Fig 6. Induction of effector functions and memory phenotypes by IL-2v asVNP.

Enhanced in vivo cytotoxicity of CD8⁺ effector T cells in mice immunized with IL-2::2Ig(F)GPI asVNP. (A) Flow cytometry analysis showing recovery of adoptively transferred and differentially CPD labeled LCMV-GP_33-41 peptide (CPD^dim) or mock peptide (CPD^bright) pulsed congenic CD45.1⁺ splenocytes from CD45.2⁺ wild-type recipient mice. Two days before target cell transfer, wt mice received 2x10⁵ purified P14 CD45.1⁺CD8⁺ effector T cells and were immunized with IL-2::GPI or IL-2::2Ig(F)GPI asVNP (black histograms) or with IL-2(A)::GPI or IL-2(A)::2Ig(F)GPI asVNP (open histograms) or were left untreated. (B) Quantification of specific target cell lysis in vivo. Horizontal lines indicate the mean. (C-E) Pre-stimulation of CD8⁺ T cells with IL-2::GPI asVNP generates higher absolute numbers of memory cells in vivo. P14 CD8⁺ T cells (CD45.2⁺) were in vitro pre-stimulated with IL-2v asVNP or αCD3/αCD28 microbeads (+100 U/ml IL-2) for six days. Equal amounts of viable naïve and activated cells (1x10⁶) were adoptively transferred into CD45.1⁺ congenic recipient mice. After 7 days engraftment of P14 CD8⁺CD45.2⁺ T cells in the inguinal lymphnodes and the spleens were determined by flow cytometry. (C) Flow cytometry analysis showing expression of the indicated markers on naïve (shaded grey histograms) and in vitro pre-activated (black line) CD8⁺CD45.2⁺ donor cells isolated from the lymph nodes of recipient mice. Absolute numbers of CD62L^low and CD62L^high donor cells recovered from (D) inguinal lymphnodes and (E) spleens of recipient mice. Data are representative (A, C) or show the summary (B, D, E) of 28 mice (seven per group) that were analyzed in four independent experiments (A, B), or of 18 mice (eight per group, except for naïve (two), IL-2::GPI (three), αCD3/αCD28 microbeads plus IL-2 (five)) (C-E) that were analyzed in two independent experiments. * p < 0.05, **, p < 0.01 ANOVA, t-test and post hoc Bonferroni correction (B); Mann-Whitney U-test (D, E).