Comparison of proliferation and activation status of naïve vs. in vivo primed T cells. (A) Schematic representation of the protocol used in this experiment. Briefly, 1.5 × 106 naïve or in vivo primed CD4+ T cells were adoptively transferred into STAT6xRAG2-/- mice and primed with OVA/alum i.p. on day 1. One group of mice was treated daily with BrdU (1 mg/mouse) i.p for 3 days before harvesting spleens on day 5. Splenocytes were pooled together and total cell counts were recorded. Cells were stained with anitbodies to CD4, KJ126, CD44 and BrdU and flow cytometry was performed. Another group of mice, that didn't receive BrdU were immunized with OVA/alum a second time on day 8. Four days later, splenocytes were harvested, counted and stimulated with PMA (50 ng/ml) and Ionomycin (1 μg/ml) for 6 h. (B) BrdU and CD44 expression in the CD4+ KJ126+ population in the naïve T cell or in vivo primed T cell transfer groups are shown. (C) CD44 expression in the CD4+ KJ126+ population in naïve vs. in vivo primed T cell transfer groups on day 12 is shown. IL-4 production by naïve and in vivo primed DO11.10 CD4 T cells was measured by intracellular cytokine staining (ICS).