Adoptive Transfer of T Cells in PKCζ-KO Mice

T cells were isolated from the spleens of WT or PKCζ-KO mice using the Pan T Cell Isolation Kit II according to the manufacturer’s instructions (Miltenyi Biotec). In brief, splenocytes were stained with Pan T Cell Biotin-Antibody Cocktail (Miltenyi Biotec), followed by addition of Pan T Cell Micro Bead Cocktail (Miltenyi Biotec) before separation using a MACS Separator (Miltenyi Biotec). After sorting, the purity of T cells was assessed by flow cytometry and confirmed to be ∼92–97% (data not shown). The 2 × 10⁶ T cells isolated from WT of PKCζ-KO mice were transferred into each PKCζ-KO mouse intravenously. The number of transferred T cells (2 × 10⁶ cells) was twice the number (10⁶ cells) that had been previously shown to initiate T cell–dependent responses sufficiently in naive C57BL/6 mice (Shaw et al., 2015 (link)). 12 h after T cell transfer, mice were intravenously infected with 5 × 10³P. berghei sporozoites.