Eur. J. Immunol. 2002. 32: 3181–3190 TCR V § restriction in Ag-specific responses 3181 Dominant TCR V > usage by virus and tumor- reactive T cells with wide affinity ranges for their specific antigens

Lydie Trautmann1, Nathalie Labarriere ` 1, Francine Jotereau1, Vaios Karanikas2,Nadine Gervois1, Thierry Connerotte2, Pierre Coulie2 and Marc Bonneville1

1 INSERM U463, Institut de Biologie, Nantes, France 2 Cellular Genetics Unit, Institute of Cellular Pathology and University of Louvain, Brussels, Belgium

We have studied the TCR features and functional responses of three sets of human cytolytic T cell (CTL) clones, recognizing antigenic peptides presented by HLA-A2 and derived from the Epstein-Barr virus proteins BMLF1 and BRLF1 and from the protein Melan-A/ MART-1. Within each set, a majority of clones used a recurrent V § region, even though they expressed highly diverse TCR g chains and V(D)J junctional sequences. Functional assays and peptide/MHC multimer binding studies indicated that this restricted V § usage was not associated with the affinity/avidity of the CTL clones. The V § dominance, which may be a frequent feature of antigen-specific T cells, likely reflects a restricted geometry of TCR/pep- tide/MHC complexes, primarily determined by V § CDR. Received 14/6/02 Accepted 3/9/02 Key words: TCR / T lymphocyte / Epstein-Barr virus / HCMV / Melanoma

1 Introduction suggest a dominant role of the V § domain in the orienta- tion of the TCR/pMHC complex [4, 5]. However, the gen- T cell recognition of antigenic peptides bound to self erality of this assumption has been put into question by MHC molecules (pMHC) is mediated by the TCR, whose the balanced contribution of V § and V g domains to § and g subunits are each encoded by randomly rear- pMHC binding in some cases [6, 7], and by the lack of ranged V, (D) and J segments [1]. The exquisite specific- conserved atomic contact points between pMHC and ity of pMHC recognition is made possible by the tremen- CDR § in the pMHC/TCR complexes resolved so far dous structural diversity of the TCR § and g chains, [4–7]. mainly concentrated in the CDR. The diversity of CDR1 and CDR2, which are completely encoded by V gene The amount of structural data recovered to date is still segments, is limited by the number of germ-line V seg- too limited to give a general understanding of the molec- ments that are available for recombination. The diversity ular mechanisms of TCR repertoire selection. In this of CDR3 is far more extensive, since these loops com- respect, the extensive TCR molecular studies performed prise both germ-line residues derived from the V(D)J on cells selected by defined pMHC complexes have segments and non-germ-line residues contributed by been useful in allowing identification of recurrent TCR nucleotides added in a template-independent fashion at structural motifs and analysis of the mechanisms under- the V(D)J joints [1]. Structural analyses of pMHC/TCR lying their selection. However, the TCR sequence diver- complexes have revealed a conserved diagonal orienta- sity of T cells reacting against a given pMHC turned out tion of the TCR over the pMHC complex, with the CDR3 § to greatly vary from one situation to another, ranging and CDR3 g loops centered in the TCR binding site and in from highly diverse to fully conserved TCR chains (see contact with the antigenic peptide [2–6]. The CDR1, 2 of ref. [8] for a review). These heterogeneous results proba- the TCR § and g chains are positioned at N and C termini bly reflect the complexity of parameters that come into of the peptide, respectively, and interact with both the play to restrict TCR diversity, such as enzymatic and peptide and MHC helices. Interactions between pMHC combinatorial constraints, TCR repertoire shaping by and residues at conserved positions of the CDR § loops intrathymic selection, cross-reactivity to environmental Ag, and affinity focusing subsequent to chronic Ag stimulation. [I 23265]

Abbreviations: pMHC: Peptide/MHC MFI: Mean fluores- In the present study, we made use of recombinant pMHC cence intensity TIL: Tumor-infiltrating lymphocytes multimers [9] to sort out T cells directed against several

© 2002 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim 0014-2980/02/1111-3181$17.50+.50/0 3182 L. Trautmann et al. Eur. J. Immunol. 2002. 32: 3181–3190

