs)2 that confer Ag specificity. CDR1 and 2 are encoded by germline V segments, whereas CDR3 is created by somatic rearrangement and makes a greater contribution to TCR diversity (2). Crystallographic analysis of several class-I-restricted and one class-II-restricted TCRs has shown that TCR-α has more contacts with peptide than TCR-β and is presumably of greater importance in peptide recognition (3, 4, 5, 6, 7, 8). Specifically, dominance of the TCRAV domain in peptide recognition has been established (4, 6). Besides, TCR-α is also important in making contacts with the α-helices of the MHC (9). In addition to structural analyses, studies of single TCR-transgenic mice and clonal TCR reconstitution systems suggest that recognition of foreign Ags is predominantly dependent on TCR-α and that multiple TCR-β-chains can create the same Ag specificity when paired with a single TCR-α during generation of the T cell repertoire (10, 11, 12, 13).

The melanocyte differentiation Ag Melan-A/MART-1 is a self protein of unknown function expressed by melanocytes and most malignant melanoma cells, but not by other tissues (14, 15). Among the several melanoma-associated Ags so far identified, it has received particular attention because of its “immunodominance” in patients carrying the HLA-A2 allele: it is recognized by 90% of tumor-infiltrating lymphocytes from HLA-A*0201 patients. This immunodominance is subject to extremely stringent requirements (16): only two largely overlapping peptides, Melan-A 26–35 and 27–35, of intermediate affinity for HLA-A*0201 (17, 18). HLA-A2 tetramers synthesized around the Melan-A A27L peptide analog have been used to identify Melan-A-specific T cells ex vivo among circulating lymphocytes from both melanoma (19, 20, 21) and vitiligo patients (22, 23). A surprising finding was a large Melan-A-specific naive T cell pool: up to 70% of HLA-A*0201 healthy donors possess discrete amounts of tetramer-positive cells, free from signs of previous Ag encounter (23, 24, 25, 26, 27). The size of the circulating Melan-A T cell pool in adults is at least 100-fold larger than current estimates for naive Ag-specific lymphocyte precursors, and approaches that of epitope-specific memory CD8 T cells. Sequence homology between the Melan-A peptide and a broad range of self-derived proteins (28, 29) suggests that cross-recognition of many ligands may play an important role in positive thymic selection and generation of a large naive T cell pool. High frequencies of Melan-A-specific T cells among CD8 single-positive (SP) thymocytes from HLA-A2 individuals (27) indicate that a high thymic output makes a major contribution to this precursor frequency.

It is thus of interest to determine whether specific TCR sequence motifs are involved in Melan-A recognition and in positive thymic selection of Melan-A-specific T cells. Previous experiments, confined to TCR-β, indicated that generation of the large Melan-A-specific T cell pool was not due to selection of a particular TCR (27, 30, 31, 32, 33). We now demonstrate that TCR-α is predominant in recognition of this self Ag and suggest that stringent allele/epitope/TCR-α requirements constitute the molecular basis for Melan-A/MART-1 responses in humans.

Materials and Methods

Cells and tissues

Peripheral blood from seven healthy donors, seven melanoma patients, and eight vitiligo patients was collected after signed informed consent. All subjects were selected on the basis of HLA-A2 expression. Molecular typing for HLA-A genes was performed by PCR-sequence-specific primers technique. PBMC were isolated by Ficoll centrifugation (Ficoll-Paque; Amersham Pharmacia Biotech, Uppsala, Sweden). Pediatric thymus tissue from 10 children undergoing corrective cardiac surgery was obtained at Ospedale Infantile Regina Margherita (Turin, Italy) in accordance with the guidelines of the local Ethical Committee. Three specimens were HLA-A2 positive.

T cell sorting and cloning

PBMC were sorted into defined populations on a FACSVantage SE using CellQuest software (BD Biosciences, Mountain View, CA). To maximize purification efficiency, the flow speed used during cell sorting corresponded to (∼2 × 103) cells per second. Purification of 1–2 × 103 A2/Melan-A+ cells required 3–4 h sorting. Immediate reanalysis of the sorted populations revealed >98% purification efficiency. For the cloning experiments, part of sorted cells were plated at 0.3 cells/well in complete RPMI medium in the presence of irradiated PBMC (5 × 105 cells/ml), 2 μg/ml PHA, and 250 U/ml rIL-2. Proliferating clones were expanded in complete RPMI medium supplemented with 5% human serum (Euroclone, Wetherby, U.K.) and 500 U/ml rIL-2. For the functional tests, part of ex vivo tetramer-sorted T lymphocytes were expanded for 2 wk with 1 μg/ml PHA, 250 U/ml rIL-2, and 0.5 × 106/ml allogenic-irradiated PBMC to obtain a sufficient number of cells. For some experiments, CD8 cells were enriched from PBMC using CD8 microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany) and either directly stained, or when necessary, expanded for 8 days with 1 μg/ml PHA. CD8 SP thymocytes were negatively enriched with CD4 microbeads.

T cell lines

For the generation of peptide-specific T cell lines, PBMC were monocyte-depleted by exclusion of plastic adherent cells. PBMC (105/well) were cultured in complete RPMI medium in the presence of 105 peptide-pulsed T2 cells (34) using 1–2 μg/ml of Melan-A A27L peptide. Low-dose (25 U/ml) IL-2 was added on day 5. Cells were stimulated at 3-wk intervals.

Peptides and tetramers

The following peptides were purchased from Primm (Milan, Italy): Melan-A26–35 (EAAGIGILTV), Melan-A A27L (an analog of the Melan-A26–35 epitope carrying a substitution of Ala for Leu at position 2 from the NH2 terminus, ELAGIGILTV) (35), influenza matrix Flu-MA58–66 (GILGFVFTL), and MAGE-3271–279 (FLWGPRALV). All peptides were >90% pure as indicated by analytical HPLC. Lyophilized peptides were diluted in DMSO and stored at −20°C. MHC/peptide tetramers built around the first three peptides were purchased from Beckman-Coulter (iTAg MHC tetramers; San Diego, CA). For some experiments, a mutated A2/Melan-A tetramer, containing the substitution Val for Ala at position 245 known to alter the interaction between class I and CD8 (36, 37), was used (38).

