Rac1 and Rac2 are required for efficient positive, but not negative, selection in the thymus. (A) Flow cytometric analysis of CD4 and CD8 expression on thymocytes from F5Rag1^−/− mice that were either Rac1^+/+Rac2^+/+hCD2-iCre (WT) or Rac1^TRac2^−/−. The top plots show all thymocytes, whereas the bottom plots show only cells expressing high levels of TCRβ. Gates indicate DP and CD8SP cells. Numbers indicate percentage of cells falling into the gates. (B) Graphs showing the mean (± SEM) ratio of CD8SP to DP cells in thymi from F5Rag1^−/− mice of the indicated Rac1 and Rac2 genotypes. Statistically significant differences between WT and the 3 Rac mutant genotypes are indicated. (C) Flow cytometric analysis of CD4 and CD8 expression on cells from fetal thymic lobes from F5Rag1^−/− mice that were either WT or Rac1^TRac2^−/−, cultured for 5 days, and then treated for 11 hours with a control peptide, or with NP68, an agonist peptide for the F5 TCR. Cell death was assessed by annexin V staining on DP cells, gated as shown in CD4/CD8 plots. Marker indicates live annexin V⁻ cells. Numbers indicate percentage of cells in indicated gates or markers. (D) Graph showing mean (± SEM) percentage of live DP cells in fetal thymic organ cultures treated and analyzed as in panel C. Percentages were normalized to the number of live DP cells in WT F5Rag1^−/− thymi cultured in medium alone (set to 100%). Shading of columns indicate Rac1 and Rac2 genotypes as in panel B. Statistically significant differences between cultures treated with NP68 or control peptides are indicated. No difference was seen in the response to NP68 between the 4 different genotypes. (*P < .05; **P < .01; ***P < .001.)