Conserved CDR3 regions in T-cell receptor (TCR) CD8(+) T cells that recognize the Tax11-19/HLA-A*0201 complex in a subject infected with human T-cell leukemia virus type 1: relationship of T-cell fine specificity and major histocompatibility complex/peptide/TCR crystal structure

Abstract

We investigated the T-cell receptor (TCR) repertoire of CD8(+) T cells that recognize the Tax11-19 immunodominant epitope of Tax protein expressed by human T-cell leukemia virus (HTLV-1) that is implicated in the disease HTLV-1-associated myelopathy (HAM/TSP). A panel of Tax11-19-reactive CD8(+) T-cell clones was generated by single-cell cloning of Tax11-19/HLA-A*0201 tetramer-positive peripheral blood lymphocytes from an HTLV-1-infected individual. The analyses of TCR usage revealed that the combination of diverse TCR alpha and beta chains could be used for the recognition of Tax11-19 but the major population of T-cell clones (15 of 24 clones) expressed the TCR V beta 13S1 and V alpha 17 chain. We found striking similarities in CDR3 regions of TCR alpha and beta chains between our major group of CD8(+) T-cell clones and those originating from different subjects as previously reported, including TCRs with resolved crystal structures. A 3-amino-acid sequence (PG-G) in the CDR3 region of the V beta chain was conserved among all the Tax11-19-reactive T-cell clones expressing V beta 13S1 and V alpha 17 chains. Conserved amino acids in the CDR3 region do not directly contact the Tax11-19 peptide, as corroborated by the crystal structure of B7-TCR, a TCR that is almost identical to VB13S1 clones isolated in this study. Analysis of fine peptide specificity using altered peptide ligands (APL) of Tax11-19 revealed a similar recognition pattern among this panel of T-cell clones. These data suggest that the PG-G amino acids in the CDR3 beta loop provide a structural framework necessary for the maintenance of the tertiary TCR structure.

FIG. 1

Similar pattern of antigen recognition among T-cell clones expressing TCR BV13S1. (A) Antigen dose-response cytotoxic activity of Tax11-19-reactive CD8⁺ T-cell clones. HLA-A*0201-expressing EBV-transformed B-cell lines were pulsed with the indicated concentrations of Tax11-19 peptide and used as target cells in a 4-h ⁵¹Cr-release assay. The effector-to-target ratio was 10:1. (B) Specific binding of HLA-A*0201/Tax11-19 multimer by CD8⁺ T-cell clones expressing TCRBV13S1. T-cell clones were incubated with the indicated dilutions of tetramer at 4°C for 1 h. The left y axis indicates the mean fluorescence intensity of multimer binding, while the right y axis shows the mean fluorescence intensity of TCR expression.

FIG. 2

Requirement of conserved amino acid positions for recognition of the Tax11-19 peptide. (Top) Crystal structure representation of interactions between TCR-B7 CDR3 regions and HLA-A*0201 complexed with Tax11-19 peptide. Only amino acids 4 to 8 of the Tax11-19 peptide are presented. Amino acids that are conserved in the CDR3 beta chain among T-cell clones are indicated by arrows. Conservation of Pro at position 97 and Gly at positions 98 and 100 in all clones expressing TCRBV13S1 suggest that these residues provide a framework for the tertiary structure of the CDR3 loop. Residues that contact MHC peptide are underlined. Amino acid numbering of TCR alpha and beta chains is as in reference . (Bottom) Comparison of CDR3 V beta amino acid sequences of Tax11-19-reactive CD8⁺ T-cell clones isolated in this study (groups I to V) and previously published (B7 and ID4) (13). Positions that are maintained in all clones are indicated by arrows.

FIG. 3

Fine specificity of CD8⁺ T-cell clones to singly substituted Tax11-19 analogues. T-cell recognition of Tax11-19 peptide analogues at TCR P5 and P8 pockets in Tax11-19-reactive CD8⁺ T-cell clones expressing strong structural similarities is shown. The peptide recognition was determined by a standard 4-h ⁵¹Cr release assay. HLA-A*0201-expressing EBV-transformed B-cell lines were pulsed with peptides at 10 μM and used as target cells. The effector-to-target ratio was 10:1. AA, amino acid.

FIG. 4

Different recognition patterns of CD8⁺ T-cell clones toward naturally occurring peptides. Differential recognition of natural peptides by CD8⁺ Tax11-19-reactive T-cell clones expressing BV13S1 is shown. Human and microbial peptides expressing the recognition motif for B7 TCR were previously published (12) and used in this study. Recognition patterns of CD8⁺ T-cell clones were determined by a ⁵¹Cr release assay at a peptide concentration of 10 μM. Of 16 peptides tested, 3 were recognized by CD8⁺ T-cell clones, and reactivity toward these 3 peptides is shown. As previously published (12), the B7 clone recognized the above peptides with a range of 18 to 20% specific lysis. a.a., amino acid.