a, Mouse strains generated and used in this study. We define wild type as littermate controls TCR TAG ;dLck-Cre;Tox+/+ or TCR TAG ;Toxfl/fl. We define knockout as TOX-deficient T cells from TCR TAG ;dLck-Cre;Toxfl/fl mice. b, Thymocytes and peripheral CD8 T cells from knockout mice develop normally. CD4 and CD8 flow staining of thymocytes isolated from knockout (red, n = 5) or littermate controls (grey, n= 3). TCR Vβ7 and CD44 expression, and enumeration of single-positive CD8+ thymocytes from knockout and wild-type mice. c, Enumeration of total splenocytes (n = 5) and CD8+ splenocytes (n = 4) of knockout and wild-type mice. d, e, TOX is not required for effector and memory CD8 T cell differentiation during acute Listeria infection. d, Approximately 1 × 105 congenically marked naive wild-type and knockout TCR TAG T cells were adoptively transferred into B6 mice, and infected with Listeria 1 day later. Flow cytometric analysis of CD44, CD62L, CD127 and KLRG1 expression directly ex vivo (inset numbers show percentage in respective quadrants) of wild-type and knockout effector TCR TAG cells isolated from spleens of LmTAG-immunized B6 mice 7 days after immunization. e, Flow cytometric analysis of CD44, CD62L, CD127 and KLRG1 expression of wild-type and knockout memory TCR TAG cells isolated from spleens of LmTAG-immunized B6 mice 3 weeks after immunization. Right, intracellular IFNγ and TNF production after 4-h ex vivo TAG peptide stimulation of wild-type (n = 4) and knockout (n = 4) memory TCR TAG T cells. Flow plots are gated on CD8+Thy1.1+ cells. Data are representative of at least three independent experiments. f–i, Phenotypic and functional characterization of TOX wild-type and knockout TCR TAG cells differentiating in developing liver tumours of AST×Cre mice. f, Top, CD44, CD69, CD25 and PD-1 expression and CellTrace Violet (CTV) dilution of adoptively transferred, CTV-labelled naive wild-type (black) or knockout (red) TCR TAG cells isolated from livers of AST×Cre mice 3 days after transfer. Data are representative of three independent experiments. Middle, expression of CD44 and proliferation (CTV dilution) of wild-type (black) or knockout (red) TCR TAG cells isolated from AST×Cre liver lesions 5 days after transfer. CTV-labelled TCR TAG cells transferred into B6 control mice are shown in grey as controls transferred and isolated at the same time points. Bottom, PD-1 and LAG-3 expression together with TOX expression of wild-type and knockout TCR TAG cells isolated from the livers of AST×Cre mice 8 days after transfer. All FACS plots are gated on CD8+ and Thy1.1+. g, Flow cytometric analysis of intracellular IFNγ and TNF production (top), CD107 degranulation (middle), and GZMB expression (bottom) of day 7–10 wild-type (black) or knockout (red) TCR TAG cells after 4-h peptide stimulation. h, i, PMA and ionomycin stimulation (h) or 4-h peptide stimulation using in vivo LPS-activated APCs (i). Each sample is gated on its respective no-peptide control. All flow plots are gated on CD8+Thy1.1+ T cells. Data are representative of three independent experiment and shown as mean ± s.e.m. P values determined by two sided Student’s t-test. Source Data