Abs and flow cytometry

PBMC were stained with tetramers-PE (0.5 μg/106 cells) for 15 min at 37°C; after two washes, indirect double-staining was performed on ice using anti-human CD8 mAb (OKT8, IgG2a) and FITC-labeled goat anti-mouse IgG2a (Southern Biotechnology Associates, Birmingham, AL) as second mAb. The specificity of each tetramer was confirmed by staining CTL lines or clones specific for HLA-A2 in association with the peptide of interest (data not shown). To minimize background staining, tetramers were titrated and used at the lowest concentration that still gave a discernible population. Triple stainings with tetramer-PE, anti-CD45RA-CYC (BD Biosciences), and either anti-CD45RO-FITC (Caltag Laboratories, Burlingame, CA) or anti-CD56-FITC (Southern Biotechnology Associates), were performed to assess the naive/effector/memory phenotype of tetramer+ cells. For the TCR repertoire studies, the following Abs (mAbs) were used: anti-AV2 (Beckman-Coulter), anti-AV24 (C15, IgG1), anti-AV12-FITC (Endogen, Woburn, MA), and anti-BV1, -3, -4, -5, -6, -8, -12, -13, -14, -16, -20, -21-FITC (Beckman-Coulter). Cells were analyzed on a FACSCalibur (BD Biosciences).

TCR down-regulation assay

T2 target cells were pulsed for 1.5 h with various concentrations of Melan-A A27L peptide in serum-free medium (X-VIVO 15; BioWhittaker, Walkersville, MD) at 37°C, washed four times, and then added to effector cells to give an E:T ratio of 1:5. After 3 h at 37°C, TCR internalization was measured by flow cytometry using HLA-A2 tetramers synthesized around the Melan-A A27L peptide. The use of the same reagent (A2/Melan-A tetramer) for identification of Ag-specific cells and evaluation of surface TCR expression poses a problem when data are analyzed, since tetramer staining intensity decreases due to TCR down-regulation and a cell may escape detection if its fluorescence intensity drops below the gate established to identify specific cells. Therefore, when Ag concentration is increased, a large proportion of the sample population is “lost” because of its undetectability. For this reason, the use of the percentage decrease of tetramer fluorescence intensity as the sole parameter is inappropriate. Therefore, two parameters where used to calculate the percentage of surface TCR within the tetramer+ population: the percentage decrease of tetramer fluorescence intensity and the percentage of tetramer+ cells still present within the gate.

Cytotoxicity assay

Sorted tetramer+ cells and peptide-specific T cell lines were assayed for specific lysis against an HLA-matched melanoma cell line (501 mel; a kind gift of L. Rivoltini, Cancer Immunotherapy Unit, Istituto Nazionale Tumori, Milan, Italy) or peptide-pulsed T2 cells, respectively, in a standard 4-h chromium release assay. Briefly, 106 target cells were labeled with 200 μCi 51Cr for either 1.5 h (melanoma cells) or 15 h (T2 cells). T2 cells were pulsed with 3 μg/ml Melan-A A27L peptide for 30 min at room temperature. T2 cells pulsed with an HLA-A*0201-restricted, irrelevant peptide (MAGE-3271–279) were used as a negative control at the same concentration as the relevant peptide. A total of 103 target cells/well were mixed with appropriate amounts of effector cells to give final E:T ratios ranging between 30:1 and 2:1. After 4 h at 37°C, 25 μl supernatant was collected, seeded in Lumaplate 96 solid scintillation plates (Packard Instruments Company, Meriden, CT), and counted in a beta counter. The percentage of specific lysis was calculated as: 100 × (experimental release − spontaneous release)/(maximum release − spontaneous release). Spontaneous release was assessed by incubating target cells in the absence of effectors and maximum release was determined in the presence of 1% Nonidet P-40 detergent (BDH Biochemicals, Poole, U.K.).

Amplification and sequencing of TCR transcripts

Total RNA from 106 cells from each clone was extracted using EUROZOL (Euroclone). First-strand cDNA was synthesized using oligo(dT) and Moloney murine leukemia virus M-MLV Reverse Transcriptase (Invitrogen, Paisley, U.K.) in 20 μl final volume. Aliquots of the cDNA were PCR amplified in parallel with a panel of 32 AV- and 24 BV-specific oligonucleotides in conjunction with one AC- (5′-GTTGCTCCAGGCCGCGGCACTGTT-3′) and one BC-specific (5′-TGCTGACCCCACTGTCGACCTCCTTCCCATT-3′) oligonucleotide. The AV-specific primers were taken from the literature (39, 40).

The BV-specific primers were as follows: BV1 5′-GCACAACAGTTCCCTGACTTGCAC-3′, BV2 5′-TCATCAACCATGCAAGCCTGACCT-3′, BV3 5′-GTCTCTAGAGAGAAGAAGGAGCGC-3′, BV4 5′-TTCCCATCAGCCGCCCAAACCTAA-3′, BV5 5′-ATACTTCAGTGAGACACAGAGAAA-3′, BV6 5′-TCTCAGGTGTGATCCAAATTCGGG-3′, BV7 5′-CACCTGAATGCCCCAACAGCTCTC-3′, BV8 5′-ATTTACTTTAACAACAACGTTCCG-3′, BV9 5′-TTCCCTGGAGCTTGGTGACTCTGC-3′, BV10 5′-CTCCAAAAACTCATCCTGTACCTT-3′, BV11 5′-TCAACAGTCTCCAGAATAAGGACG-3′, BV12 5′-TGTCACCAGACTGGGAACCACCAC-3′, BV13 5′-CACTGCGGTGTACCCAGGATATGA-3′, BV14 5′-GTCTCTCGAAAAGAGAAGAGGAAT-3′, BV15 5′-CAGGCACAGGCTAAATTCTCCCTG-3′, BV16 5′-TGAAAGAGTCTAAACAGGATGAGT-3′, BV17 5′-TCCTCTCACTGTGACATCGGCCCA-3′, BV18 5′-GATGAGTCAGGAATGCCAAAGGAA-3′, BV19 5′-TCAATGCCCCAAGAACGCACCCTG-3′, BV20 5′-ATCAGCTCTGAGGTGCCCCAGAAT-3′, BV21 5′-ATTCACAGTTGCCTAAGGATCGAT-3′, BV22 5′-GGGCAGAAAGTCGAGTTTCTGGTT-3′, BV23 5′-TTTTATGAAAAGATGCAGAGCGAT-3′, and BV24 5′-AAGTCAAGTCAGGCCCCAAAGCTG-3′.

The TCRAV-PCR profile was 2 min at 94°C, 30 cycles at 94°C for 1 min, 60°C for 30 s, 72°C for 30 s followed by a 7-min extension. The TCRBV-PCR profile was 2 min at 94°C, 35 cycles at 94°C for 30 s, 55°C for 20 s, 72°C for 1 min followed by a 7-min extension. PCR amplification products were purified using JetSorb gel extraction kit (Genomed, Bad Oeynhausen, Germany) and directly sequenced with the BigDye Terminator Cycle Sequencing kit (Applied Biosystems, Foster City, CA) using an internal AC- (5′-GTCACTGGATTTAGAGTC-3′) and BC-specific (5′-TGCTTCTGATGGCTCAA-3′) oligonucleotide according to the manufacturer’s instructions. Sequences were run on an ABI 377 DNA sequencer (Applied Biosystems). The TCR nomenclature proposed by Arden et al. (41) was adopted.

