Fig. 3: p66Shc deficiency in Eμ-TCL1 mice enhances the ability of leukemic cells to enhance PD-1 expression in CD8⁺ cells.

qRT-PCR analysis of mRNA (A) and flow cytometric analysis of surface (B) expression of PD-1 in CD8⁺ cells isolated from lymph nodes of either wild-type (n = 10) or Eμ-TCL1 (n = 18) mice. Representative flow cytometric histograms are shown (Mann Whitney Rank Sum test). C. mRNA expression of PD-1 in CD8⁺ cells from lymph nodes of Eμ-TCL1 shown in (A) were subgrouped, according to disease stage, in mild leukemia (20-39% leukemic cells in PB, 1-2 × 10⁷ WBC/ml PB, n Eμ-TCL1 mice = 10) and overt leukemia (>40% leukemic cells in PB, >2 × 10⁷ WBC/ml PB, n Eμ-TCL1 mice = 8). (Mann Whitney Rank Sum test). D. Correlation between surface (MFI) or mRNA levels of PD-1 in CD8⁺ cells isolated from lymph nodes of Eμ-TCL1 mice and the percentage of leukemic cells in peripheral blood of the same mouse. E Correlation between surface (MFI) or mRNA levels of PD-1 in CD8⁺ cells isolated from lymph nodes of wild-type mice cultured in the presence of media conditioned by splenic B cells from Eμ-TCL1 mice, and the mRNA levels p66Shc in Eμ-TCL1 cells used to generate conditioned media (n wild-type mice = 18). F qRT-PCR analysis of PD-1 mRNA in CD8⁺ cells isolated from spleens of wild-type mice (n = 10), cultured in the presence of media conditioned by splenic B cells from either wild-type, Eμ-TCL1 or Eμ-TCL1/p66^−/− mice (n independent experiments ≥5; one-way ANOVA). G. Flow cytometric analysis of PD-1⁺CD8⁺ cells in 220 μm-thick slices from spleens of wild-type mice (n = 10), cultured as in (F) (n independent experiments ≥5; one-way ANOVA). Data are expressed as mean ± SD. ****p ≤ 0.0001; ***p ≤ 0.001; **p ≤ 0.01; *p ≤ 0.05.