dendritic cells

The use of cryoablation for colorectal liver metastases (CLM) remains limited and controversial. This study aimed to investigate the antitumor immune response following cryoablation combined with granulocyte-macrophage colony-stimulating factor (GM-CSF) treatment in a CLM mouse model.

A CLM mouse model was established using BALB/c mice. The tumor-bearing mice were randomly divided into Control group, GM-CSF group, cryoablation group, and cryoablation + GM-CSF group. Tumor size, survival time, dendritic cells (DCs) count, serum cytokine levels (IL-4, IFN-γ), and the Th1/Th2 ratio (IFN-γ/IL-4) were compared among the four groups.

The combination of cryoablation and GM-CSF demonstrated synergistic effects, resulting in the smallest tumor lesion, longest mean survival time, and highest DC count on day 21 post-treatment compared to other groups. Both cryoablation alone and combined with GM-CSF significantly increased serum IFN-γ levels and suppressed IL-4 levels on day 21 compared to pre-treatment levels (P<0.05). Notably, the combination of cryoablation and GM-CSF significantly elevated the Th1/Th2 ratio (P<0.05).

Combining cryoablation with GM-CSF treatment holds promise for CLM management. It exhibits increased DC infiltration within the tumor microenvironment, enhanced immune responses, and prolonged survival in tumor-bearing mice.

COVID-19 (2019) clearly demonstrates an imbalanced immune response. Variations in the function and subtypes of dendritic cells (DCs) may have effects on immune responses in COVID-19 patients and contribute to immunopathology.

To assess the phenotype and frequency of Plasmacytoid dendritic cells (pDCs), Conventional DCs (cDCs), and double-positive DCs in COVID-19 patients admitted to the ICU and non-ICU compared to the healthy control group.

The study included 10 healthy individuals and 25 COVID-19 patients. In the second week of their illness, Peripheral blood mononuclear cells (PBMCs) were isolated from the patients and labeled with targeted antibodies for HLA-DR, CD123, and CD11c. The samples were then analyzed using flow cytometry. The COVID-19 patients were divided into two ICU and non-ICU groups and were closely monitored throughout the study.

In comparison to healthy controls, COVID-19 patients exhibited a significantly lower pDCs ratio (P=0.04). Patients were categorized into two groups: (A) the ICU group (n=11; 44%) and (B) the non-ICU group (n=14; 56%). The frequency of pDC was significantly lower in ICU patients than in non-ICU patients (P<0.01). Although not statistically significant, ICU patients had a lower frequency of cDCs and double positive DCs compared to non-ICU patients. Additionally, a significant association between the age of COVID-19 patients and cDC levels was observed (p=0.049).

SARS-CoV-2 can evade attacks from the immune response by reducing the number of DCs and suppressing their function of DCs, ultimately resulting in weakened development of both innate and adaptive immunity.

Dendritic cell-based cancer immunotherapy is considered an innovative and promising approach aimed at enhancing the host's immune response to combat tumors. Additionally, IPI-549 has been identified as a first-line therapeutic option for breast cancer treatment. The objective of this research was to develop and formulate a novel therapeutic supplement by combining dendritic cells with IPI-549 (Eganelisib) for breast cancer treatment. The concurrent administration of dendritic cells and IPI-549 (DC-IPI) was utilized to treat mice and elicit immunological responses triggered by vaccination. Tumor regression and overall survival rates were evaluated across five distinct experimental groups. The administration of tumor cell lysate alongside DC-IPI resulted in a significant reduction in tumor growth and a two- to three-fold increase in the survival duration of treated mice. DC-IPI, whether decorated with mannan or not, elicited stronger responses in terms of delayed-type hypersensitivity, lymphocyte proliferation, and CD107a expression. Moreover, our findings demonstrated a reduction in IL-4 production in the supernatants of splenocyte cultures. A significant reduction in BRCA1 mRNA levels was also observed following treatment with DC-IPI. In conclusion, our results suggest that the DC-IPI antigen delivery system exhibited substantial anti-tumor efficacy in a breast cancer mouse model, representing a potential advancement in immunotherapy for human breast cancer.

