Engineering mouse T lymphocytes specific to type II collagen by transduction with a chimeric receptor consisting of a single chain Fv and TCR zeta

The chimeric cell surface receptor scC2Fv/CD8/zeta was constructed to engin eer primary mouse T lymphocytes with antibody-type specificity to type II c ollagen (CII). Such cells could be used as gene carriers in the anti-inflam matory gene therapy of an autoimmune arthritis. This receptor includes the single chain Fv domain (scFv) of the anti-CII monoclonal antibody (mAb) C2, hinge region of CD8 alpha and the transmembrane and cytoplasmic domains of TCR zeta. The scC2Fv/CD8/zeta gene was transduced into T cell hybridomas a nd primary mouse lymphocytes using retrovirus-mediated gene transfer The ch imeric receptor scC2Fv/CD8/zeta forms covalently bound homodimers, as demon strated in T cell hybridomas and packaging fibroblasts. It does not associa te with endogenous signalling subunits of the TCR complex. When scC2Fv/CD8/ zeta-expressing clones of T cell hybridomas MD.45 and HCQ6 were stimulated with CII they produced IL-2. The level of their IL-2 response correlated wi th the expression level of the chimeric receptor on the cell surface. Splen ocytes isolated from DBA/1 mice were stimulated with Con A in vitro to faci litate retrovirus-mediated transfer of the scC2Fv/CD8/zeta gene. As a resul t of transduction, approximately 4% of the Con A-activated splenocytes expr essed the chimeric receptor scC2Fv/CD8/zeta on the cell surface. These cell s proliferated in response to stimulation with CII.