Exportin 1 governs the immunosuppressive functions of myeloid-derived suppressor cells in tumors through ERK1/2 nuclear export.

Myeloid-derived suppressor cells (MDSCs) are a main driver of immunosuppression in tumors. Understanding the mechanisms that determine the development and immunosuppressive function of these cells could provide new therapeutic targets to improve antitumor immunity. Here, using preclinical murine models, we discovered that exportin 1 (XPO1) expression is upregulated in tumor MDSCs and that this upregulation is induced by IL-6-induced STAT3 activation during MDSC differentiation. XPO1 blockade transforms MDSCs into T-cell-activating neutrophil-like cells, enhancing the antitumor immune response and restraining tumor growth. Mechanistically, XPO1 inhibition leads to the nuclear entrapment of ERK1/2, resulting in the prevention of ERK1/2 phosphorylation following the IL-6-mediated activation of the MAPK signaling pathway. Similarly, XPO1 blockade in human MDSCs induces the formation of neutrophil-like cells with immunostimulatory functions. Therefore, our findings revealed a critical role for XPO1 in MDSC differentiation and suppressive functions; exploiting these new discoveries revealed new targets for reprogramming immunosuppressive MDSCs to improve cancer therapeutic responses.

Myeloid-derived suppressor cell mitochondrial fitness governs chemotherapeutic efficacy in hematologic malignancies.

Myeloid derived suppressor cells (MDSCs) are key regulators of immune responses and correlate with poor outcomes in hematologic malignancies. Here, we identify that MDSC mitochondrial fitness controls the efficacy of doxorubicin chemotherapy in a preclinical lymphoma model. Mechanistically, we show that triggering STAT3 signaling via ß2-adrenergic receptor (ß2-AR) activation leads to improved MDSC function through metabolic reprograming, marked by sustained mitochondrial respiration and higher ATP generation which reduces AMPK signaling, altering energy metabolism. Furthermore, induced STAT3 signaling in MDSCs enhances glutamine consumption via the TCA cycle. Metabolized glutamine generates itaconate which downregulates mitochondrial reactive oxygen species via regulation of Nrf2 and the oxidative stress response, enhancing MDSC survival. Using ß2-AR blockade, we target the STAT3 pathway and ATP and itaconate metabolism, disrupting ATP generation by the electron transport chain and decreasing itaconate generation causing diminished MDSC mitochondrial fitness. This disruption increases the response to doxorubicin and could be tested clinically.

Galectin-3 expression in donor T cells reduces GvHD severity and lethality after allogeneic hematopoietic cell transplantation.

Abundant donor cytotoxic T cells that attack normal host organs remain a major problem for patients receiving allogeneic hematopoietic cell transplantation (allo-HCT). Despite an increase in our knowledge of the pathobiology of acute graft versus host disease (aGvHD), the mechanisms regulating the proliferation and function of donor T cells remain unclear. Here, we show that activated donor T cells express galectin-3 (Gal-3) after allo-HCT. In both major and minor histocompatibility-mismatched models of murine aGvHD, expression of Gal-3 is associated with decreased T cell activation and suppression of the secretion of effector cytokines, including IFN-γ and GM-CSF. Mechanistically, Gal-3 results in activation of NFAT signaling, which can induce T cell exhaustion. Gal-3 overexpression in human T cells prevents severe disease by suppressing cytotoxic T cells in xenogeneic aGvHD models. Together, these data identify the Gal-3-dependent regulatory pathway in donor T cells as a critical component of inflammation in aGvHD.

Polarization and ß-Glucan Reprogram Immunomodulatory Metabolism in Human Macrophages and Ex Vivo in Human Lung Cancer Tissues.

