Loss of original antigenic specificity in T cell hybridomas transduced with a chimeric receptor containing single-chain Fv of an anti-collagen antibody and Fc epsilon RI-signaling gamma subunit

T cell hybridomas HCQ6 and MD.45 acquired Ab-type specificity to collagen t ype II, when engrafted with a chimeric cell surface receptor, scC2Fv/gamma, which includes the single-chain Fv domain (scFv) of the anti-collagen type II mAb C2 and the signaling gamma subunit of the Fc epsilon RI. When trans duced into MD.45 cells, scC2Fv/gamma or its mutated form lacking immunorece ptor tyrosine-based activation motif (ITAM), scC2Fv/gamma IC- formed mainly homodimers, A small proportion of these molecules formed heterodimers with endogenous CD3 zeta in these hybridoma cells, By contrast, in HCQ6 cells, the majority of scC2Fv/gamma and scC2Fv/gamma IC- molecules formed heterodi mers with CD3 zeta and only a small proportion of them was expressed as hom odimers, Stimulation with plastic-immobilized collagen induced IL-2 product ion in scC2Fv/gamma-transduced MD.45 cells, but not in MD.45 cells transduc ed with the ITAM-less chimera scC2Fv/gamma IC-. HCQ6 cells transduced with scC2Fv/gamma responded to plastic-bound collagen. Due to the high content o f CD3 zeta-associated chimeras, HCQ6 cells transduced with the ITAM-less sc C2Fv/gamma IC- chimera were also responsive to plastic-bound collagen. When cells were stimulated with collagen in solution, MD.45 cells transduced wi th scC2Fv/gamma produced IL-2, whereas transduced HCQ6 cells mere unrespons ive, hence suggesting that the ability of cells transduced with scC2Fv chim eras to respond to soluble collagen correlated with predominant expression of divalent scC2Fv/gamma homodimers, but not monovalent scC2Fv/gamma-CD3 ze ta or scC2Fv/gamma IC--CD3 zeta heterodimers, Of interest, expression of CD 3 subunits in hybridomas transduced with scC2Fv chimeras was reduced, resul ting in decreased response to cognate Ags.