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-Chain in the Selection of the Preimmune Repertoire Specific for a Human Tumor-Associated Self-Antigen1
* Division of Oncology, Laboratory of Tumor Immunology, University Hospital, Geneva, Switzerland;
Division of Clinical Onco-Immunology, Ludwig Institute for Cancer Research, University Hospital, Lausanne, Switzerland;
Institut National de la Santé et de la Recherche Médicale, Unité 463, Institut de Biologie, Nantes, France;
Ludwig Institute for Cancer Research, Lausanne Branch, University of Lausanne, Epalinges, Switzerland; and
¶ Cellular Genetics Unit, Université Catholique de Louvain, Brussels, Belgium
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResults and DiscussionReferences |
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- and 
-chains. Recent experimental evidence has suggested that Ag-specific TCR repertoires may exhibit a more V- than V-restricted usage. Whether V usage is narrowed during immune responses to Ag or if, on the contrary, restricted V 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 V usage, but a preferential V 2.1 usage. Restricted V 2.1 usage was also found among single CD8+ A2/melan-A multimer+ thymocytes, indicating that V-restricted selection takes place in the thymus. V 2.1 usage, however, was independent from functional avidity of Ag recognition. Thus, interaction of the pMHC complex with selected V-chains contributes to set the broad Ag specificity, as underlined by preferential binding of A2/melan-A multimers to V 2.1-bearing TCRs, whereas functional outcomes result from the sum of these with other interactions between pMHC complex and TCR. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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- and -chains generated by somatic recombination of germline-encoded V and J gene segments for the -chain and V, D, and J gene segments for the -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 -chain and V, D, and J segments for the -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 - and -chains have been addressed. Consistent with their high degree of diversity, CDR3 and - regions have been shown to play an important role in Ag recognition. In a model of TCR - or -chain transgenic mice, immunization with altered peptide ligands resulted in changes in the CDR3 sequences of the - and -chains (3, 4, 5, 6). Interestingly, in this model, CTL used more restricted VJ than VDJ regions (4). Recently, Yokosuka and colleagues (7) have analyzed the Ag-specific TCR- pairs generated by in vitro transfection of a single TCR chain ( or ) 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 -chain, but could use a variety of TCR -chains. Together, these and other studies have suggested a dominant role of -chain in Ag recognition by TCR. Moreover, the analyses of TCR-pMHC complex crystals (8, 9, 10, 11, 12) have shown that the -chain usually makes more contacts with the peptide than the -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 V (i.e., V 2.1) and V (i.e., V 14) regions, however, has also been reported (19, 21, 22). A more recent survey of TCR V- and V-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 V 2.1 (23). Whether this V 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 V usage, but a highly preferential V 2.1 usage. Preferential V 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 V 2.1 usage and functional avidity of Ag recognition. Together, our results indicate that the interaction of V-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 V-chains, but does not determine functional outcomes.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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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 x [(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 TCRV and TCRV 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 V and 22 V 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--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 C primer or the V 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).
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Results and Discussion |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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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. 1A, four of seven clones used the V 2.1 gene segment, whereas the J segments used and the CDR3 sequences were different for each clone. Interestingly, three of the four V 2.1+ and one of the three V 2.1- clones expressed V 14 and used the same J gene segment, whereas CDR3 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 V 2.1 and V 14, recognized melan-A peptides with high avidity and exhibited high tumor reactivity (Fig. 1, A and B).
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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. 2A). 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. 2B). 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).
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2.1 gene segment (Table I). The latter was rearranged with a large number (15) of different J gene segments, albeit some of those were slightly over-represented (e.g.. J 43, J 35). Overall, there was a general lack of restriction in both CDR3 size (which ranged from three to 10 amino acids) and sequences. However, sequence similarities were found within defined V-J rearrangements to a variable extent, going from identity (e.g., the two V 2.1-J 41-using clones) to relatively large differences (e.g., the V 2.1-J 43-using clones). In several cases (e.g., J 35-using clones 2/7E8 and 2/5H9 or J 45-using clones 2/5G9 and 2/7B12), CDR3 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 V usage, the V repertoire was more heterogeneous, with 13 different V gene segments used, although a relatively more frequent usage of some V gene segments was noticeable (e.g., V 14 used by six clones and V 7 used by five clones). In addition, there was no apparent restriction in J usage or evident constraints on CDR3 size and sequence.