Immunomodulatory (IM) metabolic reprogramming in macrophages (Mϕs) is fundamental to immune function. However, limited information is available for human Mϕs, particularly in response plasticity, which is critical to understanding the variable efficacy of immunotherapies in cancer patients. We carried out an in-depth analysis by combining multiplex stable isotope-resolved metabolomics with reversed phase protein array to map the dynamic changes of the IM metabolic network and key protein regulators in four human donors' Mϕs in response to differential polarization and M1 repolarizer ß-glucan (whole glucan particles [WGPs]). These responses were compared with those of WGP-treated ex vivo organotypic tissue cultures (OTCs) of human non-small cell lung cancer. We found consistently enhanced tryptophan catabolism with blocked NAD+ and UTP synthesis in M1-type Mϕs (M1-Mϕs), which was associated with immune activation evidenced by increased release of IL-1ß/CXCL10/IFN-γ/TNF-α and reduced phagocytosis. In M2a-Mϕs, WGP treatment of M2a-Mϕs robustly increased glucose utilization via the glycolysis/oxidative branch of the pentose phosphate pathway while enhancing UDP-N-acetyl-glucosamine turnover and glutamine-fueled gluconeogenesis, which was accompanied by the release of proinflammatory IL-1ß/TNF-α to above M1-Mϕ's levels, anti-inflammatory IL-10 to above M2a-Mϕ's levels, and attenuated phagocytosis. These IM metabolic responses could underlie the opposing effects of WGP, i.e., reverting M2- to M1-type immune functions but also boosting anti-inflammation. Variable reprogrammed Krebs cycle and glutamine-fueled synthesis of UTP in WGP-treated OTCs of human non-small cell lung cancer were observed, reflecting variable M1 repolarization of tumor-associated Mϕs. This was supported by correlation with IL-1ß/TNF-α release and compromised tumor status, making patient-derived OTCs unique models for studying variable immunotherapeutic efficacy in cancer patients.

6-Phosphogluconate dehydrogenase (6PGD), a key checkpoint in reprogramming of regulatory T cells metabolism and function.

Cellular metabolism has key roles in T cells differentiation and function. CD4+ T helper-1 (Th1), Th2, and Th17 subsets are highly glycolytic while regulatory T cells (Tregs) use glucose during expansion but rely on fatty acid oxidation for function. Upon uptake, glucose can enter pentose phosphate pathway (PPP) or be used in glycolysis. Here, we showed that blocking 6-phosphogluconate dehydrogenase (6PGD) in the oxidative PPP resulted in substantial reduction of Tregs suppressive function and shifts toward Th1, Th2, and Th17 phenotypes which led to the development of fetal inflammatory disorder in mice model. These in turn improved anti-tumor responses and worsened the outcomes of colitis model. Metabolically, 6PGD blocked Tregs showed improved glycolysis and enhanced non-oxidative PPP to support nucleotide biosynthesis. These results uncover critical role of 6PGD in modulating Tregs plasticity and function, which qualifies it as a novel metabolic checkpoint for immunotherapy applications.

Innate immune activation by checkpoint inhibition in human patient-derived lung cancer tissues.

Although Pembrolizumab-based immunotherapy has significantly improved lung cancer patient survival, many patients show variable efficacy and resistance development. A better understanding of the drug's action is needed to improve patient outcomes. Functional heterogeneity of the tumor microenvironment (TME) is crucial to modulating drug resistance; understanding of individual patients' TME that impacts drug response is hampered by lack of appropriate models. Lung organotypic tissue slice cultures (OTC) with patients' native TME procured from primary and brain-metastasized (BM) non-small cell lung cancer (NSCLC) patients were treated with Pembrolizumab and/or beta-glucan (WGP, an innate immune activator). Metabolic tracing with 13C6-Glc/13C5,15N2-Gln, multiplex immunofluorescence, and digital spatial profiling (DSP) were employed to interrogate metabolic and functional responses to Pembrolizumab and/or WGP. Primary and BM PD-1+ lung cancer OTC responded to Pembrolizumab and Pembrolizumab + WGP treatments, respectively. Pembrolizumab activated innate immune metabolism and functions in primary OTC, which were accompanied by tissue damage. DSP analysis indicated an overall decrease in immunosuppressive macrophages and T cells but revealed microheterogeneity in immune responses and tissue damage. Two TMEs with altered cancer cell properties showed resistance. Pembrolizumab or WGP alone had negligible effects on BM-lung cancer OTC but Pembrolizumab + WGP blocked central metabolism with increased pro-inflammatory effector release and tissue damage. In-depth metabolic analysis and multiplex TME imaging of lung cancer OTC demonstrated overall innate immune activation by Pembrolizumab but heterogeneous responses in the native TME of a patient with primary NSCLC. Metabolic and functional analysis also revealed synergistic action of Pembrolizumab and WGP in OTC of metastatic NSCLC.

