Chronic TCR stimulation suppresses KLHL6 expression via FOXO1 inhibition to promote T cell exhaustion and impair antitumor immunity. (IMAGE)
Caption
a-b Immunoblots and quantification of KLHL6 expression in mouse and human CD8⁺ T cells after TCR stimulation. Splenic OT-I CD8⁺ T cells were initially activated by anti-CD3/CD28 antibodies, expanded in the media containing mIL-2 for 4 days, and then either three repeated stimulations in vitro using anti-CD3 (2 μg/mL) antibody treatment every 2 days until day 10 (Three restim), or no restimulations and let the cell rest in mIL-2 media until day 10 (No restim) (a). For human T cells, the CD8⁺ T cells were activated by anti-CD3/CD28 antibodies and expanded in the media containing hIL-2 for 8 days, and then either three repeated stimulations (Three restim) in vitro using anti-CD3 (2 μg/mL) antibody treatment every 2 days until day 14, or no restimulations and let the cell rest in hIL-2 media until day 14 (No restim) (b). n = 3 independent samples. c Binding sites of FOXO1 to the promoter of Klhl6 analyzed using JASPAR (left). FOXO1 occupancy on the conserved region of Klhl6 promoter in both mouse and human CD8+ T cells was analyzed (right). The activated mouse and human T cells were cultured for 4 days and then re-stimulated with anti-CD3 (2 μg/mL) antibody for 2 days. The cells with (anti-CD3) or without (No stim) re-stimulation were collected and analyzed using CUT&RUN-qPCR. IgG: negative control. n = 3 independent samples. d Assessment of AKT-FOXO axis by western blot in resting or stimulated OT-I T cells. The activated OT-I CD8+ T cells were cultured for 4 days in vitro and then re-stimulated by anti-CD3 (2 μg/mL) antibody for 2 days. The cells re-stimulated (with anti-CD3) or not re-stimulated (No stim) were collected and analyzed. n = 3 independent samples. e Immunoblot and quantification of KLHL6 in OT-I CD8⁺ T cells transduced with FOXO1WT, FOXO1AAA, or empty plasmid (Ctrl). The transduced cells were cultured for 4 days in vitro, and then re-stimulated with anti-CD3 (2 μg/mL) for 48 h for analysis. n = 3 independent samples. f Immunoblot and quantification of KLHL6 expression in shFOXO1 or shCtrl Jurkat cells after stimulation with anti-CD3 (2 μg/mL) for 24 h. n = 3 independent samples. g-j CD45.1+ OT-I CD8⁺ T cells were transduced with indicated plasmids, and adoptively transferred into CD45.2⁺ mice bearing B16-OVA tumor that had been implanted 9 days earlier. Tumor weights in tumor-bearing mice were measured on day 14 after ACT (g, n = 9 mice); TOX expression in transferred CD8⁺ TILs at day 14 after ACT (h, n = 5 mice). Representative plots (left) and percentages (right) of TIM-3⁺PD-1⁺ populations in transferred CD8⁺ TILs at day 14 after ACT (i, n = 5 mice); TNF-α and IFN-γ production in transferred CD8⁺ TILs after 4.5 h PMA + BFA stimulation at day 14 after ACT (j, n = 5 mice). K Schematic diagram illustrates chronic TCR engagement promoting CD8⁺ T cell exhaustion. Data are presented as mean ± SEM. Statistical analyses were determined by unpaired two-tailed Student’s t-test (a-d) or two-way ANOVA with Tukey’s multiple-comparisons test (e-j). *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001; ns, no significance
Credit
Prof. Guideng Li from Suzhou Institute of Systems Medicine, China Image source link: https://link.springer.com/article/10.1007/s44466-025-00023-z
Usage Restrictions
Credit must be given to the creator. Only noncommercial uses of the work are permitted. No derivatives or adaptations of the work are permitted.
License
CC BY-NC-ND