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The Journal of Immunology, 2000, 165: 2001-2011.
Copyright © 00 by The American Association of Immunologists

Frequent Contribution of T Cell Clonotypes with Public TCR Features to the Chronic Response Against a Dominant EBV-Derived Epitope: Application to Direct Detection of Their Molecular Imprint on the Human Peripheral T Cell Repertoire1

Annick Lim*, Lydie Trautmann{dagger}, Marie-Alix Peyrat{dagger}, Chrystelle Couedel{dagger}, François Davodeau{dagger}, François Romagné{ddagger}, Philippe Kourilsky* and Marc Bonneville2,{dagger}

* Unité de Biologie Moléculaire du Gène, Institut National de la Santé et de la Recherche Médicale, Unité 277, Institut Pasteur, Paris, France; {dagger} Institut National de la Santé et de la Recherche Médicale, Unité 463, Institut de Biologie, Nantes, France; and {ddagger} Immunotech SA, Marseilles, France


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
In an attempt to provide a global picture of the TCR repertoire diversity of a chronic T cell response against a common Ag, we performed an extensive TCR analysis of cells reactive against a dominant HLA-A2-restricted EBV epitope (hereafter referred to as GLC/A2), obtained after sorting PBL or synovial fluid lymphocytes from EBV-seropositive individuals using MHC/peptide multimers. Although TCR ß-chain diversity of GLC/A2+ T cells was extensive and varied greatly from one donor to another, we identified in most cell lines several recurrent Vß subsets (Vß2, Vß4, and Vß16 positive) with highly conserved TCRß complementarity-determining region 3 (CDR3) length and junctional motifs, which represented from 11 to 98% (mean, 50%) of GLC/A2-reactive cells. While TCR ß-chains expressed by these subsets showed limited CDR1, CDR2, and CDR3 homology among themselves, their TCR {alpha}-chains comprised the same TCRAV region, thus suggesting hierarchical contribution of TCR {alpha}-chain vs TCR ß-chain CDR to recognition of this particular MHC/peptide complex. The common occurrence of T cell clonotypes with public TCR features within GLC/A2-specific T cells allowed their direct detection within unsorted PBL using ad hoc clonotypic primers. These results, which suggest an unexpectedly high contribution of public clonotypes to the TCR repertoire against a dominant epitope, have several implications for the follow-up and modulation of T cell-mediated immunity.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Activation of T cell effector functions requires prior recognition of antigenic peptides bound to self-MHC molecules. This recognition process is conducted by clonotypic TCR{alpha}ßs, whose structural diversity is generated by somatic recombination of V, D, and J gene segments (1). TCR V{alpha} and Vß regions comprise three major hypervariable regions that are homologous to the Ig complementarity-determining regions (CDR).3 While CDR1 and CDR2 are exclusively encoded by germline parts of the V segment, CDR3 comprise both germline residues derived from the V(D)J segments and nongermline residues contributed by N nucleotides, which are added in a template-independent fashion at the V(D)J joints during the recombination process (1). Early models based upon the crystal structure of MHC and Ig molecules proposed that CDR1 and CDR2 primarily contact residues of the MHC {alpha}-helices, whereas CDR3 contact the antigenic peptide in the MHC groove (1, 2). The key role of CDR3 in recognition of the antigenic peptide was then confirmed experimentally through several approaches, such as peptide immunization of single-chain TCR transgenic mice (3, 4) and site-directed mutagenesis (5). More recently, crystallographic analyses of several TCR/MHC/peptide complexes have revealed a conserved diagonal orientation of the TCR over the MHC/peptide complex and identified several contact points between, respectively, the N-terminal and C-terminal halves of the peptide and the CDR3 loops of the TCR {alpha}-chain and TCR ß-chain (6, 7, 8). These structural studies also suggested a greater contribution of TCR{alpha} than TCRß CDR in contacting the MHC/peptide complex (6, 8).

