T cells elicited by effective peptide variants express Vβ8.3 TCRs with a CDR3β motif that correlates with tumor protection. **a** The average percentages of sequences encoding the Jβ2.6 gene segment were calculated for the sequences of the Vβ8.3-expressing TCRs from vaccinated mice (analyzed as in Fig. 4). *Symbols* represent individual mice analyzed by traditional sequencing methods (*black*) or high-throughput sequencing (*gray*). The* bar* indicates the mean, and groups were compared using a Student’s *t* test (**p* < 0.05, ***p* < 0.009, *n* = 4 mice). **b** As in (**a**), the average percentages of sequences encoding the common CDR3β motif were calculated for each vaccine. **c** The number of amino acids encoded in the CDR3β loop of each Vβ8.3 sequence was determined. The percentage of sequences encoding the indicated CDR3β length was calculated for each mouse, as in traditional spectratyping analysis. The *errors bars* represent the SEM (*n* = 4 mice). **d** The average length of the CDR3β chains was determined as in (**c**) for the Vβ8.3 sequences and compared using a Student’s *t* test (**p* < 0.05, ****p* = 0.007, *n* = 4 mice). **e** The average percentage of sequences encoding the Jβ2.6 gene segment (*left*) or the CDR3β motif (*right*) was calculated for the effective (A5, F1A5, and 39 in *black*), ineffective (15 and WMF in *gray*), and native peptides (*white*). *Error bars* represent the SEM. Groups were compared using a Student’s *t* test (***p* = 0.0073, **p* = 0.039). **f** The frequency of sequences encoding the Jβ2.6 gene segment (*x*-axis, from a) was plotted versus the frequency of tumor-free survival observed for each vaccine (35), and a positive correlation was found using a Spearman’s nonparametric correlation test (*r* = 0.6256, *p* = 0.0032). **g** As in (**f**), the correlation of the frequency of sequences encoding the CDR3β motif (*x*-axis, from **b**) and tumor-free survival was analyzed (*r* = 0.5582, *p* = 0.0105). **h** The estimated number of AH1-tet^{+} T cells expressing Vβ8.3 TCRs with the CDR3β motif was determined by multiplying the average frequency of CDR3β motif-containing TCR sequences (from **b**) by the number of Vβ8.3^{+} AH1-tet^{+} cells in the spleens of a separate cohort of mice (frequencies from these mice are shown in Supplementary Fig. 1a) and compared using a Student’s *t* test (****p* < 0.004)

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