HLA-A2-restricted derived from viral and tumor 2Results proteins, and systematically sequenced their expressed TCR genes. Despite extensive CDR3 § and TCR g chain 2.1 Restricted TCR V > usage by T cells directed diversity, a highly recurrent usage of a particular V § against a dominant EBV antigen region was observed in three sets of clones directed against two viral and one tumor antigen. Furthermore A previous analysis of GLC/A2-specific T cell clones functional studies indicated that this recurrent structural suggested a restricted TCR V g repertoire and a rather feature was not associated with affinity/avidity restriction high frequency of clonotypes with “public” TCR junc- of the T cell response. The generality of these findings, tional features [10]. Significantly the same V § region (AV5 as well as their functional and structural implications, will according to the nomenclature of the IMGT data base) be discussed. was used by the three prominent V g subsets (BV20–1,

Table 1. TCR features of T cells directed against three HLA-A2-restricted antigensa)

CDR3 § J § V g J g CDR3 g

Specificity n length repertoire repertoire repertoire length

GLC/A2Total615–122514116–12

AV5+ 34 5–11 16 8 9 6–12

AV12-1+ 13 8–11 3 6 4 9–12

YVL/A2 Total 11 6–11 9 7 7 6–12

AV13-1+ 8 6–9 6 5 6 6–12

ELA/A2Total574–12271596–15

AV12-2+404–12221286–13 a) For each T cell subset of defined specificity, the number (n) of distinct clones studied (i.e. expressing TCR with distinct nucleotidic sequences), the range of CDR3 § and g lengths and the number of distinct J § ,Vg or J g used are indicated. Complete TCR § and g sequences are provided in Table 3 and as supplementary information in Tables A and B. Note the extensive TCR diversity of the three dominant V § subsets (AV5+, AV13–1+ and AV12–2+) identified, respectively, within GLC/ A2, YVL/A2 and ELA/A2-specific T cells.

Table 2. Frequent occurrence of public CDR3 within EBV- and tumor-specific responsesa)

Specificity Public CDR3

TCR V Sequence fb) Interindividual recurrencec)

GLC/A2 AV5 DN/SNARL 9/61 5/14

AV5 SI/TGKL 4/61 4/14

AV12-1 NGMDSSYKL 3/61 2/14

BV29-1 GTGGTNEKL 4/61 4/14

BV14 SQSPGGTQ 4/61 3/14

YVL/A2 AV13-1 KDTDKL 3/11 2/4

ELA/A2 AV12-2 SIGFGNVL 6/57 4/4 a) Listed are only canonical CDR3 sequences detected in at least two distinct donors. b) Frequency of public CDR3 within the sets of distinct clones listed in Table 3 and as supplementary information in Table A and B. c) Frequency of donors yielding clones with the indicated CDR3 sequence. Eur. J. Immunol. 2002. 32: 3181–3190 TCR V § restriction in Ag-specific T cell responses 3183

Table 3. TCR § and g sequences expressed by MelanA/A2-reactive T cell clonesa)

a) T cell clones were derived from tumor-infiltrating lymphocytes of three melanoma patients (M138, M180 and M199). nd: not determined. Additional TCR sequences obtained from ELA-peptide stimulated PBL from another patient are provided in Table C (supplemental information).