Although resolution of the three-dimensional structure of TCR/MHC/peptide complexes has brought important insights into the structural basis of Ag recognition, the amount of data recovered to date from these studies is still too limited to give a general understanding of the molecular mechanisms of TCR repertoire central and peripheral selection. This issue has been more specifically addressed through two other approaches. On the one hand, analyses of the TCR repertoire of cells selected by defined MHC/peptide complexes in vitro and in vivo have clearly shown TCR repertoire restriction within Ag-selected T cells (9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20) and have led in some systems to the identification of highly recurrent CDR motifs associated with response to particular Ags (9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20). On the other hand studies of T cell development in in vivo situations where either the specificity of the selected TCR and/or the quality of the selecting MHC/peptide complexes could be controlled by transgenesis have recently demonstrated the key role played by self-MHC/peptide complexes in shaping the mature T cell repertoire (21, 22, 23, 24). However, several questions remain open. For instance, in systems where TCR restrictions have been identified, it has been difficult to assess the respective contributions of combinatorial/enzymatic constraints, thymic selection by self-MHC/peptide complexes, and peripheral selection by environmental Ags to the emergence of cells with public TCR features (i.e., with restricted TCR motifs shared by clonotypes from distinct individuals) (12). Moreover, since in several studies the repertoire diversity of cells selected by a given Ag was extensive and showed no evidence for selection of clonotypes with recurrent TCR motifs (25, 26), it is still unclear whether these public T cell responses are the exception or the rule. This is an important point, since identification of public TCR motifs could have major implications in immunodiagnosis (i.e., by permitting follow-up of immune responses with serological or molecular probes specific to public clonotypes) and immunotherapy (i.e., by permitting specific selection or elimination of public clonotypes with wanted or unwanted specificities, respectively).

In the present study we took advantage of several recent technical breakthroughs permitting 1) efficient isolation of Ag-specific T cells by means of multimeric MHC/peptide complexes (27) and 2) global qualitative and semiquantitative analysis of their TCR features using a combination of flow cytometric and molecular approaches (28) to study in detail the TCR structural constraints imposed by a given selecting Ag. As a model Ag, we chose the HLA-A2-restricted epitope GLCTLVAML derived from the EBV lytic protein BMLF1 (hereafter referred to as GLC/A2) because 1) this Ag is derived from a common infectious agent that infects a large fraction of the adult population (29), and 2) this particular epitope triggers strong peripheral T cell responses in infected individuals and is recognized by a relatively large fraction of EBV-specific T cells in HLA-A2 donors in various physiopathologic situations (30, 31, 32, 33). Our results indicate that several T cell clonotypes with recurrent TCR motifs (and in some cases with fully conserved TCR {alpha}ß-chains) are shared by GLC/A2+ T cell lines derived from several individuals and together account for about half of the reactive T cells. Hence, this suggests frequent selection of public clonotypes by dominant Ags, at least in the course of chronic stimulation as is presently the case. Our observations also suggest tighter interactions between this particular MHC/peptide complex and the CDR from the TCR {alpha}-chain than the TCR ß-chain, since several clonotypes bearing TCR ß-chains with weak CDR1, -2, and -3 homology shared the same TCRAV region.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Abs and reagents

The following mAbs, whose specificity is indicated in parentheses, were used for flow cytometry and immunomagnetic sorting: E2.2E7.2 (Vß2), LE89 (Vß3), IMMU157 (Vß5.1), 3D11 (Vß5.3), CRI304.3 (Vß6.2), 3G5D15 (Vß7), 56C5.2 (Vß8.1/8.2), FIN9 (Vß9), C21 (Vß11), S511 (Vß12), IMMU1222 (Vß13.1), JU74 (Vß13.6), CAS1.1.13 (Vß14), Tamaya1.2 (Vß16), E17.5F3 (Vß17), BA62.6 (Vß18), ELL1.4 (Vß20), IG125 (Vß21.3), IMMU546 (Vß22), and HUT78.1 (Vß23). Synthetic peptides were obtained from either Chiron Mimotopes Corp. (Victoria, Australia) or Genosys (Cambridgeshire, U.K.). For functional assays, lyophilized peptides were dissolved at 20 mg/ml in DMSO, diluted at 2 mg/ml in 10 mM acetic acid, and stored as stock solutions at -80°C.

Generation of recombinant MHC/peptide complexes

Soluble MHC/peptide tetramers were produced using a method similar to that described by Altman et al. (27). ß2-Microglobulin and HLA-A2 heavy chain extracellular part fused at the COOH-terminus to a 13-aa target sequence for the biotin ligase BirA enzyme were produced in Escherichia coli as inclusion bodies. HLA-A2 complexes (2 µM HLA-A2 and 2 µM ß2-microglobulin) were folded in vitro by dilution in the presence of either 10 µM of the synthetic EBV BMLF1 peptide (GLCTLVAML) (30, 31, 32, 33) or the CMV pp65 peptide (NLVPMVATV; as a control) essentially as previously described. The folded MHC complexes were diafiltrated and concentrated to 1 mg/ml in 10 mM Tris, pH 8.00, on a prep scale 3-kDa concentration cassette (Millipore, Bedford, MA). The MHC complexes were biotinylated using purified BirA enzyme at a concentration of 10 µg/ml of enzyme, 40 µM biotin, 2 mM ATP, and 10 mM MgOAc. Biotinylated complexes were then purified by anion exchange (MonoQ, Pharmacia, Piscataway, NJ) with a 0 to 0.5 M NaCl gradient and were checked by SDS-PAGE and analytical gel filtration. Biotinylated complexes were then tetramerized with PE-streptavidin (Immunotech, Westbrook, ME) at a 4/1 molar ratio and stored at 4°C at 200 µg of MHC in 10 mM Tris (pH 8.00), 50 mM NaCl, and 0.5 mM EDTA until use. Titration curves by flow cytometry on specific T cell lines were stable for at least 3 mo for the two tetramers.

