Analysis of major histocompatibility complex class I-restricted hapten recognition by mutation of the V-J joining of T cell receptor alpha chains.

Hapten-specific T cell responses are responsible for chemically induced immune disorders. However, the molecular details of hapten interactions with T cell receptors (TCR) are poorly understood. Recent studies of trinitrophenyl (TNP)-specific responses revealed major histocompatibility complex-associated TNP-peptides as dominant epitopes for CD8+ and CD4+ T cells. The present study is based on the observation that two H-2Kb/TNP-specific CTL clones (II/7 and III/1), differing exclusively in two amino acids of their TCR alpha chains, also differed in their carrier specificities for various TNP-peptides. The genes of the two alpha chains and the common beta chain were cloned into expression vectors. Transfection of the TCR alpha chain of clone III/1 into a hybridoma of clone II/7 also transferred the fine specificity of clone III/1, indicating that the small alpha chain variations were indeed responsible for the different carrier specificities. Point mutations bridging the difference between the alpha chains of clones II/7 and III/1 and functional studies of the respective TCR alpha beta transfectants into a TCR-negative hybridoma revealed an unexpected result: the two receptors did not represent examples of structural complementarity for different sets of hapten-peptide conjugates; rather, they resembled two structures of principally similar specificity but of significantly different overall affinity. This was demonstrated more directly by comparing the fine specificities of III/1 transfectants expressing or not expressing the co-receptor CD8: the CD8-negative III/1 transfectant assumed a specificity pattern indistinguishable from that of a CD8-expressing, II/7-derived transfectant. Hence, comparable alterations of antigen recognition may be induced either by subtle TCR alterations or by removal of CD8, i.e. by the presence or absence of a non-polymorphic adhesion molecule.

Carrier-reactive hapten-specific cytotoxic T lymphocyte clones originate from a highly preselected T cell repertoire: implications for chemical-induced self-reactivity.

We have recently described trinitrophenyl (TNP)-specific cytotoxic T lymphocyte (CTL) clones from C57BL/6 mice specific for hapten-modified peptides bearing a TNP-lysine in a peripheral position, i.e. in position 7 of H-2Kb-bound octapeptides. CTL recognition of such determinants is always sequence-dependent due to co-recognition of TNP as well as amino acid side chains of the carrier peptide. By the use of glycine-based designer peptides for primary induction of CTL in vitro, we have identified two sub-epitopes on individual position 7-haptenated peptides that form two TcR contact points and which can be independently recognized by cloned CTL. One of these sub-epitopes is represented by the hapten itself, the other by the amino acids tyrosine and lysine in positions 3 and 4 of the carrier peptide, respectively. Immunization with such TNP-modified peptides frequently results in the specific induction of CTL also reacting with the unmodified carrier peptides. DNA sequence analyses of the TcR revealed an extraordinary similarity of several independent TcR of CTL from individual mice and induced with different TNP-peptides. These receptor similarities clearly correlate with structural elements common to the immunizing peptides and suggest their origin from positive thymic selection of TcR on Kb-associated associated self-peptides bearing Tyr in position 3. Our data provide additional information concerning the topology of TcR binding to peptide/MHC complexes with, but also without, TNP. They also indicate a mechanism which might explain the potential of chemicals or drugs to induce autoimmune phenomena.

Purified MHC class I molecules present hapten-conjugated peptides to TNP/H-2Kb-specific T cell hybridomas.

Several trinitrophenyl (TNP)-specific mouse cytotoxic T cell (CTL) clones recognize TNP-conjugated peptides in association with class I MHC Kb-molecules. Here we show that CD8+ T cell hybridomas derived from these CTL exhibit the same pattern of antigen-specificity as their parent CTL-clones. These T cell hybridomas reacted with TNBS- or TNP-peptide modified syngeneic target cells, and also with affinity purified, immobilized Kb-molecules preloaded with TNP-peptides. These findings demonstrate most directly that MHC-associated, haptenated peptides create functional antigenic epitopes for TNP-specific CTL. Furthermore, using purified Kb-molecules and a panel of Kb-binding TNP-conjugated peptides, we demonstrated that the epitope density is a critical factor in triggering these T cell hybridomas. Chemical modification of immobilized Kb-layers resulted in poor antigenicity, implying low epitope density and therefore arguing against covalent MHC-haptenization as a major source of T cell antigenic determinants.

Antigen contact sites in class I major histocompatibility complex-restricted, trinitrophenyl-specific T cell receptors.

Cloned trinitrophenyl (TNP)-specific cytotoxic T cells (CTL) were obtained from mice transgenic for the beta chain of the antigen-specific receptor (TcR) of a Kb-restricted, TNP-specific CTL clone (BT7.4.1). The transgene-expressing CTL, specific for TNP/Kb were found to select for TcR alpha chains highly similar to that of the transgene donor clone BT7.4.1. In that way, two clones (II/7 and III/1) were identified whose TcR differed from the BT7.4.1 receptor only in their N alpha- and J alpha-sequences, i.e. within the third complementarity-determining regions of their alpha chains (CDR3 alpha). Moreover, the TcR of clones II/7 and III/1 had both rearranged the same J alpha element, thus differing from each other by only two amino acids in their V alpha/J alpha junctional regions. Functionally, however, clone III/1 exhibited unique cytolytic specificities for synthetic, Kb-binding TNP-peptides as well as for chemically TNP-modified allogeneic (H-2k) target cells. These findings demonstrate that (a) similar to "conventional" peptide antigens, synthetic hapten-peptide determinants are contacted by CDR3 alpha-determined amino acids of the TcR and (b) in contrast to current models, CDR alpha also appears to influence the major histocompatibility complex restriction specificity of a given TcR.