Immunomodulatory effects of human umbilical cord wharton's Jelly-Derived mesenchymal stem cells on differentiation, maturation and endocytosis of monocyte-derived dendritic cells

The Wharton's jelly of the umbilical cord is believed to be a source of mesenchymal stem cells (MSCs) which can be therapeutically applied in degenerative diseases. In this study, we investigated the immunomodulatory effect of umbilical cord derivedmesenchymal stem cells (UC-MSCs) and bone marrow-derived-mesenchymal stem cells (BM-MSCs) on differentiation, maturation, and endocytosis of monocyte-derived dendritic cells in a transwell culture system under laboratory conditions. Monocytes were differentiated into immature dendritic cells (iDCs) in the presence of GM-CSF and IL-4 for 6 days and then differentiated into mature dendritic cells (mDCs) in the presence of TNF-for 2 days. In every stage of differentiation, immature and mature dendritic cells were separately cocultured with UC-MSCs and BM-MSCs. The findings showed that UC-MSCs and BM-MSCs inhibited strongly differentiation and maturation of dendritic cells at higher dilution ratios (1:1). The BM-MSCs and UC-MSCs showed more inhibitory effect on CD1a, CD83, CD86 expression, and dendritic cell endocytic activity, respectively. On the other hand, these cells severely up-regulated CD14 marker expression. We concluded that UC-MSCs and BM-MSCs could inhibit differentiation, maturation and endocytosis in monocyte-derived DCs through the secreted factors and free of any cellcell contacts under laboratory conditions. As DCs are believed to be the main antigen presenting cells for naive T cells in triggering immune responses, it would be logical that their inhibitory effect on differentiation, maturation and function can decrease or modulate immune and inflammatory responses. Copyright © Spring 2013, Iran J Allergy Asthma Immunol. All rights reserved

Effects of immediate TNF-α exposure on phenotype and function of dendritic cells derived from cord blood mono nuclear cells

Objective: Effects of immediate TNF-α exposure on phenotype and function of dendritic cells (DCs) derived from cord blood mono nuclear cells Materials and Methods: Umbilical cord blood MNCs were isolated from healthy mothers and were divided into TNF(+( and TNF (-) groups. Both were cultured using SCF, Flt3L, GM-CSF and IL-4. But, three ng/ml of TNF-α was first added in the culture of TNF(+) group. All cells were cultured for 14 days and matured with TNF-α or LPS for additional four days. Light microscopic and flowcytometric analyses were performed on days 0, 7, and 14 of both cultures. MLR and cytokines assays were used to characterize the function of immature and mature DCs. Results: Co-culture of cord blood monocytes and hematopoietic stem cells led to the production of DCs with a characteristic veiled appearance and were consistent with a DC panel of surface markers. However, immediate exposure to TNF-α enhanced the survival of culturing cells in the first week of culture and produced mature DCs with higher maturation markers and IL-12 production. Addition of TNF-α as a maturation marker led to the production of matured DCs and also certain immature and hematopoietic stem cells with higher level of IL-10 production. Conclusion: This study developed a simple, easy and cost effective way to generate DCs from non fractionating mononuclear cells. It seems that primitive DCs and monocytes in the MNCs are contented in the presence of TNF. This will lead different hematopoitic stem cells to myeloid pathway and results in DCs

Highly Efficient Generation of Transgenically Augmented CAR NK Cells Overexpressing CXCR4

Natural killer (NK) cells are a noteworthy lymphocyte subset in cancer adoptive cell therapy. NK cells initiate innate immune responses against infections and malignancies with natural cytotoxicity, which is independent of foreign antigen recognition. Based on these substantive features, genetically modifying NK cells is among the prime goals in immunotherapy but is currently difficult to achieve. Recently, we reported a fully human CAR19 construct (huCAR19) with remarkable function in gene-modified T-cells. Here, we show efficient and stable gene delivery of huCAR19 to primary human NK cells using lentiviral vectors with transduction efficiencies comparable to those achieved with NK cell lines. These huCAR19 NK cells display specific and potent cytotoxic activity against target cells. To improve homing of NK cells to the bone marrow, we augmented huCAR19 NK cells with the human CXCR4 gene, resulting in transgenically augmented CAR NK cells (TRACKs). Compared to conventional CAR NK cells, TRACKs exhibit enhanced migration capacity in response to recombinant SDF-1 or bone marrow stromal cells while retaining functional and cytolytic activity against target cells. Based on these promising findings, TRACKs may become a novel candidate for immunotherapeutic strategies in clinical applications. © Copyright © 2020 Jamali, Hadjati, Madjd, Mirzaei, Thalheimer, Agarwal, Bonig, Ullrich and Hartmann

Dendritic cells loaded with exosomes derived from cancer stem cell-enriched spheroids as a potential immunotherapeutic option

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 (CSCenr-EXOs) as an antigen source in activating CSC-specific T-cell responses. HT-29 lysate, HT-29-EXOs and CSCenr lysate were independently assessed as separate antigen sources. Having confirmed CSCs enrichment in spheroids, CSCenr-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. CSCenr-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 CSCenr lysate, CSCenr-EXOs significantly increased the IL-12/IL-10 ratio in supernatants of mature DCs. CSCenr-EXO-loaded DCs effectively promoted T-cell proliferation. Importantly, T cells stimulated with CSCenr-EXOs disrupted spheroids' structure. Thus, CSCenr-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

Crosstalk between autophagy and metabolic regulation of (CAR) T cells: therapeutic implications