Generation of T cell lines and culture

T cell lines were derived from either fresh PBL of HLA-A2 EBV-seropositive healthy donors or from synovial fluid lymphocytes (SFL) of HLA-A2 rheumatoid arthritis (RA) patients with previously documented reactivity against EBV (32, 33). T cells were cultured and cloned as previously described (34, 35) in RPMI 1640 supplemented with human serum (8%) and recombinant IL2 (150 IU/ml) and were stimulated once a month with purified PHA (Sigma) and irradiated PBL and B lymphoblastoid cells. Sorting of T cells with TCR Vß-specific mAb was performed as previously described (35) using sheep anti-mouse Ig-coated immunomagnetic beads (Dynal, Oslo, Norway). The purity of TCR Vß-sorted cells was checked by flow cytometry and was always >98% (data not shown). Sorting of Ag-specific T cells using recombinant MHC/peptide complexes was performed as follows. Avidin-coated beads (Dynal) were incubated for 15 min with biotinylated MHC/peptide monomers (50 µl at 10 µg/ml for 2 x 107 beads) and then added to CD8+ synovial T cells or fresh PBL. Cells were rotated for 2 h at 4°C, immunomagnetically sorted, and washed extensively as previously described (35). Bead-coated cells were then expanded as described above.

Flow cytometric analysis

Cells were phenotyped by indirect immunofluorescence as previously described (34). In brief, cells were incubated first with unconjugated mAb for 30 min at room temperature, washed, and then incubated with FITC-conjugated rabbit anti-mouse Ig antiserum (BioAtlantic, Nantes, France) for 30 min on ice. After washing, cells were analyzed by flow cytometry on a FACScan apparatus (Becton Dickinson, Mountain View, CA) using the LYSYS II software package on a FACstation.

CD25 induction assay

HLA-A2+ stimulator cells (B lymphoblastoid cell lines) were incubated for 1 h at 37°C with 10 µM peptide and washed three times before use. Responder T cells were incubated at a 1/1 ratio with peptide-loaded stimulator cells for 18 h, stained with a CD25-specific mAb, and analyzed by flow cytometry as described above.

Immunoscope analysis

Total RNA was extracted using TRIzol reagent (Life Technologies, Gaithersburg, MD) as recommended by the manufacturer. RT-PCR amplification and run-off steps were performed as previously described (36). TCR ß-chain-specific primers were described previously (37). Fluorescent DNA products were loaded on a sequencing gel and analyzed with Immunoscope software (38).

Sequence analysis of TCR transcripts

PCR were performed as described above with 5 U of Pfu polymerase for 25 cycles. PCR products were then cloned in pCR-blunt II-TOPO vector using the Blunt TOPO PCR cloning kit (Invitrogen, Carlsbad, CA). For sequencing purposes, PCR was conducted directly on E. coli- colonies with Taq polymerase as previously described (39). Sequencing reactions were conducted directly on these products using M13(-20) primer and with the ABI PRISM Dye Terminator Cycle Sequencing Ready Reaction kit or the ABI PRISM Big Dye Terminator Cycle Sequencing Ready Reaction Kit (Perkin-Elmer Applied Biosystems, Foster City, CA). CDR3 region corresponding sequences were extracted and analyzed using a software designed for this purpose.

Clonotypic analysis

cDNA made from total RNA were extracted from unsorted PBL of HLA-A2 EBV-seropositive healthy donors and from a control thymus. Each sample was amplified (28 cycles) with TCR BV-specific sense primers and fluorescent antisense public clonotypic primers specific for consensus junctional sequences. The fluorescent products were migrated in a 373A DNA sequencer, and the raw data were analyzed with the Immunoscope software package.

The fluorescent clonotypic primers were 5'-6-Fam-CGA AGG TGT AGC CAT TCC CTG T-3' for BV2, 5'-6-Fam-CAA AAT ACT GCG TNC CNC CNG G-3' for BV16BJ2S3, and 5'-6-Fam-CGA AGT ACT GAA TNC CNC CNG G-3' for BV16BJ2S4.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Generation of T cell lines reactive against a dominant EBV epitope

GLC/A2-reactive T cell lines were generated following immunomagnetic sorting of specific T cells using streptavidin-coated beads coated with biotinylated recombinant HLA-A2/GLC complexes. Sorted cells were then expanded using polyclonal stimulators before specificity and repertoire analyses. The Ag specificity of T cell lines was documented first by flow cytometry using fluorescent HLA-A2 tetramers bound to relevant or irrelevant peptides (see representative staining profiles of unsorted vs GLC/A2-sorted lines from two donors in Fig. 1GoA). T cell line specificity was then confirmed by analyzing their cytotoxic activity and CD25 up-regulation after short term incubation with HLA-A2 target cells loaded with the relevant peptide (Fig. 1GoB and data not shown).



