Antiangiogenic Therapeutic mRNA Delivery Using Lung-Selective Polymeric Nanomedicine for Lung Cancer Treatment.

Therapeutic antibodies that block vascular endothelial growth factor (VEGF) show clinical benefits in treating nonsmall cell lung cancers (NSCLCs) by inhibiting tumor angiogenesis. Nonetheless, the therapeutic effects of systemically administered anti-VEGF antibodies are often hindered in NSCLCs because of their limited distribution in the lungs and their adverse effects on normal tissues. These challenges can be overcome by delivering therapeutic antibodies in their mRNA form to lung endothelial cells, a primary target of VEGF-mediated pulmonary angiogenesis, to suppress the NSCLCs. In this study, we synthesized derivatives of poly(ß-amino esters) (PBAEs) and prepared nanoparticles to encapsulate the synthetic mRNA encoding bevacizumab, an anti-VEGF antibody used in the clinic. Optimization of nanoparticle formulations resulted in a selective lung transfection after intravenous administration. Notably, the optimized PBAE nanoparticles were distributed in lung endothelial cells, resulting in the secretion of bevacizumab. We analyzed the protein corona on the lung- and spleen-targeting nanoparticles using proteomics and found distinctive features potentially contributing to their organ-selectivity. Lastly, bevacizumab mRNA delivered by the lung-targeting PBAE nanoparticles more significantly inhibited tumor proliferation and angiogenesis than recombinant bevacizumab protein in orthotopic NSCLC mouse models, supporting the therapeutic potential of bevacizumab mRNA therapy and its selective delivery through lung-targeting nanoparticles. Our proof-of-principle results highlight the clinical benefits of nanoparticle-mediated mRNA therapy in anticancer antibody treatment in preclinical models.

AMPK Alchemy: Therapeutic Potentials in Allergy, Aging, and Cancer.

All cells are equipped with intricate signaling networks to meet the energy demands and respond to the nutrient availability in the body. AMP-activated protein kinase (AMPK) is among the most potent regulators of cellular energy balance. Under ATP -deprived conditions, AMPK phosphorylates substrates and affects various biological processes, such as lipid/glucose metabolism and protein synthesis. These actions further affect the cell growth, death, and functions, altering the cellular outcomes in energy-restricted environments. AMPK plays vital roles in maintaining good health. AMPK dysfunction is observed in various chronic diseases, making it a promising target for preventing and alleviating such diseases. Herein, we highlight the different AMPK functions, especially in allergy, aging, and cancer, to facilitate the development of new therapeutic approaches in the future.

Shaping the "hot" immunogenic tumor microenvironment by nanoparticles co-delivering oncolytic peptide and TGF-ß1 siRNA for boosting checkpoint blockade therapy.

Induction of potent immune responses toward tumors remains challenging in cancer immunotherapy, in which it only showed benefits in a minority of patients with "hot" tumors, which possess pre-existing effector immune cells within the tumor. In this study, we proposed a nanoparticle-based strategy to fire up the "cold" tumor by upregulating the components associated with T and NK cell recruitment and activation and suppressing TGF-ß1 secretion by tumor cells. Specifically, LTX-315, a first-in-class oncolytic cationic peptide, and TGF-ß1 siRNA were co-entrapped in a polymer-lipid hybrid nanoparticle comprising PLGA, DSPE-mPEG, and DSPE-PEG-conjugated with cRGD peptide (LTX/siR-NPs). The LTX/siR-NPs showed significant inhibition of TGF-ß1 expression, induction of type I interferon release, and triggering immunogenic cell death (ICD) in treated tumor cells, indicated via the increased levels of danger molecules, an in vitro setting. The in vivo data showed that the LTX/siR-NPs could effectively protect the LTX-315 peptide from degradation in serum, which highly accumulated in tumor tissue. Consequently, the LTX/siR-NPs robustly suppressed TGF-ß1 production by tumor cells and created an immunologically active tumor with high infiltration of antitumor effector immune cells. As a result, the combination of LTX/siR-NP treatment with NKG2A checkpoint inhibitor therapy remarkably increased numbers of CD8+NKG2D+ and NK1.1+NKG2D+ within tumor masses, and importantly, inhibited the tumor growth and prolonged survival rate of treated mice. Taken together, this study suggests the potential of the LTX/siR-NPs for inflaming the "cold" tumor for potentiating the efficacy of cancer immunotherapy.

