SMAD4, activated by the TCR-triggered MEK/ERK signaling pathway, critically regulates CD8+ T cell cytotoxic function

Abstract

Transforming growth factor-β is well known to restrain cytotoxic T cell responses to maintain self-tolerance and to promote tumor immune evasion. In this study, we have investigated the role of SMAD4, a core component in the TGF-β signaling pathway, in CD8⁺ T cells. Unexpectedly, we found that SMAD4 was critical in promoting CD8⁺ T cell function in both tumor and infection models. SMAD4-mediated transcriptional regulation of CD8⁺ T cell activation and cytotoxicity was dependent on the T cell receptor (TCR) but not TGF-β signaling pathway. Following TCR activation, SMAD4 translocated into the nucleus, up-regulated genes encoding TCR signaling components and cytotoxic molecules in CD8⁺ T cells and thus reinforced T cell function. Biochemically, SMAD4 was directly phosphorylated by ERK at Ser³⁶⁷ residue following TCR activation. Our study thus demonstrates a critical yet unexpected role of SMAD4 in promoting CD8⁺ T cell-mediated cytotoxic immunity.

Fig. 1.. SMAD4-deficient CD8⁺ T cells exhibit impaired cytotoxic function.

(A) E.G7 cells (1 × 10⁶ cells per mouse) were inoculated subcutaneously into WT (n = 5) and Cd8^CreSmad4^fl/fl mice (n = 5). Tumor growth was monitored from day 9 after inoculation. (B) TILs were isolated at day 17 from E.G7-bearing mice. OVA-specific CD8⁺ T cell percentages and GZMB, TNFα, and IFN-γ expression in CD8⁺ TILs were analyzed by flow cytometry. (C) Left: Growth of E.G7 tumor in mice receiving WT or Smad4-deficient OT-I cells (3 × 10⁵ cells per mouse). Right: Representative images of tumors, 17 days after transplant. KO, knockout. (D) OVA-modified L. monocytogenes (LM-OVA) colony-forming units (CFUs) from spleens and livers were calculated at day 8 after infection. (E) The percentages of OVA-specific CD8⁺ T cells in spleens and livers from LM-OVA–infected WT (n = 5) or Cd8^CreSmad4^fl/fl mice (n = 5). Measured by flow cytometry. (F) Expression levels of GZMB and IFN-γ in CD8⁺ T cells from LM-OVA–infected WT or Cd8^CreSmad4^fl/fl mice. These experiments were repeated three times. Data are represented as means ± SEM. ns, not significant. *P < 0.05, **P < 0.01, and ***P < 0.001.

Fig. 2.. SMAD4 regulates CD8⁺ T cell activation and effector function.

(A) Heatmap of selected differentially expressed genes showing OVA-specific CD8⁺ TILs from WT mice versus that from Cd8^CreSmad4^fl/fl mice (WT versus KO). (B) Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis of SMAD4 up-regulated (SMAD4 abrogation down-regulated) genes between OVA-specific CD8⁺ TILs from WT and Cd8^CreSmad4^fl/fl mice. T_H, T helper cell. (C) Gene set enrichment analysis of effector genes in chronic LCMV infection (12). Genes from the left to right of the rank-ordered list are enriched in WT and Smad4-deficient (KO) groups, respectively. (D) Gene set enrichment of genes describing for exhausted CD8⁺ T cells in mouse autochthonous melanoma, relative to naive CD8⁺ T cells (13). (E) Gene set enrichment of genes describing for terminally exhausted CD8⁺ T cells in B16-OVA tumor model, relative to progenitor exhausted CD8⁺ T cells (14). NES, normalized enrichment score.

Fig. 3.. SMAD4-mediated transcriptional regulation of CD8⁺ T cell activation and cytotoxicity.