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  		FIGURE 1. Phenotypic and functional analysis of GLC/A2-sorted T cells. A, Unsorted and GLC/A2-sorted T cell lines from two donors (RA11 and RA15) were stained with fluorescent tetramers loaded with either the specific GLC peptide (empty histograms) or an irrelevant peptide (NLV; see Materials and Methods; plain histograms). B, CD25 expression was studied on GLC/A2-sorted T cells incubated overnight with the autologous B lymphoblastoid cells loaded with either the GLC peptide (open histograms) or an irrelevant peptide (NLV; plain histograms).

 
We failed to obtain GLC/A2 T cell lines from either HLA-A2- donors or EBV-seronegative HLA-A2+ cord blood cells (data not shown). By contrast, GLC-A2-reactive T cells were obtained from PBL of 7/7 HLA-A2+ EBV-seropositive healthy donors and from synovial lymphocytes (SL) of 9/9 HLA-A2+ RA patients with previously documented EBV reactivity (33). Taken together, these results strongly suggested that the in vivo expansion of GLC/A2-reactive T cells specifically occurred in the course of an EBV-specific T cell response. They also confirmed the marked immunodominance of the GLC/A2 epitope, which systematically elicited T cell responses in EBV-seropositive donors carrying the appropriate HLA allele (32, 33). Six PBL- and eight SL-derived cell lines that contained >98% of GCL/A2-reactive T cells were selected for further TCR repertoire analyses.

Detection by flow cytometry of several recurrent TCR Vß subsets within GLC/A2-reactive T cells derived from healthy and RA individuals

TCR Vß expression by GLC-A2+ T cells was studied by flow cytometry using a panel of 23 Vß-specific mAb. As shown in Table IGo, the frequency of T cells recognized by a given Vß-specific Ab greatly varied from one cell line to another. For instance, while the majority of GLC/A2-reactive T cells derived from donors RA1 and RA14 were stained by the Vß4-specific mAb, a large fraction of cells from, e.g., donors RA4, PBL2, RA15, RA3, and PBL6 reacted with the Vß1-, Vß2-, Vß12-, Vß14-, and Vß16-specific mAb, respectively (Table IGo). Despite this heterogeneity, there was a significant skewing of the TCR repertoire in favor of few highly recurrent Vß subsets in most cell lines regardless of their donor origin and physiopathologic context. In particular, Vß1+, -2+, -4+, -16+, and -22+ subsets were each found in at least one-third of the GLC/A2+ T cell lines and together accounted for at least 70% of the cells in 10 of 14 lines.


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  		Table I. Flow cytometry analysis of GLC/A2+ T cell lines using TCR Vß-specific mAb1

 
Highly recurrent TCR Vß subsets within GLC/A2+ T cells express TCR ß-chains with conserved CDR3 length

V(D)J composition and junctional features of TCR ß-chains expressed by GLC/A2+ T cells were further studied by the Immunoscope approach in four PBL- and seven SL-derived T cell lines. This technique, which consists of amplifying TCR transcripts with appropriate pairs of BVBC or BVBJ primers followed by a run-off labeling of the amplified products with fluorescent internal primers, permits global analysis of BVBJ region usage and distribution of TCRB junctional length within complex mixtures of T cells (28, 36, 37, 38). In agreement with flow cytometric analysis, junctional transcripts were frequently amplified from GLC/A2+ T cell lines using BV1-, BV2-, BV4-, BV16-, and BV22-specific primers (Fig. 2Go). TCRBV transcripts were recurrently amplified in several cell lines using other BV primers specific for, e.g., BV3, BV9, BV13A, and BV23, but their frequency remained <1% in most cases, as suggested by semiquantitative PCR estimations (data not shown).



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  		FIGURE 2. CDR3 length distribution analysis of TCR BV transcripts derived from GLC/A2-sorted T cell lines. TCR BV CDR3 length distribution was analyzed by the Immunoscope technique (see Materials and Methods) using pairs of BV and BC primers. Each color corresponds to a given transcript size. A large fraction of T cell lines expressed BV2, BV4, BV16, and BV22 transcripts with the same CDR3 size (indicated by arrows).