Reactive oxygen species-responsive dual-targeted nanosystem promoted immunogenic cell death against breast cancer.

The development of an optimal treatment modality to improve the therapeutic outcome of breast cancer patients is still difficult. Poor antigen presentation to T cells is a major challenge in cancer immunotherapy. In this study, a synergistic immunotherapy strategy for breast cancer incorporating immune cell infiltration, immunogenic cell death (ICD), and dendritic cell (DC) maturation through a reactive oxygen species (ROS)-responsive dual-targeted smart nanosystem (anti-PD-L1-TKNP) for the simultaneous release of DOX, R848, and MIP-3α in the tumor microenvironment is reported. Following local injection, anti-PD-L1-DOX-R848-MIP-3α/thioketal nanoparticle (TKNP) converts tumor cells to a vaccine owing to the combinatorial effect of DOX-induced ICD, R848-mediated immunostimulatory properties, and MIP-3α-induced immune cell recruitment in the tumor microenvironment. Intratumoral injection of anti-PD-L1-DOX-R848-MIP-3α/TKNP caused significant regression of breast cancer. Mechanistic studies reveal that anti-PD-L1-DOX-R848-MIP-3α/TKNP specifically targets tumor tissue, resulting in maximum exposure of calreticulin (CRT) and HMGB1 in tumors, and significantly enhances intratumoral infiltration of CD4+ and CD8+ T cells in tumors. Therefore, a combined strategy using dual-targeted ROS-responsive TKNP highlights the significant application of nanoparticles in modulating the tumor microenvironment and could be a clinical treatment strategy for effective breast cancer management.

Liposomal co-delivery of toll-like receptors 3 and 7 agonists induce a hot triple-negative breast cancer immune environment.

Triple-negative breast cancer (TNBC) is highly aggressive and has no standard treatment. Although being considered as an alternative to conventional treatments for TNBC, immunotherapy has to deal with many challenges that hinder its efficacy, particularly the poor immunogenic condition of the tumor microenvironment (TME). Herein, we designed a liposomal nanoparticle (LN) platform that delivers simultaneously toll-like receptor 7 (imiquimod, IQ) and toll-like receptor 3 (poly(I:C), IC) agonists to take advantage of the different toll-like receptor (TLR) signaling pathways, which enhances the condition of TME from a "cold" to a "hot" immunogenic state. The optimized IQ/IC-loaded LN (IQ/IC-LN) was effectively internalized by cancer cells, macrophages, and dendritic cells, followed by the release of the delivered drugs and subsequent stimulation of the TLR3 and TLR7 signaling pathways. This stimulation encouraged the secretion of type I interferon (IFN-α, IFN-ß) and CXCLl0, a T-cell and antigen-presenting cells (APCs) recruitment chemokine, from both cancer cells and macrophages and polarized macrophages to the M1 subtype in in vitro studies. Notably, systemic administration of IQ/IC-LN allowed for the high accumulation of drug content in the tumor, followed by the effective uptake by immune cells in the TME. IQ/IC-LN treatment comprehensively enhanced the immunogenic condition in the TME, which robustly inhibited tumor growth in tumor-bearing mice. Furthermore, synergistic antitumor efficacy was obtained when the IQ/IC-LN-induced immunogenic state in TME was combined with anti-PD1 antibody therapy. Thus, our results suggest the potential of combining 2 TLR agonists to reform the TME from a "cold" to a "hot" state, supporting the therapeutic efficacy of immune checkpoint inhibitors.

Transplantation of adipose tissue-derived microvascular fragments promotes therapy of critical limb ischemia.