BV29–1 and BV14), suggesting a more homogeneous V § CDR3 g lengths ranged from 5 to 11 and 8 to 12 amino than V g repertoire [10]. To extend this observation made acids, respectively. Similarly, AV12–1+ clones expressed on a small set (n =18) of clones, we studied published TCRwithatleast6distinctVg elements and highly [11] and unpublished TCR sequences of 61 anti-GLC/A2 diverse VJ § and VDJ g junctions (Table 1). In contrast, CTL clones obtained during acute and memory anti-EBV subsets defined by expression of a given V g region used responses. As shown in Table 1, 47/61 clones expressed no more than 3 distinct V § , and most of them expressed the AV5 or AV12–1 genes. A marked conservation of aV§ region (AV5 or AV12–1) also used by the other domi- V(D)J junctional sequences was noted within subsets nant V g subsets (see e.g. the BV20–1, BV29–1 and BV2 expressing similar V § V g combinations (e.g. AV5BV20–1, subsets in Table A, supplementary information). AV5BV29–1 or AV12–1BV14), even within clonotypes derived from distinct individuals [Table 2 and supple- mentary information (Table A) at the following home- page: http://www.ifr26.nantes.inserm.fr/maj/table–A- 2.2 Restricted TCR V > usage by T cells directed C=trautmann.doc]. Despite these recurrent features, the against other antigens overall diversity of TCR chains carried by GLC/A2- specific T cells expressing a given V § region was exten- We studied the structural features of panels of T cell sive. For instance, AV5+ clones used at least 16 distinct clones recognizing two other HLA-A2-restricted epi- J § ,8Vg and 9 J g elements, and their TCR CDR3 § and topes derived, respectively from the EBV lytic protein 3184 L. Trautmann et al. Eur. J. Immunol. 2002. 32: 3181–3190

Table 4. Estimation of GLC/A2- and ELA/A2-specific T cell Two sets of anti-ELA/A2 CTL clones were analyzed: the clone avidity in the absence or presence of blocking anti- M clones were derived from tumor-infiltrating lympho- CD8 mAb cytes (TIL) of three melanoma patients (Table 3), while the VER clones were obtained from blood lymphocytes of another melanoma patient (Table C, supplementary information). Fifty-seven different TCR sequences were obtained (Table 3 and C). For the g chain, the V repertoire and VDJ sequences were highly heterogeneous, as reported [16–18], even though a bias for BV27 and BV28 was noted. For the § chain, the junctional diversity was extensive: 25 J § genes were used, and CDR3 regions contained 4 to 12 amino acids. Here again, the majority (40/57) of the CTL expressed the same V § gene (AV12–2) (Table 1). The dominant usage of this V § gene, irrespec- tive of other TCR features, is illustrated by a comparison

a) The EC50 value corresponds to the Ag peptide concentration required for half maximal lysis of peptide- loaded target cells. b) These clones were tested in separate experiments.

BRLF1 (hereafter referred to as YVL/A2) [12], and the melanoma protein Melan-A/MART-1 (hereafter referred to as ELA/A2) [13–15]. Fig. 1. Cytolytic responses of AV12–2+ ELA/A2-specific The anti-YVL/A2 CTL (n =11) used seven distinct V g clones against peptide Ag-loaded target cells. (A) CTL dose regions, two of which (BV19 and BV27) were shared by 3 response curves (effector to target ratio of 10/1) of AV12–2+ ELA/A2-specific clones derived from donors M138, M180 clones (Table 1 and Table B, supplementary information). § and M199 against HLA-A2+ allogeneic B lymphoblastoid TCR V usage was more homogeneous: 8/11 clones cells loaded with various amounts of ELA/A2 peptide. (B) + expressed the AV13–1 gene. The TCR of these AV13–1 CTL response of two AV12–2+ clones (M138–33 and clones were structurally diverse, as indicated by usage M180–57) against ELA/A2-loaded targets in the absence of five distinct V g ,6J§ ,6Jg and highly heterogeneous (closed symbols) or presence (open symbols) of blocking CDR3 (Table 1 and Table B, supplementary information). anti-CD8 mAb. Eur. J. Immunol. 2002. 32: 3181–3190 TCR V § restriction in Ag-specific T cell responses 3185 of the V g usage within V § subsets with the V § usage presence of blocking anti-CD8 mAb, was more hetero- within V g subsets. While the AV12–2+ cells used at least geneous, even within sets of clones that expressed the 12 V g genes, subsets of CTL sharing the expression of a same V § gene (see e.g. the AV5+ clones A2.10 vs. 16.14 particular V g region used no more than 3 V § genes, in Table 4). always including AV12–2 (Table 3 and C).