Results

Strong TCR-α chain conservation in Melan-A-specific cell clones

Freshly isolated PBMC from one HLA-A2 vitiligo patient (PSA) were stained with HLA-A2 tetramers synthesized around the Melan-A A27L analog peptide (35) together with an anti-CD8 mAb and analyzed by flow cytometry. As shown in Fig. 1⇓A, the A2/Melan-A tetramer+ T cells constituted 0.16% of the CD8+ cells. The CD8+ tetramer+ population was isolated by flow cytometry cell sorting. Sorted cells were too few to allow directly Ag specificity testing. They were split into two parts: one was shortly expanded in the presence of PHA for functional studies, the other immediately cloned by limiting dilution. Specificity of the sorted cells was demonstrated through peptide-specific TCR down-regulation experiments and cytotoxicity assays against an HLA-matched melanoma cell line (Fig. 1⇓, B and C). To analyze the TCR repertoire displayed by the CD8+ A2/Melan-A tetramer+ T cells, RNA was extracted from 30 clones and cDNA subjected to RT-PCR using a panel of AV and BV oligonucleotides covering virtually 100% of the TCR-αβ repertoire. In line with previously published data concerning the heterogeneity of the Melan-A-specific TCR-β repertoire (27, 30, 31, 32, 33), Melan-A-specific CD8+ T cells from this vitiligo patient were found to rearrange many distinct BV gene segments (Fig. 1⇓D). In contrast, all clones expressed the same rearranged AV gene segment (AV2, 28 of 28 clones; the AV rearrangement of two clones could not be determined) (Fig. 1⇓E). Direct sequencing with an internal AC-specific primer was performed to determine the complete TCR-α CDR3 region of the Melan-A-specific clones (Fig. 2⇓). The AV2 segment was always productively rearranged and the CDR3 regions were all different, indicative of a large degree of polyclonality in the specific TCR repertoire. Twenty-two of 28 clones (78%) used the AV2.1 segment, while the remaining six used either AV2.2 (five cases) or AV2.3 (one case); these last six clones also used the same AJ segment (AJ48) and contained an identical CDR3α size (16 aa). Two clones (PSA.S.6 and PSA.S.26) possessed only two nucleotide differences in their CDR3 and their amino acids were identical. A total of 11 clones expressed a second rearranged AV segment; this also was productive in four cases, in line with figures reported by others (42, 43).

FIGURE 1.
FIGURE 1.

Ex vivo sorted Melan-A-specific T cells from one HLA-A2 vitiligo patient display a highly restricted TCRAV usage. A, Freshly isolated PBMC from one HLA-A2 vitiligo patient (PSA) were stained with A2/A27L tetramers and anti-CD8 mAb and analyzed by flow cytometry. The percentage of tetramer+ cells within the CD8+ population is shown. Cells within the gate were FACS sorted using a FACSVantage SE. B, Sorted Melan-A-specific CD8+ T cells that had been shortly expanded in vitro were tested for their peptide specificity through TCR down-regulation experiments. HLA-A2 TAP-deficient T2 cells were pulsed with various concentrations of Melan-A A27L peptide and added to effector cells at a final E:T ratio of 1:5. After 3 h at 37°C, TCR internalization was measured by flow cytometry using A2/Melan-A tetramers. The percentage of surface TCR was calculated with a modified method (see Materials and Methods). C, Sorted tetramer+ cells that had been expanded in vitro were assayed for specific lysis against a Melan-A-expressing, HLA-matched melanoma cell line (501 mel) in a standard 4-h chromium release assay. T2 cells pulsed with a HLA-A2 restricted, irrelevant peptide (MAGE-3271–279) were used as negative control. •, 501 mel; ▴, T2 + irrelevant peptide. D and E, Part of the cells were cloned by limiting dilution immediately after sorting and 30 independent clones were analyzed for their TCRBV (D) and TCRAV (E) usage by RT-PCR; the TCRAV rearrangement of two clones could not be determined.

FIGURE 2.
FIGURE 2.

TCR-α chain amino acid sequences of Melan-A-specific cell clones from one HLA-A2 vitiligo patient. CDR3α junctional amino acid sequences of 28 ex vivo sorted Melan-A-specific T cell clones from this patient are shown. Amino acids either partially or completely formed by nontemplate added nucleotides (N nucleotides) are underlined. Two clones (PSA.S.6 and PSA.S.26) possessed only two nucleotide differences in their CDR3, and their amino acids were identical. These sequence data are available from GenBank/European Molecular Biology Laboratory (EMBL)/DNA Data Base in Japan (DDBJ) under accession no. AY135832-AY135859.

Circulating Melan-A-specific cells from a second HLA-A2 patient (SCO) affected by melanoma were similarly analyzed. The percentage of A2/Melan-A tetramer+ T cells in this patient and the functional analysis of sorted cells are illustrated in Fig. 3⇓. The molecular characterization of 21 clones obtained after sorting and cloning of the tetramer+ cells confirmed a highly selective usage of AV2 segment in this patient, which contributed to form the expressed Melan-A-specific TCR in 20 of 21 clones (Figs. 3⇓D and 4). Eight clones expressed a second rearranged AV segment and this was also productive in one case.

FIGURE 3.
FIGURE 3.

Restriction of TCRAV usage in ex vivo-sorted Melan-A-specific T cells from one HLA-A2 melanoma patient. A, Freshly isolated PBMC from this patient (SCO) were stained with A2/A27L tetramers and anti-CD8 mAb and analyzed by flow cytometry. The percentage of tetramer+ cells within the CD8+ population is shown. Tetramer+ cells were FACS sorted using a FACSVantage SE. B, Sorted Melan-A-specific CD8+ T cells that had been shortly expanded in vitro were assayed for their TCR down-regulation upon peptide encounter, as described in Materials and Methods. C, Tumor reactivity of the sorted cells that had been expanded in vitro was assessed by chromium release assay against a HLA-matched melanoma cell line (501 mel). T2 cells pulsed with a HLA-A2 restricted, irrelevant peptide were used as negative control. •, 501 mel; ▴, T2 + irrelevant peptide. D, Part of the sorted cells were immediately cloned by limiting dilution after sorting and 21 independent clones were analyzed for their TCRAV usage by RT-PCR.