Blockade of 6-phosphogluconate dehydrogenase generates CD8+ effector T cells with enhanced anti-tumor function.

Although T cell expansion depends on glycolysis, T effector cell differentiation requires signaling via the production of reactive oxygen species (ROS). Because the pentose phosphate pathway (PPP) regulates ROS by generating nicotinamide adenine dinucleotide phosphate (NADPH), we examined how PPP blockade affects T cell differentiation and function. Here, we show that genetic ablation or pharmacologic inhibition of the PPP enzyme 6-phosphogluconate dehydrogenase (6PGD) in the oxidative PPP results in the generation of superior CD8+ T effector cells. These cells have gene signatures and immunogenic markers of effector phenotype and show potent anti-tumor functions both in vitro and in vivo. In these cells, metabolic reprogramming occurs along with increased mitochondrial ROS and activated antioxidation machinery to balance ROS production against oxidative damage. Our findings reveal a role of 6PGD as a checkpoint for T cell effector differentiation/survival and evidence for 6PGD as an attractive metabolic target to improve tumor immunotherapy.

Long-term tolerance of islet allografts in nonhuman primates induced by apoptotic donor leukocytes.

Immune tolerance to allografts has been pursued for decades as an important goal in transplantation. Administration of apoptotic donor splenocytes effectively induces antigen-specific tolerance to allografts in murine studies. Here we show that two peritransplant infusions of apoptotic donor leukocytes under short-term immunotherapy with antagonistic anti-CD40 antibody 2C10R4, rapamycin, soluble tumor necrosis factor receptor and anti-interleukin 6 receptor antibody induce long-term (≥1 year) tolerance to islet allografts in 5 of 5 nonsensitized, MHC class I-disparate, and one MHC class II DRB allele-matched rhesus macaques. Tolerance in our preclinical model is associated with a regulatory network, involving antigen-specific Tr1 cells exhibiting a distinct transcriptome and indirect specificity for matched MHC class II and mismatched class I peptides. Apoptotic donor leukocyte infusions warrant continued investigation as a cellular, nonchimeric and translatable method for inducing antigen-specific tolerance in transplantation.

Optimizing PLG nanoparticle-peptide delivery platforms for transplantation tolerance using an allogeneic skin transplant model.

A robust regimen for inducing allogeneic transplantation tolerance involves pre-emptive recipient treatment with donor splenocytes (SP) rendered apoptotic by 1-ethyl-3-(3'-dimethylaminopropyl)-carbodiimide(ECDI) treatment. However, such a regimen is limited by availability of donor cells, cost of cell procurement, and regulatory hurdles associated with cell-based therapies. Nanoparticles (NP) delivering donor antigens are a promising alternative for promoting transplantation tolerance. Here, we used a B6.C-H-2bm12(bm12) to C57BL/6(B6) skin transplant model involving a defined major histocompatibility antigen mismatch to investigate design parameters of poly(lactide-co-glycolide) (PLG) NPs delivering peptides containing the donor antigen for optimizing skin allograft survival. We showed that an epitope-containing short peptide (P1) was more effective than a longer peptide (P2) at providing graft protection. Importantly, the NP and P1 complex (NP-ECDI-P1) resulted in a significant expansion of graft-infiltrating Tregs. Interestingly, in comparison to donor ECDI-SP that provided indefinite graft protection, NP-ECDI-P1 targeted different splenic phagocytes and skin allografts in these recipients harbored significantly more graft-infiltrating CD8+IFN-γ+ cells. Collectively, the current study provides initial engineering parameters for a cell-free and biocompatible NP-peptide platform for transplant immunoregulation. Moreover, it also provides guidance to future NP engineering endeavors to recapitulate the effects of donor ECDI-SP as a goal for maximizing tolerance efficacy of NP formulations.

