Figure 6. Levels of SHP-1 protein and of miR-155 and miR-181a expression in CD8⁺ T cells engineered with TCRs of incremental affinities.

(A) Unstimulated at baseline (0 hours) and log₂ fold change (0–6 hour difference) expression levels of SHP1 transcripts as detected in microarray analysis. (B and C) TCR-transduced CD8⁺ T cells (B) and SUP-T1 cells (C) were stimulated with 10 μg/ml A2/NY-ESO-1_157–165 multimers for the indicated time points and assessed for SHP-1 phosphorylation and total SHP-1 levels by Western blotting. Actin (B) or α-tubulin (C) expression levels were used as loading controls between samples. Data are each representative of 3 independent experiments. For CD8⁺ transduced T cell samples, all lines were run on the same gel, but were noncontiguous. TCR-untransduced SUP-T1 cells are shown as empty controls (Ø). (D) To allow direct comparison between the engineered SUP-T1 cells, intensity of SHP-1 phosphorylation and of total SHP-1 levels was quantified and normalized to α-tubulin (n = 3 independent experiments). (E) Expression levels of miR-181a and miR-155 were determined by qRT-PCR in TCR-transduced CD8⁺ T cells following stimulation with 0.1 μg/ml A2/NY-ESO-1_157–165 multimers at the indicated time points. Data represent relative expression compared with RNU48 control and are representative of 3 independent experiments. Pri-miR-155 (BIC) mRNA expression values (inset) were retrieved from the microarray analysis.