uCDR3 number and diversity characterization
The number of unique CDR3 (uCDR3) in 8 samples including both VC and CC samples was determined by CDR3 Nucleic acid sequence, and the number of uCDR3 between PBMC and TIL was compared. The number of uCDR3 in TIL (mean = 14122.38, median = 8087) was significantly lower than that of PBMC (mean = 63679.62, median = 56350.5) (Figure 5A, P = 0.00133). In addition, the number of the uCDR3 in each TIL sample was lower than that in its paired PBMC sample (Figure 5B). The accumulate relative abundance of Top10 ranked uCDR3 in every TIL sample (mean = 50.49, median = 49.83) is higher than that in the paired PBMC sample (mean = 16.32, median = 13.72). Notably, the accumulate relative abundance of Top10 uCDR3 is more than 50% in 4 out of 8 TIL samples. Moreover, we analyzed the Shannon Entropy in each sample, which is a quantitative measure of uncertainty that simultaneously takes both richness and evenness into account. A higher Shannon entropy indicates that the TCR sample has more types of uCDR3 or a more even distribution of CDR3 reads. Shannon Entropy of TIL (mean = 4.93, median = 4.96) was significantly lower than that of PBMC (mean = 9.01, median = 9.31) (Figure 5D, P = 0.0004). The Shannon Entropy of each TIL sample was lower than that in its paired PBMC sample (Figure 5E). Collectively, the comparison of the uCDR3 number, relative abundance distribution of Top10 ranked uCDR3 and Shannon Entropy suggests that TIL samples have more enriched T cell clones than PBMC.