Smaller thymus observed in AML1-3 mice. (A) Macroscopic appearance of dissected thymi from representative 1-, 3-, and 5-week-old litters obtained from intercrossed parents heterozygous for AML1-2 or AML1-3 knock-in mutations. ^*Homozygous mutants. Photographs were taken with a digital camera (Coolpix 4300, Nikon, Tokyo, Japan). (B) The number of thymocytes per thymic lobe per body weight (g) is indicated by columns for 1-week-old AML1-3 homozygous mice compared with the numbers for wild-type littermates. Bars indicate standard deviations. Thymi from AML1-3 homozygous mice were smaller than those of controls even if reduced body size was taken into account (see “Abnormal thymus size in AMLI-3 mice.”). (C) Appearance of the spleens and kidneys of the 1-week-old AML1-3 litter together with their thymi. Most organs other than thymi, including kidneys and hearts (not shown), were proportional to body weight (BW). Spleens appeared small in infancy, but they became proportional to the body size later in their life (not shown). (D) Results of flow cytometric analysis of the CD4 and CD8 expression for thymocytes. Numbers in graph quadrants indicate fractions of the cells analyzed by percentile. (E) Hematoxylin-eosin–stained sections of the thymi. Note that slightly less developed medulla is observed in the thymus from AML1-3 mice. No obvious bleeding or degenerative lesions were detected in these specimens. A microscope (BH-2, Olympus) with a digital camera (C-5050 Zoom, Olympus) was used. Original magnification, × 100. (F) In vitro proliferation assay on the splenic T cells isolated from mice of each genotype. Cells (1.5 × 10⁵) were stimulated with appropriate antibodies and/or cytokines (light blue, 0.1 μg/mL CD3; medium blue, CD3 plus CD28; dark blue, CD3 plus CD28 plus IL-2) for 48 hours, and were pulsed with [³H]-thymidine for the last 14 hours. The [³H]-thymidine uptake (counts per minute) is shown with standard deviation. The T cells from AML1-3 animals proliferated equally upon stimulation.