SV40 immortalization of feline fibroblasts as targets for MHC-restricted cytotoxic T-cell assays

CTL assays in outbred cats have been difficult to perform because of a lack of a good source of syngeneic target cell. Primary fibroblasts from cats a re widely used as target cells for MHC-restricted cytotoxic T-cell (CTL) as says, but their limited life-spans of 8-10 culture passages can be problema tic for longitudinal studies. To circumvent the life-span limitations of pr imary fibroblast cultures, we developed a procedure for immortalizing felin e primary fibroblast cells by transfection with a molecular clone of simian virus 40 (SV40). Fibroblast cultures from skin biopsies of 28 cats were im mortalized using this procedure and have been passaged for longer than 6 mo nths without showing any phenotypic difference from the original primary ce lls. Non-SV40 transfected feline fibroblasts from a selection of animals in the same group survived for only 6-8 weeks before reaching senescence. The immortalized fibroblasts expressed SV40 T-antigen and Class I MHC protein, and were successfully used as target cells in Cr-51 release CTL assays in feline immunodeficiency virus (FIV)-infected cats and in vitro stimulated a llogeneic T-cell cultures. (C) 2001 Elsevier Science B.V. All rights reserv ed.