Clonotype analysis and clustering methodology. (A) Number of cells with the indicated ratios of TCR α to β chains per clonotype (left). Proportion of cells within each tissue with α:β clonotype ratio indicated. (B) Number of unique paired clonotypes—individual α and individual β chains at both nucleotide and amino acid levels compared with total cells in this study. (C–E) Schematic of iterative clustering approach. Initially assigned clusters in C are selected and examined for heterogeneity of key marker genes and subclustered by PCA-based clustering (D), resulting in final cluster assignments (E). (F) Stability of clusters under different levels of downsampling. Adjusted Rand Index (ARI) was computed for all clusters, peripheral CD4/T reg cell clusters (C16:C23), tumor CD4 effector clusters (C17:C20), and tumor CD8 clusters (C8:C11). dLN, draining LN.