PD-1 blockade exerts clinical efficacy against various types of cancer by reinvigorating T cells that directly attack tumor cells (tumor-specific T cells) in the tumor microenvironment (TME), and tumor-infiltrating lymphocytes (TILs) also comprise nonspecific bystander T cells. Here, using single-cell sequencing, we show that TILs include skewed T cell clonotypes, which are characterized by exhaustion (T_ex) or nonexhaustion signatures (T_non-ex). Among skewed clonotypes, those in the T_ex, but not those in the T_non-ex, cluster respond to autologous tumor cell lines. After PD-1 blockade, non-preexisting tumor-specific clonotypes in the T_ex cluster appear in the TME. Tumor-draining lymph nodes (TDLNs) without metastasis harbor a considerable number of such clonotypes, whereas these clonotypes are rarely detected in peripheral blood. We propose that tumor-infiltrating skewed T cell clonotypes with an exhausted phenotype directly attack tumor cells and that PD-1 blockade can promote infiltration of such T_ex clonotypes, mainly from TDLNs.