In agreement with previous reports [20, 21], the EC50 of ELA/A2-specific clones were highly diverse, ranging 2.3 Restricted TCR V > usage is not associated from 4.10–10 to 2.10–7 M (Table 4). This was also true for + with TCR avidity/affinity focusing the AV12–2 cells, which included clones with EC50 differ- ing by as much as 500-fold (i.e. between clones M199.38 Lytic activities of anti-GLC/A2 and anti-YVL/A2 CTL and M199.29) (Table 4 and Fig. 1A). While in most cases were analyzed with HLA-A2+ EBV-B cells loaded with antigen recognition was abrogated or strongly inhibited various amounts of antigenic peptides. In agreement by anti-CD8 mAb (see e.g. clone M180.57 in Fig. 1B), with previous analyses of anti-EBV CTL [19], the range of three clones (M138.33, M199.15 and M199.34) were peptide concentrations yielding half-maximal lysis of mildly affected by anti-CD8-blocking Ab (Table 4). CTL –8 peptide-loaded targets (EC50)wasnarrow:from10 to expressing AV12–2 were either CD8 “independent” or 10–7 Mand10–7 to 10–6 M for anti-GLC/A2 and anti-YVL/ strongly “dependent” (see e.g. M138.33 vs. M180.57 in A2 CTL, respectively (Table 4 and data not shown). The Fig. 1B and Table 4), indicating that there was no clear contribution of CD8 to the overall avidity, classically esti- link between CD8-dependency and expression of this V § mated by comparing T cell responses in the absence or gene. Altogether, the results obtained with the anti-EBV

Fig. 2. analysis of ELA/A2-specific clones using ad hoc pMHC tetramers. (A) Representative flow cytometry pro- files of 3 AV12–2+ T cell clones obtained with a control Ab (filled histogram), PE-conjugated ELA/A2 tetramers (empty histogram, thick line) or anti-CD3 mAb (empty histogram, thin line). (B) Distribution of MFI obtained with 20 ? g/ml of ELA/A2 tetramer (left panel), normalized to CD3 staining intensity (right panel), of AV12–2+ and AV12-2- ELA/A2-specific clones. (C) MFI curves obtained at various concentrations of ELA/A2 tetramer with 7 ELA/A2-specific clones (left panel) and their Scatchard representa- tion (MFI, × axis vs. MFI/tetramer concentration, y axis; arbitrary units) (right panel). 1: M199.54, 2: M180.24, 3: M180.27, 4: M180.25, 5: M138.42, 6: M199.17, and 7: M180.51 (TCR sequences provided in Table 3). 3186 L. Trautmann et al. Eur. J. Immunol. 2002. 32: 3181–3190 and the anti-Melan-A CTL suggested that restriction of AV12–2+ cells (Fig. 2A and B). Similar results were V § usage was not associated with TCR affinity focusing. obtained when normalizing the results to CD3 or TCR g expression (Fig. 2B and data not shown), indicating that As a more direct way to estimate TCR avidity, we studied the heterogeneous multimer staining was not merely the binding of fluorescent pMHC multimers. TCR affinity accounted for by variability of TCR expression levels. for monomeric pMHC complexes has been shown to Furthermore, the presence of another functional TCR § correlate with the intensity of staining with pMHC class II chain, which could have resulted in reduced multimer multimers that could not bind to CD4 coreceptor [22]. staining levels, was ruled out by isolation of out of Therefore we analyzed the binding of mutated HLA-A2 frame TCR § transcripts in most clones, including multimers that have a greatly reduced affinity for CD8 those that were poorly stained by multimers (data not coreceptors [23], thus permitting a more specific assess- shown). ment of the TCR contribution to multimer binding. Inten- sities of staining with ELA/A2-multimers varied greatly The intensity of staining with the multimers was corre- from one CTL clone to another, even within the subset of lated with the affinity/avidity of multimer binding as doc- umented by Scatchard analysis (Fig. 2C). Intensities of staining with multimers of anti-GLC/A2 CTL were more homogeneous than with multimers of the anti-ELA/A2 clones (Fig. 3A) in line with the narrower range of func- tional avidities observed with the former set of CTL (Table 4). For the anti-Melan-A CTL, intensities of multi-

mer staining correlated with EC50 estimated in lysis assays. This correlation improved significantly when only the AV12–2+ cells were taken into account (R2=0.73 vs. 0.42 for the whole population, Fig. 3B).