 
Analysis of CDR3 length distribution within recurrent Vß subsets showed lack of interindividual conservation of TCRß junctional length, except for BV2, BV4, BV16, and BV22 transcripts, whose CDR3 lengths were predominantly of 8, 9, 8, and 10 aa, respectively, in most GLC/A2+ T cell lines (Figs. 2Go and 3Go). Importantly, these four Vß subsets showed the highest interindividual recurrence as demonstrated by flow cytometric analysis, and together represented, on the average, 50% of reactive T cells (Table IGo).



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  		FIGURE 3. BV and AV15 Immunoscope profiles of GLC/A2+ T cell lines. BV2BC, BV4BC, and BV16BC Immunoscope analysis was performed on total GLC/A2+ T cell lines. AV15/AC Immunoscope analysis was performed on GLC/A2+ T cells sorted with Vß2-, Vß4-, or Vß16-specific mAb (purity >98%). Control profiles were obtained in a separate experiment from a T cell line derived from unsorted PBL.

 
Detection of clonotypes with public TCR ß-chain junctional motifs within GLC/A2-reactive T cells

The TCR ß-chain junctional features of the GLC/A2-reactive TCR Vß subsets with conserved CDR3 length were analyzed in more detail by semiquantitative BVBJ Immunoscope analyses (in the case of the Vß2+ and Vß4+ subsets) and by extensive sequencing of TCRB junctional transcripts (in the case of Vß2+, Vß4+, Vß16+, and Vß22+ subsets). As shown in Fig. 4Go, BJ usage was highly restricted within both the Vß2+ and Vß4+ GLC/A2-reactive T cells. In almost all donors the 8-aa-long BV2 junctional transcripts comprised either the BJ1S2 or the highly related BJ1S3 element. Likewise, the dominant 9-aa-long BV4 transcripts comprised almost exclusively the BJ1S4 element. Sequence analysis of BV2 and BV4 transcripts amplified with primers specific to the recurrent BJ elements demonstrated extensive conservation of the CDR3 junctional sequences in most donors. For instance, the 8-aa-long BV2BJ1S3 transcripts, which were by far the predominant ones in several donors (i.e., RA3, RA5, RA11, and RA15; see Fig. 4Go), showed full amino acid sequence conservation at all but one CDR3 position (Table IIGo). The same held true for BV2BJ1S2 transcripts (Table IIGo), which predominated in donors RA1, PBL1, and PBL2 (Fig. 4Go). More sequence variability was observed within BV4BJ1S4 transcripts, although identical CDR3 sequences were isolated from several individuals (i.e., RA1, RA5, RA11, RA14, and PBL1; Table IIIGo).



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  		FIGURE 4. Semiquantitative BVBJ Immunoscope analysis of GLC/A2+ T cell lines. Shown are the size distributions of BVBJ transcripts amplified with either BV2 (left) or BV4 (right) primers. CDR3 size is given on top of each histogram. Control histograms were obtained in a separate experiment from a T cell line derived from unsorted PBL.

 

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  		Table II. Sequence analysis of TCR junctional transcripts derived from Vß2+ GLC/A2-sorted T cells1

 

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  		Table III. Sequence analysis of TCR junctional transcripts derived from Vß4+ GLC/A2-sorted T cells1

 
The junctional features of BV16 and BV22 transcripts derived from GLC/A2+ T cell lines were directly studied by sequencing products amplified with BV and BC primers. Here again several recurrent junctional motifs were identified within sequences derived from distinct donors. BV16 transcripts derived from most donors showed identical CDR3 sequences, although we observed some flexibility in terms of BJ segment usage (Table IVGo). By contrast, BV22 transcripts frequently comprised the same BJ elements (BJ1S1 or BJ1S2), but their CDR3 sequences were quite diverse (except for a highly conserved Gly motif at position +5 relative to the V-derived Cys (Cys+5); Table VGo). Interestingly, we identified several junctional motifs that were shared by BV22 sequences derived from a given individual but were absent in other donors; in particular, BV22 sequences derived from PBL1 contained three recurrent motifs (Glu at Cys+4, Arg at Cys+6, and Ser at Cys+8) that were found in none of the BV22 sequences derived from other donors. These donor-specific motifs may reflect structural constraints imprinted by the T cell-positive selection process or following peripheral selection by cross-reactive Ags (see Discussion).


