I and Fr. 9 of 12 genes reported to be commonly elevated in both exhausted CD8 T cells and CD4 regulatory T cells (Tregs)7 were upregulated in Fr. IV compared to both Fr. I and Fr. II. Only expression levels of and among the above 21 genes were upregulated when comparing Fr. III and Fr. IV. Fourteen genes including and were consistently upregulated in Fr. IV compared to Frs. I, II, and III. Moreover, genes such as specific to na?ve CD8 T cells identified by single-cell RNA sequencing7 were consistently downregulated in Fr. IV compared to Frs. I, II, and III (Fig.?2e). TCR clonal analysis showed that diversities of T-cell receptor (TCR) clonotypes of both chain (TCRA) and chain (TCRB) were the lowest in Fr. IV among the 4 populations although the difference was not significant (Supplementary Fig.?2a,b). Clonotypes with specific proportions tended to be higher in Fr. IV among 3 patients (Supplementary Fig.?2c), and some clonotypes of TCRA and TCRB were shared among the 4 fractions within each individual (Supplementary Fig.?2d). There were no differences in the proportion of clonotypes according to the expression of CD103 as a feature of Trem cells (Supplementary Figs.?1 and 2c). The expression ratio of CD8 T cells producing all three cytokines (IL-2, TNF, and IFN) in Fr. IV was significantly lower than that of Fr. II and Fr. III (Fig.?2f). In addition, the expression ratio of CD8 T cells producing either of two kinds of cytokines was significantly higher in Fr. LSN 3213128 II compared to Fr. III and Fr. IV, and the expression ratio of CD8 T cells producing no cytokines was higher in Fr. III and Fr. IV compared to Fr. II. In terms of cytotoxic functionality, the expression ratio of CD8 T cells producing GZMB LSN 3213128 was significantly higher in LSN 3213128 Fr. II compared to Fr. III and Fr. IV (among each fraction. (g) Comparison of the expressions of CD4?+?CD25high T cells, previously defined as CD4 Tregs, is stratified by each fraction. (h) Comparison of multiple cytokine productivity (IFN, TNF, and IL-2) of CD4 TILs from 18 patients in Fr. II, Fr. III, and Fr. V. among three sample types was performed by Kruskal-Wallis analysis, and then a comparison of each sample type was Muc1 performed by Bonferroni-corrected Mann-Whitney U test. The central tendency of the box plot indicates the median of each group, and the upper and lower ranges of the box plot show the 25th and 75th percentiles of each data set, respectively. **was significantly upregulated in Fr. V along with 13 of 20 genes characterized as CD4 Treg-specific genes7 (Fig.?3e,f). Also 8 of 12 genes reported to be commonly elevated in both exhausted CD8 T cells and CD4 Tregs7 were upregulated in Fr. V compared that in to Frs. I, II, and III. Moreover, genes such as and and em PDCD1 /em , which were upregulated in Fr. III and Fr. IV of CD8 T cells compared to Fr. I in the present study. So, the combination therapy of ipilimumab and nivolumab targeting both CD4?+?PD-1lowTIM-3+ and CD8+ PD-1highTIM-3+ might be able to reverse the natural prognosis of RCC and be ideal for treating patients with higher tumour grade. Actually, this novel immune therapy was effective in metastatic RCC patients with intermediate and poor risk, but the superiority of nivolumab and ipilimumab could not be shown in metastatic RCC patients with good risk2. We speculated that one reason might be the presence of more patients with lower-grade RCC among the metastatic RCC patients with good risk, and combination therapy was unnecessary for these patients, although we could not verify the results of the clinical trial. The WHO/ISUP grading system is widely recognized to LSN 3213128 be useful for the prognostic prediction of clear cell and papillary RCC and is usually used to evaluate only these two histological types31. However, this system could be LSN 3213128 used to describe the.