Figure 3.

High-affinity CD8 variants enhance signal transduction and tetramer uptake by cancer-targeting TCRs.A, expression of NGFR and CD8α among 1E9 TCR⁺ CD8αβ⁺ JE6.1 reporter cells transduced with CD8αβ containing either wild-type (WT) CD8α (red) or mutated forms of CD8α, namely S53G (teal) or S53N (purple). B, NFAT and NFκB reporter activity among the corresponding reporter cells in response to coculture with ALL CM cells or K562 HLA-A2+CD20 cells. C, expression of NGFR and CD8α among KL14 TCR⁺ CD8αβ⁺ JE6.1 reporter cells transduced with CD8αβ containing either wild-type (WT) CD8α (red) or mutated forms of CD8α, namely S53G (teal) or S53N (purple). D, NFAT and NFκB reporter activity among the corresponding reporter cells in response to coculture with UM3 cells or U266 cells. Data are shown as mean ± SD of duplicate or triplicate measurements from one experiment in (B) and (D). E and F, fold change in geometric mean fluorescence intensity (MFI) of cognate tetramer staining among NGFR^high/NGFR⁻ populations of primary CD8⁺ T cells (E) or primary CD4⁺ T cells (F) transduced with the CMV TCR, the 1E9 TCR, or the KL14 TCR and CD8αβ containing either wild-type (WT) CD8α (red) or mutated forms of CD8α, namely S53G (teal) or S53N (purple). Data from three donors are shown as mean ± SD (E and F). Significance was determined using a one-way ANOVA with Dunnett’s post hoc test to compare each variant versus wild-type CD8.