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Cancer stem cells (CSCs) are responsible for therapeutic resistance and recurrence in colorectal cancer. Despite advances in immunotherapy, the inability to specifically eradicate CSCs has led to treatment failure. Hence, identification of appropriate antigen sources is a major challenge in designing dendritic cell (DC)-based therapeutic strategies against CSCs. Here, in an in vitro model using the HT-29 colon cancer cell line, we explored the efficacy of DCs loaded with exosomes derived from CSC-enriched colonospheres (CSC enr -EXOs) as an antigen source in activating CSC-specific T-cell responses. HT-29 lysate, HT-29-EXOs and CSC enr lysate were independently assessed as separate antigen sources. Having confirmed CSCs enrichment in spheroids, CSC enr -EXOs were purified and characterized, and their impact on DC maturation was investigated. Finally, the impact of the antigen-pulsed DCs on the proliferation rate and also spheroid destructive capacity of autologous T cells was assessed. CSC enr -EXOs similar to other antigen groups had no suppressive/negative impacts on phenotypic maturation of DCs as judged by the expression level of costimulatory molecules. Notably, similar to CSC enr lysate, CSC enr -EXOs significantly increased the IL-12/IL-10 ratio in supernatants of mature DCs. CSC enr -EXO-loaded DCs effectively promoted T-cell proliferation. Importantly, T cells stimulated with CSC enr -EXOs disrupted spheroids' structure. Thus, CSC enr -EXOs present a novel and promising antigen source that in combination with conventional tumour bulk-derived antigens should be further explored in pre-clinical immunotherapeutic settings for the efficacy in hampering recurrence and metastatic spread., (© 2021 The Authors. Journal of Cellular and Molecular Medicine published by Foundation for Cellular and Molecular Medicine and John Wiley & Sons Ltd.)

Design and application of epitope-based polyvalent vaccines have recently garnered attention as an efficient alternative for conventional vaccines. We previously have reported the in silico design of HHP antigen which encompasses the immune-dominant epitopes of Hepatitis B surface antigen (HBsAg), Hepatitis C core protein (HCVcp) and Poliovirus viral proteins (VPs). It has been shown that the HHP has desirable conformation to expose the epitopes, high antigenicity and other desired physicochemical and immunological properties. To confirm the accuracy of these predictions, the ex-vivo immunogenicity of the HHP was assessed. The HHP gene was chemically synthesized in pET28a and expressed in E. coli (BL21). The expressed protein was purified and its immunological potency was evaluated on dendritic cells (DCs) as antigen presenting cells (APCs). Functional analysis was assessed in co-cultivation of autologous T-cells with matured DCs (mDCs). T-cell activation, proliferation and cytokines secretion were evaluated using flowcytometry and ELISA methods. Our results indicated that the HHP could induce the DC maturation. The mDCs were able to trigger T-cell activation and proliferation. In silico design and ex-vivo confirmation of immunological potential could pave the way to introduce efficient immunogens for further analysis. The ability of HHP in DC maturation and T-cell activation makes it an amenable vaccine candidate for further in-vivo studies., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020. Published by Elsevier Inc.)

Introduction: Menstrual blood stromal stem Cells (MenSCs) have shown promising potential for future clinical settings. Nonetheless, data regarding their interaction with immune cells is still scarce. Here, we investigated whether MenSCs could affect the generation and/or maturation of human blood monocyte-derived dendritic cells (DCs)., Materials and Methods: MenSCs were isolated from menstrual blood of normal women through culture of adherent mononuclear cells. Magnetically-isolated peripheral blood monocytes were differentiated toward immature DCs (iDC) and mature DCs (mDCs) in the presence or absence of MenSCs. Monocyte-derived cells were assessed for the percentage of monocyte-, iDC-, and mDC-specific markers as well as the expression of costimulatory molecules. IL-6 and IL-10 levels were also determined in supernatants of MenSC-monocytes cocultures., Results: Optimal phenotypic differentiation of monocytes into iDCs was inhibited upon coculture with MenSCs. Moreover, higher levels of IL-6 and IL-10 were detected in these settings. Even though addition of MenSCs to iDC cultures could not prevent iDC maturation, coculture of MenSCs with monocytes from the beginning of differentiation process could effectively hinder generation of fully mature DCs., Conclusion: This is the first study to address the inhibitory impact of MenSCs on generation and maturation of DCs. IL-6 and IL-10 could be partly held responsible for this effect. Given the central roles of DCs in regulation of immune responses, these results highlight the importance of further research on the potential modulatory impacts of MenSCs, as rather easily accessible and expandable stem cells, on the immune system-related cells., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Aim: Tolerance to the semi-allogenic fetal graft by the maternal immune system is a medical enigma. Many aspects of immunoregulation at the feto-maternal interface have been clarified, but systemic effects of pregnancy on the immune system are still elusive. The present study was undertaken to determine whether mid-pregnancy mouse serum has an inhibitory effect on dendritic cells (DC) function., Material and Methods: Mid-gestational sera were obtained from allogenic pregnant Balb/c mice (Balb/c × C57BL/6) on days 9-11 of gestation. Splenic DC were purified from Balb/c mice, and treated with mid-pregnancy mouse serum. Antigen pulsed DC were injected into mice palms. After 5 days, draining lymph nodes were removed, cultured in the presence of cognate antigen, and proliferation of responding cells was measured by (3)H-thymidin incorporation. Interleukin (IL)-10 and interferon-gamma (IFN-γ) production by stimulated lymph node antigen-specific cells was also measured in culture supernatants using sandwich ELISA., Results: Treatment of DC with pregnant mouse serum markedly blocked their ability to induce antigen-specific lymphocyte proliferation and IFN-γ and IL-10 production by primed lymph node cells in comparison with non-pregnant serum-treated DC., Conclusion: Pregnant mouse serum has an inhibitory effect on DC capacity to induce antigen-specific proliferation and cytokine secretion by lymph node cells. The suppressive effects of pregnant serum on DC could be considered as one of the mechanisms responsible for the systemic immunomodulation observed during pregnancy., (© 2012 The Authors. Journal of Obstetrics and Gynaecology Research © 2012 Japan Society of Obstetrics and Gynecology.)