Blockade of Lactate Dehydrogenase-A (LDH-A) Improves Efficacy of Anti-Programmed Cell Death-1 (PD-1) Therapy in Melanoma.

Immunotherapy is a curable treatment for certain cancers, but it is still only effective in a small subset of patients. We have recently reported that programmed cell death protein-1 (PD-1) ligand (PD-L1) expression is regulated by lactate present at high levels in the tumor microenvironment (TME). We hypothesized that the efficacy of anti-PD-1 treatment can be improved by blocking the lactate-generating enzyme, lactate dehydrogenase-A (LDH-A). Anti-PD-1 treatment of mice harboring LDH-A deficient B16-F10 melanoma tumors led to an increase in anti-tumor immune responses compared to mice implanted with tumors expressing LDH-A. Specifically, we observed heightened infiltration of natural killer (NK) cells and CD8⁺ cytotoxic T cells in the LDH-A deficient tumors. These infiltrated cytotoxic cells had an elevated production of interferon-γ (IFN-γ) and granzyme B. Mechanistically, CD8⁺ T cells isolated from the TME of LDH-A deficient B16-F10 melanoma tumors and treated with anti-PD-1 showed enhanced mitochondrial activity and increased reactive oxygen species (ROS) levels. Moreover, infiltration of T regulatory (Treg) cells was diminished in LDH-A deficient tumors treated with anti-PD-1. These altered immune cell profiles were clinically relevant as they were accompanied by significantly reduced tumor growth. Our study suggests that blocking LDH-A in the tumor might improve the efficacy of anti-PD-1 therapy.

The Role of Lactate Metabolism in Prostate Cancer Progression and Metastases Revealed by Dual-Agent Hyperpolarized 13C MRSI.

This study applied a dual-agent, 13C-pyruvate and 13C-urea, hyperpolarized 13C magnetic resonance spectroscopic imaging (MRSI) and multi-parametric (mp) ¹H magnetic resonance imaging (MRI) approach in the transgenic adenocarcinoma of mouse prostate (TRAMP) model to investigate changes in tumor perfusion and lactate metabolism during prostate cancer development, progression and metastases, and after lactate dehydrogenase-A (LDHA) knock-out. An increased Warburg effect, as measured by an elevated hyperpolarized (HP) Lactate/Pyruvate (Lac/Pyr) ratio, and associated Ldha expression and LDH activity were significantly higher in high- versus low-grade TRAMP tumors and normal prostates. The hypoxic tumor microenvironment in high-grade tumors, as measured by significantly decreased HP 13C-urea perfusion and increased PIM staining, played a key role in increasing lactate production through increased Hif1α and then Ldha expression. Increased lactate induced Mct4 expression and an acidic tumor microenvironment that provided a potential mechanism for the observed high rate of lymph node (86%) and liver (33%) metastases. The Ldha knockdown in the triple-transgenic mouse model of prostate cancer resulted in a significant reduction in HP Lac/Pyr, which preceded a reduction in tumor volume or apparent water diffusion coefficient (ADC). The Ldha gene knockdown significantly reduced primary tumor growth and reduced lymph node and visceral metastases. These data suggested a metabolic transformation from low- to high-grade prostate cancer including an increased Warburg effect, decreased perfusion, and increased metastatic potential. Moreover, these data suggested that LDH activity and lactate are required for tumor progression. The lactate metabolism changes during prostate cancer provided the motivation for applying hyperpolarized 13C MRSI to detect aggressive disease at diagnosis and predict early therapeutic response.

Enhanced CD40 and ICOSL expression on dendritic cells surface improve anti-tumor immune responses; effectiveness of mRNA/chitosan nanoparticles.

