Fig 1: Tmed4 deficiency alters the CD8+ T cell transcription profile and reduces terminal exhaustion.(A) Uniform manifold approximation and projection (UMAP) visualization of tumor-infiltrating CD8+ T cell subsets from Tmed4f/f and Tmed4ΔT MC38 tumor–bearing mice. (B) Dot plot depicting the expression of key marker genes across CD8+ T cell clusters. The size of each dot represents the percentage of cells expressing the gene, while the color intensity indicates the average expression level. (C and D) Quantitation (C) and density distribution (D) of CD8+ T cell subsets in Tmed4f/f and Tmed4ΔT MC38 tumor–bearing mice. (E) UMAP visualization of pseudotime analysis for CD8+ T cells. (F) Cell density changes along pseudotime, illustrating the transition from naïve to terminally exhausted CD8+ T cells in Tmed4f/f and Tmed4ΔT MC38 tumor–bearing mice. The Kolmogorov–Smirnov test was used to assess statistical significance. (G) FCM plots and quantification of TCF1 and TIM3 expression in tumor-infiltrating PD1+CD8+ T cells from B16F10 tumor–bearing mice (n = 4 to 5). (H and I) Workflow and FCM analysis of PD1+TIM3+ Tex CD8+ T cells in Tmed4f/f and Tmed4ΔT mice (n = 4). CD8+ T cells were stimulated with anti-CD3/CD28 antibodies (1 μg/ml) with IL-2 (100 U/ml) for 7 days and were changed with fresh culture medium every 2 days. Data are presented as means ± SEM. Statistical significance was determined using a two-tailed unpaired Student’s t test (**P < 0.01. All data are representative of at least three independent experiments.
Fig 2: Loss of Tmed4 enhances CD8+ T cell antitumor immunity.(A) Volcano plot depicting differentially expressed genes (DEGs) in WT and Tmed4−/− CD8+ T cells. DEGs were identified using DESeq2 with the threshold |log2(fold change)| ≥ 1.5; P < 0.05. (B and C) Heatmaps illustrating the relative expression of cytotoxic mediators (B) and chemokines along with their respective receptors (C) in WT and Tmed4−/− CD8+ T cells (n = 3). (D and E) Tumor growth (n = 3 to 5) (D) and survival curves (n = 5) (E) of MC38 tumors in Tmed4f/f and Tmed4ΔT mice. A total of 5 × 105 MC38 cells were injected subcutaneously in 100 μl of phosphate-buffered saline (PBS), and tumor volume was measured every 2 days. (F and G) Tumor growth (n = 4) (F) and survival curves (n = 5 to 6) (G) of B16F10 tumors in Tmed4f/f and Tmed4ΔT mice. A total of 3 × 105 B16F10 cells were injected subcutaneously, and tumor volume was measured every 2 days. (H) Quantification of CD8+ TILs in MC38 tumor–bearing mice on day 21 (n = 5). (I) FCM plot and analysis of Ki67+ tumor-infiltrating CD8+ T cells (n = 4). (J) FCM plot and analysis of IFN-γ+, TNF-α+, and GzmB+ tumor-infiltrating CD8+ T cells (WT: n = 5; cKO: n = 3). (K) Workflow diagram of the in vivo ovalbumin (OVA)–specific cytotoxic CD8+ T cell killing assay. iv, intravenously. (L) Quantification of specific lysis percentage in the OVA-specific cytotoxic T cell killing assay: % specific lysis = 100 − [100 × (% CFSEhigh immunized mouse/% CFSElow immunized mouse) / (% CFSEhigh naïve mouse/% CFSElow naïve mouse)] (n = 5 to 6). Data are presented as means ± SEM. Statistical significance was determined using a two-tailed unpaired Student’s t test (*P < 0.05, **P < 0.01, and ***P < 0.001). All data are representative of at least three independent experiments. CFSE, carboxyfluorescein diacetate succinimidyl ester.