Dendritic cells (DCs) are remarkable professional antigen-presenting cells (APCs) that are pivotal in bridging the gap between innate and adaptive immunity (1). Given this unique ability, these cells are a promising target for immunotherapy of various diseases (2). Monocytes serve as a valuable resourcefor generating DCs in vitro. To generate monocyte-derived DCs and investigate morphological changes during the differentiation phenomenon, following the reception of written informed consent, peripheral blood samples were obtained from healthy donors using falcon tubes containing heparin. Peripheral blood mononuclear cells (PBMCs) were isolated using Ficoll solution with a density of 1.077 g/ml and density gradient centrifugation. Monocytes were subsequently extracted from PBMCs through the magnetic-activated cell sorting method (MACS) following the instructions provided with the kit. The culture of monocytes was carried out in a concentration of 1.5 × 106/mLin a 6-well plate using complete culture media (RPMI-1640 supplemented with 15% FBS, 100 μg/mL streptomycin, 100 IU/mL penicillin, and 2 mM L-glutamine), in conjunction with 50 μM 2-mercaptoethanol solution and recombinant human GM-CSF and IL-4 cytokines at concentrations of 40 and 25 ng/mL, respectively. The plate was incubated at 37°C with 5% CO2 and humidification. After three days, half of the culture media was substituted with a new mixture containing GM-CSF and IL-4 cytokines. Immature DC (iDCs) cells were collected on the fifth day. Following this, iDCs were treated with 100 ng/mL of lipopolysaccharide (LPS) and incubated for 24 hours to induce the maturation of iDCs. On the sixth day, mature DCs were harvested. Monocytes and DCs were examined for their morphology, with images captured utilizing an inverted light microscope. In this regard, microscopic analysis revealed morphological alterations between monocytes derived from PBMCs at the initiation of culture and differentiated mature DCs acquired on day 6. Monocytes appeared as round cells with no visible dendrites, in contrast to DCs whichwere distinguishable by their visible dendrites (Figure1).

Rheumatoid arthritis (RA) is a type of autoimmune disease that results in immune disorder and excessive inflammatory response due to a reduction of self-tolerance. Invariant natural killer T (iNKT) cells can effectively alleviate clinical symptoms and hyper-inflammation in RA, but their mechanism of action is not well-defined. This study aims to investigate the mechanism of iNKT cell therapy for RA. We established a DBA/1 mouse model for RA and treated it with specific iNKT cells. A cytometric bead array was used to measure the amounts of cytokines in the serum. Flow cytometry was then employed to identify different subsets of helper T cells (Th), the frequency of conventional dendritic cells (cDC), the expression of CD80, CD86, programmed cell death ligand 1 (PD-L1), and PD-L2 on cDC surfaces, and associated pathway proteins. iNKT cell treatment reduced Th1/Th2 and Th17/ regulatory T (Treg) cell ratios while increasing interleukin-4 (IL-4) and IL-10. It enhanced the generation of immature cDCs, and it upregulated the level of PD-L2 by stimulating the signal transducer and activator of transcription 3 (STAT3) signaling pathway. Meanwhile, it activated the extracellular signal-regulated kinase 1/2 (ERK1/2) pathway and inhibited the nuclear factor kappa B (NF-κB) pathway. According to our findings, iNKT cell treatment increased the expression of phosphates STAT3  in lymph node cDC, causing them to upregulate PD-L2 molecules. While activating the ERK1/2 pathway and inhibiting the NF-κB pathway, tolerogenic cDC was produced, restoring immune homeostasis and correcting excessive inflammation. These results deliver new insights into the treatment of RA by iNKT cells.

