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Fig. 3 | Journal for ImmunoTherapy of Cancer

Fig. 3

From: Chronic TCR-MHC (self)-interactions limit the functional potential of TCR affinity-increased CD8 T lymphocytes

Fig. 3

Basal expression of co-activating/inhibitory receptors upon affinity-increased TCR transduction in relation to HLA-A2. a Schematic representation of the experimental design using A2-KO CD8 T cells. A2pos primary CD8 T cells were first knocked-out for HLA-A2 by CRISPR/Cas9, expanded by PHA/A2neg feeder cells and transduced with affinity-increased TCRs, before being characterized by flow cytometry (from day 8 to day 14). b For functional analyses, TCR-redirected A2pos and A2neg primary CD8 T cells were further purified by FACS-sorting (between D15–21) and expanded using PHA/A2neg feeder cells. c Quantification of the expression of co-activating/inhibitory receptors on A2pos (CRISPR/GFP) and A2neg (CRISPR/A2) primary CD8 T cells from day 8 to day 14 post-TCR transduction, independently of antigen-specific stimulation. Data are depicted as means ± SD (relative to the WT TCR variant) and representative of 4 to 5 independent experiments. * P ≤ 0.05, ** P ≤ 0.01, and *** P ≤ 0.001. d Co-expression of 0 to 3 co-inhibitory (PD-1, TIM-3 and 2B4) receptors of A2pos (CRISPR/GFP) and A2neg (CRISPR/A2) primary CD8 T cells. e TCR-pMHC off-rate measurements of two CDR3-based TCR variants (α95:LYm and α95:LYm/A97L). f Quantification of PD-1, TIM-3, CD69 and CD28 expression levels in primary CD8 T cells redirected with CDR3-based TCR variants, in the absence of antigen-specific stimulation. g Expression levels of TCR/CD3 complex and of CD5 in redirected J76 CD8αβ cells with CDR3-based TCR variants, analyzed under steady-state culture conditions. e-g Data are depicted in comparison to WT (blue dotted line), DMβ (green line) and TMβ (orange line) TCR variants and are representative of 2 to 4 independent experiments

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