Abstract
The specificity of recognition of pMHC complexes by T lymphocytes is determined by the V regions of the TCR α- and β-chains. Recent experimental evidence has suggested that Ag-specific TCR repertoires may exhibit a more Vα- than Vβ-restricted usage. Whether Vα usage is narrowed during immune responses to Ag or if, on the contrary, restricted Vα usage is already defined at the early stages of TCR repertoire selection, however, has remained unexplored. Here, we analyzed V and CDR3 TCR regions of single circulating naive T cells specifically detected ex vivo and isolated with HLA-A2/melan-A peptide multimers. Similarly to what was previously observed for melan-A-specific Ag-experienced T cells, we found a relatively wide Vβ usage, but a preferential Vα 2.1 usage. Restricted Vα 2.1 usage was also found among single CD8+ A2/melan-A multimer+ thymocytes, indicating that Vα-restricted selection takes place in the thymus. Vα 2.1 usage, however, was independent from functional avidity of Ag recognition. Thus, interaction of the pMHC complex with selected Vα-chains contributes to set the broad Ag specificity, as underlined by preferential binding of A2/melan-A multimers to Vα 2.1-bearing TCRs, whereas functional outcomes result from the sum of these with other interactions between pMHC complex and TCR.
T cells play a pivotal role in the adaptive immune responses by recognizing Ag-derived peptides bound to MHC molecules (pMHC complexes). The specificity of Ag recognition is provided by the TCR, a heterodimeric transmembrane complex composed of α- and β-chains generated by somatic recombination of germline-encoded V and J gene segments for the α-chain and V, D, and J gene segments for the β-chain (1, 2). Variability is concentrated in three complementarity-determining regions (CDRs)4; CDR1 and CDR2 are encoded within the V segments, whereas CDR3 is generated from the junction of the V and J elements for the α-chain and V, D, and J segments for the β-chain. Because of the imprecise juxtaposition of these latter, with variable deletion and random addition of nucleotides at the joined ends, the variability of the CDR3s is much greater than that in other variable regions.
The impact of TCR diversity on recognition of single antigenic pMHC complexes has been extensively investigated, and the relative contributions of TCR α- and β-chains have been addressed. Consistent with their high degree of diversity, CDR3α and -β regions have been shown to play an important role in Ag recognition. In a model of TCR α- or β-chain transgenic mice, immunization with altered peptide ligands resulted in changes in the CDR3 sequences of the α- and β-chains (3, 4, 5, 6). Interestingly, in this model, CTL used more restricted VJα than VDJβ regions (4). Recently, Yokosuka and colleagues (7) have analyzed the Ag-specific TCR-αβ pairs generated by in vitro transfection of a single TCR chain (α or β) with a variety of the other TCR gene chains. Their findings show that to recognize Ag in their selected antigenic systems, CTL had to use a certain TCR α-chain, but could use a variety of TCR β-chains. Together, these and other studies have suggested a dominant role of α-chain in Ag recognition by TCR. Moreover, the analyses of TCR-pMHC complex crystals (8, 9, 10, 11, 12) have shown that the α-chain usually makes more contacts with the peptide than the β-chain, thus providing a structural basis for its dominance.
Most studies that have addressed the above-mentioned issues have been performed in transgenic mouse models, on Ag-specific clones obtained from Ag-experienced T cell populations, or through in vitro selection procedures. Because of the low precursor frequency of naive T cells specific for most single Ags, which generally precludes their direct identification and isolation, the structural diversity of preimmune TCR repertoires available for single Ags has remained largely unexplored. Melan-A is a self-protein of unknown function that is expressed by the majority of malignant melanoma cells and by cells of the melanocytic lineage, but not by other normal cells (13, 14). HLA-A2 (A2)-restricted, melan-A-specific CD8+ T cells have been shown to recognize primarily the melan-A26–35 region (15). Using A2/peptide multimers incorporating the melan-A26–35 A27L analog (A2/melan-A multimers hereafter), we have previously shown that a significant proportion of A2/melan-A multimer+ CD8+ T cells can often be directly visualized ex vivo in both tumor-infiltrated lymph nodes and circulating lymphocytes of melanoma patients as well as in healthy individuals (16, 17). In the latter case, circulating A2/melan-A multimer+ CD8+ T cells are phenotypically naive (CD45RA+ CD45RO− CCR7+) and may constitute up to 10−3 of circulating CD8+ T cells. We have recently reported that this large pool is not generated through peripheral T cell expansion, but is mainly the result of the thymic output of a high number of specific T cell precursors (18). This represents the only known naive Ag-specific T cell repertoire directly identifiable ex vivo in humans and is, therefore, particularly attractive for comprehensive functional and molecular analyses at a clonal level.
We and others have previously observed that melan-A-specific T cells isolated from melanoma patients exhibit a large and diverse TCR repertoire in terms of both V regions usage and clonal composition (15, 19, 20, 21, 22). Frequent usage of selected Vα (i.e., Vα 2.1) and Vβ (i.e., Vβ 14) regions, however, has also been reported (19, 21, 22). A more recent survey of TCR Vα- and Vβ-chain usage by a large panel of melan-A-specific T cells derived from tumor-infiltrating and peripheral blood lymphocytes of melanoma patients has underlined the frequent usage (70%) of Vα 2.1 (23). Whether this Vα restriction results from narrowing of the TCR repertoire by affinity focusing during Ag-driven immune responses or reflects instead a structural constraint already present in the preimmune TCR repertoire, however, has remained unexplored. To address this question here, we analyzed V and CDR3 TCR regions of A2/melan-A multimer+ naive T cells isolated from circulating CD8+ T lymphocytes of A2+ healthy donors at the clonal level. We found a relatively large Vβ usage, but a highly preferential Vα 2.1 usage. Preferential Vα 2.1 usage was not due to peripheral homeostatic expansion, since a similar restriction was also found among cord blood lymphocytes and single CD8+ A2/melan-A multimer+ thymocytes. However, there was no correlation between Vα 2.1 usage and functional avidity of Ag recognition. Together, our results indicate that the interaction of Vα-chain with pMHC complex contributes to set the broad TCR specificity for Ag, as underlined by Ag-specific binding of A2/peptide multimers to selected Vα-chains, but does not determine functional outcomes.
Materials and Methods
Cells and Ag recognition assay
A2/melan-A multimer+ CD8+ T cells were purified ex vivo from PBMC, cord blood cells, or single CD8+ thymocytes of HLA-A*0201 (A2)-expressing donors by flow cytometry cell sorting and were cloned by limiting dilution culture in the presence of PHA, allogenic irradiated PBMC, and recombinant human IL-2 as previously described (24). Clones were subsequently expanded by periodic (3–4 wk) restimulation into microtiter plates. Ag recognition was assessed using a chromium release assay (CTL assay). The A2+ human mutant cell line CEMx721.T2 (T2) (25) or the melanoma cell lines Me 275 (A2+ melan-A+) and NA8-MEL (A2+ melan-A−) were used as targets. Briefly, after labeling with 51Cr for 1 h at 37°C, followed by extensive washing, target cells (1000/well) were incubated with effector cells at the indicated E:T cell ratio for 4 h at 37°C in V-bottom microwells in the absence or the presence of the indicated synthetic peptide (1 μM). In peptide titration experiments, target cells were incubated with effectors at an E:T cell ratio of 10:1 in the presence of serial dilutions of the indicated peptide. Chromium release was measured in the supernatant of the cultures using a gamma counter. The percent specific lysis was calculated as: 100 × [(experimental − spontaneous release)/(total − spontaneous release)].