Asthma, characterized by persistent inflammation and increased sensitivity of the airway, is the most common chronic condition among children. Novel, safe, and reliable treatment strategies are the focus of current research on pediatric asthma. Amygdalin, mainly present in bitter almonds, has anti-inflammatory and immunoregulatory potential, but its effect on asthma remains uninvestigated. Here, the impact of amygdalin on the thymic stromal lymphopoietin (TSLP)–dendritic cell (DC)–OX40L axis was investigated. A BALB/c mouse model for allergic asthma was established using the ovalbumin-sensitization method. Amygdalin treatment was administered between days 21 and 27 of the protocol. Cell numbers and hematoxylin and eosin (H&E) staining in bronchoalveolar lavage fluid (BALF) were used to observe the impact of amygdalin on airway inflammation. TSLP, IL-4, IL-5, IL-13, and IFN-γ concentrations were determined via Enzyme-linked immunosorbent assay (ELISA). TSLP, GATA-3, and T-bet proteins were measured using western blotting. Cell-surface receptor expression on DCs (MHC II, CD80, and CD86) was assessed via flow cytometry. OX40L mRNA and protein levels were detected using western blotting and qRT-PCR, respectively. Amygdalin treatment attenuated airway inflammation decreased BALF TSLP levels, inhibited DC maturation, restrained TSLP-induced DC surface marker expression (MHCII, CD80, and CD86), and further decreased OX40L levels in activated DCs. This occurred together with decreased Th2 cytokine levels (IL-4, IL-5, and IL-13) and GATA3 expression, whereas Th1 cytokine (IFN-γ) levels and T-bet expression increased. Amygdalin thus regulates the Th1/Th2 balance through the TSLP–DC–OX40L axis to participate in inflammation development in the airways, providing a basis for potential allergic asthma treatments.

Different subtypes of dendritic cells (DCs) can induce different types of immune responses. Our previous study found that Echinococcus granulosus (E. granulosus) antigens (Eg.ferritin, Eg.mMDH and Eg.10) stimulated DC differentiation to different subtypes and produced different immune responses. To further understand whether Eg.ferritin, Eg.mMDH and Eg.10 affect the DC-mediated immune response by promoting the differentiation of monocytes to DCs. Bone marrow-derived monocytes were exposed to three antigens of E. granulosus on days 0, 3, 5, and 7. The percentage of monocyte-derived DCs (moDCs), DCs subsets, and the expression of surface molecules of DCs at different time points in different groups were assessed by flow cytometry. The levels of cytokines of IL-1β, IL-4, IL-6, IL-10, IL-13, IFN-γ, TNF-α, IL-12p70, IL-18, IL-23, and IL-27 in the cell culture supernatant were detected by multi-factorial detection technology. The percentage of moDCs revealed that none of the three antigens blocked monocyte differentiation to DCs. The monocytes of 7-day-old cultures showed increased sensitivity to these antigens. The Eg.ferritin induced more mature DCs, which expressed high levels of MHC II and costimulatory molecules, and secreted Th1 cytokines. Eg10 and Eg.mMDH induced lower degrees of DC maturation, however differentiated DCs were in a semi-mature state due to low expression of MHC II and costimulatory molecules and secretion of higher Th2 and lower Th1 cytokines. Eg.ferritin promotes full maturation of DCs and induces Th1 immune response, whereas Eg.10 and Eg.mMDH induce semi-mature DCs producing higher levels of Th2 cytokines.

The maturation faith of dendritic cells is restrained by the inflammatory environment and cytokines, such as interleukin-6 and its downstream component. Therefore, introducing the suitable antigen to dendritic cells is crucial. However, reducing the severity of the suppressive tumor microenvironment is indispensable. The present study examined the combination therapy of lymphocyte antigen 6 family member E (LY6E) pulsed mature dendritic cells (LPMDCs) and pioglitazone against colorectal cancer (CRC) to elevate the effectiveness of cancer treatment through probable role of pioglitazone on inhibiting IL-6/STAT3 pathway.

Dendritic cells were generated from murine bone marrow and were pulsed with lymphocyte antigen 6 family member E peptide to assess antigen-specific T-cell proliferation and cytotoxicity assay with Annexin/PI. The effect of pioglitazone on interleukin (IL)-6/STAT3 was evaluated in vitro by real-time polymerase chain reaction (PCR). Afterward, the CRC model was established by subcutaneous injection of CT26, mouse colon carcinoma cell line, in female mice. After treatment, tumor, spleen, and lymph nodes samples were removed for histopathological, ELISA, and real-time PCR analysis.