Despite recent attempts to take advantage of dendritic cell (DC)-based vaccines for cancer immunotherapy, the results of clinical studies have been disappointing. This is mainly as a result of the diverse immune escape mechanisms used by the tumor together with the insufficient ability of DCs to mount an effective immune response against these mechanisms. In this regard, several approaches have been devised to improve the efficacy of DC-based vaccines. However, the application of each individual approach per se might not be sufficient to overwhelm the diverse immune escape mechanisms. In this review, we focus on current strategies for the ex vivo potentiation of DC-based vaccines, with an emphasis on combinational therapy methods as a promising alternative for tumor immunotherapy., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Garlic is used all over the world for treatment of different diseases. A wide range of biological activities of garlic has been verified in vitro and in vivo. One of major proteins of garlic which has been isolated and purified is the 14 kDa protein. This protein has been shown to have immunomodulatory effects. In this study, the effect of the 14 kDa protein isolated from aged garlic extract (AGE) was investigated on maturation and immunomodulatory activity of dendritic cells (DC). Proteins were purified from AGE by biochemical method; the semi-purified 14 kDa protein was run on gel filtration Sephadex G50 and its purity was checked by SDS-PAGE. DC were isolated from spleen of BALB/c mice by Nycodenz centrifugation and their adhesiveness to plastic dish. 14 kDa protein from AGE was added to overnight culture of DC medium and the expression percentage of CD40, CD86, and MHC-II was evaluated by flowcytometric analysis. Also, proliferation of T-cells was measured by allogenic mixed lymphocyte reaction (MLR) test. The purified 14 kDa protein isolated from AGE increased the expression of CD40 molecule on DC, but it did not influence CD86 and MHCII molecules. Furthermore, no significant differences were noticed in the pulsed-DC with 14 kDa protein and non-pulsed DC on the MLR., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Background: Candida albicans is a member of the normal human microflora. C. albicans cell wall is composed of several protein and carbohydrate components which have been shown to play a crucial role in C. albicans interaction with the host immune system. Major components of C. albican cell wall are carbohydrates such as mannans, beta glucans and chitins, and proteins that partially modulate the host immune responses. Dendritic cells (DC), as the most important antigen-presenting cells of the immune system, play a critical role in inducing immune responses against different pathogens., Objective: We investigated the effect of the cell wall protein fraction (CPF) of C. albicans on DC maturation., Methods: The CPF of C. albicans cells was extracted by a lysis buffer containing sodium dodecyl sulphate, 2-mercaptoethanol and phosphate-buffered saline. The extract was dialyzed and its protein pattern was evaluated by electrophoresis. Dendritic cells were purified from Balb/c mice spleens through a three-step method including mononuclear cell separation, as well as 2-h and overnight cultures. The purified CPF was added at different concentrations to DC. The purity and maturation status of DC were determined by flow cytometry using monoclonal antibodies against CD11c, MHC-II, CD40 and CD86., Results: Treatment of DC with 10 microg/ml of CPF increased the expression of maturation markers including MHC-II, CD86 and CD40 on DC compared to the control group., Conclusion: In this study we used C. albicans CPF with the molecular weight of 40-45 kDa for pulsing and maturation of dendritic cells. Since according to our results CPF significantly increased the expression of maturation markers on DC, we suggest that CPF may act as an efficient immunomodulator, or may be used as a potential adjuvant to boost the host immune system against infections.