Fig 3: Tmed4 deletion specifically enhances CD8+ T cell effector function.(A and B) Flow cytometry (FCM) plots (A) and quantification (B) of CD62LloCD44hi (effector, Teff), CD62LhiCD44hi (central memory, Tcm), and CD62LhiCD44lo (naïve, Tna) subsets in CD4+ and CD8+ T cells from Tmed4f/f [wild-type (WT)] and Tmed4ΔT (cKO) spleen and peripheral lymph nodes (pLN) (n = 4 to 5). NS, not significant. (C) FCM analysis of IFN-γ+, TNF-α+, IL-2+, IL-4+, and IL-17+ cytokine-producing CD4+ T cells from Tmed4f/f and Tmed4ΔT mice (n = 4 to 5). (D) FCM analysis of IFN-γ+, TNF-α+, and IL-2+ cytokine-producing CD8+ T cells from Tmed4f/f and Tmed4ΔT mice (n = 4 to 5). (E and F) ELISA quantification of IFN-γ (E) and IL-2 (F) levels in CD4+ and CD8+ T cells stimulated with anti-CD3/CD28 (1 μg/ml) for 24 hours (n = 3). (G) FCM plot and analysis of CD8+ T cell apoptosis from the spleen. Early apoptosis is defined as annexin V+PI−, and late apoptosis as annexin V+PI+ (n = 4). (H) Proliferation assay of CD8+ T cells based on eFluor 450 (E450) dilution. Purified CD8+ T cells were stimulated with anti-CD3/CD28 (1 μg/ml) for 3 days and performed gating strategy based on unstimulated control (n = 4). Data are presented as means ± SEM. Statistical significance was determined using a two-tailed unpaired Student’s t test (*P < 0.05,***P < 0.001, and ****P < 0.0001). All data are representative of at least three independent experiments.
Fig 4: Targeting Tmed4 enhances the antitumor activity of CAR T cells.(A to F) Schematic workflow [(A) and (D)], tumor growth [(B) and (E)], and survival analysis [(C) and (F)] for the MC38-CLDN18.2 (top) and B16F10-CLDN18.2 (bottom) tumor models [n = 15 for survival analysis (F), n = 5 to 6 for others]. sc, subcutaneously; iv, intravenously. (G to L) Experimental schematic [(G) and (J)], tumor growth [(H) and (K)], and survival analyses [(I) and (L)] for the MC38-HER2 (top) and B16F10-HER2 (bottom) tumor models (n = 4 to 5). Data are presented as means ± SEM of biologically independent samples. Each experiment represents at least three independent replicates. Statistical significance was determined using a two-tailed unpaired Student’s t test (*P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001).
Fig 5: Targeting Tmed4 potentiates immunotherapy.(A to C) Experimental schematic (A), tumor growth (B), and tumor weight (C) of ASO-treated CD8+ OT-I T cells. No transfer group (black) (n = 7), WT (blue) (n = 7), and Tmed4-knockdown (red) (n = 6). (D to F) Schematic workflow (D), tumor growth (E), and survival analysis (F) for the ASO-treated anti-CLDN18.2 CAR T cells in MC38-CLDN18.2 tumor model (n = 7). (G and H) Schematic workflow (G) and tumor growth (H) of anti-PD1 combination therapy (n = 6). ip, intraperitoneally. Data are presented as means ± SEM of biologically independent samples. Statistical significance was determined using a two-tailed unpaired Student’s t test. [(B),(C),(E), and (F)] *P < 0.05, and **P < 0.01, ASO-Control (ASO-Ctrl) vs ASO-Tmed4; #P < 0.05, and ##P < 0.01, No transfer vs ASO-Tmed4. (H) **P < 0.01, ***P < 0.001, and ****P < 0.0001, WT+Ctrl vs KO+Ctrl; $P < 0.05, and $$$P < 0.001, WT+Anti-PD1 vs KO+Anti-PD1; ###P < 0.001, and ####P < 0.0001, WT+Ctrl vs KO+Anti-PD1.
Supplier Page from Abcam for Anti-TMED4 antibody