Reza Mirzaei

Inhibition of HIF-1α enhances anti-tumor effects of dendritic cell-based vaccination in a mouse model of breast cancer

Considerable evidence shows that the tumor microenvironment is an active participant in preventing immunosurveillance and limiting the efficacy of anticancer therapies. Hypoxia is a prominent characteristic of the solid tumor microenvironment. The transcription factor hypoxia-inducible factor-1α (HIF-1α) is an important mediator of hypoxic response of tumor cells that modulates the expression of specific genes involved in tumor immunosuppression. Using a 4T1 breast cancer model, we show that in vivo administration of PX-478, an inhibitor of oxygen-sensitive HIF-1α, led to reduced expression of Foxp3 and VEGF transcript and/or protein, molecules that are directly controlled by HIF-1. When combined with dendritic cell (DC)-based vaccination, HIF-1α inhibition resulted in an augmented cytotoxic T lymphocyte effector function, improved proliferation status of T cells, increased production of inflammatory cytokine IFN-γ, as well as reduced regulatory function of T cells in association with slower tumor growth. Taken together, our findings indicate that the use of HIF-1α inhibition provides an immune adjuvant activity, thereby improves the efficacy of tumor antigen-based DC vaccine.

Evaluation of TLR9 expression on PBMCs and CpG ODN-TLR9 ligation on IFN-α production in SLE patients

Abstract Context: Systemic lupus erythematosus (SLE) is a systemic autoimmune disease characterized by autoreactive antibodies. Recent findings revealed the importance of innate immune responses, especially Toll-like receptors (TLRs) in the pathogenesis of SLE. Objective: In this study, the level of TLR9 expression on peripheral blood mononuclear cells (PBMCs) was analyzed. The levels of produced IFN-α were also measured in supernatant of PBMCs from SLE patients and healthy controls after stimulation with CpG ODN2216 which is a plasmocytoid dendritic cell (pDC)-specific TLR9 ligand. Materials and methods: TLR9 expression was analyzed by real-time polymerase chain reaction (PCR) and flow cytometry in 35 SLE patients and 38 healthy controls and IFN-α concentration was measured in supernatants using enzyme-linked immunosorbent assay (ELISA). Results: The results showed that the TLR9 expression in the mRNA and the protein level was significantly higher in PBMCs from SLE patients. However, IFN-α concentration in patients and controls significantly increased in response to CpG stimulation but this increase was significantly higher in healthy controls compared with SLE patients. Our results do not show any association between taking hydroxychloroquine and reduction in IFN-α production in SLE patients. Discussion and conclusions: Regarding the findings of the study, there is the possibility that TLR9 has played a role in SLE pathogenesis, and consequently it implies that TLRs can be considered to be the therapeutic targets for systemic autoimmunity. We may conclude that PBMCs in patients are functionally impaired in response to TLR ligation via innate response stimulating pathogen-associated molecular patterns (PAMPs).

Lactobacillus crispatus strain SJ-3C-US induces human dendritic cells (DCs) maturation and confers an anti-inflammatory phenotype to DCs.

Lactobacillus crispatus is one of the most predominant species in the healthy vagina microbiota. Nevertheless, the interactions between this commensal bacterium and the immune system are largely unknown. Given the importance of the dendritic cells (DCs) in the regulation of the immunity, this study was performed to elucidate the influence of vaginal isolated L. crispatus SJ‐3C‐US from healthy Iranian women on DCs, either directly by exposure of DCs to ultraviolet‐inactivated (UVI) and heat‐killed (HK) L. crispatus SJ‐3C‐US or indirectly to its cell‐free supernatant (CFS), and the outcomes of immune response. In this work we showed that L. crispatus SJ‐3C‐US induced strong dose‐dependent activation of dendritic cells and production of high levels of IL‐10, whereas IL‐12p70 production was induced at low level in an inverse dose‐dependent manner. This stimulation skewed T cells polarization toward CD4+ CD25+ FOXP3+ Treg cells and production of IL‐10 in a dose‐dependent manner in mixed leukocyte reaction (MLR) test. The mode of bacterial inactivation did not affect the DCs activation pattern, upon encounter with L. crispatus SJ‐3C‐US. Moreover, while DCs stimulated with CFS showed moderate phenotypic maturation and IL‐10 production, it failed to skew T cells polarization toward CD4+ CD25+ FOXP3+ regulatory T cells (Treg) and production of IL‐10. This study showed that L. crispatus SJ‐3C‐US confers an anti‐inflammatory phenotype to DCs through up‐regulation of anti‐inflammatory/regulatory IL‐10 cytokine production and induction of CD4+ CD25+ FOXP3+ T cells at optimal dosage. Our findings suggest that L. crispatus SJ‐3C‐US could be a potent candidate as protective probiotic against human immune‐mediated pathologies, such as chronic inflammation, vaginitis or pelvic inflammatory disease (PID).

