Extended Data Fig. 8: Enforced KLHL6 expression restrains exhaustion and boosts anti-viral T cell responses.

a, Experimental design. Activated CD45.1⁺ P14 T cells transduced with KLHL6-OE or Control retrovirus were separately transferred into CD45.2⁺ recipients (5×10³ cells/recipient), followed by LCMV-CL13 infection one day later. Mice were sacrificed at days 8 and 21 p.i. b,c, Representative plots and frequencies of “effector-like” (TCF-1⁻GzmB⁺ or Ly108⁻TIM-3⁺) and Tex^prec (TCF-1⁺GzmB⁻ or Ly108⁺TIM-3⁻) subsets in transferred Control and KLHL6-OE P14 T cells from spleens at day 8 p.i. (n = 6 mice). d, Numbers of TCF-1⁺GzmB⁻, TCF-1⁻GzmB⁺ and total P14 T cells from spleen at day 8 p.i. (n = 6 mice). e, Quantification of Annexin V⁺ P14 cells at day 8 p.i. (n = 6 mice). f,g, Cytokine production (TNFα and IFNγ) (f) and relative MFI of TIM-3, PD-1 and TCF-1 (g) in KLHL6-OE versus Control P14 T cells from spleens at day 21 p.i. (n = 6 mice). h-j, Frequencies (h) and absolute numbers (i) of Tpex1, Tpex2, Tex^int and Tex^term subsets, and TOX expression in these subsets (j) (n = 6 mice). k,l, Frequencies (k) and numbers (l) of Tpex (Ly108⁺CX3CR1⁻), Tex^int (Ly108⁻CX3CR1⁺), and Tex^term (Ly108⁻CX3CR1⁻) subsets in spleens at day 21 p.i. (n = 6 mice). m, Mice receiving PBS, Control, and KLHL6-OE P14 T cells were infected with LCMV-CL13 one day after adoptive transfer, and LCMV viral loads in liver and lungs were measured on day 15 p.i., normalized to the PBS group. LCMV titers were assessed by qPCR relative to HPRT (PBS, n = 5 mice; Control and KLHL6-OE, n = 8 mice). Diagram in a created in BioRender. Li, G. (2025) https://BioRender.com/l0bgjp0. Data are presented as mean ± s.e.m. Statistical analyses were determined by unpaired two-tailed Student’s t-test (b-l), or two-way ANOVA with Tukey’s multiple-comparisons test (m). *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001; ns, not significant.

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