Immunotherapy represented by programmed cell death protein 1 (PD-1)/programmed death ligand 1 (PD-L1) monoclonal antibodies has led tumor treatment into a new era. However, the low overall response rate and high incidence of drug resistance largely damage the clinical benefits of existing immune checkpoint therapies. Recent studies correlate the response to PD-1/PD-L1 blockade with PD-L1 expression levels in tumor cells. Hence, identifying molecular targets and pathways controlling PD-L1 protein expression and stability in tumor cells is a major priority. In this study, we performed a Stress and Proteostasis CRISPR interference screening to identify PD-L1 positive modulators. Here, we identified TRAF6 as a critical regulator of PD-L1 in melanoma cells. As a non-conventional E3 ubiquitin ligase, TRAF6 is inclined to catalyze the synthesis and linkage of lysine-63 (K63) ubiquitin which is related to the stabilization of substrate proteins. Our results showed that suppression of TRAF6 expression down-regulates PD-L1 expression on the membrane surface of melanoma cells. We then used in vitro and in vivo assays to investigate the biological function and mechanism of TRAF6 and its downstream YAP1/TFCP2 signaling in melanoma. TRAF6 stabilizes YAP1 by K63 poly-ubiquitination modification, subsequently promoting the formation of YAP1/TFCP2 transcriptional complex and PD-L1 transcription. Inhibition of TRAF6 by Bortezomib enhanced cytolytic activity of CD8+ T cells by reduction of endogenous PD-L1. Notably, Bortezomib enhances anti-tumor immunity to an extent comparable to anti-PD-1 therapies with no obvious toxicity. Our findings reveal the potential of inhibiting TRAF6 to stimulate internal anti-tumor immunological effect for TRAF6-PD-L1 overexpressing cancers.

Heart transplantation offers life-saving treatment for patients with end-stage heart failure; however, ischemia-reperfusion injury (IRI) and subsequent immune responses remain significant challenges. Current therapies primarily target adaptive immunity, with limited options available for addressing IRI and innate immune activation. Although plant-derived vesicle-like nanoparticles show promise in managing diseases, their application in organ transplantation complications is unexplored. Here, this work develops a novel reactive oxygen species (ROS)-responsive multifunctional fusion extracellular nanovesicles carrying rapamycin (FNVs@RAPA) to address early IRI and Ly6C+Ly6G- inflammatory macrophage-mediated rejection in heart transplantation. The FNVs comprise Exocarpium Citri grandis-derived extracellular nanovesicles with anti-inflammatory and antioxidant properties, and mesenchymal stem cell membrane-derived nanovesicles expressing calreticulin with macrophage-targeting ability. A novel ROS-responsive bio-orthogonal chemistry approach facilitates the active targeting delivery of FNVs@RAPA to the heart graft site, effectively alleviating IRI and promoting the polarization of Ly6C+Ly6G- inflammatory macrophages toward an anti-inflammatory phenotype. Hence, FNVs@RAPA represents a promising therapeutic approach for mitigating early transplantation complications and immune rejection. The fusion-targeted delivery strategy offers superior heart graft site enrichment and macrophage-specific targeting, promising improved transplant outcomes.

Excessive inflammatory response after severe scalding is an important cause of delayed wound healing and is even life-threatening. Tumor necrosis factor α (TNF-α) is a key pro-inflammatory factor of skin trauma. Interacting with tumor necrosis factor receptor 1 (TNF-R1), TNF-α causes excessive inflammation and poor prognosis by activating NF-κB pathway. Antagonizing high levels of TNF-α is one of the therapeutic approaches for diseases associated with the overactivation of inflammatory responses. However, the available monoclonal antibodies are limited in their application due to their complex preparation process, high price, and the lack of cell targeting ability leading to systemic toxicity and side effects. In this study, by using a genetic bioengineering technique, we modified TNF-R1 on the cell membrane surface-derived nanovesicles (NVs). We confirmed that TNF-R1 NVs stably expressed TNF-R1 on the membrane surface and interacted with its ligand TNF-α. Furthermore, TNF-R1 NVs competitively antagonized the effect of TNF-α in the wound healing assay in vitro. In the scalded mouse model, TNF-R1 NVs were released continuously from the thermosensitive hydrogel Pluronic F-127, resulting in less inflammation and better wound healing. Our results revealed TNF-R1 NVs as promising cell-free therapeutic agents in alleviating TNF-α-mediated pro-inflammatory signaling and promoting wound repair.