(A) KEGG pathway analysis of SMAD4-bound gene loci in activated CD8⁺ T cells. (B) Transcription factor motif analysis following SMAD4 ChIP-seq experiment on activated CD8⁺ T cells. (C) Venn diagram of SMAD4-regulated genes and SMAD4-bound genes. (D) SMAD4 peaks at the Cd247, Cd27, Cd8a, Gzmb, Prf1, and Fasl gene loci in activated CD8⁺ T cells (versus control input DNA).

Fig. 4.. SMAD4 regulation of CD8⁺ T cell cytotoxicity is independent of TGF-β signaling.

(A) Expression levels of GZMB, IFN-γ, Ki-67, and TNFα in WT and Smad4-deficient CD8⁺ T cells. Cells were activated with plate-bound anti-CD3 (5 μg/ml) plus anti-CD28 (5 μg/ml) and cultured in the presence of IL-2 (1 ng/ml) in vitro for 3 to 4 days. Measured by flow cytometry. FSC, forward scatter. (B) WT or Smad4-deficient OT-I cells were activated in vitro for 3 days and mixed with E.G7 cells at 1:1 ratio. Intracellular cleaved caspase-3 was measured by anti–cleaved caspase 3 (1:100) at 5 hours after coculture. GZMB was measured by anti-GZMB (1:400) at 24 hours after coculture. (C) Top: Expression levels of GZMB, IFN-γ, and Ki-67 in WT and Smad4-deficient CD8⁺ T cells in the presence of TGF-β1 (2 ng/ml). Bottom: Expression of GZMB, IFN-γ, and TNFα in WT and Smad4-deficient CD8⁺ T cells in the presence of anti–TGF-β (1 μg/ml). Measured by flow cytometry after 3 days of culture. (D) Expression levels of effector molecules in WT and Smad4-deficient CD8⁺ T cells in the presence of TGF-β receptor inhibitor (SB431542; 1 μM) or BMP receptor inhibitor (K02288; 10 μM). Measured by flow cytometry after 3 to 4 days of culture. These experiments were repeated three times. Data are represented as means ± SEM. *P < 0.05 and ***P < 0.001. DMSO, dimethyl sulfoxide.

Fig. 5.. SMAD4 nuclear translocation in CD8⁺ T cells is triggered by TCR signaling.

(A) Expression levels of SMAD4 in cytoplasm and nucleus between naive and activated CD8⁺ T cells by Western blotting. Cytoplasmic and nuclear protein was extracted from naive and in vitro activated CD8⁺ T cells. The cells were activated by αCD3/CD28 for 24 to 48 hours. Cyt, cytoplasm; Nuc, nucleus; n, naive; a, activated; GAPDH, glyceraldehyde-3-phosphate dehydrogenase. (B) Subcellular localization of SMAD4 in naive and αCD3/CD28 activated CD8⁺ T cells (in the presence of DMSO control, Itk inhibitor, PI3K inhibitor, Lck inhibitor, and combination of Lck/Itk inhibitors, respectively). The cells were collected, spun down to a cytospin microscope slide, and fixed and stained with αSMAD4 followed by staining with Alexa Fluor 488–conjugated secondary antibody. The results shown here represent the merged photos of SMAD4 (green) and 4′ (green) andcondary antibod (DAPI) (blue; indicated for nuclear location) staining. Scale bars, 10 μm. Right: Statistic data of SMAD4 nuclear translocation ratio, which was determined by manually counting the percentage of cells containing higher SMAD4 staining intensity in the nucleus versus cytoplasm in three representative fields revealed by Image-Pro Plus software. (C) Subcellular localization of SMAD4 in αCD3/CD28 activated CD8⁺ T cells in the presence of DMSO control, p38α/β inhibitor, c-Jun N-terminal kinase 1/2 inhibitor, MEK1/2 inhibitor, and ERK1/2 inhibitor, respectively. All the groups were added TGF-β receptor inhibitor. These experiments were repeated two or three times with consistent results. The statistics were performed by Student’s t test. *P < 0.05, **P < 0.01, and ****P < 0.0001.

