Strong CD8⁺ T cell effector functions induced by IL-2::2Ig(F)GPI asVNP.

<p>(<b>A</b>) Flow cytometry analysis showing proliferation and intracellular IFN-γ-expression of CFSE-labeled CD8^<b>+</b> TCR Vα2^<b>+</b> T-cells. Markers set according to negative stainings and no-stimulation control. Numbers indicate percentage of cells in respective quadrants. (<b>B</b>) Flow cytometry analysis, (<b>C</b>) percentage and (<b>D</b>) fold-induction of IFN-γ producing CD8^<b>+</b> T cells in the absence or presence of indicated stimuli. (<b>E</b>) Amounts of IFN-γ (pg/ml) secreted upon co-culture of CD8^<b>+</b> T cells with indicated IL-2v asVNP or αCD3/αCD28 coated microbeads plus IL-2 (10 or 100 U/ml). (<b>F-H</b>) IL-2::2Ig(F)GPI asVNP increase the cytotoxic potential of antigen-specific T cells. P14 splenocytes were stimulated with IL-2v asVNP for 48 hours or left untreated and were subsequently co-cultured with 1x10^<b>4</b> wt (CPD^{<b>bright</b>}) or LCMV-GP expressing (CPD^<b>dim</b>) EL-4 target cells. Specific target cell lysis was analyzed after 20 hours by flow cytometry. (<b>F</b>) Flow cytometry analysis showing specific lysis of LCMV-GP^<b>+</b> but not wt target cells at an effector/target ratio of 10. (<b>G</b>) Quantification by flow cytometric analysis of specific target cell lysis relative to maximum lysis (LCMV-GP_33-41 stimulated effector cells, E:T ratio 20:1). (<b>H</b>) Determination of cytotoxic potential in ^<b>51</b>Cr-release assays of P14 splenocytes, which were stimulated with IL-2v asVNP or left untreated for 48 hours. Effector cells were co-cultured with ^<b>51</b>Cr-labelled wildtype or LCMV-GP-expressing EL-4 target cells (1x10^<b>4</b>) and specific lysis was analyzed. Data are representative (A, B, F, H) or show the summary (C, D, E, G) of three (A, B, F, G), five (except three for αCD3/αCD28 microbeads) (C, D), four (except two for 48 and 72 hrs) (E) and triplicates of one (H) independent experiments. * p < 0.05, **, p < 0.01, ***, p < 0.001; ANOVA and Tukey’s multiple comparison test (C, D), Kruskal-Wallis test and Mann-Whitney U-test and Bonferroni correction (E), t-test comparing IL-2::GPI with IL-2::2Ig(F)GPI asVNP (G, H).