BACKGROUND: Adipose tissue-derived microvascular fragments are functional vessel segments derived from arterioles, capillaries, and veins. Microvascular fragments can be used as vascularization units in regenerative medicine and tissue engineering containing microvascular networks. However, the in vivo therapeutic and vascularization properties of human microvascular fragments have not been investigated. METHODS: In this study, we isolated microvascular fragments, stromal vascular fractions, and mesenchymal stem cells from human lipoaspirate and studied their therapeutic efficacy and in vivo vasculogenic activity in a murine model of hindlimb ischemia. In addition, in vivo angiogenic activity and engraftment of microvascular fragments into blood vessels were measured using Matrigel plug assay. RESULTS: Both microvascular fragments and stromal vascular fractions contain not only mesenchymal stem cells but also endothelial progenitor cells. In a Matrigel plug assay, microvascular fragments increased the number of blood vessels containing red blood cells more than mesenchymal stem cells and stromal vascular fractions did. The engraftment of the microvascular fragments transplanted in blood vessels within the Matrigel plug significantly increased compared to the engraftment of mesenchymal stem cells and stromal vascular fractions. Moreover, intramuscular injection of microvascular fragments markedly increased blood flow in the ischemic hindlimbs and alleviated tissue necrosis compared to that of mesenchymal stem cells or stromal vascular fractions. Furthermore, transplanted microvascular fragments formed new blood vessels in ischemic limbs. CONCLUSIONS: These results suggest that microvascular fragments show improved engraftment efficiency and vasculogenic activity in vivo and are highly useful for treating ischemic diseases and in tissue engineering. Adipose tissue-derived microvascular fragments are vascularization units in regenerative medicine and tissue engineering containing microvascular networks. Intramuscular injection of microvascular fragments markedly increased blood flow in the ischemic hindlimbs and alleviated tissue necrosis. The present study suggests that microvascular fragments show improved engraftment efficiency and vasculogenic activity in vivo and are highly useful for treating ischemic diseases and in tissue engineering.

Methionine consumption by cancer cells drives a progressive upregulation of PD-1 expression in CD4 T cells.

Programmed cell death protein 1 (PD-1), expressed on tumor-infiltrating T cells, is a T cell exhaustion marker. The mechanisms underlying PD-1 upregulation in CD4 T cells remain unknown. Here we develop nutrient-deprived media and a conditional knockout female mouse model to study the mechanism underlying PD-1 upregulation. Reduced methionine increases PD-1 expression on CD4 T cells. The genetic ablation of SLC43A2 in cancer cells restores methionine metabolism in CD4 T cells, increasing the intracellular levels of S-adenosylmethionine and yielding H3K79me2. Reduced H3K79me2 due to methionine deprivation downregulates AMPK, upregulates PD-1 expression and impairs antitumor immunity in CD4 T cells. Methionine supplementation restores H3K79 methylation and AMPK expression, lowering PD-1 levels. AMPK-deficient CD4 T cells exhibit increased endoplasmic reticulum stress and Xbp1s transcript levels. Our results demonstrate that AMPK is a methionine-dependent regulator of the epigenetic control of PD-1 expression in CD4 T cells, a metabolic checkpoint for CD4 T cell exhaustion.

Modulation of NLRP3 inflammasomes activation contributes to improved survival and function of mesenchymal stromal cell spheroids.

Mesenchymal stem cells (MSCs) are ubiquitous multipotent cells that exhibit significant therapeutic potentials in a variety of disorders. Nevertheless, their clinical efficacy is limited owing to poor survival, low rate of engraftment, and impaired potency upon transplantation. Spheroidal three-dimensional (3D) culture of MSCs (MSC3D) has been proven to better preserve their in vivo functional properties. However, the molecular mechanisms underlying the improvement in MSC function by spheroid formation are not clearly understood. NLRP3 inflammasomes, a key component of the innate immune system, have recently been shown to play a role in cell fate decision of MSCs. The present study examined the role of NLRP3 inflammasomes in the survival and potency of MSC spheroids. We found that MSC3D led to decreased activation of NLRP3 inflammasomes through alleviation of ER stress in an autophagy-dependent manner. Importantly, downregulation of NLRP3 inflammasomes signaling critically contributes to the enhanced survival rate in MSC3D through modulation of pyroptosis and apoptosis. The critical role of NLRP3 inflammasome suppression in the enhanced therapeutic efficacy of MSC spheroids was further confirmed in an in vivo mouse model of DSS-induced colitis. These findings suggest that 3D culture confers survival and functional advantages to MSCs by suppressing NLRP3 inflammasome activation.

T-cell engaging poly(lactic-co-glycolic acid) nanoparticles as a modular platform to induce a potent cytotoxic immunogenic response against PD-L1 overexpressing cancer.

