Extended Data Fig. 4: TCR stimulation downregulates KLHL6 level.

a, Relative Klhl6 mRNA levels in adoptively transferred OT-I T cells from spleens and PD-1⁻TIM-3⁻, PD-1⁺TIM-3⁻ or PD-1⁺TIM-3⁺ CD8⁺ TILs from B16-OVA tumours at day 14 after ACT (n = 3 independent samples). b,c, Schematic of OT-I T cell restimulation in vitro. OT-I T cells were isolated from spleens, activated with anti-CD3/CD28, and cultured in mIL-2-containing media for 4 days. Cells were restimulated on day 4 with anti-CD3 (2 μg/mL, 24 h), OVA (1 μg/mL, 24 h), or PMA (50 ng/mL, 12 h) and compared to the un-restimulated Controls (b). Immunoblot and quantification of KLHL6 protein level in treated cells. Actin was used as a loading control (c, n = 3 independent samples). d,e, OT-I T cells were activated and expanded in the media containing mIL-2 for 4 days, then subjected to either repeated anti-CD3 restimulation every 2 days until day 9 (three restim) or a single restimulation on day 4 followed by resting in mIL-2 media until day 9 (one restim) (d). Western blot and quantification of KLHL6 expression were performed (e, n = 3 independent samples). f, Transcriptional expression of Tcf7, Pdcd1, and Klhl6 in P14 T cells during acute and chronic LCMV infection (GSE41867) (n = 4 independent samples). The boxplot spans from the first to the third quartile of the distribution, with the median positioned in the center. Whiskers represent the minimum and maximum values, excluding outliers. g, Klhl6 mRNA expression in antigen-specific T cells over time during acute infection and tumour progression (GSE89307) (n = 2-3 independent samples). h-j, Naïve CD45.1⁺P14⁺CD8⁺ T cells were adoptively transferred into CD45.2⁺ recipients (5×10³ cells/mouse for LCMV-Clone 13 or 5×10⁴ cells/mouse for LCMV-Armstrong). One day later, mice were infected with either virus. CD45.1⁺P14⁺ T cells were sorted from the spleen at the indicated time points post-infection for analysis. Experimental design (h). mRNA (i, n = 3 independent samples) and protein (j) levels of KLHL6 in CD45.1⁺P14⁺ T cells were analyzed at indicated time points. Diagram in b created in BioRender. Li, G. (2025) https://BioRender.com/9zoxyuh. Diagram in d created in BioRender. Li, G. (2025) https://BioRender.com/5se3g09. Diagram in h created in BioRender. Li, G. (2025) https://BioRender.com/l0bgjp0. Data are presented as mean ± s.e.m. Statistical analyses were determined by two-way ANOVA with Tukey’s multiple-comparisons test (a,c,e). **P < 0.01, ***P < 0.001, and ****P < 0.0001.

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