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  		Table IV. Sequence analysis of TCR junctional transcripts derived from Vß16+ GLC/A2-sorted T cells1

 

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  		Table V. Sequence analysis of TCR BV22 junctional transcripts derived from GLC/A2-sorted T cells1

 
Dominant expression of the same TCRAV region by the three GLC/A2-reactive Vß subsets showing public TCR ß-chain junctional features

The above results indicated that T cell clonotypes bearing public TCR Vß2, Vß4, and Vß16 chains (i.e., showing extensive CDR3 sequence homology in distinct individuals) significantly contributed to the response against the GLC/A2 Ag. To assess the extent of TCR structural conservation within these public T cell subsets, we then analyzed TCRA chain transcripts derived from Vß2+, Vß4+, and Vß16+ T cells obtained from several GLC/A2+ cell lines. Strikingly, sequencing of TCRA transcripts derived from T cell clones expressing Vß2, Vß4, and Vß16 TCR chains with the consensus junctional motifs described in Tables IIGo, IIIGo, and IVGo revealed predominant usage of the AV15 region by all T cell clones regardless of the BV region used (Table VIGo). Accordingly, AV15 transcripts were amplified from all GLC/A2+ T cell lines sorted with Vß2-, Vß4-, and Vß16-specific mAbs (Fig. 3Go). AV15 transcripts showed a highly conserved CDR3 length within a given Vß subset, which differed from one Vß subset to another (Fig. 3Go). Despite CDR3 length restriction, AV15 transcripts derived from Vß2 and Vß4 subsets showed significant junctional diversity in terms of both AJ usage and CDR3 sequence (Tables IIGo and IIIGo). By contrast, Vß16 populations derived from three of four GLC/A2+ cell lines studied (RA2, RA4, and PBL3) expressed AV15 TCR chains with identical CDR3 sequence (Table IVGo). None of the clones bearing other Vß regions (i.e., Vß1, Vß14, and Vß22 from donors RA5 and PB1) expressed AV15 transcripts, thus strongly suggesting that expression of this particular V{alpha} region was mainly restricted to the Vß2, Vß4, and Vß16 subsets. Interestingly, most (four of five) of these V{alpha}15-negative clones used the same V{alpha} region (AV2S3) despite expression of highly heterogeneous Vß regions, thus suggesting here again a hierarchical contribution of V{alpha} vs Vß regions to recognition of the GLC/A2 complex.


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  		Table VI. Junctional amino acid sequence of TCRA and TCRB transcripts derived from GLC/A2-reactive T cell clones1

 
Direct detection within unsorted PBL populations of the molecular imprint of the GLC/A2-specific T cell response on the peripheral TCR repertoire

The existence of a significant fraction of T cells with highly conserved CDR3 sequences in most GLC/A2-sorted T cell lines should lead to TCR repertoire alterations readily detectable within unsorted PBL using appropriate clonotypic primers. To test this, we synthesized primers specific for three consensus junctional sequences that predominated in GLC/A2+ cell lines derived from PBL1 (BV16BJ2S4), PBL2 (BV2BJ1S2), and PBL3 (BV16BJ2S3; see Tables IIGo and IVGo). We then performed successive nested PCR amplifications on total PBL or control thymus samples using firstly BV and BC primers, secondly BV and internal BJ primers, and thirdly BV and clonotypic primers. As shown in Fig. 5Go, peaks with the expected size were amplified using the relevant clonotypic primers in the three PBL samples studied, but were absent in the control sample. In parallel, we could directly estimate the frequency of the corresponding clonotypes by multiplying the percentage of GLC/A2+ T cells assessed by flow cytometry using the relevant fluorescent HLA/peptide tetrameric complexes (data not shown) by the frequency of T cells bearing the appropriate junctional sequence within GLC/A2+ T cells deduced from flow cytometry and junctional sequence analyses of GLC/A2-sorted T cells (Tables IGo, IIGo, and IVGo and Fig. 4Go). While the frequency of these clonotypes was ~5 x 10-4 in two-thirds of the samples studied, it was in the range of 10-5 or less in one sample (PBL3), thus demonstrating the high sensitivity of the present approach for detecting TCR repertoire biases associated with responses to dominant peripheral Ags.