Bispecific nanoparticles (NPs) are conjugated with two antibodies that enhance T cell cytotoxicity by sequentially targeting CD3 and tumor-specific proteins. This interaction redirects T cells to specific tumor antigens and activates them to lyse tumor cells by blocking two different signaling pathways simultaneously. This study developed NP-based bispecific T-cell engagers (nanoBiTEs), which are R848-loaded bispecific poly(lactic-co-glycolic acid) NPs decorated with anti-CD3 antibody targeting T cells and anti-PD-L1 antibody targeting PD-L1 ligands (bis-R848-PLGA-NPs). Bis-R848-PLGA-NPs enhance the immunogenic response in destroying cancer cells by restoring the T cell effector functions. These interactions allow T cells to come in close proximity to the tumor cells. Finally, the release of R848 from PLGA-NPs activates dendritic cells, enhancing T cell activation. In vitro results show maximum internalization of bis-R848-PLGA-NPs in SK-OV3 and B16F10 cell lines, attributed to high PD-L1 expression in both cells. Furthermore, bis-R848-PLGA-NPs-treated CD8+ T cells exhibit a significantly increased total amount of CD8+/CD25+, CD8+/CD69+, and cytokine expression that leads to the robust inhibition of PD-L1 expressed cancer cells. Additionally, tumor growth is significantly inhibited by bis-R848-PLGA-NPs in the B16F10 xenograft mouse model and significantly enhanced intratumoral infiltration of CD4+ and CD8+ T cells, as well as tumor-infiltrated cytokines.

AMPK Amplifies IL2-STAT5 Signaling to Maintain Stability of Regulatory T Cells in Aged Mice.

AMP-activated protein kinase (AMPK), an important regulator of the aging process, is expressed in various immune cells. However, its role in regulatory T cell (Treg) stability during aging is poorly understood. Here, we addressed the role of AMPK in Treg function and stability during aging by generating Treg-specific AMPKα1 knockout mice. In this study, we found that AMPKα1-deficient Tregs failed to control inflammation as effectively as normal Tregs did during aging. AMPK knockout from Tregs reduces STAT5 phosphorylation in response to interleukin (IL)-2 stimulation, thereby destabilizing Tregs by decreasing CD25 expression. Thus, our study addressed the role of AMPK in Tregs in sensing IL-2 signaling to amplify STAT5 phosphorylation, which, in turn, supports Treg stability by maintaining CD25 expression and controlling inflamm-aging.

AMPK suppresses Th2 cell responses by repressing mTORC2.

Allergic inflammation is a T helper 2 (Th2) cell-driven pathophysiological phenomenon, but the mechanism by which the metabolic cascade affects Th2 cell differentiation remains unclear. In this study, we investigated the roles of AMP-activated protein kinase (AMPK) and intracellular energy sensors in Th2 cell differentiation and the pathogenesis of allergic inflammation. Accordingly, T-cell-specific AMPK or Sirtuin 1 (Sirt1)-knockout mice were subjected to allergic inflammation, and their Th2 cell responses were investigated. The results demonstrated that inducing allergic inflammation in AMPK- and Sirt1-knockout mice increased Th2 cell responses and exacerbated allergic phenotypes. Furthermore, treatment with 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR), an activator of AMPK, ameliorated allergic inflammation in mice. Mechanistically, our findings revealed that AMPK repressed mechanistic target of rapamycin complex 2 (mTORC2), which downregulated the expression of suppressor of cytokine signaling 5 (SOCS5) in CD4+ T cells. In addition, the loss of AMPK signaling reduced SOCS5 expression and increased interleukin-4-STAT6-GATA3 axis-mediated Th2 cell differentiation. Finally, the T-cell-specific deletion of Rictor, a member of mTORC2, in Sirt1T-KO mice led to the reversal of allergic exacerbation to the level in control mice. Overall, our findings suggest that AMPK in CD4+ T cells inhibits the differentiation of Th2 cells by repressing mTORC2 and thus serves as a potential target for Th2 cell-associated diseases.

Engineering of hybrid spheroids of mesenchymal stem cells and drug depots for immunomodulating effect in islet xenotransplantation.