In vitro results revealed the potential of lysate-pulsed dendritic cells in the proliferation of double-positive CD3-8 splenocytes and inducing immunogenic cell death responses, whereas pioglitazone declined the expression of IL-6/STAT3 in colorectal cell lines. In animal models, the recipient of LPMDCs combined with pioglitazone demonstrated high tumor-infiltrating lymphocytes. Elevating the IL-12 and interferon-gamma (IFN-γ) levels and prolonged survival in lysate-pulsed dendritic cell and combination groups were observed.

Pioglitazone could efficiently ameliorate the immunosuppressive feature of the tumor microenvironment, mainly through IL-6. Accordingly, applying this drug combined with LPMDCs provoked substantial CD8 positive responses in tumor-challenged animal models.

The functions of dendritic cells (DCs) are influenced by their intracellular metabolism, in which liver kinase B1 (LKB1) plays an important role. However, due to the difficulty in isolating the DCs, the roles of LKB1 in DC maturation and functions in tumor settings have been poorly characterized. To investigate the roles of LKB1 in DC functions including phagocytosis and presentation of antigens, activation, T cell differentiation, and ultimately tumor eradication. Genetic modification of Lkb1 in the DCs was made by lentiviral transduction, and their impacts on T cell proliferation, differentiation, activity, or B16 melanoma metastasis were examined by flow cytometry, qPCR, or lung tumor nodule counting. LKB1 did not affect antigen uptake and presentation by the DCs, but facilitated the stimulation of T cell proliferation. Interestingly, following T cell activation, Foxp3-expressing regulatory T cells (Treg) were increased (P=0.0267) or decreased (P=0.0195) in mice injected with Lkb1 knockdown DCs or overexpressing DCs, respectively. Further exploration revealed that LKB1 inhibited OX40L (P=0.0385) and CD86 (P=0.0111) expression, and these co-stimulatory molecules enhanced Treg proliferation, and downregulated immune suppressive cytokine IL-10 (P=0.0315). Moreover, we found that the injection of the DCs with limited LKB1 expression before tumor inoculation could reduce their production of granzyme B (P<0.0001) and perforin (P=0.0042) from CD8+T cells, thereby impairing their cytotoxicity and promoting tumor growth. Our data suggest that LKB1 can enhance DC-mediated T cell immunity by restraining Treg development and thereby suppressing tumor growth.

Double-stranded fragmented extracellular DNA is a participant, inducer, and indicator of various processes occurring in the organism. When investigating the properties of extracellular DNA, the question regarding the specificity of exposure to DNA from different sources has always been raised. The aim of this study was to perform comparative assessment of biological properties of double-stranded DNA obtained from the human placenta, porcine placenta and salmon sperm.

The intensity of leukocyte-stimulating effect of different dsDNA was assessed in mice after cyclophosphamide-induced cytoreduction. The stimulatory effect of different dsDNA on maturation and functions of human dendritic cells and the intensity of cytokine production by human whole blood cells was analyzed ex vivo. The oxidation level of the dsDNA was also compared.

Human placental DNA exhibited the strongest leukocyte-stimulating effect. DNA extracted from human and porcine placenta exhibited similar stimulatory action on maturation of dendritic cells, allostimulatory capacity, and ability of dendritic cells to induce generation of cytotoxic CD8+CD107a+ T cells in the mixed leukocyte reaction. DNA extracted from salmon sperm stimulated the maturation of dendritic cells, while having no effect on their allostimulatory capacity. DNA extracted from human and porcine placenta was shown to exhibit a stimulatory effect on cytokine secretion by human whole blood cells. The observed differences between the DNA preparations can be caused by the total methylation level and are not related to differences in oxidation level of DNA molecules.

Human placental DNA exhibited the maximum combination of all biological effects.

Leishmania is a parasite causing leishmaniasis with different clinical manifestations depending on the infectious species in many countries worldwide. Although different studies have been taken place to clear the interaction of the parasite with the immune system, many aspects of immunology of leishmaniasis is remained uncertain.

Bone marrow derived dendritic cells (DCs) were cultured in vitro and divided into different groups (Nottingham Trent University, Nottingham, UK). The groups were separately infected with live or autoclaved L. mexicana or loaded with Soluble Leishmania Antigen (SLA). The expression of major histocompatibility complex class I (MHC-I) molecule was checked and compared on the cultured DCs using flow cytometry.