Despite chimeric antigen receptor (CAR) T cell therapy’s extraordinary success in subsets of B-cell lymphoma and leukemia, various barriers restrict its application in solid tumors. This has prompted investigating new approaches for producing CAR T cells with superior therapeutic potential. Emerging insights into the barriers to CAR T cell clinical success indicate that autophagy shapes the immune response via reprogramming cellular metabolism and vice versa. Autophagy, a self-cannibalization process that includes destroying and recycling intracellular components in the lysosome, influences T cell biology, including development, survival, memory formation, and cellular metabolism. In this review, we will emphasize the critical role of autophagy in regulating and rewiring metabolic circuits in CAR T cells, as well as how the metabolic status of CAR T cells and the tumor microenvironment (TME) alter autophagy regulation in CAR T cells to restore functional competence in CAR Ts traversing solid TMEs

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

Abstract 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. Keywords Regulatory T cells Myeloid-derived suppressor cells Foxp3 Melanom

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.. © 2017, Springer-Verlag GmbH Germany, part of Springer Nature

Prolonged Persistence of Chimeric Antigen Receptor (CAR) T Cell in Adoptive Cancer Immunotherapy: Challenges and Ways Forward

CAR T cell qualities, such as persistence and functionality play important roles in determining the outcome of cancer immunotherapy. In spite of full functionality, it has been shown that poor persistence of CAR T cells can limit an effective antitumor immune response. Here, we outline specific strategies that can be employed to overcome intrinsic and extrinsic barriers to CAR T cell persistence. We also offer our viewpoint on how growing use of CAR T cells in various cancers may require modifications in the intrinsic and extrinsic survival signals of CAR T cells. We anticipate these amendments will additionally provide the rationales for generation of more persistent, and thereby, more effective CAR T cell treatments. CAR T cell qualities, such as persistence and functionality play important roles in determining the outcome of cancer immunotherapy. In spite of full functionality, it has been shown that poor persistence of CAR T cells can limit an effective antitumor immune response. Here, we outline specific strategies that can be employed to overcome intrinsic and extrinsic barriers to CAR T cell persistence. We also offer our viewpoint on how growing use of CAR T cells in various cancers may require modifications in the intrinsic and extrinsic survival signals of CAR T cells. We anticipate these amendments will additionally provide the rationales for generation of more persistent, and thereby, more effective CAR T cell treatments

Sodium Butyrate as a Histone Deacetylase Inhibitor Affects Toll-Like Receptor 4 Expression in Colorectal Cancer Cell Lines

We assessed the effect of sodium butyrate (SB) as a histone deacetylase inhibitor (HDACi) on Toll-like receptor 4 (TLR4) gene expression levels, in low TLR4 expressing (HCT116) and high TLR4 expressing (SW480) colorectal cancer cells. The cytotoxic effect of SB was assessed by culturing SW480 and HCT116 cell lines using a broad spectrum of times and concentrations of SB. The MTT assay was done to check the cytotoxic properties of different SB concentrations. Gene expression levels of TLR4 was then evaluated for non-cytotoxic SB concentrations. Morphological analysis and MTT assay confirmed that SB concentrations equal to or less than 5mM were not cytotoxic for both cell lines. At 5mM concentration of SB in SW480 cell line and 1mM concentration of SB in HCT116 cell line, TLR4 gene expression level significantly increased from 24 to 48 hrs and decreased significantly from 48 to 72 hrs with an �early increased and late decreased pattern�. At 1mM concentration of SB in SW480 cell line and 5mM concentration of SB in HCT116 cell line, TLR4 expression had a �gradually increased pattern�. This study focuses on the dose-time-effect of SB in the pathogenesis of colorectal cancer. SB alters the expression level of TLR4 in colorectal cancer cells. This effect may depend on the cell type, treatment duration and SB concentration. The alterations in TLR4 expression may be due to the direct effect of SB on TLR4 and/or the expression changes of in other genes which may indirectly affect the TLR4 expression. Abbreviations: TLR4: Toll-like receptor 4; HDACi: histone deacetylase inhibitor; SB: sodium Butyrate; CRC: colorectal cancer; SCFA: short-chain fatty acid; hrs: hours. © 2019, © 2019 Taylor & Francis Group, LLC

In vitro induction of potent tumor-specific cytotoxic T lymphocytes using TLR agonist-activated AML-DC

Dendritic cells (DCs) are recognized as key regulators of the immune system. Active DC immunization protocols are quickly obtaining interest as an alternative therapeutic approach in acute myeloid leukemia patients. Despite apparent progress in DC-based immunotherapy, some discrepancies were reported in generating potent DCs and their source. In addition to monocytes, DCs can be differentiated from leukemic blasts of acute myeloid leukemia (AML) patients (AML-DC) possessing the ability of stimulating anti-leukemic immune response. In this study, we differentiated peripheral blood blasts of 16 out of 20 AML patients in vitro in the presence of GM-CSF and IL-4 into immature AML-DC. Then, DCs matured using different combinations of Toll-like receptor (TLR) ligands to obtain functional DCs as demonstrated by cell morphology, immunophenotype, and functional activity. Autologous cytotoxic T cell induction of matured DCs was evaluated in four patients and compared with immature counterparts. Our results showed that although the TLR3 agonist (Poly I:C) has a synergistic effect on the TLR4 agonist (lipopolysaccharide, LPS), the addition of the TLR7/8 agonist (R848) is necessary to reinforce the effect of LPS or LPS + POLY(I:C) to produce efficient DCs with the higher level of IL-12 (30 to 90 times). Such DCs activate allogeneic T cells and effectively prime autologous cytotoxic T cells in vitro. In contrast, FSL-1 as a TLR2/6 agonist has a negative effect on LPS + Poly(I:C) and LPS + R848 to produce IL-12. Thus, DCs prepared using a maturation mixture including a TLR7/8 agonist may be used as a potential tool for DC-based immunotherapy purposes in leukemic patients. © 2013 Springer-Verlag France