A2/peptide multimers and flow cytometry immunofluorescence analysis
PE-conjugated multimeric A2/peptide complexes containing the melan-A-enhanced peptide analog 26–35 A27L (ELAGIGILTV) (26) were synthesized as previously described (16, 27). Samples were stained with multimers at the indicated dose in PBS containing 0.2% BSA for 1 h at room temperature, washed once in the same buffer, stained with mAbs where indicated for 30 min at 4°C, washed again, and analyzed by flow cytometry. Anti-CD8 (SK1) and anti-CD45RA mAbs were purchased from BD Biosciences (San Jose, CA). Anti-CCR7 mAb 3D12 was provided by Dr. M. Lipp (Berlin, Germany). Data analysis was performed using CellQuest software (BD Biosciences).
Amplification of TCRVα and TCRVβ transcripts and sequencing of the PCR products
Total mRNA was prepared from monoclonal or polyclonal CD8+ A2/melan-A multimer+ populations using TRIzol (Life Technologies, Paisley, U.K.) and converted to cDNA by standard methods using reverse transcriptase and an oligo(dT) primer as previously described (22, 28). cDNAs were amplified in nonsaturating PCR conditions (30 cycles) with a panel of previously validated 5′ sense primers specific for 29 Vα and 22 Vβ subfamilies and one 3′ antisense primer specific for the corresponding C gene segment (29). PCR products were cloned with the TOPO TA cloning kit (Invitrogen, Carlsbad, CA). One Shot TOP10 chemically competent Escherichia coli (Invitrogen, San Diego, CA) were transformed and plated for blue/white color selection on medium containing 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside. Plasmid DNA was extracted from white colonies using the Qiagen Plasmid Mini Kit (Qiagen, Hilden, Germany) and sequenced using a Thermo Sequenase fluorescent-labeled primer cycle sequencing kit with 7-deaza-dGT7 (Amersham Pharmacia Biotech, Little Chafont, U.K.). For each plasmid, the sequencing reaction was performed in both directions using a Cα primer or the Vα 2-specific primer, respectively. A fraction of sequence analyses was performed by Microsynth. The sequence of highly homologous TCR clonotypes (i.e., the ones with only one or two nucleotide differences) was checked by repeating the sequence analysis starting from the total mRNA, to avoid artifacts. The TCR nomenclature used was according to Arden et al. (30).
Results and Discussion
To substantiate the frequent Vα 2.1 usage by Ag-experienced melan-A-specific CTL, we analyzed seven CTL clones representative of distinct melan-A-specific T cell clonotypes that were predominant among Ag-experienced (CD45RA− CD45RO+) A2/melan-A multimer+ T cells from a melanoma patient vaccinated with peptide melan-A26–35 (31). As illustrated in Fig. 1,A, four of seven clones used the Vα 2.1 gene segment, whereas the Jα segments used and the CDR3α sequences were different for each clone. Interestingly, three of the four Vα 2.1+ and one of the three Vα 2.1− clones expressed Vβ 14 and used the same Jβ gene segment, whereas CDR3β sequences were different for each clone. It is of note that all CTL clones corresponding to the different clonotypes, including those using V segments other than Vα 2.1 and Vβ 14, recognized melan-A peptides with high avidity and exhibited high tumor reactivity (Fig. 1, A and B).
TCR Vα and Vβ usage, CDR3s sequence, functional avidity of Ag recognition, and tumor recognition of melan-A-specific CTL clones from LAU 337. A, TCR Vα, Vβ usage, and CDR3α and -β size and sequence were assessed as detailed in Materials and Methods. Peptide recognition was assessed in a standard 4-h chromium release assay using T2 cells as targets at a E:T cell ratio of 10:1. Tumor recognition was similarly assessed using Me 275 (A2+ melan-A+) and NA8-MEL (A2+ melan-A−) tumor cell lines as targets at the indicated lymphocyte to target cell ratio. Data obtained for clones representative of seven distinct clonotypes are summarized. B, Recognition of peptides melan-A26–35 and melan-A27–35 is shown for two representative clones: LAU 337 1D3 (Vα 2.1+) and LAU 337 5B3 (Vα 2.1−).
TCR Vα and Vβ usage, CDR3s sequence, functional avidity of Ag recognition, and tumor recognition of melan-A-specific CTL clones from LAU 337. A, TCR Vα, Vβ usage, and CDR3α and -β size and sequence were assessed as detailed in Materials and Methods. Peptide recognition was assessed in a standard 4-h chromium release assay using T2 cells as targets at a E:T cell ratio of 10:1. Tumor recognition was similarly assessed using Me 275 (A2+ melan-A+) and NA8-MEL (A2+ melan-A−) tumor cell lines as targets at the indicated lymphocyte to target cell ratio. Data obtained for clones representative of seven distinct clonotypes are summarized. B, Recognition of peptides melan-A26–35 and melan-A27–35 is shown for two representative clones: LAU 337 1D3 (Vα 2.1+) and LAU 337 5B3 (Vα 2.1−).
To determine whether the frequent Vα 2.1 usage by Ag-experienced melan-A-specific CTL is the consequence of repertoire selection occurring during immune response or reflects instead a bias already present in the preimmune repertoire, we analyzed V and CDR3 TCR regions of A2/melan-A multimer+ T cells isolated from circulating CD8+ T cells from a healthy donor (HD 421) by ex vivo multimer-guided cell sorting and cloned by limiting dilution in the presence of PHA as described previously (24). CD8+ A2/melan-A multimer+ T cells constituted ∼0.1% of the total CD8+ T cells from HD 421 and exhibited a naive (CD45RA+ CCR7+) phenotype (Fig. 2,A). The 30 HD 421-derived T cell clones analyzed here were specifically stained by A2/melan-A multimers compared with background staining on clones of unrelated specificity (e.g., clone Flu MA NM55 specific for peptide Flu-MA58–66), albeit the intensity of A2/melan-A multimer staining was variable among clones (Fig. 2,B). However, as reported recently, the clones largely differed in terms of both functional avidity of peptide recognition and tumor reactivity (32) and could be divided, according to these characteristics, into three functional groups (Fig. 2, C and D): group 1, clones that efficiently recognized melan-A26–35 A27L analog and melan-A parental peptides in the CTL assay and specifically lysed melan-A+ tumors (e.g., clone 2/4A12); group 2, clones that efficiently recognized melan-A26–35 A27L analog, but only poorly or did not recognize melan-A parental peptides and failed to lyse melan-A+ tumors (e.g., clone 2/5G9); and group 3, clones that very poorly or did not recognize melan-A26–35 A27L analog, did not recognize melan-A parental peptides, and failed to lyse melan-A+ tumors (e.g., clone 2/6F7). Clones from the three groups were efficiently cross-stained with multimers incorporating melan-A parental peptides (32). It is noteworthy that the lytic potential of clones in group 3 was comparable to that of clones in the other groups, as assessed in redirected lysis experiments (not shown). Thus, these clones specifically bind A2/melan-A multimers, but display a functional avidity of peptide recognition that is only poorly or not detectable in CTL assay. This suggests that their TCRs share a certain level of similarity with other melan-A-specific TCRs that is sufficient for A2/melan-A multimer binding, but not for detectable peptide recognition. In support of this interpretation, several clones of group 3 were able to efficiently cross-recognize melan-A-related sequences retrieved through a combinatorial peptide library-based approach (32).