3Discussion

The most striking and unexpected observation of this studyistheV§ dominance, namely the usage of a recur- rent V § region, combined with diverse CDR3 § and CDR g sequences, by three sets of CTL. The fact that these CTL were derived from several donors and selected under various physiopathological contexts, namely acute or chronic in vivo stimulation by either viral or tumor anti- gens, suggests that the V § dominance is a frequent phe- nomenon. This is supported by preliminary repertoire analyses of T cell responses directed against three other antigens: the viral antigens LMP2/A2 from EBV and IE1/ Fig. 3. Correlation between pMHC tetramer staining inten- B18 from human , and the tumor anti- gen Mage-3/A2. Indeed in all cases, we identified clono- sity and T cell clone EC50 deduced from functional assays. MFI obtained with either GLC/A2- or ELA/A2-tetramers were types expressing identical V § and distinct V g but not the plotted against the EC50 estimated from CTL assays reverse(L.T.,M.B.,V.Prodhomme,M.M.Hallet,K.Ber- against Ag-loaded targets. For each T cell response, clones nardeau and F. Davodeau; unpublished). These results were split into two subsets expressing (closed symbols) or are reminiscent to several previous studies describing a § not expressing (open symbols) the dominant V region (i.e. restricted V § but diverse V g repertoire within Ag-specific AV5 for GLC/A2 and AV12–2 for ELA/A2-reactive clones). T cells in various models [24–27]. Our results are also in Correlation between pMHC tetramer MFI and EC was 50 line with a recent report from Yokosuka and colleagues highly significant for ELA/A2-specific T cells (p X 0.001) but note the much higher regression coefficient obtained when [28], which demonstrated a dominant contribution of the § only AV12–2+ clones are taken into account. TCR chain to recognition of two pMHC complexes through analysis of TCR transfectants and single TCR- transgenic mice. Quite strikingly, this study showed that the original TCR specificity could be reconstituted by co- Eur. J. Immunol. 2002. 32: 3181–3190 TCR V § restriction in Ag-specific T cell responses 3187 transfecting the TCR § chain from an HIV-specific T cell previously on activated T cells using pMHC dimers, clone with TCR g chains sharing the same V g with the could reflect variable degrees of multimer cooperative reactive clone but carrying “random” CDR3 sequences. binding, possibly due to heterogeneous TCR distribution However, in this particular experimental setting, the within and without lipid rafts [31]. Although the correla-