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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 V 2.1 gene segment. Similarly to what was observed with A2/melan-A multimer+ clones from HD 421, we found a diverse J usage (10 different J used) together with over-representation of certain J (e.g., J 45 expressed by five clones). CDR3 size ranged from five to 11 amino acids, and CDR3 sequences were rather diverse. However, again, more or less prominent sequence similarities were found within defined V-J rearrangements. V usage was rather diverse (eight different V gene segments used), albeit a high proportion of clones (seven of 18, 38%) used in this case a single V (V 3.1). Also, J segment usage was diverse; however, in contrast with what observed when analyzing the CDR3, no significant similarity was found when comparing sequences of defined V-J rearrangements.
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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 V usage of A2/melan-A multimer+ CD8+ T cells isolated from cord blood and thymus of A2+ individuals. As shown in Fig. 3A, 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 V 2, whereas minor signals were found for some of the other V elements, suggesting frequent V 2.1 usage by T cells composing the lines (Fig. 3B and data not shown). In addition, from T12 we obtained seven A2/melan-A multimer+ CD8+ clones. Six of the seven clones (85%) used V 2.1. (shown in Fig. 3C). 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 J, V, and J usage; CDR3/ size; and sequence.
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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 -chain in TCR-mediated Ag recognition and provide the first formal demonstration that a marked V selection occurs in a normal Ag-specific preimmune TCR repertoire. TCR- dominance would make physiologic sense; indeed, during T cell development TCR- rearrangements take place in thymocytes that have already undergone TCR- rearrangement and proliferation. It follows that under physiological conditions a given TCR- can associate with multiple TCR-, whereas the opposite does not occur. Therefore, if the TCR specificity for Ag was mostly contributed for by the -chain, TCRs bearing the same -chain and different -chains would display similar specificity. In this context, the dominant role of TCR -chain in Ag recognition most likely reflects a mechanism that ensures maximal diversity of the TCR repertoire.
Interestingly, the V 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 V-J rearrangements, we found some CDR3 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 CDR3. Interestingly, recurrent or homologous V 2.1-J 35 sequences seemed to preferentially pair with the V 14 gene segment. Interestingly, most of these clones belong to group 1 (tumor-reactive). In contrast, preferential V pairing was not evident for other recurrent or homologous sequences (i.e., V 2.1-J 48, V 2.1-J 45). Remarkably, the four V 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 V 14-J 2.5 chains. In conclusion, despite the limited number of sequences available to date, we identified here some highly conserved structural features in the CDR3 region of the V 2.1 chain whose presence correlates with high functional avidity of Ag recognition. This finding reconciles the apparent discrepancy between V 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 CDR3 loop and the pairing with selected V (e.g., V 14) chains.
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2.1-restricted repertoire of melan-A-specific TCR ischaracterized by an extraordinary diversity of the CDR3 region (with a 3- to 12-amino acids variable length and >20 distinct J used) and of CDR1, -2, and -3 regions. In this context, it is conceivable that in the case of melan-A-specific TCRs, the CDR1 and CDR2 regions of the V 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 CDR3 and CDR3 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 -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 (V 2.1, V 14) elements. In this study we show that diverse V usage, but highly restricted V 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, V 2.1 usage conservation encompasses T cells displaying various functional avidities of Ag recognition. Thus, interaction of the A2/melan-A peptide complex with V 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 V 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 CDR3 loop and by pairing with selected V (e.g., V 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.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Danila Valmori, Division of Clinical Onco-Immunology, Ludwig Institute for Cancer Research, Hôpital Orthopédique, avenue Pierre-Decker 4, 1011 Lausanne, Switzerland. E-mail address: valmori{at}cancercenter.columbia.edu; or Dr. Pierre-Yves Dietrich, Division of Oncology, Laboratory of Tumor Immunology, University Hospital, 1211 Genève 14, Switzerland. E-mail address: pierre-yves.dietrich{at}hcuge.ch
3 F.-A.L.G. and V.D. contributed equally to this work.
4 Abbreviation used in this paper: CDR, complementarity-determining region.
Received for publication December 6, 2002. Accepted for publication February 27, 2003.
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References |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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and V variable region domains and chain junctional region in viral antigen recognition. Eur. J. Immunol. 21:2195.[Medline]
chain in forming preimmune TCR repertoire revealed by clonal TCR reconstitution system. J. Exp. Med. 195:991. T cell receptor structure at 2.5 A and its orientation in the TCR-MHC complex. Science 274:209. T-cell receptor, influenza HA peptide and MHC class II molecule, HLA-DR1. EMBO J. 19:5611.[Medline]
chain sequence used by an HLA-A2-restricted and melanocyte-lineage-specific CTL clone. J. Exp. Med. 178:1231. usage by virus and tumor-reactive T cells with wide affinity ranges for their specific antigens. Eur. J. Immunol. 32:3181.[Medline]
1-w29/V1-w24) for the study of human T cell receptor variable V gene segment usage by polymerase chain reaction. Eur. J. Immunol. 22:1261.[Medline]
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