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Usage by Virus and Tumor-Reactive T Cells with Wide Eur. J. Immunol. 2002. 32: 3181–3190 TCR V § restriction in Ag-specific T cell 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 melanoma 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 epitopes 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).
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