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 Vbeta2 germ-line segment which paired with several Valpha 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 Vbeta germ-line segment (Vbeta2), two expressing Valpha21.1, two Valpha17.1 and one Valpha8.1 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 differences 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 Valpha17.1 could functionally replace Valpha21.1, as they appear to be related in their primary sequence. However, as in the case of CDR3 exchanges, Valpha 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.