HY-specific memory CD28Y170F T cells display defective localization to antigenic sites in vivo. (A) Percentage of Uty-specific CD8⁺ T cells (as determined by D^b/Uty tetramer binding) detected in WT and CD28Y170F female mice after 2 immunizations with male splenocytes (cells are gated based on CD8 expression). (B) Average percentage of Uty-specific T cells in 3 samples. Standard error bars are shown. (C-D) Localization of CFSE-labeled T cells obtained from immunized WT and CD28Y170F female mice and injected in a female recipient that had either received an intraperitoneal injection of IFNγ only (No Ag) or that had also been injected with male splenocytes on the same day and Uty peptide 48 hours later (+ Ag). Cells were visualized in the peritoneal membrane (C; scale bar: 40 μm) and in the organs indicated in panels E-F 24 hours later by wide-field fluorescence microscopy and quantified as described in “Materials and methods.” Panels D-F represent data averaged from at least 3 animals, and standard error bars are shown. (D) *P < .03; (F) *P < .04.