Dominant usage of TCRAV2 is widely conserved in the Melan-A-specific peripheral repertoire

To assess whether the selective use of this AV segment is a generalized feature of Melan-A-specific cells, PBMC from 14 HLA-A2 patients (seven with vitiligo and seven with melanoma) were analyzed. PBMC were enriched in CD8+ cells with magnetic beads and stained with A2/Melan-A tetramers in association with an anti-AV2 mAb either directly or, when the number of CD8+ cells was too low, following short expansion in the presence of PHA. It is important to note that the mAb we used is specific for the AV2.1 segment and does not cross-react with AV2.2 or AV2.3 (data not shown). Thus, the number of AV2-expressing cells is underestimated when this reagent is used. The high percentages of AV2.1-expressing cells in the tetramer+ population of all our patients (range 50–87%; mean ± SD, 67 ± 10.9%) was consistent with a highly preferential use of AV2 in forming the Melan-A-specific TCR (Fig. 5⇓), whereas AV12- and AV24-expressing cells were always undetectable (Fig. 5⇓B). No other AV families could be tested due to lack of other commercialized AV-specific Abs. We repeated the same stainings using HLA-A2 tetramers built around the natural 26–35 peptide, as well as tetramers containing a mutation known to reduce the nonspecific binding between HLA-A2 and CD8 (38). In both cases, we obtained comparable percentages of tetramer+ AV2+ cells (data not shown). HLA-A2 tetramers built around the Flu-MA peptide were also used in conjunction with the AV2.1 mAb to stain these samples. None of the A2/Flu-MA tetramer+ cells turned out to be AV2.1+, indicating that preferential usage of this AV segment is specifically relevant for recognition of the self Ag Melan-A (Fig. 5⇓).

FIGURE 4.
FIGURE 4.

TCR-α chain amino acid sequences of Melan-A-specific cell clones from one HLA-A2 melanoma patient. CDR3α junctional amino acid sequences of 21 ex vivo-sorted Melan-A-specific T cell clones from this patient are shown. Amino acids either partially or completely formed by nontemplate added nucleotides (N nucleotides) are underlined. These sequence data are available from GenBank/EMBL/DDBJ under accession no. AF532837-AF532857.

FIGURE 5.
FIGURE 5.

Dominant usage of TCRAV2 is a conserved feature of the Melan-A-specific peripheral repertoire. A, PBMC from seven HLA-A2 vitiligo patients, seven HLA-A2 melanoma patients, and seven HLA-A2 healthy donors were enriched for CD8+ cells with magnetic beads and analyzed by double staining with A2/Melan-A tetramers and anti-AV2.1 mAb. Only the cases containing detectable A2/Melan-A positive populations are shown. Bars represent the total percentages of tetramer+ cells within the CD8+ population; ▪ represent the percentages of AV2.1+ cells and ▦ the percentages of AV2.1− cells within the tetramer+ CD8+ population. Lower panel, The same samples analyzed by double staining with A2/Flu-MA tetramers and anti-AV2.1 mAb. B, Representative double stainings of CD8+ cells from one patient (SDB) and one healthy donor (HD3) with different combinations of tetramer/anti-AV mAbs.

Consistent with previous data indicating that Melan-A-specific CTL from both melanoma and vitiligo patients frequently show signs of an in vivo Ag encounter (23, 25, 44), all patients analyzed in this part of the work contained variable proportions of A2/Melan-A tetramer+ lymphocytes displaying an effector/memory phenotype (between 6 and 59%; mean ± SD, 31.3 ± 17.6%, data not shown). To define whether the predominant use of AV2 by the Melan-A-specific repertoire is defined at the preimmune level or reflects a postimmune selection, we analyzed the Melan-A-specific repertoire by tetramer staining PBMC in seven HLA-A2 healthy subjects. PBMC were enriched in CD8+ cells with magnetic beads and stained with A2/Melan-A tetramers. A detectable population was found in five cases (71%) (Fig. 5⇑). Virtually all tetramer+ cells exhibited a CD45RA+CD56− phenotype, indicative of a naive repertoire (data not shown). These data are in agreement withprevious reports of high frequencies of circulating Melan-A-specific CD8 T cells with a bona fide naive phenotype (CD45RA+CD45RO−CCR7+CD27+CD28+CD56−CD57−) in healthy HLA-A2 individuals (23, 24, 25, 26, 27). Once again (Fig. 5⇑) a large fraction of tetramer+ cells, comparable to that found in the patients, expressed the AV2 segment (range 55–90%; mean ± SD, 73 ± 15.4% of the CD8+ cells).

Requirement of single TCRAV and heterogeneous TCRBV segments for Melan-A recognition during expansion of Ag-specific cell lines

Next, peptide-specific cell lines were established from the PBMC of two vitiligo, two melanoma, and one healthy subjects. In all cases, enrichment of tetramer+ cells upon two rounds of stimulation correlated with similar expansion rates of AV2.1 cells (Fig. 6⇓A for one representative case) as well as with enhanced TCR down-regulation upon peptide engagement (Fig. 6⇓B) and enhanced peptide-specific cytotoxicity (Fig. 6⇓C). The percentage of AV12 cells increased only slightly or not at all during stimulation (Fig. 6⇓A). At the highest expansion levels, virtually all tetramer+ cells in the peptide-specific lines were AV2.1+ (Fig. 6⇓A), whereas their BV repertoire as assessed by the use of mAbs specific for 12 BV segments was still highly heterogeneous (Fig. 6⇓D). These experiments demonstrate that AV2 has a predominant role in determining Melan-A-recognition and that the same Ag specificity can be created by pairing of a conserved TCR-α chain with multiple TCR-β chains. In addition, they rule out the possibility that A2/Melan-A tetramers cross-react with AV2-containing TCR, irrespective of their Ag specificity.

FIGURE 6.
FIGURE 6.

CD8 T lymphocytes selected during expansion of Melan-A-specific cell lines express TCRAV2 paired to heterogeneous TCRBV segments. A, PBMC from two vitiligo, two melanoma, and one healthy donors were in vitro expanded by two rounds of peptide-specific stimulations and stained with A2/Melan-A tetramers, anti-AV2.1, and anti-AV12 mAbs at each time point through either single or double stainings. The results for patient SDB are shown. I-st, first stimulation; II-st, second stimulation. B, Peptide specificity of the cell lines was assessed by TCR down-regulation experiments 2 wk after each stimulation, as described in Materials and Methods. C, Functional activity of the cell lines was assessed by chromium release assay against T2 cells pulsed with Melan-A A27L peptide. T2 cells pulsed with a HLA-A2 restricted, irrelevant peptide were used as negative control and nonspecific lyses were subtracted from each point. D, The TCRBV repertoire expressed by the Melan-A-specific cell lines was assessed with mAbs specific for 12 BV in conjunction with A2/Melan-A tetramers.

