Dose-response curves were fitted to a log agonist versus stimulus with four parameters [Span, Baseline, Hill coefficient, and EC50 (median effective concentration)] with the minimum baseline constrained to zero using Prism 7.0 software (GraphPad). To compare ligands in concentration-response assays or time-response assays, a two-way ANOVA of ligand and concentration or ligand and time, respectively, was conducted. If a statistically significant interaction or main effect of treatment, depending on the experiment, was observed (P < 0.05), then comparative two-way ANOVAs between individual ligands were performed. Further details of statistical analysis and replicates are included in the figure legends. For mouse ear T cell counts, two mice corresponding to the highest and lowest values in the WT VUF10661 treatment group were excluded from analysis (CD8+CD44+ counts 79,515 and 2709, respectively). Lines indicate the mean, and error bars signify the SEM throughout the manuscript, unless otherwise noted. Asterisk indicates P < 0.05 throughout the paper to indicate statistical significance from pertinent comparisons detailed in the figure legends, unless otherwise noted.

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