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  		FIGURE 5. Direct detection within unsorted PBL of public TCR transcripts derived from GLC/A2-reactive T cell clonotypes. Shown are the Immunoscope profiles obtained from PBL1, -2, and -3 and from a control thymus sample using primers mentioned on top of each histogram (left, BV/BC; middle, BV/BJ; right, BV/clonotypic). In parallel, the percentage of cells stained by fluorescent A2/GLC tetramers was directly estimated by flow cytometry. The frequency of the corresponding public clonotype was then calculated by multiplying the percentage of GLC/A2+ T cells by the frequency of public transcripts within GLC/A2+ T cells, deduced from flow cytometry and molecular analysis of the GLC/A2 TCR repertoire.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
This study describes an extensive analysis of the TCR repertoire features of cells chronically stimulated by a dominant Ag. At first sight, repertoire diversity of GLC/A2-specific T cell lines seemed extensive, as suggested in particular by analysis of CDR3 size distribution of TCR ß-chains (Fig. 2Go). However, an in-depth analysis revealed the presence of clonotypes with recurrent TCR motifs within GLC/A2+ cells derived from most donors. While CDR1{alpha} and CDR2{alpha} (derived from the TCR AV15 gene segment) seemed to be shared by the largest fraction of reactive T cells, restriction of CDR1ß and CDR2ß usage was also obvious, although less pronounced, with seven Vß covering most of the GLC/A2 repertoire. Finally within several recurrent Vß subsets (Vß2, -4, -16, and -22) we identified conserved junctional motifs on either the CDR3ß and/or the CDR3{alpha} loops. The extent of CDR3 homology greatly varied from one subset to another, ranging from the presence of a conserved amino acid at a single position on the CDR3 loop (e.g., Gly at Cys+6 within BV22 sequences; Table VGo) to conservation of the whole CDR3{alpha} and/or -ß sequences. Interestingly, clonotypes expressing strictly identical TCR were identified in several cell lines (see, e.g., the BV4AV15 sequences from RA1, RA11 and RA5, RA14 donors (Table IIIGo) or BV16AV15 sequences from RA2, RA4 and PBL3 donors; Table IVGo).

Given the rather frequent occurrence of these public clonotypes within GLC/A2-reactive cells, these results raise the possibility that cells with similarly conserved features will be found in other antigenic responses. How, then, could we reconcile this assumption with the relatively seldom occurrence of public clonotypes suggested by previous analyses of Ag-specific T cells in many other models (9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20)? A possible explanation may rely on differences between the Ag selection conditions of the T cell populations studied. The extent of TCR repertoire restriction within Ag-selected T cells may be greatly affected by the avidity of the TCR/MHC/peptide interactions (which depends itself on the number of MHC/peptide complexes displayed by the presenting cell and the affinity of the TCR for these ligands). In particular, several groups have shown a more pronounced TCR repertoire restriction within cells stimulated at low Ag density and/or subjected to several rounds of antigenic stimulation, which should favor in both cases preferential expansion of subsets showing the highest affinity for the selecting Ag (40, 41, 42). In this regard, since EBV has been shown to be frequently reactivated through the lifetime of the individual (29), it is likely that GLC/A2-reactive cells, which were derived in most cases from old donors, have been chronically exposed to their Ag. Such a chronic stimulation could then explain the relatively common occurrence of public clonotypes within cells reactive against this particular Ag. Along this line, clonotypes with similarly highly conserved TCR features were previously identified in another EBV-specific T cell response (15). However, the chronic nature of the antigenic stimulus cannot alone explain the common occurrence of T cell with recurrent TCR features, as suggested by a recent study identifying public T cell clonotypes even during the acute phase of the GLC/A2-specific T cell response.4

These observations naturally raise several questions about the nature of the combinatorial and/or selective constraints that could favor the expansion of clonotypes with public TCR features. Comparison of the nucleotide sequences of TCR rearrangements coding for identical TCR chains in distinct individuals indicated rather limited "N" additions within most public V(D)J junctions (Table VIIGo). In particular, several public BV4, BV16, and AV15 transcripts comprised VD, DJ, or VJ joints devoid of nongermline nucleotides; among them, one BV4BD2BJ1S4 transcript shared by three donors was totally devoid of N nucleotides at both VD and DJ joints, and one AV15S1AJ37 joint shared by two donors was generated by so-called homologous region guided recombination (43) (Table VIIGo). Therefore, enzymatic constraints linked to the recombination machinery may favor the generation of some public rearrangements before Ag exposure, as suggested by several previous studies (12, 13, 14, 15). However, several public sequences also comprised highly conserved residues that were mainly N encoded; this was typically the case for public BV16 sequences, which showed highly diverse nucleotide sequences due to extensive N addition at the VD joints despite full conservation of their amino acid sequence (Table VIIGo). Hence, this indicates the occurrence of an Ag-driven selection of these public sequences, either intrathymically in the course of positive selection of the naive T cell repertoire or in the periphery, following exposure to one or several related environmental Ags. In this regard it has been previously proposed that public clonotypes might correspond to T cells that have been selected by self-MHC peptides highly related to the selecting peripheral Ags (44). According to this hypothesis, which is consistent with recent evidence for a key role played by self peptides in the positive selection process (21, 22, 23, 24), it is assumed that the public clonotypes will correspond to subsets establishing tight interactions with the selecting peptide, thus limiting the peptide degeneracy of the positive selection process. Another nonexclusive possibility is that the extent of TCR repertoire restriction of T cells reactive against a given Ag may be affected by peripheral selection driven by related peripheral Ags, and thus may depend on the immunological history of each individual (45). Either hypothesis could explain the occurrence of specific junctional motifs that were highly recurrent within BV22 sequences derived from donor PBL1, but were absent in other individuals (Table VGo).


