A HUMAN TCR-IG CHIMERIC PROTEIN USED TO GENERATE A TCR-ALPHA CHAIN VARIABLE REGION-SPECIFIC MAB

In a recent report, a construction containing the α chain-variable region (Vα) coding sequence of a cDNA clone derived from a diphtheria toxoid-specific human T cell (P28), fused to a human immunoglobulin kappa light chain constant region (Ck), was used stably to transfect a murine myeloma cell. In the present study, these transfected cells were employed as an immunogen to raise a mAb, termed 1C5Vα, specific both for the VαCk chimeric protein secreted by the transfectant and the P28 T cell antigen receptor-Vα region. mAb 1C5Vα specifically immunoprecipitates the VαCk protein as a family of 32-35 kDa bands present in the 35S-methionine-labeled culture supernatant from the transfected cells. It specifically binds clone P28. Surface molecules recognized by mAb 1C5Vα are physically linked to the CD3 molecules since cell treatment with either 1C5Vα or anti-CD3 mAbs caused the simultaneous down-regulation of the CD3/TCR molecular complex. This link is further supported by immunoprecipitation experiments. Thus, both the 1C5Vα and the anti-CD3 mAbs precipitate the 16-28 kDa CD3 molecules and the disulfide-linked form of P28 TCR from 125I-labeled P28 T cells. Studies performed in order to define whether a stimulus directly acting on the TCR-Vα region may trigger the intracellular events observed during human T cell activation showed that (a) mAb 1C5Vα efficiently triggers the phospholipase C transduction pathway revealed by an accelerated phosphoinositides turn-over and an increased production of phosphorylated derivatives of inositol phosphates; (b) mAb 1C5Vα induces an up-regulation of IL2R mRNA, accompanied by a slight increase of IL2 and IFNα mRNA transcripts evidently amplified in the presence of PMA; (c) soluble mAb 1C5Vα is strongly mitogenic together with PMA. These results provide the first evidence for the structural authenticity of a secreted water-soluble chimeric form of the variable region of a human TCR α chain. They further demonstrate that such chimeric proteins may be valuable tool to further dissect the various functional structure of the human TCR. © 1990.