TCR-α dominates in shaping the intrathymic Melan-A-specific repertoire

The origin of the AV2+, Melan-A-specific T cells, was directly investigated by tetramer staining T cells from the human thymus. High percentages of A2/Melan-A-, but not influenza-specific, cells are detected in the pool of CD8 SP, but not double-positive thymocytes (27). SP thymocytes constituted 1–5% of thymocytes that were rescued by TCR engagement and matured to express either CD4 or CD8. CD8 SP cells were enriched from the thymus of three HLA-A2+ and three HLA-A2− individuals through magnetic depletion of CD4-expressing cells. Staining detected significant numbers of A2/Melan-A tetramer+ cells in all three HLA-A2+ cases (mean ± SD, 0.05 ± 0.006%; Fig. 7⇓A), but in none of the HLA-A2− samples (data not shown). A2/Flu-MA tetramer+ cells were never detected (Fig. 7⇓B). Interestingly, most of the A2/Melan-A tetramer+ thymocytes expressed AV2.1 (mean ± SD, 78 ± 20.2%) (Fig. 7⇓, A and C), but neither AV12+ nor AV24+ (Fig. 7⇓C). Altogether, these results indicate that the preferential usage of AV2 in forming the Melan-A-specific TCR is mostly imposed by intrathymic-positive selection.

FIGURE 7.
FIGURE 7.

Preferential use of TCRAV2 by the Melan-A-specific CD8 T cells in thymus. A and B, CD4-depleted thymocytes from three HLA-A2 thymus specimens were stained with anti-CD8, anti-AV2.1 mAbs, and A2/Melan-A (A) or A2/Flu-MA (B) tetramers. Bars represent the total percentages of tetramer+ cells; ▪ represent the percentages of AV2.1+ cells and ▦ the percentages of AV2.1− cells within the tetramer+ population. C, Representative double stainings of CD8 SP thymocytes from one HLA-A2 donor with anti-AV2.1, anti-AV12, anti-AV24 mAbs, and A2/Melan-A tetramers.

Discussion

The data reported in this study show that TCR-α is dominant in formation of the TCR repertoire specific for the human self Ag Melan-A/MART-1 and support the view that Ag recognition is mediated predominantly by TCR-α. The most substantial preservation of TCR-α structure was the strongly preferential use of a particular AV segment, whereas the CDR3 regions were less conserved. In fact, crystallography of the TCR-αβ/peptide-MHC complex has revealed that the CDR1, CDR2, and CDR3 αβ regions generally contribute to the buried surface area in the interface. However, dominance of the AV-encoded CDR1 and CDR2 domains in peptide recognition has since been established. This is exemplified by the D10 TCR, where 23 of 27 atomic contacts with the peptide involved AV and only four involved BV (6). This dominance had not been appreciated previously, although a generalized complex orientation mediated by the AV domain had been proposed (2). This is consistent with our results concerning recognition of a human self Ag and those of Yokosuka et al. (13) regarding recognition of foreign Ags. In single TCR transgenic mice models, those authors observed that a single TCR-α chain generates the same functional Ag specificity when paired to different TCR-β chains. Analogously, when we used Melan-A peptides to in vitro stimulate PBMC from various HLA-A2 patients, we observed that AV2-expressing cells were selectively enriched, whereas TCR-β chains remained highly polyclonal.

The high precursor frequency of naive Melan-A-specific cells provided a unique situation for assessment of the structural relationship between TCR and peptide/MHC ligand at the preimmune level. The number of Melan-A-specific cells entering the peripheral repertoire is exceptionally high, although many self mimics can contribute to this large precursor population predominantly determined by a high thymic output (27). The composition of self-peptide ligands in the thymus is critical in shaping the preimmune T cell repertoire, but is Melan-A expressed and processed intrathymically? There are divergent opinions regarding this question. It was demonstrated that the immunoproteosome does not process the Melan-A 26–35 peptide (45), but the standard proteosome should also be expressed by thymic dendritic cells. If Melan-A peptides are processed in the thymus, as negatively selecting ligands they would narrow the structural diversity of self-specific TCR. Interestingly, the preferential use of a highly selected AV segment, as we observed for Melan-A-specific cells, has been reported as a feature of the “tolerant” repertoire (46). This raises the question of whether AV2 is one of the few segments capable of conferring Melan-A specificity to a TCR, or one of the few AV segments determining a low enough TCR affinity for Melan-A to allow positive selection. In any case, from our data the intrathymic generation of the large Melan-A-specific T cell pool appears to be favored by the selection of a particular TCR.

Preferential use of AV2 by Melan-A-specific T cells has been occasionally documented in HLA-A2 melanoma patients. Impressive dominance of this segment in forming the Melan-A-specific TCR was shown in one report where six of nine CTL clones obtained from five patients expressed TCRAV2.1 (47). However, TCRBV-restricted usage was also noted and practically all subsequent studies regarding the Melan-A-specific T cell repertoire have concentrated their attention on the TCR-β alone (30, 31, 32, 33, 48, 49), presumably due to both technical reasons, i.e., lack of a large panel of AV-specific mAbs, and the persistent assumption that TCR-β is the main contributor to peptide recognition despite its recent rebuttal by crystallography. The only exception is one paper in which the clonally expanded Melan-A-specific TCR repertoire was monitored in a melanoma patient during immunization with melanoma-associated peptides (50); AV2-expressing cells were not detected among the clonally expanded populations. However, this patient already displayed a single monoclonal TCR-α transcript before immunization and all the repertoire studies were focused on clonally expanded cells, while in our case the AV2-expressing lymphocytes were mainly polyclonal even upon peptide-specific stimulation in vitro. A certain degree of heterogeneity among donors must also be taken in consideration. Our data suggest that AV2-containing TCRs are not the only ones involved in Melan-A recognition, and a few clones expressing other AV segments have been described (Ref. 51 and clone SCO126). Rather, it would seem more likely that AV2-containing TCRs have either a structural or a functional advantage during intrathymic-positive selection and peripheral expansion.

The immunodominance of Melan-A/MART-1 Ag in association with HLA-A2 has received particular attention from the immunotherapeutic standpoint, since this HLA allele is predominant in the North American Caucasian population and Melan-A reactivity is frequent in tumor-infiltrating lymphocytes from HLA-A2 melanoma patients. A specific allele/ligand combination, whose minimal terms are HLA-A*0201 and a single conserved glycine in position 5 shared by Melan-A peptides as well as by the large number of Melan-A epitope mimics so far described (29), has been proposed as the major contributor to this immunodominance (16). Our data now indicate that an additional structural constraint is imposed on the TCR for engagement with Melan-A peptides presented by HLA-A2. We would thus like to extend this previous observation by proposing that a unique allele/ligand/TCR-α combination, which includes TCRAV2, may be the molecular basis for Melan-A immunodominance.

In the light of our findings, the important role of TCR-α in determining Ag specificity, structurally suggested by crystallography, but functionally limited to “artificial” models, is now demonstrated for the first time in the case of a natural Ag response in humans and could represent the general molecular basis for recognition of both self and non-self Ags.