Immunomodulation is an essential consideration for cell replacement procedures. Unfortunately, lifelong exposure to nonspecific systemic immunosuppression results in immunodeficiency and has toxic effects on nonimmune cells. Here, we engineered hybrid spheroids of mesenchymal stem cells (MSCs) with rapamycin-releasing poly(lactic-co-glycolic acid) microparticles (RAP-MPs) to prevent immune rejection of islet xenografts in diabetic C57BL/6 mice. Hybrid spheroids were rapidly formed by incubating cell-particle mixture in methylcellulose solution while maintaining high cell viability. RAP-MPs were uniformly distributed in hybrid spheroids and sustainably released RAP for ~3 weeks. Locoregional transplantation of hybrid spheroids containing low doses of RAP-MPs (200- to 4000-ng RAP per recipient) significantly prolonged islet survival times and promoted the generation of regional regulatory T cells. Enhanced programmed death-ligand 1 expression by MSCs was found to be responsible for the immunomodulatory performance of hybrid spheroids. Our results suggest that these hybrid spheroids offer a promising platform for the efficient use of MSCs in the transplantation field.

LKB1 in Intestinal Epithelial Cells Regulates Bile Acid Metabolism by Modulating FGF15/19 Production.

BACKGROUND & AIMS: Liver kinase B1 (LKB1) is a master upstream protein kinase involved in nutrient sensing and glucose and lipid metabolism in many tissues; however, its metabolic role in intestinal epithelial cells (IEC) remains unclear. In this study, we investigated the regulatory role of LKB1 on bile acid (BA) homeostasis. METHODS: We generated mice with IEC-specific deletion of LKB1 (LKB1ΔIEC) and analyzed the characteristics of IEC development and BA level. In vitro assays with small interfering RNA, liquid chromatography/mass spectrometry, metagenomics, and RNA-sequencing were used to elucidate the regulatory mechanisms underlying perturbed BA homeostasis. RESULTS: LKB1 deletion resulted in abnormal differentiation of secretory cell lineages. Unexpectedly, BA pool size increased substantially in LKB1ΔIEC mice. A significant reduction of the farnesoid X receptor (FXR) target genes, including fibroblast growth factor 15/19 (FGF15/19), known to inhibit BA synthesis, was found in the small intestine (SI) ileum of LKB1ΔIEC mice. We observed that LKB1 depletion reduced FGF15/19 protein level in human IECs in vitro. Additionally, a lower abundance of bile salt hydrolase-producing bacteria and elevated levels of FXR antagonist (ie, T-ßMCA) were observed in the SI of LKB1ΔIEC mice. Moreover, LKB1ΔIEC mice showed impaired conversion of retinol to retinoic acids in the SI ileum. Subsequently, vitamin A treatment failed to induce FGF15 production. Thus, LKB1ΔIEC mice fed with a high-fat diet showed improved glucose tolerance and increased energy expenditure. CONCLUSIONS: LKB1 in IECs manages BA homeostasis by controlling FGF15/19 production.

TPH1 and 5-HT7 Receptor Overexpression Leading to Gemcitabine-Resistance Requires Non-Canonical Permissive Action of EZH2 in Pancreatic Ductal Adenocarcinoma.

In the present study, we investigated the regulatory mechanisms underlying overexpression of EZH2, tryptophan hydroxylase 1 (TPH1), and 5-HT7, in relation to gemcitabine resistance and CSC survival in PDAC cells. In aggressive PANC-1 and MIA PaCa-2 cells, knock-down (KD) of EZH2, TPH1, or HTR7 induced a decrease in CSCs and recovery from gemcitabine resistance, while preconditioning of less aggressive Capan-1 cells with 5-HT induced gemcitabine resistance with increased expression of EZH2, TPH1, and 5-HT7. Such effects of the gene KD and 5-HT treatment were mediated through PI3K/Akt and JAK2/STAT3 signaling pathways. EZH2 KD or GSK-126 (an EZH2 inhibitor) inhibited activities of these signaling pathways which altered nuclear level of NF-kB, Sp1, and p-STAT3, accompanied by downregulation of TPH1 and 5-HT7. Co-immunoprecipation with EZH2 and pan-methyl lysine antibodies revealed that auto-methylated EZH2 served as a scaffold for binding with methylated NF-kB and Sp1 as well as unmethylated p-STAT3. Furthermore, the inhibitor of EZH2, TPH1, or 5-HT7 effectively regressed pancreatic tumor growth in a xenografted mouse tumor model. Overall, the results revealed that long-term exposure to 5-HT upregulated EZH2, and the noncanonical action of EZH2 allowed the expression of TPH1-5-HT7 axis leading to gemcitabine resistance and CSC population in PDAC.

