Figure 5. Frequencies of TCR 46β residues in TCR library-transduced T cells isolated from tumor and other tissues (in vivo selection).

(a) Comparison of relative frequencies of TCR 46β residues selected for binding SIY/K^b in vitro (left panels) with TCR 46β residues isolated from B16-SIY tumors in mice treated with CD8⁺ (middle panels) or CD4⁺ (right panels) T cells transduced with the 2C (top row) or m33 (bottom row) libraries. The averages of two independent mice are shown, and data for individual mice can be seen in Supplementary Fig. 3. For the 2C library in CD8⁺ T cells, analysis of signature residues (Met, Phe, Trp, Tyr, and His) appearing in the tumor showed that Tyr, Phe, Met, and His were significantly enhanced. (p<0.001). Signature residues located in the tumor from the m33 library in CD4⁺ T cells that are significantly enhanced are Phe, His, and Met (p<0.001) compared to a significant reduction of residues Tyr, Phe, Met, His, and Trp in the tumor of m33 library CD8⁺ T cells (p<0.001). (b) Relative frequencies of TCR 46β residues for T cells isolated from the draining lymph node (first and third columns) or spleen (second and fourth columns) of B16-SIY tumor-bearing mice treated with CD8⁺ (left two columns) or CD4⁺ (right two columns) T cells transduced with the 2C TCR library (top row) or the m33 TCR library (bottom row). The averages of two independent mice are shown for all samples, except for the 2C CD8⁺ library in the draining lymph node (6,629 sequences; one mouse) and spleen (8,809 sequences; one mouse), and for the m33 CD8⁺ library in the spleen (15,400; one mouse). There is a significant enhancement of Phe, Trp (p<0.001) and Tyr, His (p<0.01) found in the tumor draining lymph node from the 2C library in CD8⁺ T cells while Tyr, Phe and His from the same library were significantly enhanced in the spleen (p<0.001). For the 2C library in CD4⁺ T cells, a significant enhancement of Tyr, Phe, Met and His was found in the tumor draining lymph node (p<0.001). For the m33 library in CD8⁺ T cells, there is a significant reduction of residues Tyr, Phe, Met, His and Trp found in the tumor draining lymph node (p<0.001). Graphs for individual mice averaged in (a) and (b), along with the number of sequences analyzed per sample are shown in Supplementary Figure 3. A statistical approach to determine significance of amino acid selections in vivo is described in Supplementary Figures 4 and 5. The approach uses the frequencies of all 20 amino acid frequencies and the single-stop codon for each in vivo sample, in comparison with the in vitro SIY/K^b-selected frequencies.