Objective: To improve dendritic cells (DCs) function, we targeted DCs to over express CD40 and inducible costimulator ligand (ICOSL) costimulatory molecules along with total messenger RNA (mRNA) of tumor cells to achieve a safe and effective system for treatment of tumor. Materials and methods: We generated CD40 and ICOSL mRNA in vitro and manipulated DCs using chitosan nanoparticles and also lipofectamine transfection system then examined in vitro and in vivo. Results: Mice bone marrow derived DCs pulsed with total tumor mRNA/CD40 mRNA or ICOSL mRNA showed higher expression of DCs maturation markers (CD40, ICOSL, CD86, and MHC-II) and accelerated secretion of pro-inflammatory cytokines. Co-culture of DCs with T cells enhanced proliferation of T cells and shift toward stronger Th1 cytokine responses especially in presence of CD40 over expressed DCs. Intra-tumor administration of manipulated DCs to 4T1 tumor mice model showed delay in growth of tumor volume, trend to increase in mice survival, and stronger anti-tumor cytokines production in splenocytes of mice model (with higher efficacy of mRNA/chitosan nanoparticle system). Conclusions: Hence, we suggest that targeting intra-tumor DCs to elicit expression of CD40 and ICOSL and present broad range of tumor antigens could yield effective anti-tumor responses. In this regard, CD40 molecule manipulation trigger stronger functions, while mRNA/chitosan nanoparticles system could provide a high potent tool for targeting strategies.

Targeting T Cell Metabolism for Improvement of Cancer Immunotherapy.

There has been significant progress in utilizing our immune system against cancer, mainly by checkpoint blockade and T cell-mediated therapies. The field of cancer immunotherapy is growing rapidly but durable clinical benefits occur only in a small subset of responding patients. It is currently recognized that cancer creates a suppressive metabolic microenvironment, which contributes to ineffective immune function. Metabolism is a common cellular feature, and although there has been significant progress in understanding the detrimental role of metabolic changes of the tumor microenvironment (TEM) in immune cells, there is still much to be learned regarding unique targetable pathways. Elucidation of cancer and immune cell metabolic profiles is critical for identifying mechanisms that regulate metabolic reprogramming within the TEM. Metabolic targets that mediate immunosuppression and are fundamental in sustaining tumor growth can be exploited therapeutically for the development of approaches to increase the efficacy of immunotherapies. Here, we will highlight the importance of metabolism on the function of tumor-associated immune cells and will address the role of key metabolic determinants that might be targets of therapeutic intervention for improvement of tumor immunotherapies.

Metabolic Switch in the Tumor Microenvironment Determines Immune Responses to Anti-cancer Therapy.

Tumor-induced immune tolerance permits growth and spread of malignant cells. Cancer cells have strong influence on surrounding cells and shape the hypoxic tumor microenvironment (TME) facilitating cancer progression. A dynamic change in glucose metabolism occurring in cancer cells and its influence on the TME are still poorly understood. Indeed, cancer and/or immune cells undergo rapid adaptation in metabolic pathways during cancer progression. Metabolic reprograming affects macrophages, T cells, and myeloid derived suppressor cells (MDSCs) among other immune cells. Their role in the TME depends on a nature and concentration of factors, such as cytokines, reactive oxygen species (ROS), growth factors, and most importantly, diffusible metabolites (i.e., lactate). Further, the amounts of available nutrients and oxygen as well as activity of microbiota may influence metabolic pathways in the TME. The roles of metabolites in regulating of the interaction between immune and cancer cell are highlighted in this review. Targeting metabolic reprogramming or signaling pathways controlling cell metabolism in the TME might be a potential strategy for anti-cancer therapy alone or in combination with current immunotherapies.

Sesame seeds essential oil and Sesamol modulate the pro-inflammatory function of macrophages and dendritic cells and promote Th2 response.

