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-DRB11302) response to a tetanus toxi n peptide (tt830-843) frequently used the Vbeta2 germ-line segment whi ch paired with several Valpha segments and that the putative CDR3 of b oth alpha and beta chains showed remarkable heterogeneity To analyze t he structural basis for recognition of the tt830-843/DR complex, five of these TcR were reconstituted into a murine T cell hybridoma, 58 alp ha-beta-, by expressing the human alpha and beta variable regions join ed to the mouse alpha and beta constant regions, respectively. The chi meric TcR, expressing the same Vbeta germ-line segment (Vbeta2), two e xpressing Valpha21.1, two Valpha17.1 and one Valpha8.1 were shown to h ave the expected antigen specificity and DR restriction. Two lines of evidence suggested that the putative CDR3, although not conserved in t hese TcR, played a key role in recognition. First, two TcR with identi cal V germ-line segments but distinct CDR3 showed large differences in their capacity to react with the ligand. Second, interchanging the al pha 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 Valpha2 1.1, as they appear to be related in their primary sequence. However, as in the case of CDR3 exchanges, Valpha replacement abrogated TcR rea ctivity. Taken together, these data underline the fine interdependence of the structural components of the TcR binding site in defining a gi ven specificity. Four of the TcR studied displaying promiscuous recogn ition 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.