(A) Scheme of GP61 wt and altered peptide ligand (APL) sequences with mutations highlighted in red ordered hierarchically according to T cell receptor (TCR) signal strength. (B) Peptide dose–activation curves of overnight cultured SMARTA cells with peptide pulsed splenocytes using the percentage of CD69+ SMARTA cells as a readout for activation. EC50 values are ~5 nM for GP61 wt, ~0.1 µM for V71S, and ~1 µM for Y72F. (C) In vitro growth kinetics depicting the viral load in the culture medium (focus forming units [FFU]/ml) of GP61 wt or V71S and Y72F variants of Armstrong (left) and Clone-13 (right) variant infection on BHK21 cells over time. Data are displayed as mean ± SD. (D) Early splenic viral load day 3 post infection (p.i.) in Armstrong variants. Bars represent the mean and symbols represent individual mice. (E) Viral load (FFU) in indicated organs per gram tissue over time in C57BL/6 mice. The dotted line represents the limit of detection. Data are displayed as mean ± SEM of 7–10 samples. (F) Peptide dose–response curves depicting the out-competition of the GP61 FITC signal by unlabeled GP61 wt or APLs on B220+ B cells. Data are displayed as mean ± SD of two to three technical replicates. Data represent one of n = 2 independent experiments (B, D, F) or pooled data from n = 2 independent experiments (C, E).