Fig. 1.

Naïve CD4⁺ T cells in aged mice respond poorly to stimulation. (A) Splenocytes were isolated from young and aged mice, enriched for CD4⁺ T cells using anti-CD4 magnetic beads, and sorted by flow cytometry to yield a pure naïve CD4⁺ T cell population (CD4⁺CD25⁻CD62L⁺CD44^lo; purity >99%) and stimulated ex vivo using plate-bound anti-CD3/anti-CD28. Cultured cells were harvested at 24 h and analyzed by flow cytometry to assess (B) cell size, (C) IL-2 concentration in culture media, (D) expression of early activation markers: CD69 and CD25, and (E) cell death by 7-AAD incorporation. To assess proliferation, cells were loaded with CFSE and analyzed by flow cytometry at 48 h postactivation. Cell divisions were detected by dilution of the CFSE. (F) Quantitation of cell division, showing the percentage of cells in each generation. *P < 0.05, **P < 0.01, ***P < 0.001 (Student’s t test comparing young vs. aged T cells). Data are representative of at least two independent experiments.