Transforming growth factor beta orchestrates PD-L1 enrichment in tumor-derived exosomes and mediates CD8 T-cell dysfunction regulating early phosphorylation of TCR signalome in breast cancer.
Tumor cells promote immune evasion through upregulation of programmed death-ligand 1 (PD-L1) that binds with programmed cell death protein 1 (PD1) on cytotoxic T cells and promote dysfunction. Though therapeutic efficacy of anti-PD1 antibody has remarkable effects on different type of cancers it is less effective in breast cancer (BC). Hence, more details understanding of PD-L1-mediated immune evasion is necessary. Here, we report BC cells secrete extracellular vesicles in form of exosomes carry PD-L1 and are highly immunosuppressive. Transforming growth factor beta (TGF-β) present in tumor microenvironment orchestrates BC cell secreted exosomal PD-L1 load. Circulating exosomal PD-L1 content is highly correlated with tumor TGF-β level. The later also found to be significantly associated with CD8+CD39+, CD8+PD1+ T-cell phenotype. Recombinant TGF-β1 dose dependently induces PD-L1 expression in Texos in vitro and blocking of TGF-β dimmed exosomal PD-L1 level. PD-L1 knocked down exosomes failed to suppress effector activity of activated CD8 T cells like tumor exosomes. While understanding its effect on T-cell receptor signaling, we found siPD-L1 exosomes failed to block phosphorylation of src family proteins, linker for activation of T cells and phosphoinositide phospholipase Cγ of CD8 T cells more than PD-L1 exosomes. In vivo inhibition of exosome release and TGF-β synergistically attenuates tumor burden by promoting Granzyme and interferon gamma release in tumor tissue depicting rejuvenation of exhausted T cells. Thus, we establish TGF-β as a promoter of exosomal PD-L1 and unveil a mechanism that tumor cells follow to promote CD8 T-cell dysfunction.
Chatterjee S
，Chatterjee A
，Jana S
，Dey S
，Roy H
，Das MK
，Alam J
，Adhikary A
，Chowdhury A
，Biswas A
，Manna D
，Bhattacharyya A
... -
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Endoplasmic reticulum stress-induced exosomal miR-27a-3p promotes immune escape in breast cancer via regulating PD-L1 expression in macrophages.
Immune escape of breast cancer cells contributes to breast cancer pathogenesis. Tumour microenvironment stresses that disrupt protein homeostasis can produce endoplasmic reticulum (ER) stress. The miRNA-mediated translational repression of mRNAs has been extensively studied in regulating immune escape and ER stress in human cancers. In this study, we identified a novel microRNA (miR)-27a-3p and investigated its mechanistic role in promoting immune evasion. The binding affinity between miR-27a-3p and MAGI2 was predicted using bioinformatic analysis and verified by dual-luciferase reporter assay. Ectopic expression and inhibition of miR-27a-3p in breast cancer cells were achieved by transduction with mimics and inhibitors. Besides, artificial modulation of MAGI2 and PTEN was done to explore their function in ER stress and immune escape of cancer cells. Of note, exosomes were derived from cancer cells and co-cultured with macrophages for mechanistic studies. The experimental data suggested that ER stress biomarkers including GRP78, PERK, ATF6, IRE1α and PD-L1 were overexpressed in breast cancer tissues relative to paracancerous tissues. Endoplasmic reticulum stress promoted exosome secretion and elevated exosomal miR-27a-3p expression. Elevation of miR-27a-3p and PD-L1 levels in macrophages was observed in response to exosomes-overexpressing miR-27a-3p in vivo and in vitro. miR-27a-3p could target and negatively regulate MAGI2, while MAGI2 down-regulated PD-L1 by up-regulating PTEN to inactivate PI3K/AKT signalling pathway. Less CD4+ , CD8+ T cells and IL-2, and T cells apoptosis were observed in response to co-culture of macrophages and CD3+ T cells. Conjointly, exosomal miR-27a-3p promotes immune evasion by up-regulating PD-L1 via MAGI2/PTEN/PI3K axis in breast cancer.
Yao X
，Tu Y
，Xu Y
，Guo Y
，Yao F
，Zhang X
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Disruption of SIRT7 Increases the Efficacy of Checkpoint Inhibitor via MEF2D Regulation of Programmed Cell Death 1 Ligand 1 in Hepatocellular Carcinoma Cells.
