HEK293T (American Type Culture Collection, ATCC; CRL-3216), FeFAB [40 (link)], CrFK (ATCC, CCL-944), and KE-R (Depository for Cell Lines in Veterinary Medicine, Federal Research Institute for Viral Animal Diseases, Riems, Germany) were propagated under standard conditions in DMEM containing 10% heat-inactivated fetal calf serum plus antibiotics [40 (link)]. CrFK-derived FeFAB (FFV-activated β-galactosidase) cells contain the β-galactosidase gene under the control of the FFV internal promoter [40 (link)]. All cells were regularly screened for the absence of mycoplasma and other agents and for cell authenticity (Multiplexion, Immenstaad, Germany).
The C57BL/6-derived tumor cell lines EL4 [41 (link)] and RMA [42 (link)] were cultured in RPMI 1640 supplemented with Glutamax, 10% FCS, and 1% penicillin-streptomycin. The transfectants E.G7 (EL4 cells expressing chicken ovalbumin) [43 (link)], 2F11 (RMA cells expressing the HPV16-derived oncoprotein E7) [44 (link)] and RMA/TRP-2 (expressing human tyrosinase related protein 2) (kindly provided by A. Paschen), were grown in the same medium containing 0.8 μg/ml G418 (Gibco). The following antigen-specific CTL lines were used: OVA-specific CTL line [45 (link)] recognizing the H2Kb-restricted OVA-derived epitope aa257-264 (SIINFEKL) [46 (link)]; E7-specific CTL line [47 (link)], reacting against the E7-derived Db-restricted epitope RAHYNIVTF (amino acids, aa, 49–57 [48 (link)], and a TRP-2-specific CTL line obtained upon TRP-2-specfic DNA immunization of C57BL/6 mice (Osen, unpublished), recognizing the epitope SVYDFFVWL (aa 180–188) [49 (link)]. All CTL lines were expanded in vitro upon periodical re-stimulation with the transfectant clones expressing the respective target antigen in CTL medium, according to the protocol described in [50 (link)].
The C57BL/6-derived tumor cell lines EL4 [41 (link)] and RMA [42 (link)] were cultured in RPMI 1640 supplemented with Glutamax, 10% FCS, and 1% penicillin-streptomycin. The transfectants E.G7 (EL4 cells expressing chicken ovalbumin) [43 (link)], 2F11 (RMA cells expressing the HPV16-derived oncoprotein E7) [44 (link)] and RMA/TRP-2 (expressing human tyrosinase related protein 2) (kindly provided by A. Paschen), were grown in the same medium containing 0.8 μg/ml G418 (Gibco). The following antigen-specific CTL lines were used: OVA-specific CTL line [45 (link)] recognizing the H2Kb-restricted OVA-derived epitope aa257-264 (SIINFEKL) [46 (link)]; E7-specific CTL line [47 (link)], reacting against the E7-derived Db-restricted epitope RAHYNIVTF (amino acids, aa, 49–57 [48 (link)], and a TRP-2-specific CTL line obtained upon TRP-2-specfic DNA immunization of C57BL/6 mice (Osen, unpublished), recognizing the epitope SVYDFFVWL (aa 180–188) [49 (link)]. All CTL lines were expanded in vitro upon periodical re-stimulation with the transfectant clones expressing the respective target antigen in CTL medium, according to the protocol described in [50 (link)].
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