Analysis of tetanus toxin peptide/DR recognition by human T cell receptors reconstituted into a murine T cell hybridoma.
We have previously reported that human T cell receptors (TcR) selected in the class II-restricted (HLA-DRB1*1302) response to a tetanus toxin peptide (tt830-843) frequently used the V beta 2 germ-line segment which paired with several V alpha segments and that the putative CDR3 of both alpha and beta chains showed remarkable heterogeneity. To analyze the structural basis for recognition of the tt830-843/DR complex, five of these TcR were reconstituted into a murine T cell hybridoma, 58 alpha- beta-, by expressing the human alpha and beta variable regions joined to the mouse alpha and beta constant regions, respectively. The chimeric TcR, expressing the same V beta germ-line segment (V beta 2), two expressing V alpha 21.1, two V alpha 17.1 and one V alpha 8.1 were shown to have the expected antigen specificity and DR restriction. Two lines of evidence suggested that the putative CDR3, although not conserved in these TcR, played a key role in recognition. First, two TcR with identical V germ-line segments but distinct CDR3 showed large difference in their capacity to react with the ligand. Second, interchanging the alpha and beta chains from tt830-843/DR1302-specific TcR which differed in their CDR3 sequences invariably led to loss of recognition. We also asked whether germ-line V alpha 17.1 could functionally replace V alpha 21.1, as they appear to be related in their primary sequence. However, as in the case of CDR3 exchanges, V alpha replacement abrogated TcR reactivity. Taken together, these data underline the fine interdependence of the structural components of the TcR binding site in defining a given specificity. Four of the TcR studied displaying promiscuous recognition were also tested against different DR alleles and site-directed mutants. The results of these experiments suggested that, in spite of their structural heterogeneity, anti-tt830-843 TcR may have a similar orientation with respect to the peptide/DR complex. The reconstitution system described herein should represent a valuable tool for detailed studies of human TcR specificity.
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Laboratory of Molecular Immunology, Pasteur Institute, Paris, France.
Boitel
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Mège
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Ermonval
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Acuto
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D000595:Amino Acid Sequence; D000818:Animals; D001427:Bacterial Toxins; D001483:Base Sequence; D002678:Chimera; D004768:Enterotoxins / Q000276:immunology; D006684:HLA-DR Antigens / Q000276:immunology*; D059811:HLA-DRB1 Chains; D000949:Histocompatibility Antigens Class II / Q000276:immunology*; D006801:Humans; D006825:Hybridomas / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000276:immunology*; D016693:Receptors, Antigen, T-Cell, alpha-beta / Q000235:genetics / Q000276:immunology / Q000502:physiology*; D013329:Structure-Activity Relationship; D018089:Superantigens; D013744:Tetanus Toxin / Q000276:immunology*; D014162:Transfection
D001427:Bacterial Toxins; D004768:Enterotoxins; D006684:HLA-DR Antigens; D059811:HLA-DRB1 Chains; D000949:Histocompatibility Antigens Class II; D010446:Peptide Fragments; D016693:Receptors, Antigen, T-Cell, alpha-beta; D018089:Superantigens; D013744:Tetanus Toxin; C030107:enterotoxin F, Staphylococcal