Reduced efficacy of multiple doses of CpG-matured dendritic cell tumor vaccine in an experimental model

CpG motifs have been advanced as agents that stimulate the maturation of DCs for immunotherapy. The present study tested the hypothesis that multiple doses of CpG-matured DC vaccine would be efficacious for complete eradication of experimentally-induced tumor. Accordingly, WEHI164 cells were implanted subcutaneously in the flank of BALB/c mice. During DC culture, tumor lysate was added to immature DCs followed by addition of CpG or non-CpG control 4-6h later. A total of three doses of CpG or non-CpG control-matured DCs were injected around tumors. The results showed that multiple doses of CpG-matured DCs led to considerable decrease in cytotoxicity of lymphocytes and significantly increased tumor growth rate compared to a single dose. Further, mice which received three doses of the vaccine also displayed significant FoxP3 in tumor tissue. In conclusion, multiple doses of CpG-matured DCs exhibited decreased anti-tumor immunity in association with increased expression of FoxP3.

Increased efficacy of a dendritic cell–based therapeutic cancer vaccine with adenosine receptor antagonist and CD73 inhibitor:

Dendritic cells are important in initiating immune responses; therefore, a range of dendritic cell–based approaches have been established to induce immune response against cancer cells. However, the presence of immunosuppressive mediators such as adenosine in the tumor microenvironment reduces the efficacy of dendritic cell–based cancer immunotherapy. In this study, we investigated whether blockade of the A2A adenosine receptor with a selective antagonist and a CD73 inhibitor may increase the efficacy of a dendritic cell–based cancer vaccine. According to the findings, this therapeutic combination reduced tumor growth, prolonged survival of tumor-bearing mice, and enhanced specific antitumor immune responses. Thus, we suggest that targeting cancer-derived adenosine improves the outcomes of dendritic cell–based cancer immunotherapy.

Identification of Toxoplasma gondii protein fractions induce immune response against melanoma in mice

Dendritic cells (DCs) play a crucial role in the initiation of adaptive immune responses against tumor cells. We recently found that protein components of Toxoplasma gondii (T. gondii) could mature DCs efficiently. Therefore, in this study, we aimed to find the most effective protein components of T. gondii which are able to mature DCs and consequently instruct immune responses in tumor‐bearing mice. Soluble tachyzoite antigens (STAgs) were fractionated by ammonium sulfate precipitation and subsequently by anion‐exchange HPLC. Immature DCs (iDCs) were treated by these protein fractions and were monitored for IL‐12p70 and IL‐10 production. Moreover, the capacity of mature DCs (mDCs) to induce lymphocyte proliferation was investigated. Ultimately, we analyzed the ability of mDCs in instructing immune responses in tumor‐bearing mice. We found that ammonium sulfate fraction one (A1) matured‐DCs produced higher IL‐12 level and IL‐12/IL‐10 ratio; therefore, this fraction was selected for further fractionation by anion‐exchange HPLC. The results showed that anion‐exchange HPLC fraction 14 (C14) matured‐DCs secrete higher levels of IL‐12p70 and IL‐12p70/IL‐10 ratio. Survival of the mice matured by A1 fraction increased significantly compared to other groups. Moreover, SDS‐PAGE electrophoresis showed that different obtained fractions have distinct proteins based on their size. These results demonstrate that two protein fractions of T. gondii are able to mature DCs more efficient.

Prophylactic DNA vaccine targeting Foxp3+ regulatory T cells depletes myeloid-derived suppressor cells and improves anti-melanoma immune responses in a murine model

Regulatory T cells (Treg) and myeloid-derived suppressor cells (MDSC) are the two important and interactive immunosuppressive components of the tumor microenvironment that hamper anti-tumor immune responses. Therefore, targeting these two populations together might be beneficial for overcoming immune suppression in the tumor microenvironment. We have recently shown that prophylactic Foxp3 DNA/recombinant protein vaccine (Foxp3 vaccine) promotes immunity against Treg in tumor-free conditions. In the present study, we investigated the immune modulatory effects of a prophylactic regimen of the redesigned Foxp3 vaccine in the B16F10 melanoma model. Our results indicate that Foxp3 vaccination continuously reduces Treg population in both the tumor site and the spleen. Surprisingly, Treg reduction was associated with a significant decrease in the frequency of MDSC, both in the spleen and in the tumor environment. Furthermore, Foxp3 vaccination resulted in a significant reduction of arginase-1(Arg-1)-induced nitric oxide synthase (iNOS), reactive oxygen species (ROS) and suppressed MDSC activity. Moreover, this concurrent depletion restored production of inflammatory cytokine IFN-γ and enhanced tumor-specific CTL response, which subsequently resulted in the reduction of tumor growth and the improved survival rate of vaccinated mice. In conclusion, our results revealed that Foxp3 vaccine promotes an immune response against tumor by targeting both Treg and MDSC, which could be exploited as a potential immunotherapy approach.