To develop an efficient dendritic cell (DC)-based immunotherapy protocol, we examined whether simultaneous pulsing of DCs with a given antigen and a third-party antigen could enhance their antigen presentation capacity. Purified splenic DCs of Balb/c mice were pulsed separately with immunoglobulin G, ovalbumin, conalbumin, P15 peptide of Mycobacterium tuberculosis, and prostate-specific antigen or double combinations of the aforementioned antigens. In some settings, DCs pulsed with 1 antigen were mixed equally with those pulsed with another antigen. Antigen-pulsed DCs were injected into the footpad of syngeneic mice and proliferation of whole, CD4 and CD8 depleted lymph node cells was measured after restimulation with cognate antigen. Antigen-specific production of interferon-gamma (IFNgamma) was tested in culture supernatants. Frequency of responding lymph node cells was determined by IFNgamma enzyme-linked immunosorbent spot assay. Our results showed that copulsing of DCs with 2 unrelated antigens increased the capacity of DCs to induce antigen-specific T-cell proliferation against both antigens up to 16-fold. Injection of 2 populations of DCs each pulsed with a different antigen, increased proliferation of primed T cells significantly as well. Both CD4 and CD8 depleted populations showed vigorous proliferative response in copulsing system. In addition, copulsing of DCs with 2 antigens resulted in higher frequency of antigen-specific responding cells and significantly more IFNgamma production. Our results clearly showed that unrelated peptides and proteins could be used to enhance efficacy of DC-based vaccines and in this system, each antigen served to help the other one, a condition that we termed as "mutual helper effect."

In normal pregnancy, the maternal immune system is directed towards tolerance or suppression in order to prevent rejection of the semi-allogenic fetus. Antigen-presenting cells, especially dendritic cells (DCs), are key cells in initiation and regulation of immune responses. The presence of potent immunostimulatory DCs in the decidual tissue of pregnancy has been demonstrated. The aim of this study was to determine how allostimulatory activity of DCs could be affected during pregnancy. DCs were isolated from spleen of pregnant or non-pregnant Balb/c mice and co-cultured with allogenic T lymphocytes prepared from brachial lymph nodes of C57BL/6 mice. Some cultures of non-pregnant female DCs were treated by 2.5% serum obtained from pregnant mice at early, middle or late gestational periods, and were used in the same mixed lymphocyte reaction (MLR) settings. Cell proliferation was measured by 3H-thymidine incorporation, and cytokine production measured in supernatants of MLR cultures using ELISA. The effect of pregnant mouse serum on expression of DC surface markers was evaluated by flow cytometry. No significant difference was found between stimulatory potential of splenic DCs from pregnant and non-pregnant mice in induction of allogenic T cell proliferative response. Moreover, serum of early or late pregnancy did not have any effect on DC function in comparison with non-pregnant mouse serum, while mid-pregnancy serum significantly inhibited allostimulatory activity of DCs. IFNgamma production in co-culture of DCs treated with pregnant mouse serum was significantly lower than that of the control group; however, no significant difference in IL-10 production was observed. Treatment of DCs with pregnant mouse serum did not influence the percentage of cells expressing MHC-II, CD86, CD8alpha or CD11b. However, a marked reduction of the mean fluorescence intensity of MHC-II was observed. Collectively, our results concerning the diminished capacity of DCs to induce production of Th1 cytokines and allogenic T cell proliferation after treatment with pregnant mouse serum reveal a new way of immunologic tolerance against the semi-allogenic fetus.

Introduction: In normal pregnancy the maternal immune system is directed towards tolerance or suppression in order to prevent the rejection of the semi allogenic fetus. Antigen presenting cells especially dendritic cells (DCs) are the key cells in initiation and regulation of immune responses. The presence of potent immunostimulatory dendritic cells in the decidual tissue of pregnancy has been demonstrated. The aim of this study was to determine how allostimulatory activity of DCs could be affected during pregnancy. Materials and Methods: DCs were isolated from spleen of pregnant or non-pregnant Balb/c mice and co-cultured with allogenic C57BL/6 T lymphocytes prepared by nylon wool method from brachial lymph nodes. Some cultures of non-pregnant female DCs were treated by 2.5% serum obtained from pregnant mice at early, middle or late gestational periods and were used in the same mixed lymphocyte reaction (MLR) settings. Cell proliferation was measured by ³H thymidin incorporation and cytokine production was measured in supernatants of MLR cultures using ELISA method. Effect of pregnant mouse serum on expression of DC surface markers was evaluated by flow cytometry. Results: No significant difference was found between stimulatory potential of splenic DCs from pregnant and non-pregnant mice in induction of allogenic T cell proliferative response. Moreover, serum of early or late pregnancy did not have any effect on DCs function in comparison with non-pregnant mouse serum, while mid-pregnancy serum significantly inhibited allostimulatory activity of DCs. IFNγ production in co-culture of DCs treated with pregnant mouse serum was significantly lower than that of control group, however, no significant difference in IL-10 production was observed. Treatment of DCs with pregnant mouse serum did not influence the percentage of cells expressing MHCII, CD86, CD8α or CD11b. However a marked reduction of the mean fluorescence intensity of MHC-II was observed. Conclusion: Our results concerning the diminished capacity of DCs to induce production of TH1 cytokines and allogenic T cell proliferation after treatment with pregnant mouse serum reveals a new way of immunologic tolerance against semi-allogenic fetus. [ABSTRACT FROM AUTHOR]