Fig. 6.. SMAD4 phosphorylation at Ser³⁶⁷ by ERK regulates SMAD4 nuclear translocation.

(A) Expression levels of total SMAD4 and phosphorylated SMAD4 at Thr²⁷⁶ in naive and αCD3/CD28 activated CD8⁺ T cells (in the presence of DMSO control, PI3K inhibitor, ERK inhibitor, respectively). ERKi, ERK1/2 inhibitor; PI3Ki, PI3K inhibitor. (B) Three candidate serine/threonine phosphorylation sites (Ser³⁶⁷, Thr³⁷², and Ser⁴³¹) on SMAD4. Cell lysates from αCD3/CD28 activated CD8⁺ T cells were subjected to anti-SMAD4 purification via immunoprecipitation followed by mass spectrometry analysis. (C) Immunofluorescence staining data of SMAD4 (red; Alexa Fluor 594) in naive or αCD3/CD28 activated CD8⁺ T cells infected with retrovirus harboring WT Smad4 or the three mutant Smad4 gene (24 hours after retrovirus infection). Statistic data of SMAD4 nuclear translocation ratio were determined by manually counting the percentage of cells containing higher SMAD4 staining intensity in the nucleus versus cytoplasm in three representative fields. (D) Co-overexpressed MEK1 and ERK2 phosphorylated SMAD4 at Ser³⁶⁷ site. FLAG-SMAD4-WT or FLAG-SMAD4-S367F and MEK1-ERK2 were cotransfected into HEK-293T cells. Immunoprecipitation by anti-FLAG and Western blotting were carried out. (E) Western blotting results of in vitro phosphorylation assay by using recombinant active ERK2. FLAG-SMAD4-WT and FLAG-SMAD4-S367F were immunoprecipitation-purified with anti-FLAG affinity gel, washed by 3× FLAG peptide. Phospho-serine (p-Ser) levels (relative to total SMAD4) were quantified by ImageJ software. These experiments were repeated two or three times with consistent results.

Fig. 7.. SMAD4-S367F mutant CD8⁺ T cells exhibit impaired cytotoxicity.

(A) Expression levels of GZMB in empty control, SMAD4-WT, or SMAD4-S367F retrovirus–infected CD8⁺ T cells. CD8⁺ T cells from Cd8^CreSmad4^fl/fl mice were isolated and infected with empty control retrovirus or retrovirus containing either WT or S367F mutant Smad4. Forty-eighty hours after infection, GZMB expression was measured by flow cytometry. (B) LM-OVA CFUs from spleens and livers were calculated at day 7 after infection. OT-I cells isolated from Cd8^CreSmad4^fl/fl OT-I mice were infected with empty control retrovirus or retrovirus containing either WT or S367F mutant Smad4. The infected OT-I cells (EGFP⁺ cells) were sorted and intravenously transferred into C57BL/6 mice. Twelve hours later, LM-OVA (2 × 10⁴ CFUs per mouse) was also intravenously injected into the recipient mice. (C) Expression of GZMB in splenic OT-I cells. Measured by flow cytometry. (D) E.G7 cells (1 × 10⁶ cells per mouse) were inoculated subcutaneously into TCRbd^−/− mice. Tumor growth were monitored from day 9 after inoculation. OT-I cells isolated from Cd8^CreSmad4^fl/fl OT-I mice were infected with retrovirus containing either WT or S367F mutant Smad4. The infected OT-I cells (EGFP⁺ cells) were sorted and intravenously transferred into E.G7-bearing TCRbd^−/− mice at day 9 after E.G7 cells inoculation. (E) Expression of GZMB, TNFα, and IFN-γ in CD8⁺ TILs from E.G7-bearing TCRbd^−/− mice was measured by flow cytometry. Data are represented as means ± SEM. The statistics were performed by Student’s t test. *P < 0.05, ***P < 0.001, and ****P < 0.0001.