ATM deficiency leads to a severe deficit in TCRβ^int and TCRβ^hi thymocytes and diminished responses to TCR engagement in vitro. (A) Frequency of SP thymocytes in Atm^−/−, Atm^+/−, and Atm^+/+ mice. Flow cytometric analysis of T-cell development was performed in 3- to 4-week-old mice segregating the ATM mutant allele. Thymocytes were stained with anti–CD4–phycoerithryn (PE), anti–CD8–fluorescein isothyocyanate (FITC), and anti–TCRβ-biotin, revealed with streptavidin-allophycocyanin (SAv-APC). Representative CD4 versus CD8 staining profiles of each genotype are shown, together with the quadrant gates used to identify DN, DP, and SP thymocytes. (B) TCRβ expression on thymocyte subsets from Atm^−/− and Atm^+/+ mice. TCRβ levels (solid) are compared with staining of isotype-matched control antibodies (dashed). (C) Percentages and absolute numbers of thymocyte subsets in Atm^−/− mice and littermates. The bar graphs depict percentages (i) and absolute numbers (ii) of DN, DP (TCRβ^low, TCRβ^int, and TCRβ^hi) and CD4 or CD8 SP thymocytes in Atm^−/− mice (□) and littermates (▪). The numbers above each bar represent the mean. The graphs display the mean ± SD for 9 Atm^+/+ and 12 Atm^−/− mice. Percentages and absolute numbers of CD8⁺ SP cells were determined by gating on TCRβ^hi CD8⁺ SP cells to eliminate immature single-positive cells. All differences were statistically significant (P < .001 by Student t test) except for the DN frequency, DN, and TCRβ^low DP absolute number comparisons. (D) CD4 versus CD8 expression on thymocytes from 3-week-old Atm^+/+ or Atm^−/− mice was assessed after overnight culture in the presence of plate-bound anti-TCRβ antibody. Fluorescence signals were gated to display CD69 or CD5 staining on CD4⁺CD8⁺ DP thymocytes. Expression of the early activation markers CD69 and CD5 by DP thymocytes from Atm^−/− mice and littermate controls was evaluated by flow cytometry after overnight culture in the presence (shaded) or absence (open) of stimulation with anti-TCRβ.