Infection of L. mexicana caused a significant downregulation in expression of molecules where killed Leishmania or SLA could not induce suppression in expression of these molecules.

L. mexicana infection results in downregulation of MHC-I expression on bone marrow-derived dendritic cells.

Dendritic cells (DCs) are a group of bone marrow-derived cells that play a crucial role in innate and acquired immune responses. Bone marrow-derived dendritic cells (BMDC) are used in many studies, so the efficiency and purity of the differentiated cells are essential. This study aimed to investigate the effect of several parameters, including the age of mice, cell culture medium, and swirling of the culture plate, to increase the efficiency of the induced cells, considering the standard protocols. Bone marrow-derived dendritic cells were induced from both juvenile and adult mice bone marrow cells. Then, the purity of CD11c+ cells was compared between juvenile mice BMDCs and adult mice BMDCs. Cells were cultured in an enriched and non-enriched medium, and some wells were swirled when changing the medium on the 3rd day. Then the effect of enriched medium and swirling before medium replacement were evaluated based on the expression of the CD11c marker. The efficiency of DCs differentiation (CD11c+ cells) was higher when juvenile mouse bone marrow precursors were used compared to adult mice; using the enriched media with supplements and swirling the well before media replacement significantly affected the purity of immature CD11c+ cells. Due to our results, using juvenile mice, an enriched culture medium, and physical removal of granulocyte cells could significantly improve the purity and efficiency of CD11c+ cells. Therefore, considering these three items in the production protocol of these cells can probably reduce the use of lymphocyte-removing antibodies and purification methods.

Vaccination is one of the important approaches in the prevention and control of diseases. Although the capacity to present antigens other than the disease-specific antigen in the traditional vaccine composition provides a potential benefit by increasing its protective efficacy, many components that are not needed for the related disease are also transferred. These components can reduce vaccine activity by lowering immunity against protective antigens. The reasons such as the low effectiveness of traditional vaccines and the high cost of production and time-consuming reasons show that it is necessary to develop a new vaccine method for our world, which is struggling with epidemics almost every year. Among nucleic acids, mRNA has many advantages, such as genomic integration, induction of anti-DNA autoantibodies, and immune tolerance induced by long-term antigen expression. mRNA vaccines have become a therapeutic target for reasons such as efficacy, safety, fast and non-expensive production. The fact that mRNA triggers both humoral and cellular immunity and goes only to the cytoplasm, not to the nucleus, makes it highly efficient. The mRNA must cross the lipid bilayer barrier and entry to the cytoplasm where it is translated into protein. There are two main ways of mRNA vaccine delivery for this: ex vivo loading of mRNA into dendritic cells and direct injection of mRNA with or without a carrier. Studies continue to understand which delivery system is therapeutically more efficient. Preclinical and clinical trials showed that mRNA vaccines trigger a long-lasting and safe immune response.

Mathematical modeling offers the possibility to select the optimal dose of a drug or vaccine. Considerable evidence show that many bacterial components can activate dendritic cells (DCs). Our previous report showed that multiple doses of DCs matured with Listeria monocytogenes led to tumor regression whereas multiple doses of CpG-matured DCs affected tumor reversely. To assess a combined pattern of DC vaccination proposed by a mathematical model for tumor regression. WEHI164 cells were inoculated subcutaneously in the right flank of BALB/c mice. Bone marrow-derived DCs were matured by Listeria monocytogenes and CpG motifs. DCs were injected using specific patterns and doses predicted by mathematical modeling. Effector cell-mediated cytotoxicity, gene expression of T cell-related transcription factors, as well as tumor growth and survival rate, were assessed in different groups. Our study indicated that the proposed mathematical model could simulate the tumor and immune system interaction, and it was verified by decreasing tumor size in the List+CpG group. However, comparing the effect of different treatment modalities on Th1/Treg transcription factor expression or cytotoxic responses revealed no advantage for combined therapy over other treatment modalities. These results suggest that finding new combinations of DC vaccines for the treatment of tumors will be promising in the future. The results of this study support the mathematical modelling for DC vaccine design. However, some parameters in this model must be modified to provide a more optimized therapy approach.