Immune checkpoint inhibitors have some efficacy in the treatment of hepatocellular carcinoma (HCC). Programmed cell death 1 ligand 1 (PD-L1), expressed on some cancer cells, binds to the receptor programmed cell death 1 (PDCD1, also called PD1) on T cells to prevent their proliferation and reduce the antigen-tumor immune response. Immune cells that infiltrate some types of HCCs secrete interferon gamma (IFNG). Some HCC cells express myocyte enhancer factor 2D (MEF2D), which has been associated with shorter survival times of patients. We studied whether HCC cell expression of MEF2D regulates expression of PD-L1 in response to IFNG.
We analyzed immune cells from 20 fresh HCC tissues by flow cytometry. We analyzed 225 fixed HCC tissues (from 2 cohorts) from patients in China by immunohistochemistry and obtained survival data. We created mice with liver-specific knockout of MEF2D (MEF2DLPC-KO mice). We knocked out or knocked down MEF2D, E1A binding protein p300 (p300), or sirtuin 7 (SIRT7) in SMMC-7721, Huh7, H22, and Hepa1-6 HCC cell lines, some incubated with IFNG. We analyzed liver tissues from mice and cell lines by RNA sequencing, immunoblot, dual luciferase reporter, and chromatin precipitation assays. MEF2D protein acetylation and proteins that interact with MEF2D were identified by coimmunoprecipitation and pull-down assays. H22 cells, with MEF2D knockout or without (controls), were transplanted into BALB/c mice, and some mice were given antibodies to deplete T cells. Mice bearing orthotopic tumors grown from HCC cells, with or without knockout of SIRT7, were given injections of an antibody against PD1. Growth of tumors was measured, and tumors were analyzed by immunohistochemistry and flow cytometry.
In human HCC specimens, we found an inverse correlation between level of MEF2D and numbers of CD4+ and CD8+ T cells; level of MEF2D correlated with percentages of PD1-positive or TIM3-positive CD8+ T cells. Knockout of MEF2D from H22 cells reduced their growth as allograft tumors in immune-competent mice but not in immune-deficient mice or mice with depletion of CD8+ T cells. When MEF2D-knockout cells were injected into immune-competent mice, they formed smaller tumors that had increased infiltration and activation of T cells compared with control HCC cells. In human and mouse HCC cells, MEF2D knockdown or knockout reduced expression of PD-L1. MEF2D bound the promoter region of the CD274 gene (encodes PD-L1) and activated its transcription. Overexpression of p300 in HCC cells, or knockout of SIRT7, promoted acetylation of MEF2D and increased its binding, along with acetylated histones, to the promoter region of CD274. Exposure of HCC cells to IFNG induced expression of p300 and its binding MEF2D, which reduced the interaction between MEF2D and SIRT7. MEF2D-induced expression of PD-L1 upon IFNG exposure was independent of interferon-regulatory factors 1 or 9. In HCC cells not exposed to IFNG, SIRT7 formed a complex with MEF2D that attenuated expression of PD-L1. Knockout of SIRT7 reduced proliferation of HCC cells and growth of tumors in immune-deficient mice. Compared with allograft tumors grown from control HCC cells, in immune-competent mice, tumors grown from SIRT7-knockout HCC cells expressed higher levels of PD-L1 and had reduced infiltration and activation of T cells. In immune-competent mice given antibodies to PD1, allograft tumors grew more slowly from SIRT7-knockout HCC cells than from control HCC cells.
Expression of MEF2D by HCC cells increases their expression of PD-L1, which prevents CD8+ T-cell-mediated antitumor immunity. When HCC cells are exposed to IFNG, p300 acetylates MEF2D, causing it to bind the CD274 gene promoter and up-regulate PD-L1 expression. In addition to promoting HCC cell proliferation, SIRT7 reduced acetylation of MEF2D and expression of PD-L1 in HCC cells not exposed to IFNG. Strategies to manipulate this pathway might increase the efficacy of immune therapies for HCC.
Xiang J
，Zhang N
，Sun H
，Su L
，Zhang C
，Xu H
，Feng J
，Wang M
，Chen J
，Liu L
，Shan J
，Shen J
，Yang Z
，Wang G
，Zhou H
，Prieto J
，Ávila MA
，Liu C
，Qian C
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