Fig. 2: PD-1 overexpression induced in CTLs by CLL culture supernatants is caused by p66Shc deficiency.

A Correlation between either surface (MFI) or mRNA levels of PD-1 in CD8⁺ cells isolated from CLL patients and the mRNA levels of p66Shc in B cells purified from the respective CLL patients (n CLL patients ≥ 10). B Correlation between either surface (MFI) or mRNA levels of PD-1 in healthy CTLs activated for 48 h in media conditioned by CLL-B cells, and the mRNA levels of p66Shc in B cells purified from the respective CLL patients used to generate conditioned media (n = 9). C qRT-PCR analysis of p66Shc mRNA in B cells purified from CLL patients (n = 8) and transfected with either empty (CLL/vect) or p66Shc-encoding (CLL/p66) vectors (paired t test). D, E qRT-PCR analysis of mRNA (E) and flow cytometric analysis of surface (F) expression of PD-1 in CTLs from healthy donors (n = 8), activated for 48 h in the presence of media conditioned by CLL B cell transfectants. Representative flow cytometric histograms are shown (paired t test). F Immunofluorescence analysis of pTyr, F-actin, CD3ζ, and PCNT in CTLs activated in media conditioned by CLL-B cell transfectants, mixed with SAg-loaded Raji cells (APCs), and incubated for 15 min at 37 °C. Data are expressed as % of 15-min SAg-specific conjugates harboring pTyr, F-actin, CD3ζ, and PCNT staining at the IS ( ≥50 cells/sample, n independent experiments = 3, one-way ANOVA test). Data are expressed as mean ± SD. ****p ≤ 0.0001; ***p ≤ 0.001; **p ≤ 0.01; *p ≤ 0.05.