Dendritic Cells In Mixed Lymphocyte Reaction Research Articles

Amlexanox enhances the antitumor effect of anti-PD-1 antibody

Cancer immunotherapy, especially treatment with monoclonal antibodies (mAbs) that block programmed cell death-1 (PD-1)/programmed cell death-ligand 1 (PD-L1) signaling, has attracted attention as a new therapeutic option for cancer. However, only a limited number of patients have responded to this treatment approach. In this study, we searched for compounds that enhance the efficacy of anti-PD-1 mAb using mixed lymphocyte reaction (MLR), which is a mixed culture system of the two key cells (dendritic and T cells) involved in tumor immunity. We found that amlexanox enhanced production of interferon (IFN)-γ, an indicator of T cell activation, by anti-PD-1 mAb. Amlexanox also induced PD-L1 expression in dendritic cells in MLR, whereas it did not stimulate interleukin-2 production by Jurkat T cells. These results suggest that amlexanox acts on dendritic cells, not T cells, in MLR. Furthermore, it enhanced the antitumor effect of the anti-PD-1 mAb in vivo in a mouse tumor-bearing model. The combination of amlexanox and anti-PD-1 mAb increased the expression of Ifng encoding IFN-γ, IFN-γ-related genes, Cd274 encoding PD-L1, and cytotoxic T cell-related genes in tumors. In conclusion, amlexanox stimulates the antitumor effect of anti-PD-1 mAb by acting on dendritic cells, which in turn activates cytotoxic T cells in tumors.

Efficient generation of highly immunocompetent dendritic cells from peripheral blood of patients with hepatitis C virus-related hepatocellular carcinoma.

Immunotherapy using dendritic cells (DCs) is a promising cancer therapy. The success of this therapy depends on the function of induced DCs. However, there has been no consensus on optimal conditions for DC preparation in vitro for immunotherapy of hepatocellular carcinoma (HCC) patients. To address relevant issues, we evaluated the procedures to induce DCs that efficiently function in hepatitis C virus (HCV)-related HCC. We studied immunological data from 14 HCC patients. The DC preparation and the surface markers were assessed by flow cytometric analysis. Four different additional activation stimuli (Method I, medium alone; Method II, with OK-432; Method III, with IL-1β+IL-6+TNF-α; Method IV, with IL-1β+IL-6+TNF-α+PGE2) were tested and the functions of DCs were confirmed by examination of the ability of phagocytosis, cytokine production and allogeneic mixed lymphocyte reaction (MLR). The numbers of DCs induced and their cytokine production ability were not different between healthy controls and HCC patients. T-cell stimulatory activity of DCs in MLR was significantly lower in HCC patients than in healthy controls. The maturation of DCs with OK-432 boosted production of cytokines and chemokines, such as IL-2, IL-12p70, IFN-γ, TNF-α, IL-13 and MIP1α, and restored T-cell stimulatory activity of DCs in MLR. The clinically approved compound OK-432 is a candidate for highly immunocompetent DC preparation and may be considered as a key drug for immunotherapy of HCV-related HCC patients.

Autoimmunity and thrombophilia in women treated with lymphocyte immunotherapy

able. Knowing that T cells infiltrate human decidua, we investigated the functional communications among DSC, dendritic cells (DC) and T cells, and particularly, the role of decidualized DSC on allogeneically triggered T cells by mature DC in mixed lymphocyte reaction (MLR). Methods: Decidual stromal cells from patients undergoing elective abortionswereharvested and expanded. The cellsweredecidualized in vitrowithmedroxyprogesterone, beta-estradiol and cAMP according to a standard protocol. These DSC were co-cultured with MLR, and proliferation and expression of CD25 (high affinity -chain of IL-2 receptor) and CD45RO (effector T cell isoform) on T cells were analyzed by FACS. Results: In previous reportswe showed thatDSC induce a slight proliferation of allogeneic T cells, which was even weaker when DSC were decidualized. Now, further assessment of the effect of the decidual microenvironment on effector T cells activated in MLR showed that when in a decidualized state, DSC suppress the proliferation rate of T cells to 70%. Furthermore, in contrast to CD4, cytotoxic CD8 T cells revealed a significant reduction inCD25andCD45RO markers suggesting amilder effector phenotype compared with MLR alone. Conclusion: Decidualized DSC can restrict proliferation and modulate the effector function of reactive T cells, and thus undoubtedly synergize with other physiological mechanisms for immune suppression. This study was supported by the projects: DOO250/2008 National Science Fund, Ministry of Education and Science; FP7-REGPOT-2009-1 ReProForce and BG051PO001-3.3.06-0059 the European Social Fund.

