Tamoxifen treatment 30 days after LCMV infection results in efficient deletion of SLP-76 in a normal memory T-cell population. (A) Schematic diagram of SLP-76 conditional deletion. cHET and cKO mice contain LoxP sites flanking exon 3 of the SLP-76 gene. The second SLP-76 allele in cHET mice is a wild-type allele and is a germline-null allele in cKO mice. All mice have one copy of the R26R-YFP Cre reporter. All mice express a transgene with the CreT2 cDNA under the transcriptional control of the ubiquitin promoter. Cre^T2 recombinase activity is induced by tamoxifen administration, resulting in excision of the lox-STOP-lox cassette in the reporter construct and the floxed SLP-76 alleles. (B) Intracellular staining of CD8⁺ T cells for SLP-76 in cHET and cKO mice. After tamoxifen treatment, splenocytes were permeabilized and stained for SLP-76. The dot plots show an overlay of CD8-gated cells from cKO (left panel) and cHET (right panel) mice. The numbers in the upper right quadrant are relative percentages for cKO cells in the gated areas. Results are representative of 4 experiments. (C) Schematic of LCMV infection and Cre-mediated deletion regimen. cKO and cHET mice were infected with LCMV-Armstrong on day 0, allowed to clear the virus, and contracted to a memory T-cell population before Cre-mediated deletion. More than 30 days later, tamoxifen was administered for 5 days with subsequent SLP-76 deletion and YFP expression. The solid line represents the relative abundance of LCMV-specific cells and the dashed line represents the relative abundance of YFP⁺ cells. (D) Representative FACS plots of cells from cHET and cKO mice after infection but before tamoxifen treatment. Mice were bled at day 8 and day 20 after infection, and the peripheral blood was assayed using polychromatic flow cytometry. Contour plots are gated on CD8⁺ T cells (left panels). Histogram overlays of CD8⁺CD44^hiH-2D^b:GP33⁺-gated cells from cHET mice (solid gray) and cKO mice (black line; right panels). Results are representative of 2 experiments.