Extended Data Figure 4: TCR clonotypes associated with radiation are not observed in random clones from post-treatment blood and have distinct CDR3 features.

a, Boxplot of the bootstrap variance explained by multivariable random forest regression model for effect of RT, anti-CTLA4, and/or anti-PD-L1 on immune variables from TILs. b, k-means clustering (k = 2) was used on the average CDR3 amino acid features of randomly sampled clones from post-treatment blood after anti-CTLA4, anti-PD-L1, and/or radiation treatment (RT). Membership into each cluster was determined and the P value for separation into treatment groups with and without RT was calculated by Fisher’s exact test. Boxplot shows log₁₀(P values) from 1,000 random iterations. Comparison to the P value from the observed data (red dotted line) gives a simulated P < 0.001. c, log₁₀(P values) for separation into treatment groups with and without RT vs cut-off value used to select the most frequent clones. The 0.05 significance level is indicated (red dotted line). d, Average % occupancy in the CDR3 of the most frequent T cell clonotypes after RT ± checkpoint blockade (+RT, red line) or checkpoint blockade alone (NoRT, orange line) by contiguous short amino acid sequences of length three (3-tuples) belonging to e, subsets with distinct treatment-related amino acid properties. These properties are characterized by Atchley factors, which measure (1) PAH: accessibility, polarity, and hydrophobicity, (2) PSS: propensity for secondary structure, (3) MS: molecular size, (4) CC: codon composition, and (5) EC: electrostatic charge. Shown (right) are the average values of each Atchley factor for amino acids that comprise the 3-tuples from the indicated subset (red) compared to all unselected 3-tuples (blue). Boxplots (left) show the proportion of 3-tuples from each of these subsets that are found in the CDR3s of the five most frequent clones after treatment. Compared to pre-treatment samples (Pre-Tx), subset 6 is associated with RT ± checkpoint blockade (+RT) or checkpoint blockade alone (NoRT). Subset 1 is primarily associated with checkpoint blockade alone, and subset 16 is primarily associated with RT ± checkpoint blockade.