Reduced STAT1 levels in proliferating CD8 T cells expanding ex vivo. (A) Splenic CD8 T cells derived from day 5 LCMV-infected mice cultured ex vivo are insensitive to the antiproliferative effects of IFNα. Total splenic leukocytes were prepared, CFSE-labeled, and incubated 1 or 3 days with or without IFNα (1 × 10⁴ U/mL). Histograms of CFSE dilution within electronically gated CD8 populations are shown. The experiment has been repeated more than 2 times with similar results. The overall averages ± SDs for the combined experiments were, respectively without or with IFNα, 2.2 ± 0.5 and 1.9 ± 0.7 on day 1 and 58.9 ± 3.9 and 59.2 ± 2.0 on day 3. (B) Cultured CD8 T cells were separated into high and low proliferating, based on dilution of CFSE. CD8 T cells were enriched from a 3-day culture of CFSE-labeled splenic populations prepared from day 5 LCMV-infected mice and then sorted into CFSE-low and -high subsets. CFSE staining intensities of the different samples are shown. (C) The STAT1 protein levels are lower in proliferating than nonproliferating CD8 T cells. Western blot analysis of STAT1 levels in the populations prepared in panel B. Western blot analysis of STAT4 was used as loading control. Also tested was β-actin (not shown). This figure is representative of 3 experiments. (D) STAT1 RNA levels are lower in proliferating CD8 T cells. Real-time PCR analysis of STAT1 RNA levels prepared from sorted CFSE-low (○) and CFSE-high (▪) CD8 T cells is shown. The ratio of STAT expression was calculated using amplification efficiencies (1.93 for STAT1 and 1.99 for β-actin). The experiment was repeated twice. The average C_T values ± SDs for the low CFSE samples were 29.0 ± 4.4 for STAT1 and 20.9 ± 0.4 for β-actin, and for the high CFSE samples were, respectively, 25.6 ± 1.6 and 22.8 ± 0.6.