Direct administration of lentivector results in the induction of Cw3 CTL responses. (a) Schematic representation of the constructs used to generate lentiviral vectors. (b) Phenotypic characterization of peak response (day 9). PBLs were analyzed for the cell surface markers CD8, CD62L (downregulated upon activation), and TCR Vβ10. The T cell receptor specificity was examined using H-2K^d/Cw3 tetramers in combination with Vβ10 staining. The proportion of Cw3-specific cells among total PBLs obtained after direct in vivo administration of lentivector is depicted. As a control, an irrelevant (miniMelan-A) lentivector was administered. This is followed by a detailed analysis of the immune response, featuring the proportion of activated CD8⁺ cells, the proportion of Cw3-specific cells in the activated compartment, and the proportion of Cw3-specific cells among total CD8⁺ cells. (c) CTL assay showing the in vivo elimination of target cells transferred to vaccinated mice. Syngeneic splenocytes, pulsed with Cw3 antigenic peptide (RYLKNGKETL) and labeled with CFSE at high concentration, were transferred to vaccinated mice 1 day before peak response along with the same number of nonpulsed splenocytes labeled with CFSE at a lower concentration. Twelve hours later, the disappearance of peptide-pulsed cells was determined by FACS analysis in PBL, spleen, and liver. By a comparison of the ratio of pulsed to nonpulsed cells, the percentage of specific killing was calculated. Control lv, lentivector expressing an irrelevant CTL epitope; miniMelan-A lv, ELA_26–35 minigene lentivector.