Acknowledgments

We thank Drs. Valeria Brazzelli and Giovanni Borroni (Instituto de Ricovero e Cura a Carattere Scientifico Policlinico San Matteo, Pavia, Italy); and Drs. Ausilia M. Manganoni and Graziella Carella (Spedali Civili, Brescia, Italy) for providing biological samples from melanoma and vitiligo patients; Divisione di Chirurgia Pediatrica (Ospedale Infantile Regina Margherita, Turin, Italy) for thymic biopsies; and Dr. Miriam Martinetti (IRCCS Policlinico San Matteo) for HLA-typing. We also thank Dr. Guido Forni (Department of Clinical and Biological Sciences, University of Turin, Orbassano, Italy) for careful critical reading.

Footnotes

  • ↵1 Address correspondence and reprint requests to Dr. Claudia Giachino, Department of Clinical and Biological Sciences, University of Turin, Regione Gonzole 10, 10043 Orbassano, Italy. E-mail address: claudia.giachino{at}unito.it

  • ↵2 Abbreviations used in this paper: CDR, complementarity-determining region; SP, single positive.

  • Received August 7, 2002.
  • Accepted September 27, 2002.

References

  1. ↵
    Kreuwel, H. T., L. A. Sherman. 2001. The T-cell repertoire available for recognition of self-antigens. Curr. Opin. Immunol. 13: 639
    OpenUrlCrossRefPubMed
  2. ↵
    Davis, M. M., P. J. Bjorkman. 1988. T-cell antigen receptor genes and T-cell recognition. Nature 334: 395
    OpenUrlCrossRefPubMed
  3. ↵
    Garcia, K. C., M. Degano, R. L. Stanfield, A. Brunmark, M. R. Jackson, P. A. Peterson, L. Teyton, I. A. Wilson. 1996. An αβ T cell receptor structure at 2.5 A and its orientation in the TCR-MHC complex. Science 274: 209
    OpenUrlAbstract/FREE Full Text
  4. ↵
    Garcia, K. C., M. Degano, L. R. Pease, M. Huang, P. A. Peterson, L. Teyton, I. A. Wilson. 1998. Structural basis of plasticity in T cell receptor recognition of a self peptide-MHC antigen. Science 279: 1166
    OpenUrlAbstract/FREE Full Text
  5. ↵
    Ding, Y. H., K. J. Smith, D. N. Garboczi, U. Utz, W. E. Biddison, D. C. Wiley. 1998. Two human T cell receptors bind in a similar diagonal mode to the HLA-A2/Tax peptide complex using different TCR amino acids. Immunity 8: 403
    OpenUrlCrossRefPubMed
  6. ↵
    Reinherz, E. L., K. Tan, L. Tang, P. Kern, J. Liu, Y. Xiong, R. E. Hussey, A. Smolyar, B. Hare, R. Zhang, et al 1999. The crystal structure of a T cell receptor in complex with peptide and MHC class II. Science 286: 1913
    OpenUrlAbstract/FREE Full Text
  7. ↵
    Ding, Y. H., B. M. Baker, D. N. Garboczi, W. E. Biddison, D. C. Wiley. 1999. Four A6-TCR/peptide/HLA-A2 structures that generate very different T cell signals are nearly identical. Immunity 11: 45
    OpenUrlCrossRefPubMed
  8. ↵
    Hennecke, J., A. Carfi, D. C. Wiley. 2000. Structure of a covalently stabilized complex of a human αβ T-cell receptor, influenza HA peptide and MHC class II molecule, HLA-DR1. EMBO J. 19: 5611
    OpenUrlAbstract
  9. ↵
    Wilson, I. A.. 1999. Perspectives: protein structure: class-conscious TCR?. Science 286: 1867
    OpenUrlFREE Full Text
  10. ↵
    Brandle, D., K. Burki, V. A. Wallace, U. H. Rohrer, T. W. Mak, B. Malissen, H. Hengartner, H. Pircher. 1991. Involvement of both T cell receptor Vα and Vβ variable region domains and α chain junctional region in viral antigen recognition. Eur. J. Immunol. 21: 2195
    OpenUrlCrossRefPubMed
  11. ↵
    Brandle, D., K. Brduscha-Riem, A. C. Hayday, M. J. Owen, H. Hengartner, H. Pircher. 1995. T cell development and repertoire of mice expressing a single T cell receptor α chain. Eur. J. Immunol. 25: 2650
    OpenUrlCrossRefPubMed
  12. ↵
    Zhang, W., S. Honda, F. Wang, T. P. DiLorenzo, A. M. Kalergis, D. A. Ostrov, S. G. Nathenson. 2001. Immunobiological analysis of TCR single-chain transgenic mice reveals new possibilities for interaction between CDR3α and an antigenic peptide bound to MHC class I. J. Immunol. 167: 4396
    OpenUrlAbstract/FREE Full Text
  13. ↵
    Yokosuka, T., K. Takase, M. Suzuki, Y. Nakagawa, S. Taki, H. Takahashi, T. Fujisawa, H. Arase, T. Saito. 2002. Predominant role of T cell receptor (TCR)-α chain in forming preimmune TCR repertoire revealed by clonal TCR reconstitution system. J. Exp. Med. 195: 991
    OpenUrlAbstract/FREE Full Text
  14. ↵
    Coulie, P. G., V. Brichard, A. Van Pel, T. Wolfel, J. Schneider, C. Traversari, S. Mattei, E. De Plaen, C. Lurquin, J. P. Szikora. 1994. A new gene coding for a differentiation antigen recognized by autologous cytolytic T lymphocytes on HLA-A2 melanomas. J. Exp. Med. 180: 35
    OpenUrlAbstract/FREE Full Text
  15. ↵
    Kawakami, Y., S. Eliyahu, C. H. Delgado, P. F. Robbins, L. Rivoltini, S. L. Topalian, T. Miki, S. A. Rosenberg. 1994. Cloning of the gene coding for a shared human melanoma antigen recognized by autologous T cells infiltrating into tumor. Proc. Natl. Acad. Sci. USA 91: 3515
    OpenUrlAbstract/FREE Full Text
  16. ↵
    Bettinotti, M. P., C. J. Kim, K. H. Lee, M. Roden, J. N. Cormier, M. Panelli, K. K. Parker, F. M. Marincola. 1998. Stringent allele/epitope requirements for MART-1/Melan A immunodominance: implications for peptide-based immunotherapy. J. Immunol. 161: 877
    OpenUrlAbstract/FREE Full Text
  17. ↵