AMPK promotes antitumor immunity by downregulating PD-1 in regulatory T cells via the HMGCR/p38 signaling pathway.

BACKGROUND: AMP-activated protein kinase (AMPK) is a metabolic sensor that maintains energy homeostasis. AMPK functions as a tumor suppressor in different cancers; however, its role in regulating antitumor immunity, particularly the function of regulatory T cells (Tregs), is poorly defined. METHODS: AMPKα1fl/flFoxp3YFP-Cre, Foxp3YFP-Cre, Rag1-/-, and C57BL/6 J mice were used for our research. Flow cytometry and cell sorting, western blotting, immuno-precipitation, immuno-fluorescence, glycolysis assay, and qRT-PCR were used to investigate the role of AMPK in suppressing programmed cell death 1 (PD-1) expression and for mechanistic investigation. RESULTS: The deletion of the AMPKα1 subunit in Tregs accelerates tumor growth by increasing the expression of PD-1. Metabolically, loss of AMPK in Tregs promotes glycolysis and the expression of 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR), a key enzyme of the mevalonate pathway. Mechanistically, AMPK activates the p38 mitogen-activated protein kinase (MAPK) that phosphorylates glycogen synthase kinase-3ß (GSK-3ß), inhibiting the expression of PD-1 in Tregs. CONCLUSION: Our study identified an AMPK regulatory mechanism of PD-1 expression via the HMGCR/p38 MAPK/GSK3ß signaling pathway. We propose that the AMPK activator can display synergic antitumor effect in murine tumor models, supporting their potential clinical use when combined with anti-PD-1 antibody, anti-CTLA-4 antibody, or a HMGCR inhibitor.

Preparation and evaluation of dabrafenib-loaded, CD47-conjugated human serum albumin-based nanoconstructs for chemoimmunomodulation.

The transmembrane proteins, CD47 and signal-regulatory protein α are overexpressed in cancer cells and macrophages, respectively, and facilitate the escape of cancer cells from macrophage-mediated phagocytosis. The immunomodulatory and targeting properties of CD47, the chemotherapeutic effects of dabrafenib (D), and the anti-programmed death-1 antibodies (PD-1) pave the way for effective chemoimmunomodulation-mediated anticancer combination therapy. In this study, CD47-conjugated, D-loaded human serum albumin (HSA) nanosystems were fabricated by modified nanoparticle albumin-bound technology. Cis-aconityl-PEG-maleimide (CA), an acid-labile linker, was used to conjugate D@HSA and CD47; the resultant CD47-CA@D@HSA exhibited tumor-specificity through receptor targeting, as well as preferential cleavage and drug release in the acidic tumor microenvironment (pH 5) compared to normal physiological pH conditions (pH 6.5, 7.4). The successful preparation of nanosized (∼220 nm), narrowly dispersed (∼0.13) CD47-CA@D@HSA was proven by physicochemical characterization. In vitro and in vivo internalization, accumulation, cytotoxicity, and apoptosis were observed to be higher with CD47-conjugated nanoconstructs, than with free D or non-targeted nanoconstructs. CD47-CA@D@HSA was found to promote the infiltration of cytotoxic T cells and tumor-associated macrophages into tumors and improve in vivo tumor inhibition. Administration in combination with PD-1 further improved antitumor efficacy by promoting immune responses that blocked the immune checkpoint. No signs of toxicity were seen in mice treated with the nanoconstructs; the formulation was, therefore, thought to be biocompatible and as having potential for clinical use. The targeted chemoimmunomodulation achieved by this combination therapy was found to combat major immunosuppressive facets, making it a viable candidate for use in the treatment of cancer.

Combination chemotherapeutic and immune-therapeutic anticancer approach via anti-PD-L1 antibody conjugated albumin nanoparticles.