CDR3 § was fixed, as well as the CDR1 and 2 g .Ourpres- tion between EC50 and multimer staining was highly sig- ent results clearly extend these findings as they strongly nificant, the regression coefficient remained rather low suggest, in light of the extensive TCR § and g junctional (R2=0.4), indicating that additional parameters affected T diversity observed within MelanA-reactive T cells, a hier- cell tetramer binding. In this respect, our results suggest archal contribution of CDR1 § and CDR2 § over the other that within clones showing similar avidities, tetramer CDR including CDR3 § to recognition of this particular avidity might be also affected by V § usage. Since trivial Ag. Interestingly, such a dominant contribution of two explanations such as heterogeneous TCR expression out of six CDR3 could possibly explain the previously levels, differences in terms of CD8 dependency or pres- reported high frequency of MelanA-reactive T cells in the ence of another functional § chain were all ruled out, it is naive repertoire of healthy donors [17]. possible that particular topologies of pMHC/TCR com- plexes, primarily established through interactions with Our results suggest that V § dominance is not associated V § CDR [3], interfere with an efficient cooperative bind- with TCR affinity focusing. Indeed T cell avidities for ing of pMHC multimers, consequently leading to a APC, deduced from CTL assays using peptide-loaded decreased multimer avidity. targets, greatly varied from one clone to another, even for clones sharing the same TCR V § region. Although CTL Although fully conserved (so-called “public”) TCR responses result from interactions involving not only the sequences were identified in several repertoire studies TCR but also adhesion and costimulatory molecules, the (see e.g. [32–35]), the generality of these observations average contribution of the latter receptors tends to be has remained unclear. Usually, these public CDR3 the same when analyses are performed on a sufficiently sequences have been observed for populations of cells large set of clones selected under similar conditions [19, that had been chronically exposed to their selecting anti- 21]. Therefore CTL assays can give an indirect but reli- gen and that displayed similar TCR avidities [34, 35], able estimate of the avidity of interactions involving suggesting that CDR3 restriction resulted from TCR pMHC and TCR/coreceptor complexes. TCR contribu- affinity focusing. This is consistent with our analysis of tion can be estimated further by analysis of T cell EBV-specific T cells, with several public clonotypes responses in the presence of blocking anti-CD8 mAb. whose avidity and CD8 dependency were very similar. Such responses proved to be highly heterogeneous However, anti-Melan-A clones with fully conserved –9 within a given V § subset, with EC50 ranging from 10 to CDR3 § were also identified (Table 2 and 3), even though more than 10–6 M in the presence of blocking anti-CD8 these cells displayed avidities differing by as much as mAb, supporting further the wide affinity range of TCR 50-fold (e.g. between clones M199.16 vs. M199.54 or expressed by these cells. However, conclusions drawn M199.29 vs. M199.18, Table 3 and 4). Considering that from CD8-blocking data must be taken with caution, these canonical sequences contained non-germ-line since they could reflect not only differential contribution nucleotides (data not shown), their over-representation is of CD8 ectodomains to TCR/pMHC interaction but also probably not accounted for by trivial enzymatic/recombi- variable degrees of CD8/TCR co-clustering and/or gen- nation constraints. Their recurrence could result from eration of CD8-mediated inhibitory signals [29]. It was repertoire remolding following thymic selection and/or therefore important to document the heterogeneous homeostatic peripheral expansion, which is associated TCR affinities of the above T cell clones through inde- with frequent over-representation of particular CDR3 pendent approaches. The intensities of staining of ELA/ within the naive T cell repertoire [36, 37]. Whatever the A2-specific clones with mutated HLA-A2 multimers with proper explanation, the rather common occurrence of greatly reduced avidity for CD8 coreceptors [30] corre- canonical CDR3 sequences demonstrated here could lated well with the EC50 estimated in functional assays. have implications for the follow up of tumor-specific T As suggested by multimer titration experiments per- cells in various clinical settings. In particular, sensitive formed with or without elimination of unbound molecules molecular approaches targeting the canonical sequence (Fig. 2C and data not shown), this correlation is most SIGFGNVL, found in all four individuals tested here likely explained by the fact that multimer binding is per- (Table 3) and in more than 10% of ELA/A2-specific formed under non-saturating conditions. It is significant clones, could be designed to follow Melan-A-specific that for almost all clones studied, Scatchard plots responses in melanoma patients. deduced from pMHC multimer titrations were curvilinear, indicating the presence of several binding components In the absence of crystallographic data obtained with with distinct affinities. This peculiar behavior, observed pMHC bound to TCR with similar specificity and V § 3188 L. Trautmann et al. Eur. J. Immunol. 2002. 32: 3181–3190 usage, one can only but speculate about the structural viously documented reactivity against EBV [12] or tumor basis of V § dominance. It may reflect a major role played infiltrating lymphocytes (TIL) of HLA-A2 melanoma patients. by V § in determining the general orientation of the TCR T cells were cultured, expanded and cloned as described over the pMHC complex, whose avidity might then be [40]. finely tuned by the other CDR [4, 5]. According to this hypothesis, the fine geometry of TCR/pMHC complexes Sorting of antigen-specific T cells using recombinant pMHC would be the same for TCR directed against a given complexes GLC/A2, YLV/A2 or ELA/A2 was performed on + pMHC complex and sharing the same V § region, irre- CD8 sorted T lymphocytes using streptavidin-coated spective of other CDR. This could explain our preliminary immunomagnetic beads (Dynal, Compiegne, France) as described [30]. data suggesting a significant influence of V § usage on pMHC tetramer binding and the common pattern of reactivity to some mutated peptides of clones bearing a 4.3 Flow cytometric analysis particular V § (data not shown). Furthermore this model, § which implies conserved interactions between CDR1 T cell lines and T cell clones were phenotyped by indirect § and/or CDR2 and pMHC, would fit with the key role immunofluorescence with mAb against TCR V g regions [40]. played by some CDR1 § and CDR2 § residues in skewing Stainings with GLC/A2, YLV/A2 or ELA/A2 tetramers were the MHC class specificity of some V § subsets [38, 39]. In performedbyincubatingcellsfor1hatroomtemperature the above studies, interactions involving these CDR with PE-labeled MHC/peptide tetramers at 20 ? g/ml in PBS- regions may not be affected by the antigenic peptide BSA 0.1%. After staining, cells were analyzed by flow cyto- (see, however, discussion in ref. [36]). In our study the metry on a FACScan apparatus (Becton Dickinson, Gre- dominant V § differed from one pMHC complex to noble, France). another, even though the HLA context remained the same. This suggests that the interactions that determine the topology of the TCR/pMHC complex, and that pre- 4.4 Functional assays sumably involve CDR1 § or CDR2 § residues, are strongly ? influenced by the antigenic peptide. Synthetic peptides were dissolved at 20 g/ml in DMSO, diluted at 2 mg/ml in 10 mM acetic acid and then diluted at the requested concentration in RPMI 10% SVF. Cytotoxicity was measured in a standard 4-h 51Cr-release assay [19]. For 4 Materials and methods CD8 blocking experiments, T cells were incubated with the anti-CD8 mAb B9–11 (Immunotech, Marseille, France) at 4.1 Generation of recombinant pMHC complexes 10 ? g/ml for 15 min before incubation with target cells.