    Kawakami, Y., S. Eliyahu, K. Sakaguchi, P. F. Robbins, L. Rivoltini, J. R. Yannelli, E. Appella, S. A. Rosenberg. 1994. Identification of the immunodominant peptides of the MART-1 human melanoma antigen recognized by the majority of HLA-A2-restricted tumor infiltrating lymphocytes. J. Exp. Med. 180: 347
    OpenUrlAbstract/FREE Full Text
  18. ↵
    Romero, P., N. Gervois, J. Schneider, P. Escobar, D. Valmori, C. Pannetier, A. Steinle, T. Wolfel, D. Lienard, V. Brichard, et al 1997. Cytolytic T lymphocyte recognition of the immunodominant HLA-A*0201-restricted Melan-A/MART-1 antigenic peptide in melanoma. J. Immunol. 159: 2366
    OpenUrlAbstract/FREE Full Text
  19. ↵
    Pittet, M. J., A. Zippelius, D. Valmori, D. E. Speiser, J. C. Cerottini, P. Romero. 2002. Melan-A/MART-1-specific CD8 T cells: from thymus to tumor. Trends Immunol. 23: 325
    OpenUrlCrossRefPubMed
  20. ↵
    Palermo, B., R. Campanelli, S. Mantovani, E. Lantelme, A. M. Manganoni, G. Carella, G. Da Prada, G. R. della Cuna, F. Romagne, L. Gauthier, et al 2001. Diverse expansion potential and heterogeneous avidity in tumor-associated antigen-specific T lymphocytes from primary melanoma patients. Eur. J. Immunol. 31: 412
    OpenUrlCrossRefPubMed
  21. ↵
    Palermo, B., R. Campanelli, S. Garbelli, S. Mantovani, G. Robustelli della Cuna, A. Necker, A. M. Manganoni, G. Carella, L. Rivoltini, E. Lantelme, and C. Giachino. 2002. CTL responses in melanoma through in vitro stimulation with the Melan-A peptide analogue A27.L: a qualitative analysis. Melanoma Res. In press.
  22. ↵
    Ogg, G. S., P. Rod Dunbar, P. Romero, J. L. Chen, V. Cerundolo. 1998. High frequency of skin-homing melanocyte-specific cytotoxic T lymphocytes in autoimmune vitiligo. J. Exp. Med. 188: 1203
    OpenUrlAbstract/FREE Full Text
  23. ↵
    Palermo, B., R. Campanelli, S. Garbelli, S. Mantovani, E. Lantelme, V. Brazzelli, M. Ardigo, G. Borroni, M. Martinetti, C. Badulli, et al 2001. Specific cytotoxic T lymphocyte responses against Melan-A/MART1, tyrosinase and gp100 in vitiligo by the use of major histocompatibility complex/peptide tetramers: the role of cellular immunity in the etiopathogenesis of vitiligo. J. Invest. Dermatol. 117: 326
    OpenUrlCrossRefPubMed
  24. ↵
    Pittet, M. J., D. Valmori, P. R. Dunbar, D. E. Speiser, D. Lienard, F. Lejeune, K. Fleischhauer, V. Cerundolo, J. C. Cerottini, P. Romero. 1999. High frequencies of naive Melan-A/MART-1-specific CD8+ T cells in a large proportion of human histocompatibility leukocyte antigen (HLA)-A2 individuals. J. Exp. Med. 190: 705
    OpenUrlAbstract/FREE Full Text
  25. ↵
    Dunbar, P. R., C. L. Smith, D. Chao, M. Salio, D. Shepherd, F. Mirza, M. Lipp, A. Lanzavecchia, F. Sallusto, A. Evans, et al 2000. A shift in the phenotype of melan-A-specific CTL identifies melanoma patients with an active tumor-specific immune response. J. Immunol. 165: 6644
    OpenUrlAbstract/FREE Full Text
  26. ↵
    Pittet, M. J., A. Zippelius, D. E. Speiser, M. Assenmacher, P. Guillaume, D. Valmori, D. Lienard, F. Lejeune, J. C. Cerottini, P. Romero. 2001. Ex vivo IFN-γ secretion by circulating CD8 T lymphocytes: implications of a novel approach for T cell monitoring in infectious and malignant diseases. J. Immunol. 166: 7634
    OpenUrlAbstract/FREE Full Text
  27. ↵
    Zippelius, A., M. J. Pittet, P. Batard, N. Rufer, M. de Smedt, P. Guillaume, K. Ellefsen, D. Valmori, D. Lienard, J. Plum, et al 2002. Thymic selection generates a large T cell pool recognizing a self-peptide in humans. J. Exp. Med. 195: 485
    OpenUrlAbstract/FREE Full Text
  28. ↵
    Loftus, D. J., C. Castelli, T. M. Clay, P. Squarcina, F. M. Marincola, M. I. Nishimura, G. Parmiani, E. Appella, L. Rivoltini. 1996. Identification of epitope mimics recognized by CTL reactive to the melanoma/melanocyte-derived peptide MART-1(27–35). J. Exp. Med. 184: 647
    OpenUrlAbstract/FREE Full Text
  29. ↵
    Loftus, D. J., P. Squarcina, M. B. Nielsen, C. Geisler, C. Castelli, N. Odum, E. Appella, G. Parmiani, L. Rivoltini. 1998. Peptides derived from self-proteins as partial agonists and antagonists of human CD8+ T-cell clones reactive to melanoma/melanocyte epitope MART1(27–35). Cancer Res. 58: 2433
    OpenUrlAbstract/FREE Full Text
  30. ↵
    Becker, J. C., P. Guldberg, J. Zeuthen, E. B. Brocker, P. T. Straten. 1999. Accumulation of identical T cells in melanoma and vitiligo-like leukoderma. J. Invest. Dermatol. 113: 1033
    OpenUrlCrossRefPubMed
  31. ↵
    Valmori, D., V. Dutoit, D. Lienard, F. Lejeune, D. Speiser, D. Rimoldi, V. Cerundolo, P. Y. Dietrich, J. C. Cerottini, P. Romero. 2000. Tetramer-guided analysis of TCR β-chain usage reveals a large repertoire of melan-A-specific CD8+ T cells in melanoma patients. J. Immunol. 165: 533
    OpenUrlAbstract/FREE Full Text
  32. ↵
    Schrama, D., M. H. Andersen, P. Terheyden, L. Schroder, L. O. Pedersen, P. thor Straten, J. C. Becker. 2001. Oligoclonal T-cell receptor usage of melanocyte differentiation antigen-reactive T cells in stage IV melanoma patients. Cancer Res. 61: 493
    OpenUrlAbstract/FREE Full Text
  33. ↵
    Dietrich, P. Y., P. R. Walker, A. L. Quiquerez, G. Perrin, V. Dutoit, D. Lienard, P. Guillaume, J. C. Cerottini, P. Romero, D. Valmori. 2001. Melanoma patients respond to a cytotoxic T lymphocyte-defined self-peptide with diverse and nonoverlapping T-cell repertoires. Cancer Res. 61: 2047
