Cancer immunotherapy, represented by immune checkpoint inhibitors, has proven to be effective to various types of cancer, but its efficacy is still insufficient. T cells, especially those that attack tumors directly, play crucial roles in antitumor immunity, and we are working on the analysis of such T cells. We have found several findings by analyzing the heterogeneous tumor microenvironment at the single-cell level, which is limited by the bulk analysis alone. We are also working on single cell sequencing for detailed analysis. In particular, T cell receptors (TCR) are also analyzed at the same time, and by reacting the obtained TCR sequences with tumor cell lines derived from the same patient, it was revealed that the exhausted CD8+ T cell clones that highly express so-called exhaustion-related molecules such as PD-1 and CD39 are the T cell clones that attack tumor cells directly. From comprehensive gene expression data, we have identified molecules that are more specifically expressed in such T cells and have also revealed their functions. Furthermore, some CD4+ T-cell clones had cytotoxicity, which also seemed to attack tumor cells directly. By identifying their phenotype, we are redefining CD4+ T-cell exhaustion. We believe that these may become new biomarkers and therapeutic targets in the future.