Generation and functional characterization of preimmune ex vivo-derived A2/melan-A multimer+ T cell clones. A, PBMC from healthy donor HD 421 were stained ex vivo with PE-labeled A2/melan-A multimers (4.5 μg/ml) together with anti-CD8-FITC, anti-CD45RA-PE-Texas Red, and anti-CCR7-allophycocyanin mAbs. Left panel, The number in the upper right quadrant represents the percentage of multimer+ cells among CD8+ T cells. Right panel, Numbers in quadrants represent the percentage of multimer+ CD8+ T cells with the corresponding phenotype. B, CD8+ T cell clones isolated from PBMC of healthy donor HD 421 by ex vivo multimer-guided FACS sorting were simultaneously stained (using PE) with A2/melan-A multimers (4.5 μg/ml) for 1 h at room temperature. The influenza matrix peptide FLU-MA58–66-specific clone (NM55) was similarly stained as an internal control. The mean fluorescence intensity (MFI) is shown for all populations. C, Recognition of peptides melan-A27–35, melan-A26–35, and melan-A26–35 A27L by clones 2/4A12, 2/5G9, and 2/6F7 was assessed as described in Fig. 1. D, Tumor recognition was assessed as described in Fig. 1 in the presence (+P) or the absence (−P) of exogenously added melan-A26–35 A27L peptide.
Generation and functional characterization of preimmune ex vivo-derived A2/melan-A multimer+ T cell clones. A, PBMC from healthy donor HD 421 were stained ex vivo with PE-labeled A2/melan-A multimers (4.5 μg/ml) together with anti-CD8-FITC, anti-CD45RA-PE-Texas Red, and anti-CCR7-allophycocyanin mAbs. Left panel, The number in the upper right quadrant represents the percentage of multimer+ cells among CD8+ T cells. Right panel, Numbers in quadrants represent the percentage of multimer+ CD8+ T cells with the corresponding phenotype. B, CD8+ T cell clones isolated from PBMC of healthy donor HD 421 by ex vivo multimer-guided FACS sorting were simultaneously stained (using PE) with A2/melan-A multimers (4.5 μg/ml) for 1 h at room temperature. The influenza matrix peptide FLU-MA58–66-specific clone (NM55) was similarly stained as an internal control. The mean fluorescence intensity (MFI) is shown for all populations. C, Recognition of peptides melan-A27–35, melan-A26–35, and melan-A26–35 A27L by clones 2/4A12, 2/5G9, and 2/6F7 was assessed as described in Fig. 1. D, Tumor recognition was assessed as described in Fig. 1 in the presence (+P) or the absence (−P) of exogenously added melan-A26–35 A27L peptide.
As assessed by RT-PCR analysis, 25 of the 30 clones analyzed (83%) expressed the Vα 2.1 gene segment (Table I). The latter was rearranged with a large number (15) of different Jα gene segments, albeit some of those were slightly over-represented (e.g.. Jα 43, Jα 35). Overall, there was a general lack of restriction in both CDR3α size (which ranged from three to 10 amino acids) and sequences. However, sequence similarities were found within defined Vα−Jα rearrangements to a variable extent, going from identity (e.g., the two Vα 2.1-Jα 41-using clones) to relatively large differences (e.g., the Vα 2.1-Jα 43-using clones). In several cases (e.g., Jα 35-using clones 2/7E8 and 2/5H9 or Jα 45-using clones 2/5G9 and 2/7B12), CDR3α sequences differed by only one amino acid, as the result, in each case, of a single nucleotide difference (not shown). In contrast with the highly restricted Vα usage, the Vβ repertoire was more heterogeneous, with 13 different Vβ gene segments used, although a relatively more frequent usage of some Vβ gene segments was noticeable (e.g., Vβ 14 used by six clones and Vβ 7 used by five clones). In addition, there was no apparent restriction in Jβ usage or evident constraints on CDR3 size and sequence.
TCR Vα/β usage of A2/melan-A multimer+ CD8+ T cells in healthy donor HD 421
Clone . | Vα . | CDR3α . | . | . | Jα . | Vβ . | CDR3β . | . | . | Jβ . | ||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
Group 1 | ||||||||||||||
2/3B9 | 2.1 | CAV | NVWGAGNML | TFG | 39 | 7.2 | CAS | SGQGVGNEQ | FFG | 2.1 | ||||
2/1D5 | 2.1 | CAV | SNSGYAL | NFG | 41 | 14.1 | CAS | SRSPDTQ | YFG | 2.3 | ||||
2/4A12 | 2.1 | CAP | HSGGGADGL | TFG | 45 | 3.1 | CAS | RMIGYEQ | YFG | 2.7 | ||||
2/7B12 | 2.1 | CAV | GPGGFKT | IFG | 9 | 15.1 | CAS | SDPTSPNEQ | FFG | 2.1 | ||||
2/4G7 | 2.1 | CAV | SPDYKL | SFG | 20 | 2.1 | CAS | ALPGPITGSEA | FFG | 1.1 | ||||
2/1A7 | 2.1 | CAV | NKGFGNVL | HCG | 35 | 17.1 | CAS | STRDRGYEQ | YFG | 2.7 | ||||
2/7C2 | 2.1 | CAL | SNSGYAL | NFG | 41 | 14.1 | CAS | SQEGGAFVDTQ | YFG | 2.3 | ||||
2/7E8 | 2.1 | CAV | MIGFGNVL | HCG | 35 | 14.1 | CAS | SLGAGIVETQ | YFG | 2.5 | ||||
2/5H9 | 2.1 | CAV | SIGFGNVL | HCG | 35 | 14.1 | CAS | SLLGGSTDTQ | YFG | 2.3 | ||||
4B4 | 2.1 | CAV | RDDM | RFG | 43 | 2.1 | CAS | ASAQLGNTI | YFG | 1.3 | ||||
2/4D7 | 2.1 | CAV | TGANNL | FFG | 36 | 3.1 | CAS | SLLGPGQPQ | HFG | 1.5 | ||||
2/2A1 | nda | 9.1 | CAS | SRSPDTQ | YFG | 2.3 | ||||||||
Group 2 | ||||||||||||||
2/5F1 | 2.1 | CAF | FPREDKI | IFG | 30 | 14.1 | CAS | SPPGVNTEA | FFG | 1.1 | ||||
2/5B9 | 2.1 | CAV | RDDKI | IFG | 30 | 13.1 | CAS | MTSGIVADTQ | YFG | 2.3 | ||||
2/4G9 | 2.1 | CAV | GDYKL | SFG | 20 | 2.1 | ICS | ASPGGPNEQ | FFG | 2.1 | ||||
