Fig. 3
mmHERA-GITRL significantly boosts the antigen-specific T cell response in vivo without affecting endogenous T cells. a, b C57Bl/6 mice were adoptively transferred with 2 × 106 (each) CD8+ OT-I T cells and CD4+ OT-II T cells, challenged with OVA protein (or PBS) and treated with a single injection of mmHERA-GITRL (1 or 5 mg/kg b.w.) or vehicle control (PBS). Serial blood samples were obtained from each animal over a two-week period and OT-I/OT-II T cells were quantified by FCM using CD8/CD4 and a congenic marker (CD45.2). A representative time point is shown for day 6 (a) and the entire time course is quantified (b). a Gated regions indicate transferred T cells (right polygon) and endogenous/recipient T cells (left polygon). Numbers indicate the percentage of cells within the OT-I/OT-II T cell region. Representative contour plots, gated on live single CD8+ or CD4+ T cells, from the median sample of triplicates from at least three independent experiments are shown. b Day 0 value represents historical adoptive transfer data. Each symbol represents the mean (n = 3) ± S.D. A two-way ANOVA plus post hoc Bonferroni multiple comparisons analysis was conducted to compare the effects of treatment and time on antigen-specific T cell clonal expansion. The p values represent comparisons between 5 mg/kg mmHERA-GITRL and PBS. *p < 0.05, ****p < 0.0001. c In a separate experiment, which included mmHERA-GITRL (8 mg/kg b.w.) treatment alone, blood samples were obtained from each animal on day six, cells were stained for surface expression of CD44 and the percentage CD44high (mean ± S.D.) is shown. One-way ANOVA plus post hoc Bonferroni multiple comparisons analysis was conducted on all treatment groups. No significant changes in CD44 expression were seen in the endogenous T cell populations (“Endo.”). Although not labeled, CD44 expression on OT-I/OT-II T cells in all groups treated with OVA are significantly different from non-OVA treated groups