Functional validation of BTNL8 variants. Summary flow cytometry data of the surface expression BTNL3 (first column) and BTNL8 (second column) 48 h after transfection in 293T cells, and of TCR downregulation (third column, normalized to co-culture with cells transfected with EV control) and CD69 upregulation (fourth column, normalized to co-culture with cells transfected with EV control) by J76 cells expressing a Vg4Vg1 TCR (clone hu17) following a 5 h co-culture with transfected 293T cells. Grey symbols with dashed lines indicate WT Reference (Ref) BTNL8 sequence; red symbols with solid lines, indicate BTNL8 variants. Data points are the median of three transfections and co-cultures for each plasmid quantity. Confidence intervals (defined by the range of the replicate measurements) are represented in shaded grey and red areas. ns, not significant; *P < 0.05, **P < 0.01; ***P < 0.001 (unpaired two-tailed t test, comparing each BTNL8 variant to the WT Ref sequence).