Study of the tumor microenvironment during breast cancer progression

BackgroundDifferent cells and mediators in the tumor microenvironment play important roles in the progression of breast cancer. The aim of this study was to determine the composition of the microenvironment during tumor progression in order to discover new related biomarkers and potentials for targeted therapy.MethodsIn this study, breast cancer biopsies from four different stages, and control breast biopsies were collected. Then, the mRNA expression of several markers related to different CD4+ T cell subsets including regulatory T cells (Treg), T helper (Th) type 1, 2 and 17 were determined. In addition, we investigated the expression of two inflammatory cytokines (TNF-α and IL-6) and inflammatory mediators including FASL, IDO, SOCS1, VEGF, and CCR7.ResultsThe results showed that the expression of Th1 and Th17 genes was decreased in tumor tissues compared to control tissues. In addition, we found that the gene expression related to these two cell subsets decreased during cancer progression. Moreover, the expression level of TNF-α increased with tumor progression.ConclusionWe conclude that the expression of genes related to immune response and inflammation is different between tumor tissues and control tissues. In addition, this difference was perpetuated through the different stages of cancer.

Identification of proteins derived from Listeria monocytogenes inducing human dendritic cell maturation

Dendritic cells (DCs) are potent antigen-presenting cells (APCs) that can promote antitumor immunity when pulsed with tumor antigens and then matured by stimulatory agents. Despite apparent progress in DC-based cancer immunotherapy, some discrepancies were reported in generating potent DCs. Listeria monocytogenes as an intracellular microorganism is able to effectively activate DCs through engaging pattern-recognition receptors (PRRs). This study aimed to find the most potent components derived from L. monocytogenes inducing DC maturation. The preliminary results demonstrated that the ability of protein components is higher than DNA components to promote DC maturation and activation. Protein lysate fractionation demonstrated that fraction 2 HIC (obtained by hydrophobic interaction chromatography) was able to efficiently mature DCs. F2HIC-matured DCs are able to induce allogeneic CD8+ T cells proliferation better than LPS-matured DCs and induce IFN-γ producing CD8+ T cells. Mass spectrometry results showed that F2HIC contains 109 proteins. Based on the bioinformatics analysis for these 109 proteins, elongation factor Tu (EF-Tu) could be considered as a PRR ligand for stimulating DC maturation.

The Safety Property of β-D-Mannuronic Acid (M2000) as a Novel Immunosuppressive Agent on Differentiation, Maturation and Function of Human Dendritic Cells

The studys background and aim: In this investigation, the safety property of M2000 (β-D-mannuronic acid) on differentiation, maturation and function of dendritic cells, was determined. β-D-mannuronic acid, as a novel immunosuppressive and anti-inflammatory agent, has been tested in various experimental models. In addition, DC-based immunosuppressive drugs can suppress the progression of autoimmune diseases, although, their notable side effects in increasing the risk of infectious diseases and cancers should be considered. MATERIALS AND METHODS The effect of M2000 on differentiation, maturation and function of dendritic cells was examined. To investigate how M2000 affects human dendritic cells (DC) in a defined inflammatory environment, human peripheral blood mononuclear cells (PBMC) were isolated from healthy blood and monocytes were purified using anti-CD14 microbeads. Monocytes were incubated with M2000 in two different doses (6 and 12 J.g/well) along with adding the granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-4 for inducing monocytes to immature DC and lipopolysaccharide for running DC maturation. The differentiation, maturation and function of dendritic cells were examined with flow cytometry and ELISA method. RESULT The results demonstrate that M2000 has no significant side on differentiation, maturation and function of dendritic cells in immature DC and mature DC process in vitro. CONCLUSION Our findings show that β-D-mannuronic acid (m2000) as a safe agent had no adverse effect on differentiation, maturation and function of dendritic cells which might be recommended as a novel immunosuppressive agent with no or fewer side effects in increasing the risk of infectious diseases and cancers.