Additional lymphoma antigens have not been defined. Therefore, one general approach to active immunotherapy is to utilize genetically modified autologous tumor cells as a source of Ag. Expression of various cytokine genes in tumor cells may enhance the immunogenicity and potency of cell-based vaccines. Tumor cells are modified/transduced in vitro to express immuno- modulatory molecules such as cytokines (GM-CSF, IL-2, and IL-4), chemokines (IP-10), or costimulatory molecules (B7.1 and B7.2). Although transiently transfected cells can be used, stable transfectant tumor cells are selected using selective markers, such as the neomycin analog G418 or hygromycin. Stable transfectants are characterized by flow cytometry (unit5.4) and ELISA (unit 10.17). Herein we describe a brief strategy for producing and testing stably transduced A20 lymphoma cells expressing murine GM-CSF which can elicit T cell-dependent protective and therapeutic immunity against early pre-established tumors (Levitsky et al., 1996).
1 mg/ml mammalian expression plasmid, encoding murine GM-CSF, dissolved in TE buffer, pH 8.0 (linearized by single restriction cut, purified by phenol/chloroform extraction and ethanol precipitation; see Ausubel et al., 2001, and Chapter 10 in this manual) A20 cells (ATCC #TIB-208) growing in exponential phase RPMI 1640 medium
Complete RPMI medium with 10% FBS (appendix 2a)
MPRO cells (ATCC # CRL-11422; Tsai and Collins, 1993) growing in exponential phase
Complete DMEM medium with 10% FBS (appendix 2a) Hanks' balanced salt solution (appendix2a) without Ca2+ and Mg2+ 6- to 12-week-old BALB/c female mice
Gene Pulser II System for electroporation (Bio-Rad)
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