Vimentin has been implicated in wound healing, inflammation, and cancer, but its functional contribution to intestinal diseases is poorly understood. To study how vimentin is involved during tissue injury and repair of simple epithelium, we induced colonic epithelial cell damage in the vimentin null (Vim −/− ) mouse model. Vim −/− mice challenged with dextran sodium sulfate (DSS) had worse colitis manifestations than wild-type (WT) mice. Vim −/− colons also produced more reactive oxygen and nitrogen species, possibly contributing to the pathogenesis of gut inflammation and tumorigenesis than in WT mice. We subsequently describe that CD11b + macrophages served as the mainly cellular source of reactive oxygen species (ROS) production via vimentin-ROS-pSTAT3–interleukin-6 inflammatory pathways. Further, we demonstrated that Vim −/− mice did not develop colitis-associated cancer model upon DSS treatment spontaneously but increased tumor numbers and size in the distal colon in the azoxymethane/DSS model comparing with WT mice. Thus, vimentin has a crucial role in protection from colitis induction and tumorigenesis of the colon.

Mutations in the plant homeodomain-like finger protein 6 (PHF6) gene are strongly associated with acute myeloid (AML) and T-cell acute lymphoblastic leukemia (T-ALL). In this study, we demonstrated that PHF6 can bind to H3K9me3 and H3K27me1 on the nucleolar chromatin and recruit histone methyltransferase SUV39H1 to the rDNA locus. The deletion of PHF6 caused a decrease in the recruitment of SUV39H1 to rDNA gene loci, resulting in a reduction in the level of H3K9me3 and the promotion of rDNA transcription. The knockdown of either SUV39H1 or PHF6 significantly attenuated the effects of increase in H3K9me3 and suppressed the transcription of rDNA induced by the overexpression of the other interacting partner, thereby establishing an interdependent relationship between PHF6 and SUV39H1 in their control of rRNA transcription. The PHF6 clinical mutants significantly impaired the ability to bind and recruit SUV39H1 to the rDNA loci, resulting in an increase in rDNA transcription activity, the proliferation of in vitro leukemia cells, and the growth of in vivo mouse xenografts. Importantly, significantly elevated levels of pre-rRNA were observed in clinical AML patients who possessed a mutated version of PHF6. The specific rDNA transcription inhibitor CX5461 significantly reduced the resistance of U937 AML cells deficient in PHF6 to cytarabine, the drug that is most commonly used to treat AML. Collectively, we revealed a novel molecular mechanism by which PHF6 recruits methyltransferase SUV39H1 to the nucleolar region in leukemia and provided a potential therapeutic target for PHF6-mutant leukemia.

Small Extracellular Vesicles The simultaneous high expression of LAG-3 and PD-1 on T cells in cardiac transplantation mouse models and clinical renal transplantation patients provides the possibility to establish immune tolerance by giving their ligand molecules. In article number 2102634 by Bo Jia, Fang Cheng, Hongbo Chen, and co-workers, small extracellular vesicles derived from bioengineered PD-L1/FGL1-expressed mesenchymal stem cells encapsulated with low-dose FK506 exerted a powerful synergistic inhibitory effect on T cell activation and, therefore, prolonged grafts survival.

There is an urgent need for developing new immunosuppressive agents due to the toxicity of long-term use of broad immunosuppressive agents after organ transplantation. Comprehensive sample analysis revealed dysregulation of FGL1/LAG-3 and PD-L1/PD-1 immune checkpoints in allogeneic heart transplantation mice and clinical kidney transplant patients. In order to enhance these two immunosuppressive signal axes, a bioengineering strategy is developed to simultaneously display FGL1/PD-L1 (FP) on the surface of small extracellular vesicles (sEVs). Among various cell sources, FP sEVs derived from mesenchymal stem cells (MSCs) not only enriches FGL1/PD-L1 expression but also maintain the immunomodulatory properties of unmodified MSC sEVs. Next, it is confirmed that FGL1 and PD-L1 on sEVs are specifically bound to their receptors, LAG-3 and PD-1 on target cells. Importantly, FP sEVs significantly inhibite T cell activation and proliferation in vitro and a heart allograft model. Furthermore, FP sEVs encapsulated with low-dose FK506 (FP sEVs@FK506) exert stronger effects on inhibiting T cell proliferation, reducing CD8+ T cell density and cytokine production in the spleens and heart grafts, inducing regulatory T cells in lymph nodes, and extending graft survival. Taken together, dual-targeting sEVs have the potential to boost the immune inhibitory signalings in synergy and slow down transplant rejection.