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  		Table VII. Nucleotide sequences of the V(D)J junctions of public TCR chains derived from A2/GLC-sorted T cells1

 
A striking observation was the dominant usage of a particular V{alpha} region by several GLC/A2-reactive T cells that expressed TCR ß-chains with rather weak CDR1, CDR2, and CDR3 homology (i.e., V{alpha}15 by Vß2 or Vß4 vs Vß16 subsets and V{alpha}2.3 by Vß1, Vß2, Vß14, and Vß22 subsets; Table VIGo). This finding is reminiscent of previous studies suggesting a similar V{alpha} dominance within I-E/Cytc-reactive murine T cells (9, 10, 41) and with a recent analysis of the GLC/A2-specific T cell repertoire, which showed the use of a much more restricted set of V{alpha} than Vß regions by reactive T cell clones.4 Whether this observation can be generalized to other systems is unclear. Both the limited number of available V{alpha}-specific mAb and the lack of genotypic allelic exclusion of TCRA gene rearrangements have hampered detailed analysis of the TCR{alpha} repertoire by classical flow cytometric and molecular approaches. It is therefore possible that V{alpha} dominance may have been missed in many systems because of these mere technical limitations. On a structural basis, the dominant V{alpha} usage by GLC/A2+ T cells would be fully consistent with data drawn from recent crystallographic studies of several TCR/MHC/peptide complexes suggesting greater contribution of CDR{alpha} that CDRß in contacting the MHC/peptide (7, 8).

Another aim of the present study was to compare the TCR repertoire of EBV-reactive T cells expanded in an immunopathologic situation (in the present case, in patients with RA) to that of cells directed against the same viral epitope but derived from healthy donors. There is still a controversy regarding the physiopathologic significance of the dramatic enrichment for EBV-reactive T cells found within inflamed joints of most RA patients (26, 28). The strong similarities between the TCR repertoire of RA-derived synovial lymphocytes and PBL from healthy donors suggest that the massive infiltration of inflamed joints by EBV-reactive T cells is an epiphenomenon linked to the inflammatory process rather than the consequence of a local expansion of T cells cross-reacting with joint-derived Ags. Such a hypothesis, which was recently corroborated by an analysis of EBV reactivity of cells infiltrating inflammatory lesions with or without autoimmune features (28, 46), would rule out a primary etiological role for EBV-specific T cells in RA. However, given the high frequency of cells with such a reactivity (46) and the presence of the EBV genome within joint tissues of most RA patients (47), EBV-reactive cells may still contribute to perpetuation of joint erosion through local release of proinflammatory cytokines upon recognition of EBV+ targets.

The frequent expansion of T cell clonotypes with public TCR features during antigenic responses could have important implications in both immunodiagnosis and immunotherapy. In the former case, identification of a large number of public junctional motifs selected by defined Ags may permit a direct molecular follow-up of T cell responses from DNA or RNA samples, with no need for in vitro T cell manipulation and functional studies. This is illustrated by the Immunoscope analysis performed on PBL cDNA using primers specific for public clonotypic junctions, which allowed detection of public GLC/A2-reactive cells at frequencies as low as 10-5 (Fig. 5Go). It is likely that with the development of more sensitive molecular techniques permitting screening of a large number of DNA sequences (e.g., through DNA chips), such an approach could be applied for routine detection of T cell responses directed against a broad set of common infectious or tumor Ags. On an immunotherapeutic point of view, the frequent occurrence of T cell subsets bearing recurrent V{alpha}/Vß combinations should allow selective expansion or depletion of cells with respectively protective or pathogenic properties.


   Acknowledgments
 
We thank C. Tournay (Immunotech, Marseilles, France) for technical assistance with the generation of MHC/peptide tetramers, and A. B. Rickinson (Birmingham, U.K.) for critical reading of the manuscript.


   Footnotes
 
1 This work was supported in part by the Association pour la Recherche sur le Cancer, the Ligue Nationale contre le Cancer, and institutional grants from Institut National de la Santé et de la Recherche Médicale. Back

2 Address correspondence and reprint requests to Dr. Marc Bonneville, Institut National de la Santé et de la Recherche Médicale, Unité 463, Institut de Biologie, 9 quai Moncousu, 44035 Nantes Cedex 01, France. Back

3 Abbreviations used in this paper: CDR, complementarity-determining region; SFL, synovial fluid lymphocytes; RA, rheumatoid arthritis. Back

4 N. E. Annels, M. F. C. Callan, L. Tan, and A. B. Rickinson. Changing patterns of dominant T cell receptor usage with maturation of an Epstein-Barr virus-specific cytotoxic T cell response. Submitted for publication. Back

Received for publication February 17, 2000. Accepted for publication May 30, 2000.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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