Anticancer regimens have been substantially enriched through monoclonal antibodies targeting immune checkpoints, programmed cell death-1/programmed cell death-ligand 1 (PD-L1) and cytotoxic T-lymphocyte antigen-4. Inconsistent clinical efficacy after solo immunotherapy may be compensated by nanotechnology-driven combination therapy. We loaded human serum albumin (HSA) nanoparticles with paclitaxel (PTX) via nanoparticle albumin-bound technology and pooled them with anti-PD-L1 monoclonal antibody through a pH-sensitive linker for targeting and immune response activation. Our tests demonstrated satisfactory preparation of paclitaxel-loaded, PD-L1-targeted albumin nanoparticles (PD-L1/PTX@HSA). They had small particle size (~200 nm) and polydispersity index (~0.12) and successfully incorporated each constituent. Relative to normal physiological pH, the formulation exhibited higher drug-release profiles favoring cancer cell-targeted release at low pH. Modifying nanoparticles with programmed cell death-ligand 1 increased cancer cell internalization in vitro and tumor accumulation in vivo in comparison with non-PD-L1-modified nanoparticles. PD-L1/PTX@HSA constructed by nanoparticle albumin-bound technology displayed successful tumor inhibition efficacy both in vitro and in vivo. There was successful effector T-cell infiltration, immunosuppressive programmed cell death-ligand 1, and regulatory T-cell suppression because of cytotoxic T-lymphocyte antigen-4 synergy. Moreover, PD-L1/PTX@HSA had low organ toxicity. Hence, the anti-tumor immune responses of PD-L1/PTX@HSA combined with chemotherapy and cytotoxic T-lymphocyte antigen-4 is a potential anti-tumor strategy for improving quantitative and qualitative clinical efficacy.

LKB1-PTEN axis controls Th1 and Th17 cell differentiation via regulating mTORC1.

Immuno-environmental change triggers CD4+ T cell differentiation. T cell specialization activates metabolic signal pathways to meet energy requirements. Defective T cell-intrinsic metabolism can aggravate immunopathology in chronic diseases. Liver kinase B1 (LKB1) deletion in T cell or Treg cell results in systemic inflammatory symptoms, indicating a crucial role of LKB1 in T cells. However, the mechanism underlying the development of inflammation is unclear. In our study, LKB1-deficient T cells were differentiated preferentially into Th1 and Th17 cells in the absence of inflammation. Mechanistically, LKB1 directly binds and phosphorylates phosphatase and tensin homolog (PTEN), an upstream regulator of mammalian target of rapamycin complex 1 (mTORC1), which is independent of AMP-activated protein kinase (AMPK). As a result, LKB1 deficiency was associated with increased mTORC1 activity and hypoxia-inducible factor (HIF)1α-mediated glycolysis. Inhibition of glycolysis or biallelic disruption of LKB1 and HIF1α abrogated this phenotype, suggesting Th1- and Th17-biased differentiation in LKB1-deficient T cells was mediated by glycolysis. Our study indicates that LKB1 controls mTORC1 signaling through PTEN activation, not AMPK, which controls effector T cell differentiation in a T cell-intrinsic manner. KEY MESSAGES: • LKB1 maintains T cell homeostasis in a cell intrinsic manner. • Glycolysis is involved in the LKB1-mediated T cell differentiation. • LKB1 phosphorylates PTEN, not AMPK, to regulate mTORC1.

Geranylgeranyl pyrophosphate amplifies Treg differentiation via increased IL-2 expression to ameliorate DSS-induced colitis.

Blocking the mevalonate pathway for cholesterol reduction by using statin may have adverse effects including statin-induced colitis. Moreover, one of the predisposing factors for colitis is an imbalanced CD4+ T cell, which can be observed on the complete deletion of HMG-CoA reductase (HMGCR), a target of statins. In this study, we inquired geranylgeranyl pyrophosphate (GGPP) is responsible for maintaining the T-cell homeostasis. Following dextran sulfate sodium (DSS)-induced colitis, simvastatin increased the severity of disease, while cotreatment with GGPP, but not with cholesterol, reversed the disease magnitude. GGPP ameliorated DSS-induced colitis by increasing Treg cells. GGPP amplified Treg differentiation through increased IL-2/STAT 5 signaling. GGPP prenylated Ras protein, a prerequisite for extracellular signal-regulated kinase (ERK) pathway activation, leading to increased IL-2 production. Higher simvastatin dose increased the severity of colitis. GGPP ameliorated simvastatin-increased colitis by increasing Treg cells. Treg cells, which have the capacity to suppress inflammatory T cells and were generated through IL-2/STAT5 signaling, increased IL-2 production through prenylation and activation of the Ras/ERK pathway.