Single-cell TCR VDJ analysis

Eight TIL (RTIL1-4 and NRTIL1-4) and eight spleen samples (RSP1-4 and NRSP1-4) were sequenced for TCR VDJ region ( Supplemental Table 1 ). Filtered_contig_annotations output files from the 10×Genomics CellRanger VDJ pipeline were used for further VDJ analysis using R (version 4.1.0) or for analysis using immunarch (version 0.6.7) package (43) of R. Cells were filtered in R as follows: firstly, including cells with only full length, productive, high-confidence V and J segments and secondly, including cells containing only 1 TCRβ chain, and only 1 or 2 TCRα chains (due to lack of allelic exclusion in TCRα locus). CD8 T cells from each sample were grouped into clones by identical nucleotide sequences of the TCRα and TCRβ CDR3 chains, while cells with the same a.a. sequence for the TCRα and TCRβ CDR3 chains were grouped into the same clonotype. Clonotypes were quantified to calculate the percent in each sample (% of a given clonotype = # of cells with that clonotype/# of total cells in the sample) and clonotypes above 1% in any of the 8 TIL samples were used for analysis of “top clonotypes” in further VDJ analyses. Clonotypes above 0.65% in R TIL samples (RTIL1-4) and clonotypes above 1% in NR TIL samples (NRTIL1-4) were used for analysis of “top clonotypes” in single-cell gene expression analyses to include equal numbers of clonotypes from R and NR.