LTR-Tax CD4⁺ T cells are hyper-responsive to immune stimulation. CD4⁺ T cells enriched from splenocytes of LTR-Tax transgenic or littermate control mice were stimulated with plastic-coated anti-CD3 and soluble anti-CD28 in vitro (1 × 10⁶ cells/mL) and compared after 24 hours. The LTR-Tax CD4⁺ T cells exhibited enhanced activation compared with control CD4⁺ T cells as demonstrated by: (A) physical clustering of cells; (B) increased proliferation, as measured by ³H-thymidine incorporation (results for individual animals shown); (C) greater percentages of cells expressing the surface activation markers CD25 or CD69, as detected by immunofluorescence and flow cytometry after activation with anti-CD3 and anti-CD28 (stimulated, red line; unstimulated, blue line; isotype control, gray line) in a representative animal; (D) greater percentages of cells expressing CD25 or CD69 after activation with anti-CD3 and anti-CD28 (all animals shown, lines represent means); and (E) relative Q-PCR analysis of Tax mRNA in LTR-Tax CD4⁺ T cells, 24 hours after immune stimulation, compared with RNA from time point = 0 (prior to activation). Tax mRNA levels in each sample were normalized to RNA levels of the housekeeping control gene, ASL. Solid lines show normalized Tax mRNA levels for T cells derived from individual mice. Micrographs were obtained by visualization with a 20× objective lens, using a Nikon EclipseTE2000-S microscope, Roper Scientific Photometrics imaging system, and Image Pro plus image acquisition software Version 4.5.0.29.