Dendritic cells (DCs) play crucial roles in cellular immunity as the most powerful antigen presenting cells. They have been widely used for antigen delivery in vivo and in vitro. There are different ways to generate DCs and also gene transduction. In this study we introduce some optimization in order to produce high amount of well differentiated murine DCs for potential immunotherapy and vaccine development applications.

Murine bone marrow cells were isolated from male BALB/c mice and the cells were cultured with complete RPMI in presence of the same ratio of IL-4 and GM-CSF. Some changes were made in the medium and the lysis buffer applications to increase the differentiation rate of the cells. Lentiviral virions were applied to transfer the genes of interest to DCs with no pre-maturation steps. CD11c, MHC-II, CD80 and CD86 were assessed by flow cytometry.

The optimized steps led to significant increase in number of the isolated cells. IL-4 usage in a similar dose to GM-CSF led to macrophage formation inhibition. Lentiviral virions resulted in successful gene delivery along with well-maturated DCs.

The introduced optimized steps could be followed in different DC applications by using lentiviral virions to transduce DCs, independent of the pre-maturation steps.

Dendritic cells (DCs) play key roles in regulating the immune response using the specialized function of processing and presenting antigens. Prolactin (PRL), a hormone produced by the pituitary gland, participates in DC maturation and function. The present study was aimed to determine the frequencies of peripheral blood DC subpopulations of myeloid DC (MDC) and plasmacytoid DC (PDC) in hyperprolactinemic (HPRL) women compared to normal healthy volunteers. This study was conducted on 70 women, including 35 HPRL patients and 35 matched healthy controls, whose PRL serum levels were in the normal range (lower than 25 ng/mL). Serum thyroid-stimulating hormone (TSH) levels were measured in both groups as an indicator of normal thyroid function. The electrochemiluminescence immunoassay method was applied to measure the serum levels of TSH and PRL. The frequencies of MDC and PDC in the peripheral blood samples of both groups were determined by flow cytometry. The mean serum PRL levels in the HPRL patients and healthy individuals were 46.41±21.96 and 13.75±11.19, respectively (p<0.0001); however TSH levels in both groups were similar and within the normal range (0.4–4.5 mIU/mL) (p=0.2). The frequencies of both MDC and PDC subpopulations in the peripheral blood of HPRL patients were significantly lower than they were in the healthy controls. However, the ratio of MDCs/PDCs in HPRL patients was not significantly different between the two groups (p=0.8). Our study revealed that an increased level of serum PRL may lead to a reduction in the number of MDC and PDC subpopulations. These results could help clarify the complex relationship between the immune system and the neuroendocrine axis and may be of potential use in understanding the pathogenesis of endocrine and immune disorders.

The amniotic membrane plays an important role in maintaining a healthy pregnancy. The main population cells from amniotic membrane include human amnion epithelial cells (hAECs) which have been shown to possess immunomodulatory properties.

The proximity of hAECs with monocyte leads to the generation of tollerogenic dendritic cells.

hAECs were obtained from normal pregnancy. Peripheral blood monocytes were isolated by anti-CD14 MACS method. Co-cultures of monocytes and hAECs were established in Transwell chambers supplemented with granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4) in the absence and presence of lipopolysaccharide (LPS) to produce immature and mature DCs, respectively. Immunophenotyping of the obtained DCs was done through flow cytometry and the production of cytokines was measured by ELISA. Mixed leukocyte Reaction (MLR) was also performed for the functional assessment of DCs.

Immunophenotyping of [hAECs - Immature DC (iDC)] and [hAECs - iDC] + LPS cells revealed that the expression of CD1a, CD80, CD86, CD40, HLA-DR, and CD83 markers showed no significant difference as compared with the control group (iDCs and mDCs alone). In the [hAECs-iDCs] + LPS cells, the percentage of CD14 cells at the ratio of 1:2.5 showed significant differences compared to the control group. The production of IL-10 and IL-12 showed no significant difference in any of the cultures as compared to the control groups. Also, co-cultured DCs did not inhibit proliferation of lymphocyte.

Our findings show that factors secreted from cultured hAECs are unable to generate of tollerogenic dendritic cells. To achieve a better understanding of other mechanisms more investigations are needed.