2/5B6 | 2.1 | CAG | NTGFQKL | VFG | 8 | 1.1 | CAS | SVDSGSSYNEQ | FFG | 2.1 | ||||
2/5G1 | 2.1 | CAV | SRNNNDM | RFG | 43 | 14.1 | CAS | SELAGGTDTQ | YFG | 2.3 | ||||
2/7G1 | 2.1 | CAM | SQSNFGNEKL | TFG | 48 | 13.1 | CAS | KGGEGLDTQ | YFG | 2.3 | ||||
7F9 | 2.1 | CAV | SQGANNL | FFG | 36 | nd | ||||||||
2/3A4 | 2.1 | CAV | RTDSWGKL | QFG | 24 | 23.1 | CAS | SGGSTTETQ | YFG | 2.5 | ||||
2/5G9 | 2.1 | CAA | DSGGGADGL | TFG | 45 | 1.1 | CAS | SAGLLGGNTI | YFG | 1.3 | ||||
1E3 | 2.1 | CAV | MGNTPL | VFG | 29 | 16.1 | CAS | SQDSWVSGNTI | YFG | 1.3 | ||||
2/6C7 | 21.1 | CAA | SANGGKI | IFG | 30 | 7.1 | CAS | SQGPLAGEAQ | FFG | 2.1 | ||||
2/2C3 | 10.1 | CAG | SMNYGGSQGNL | IFG | nd | 7.3 | CAS | SQEGFGVSPQPQ | HFG | 1.5 | ||||
3E3 | 14.1 | CAF | GGSQGNL | IFG | nd | 7.3 | CAS | SQESFGLGLYEQ | YFG | 2.7 | ||||
Group 3 | ||||||||||||||
2/2C12 | 2.1 | CAM | SRSNFGNEKL | TFG | 48 | 9.1 | CAS | SQGGITHNEQ | FFG | 2.1 | ||||
10D4 | 2.1 | CAV | PPNTGNQF | YFG | 49 | 7.1 | CAS | SQEDNRAQ | YFG | 2.5 | ||||
2/6F7 | 2.1 | CAV | GDM | RFG | 43 | 13.1 | CAS | SYSPANYGY | HFG | nd | ||||
2/2H11 | 2.1 | CAV | KDKL | IFG | 34 | 17.1 | CAS | SQTGVGETQ | YFG | 2.5 | ||||
2/2G11 | nd | 5.2 | CAS | SPPLSNYGS | HFG | nd |
Clone . | Vα . | CDR3α . | . | . | Jα . | Vβ . | CDR3β . | . | . | Jβ . | ||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
Group 1 | ||||||||||||||
2/3B9 | 2.1 | CAV | NVWGAGNML | TFG | 39 | 7.2 | CAS | SGQGVGNEQ | FFG | 2.1 | ||||
2/1D5 | 2.1 | CAV | SNSGYAL | NFG | 41 | 14.1 | CAS | SRSPDTQ | YFG | 2.3 | ||||
2/4A12 | 2.1 | CAP | HSGGGADGL | TFG | 45 | 3.1 | CAS | RMIGYEQ | YFG | 2.7 | ||||
2/7B12 | 2.1 | CAV | GPGGFKT | IFG | 9 | 15.1 | CAS | SDPTSPNEQ | FFG | 2.1 | ||||
2/4G7 | 2.1 | CAV | SPDYKL | SFG | 20 | 2.1 | CAS | ALPGPITGSEA | FFG | 1.1 | ||||
2/1A7 | 2.1 | CAV | NKGFGNVL | HCG | 35 | 17.1 | CAS | STRDRGYEQ | YFG | 2.7 | ||||
2/7C2 | 2.1 | CAL | SNSGYAL | NFG | 41 | 14.1 | CAS | SQEGGAFVDTQ | YFG | 2.3 | ||||
2/7E8 | 2.1 | CAV | MIGFGNVL | HCG | 35 | 14.1 | CAS | SLGAGIVETQ | YFG | 2.5 | ||||
2/5H9 | 2.1 | CAV | SIGFGNVL | HCG | 35 | 14.1 | CAS | SLLGGSTDTQ | YFG | 2.3 | ||||
4B4 | 2.1 | CAV | RDDM | RFG | 43 | 2.1 | CAS | ASAQLGNTI | YFG | 1.3 | ||||
2/4D7 | 2.1 | CAV | TGANNL | FFG | 36 | 3.1 | CAS | SLLGPGQPQ | HFG | 1.5 | ||||
2/2A1 | nda | 9.1 | CAS | SRSPDTQ | YFG | 2.3 | ||||||||
Group 2 | ||||||||||||||
2/5F1 | 2.1 | CAF | FPREDKI | IFG | 30 | 14.1 | CAS | SPPGVNTEA | FFG | 1.1 | ||||
2/5B9 | 2.1 | CAV | RDDKI | IFG | 30 | 13.1 | CAS | MTSGIVADTQ | YFG | 2.3 | ||||
2/4G9 | 2.1 | CAV | GDYKL | SFG | 20 | 2.1 | ICS | ASPGGPNEQ | FFG | 2.1 | ||||
2/5B6 | 2.1 | CAG | NTGFQKL | VFG | 8 | 1.1 | CAS | SVDSGSSYNEQ | FFG | 2.1 | ||||
2/5G1 | 2.1 | CAV | SRNNNDM | RFG | 43 | 14.1 | CAS | SELAGGTDTQ | YFG | 2.3 | ||||
2/7G1 | 2.1 | CAM | SQSNFGNEKL | TFG | 48 | 13.1 | CAS | KGGEGLDTQ | YFG | 2.3 | ||||
7F9 | 2.1 | CAV | SQGANNL | FFG | 36 | nd | ||||||||
2/3A4 | 2.1 | CAV | RTDSWGKL | QFG | 24 | 23.1 | CAS | SGGSTTETQ | YFG | 2.5 | ||||
2/5G9 | 2.1 | CAA | DSGGGADGL | TFG | 45 | 1.1 | CAS | SAGLLGGNTI | YFG | 1.3 | ||||
1E3 | 2.1 | CAV | MGNTPL | VFG | 29 | 16.1 | CAS | SQDSWVSGNTI | YFG | 1.3 | ||||
2/6C7 | 21.1 | CAA | SANGGKI | IFG | 30 | 7.1 | CAS | SQGPLAGEAQ | FFG | 2.1 | ||||
2/2C3 | 10.1 | CAG | SMNYGGSQGNL | IFG | nd | 7.3 | CAS | SQEGFGVSPQPQ | HFG | 1.5 | ||||
3E3 | 14.1 | CAF | GGSQGNL | IFG | nd | 7.3 | CAS | SQESFGLGLYEQ | YFG | 2.7 | ||||
Group 3 | ||||||||||||||
2/2C12 | 2.1 | CAM | SRSNFGNEKL | TFG | 48 | 9.1 | CAS | SQGGITHNEQ | FFG | 2.1 | ||||
10D4 | 2.1 | CAV | PPNTGNQF | YFG | 49 | 7.1 | CAS | SQEDNRAQ | YFG | 2.5 | ||||
2/6F7 | 2.1 | CAV | GDM | RFG | 43 | 13.1 | CAS | SYSPANYGY | HFG | nd | ||||
2/2H11 | 2.1 | CAV | KDKL | IFG | 34 | 17.1 | CAS | SQTGVGETQ | YFG | 2.5 | ||||
2/2G11 | nd | 5.2 | CAS | SPPLSNYGS | HFG | nd |
nd, not defined.
A similar analysis was performed for another A2+ healthy donor (HD 009). In this case all A2/melan-A multimer+ CD8+ T cell clones analyzed (a total of 18, generated and functionally characterized as previously described for HD 421) expressed the Vα 2.1 gene segment (Table II). It is noteworthy that six of 43 clones (14%) obtained from the total naive CD8+ (CCR7+CD45RA+) population of HD 009 and analyzed as an internal experimental control expressed the Vα 2.1 gene segment. Similarly to what was observed with A2/melan-A multimer+ clones from HD 421, we found a diverse Jα usage (10 different Jα used) together with over-representation of certain Jα (e.g., Jα 45 expressed by five clones). CDR3α size ranged from five to 11 amino acids, and CDR3α sequences were rather diverse. However, again, more or less prominent sequence similarities were found within defined Vα−Jα rearrangements. Vβ usage was rather diverse (eight different Vβ gene segments used), albeit a high proportion of clones (seven of 18, 38%) used in this case a single Vβ (Vβ 3.1). Also, Jβ segment usage was diverse; however, in contrast with what observed when analyzing the CDR3α, no significant similarity was found when comparing sequences of defined Vβ−Jβ rearrangements.