Soluble pMHC monomers GLC/A2, YLV/A2 and ELA/A2 g were generated as described [9]. 2-Microglobulin and HLA- 4.5 Sequence analysis of TCR transcripts A2 heavy chain were produced as inclusion bodies in Esche- richia Coli, dissolved in 8 M urea and refolded in the pres- RNA from 5×106 T cell clones was extracted with TRIZol ? ence of 15 g/ml of the following synthetic peptides: reagent (Invitrogen, Cergy Pontoise, France) according to GLCTLVAML derived from the EBV lytic early protein BMLF1 the supplier’s instructions and dissolved in 15 ? l of water. [9], YVLDHLIVV derived from the EBV lytic early protein Reverse transcriptions, PCR amplifications and sequencing BRLF1 [20] or the mutated melanA-MART1 peptide ELAGI- were performed as described [18, 40]. We have followed GILTV [13]. The folded pMHC complexes were diafiltrated throughout the manuscript the TCR nomenclature of the and concentrated in 10 mM Tris pH8 on a prep scale 3-kDa IMGT data base (http://imgt.cines.fr) concentration cassette (Millipore, Saint Quentin en Yvelines, France). pMHC complexes were biotinylated using BirA enzyme, purified by anion exchange on a MonoQ column Acknowledgements: We thank K. Echasserieau and M. A. with a 0 to 0.5 M NaCl gradient, and checked by gel filtration Peyrat for expert technical assistance, F. Lang, M. Bodinier on a Superdex 200 column. For flow cytometry analyses, and D. Colau for providing recombinant pMHC molecules, pMHC monomers were tetramerized with phycoerythrin C. Lurquin for several anti-Melan-A TCR sequences, V. (PE)-labeled streptavidin at a molar ratio of 4:0.8. Prod’homme, M. M. Hallet, K. Bernardeau, F. Davodeau (InsermU463,Nantes,France)andN.AnnelsandA.B.Rick- inson (CRC for clinical studies, Birmingham, GB) for allowing 4.2 Generation of T cell clones us to cite their published and unpublished repertoire data. This work was supported in part by the European Commu- T cell lines were derived from either fresh PBL of HLA-A2 nity, la Ligue departementale ´ (Loire-Atlantique) contre le EBV seropositive healthy donors, synovial fluid lymphocytes , the FB Assurances and VIVA (Belgium), and institu- (SFL) of HLA-A2 rheumatoid arthritis (RA) patients with pre- tional grants from Institut National de la Santeetdela ´ Eur. J. Immunol. 2002. 32: 3181–3190 TCR V § restriction in Ag-specific T cell responses 3189

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