Background: Herbal medicine is becoming progressively accepted treatment for management of different diseases worldwide. Recognition of the active ingredients and mechanisms of herbal medicine against the immune system and related anomalies is highly favorable. This experimental study aimed to investigate the effects of Sesame (Sesamum indicum L.) essential oil and sesamol as effective components on mouse splenocytes subsets, macrophages and dendritic cells (DCs). Methods: Effective components of sesame were extracted and used to treat splenocytes, PHA (5µg/ml) and LPS (10 µg/ml) stimulated splenocytes, macrophages and DCs in different concentration (0.01-100 µg/ml). The cell proliferation/viability was measured using the MTT assay and nitrite levels were measured by the diazotization method. Moreover, TNF-α and IL-1ß cytokines concentration were assayed by ELISA. Treated DCs also analysed for maturation marker levels and cytokine production. Results: Analysis of the results indicated that sesame components suppress PHA-stimulated splenocytes with no effect on LPS-stimulated subsets. Furthermore, the sesame ingredients reduced the release of IFN-γ and increased secretion of IL-4 from lymphocytes. Macrophages viability was not affected and production of NO, TNF-α, and IL-1ß were inhibited using sesame essential oil and sesamol. DCs phenotype skewed to immature and release of TNF-α and IL-1ß were abrogated form DCs. Conclusion: These results indicate that sesame essential oil and its effective component as sesamol may capable of suppressing the response of cellular immunity with the domination of Th2 responses and also could modulate macrophages and the dendritic cells proinflammatory functions.

Eliciting Th1 Immune Response Using Casein- (Alpha S1) loaded Dendritic Cells.

Allergen-specific immunotherapy (AIT) has been recently considered as an alternative approach to ameliorate the symptoms of allergen exposure and improvement the patients' quality of life. Dendritic cells (DC) in the forms of tolerogenic or Th1-induced cells have been investigated in several studies as one of the promising approaches of AIT in allergic diseases. The aim of this study was to evaluate the potency of casein-loaded DCs in eliciting the Th1 immune responses in Balb/c mice as a potential therapeutic approach in allergic condition. Immature bone marrow-derived DCs were loaded with casein (protein or mRNA) or green fluorescent protein (GFP) mRNA. DCs were evaluated based on the expression of specific markers and production of proinflammatory cytokines.  Expression of DC markers in all groups was significantly higher than immature DCs, but lower than LPS-activated DCs. Despite an increase in TNF-α and IL-12, IL-6 was decreased in casein-DC treatments. Caseinloaded DCs could induce proliferation in lymphocytes and stimulate them to produce higher amounts of IFN-γ and in some extent IL-10 and TGF-ß, while they could not stimulate IL-4 secretion. Casein-loaded DCs could partially elicit the Th1 responses; this would be a promising approach to use them as an allergic protective way for applying immune cell therapy in cow's milk allergy.

TGF-ß1 Transduced Mesenchymal Stem Cells Have Profound Modulatory Effects on DCs and T Cells.

BACKGROUND: Mesenchymal stem cells (MSCs) and transforming growth factor-ß1 (TGF-ß1) molecules are well known for their immunomodulatory properties and their function in tissue regeneration and remodeling. OBJECTIVES: To evaluate the interaction of TGF-ß1 engineered MSCs with T cells and dendritic cells (DCs) and their modulatory effect on the immune response. METHODS: MSCs and DCs were generated from bone marrow of Balb/c mice and T cells were generated from mice lymph nodes. TGF-ß1 expressing lentiviruses were used for MSCs transduction, and then these engineered MSCs were co-cultured with T cells and DCs. T cells proliferation and cytokines release and also DCs maturation, TNF-α release, and stimulation of allogeneic T cells were evaluated. RESULTS: T cells proliferation and IFN-γ release were suppressed by TGF-ß1/MSCs while IL-4 secretion was enhanced. Co-cultured DCs with TGF-ß1/MSCs showed reduced expression of CD40, CD86, and MHC II and also lower level of TNF-α secretion. Co-cultured DCs could also induce lower levels of allogeneic T cells proliferation and IFN-γ release in comparison to control DCs. CONCLUSION: Engineered TGF-ß1/MSC cells showed collaborative immune suppressive functions between TGF-ß1 and MSCs to modulate T cells and DCs immune responses. We therefore suggest that TGF-ß1/MSC cells could provide a promising tool for treatment of clinical conditions such as organ transplantation, GVHD, and autoimmune disorders.

