(A) Naive CD4⁺ T cells (CD44^loCD62L^hi) were isolated from WT and USP16-KO mice by FACS sorter. These T cells were labeled with 5 μM CFSE and stimulated with αCD3/αCD28 as indicated. The proliferative ratio was assessed as CFSE dilution by FACS. (B) USP16-deficient CD4⁺ T cells were infected with retrovirus carrying WT (USP16-WT) or inactive USP16 (USP16-M) for 48 hours, and were selected with puromycin. After resting for 6 hours, the CFSE assay was used for measuring the proliferative capacity of these T cells stimulated with αCD3/αCD28 for 72 hours. (C and D) Cell-cycle processes and genome replication of naive WT or USP16-deficient CD4⁺ T cells in vivo (C) and in vitro (D)were evaluated by bromodeoxyuridine (BrdU) staining. (E) Naive CD4⁺ T cells were isolated from WT and USP16-KO mice, and were stimulated for 4 days with αCD3/αCD28 under different polarization conditions. The frequency of T cell differentiation was analyzed by FACS based on intracellular staining of the indicated cytokines or specific transcriptional factors. (F) WT or USP16-deficient CD4⁺ T cells were infected with retrovirus carrying constitutively activated NFAT2 (Ca-NFAT2), and were selected with puromycin. After stimulation with anti-CD3/CD28 for 72 hours, the proliferative capacity of these T cells was measured by CFSE assay. Data are representative of 3 independent experiments with 3 mice in each group (A, C–F) and 4 independent experiments with 4 mice in each group (B). The error bars show the mean ± SEM. The significance of difference in B was determined by Dunnett’s multiple comparisons test. The significances of differences in all other 2-group comparisons were determined by 2-tailed Student’s t test. *P < 0.05, **P < 0.01, ***P < 0.001.