Extended Data Fig. 7: KLHL6 loss promotes Tex^term cell differentiation during chronic infection.

a, Naïve Klhl6^+/+ (WT) and Klhl6^−/− (KO) CD8⁺ P14 T cells were mixed at a 1:1 ratio (5×10³ for LCMV-CL13 or 5×10⁴ for LCMV-Arm) and transferred into CD45.2⁺ recipients, followed by intravenous LCMV infection one day later. Activation markers in transferred WT and KO P14 T cells from spleen were assessed at days 4 and 6 post-infection (p.i.). b,c, Naïve CD45.1/2⁺ WT and CD45.1⁺ KO P14 cells were cotransferred (2.5×10³ each) into CD45.2⁺ mice, followed by LCMV-CL13 infection one day later. Mice were sacrificed on days 8 and 28 p.i. (n = 6 mice). Experimental design (b), and percentages (left) and absolute numbers (right) of WT and KO P14 cells in spleen (c). d,e, Frequencies and numbers of “effector-like” (TCF-1⁻GzmB⁺ or Ly108⁻TIM-3⁺) and Tex^prec (TCF-1⁺GzmB⁻ or Ly108⁺TIM-3⁻) subsets in transferred WT and KO P14 T cells from spleens at day 8 p.i. (n = 6 mice). f-h, Percentages of TNFα⁺IFNγ⁺ cells (f), (MTDR/MTG)^lo subsets (g), and apoptotic cells (Annexin V⁺PI⁺; Annexin V⁺PI⁻) (h) in transferred WT and KO P14 cells from spleens at day 8 p.i. of CL13-infected mice (n = 6 mice). i, Quantification of TIM-3, PD-1, TOX and GzmB expression and the percentage of TCF-1⁺ cells at day 28 p.i. (n = 6 mice). j,k, Frequencies and numbers of Tpex (Ly108⁺CX3CR1⁻), Tex^int (Ly108⁻CX3CR1⁺) and Tex^term (Ly108⁻CX3CR1⁻) subsets (j), and Bcl-2/Bim expression in these subsets (k) at day 28 p.i. (n = 6 mice). l-n, Frequencies and numbers of Tpex1 (Ly108⁺CD69⁺), Tpex2 (Ly108⁺CD69⁻), Tex^int (Ly108⁻CD69⁻), and Tex^term (Ly108⁻CD69⁺) subsets (l), TOX expression in these subsets (m), and cytokine production (TNFα and IFNγ) after PMA/BFA stimulation (n) in transferred WT and KO P14 cells at day 28 p.i. (n = 6 mice). o, Mice receiving PBS, WT P14, or KO P14 cells were infected with LCMV-CL13 one day after 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 = 6 mice; WT and KO, n = 8 mice). Diagram in b 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 (c-n), or two-way ANOVA with Tukey’s multiple-comparisons test (o). *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001; ns, not significant.

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