For in vivo evaluation of IVTT products, splenocytes were stimulated with 2610 4 cells/well of A20 cells transfected with VR2516 PyCSP DNA or VR1020 negative control using the AMAXA Nucleofector transfection kit (AMAXA, Cologne, Germany) according to manufacturer's manual, or with 1610 5 cells/well of A20 cells pulsed with the PyCSP peptide pool. A20 cells alone and medium were used as negative controls and ConA used as mitogen control. AMAXA transfections included parallel reactions with GFP plasmid as a positive control (data not presented); additionally, our laboratory routinely uses AMAXAtransfected A20 cells as APCs for in vitro T cell assays [55, 56] .