    OpenUrlAbstract/FREE Full Text
  34. ↵
    Ljunggren, H. G., N. J. Stam, C. Ohlen, J. J. Neefjes, P. Hoglund, M. T. Heemels, J. Bastin, T. N. Schumacher, A. Townsend, K. Karre. 1990. Empty MHC class I molecules come out in the cold. Nature 346: 476
    OpenUrlCrossRefPubMed
  35. ↵
    Valmori, D., J. F. Fonteneau, C. M. Lizana, N. Gervois, D. Lienard, D. Rimoldi, V. Jongeneel, F. Jotereau, J. C. Cerottini, P. Romero. 1998. Enhanced generation of specific tumor-reactive CTL in vitro by selected Melan-A/MART-1 immunodominant peptide analogues. J. Immunol. 160: 1750
    OpenUrlAbstract/FREE Full Text
  36. ↵
    Salter, R. D., A. M. Norment, B. P. Chen, C. Clayberger, A. M. Krensky, D. R. Littman, P. Parham. 1989. Polymorphism in the α3 domain of HLA-A molecules affects binding to CD8. Nature 338: 345
    OpenUrlCrossRefPubMed
  37. ↵
    Salter, R. D., R. J. Benjamin, P. K. Wesley, S. E. Buxton, T. P. Garrett, C. Clayberger, A. M. Krensky, A. M. Norment, D. R. Littman, P. Parham. 1990. A binding site for the T-cell co-receptor CD8 on the α3 domain of HLA-A2. Nature 345: 41
    OpenUrlCrossRefPubMed
  38. ↵
    Bodinier, M., M. A. Peyrat, C. Tournay, F. Davodeau, F. Romagne, M. Bonneville, F. Lang. 2000. Efficient detection and immunomagnetic sorting of specific T cells using multimers of MHC class I and peptide with reduced CD8 binding. Nat. Med. 6: 707
    OpenUrlCrossRefPubMed
  39. ↵
    Genevée, C., A. Diu, J. Nierat, A. Caignard, P. Y. Dietrich, L. Ferradini, S. Roman-Roman, F. Triebel, T. Hercend. 1992. An experimentally validated panel of subfamily-specific oligonucleotide primers (Vαβ1-w29/Vβ1-w24) for the study of human T cell receptor variable V gene segment usage by polymerase chain reaction. Eur. J. Immunol. 22: 1261
    OpenUrlCrossRefPubMed
  40. ↵
    Han, M., L. Harrison, P. Kehn, K. Stevenson, J. Currier, M. A. Robinson. 1999. Invariant or highly conserved TCR α are expressed on double-negative (CD3+CD4−CD8−) and CD8+ T cells. J. Immunol. 163: 301
    OpenUrlAbstract/FREE Full Text
  41. ↵
    Arden, B., S. P. Clark, D. Kabelitz, T. W. Mak. 1995. Human T-cell receptor variable gene segment families. Immunogenetics 42: 455
    OpenUrlPubMed
  42. ↵
    Heath, W. R., F. R. Carbone, P. Bertolino, J. Kelly, S. Cose, J. F. Miller. 1995. Expression of two T cell receptor α chains on the surface of normal murine T cells. Eur. J. Immunol. 25: 1617
    OpenUrlCrossRefPubMed
  43. ↵
    Alam, S. M., N. R. Gascoigne. 1998. Posttranslational regulation of TCR Vα allelic exclusion during T cell differentiation. J. Immunol. 160: 3883
    OpenUrlAbstract/FREE Full Text
  44. ↵
    D’Souza, S., D. Rimoldi, D. Lienard, F. Lejeune, J. C. Cerottini, P. Romero. 1998. Circulating Melan-A/Mart-1 specific cytolytic T lymphocyte precursors in HLA-A2+ melanoma patients have a memory phenotype. Int. J. Cancer 78: 699
    OpenUrlCrossRefPubMed
  45. ↵
    Van den Eynde, B. J., S. Morel. 2001. Differential processing of class-I-restricted epitopes by the standard proteasome and the immunoproteasome. Curr. Opin. Immunol. 13: 147
    OpenUrlCrossRefPubMed
  46. ↵
    Bouneaud, C., P. Kourilsky, P. Bousso. 2000. Impact of negative selection on the T cell repertoire reactive to a self-peptide: a large fraction of T cell clones escapes clonal deletion. Immunity 13: 829
    OpenUrlCrossRefPubMed
  47. ↵
    Sensi, M., C. Traversari, M. Radrizzani, S. Salvi, C. Maccalli, R. Mortarini, L. Rivoltini, C. Farina, G. Nicolini, T. Wolfel, et al 1995. Cytotoxic T-lymphocyte clones from different patients display limited T-cell-receptor variable-region gene usage in HLA-A2-restricted recognition of the melanoma antigen Melan-A/MART-1. Proc. Natl. Acad. Sci. USA 92: 5674
    OpenUrlAbstract/FREE Full Text
  48. ↵
    Strohal, R., C. Brna, U. Mossbacher, G. Fischer, H. Pehamberger, G. Stingl. 1998. First comparative delineation of the T cell receptor repertoire in primary and multiple subsequent/coexisting metastatic melanoma sites. J. Invest. Dermatol. 111: 1085
    OpenUrlCrossRefPubMed
  49. ↵
    Pisarra, P., R. Mortarini, S. Salvi, A. Anichini, G. Parmiani, M. Sensi. 1999. High frequency of T cell clonal expansions in primary human melanoma: involvement of a dominant clonotype in autologous tumor recognition. Cancer Immunol. Immunother. 48: 39
    OpenUrlCrossRefPubMed
  50. ↵
    Jager, E., M. Maeurer, H. Hohn, J. Karbach, D. Jager, Z. Zidianakis, A. Bakhshandeh-Bath, J. Orth, C. Neukirch, A. Necker, et al 2000. Clonal expansion of Melan A-specific cytotoxic T lymphocytes in a melanoma patient responding to continued immunization with melanoma-associated peptides. Int. J. Cancer 86: 538
    OpenUrlCrossRefPubMed
  51. ↵
    Cole, D. J., D. P. Weil, P. Shamamian, L. Rivoltini, Y. Kawakami, S. Topalian, C. Jennings, S. Eliyahu, S. A. Rosenberg, M. I. Nishimura. 1994. Identification of MART-1-specific T-cell receptors: T cells utilizing distinct T-cell receptor variable and joining regions recognize the same tumor epitope. Cancer Res. 54: 5265
    OpenUrlAbstract/FREE Full Text
View Abstract
Back to top

In this issue

Print
Download PDF
Article Alerts
Email Article
Citation Tools

Jump to section