TCR Vα/β usage of A2/melan-A multimer+ CD8+ T cells in healthy donor HD 009
Clone . | Vα . | CDR3α . | . | . | Jα . | Vβ . | CDR3β . | . | . | Jβ . | ||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
Group 1 | ||||||||||||||
15 | 2.1 | CAV | KGWSGGGADGL | TFG | 45 | 13.2 | VYFCAS | SSGSGSPL | HFG | 1.6 | ||||
18 | 2.1 | CAV | GAGGGSQGNL | IFG | 28 | 3.1 | MYLCAS | SPAVGWGY | TFG | 1.2 | ||||
Group 2 | ||||||||||||||
1 | 2.1 | CAA | TGRDGQKL | LFG | 16 | 13.1 | VYFCAS | GQGTGQPQ | HFG | 1.5 | ||||
4 | 2.1 | CAV | DSGGGADGL | TFG | 45 | 3.1 | MYLCAS | SLPLSLDNEQ | FFG | 2.1 | ||||
3 | 2.1 | CAV | TTGGSYIP | TFG | 6 | 5 | YLCAS | SLGPGGDGY | TFG | 1.2 | ||||
10 | 2.1 | CAV | NVGGGADGL | TFG | 45 | 3.1 | MYLCAS | RVGGLNTGEL | FFG | 2.2 | ||||
11 | 2.1 | CAP | NSGGGADGL | TFG | 45 | 5.2 | LYLCAS | SPGQGLYEQ | YFG | 2.7 | ||||
14 | 2.1 | CAV | KMYSGGGADGL | TFG | 45 | 17.1 | FYLCAS | SIGGGGYEQ | YFG | 2.7 | ||||
16 | 2.1 | CAV | GGGADGL | TFG | 45 | 5 | LYLCAS | SLAISGAGEL | FFG | 2.2 | ||||
6 | 2.1 | CAV | GGGSQGNL | IFG | 28 | 3.1 | MYLCAS | SLMALNANEQ | YFG | 2.7 | ||||
7 | 2.1 | CAV | NANTNAGKS | TFG | 27 | 3.1 | MYLCAS | SLTMGSGY | TFG | 1.2 | ||||
8 | 2.1 | CAA | PNYGQNF | VFG | 26 | 3.1 | MYLCAS | SPGGVGSPL | HFG | 1.6 | ||||
9 | 2.1 | CAV | NNARL | MFG | 31 | 14.1 | LYFCAS | SESTGWDTQ | YFG | 2.3 | ||||
13 | 2.1 | CAV | YTGGGNKL | TFG | 10 | 3.1 | MYLCVS | ALGLGQPQ | HFG | 1.5 | ||||
Group 3 | ||||||||||||||
2 | 2.1 | CAV | AISNFGNEKL | TFG | 48 | 2 | FYICS | ARGSNQPQ | HFG | 1.5 | ||||
5 | 2.1 | CAV | GSQGNL | IFG | 28 | 5.2 | LYLCAS | SFSAAYEQ | YFG | 2.7 | ||||
17 | 2.1 | CAV | NMDFGNEKL | TFG | 48 | 5 | LYFCAS | SLDAGANVL | TFG | 2.6 | ||||
12 | 2.1 | CAG | GAGGTSYGKL | TFG | 52 | 1.1 | LYFCAS | NLGEGETQ | YFG | 2.5 |
Clone . | Vα . | CDR3α . | . | . | Jα . | Vβ . | CDR3β . | . | . | Jβ . | ||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
Group 1 | ||||||||||||||
15 | 2.1 | CAV | KGWSGGGADGL | TFG | 45 | 13.2 | VYFCAS | SSGSGSPL | HFG | 1.6 | ||||
18 | 2.1 | CAV | GAGGGSQGNL | IFG | 28 | 3.1 | MYLCAS | SPAVGWGY | TFG | 1.2 | ||||
Group 2 | ||||||||||||||
1 | 2.1 | CAA | TGRDGQKL | LFG | 16 | 13.1 | VYFCAS | GQGTGQPQ | HFG | 1.5 | ||||
4 | 2.1 | CAV | DSGGGADGL | TFG | 45 | 3.1 | MYLCAS | SLPLSLDNEQ | FFG | 2.1 | ||||
3 | 2.1 | CAV | TTGGSYIP | TFG | 6 | 5 | YLCAS | SLGPGGDGY | TFG | 1.2 | ||||
10 | 2.1 | CAV | NVGGGADGL | TFG | 45 | 3.1 | MYLCAS | RVGGLNTGEL | FFG | 2.2 | ||||
11 | 2.1 | CAP | NSGGGADGL | TFG | 45 | 5.2 | LYLCAS | SPGQGLYEQ | YFG | 2.7 | ||||
14 | 2.1 | CAV | KMYSGGGADGL | TFG | 45 | 17.1 | FYLCAS | SIGGGGYEQ | YFG | 2.7 | ||||
16 | 2.1 | CAV | GGGADGL | TFG | 45 | 5 | LYLCAS | SLAISGAGEL | FFG | 2.2 | ||||
6 | 2.1 | CAV | GGGSQGNL | IFG | 28 | 3.1 | MYLCAS | SLMALNANEQ | YFG | 2.7 | ||||
7 | 2.1 | CAV | NANTNAGKS | TFG | 27 | 3.1 | MYLCAS | SLTMGSGY | TFG | 1.2 | ||||
8 | 2.1 | CAA | PNYGQNF | VFG | 26 | 3.1 | MYLCAS | SPGGVGSPL | HFG | 1.6 | ||||
9 | 2.1 | CAV | NNARL | MFG | 31 | 14.1 | LYFCAS | SESTGWDTQ | YFG | 2.3 | ||||
13 | 2.1 | CAV | YTGGGNKL | TFG | 10 | 3.1 | MYLCVS | ALGLGQPQ | HFG | 1.5 | ||||
Group 3 | ||||||||||||||
2 | 2.1 | CAV | AISNFGNEKL | TFG | 48 | 2 | FYICS | ARGSNQPQ | HFG | 1.5 | ||||
5 | 2.1 | CAV | GSQGNL | IFG | 28 | 5.2 | LYLCAS | SFSAAYEQ | YFG | 2.7 | ||||
17 | 2.1 | CAV | NMDFGNEKL | TFG | 48 | 5 | LYFCAS | SLDAGANVL | TFG | 2.6 | ||||
12 | 2.1 | CAG | GAGGTSYGKL | TFG | 52 | 1.1 | LYFCAS | NLGEGETQ | YFG | 2.5 |
The large peripheral pool of phenotypically naive A2/melan-A multimer+ CD8+ T cells detectable in A2+ donors could in principle be explained by selection in the thymus of a high number of precursors or instead be the result of the homeostatic peripheral proliferation of a lower number of selected precursors. Indeed, during homeostasis-driven proliferation (i.e., in a lymphopenic environment) naive T cells can transiently change their phenotype into a memory-like form and, after ceasing division, regain naive phenotypic and functional characteristics (33, 34). We have recently addressed this question by analyzing A2/melan-A multimer+ CD8+ T cells in both cord blood and thymus (18). From the results of this study we have concluded that homeostasis-driven proliferation does not significantly impact on the frequency of circulating naive A2/melan-A multimer+ CD8+ T cells in adults, as these cells can be detected at similar frequency among both cord blood lymphocytes and single CD8+ thymocytes. In addition, assessment of the replicative history of circulating A2/melan-A multimer+ CD8+ T cells from healthy donors by ex vivo measurement of TCR excision circles’ content and telomere length provided further evidence of the truly naive nature of this population. Thus, it may be expected that the highly restricted Vα 2.1 usage found among circulating naive A2/melan-A multimer+ CD8+ T cells would reflect a bias already present at birth and at the earlier stages of lymphocyte development. To corroborate this hypothesis, we analyzed the TCR Vα usage of A2/melan-A multimer+ CD8+ T cells isolated from cord blood and thymus of A2+ individuals. As shown in Fig. 3,A, A2/melan-A multimer+ CD8+ T cells represented 0.07, 0.09, 0.05, and 0.04% in CB7, CB8, T02, and T12, respectively. These populations were sorted and expanded in vitro by PHA stimulation. For CB7, CB8, and T2 we obtained polyclonal monospecific CTL lines containing >99% A2/melan-A multimer+ CD8+ T cells (not shown). RT-PCR analysis of these lines revealed in each case a prominent signal for Vα 2, whereas minor signals were found for some of the other Vα elements, suggesting frequent Vα 2.1 usage by T cells composing the lines (Fig. 3,B and data not shown). In addition, from T12 we obtained seven A2/melan-A multimer+ CD8+ clones. Six of the seven clones (85%) used Vα 2.1. (shown in Fig. 3 C). Four clones were both peptide- and tumor-reactive (group 1), whereas the remaining three were peptide-reactive, but not tumor-reactive (group 2). Similarly to what was previously observed for circulating A2/melan-A multimer+ CD8+, we found a large diversity in Jα, Vβ, and Jβ usage; CDR3α/β size; and sequence.
