Effect of the CD8 transgene on thymocyte differentiation. (A) Decreased total thymocytes. Scatter plot depicting total thymocytes from six independent littermate groups, each containing one nontransgenic, one CD8 Tg/+, and one or two CD8 Tg/Tg mice. Littermates analyzed on the same day are represented by identical symbols. Paired Student's t test p-values are shown, with significant differences indicated by asterisks (***, P < 0.001). (B) Decreased DP thymocytes. CD4 and CD8 staining of thymocytes from two CD8 Tg/Tg mice along with littermates that were either CD8 Tg/+ or nontransgenic littermate controls (NLCs). The antibody used against CD8β.2 detects only the endogenous CD8β allele. (C) Decreased distal Jα rearrangements. Genomic DNA prepared from thymocytes from a CD8 Tg/+ mouse, a littermate control (NLC), and two CD8 Tg/Tg mice, as well as Rag^−/− splenic DNA, was amplified in separate reactions with primers specific for Vα14 and four different Jα segments from either the proximal or distal region of the Jα locus. Samples of each reaction were subjected to agarose gel electrophoresis and analyzed by Southern blotting. Rearrangement products were identified by hybridization with a ³²P-labeled Vα14 probe. Numbers designate bands resulting from rearrangement of Vα14 to the indicated Jα elements, and the proximity of the Jα segments allows the detection of several bands with one primer. All data in this figure are representative of at least three independent experiments.