TGF-ß engineered mesenchymal stem cells (TGF-ß/MSCs) for treatment of Type 1 diabetes (T1D) mice model.

OBJECTIVE: Mesenchymal stem cells (MSCs) are advantageous candidates for cell therapy of Type 1 diabetes (T1D). Considering immunomodulatory effect of MSC, in this study, we engineered MSCs with TGF-ß gene to increase MSC potency for T1D therapy in mouse model. MATERIALS AND METHODS: Two plans were designed for prevention and treatment of diabetes, respectively. In both of them, MSCs were injected i.v. and then, the diabetes features including serum insulin, blood glucose, glucose tolerance, splenocytes proliferation, and IL-4/IFN-γ production were evaluated. RESULTS: TGF-ß/MSCs treatment program resulted in the restoration of serum glucose after 3weeks, while prevention program could delay diabetes progression for two weeks. TGF-ß/MSCs treatment elevated the levels of serum insulin and Th2 cytokine shift on 5th week after start of treatment. TGF-ß/MSCs (and MSCs alone) could also diminish body weight and enhance mice survival comparing to untreated diabetic mice. CONCLUSION: Engineered TGF-ß/MSCs could restore some T1D features, including the regulation of adverse immune responses and could be potent tools for cell therapy of T1D comparing MSCs alone.

Effects of 3-dimensional culture conditions (collagen-chitosan nano-scaffolds) on maturation of dendritic cells and their capacity to interact with T-lymphocytes.

In the body, there is a natural three-dimensional (3D) microenvironment in which immune cells, including dendritic cells (DC), play their functions. This study evaluated the impact of using collagen-chitosan 3D nano-scaffolds in comparisons to routine 2D culture plates on DC phenotype and functions. Bone marrow-derived DC were cultured on scaffolds and plates and then stimulated with lipopolysaccharide (LPS) or chitosan-based nanoparticles (NP) for 24 h. Thereafter, DC viability, expression of maturation markers and levels of cytokines secretion were evaluated. In another set of studies, the DC were co-cultured with allogenic T-lymphocytes in both the 2D and 3D systems and effects on DC-induction of T-lymphocyte proliferation and cytokine release were analyzed. The results indicated that CD40, CD86 and MHC II marker expression and interleukin (IL)-12, IL-6 and tumor necrosis factor (TNF)-α secretion by DC were enhanced in 3D cultures in comparison to by cells maintained in the 2D states. The data also showed that DNA/chitosan NP activated DC more than LPS in the 3D system. T-Lymphocyte proliferation was induced to a greater extent by DNA/NP-treated DC when both cell types were maintained on the scaffolds. Interestingly, while DC induction of T-lymphocyte interferon (IFN)-γ and IL-4 release was enhanced in the 3D system (relative to controls), there was a suppression of transforming growth factor (TGF)-ß production; effects on IL-10 secretion were variable. The results here suggested that collagen-chitosan scaffolds could provide a pro-inflammatory and activator environment to perform studies to analyze effects of exogenous agents on the induction of DC maturation, NP uptake and/or cytokines release, as well as for the ability of these cells to potentially interact with other immune system cells in vitro.

PDL-1/PDL-2 blockade in mice dendritic cells by RNAi techniques to induce antitumor immunity.

AIM: We provided potent dendritic cells (DCs) for induction of stronger antitumor immune responses. MATERIALS & METHODS: Using siRNA and shRNA systems, PDL-1 and PDL-2 were knocked down and then DC in vitro and in vivo properties were evaluated. RESULTS: Mild suppression of PDL-1/PDL-2 molecules was accompanied by appropriate expression of DCs co-stimulatory molecules and release of proinflammatory cytokines. In vitro T-cell engagement induced the proliferation and secretion of Th1 cytokines. Injection of DCs to a 4T1 mice model induced intratumor CD8(+) infiltrating lymphocytes, splenocytes expansion, Th1 cytokine profile shift, and a mild drift to tumor growth inhibition and mice survival. CONCLUSION: Manipulated DCs induced significant antitumor immunity, but this subject needs further evaluation in different animals.