Biased Vα usage by A2/melan-A multimer+ CD8+ T cells in cord blood and thymus of A2-expressing individuals. A, Cord blood (CB) cells and thymocytes (T) from A2-expressing donors were stained with PE-labeled A2/melan-A multimers together with anti-CD8-FITC. B, Amplified TCR Vα 1–29/Cα PCR products were resolved on a 1% agarose gel and revealed by ethidium bromide. Vα 1–5/Cα-amplified products are shown. The positive control consisted of constant region-amplified products (180 bp), and the negative control included reactions without cDNA. L, m.w. ladder. C, TCR α/β usage and CDR3 α/β length and sequence of A2/melan-A multimer+ T cell clones derived from single CD8+ thymocytes from T12.
Biased Vα usage by A2/melan-A multimer+ CD8+ T cells in cord blood and thymus of A2-expressing individuals. A, Cord blood (CB) cells and thymocytes (T) from A2-expressing donors were stained with PE-labeled A2/melan-A multimers together with anti-CD8-FITC. B, Amplified TCR Vα 1–29/Cα PCR products were resolved on a 1% agarose gel and revealed by ethidium bromide. Vα 1–5/Cα-amplified products are shown. The positive control consisted of constant region-amplified products (180 bp), and the negative control included reactions without cDNA. L, m.w. ladder. C, TCR α/β usage and CDR3 α/β length and sequence of A2/melan-A multimer+ T cell clones derived from single CD8+ thymocytes from T12.
The data reported above show a high degree of conservation of TCR Vα 2.1 usage by naive A2/melan-A multimer+ CD8+ T cells that is already determined at the early stages of T cell development. Together, the results of this study strongly support the dominance of the α-chain in TCR-mediated Ag recognition and provide the first formal demonstration that a marked Vα selection occurs in a normal Ag-specific preimmune TCR repertoire. TCR-α dominance would make physiologic sense; indeed, during T cell development TCR-α rearrangements take place in thymocytes that have already undergone TCR-β rearrangement and proliferation. It follows that under physiological conditions a given TCR-β can associate with multiple TCR-α, whereas the opposite does not occur. Therefore, if the TCR specificity for Ag was mostly contributed for by the β-chain, TCRs bearing the same β-chain and different α-chains would display similar specificity. In this context, the dominant role of TCR α-chain in Ag recognition most likely reflects a mechanism that ensures maximal diversity of the TCR repertoire.
Interestingly, the Vα 2.1 usage conservation reported here for A2/melan-A multimer+ CD8+ T cells encompasses T cells displaying a wide range of functional avidity of Ag recognition, including some of functional avidity too low to be detected in functional Ag recognition assays (group 3). To identify additional sequence constraints that could predict the functional avidity of Ag recognition of A2/melan-A multimer+ CD8+ T cells we compared TCR sequences from A2/melan-A multimer+ clones described in this study and recently analyzed TCR sequences of melan-A-specific CTL clones from melanoma patients (23). Within defined Vα-Jα rearrangements, we found some CDR3α sequences (Table III) that were either identical (public sequences) or highly homologous among clones derived from different donors, as previously reported for high affinity T cells selected by chronic exposure to Ag (35, 36). In contrast, no public or highly homologous sequences were found in the CDR3β. Interestingly, recurrent or homologous Vα 2.1-Jα 35 sequences seemed to preferentially pair with the Vβ 14 gene segment. Interestingly, most of these clones belong to group 1 (tumor-reactive). In contrast, preferential Vβ pairing was not evident for other recurrent or homologous sequences (i.e., Vα 2.1-Jα 48, Vα 2.1-Jα 45). Remarkably, the four Vα 2.1+ clonotypes from LAU 337 (group 1) displayed either a public or a homologous sequence (Fig. 1 and Table III), and three among them paired with Vβ 14-Jβ 2.5 chains. In conclusion, despite the limited number of sequences available to date, we identified here some highly conserved structural features in the CDR3α region of the Vα 2.1 chain whose presence correlates with high functional avidity of Ag recognition. This finding reconciles the apparent discrepancy between Vα 2.1-restricted usage by A2/melan-A multimer+ T cells and its lack of correlation with functional avidity of Ag recognition by indicating that the latter could be finely tuned by both the CDR3α loop and the pairing with selected Vβ (e.g., Vβ 14) chains.
Public and homologous Vα 2.1 sequencesa
Donor . | Clone . | Group . | Vα . | CDR3α . | . | . | Jα . | Vβ . | CDR3β . | . | . | Jβ . | ||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
HD 421 | 1A7 | 1 | 2.1 | CAV | NKGFGNVL | HCG | 35 | 17.1 | CAS | STRDRGYEQ | YFG | 2.7 | ||||
7E8 | 1 | 2.1 | CAV | MIGFGNVL | HCG | 35 | 14.1 | CAS | SLGAGIVETQ | YFG | 2.5 | |||||
5H9 | 1 | 2.1 | CAV | SIGFGNVL | HCG | 35 | 14.1 | CAS | SLLGGSTDTQ | YFG | 2.3 | |||||
LAU 337 | 4C8 | 1 | 2.1 | CAV | SIGFGNVL | HCG | 35 | 14.1 | CAS | SPIDGLNTEA | FFG | 1.1 | ||||
VER | 31 | nd | 2.1 | CAV | SIGFGNVL | HCG | 35 | 14.1 | CAS | SPSQGGNTEA | FFG | 2.1 | ||||
16 | nd | 2.1 | CAV | SIGFGNVL | HCG | 35 | 14.1 | CAS | SDSTASSEQ | FFG | 2.1 | |||||
43 | nd | 2.1 | CAA | SMGFGNVL | HCG | 35 | 14.1 | CAS | SSTSGAQGDTQ | YFG | 2.3 | |||||
M138 | 31 | 1 | 2.1 | CAV | SIGFGNVL | HCG | 35 | 14.1 | CAS | SFNDEQ | FFG | 2.1 | ||||
M199 | 29 | 1 | 2.1 | CAV | SIGFGNVL | HCG | 35 | 5.1 | CAS | SLSGSGDEQ | FFG | 2.1 | ||||
18 | 1 | 2.1 | CAA | SIGFGNVL | HCG | 35 | 14.1 | CAS | SLGGSNYEQ | YFG | 2.7 | |||||
M180 | 25 | 1 | 2.1 | CAA | TIGFGNVL | HCG | 35 | 14.1 | CAS | SMTSYNEQ | FFG | 2.1 | ||||
HD 421 | 2C12 | 3 | 2.1 | CAM | SRSNFGNEKL | TFG | 48 | 9.1 | CAS | SQGGITHNEQ | FFG | 2.1 | ||||
7G1 | 2 | 2.1 | CAM | SQSNFGNEKL | TFG | 48 | 13.1 | CAS | KGGEGLDTQ | YFG | 2.3 | |||||
HD 009 | 2 | 3 | 2.1 | CAV | AISNFGNEKL | TFG | 48 | 2.1 | ICS | ARGSNQPQ | HFG | 1.5 | ||||
17 | 3 | 2.1 | CAV | NMDFGNEKL | TFG | 48 | 5.6 | CAS | SLDAGANVL | TFG | 2.6 | |||||
LAU 337 | 6C3 | 1 | 2.1 | CAV | NLFFGNEKL | TFG | 48 | 5 | CAS | SLDSNQPQ | HFG | 2.1 | ||||
M180 | 27 | 1 | 2.1 | CAG | GGGGADGL | TFG | 45 | 3.1 | CAS | TLTGLGQPQ | HFG | 1.5 | ||||
43 | 1 | 2.1 | CAL | EGGGADGL | TFG | 45 | 2.1 | CAS | RDGLNEQ | FFG | 2.1 | |||||
LAU 337 | 1D3 | 1 | 2.1 | CAL | GGGADGL | TFG | 45 | 14.1 | CAS | SLSGTTADEQ | YFG | 2.5 | ||||
M199 | 16 | 1 | 2.1 | CAG | GGGADGL | TFG | 45 | 17.1 | CAS | SQGLAGAGEL | FFG | 2.2 | ||||
34 | 1 | 2.1 | CAA | GGGADGL | TFG | 45 | 7.3 | CAS | SQTLVADTQ | YFG | 2.3 | |||||
54 | 1 | 2.1 | CAV | GGGADGL | TFG | 45 | 13.1 | CAS | SYSATGGEQ | YFG | 2.7 | |||||
HD 009 | 16 | 2 | 2.1 | CAV | SGGGADGL | TFG | 45 | 5 | CAS | SLAISGAGEL | FFG | 2.2 | ||||
10 | 2 | 2.1 | CAV | NVGGGADGL | TFG | 45 | 3.1 | CAS | RVGGLNTGEL | FFG | 2.2 | |||||
11 | 2 | 2.1 | CAV | NSGGGADGL | TFG | 45 | 5.2 | CAS | SPGQGLYEQ | YFG | 2.7 | |||||
4 | 2 | 2.1 | CAV | DSGGGADGL | TFG | 45 | 3.1 | CAS | SLPLSLDNEQ | FFG | 2.1 | |||||
HD 421 | 5G9 | 2 | 2.1 | CAA | DSGGGADGL | TFG | 45 | 1.1 | CAS | SAGLLGGNTI | YFG | 1.3 | ||||
4A12 | 1 | 2.1 | CAP | HSGGGADGL | TFG | 45 | 3.1 | CAS | RMIGYEQ | YFG | 2.7 | |||||
M180 | 22 | 2 | 2.1 | CAL | IDPGSGGGADGL | TFG | 45 | 13.3 | CAS | SETQMNTEA | FFG | 1.1 | ||||
HD 009 | 9 | 2 | 2.1 | CAV | NNARL | MFG | 31 | 14.1 | CAS | SESTGWDTQ | YFG | 2.3 | ||||
LAU 337 | 2C2 | 1 | 2.1 | CAV | NNARL | MFG | 31 | 14.1 | CAS | SLSGTGGQETQ | YFG | 2.5 | ||||
M138 | 33 | 1 | 2.1 | CAV | NNARL | MFG | 31 | 14.1 | CAS | SPSGLAGGHTQ | YFG | 2.3 | ||||
HD 421 | 1D5 | 1 | 2.1 | CAV | SNSGYAL | NFG | 41 | 14.1 | CAS | SFGGQGQPQ | HFG | 1.5 | ||||
7C2 | 1 | 2.1 | CAL | SNSGYAL | NFG | 41 | 14.1 | CAS | SQEGGAFVDTQ | YFG | 2.3 | |||||
VER | 47 | nd | 2.1 | CAV | RDDKI | IFG | 30 | 3.1 | CAS | SSSGTSGVYEQ | YFG | 2.7 | ||||
HD 421 | 5B9 | 2 | 2.1 | CAV | RDDKI | IFG | 30 | 13.1 | CAS | MTSGIVADTQ | YFG | 2.3 | ||||
HD 421 | 5B6 | 2 | 2.1 | CAG | NTGFQKL | VFG | 8 | 1.1 | CAS | SVDSGSSYNEQ | FFG | 2.1 | ||||
VER | 7 | nd | 2.1 | CAV | NTGFQKL | VFG | 8 | 7 | CAS | SPATGSNQPQ | HFG | 1.5 | ||||
37 | nd | 2.1 | CAV | NMGFQKL | VFG | 8 | 14.1 | CAS | SFSSGSPHEQ | FFG | 2.1 | |||||
HD 009 | 3 | 2 | 2.1 | CAV | TTGGSYIP | TFG | 6 | 5.1 | CAS | SLGPGGDGY | TFG | 1.2 | ||||
Thymus | T4 | 2 | 2.1 | CAV | TVGSYIP | TFG | 6 | 1.1 | CAS | SDSLIGQVG | TFG | 1.2 |
Donor . | Clone . | Group . | Vα . | CDR3α . | . | . | Jα . | Vβ . | CDR3β . | . | . | Jβ . | ||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
HD 421 | 1A7 | 1 | 2.1 | CAV | NKGFGNVL | HCG | 35 | 17.1 | CAS | STRDRGYEQ | YFG | 2.7 | ||||
7E8 | 1 | 2.1 | CAV | MIGFGNVL | HCG | 35 | 14.1 | CAS | SLGAGIVETQ | YFG | 2.5 | |||||
5H9 | 1 | 2.1 | CAV | SIGFGNVL | HCG | 35 | 14.1 | CAS | SLLGGSTDTQ | YFG | 2.3 | |||||
LAU 337 | 4C8 | 1 | 2.1 | CAV | SIGFGNVL | HCG | 35 | 14.1 | CAS | SPIDGLNTEA | FFG | 1.1 | ||||
VER | 31 | nd | 2.1 | CAV | SIGFGNVL | HCG | 35 | 14.1 | CAS | SPSQGGNTEA | FFG | 2.1 | ||||
16 | nd | 2.1 | CAV | SIGFGNVL | HCG | 35 | 14.1 | CAS | SDSTASSEQ | FFG | 2.1 | |||||
43 | nd | 2.1 | CAA | SMGFGNVL | HCG | 35 | 14.1 | CAS | SSTSGAQGDTQ | YFG | 2.3 | |||||
M138 | 31 | 1 | 2.1 | CAV | SIGFGNVL | HCG | 35 | 14.1 | CAS | SFNDEQ | FFG | 2.1 | ||||
M199 | 29 | 1 | 2.1 | CAV | SIGFGNVL | HCG | 35 | 5.1 | CAS | SLSGSGDEQ | FFG | 2.1 | ||||
18 | 1 | 2.1 | CAA | SIGFGNVL | HCG | 35 | 14.1 | CAS | SLGGSNYEQ | YFG | 2.7 | |||||
M180 | 25 | 1 | 2.1 | CAA | TIGFGNVL | HCG | 35 | 14.1 | CAS | SMTSYNEQ | FFG | 2.1 | ||||
HD 421 | 2C12 | 3 | 2.1 | CAM | SRSNFGNEKL | TFG | 48 | 9.1 | CAS | SQGGITHNEQ | FFG | 2.1 | ||||
7G1 | 2 | 2.1 | CAM | SQSNFGNEKL | TFG | 48 | 13.1 | CAS | KGGEGLDTQ | YFG | 2.3 | |||||
HD 009 | 2 | 3 | 2.1 | CAV | AISNFGNEKL | TFG | 48 | 2.1 | ICS | ARGSNQPQ | HFG | 1.5 | ||||
17 | 3 | 2.1 | CAV | NMDFGNEKL | TFG | 48 | 5.6 | CAS | SLDAGANVL | TFG | 2.6 | |||||
LAU 337 | 6C3 | 1 | 2.1 | CAV | NLFFGNEKL | TFG | 48 | 5 | CAS | SLDSNQPQ | HFG | 2.1 | ||||
M180 | 27 | 1 | 2.1 | CAG | GGGGADGL | TFG | 45 | 3.1 | CAS | TLTGLGQPQ | HFG | 1.5 | ||||
43 | 1 | 2.1 | CAL | EGGGADGL | TFG | 45 | 2.1 | CAS | RDGLNEQ | FFG | 2.1 | |||||
LAU 337 | 1D3 | 1 | 2.1 | CAL | GGGADGL | TFG | 45 | 14.1 | CAS | SLSGTTADEQ | YFG | 2.5 | ||||
M199 | 16 | 1 | 2.1 | CAG | GGGADGL | TFG | 45 | 17.1 | CAS | SQGLAGAGEL | FFG | 2.2 | ||||
34 | 1 | 2.1 | CAA | GGGADGL | TFG | 45 | 7.3 | CAS | SQTLVADTQ | YFG | 2.3 | |||||
54 | 1 | 2.1 | CAV | GGGADGL | TFG | 45 | 13.1 | CAS | SYSATGGEQ | YFG | 2.7 | |||||
HD 009 | 16 | 2 | 2.1 | CAV | SGGGADGL | TFG | 45 | 5 | CAS | SLAISGAGEL | FFG | 2.2 | ||||
10 | 2 | 2.1 | CAV | NVGGGADGL | TFG | 45 | 3.1 | CAS | RVGGLNTGEL | FFG | 2.2 | |||||
11 | 2 | 2.1 | CAV | NSGGGADGL | TFG | 45 | 5.2 | CAS | SPGQGLYEQ | YFG | 2.7 | |||||
4 | 2 | 2.1 | CAV | DSGGGADGL | TFG | 45 | 3.1 | CAS | SLPLSLDNEQ | FFG | 2.1 | |||||
HD 421 | 5G9 | 2 | 2.1 | CAA | DSGGGADGL | TFG | 45 | 1.1 | CAS | SAGLLGGNTI | YFG | 1.3 | ||||
4A12 | 1 | 2.1 | CAP | HSGGGADGL | TFG | 45 | 3.1 | CAS | RMIGYEQ | YFG | 2.7 | |||||
M180 | 22 | 2 | 2.1 | CAL | IDPGSGGGADGL | TFG | 45 | 13.3 | CAS | SETQMNTEA | FFG | 1.1 | ||||
HD 009 | 9 | 2 | 2.1 | CAV | NNARL | MFG | 31 | 14.1 | CAS | SESTGWDTQ | YFG | 2.3 | ||||
LAU 337 | 2C2 | 1 | 2.1 | CAV | NNARL | MFG | 31 | 14.1 | CAS | SLSGTGGQETQ | YFG | 2.5 | ||||
M138 | 33 | 1 | 2.1 | CAV | NNARL | MFG | 31 | 14.1 | CAS | SPSGLAGGHTQ | YFG | 2.3 | ||||
HD 421 | 1D5 | 1 | 2.1 | CAV | SNSGYAL | NFG | 41 | 14.1 | CAS | SFGGQGQPQ | HFG | 1.5 | ||||
7C2 | 1 | 2.1 | CAL | SNSGYAL | NFG | 41 | 14.1 | CAS | SQEGGAFVDTQ | YFG | 2.3 | |||||
VER | 47 | nd | 2.1 | CAV | RDDKI | IFG | 30 | 3.1 | CAS | SSSGTSGVYEQ | YFG | 2.7 | ||||
HD 421 | 5B9 | 2 | 2.1 | CAV | RDDKI | IFG | 30 | 13.1 | CAS | MTSGIVADTQ | YFG | 2.3 | ||||
HD 421 | 5B6 | 2 | 2.1 | CAG | NTGFQKL | VFG | 8 | 1.1 | CAS | SVDSGSSYNEQ | FFG | 2.1 | ||||
VER | 7 | nd | 2.1 | CAV | NTGFQKL | VFG | 8 | 7 | CAS | SPATGSNQPQ | HFG | 1.5 | ||||
37 | nd | 2.1 | CAV | NMGFQKL | VFG | 8 | 14.1 | CAS | SFSSGSPHEQ | FFG | 2.1 | |||||
HD 009 | 3 | 2 | 2.1 | CAV | TTGGSYIP | TFG | 6 | 5.1 | CAS | SLGPGGDGY | TFG | 1.2 | ||||
Thymus | T4 | 2 | 2.1 | CAV | TVGSYIP | TFG | 6 | 1.1 | CAS | SDSLIGQVG | TFG | 1.2 |
Melan-A specific clones from M138, M199, M180 and VER melanoma patients were derived from either circulating or tumor-infiltrating lymphocytes. Their Vα2.1 sequences have been reported (L. Troutman, N. Labarriere, F. Jetereau, V. Karanikas, N. Gervois, T. Connerotte, P. Coulie, and M. Bonneville. Dominant TCR vα usage by virus and tumor reactive T cells with wide affinity ranges for their specific antigens. Submitted for publication.). Public sequences are underlined. nd, not determined.
In comparison with other TCR repertoires specific for viral Ags (23), the Vα 2.1-restricted repertoire of melan-A-specific TCR ischaracterized by an extraordinary diversity of the CDR3α region (with a 3- to 12-amino acids variable length and >20 distinct Jα used) and of CDR1, -2, and -3β regions. In this context, it is conceivable that in the case of melan-A-specific TCRs, the CDR1 and CDR2 regions of the Vα 2.1 chain could substantially contribute to the total TCR/pMHC binding energy by establishing strong interactions with both the HLA-A2 molecule and amino-terminal residues of the antigenic peptide. This could explain not only the large diversity in CDR3α and CDR3β regions, but also the large degeneracy of Ag recognition that results in a high degree of cross-reactivity to related peptides (32) as well as the highly efficient positive selection of these TCRs that ensues in the high frequency of these populations within the naive T cell repertoire (17).
General conclusions
Together, the results of this study underline the prevalent role of TCR α-chain in selection of the preimmune TCR repertoire specific for the human self-Ag melan-A. Previous studies have emphasized the large diversity of the TCR repertoire of Ag-experienced, melan-A-specific CTL together with the frequent usage of some V (Vα 2.1, Vβ 14) elements. In this study we show that diverse Vβ usage, but highly restricted Vα 2.1 usage, are found in the preimmune repertoire of circulating A2/melan-A multimer+ CD8+ T cells as well as at the early stages of T cell development. Interestingly, Vα 2.1 usage conservation encompasses T cells displaying various functional avidities of Ag recognition. Thus, interaction of the A2/melan-A peptide complex with Vα 2.1 chains contributes to set the broad TCR specificity for Ag, as underlined by preferential binding of A2/melan-A multimers to a subset of Vα 2.1-bearing TCRs, whereas functional outcome results from the sum of this with other interactions between pMHC complex and TCR. The close comparison of the available A2/melan-A multimer+ T cell-derived TCR sequence indicates that these additional interactions could be determined both by the CDR3α loop and by pairing with selected Vβ (e.g., Vβ 14) chains. It is hoped that the analysis of a much larger number of A2/melan-A multimer+ CD8+ T cell-derived TCR sequences will, in the near future, allow a better definition of these structural features and, in combination with crystallographic studies, their correlation with T cell function.
Acknowledgements
We thank Nicole Montandon for technical assistance. We are deeply grieved by the recent loss of our dear colleague, Dr. Pascal Batard, who assisted us with flow cytometry immunofluorescence cell sorting.
Footnotes
This work was supported in part by Swiss National Science Foundation Grant 067022.01 (to P.-Y.D.), and NCCR Molecular Oncology (to F.-A.L.G.), the Ligue Genevoise contre le Cancer, and the Fondation pour la Lutte contre le Cancer.
Abbreviation used in this paper: CDR, complementarity-determining region.