Immunosuppressive Drugs Upregulate the Immunoinhibitory Receptor Osteoactivin in Monocyte-Derived Dendritic Cells,

Abstract 3232Osteoactivin, also known as transmembrane glycoprotein NMB (GPNMB) and dendritic cell-associated transmembrane protein (DC-HIL), is a type I transmembrane glycoprotein that is detected abundantly in dendritic cells (DC), but not or only at low levels in monocytes. Its expression on antigen-presenting cells (APC) can inhibit T cell activation by binding the type I transmembrane proteoglycan syndecan-4 (SD-4) on T cells. We here studied the influence of different therapeutically used immunosuppressive agents such as the corticosteroid prednisolone and the calcineurin inhibitors tacrolimus or cyclosporine A on the expression of osteoactivin in human monocyte-derived DC. Therefore, DC were generated from blood monocytes isolated by plastic adherence and exposed to GM-CSF and IL-4. Prednisolone, tacrolimus or cyclosporine A were added to the culture medium at concentrations obtained upon clinical application in patients (cyclosporine A: 1μg/ml, prednisolone: 3.5μg/ml, tacrolimus: 10ng/ml) every second day starting from the first day of culture. Cells were harvested for immunophenotyping and osteoactivin expression analysis by immunostaining, western-blotting and real-time RT-PCR or for functional analyses in mixed lymphocyte reactions (MLR) on day 7 of culture. DC generated in the presence of therapeutic concentrations of the immunosuppressant drugs showed a reduced expression of CD1a. Furthermore, we observed a clear upregulation of osteoactivin on DC that had been treated with the immunosuppressive agents. In line with osteoactivin up-regulation, exposure to the immunosuppressive drugs resulted in reduced stimulatory capacity of the DC in MLR with allogenic T cells, and this could be restored by addition of blocking osteoactivin antibody. In summary, our results demonstrate that upregulation of osteoactivin is critically involved in the inhibition of DC function upon exposure to different classes of immunosuppressive agents. These findings suggest that upregulation of osteoactivin in DC constitutes a ubiquitous mechanism that may underlie inhibition of T cell activation by these drugs. Disclosures:No relevant conflicts of interest to declare.

Effects of simvastatin on the function of dendritic cells in patients with rheumatic arthritis

The present study examined the functional profile of dendritic cells (DCs) in patients with rheumatoid arthritis (RA) and the effects of simvastatin on the function of DCs. A total of 40 patients who was recently diagnosed as having RA were equally assigned to two groups: the routine treatment group (group R) and the routine treatment plus simvastatin group (group R+S). Twenty healthy individuals served as control. The peripheral blood mononuclear cells (PBMCs) were isolated before and 4 weeks after the treatment and then cultured with interleukin-4 (IL-4) and granulocyte-macrophage colony stimulatory factor (GM-CSF) to prepare mature DCs. The expression of co-stimulating factor CD86 on the surface of DCs was assessed by flow cytometry. And the stimulating capacity of DCs was measured by mixed lymphocyte reaction (MLR). The contents of cytokines in culture supernatants of DCs in MLR were detected by ELISA. Blood lipids and high-sensitivity C-reactive protein (hs-CRP) were detected. The relationship between the expression of CD86 and the blood CRP level was also investigated. The results showed that, as compared with the control group, the CD86 expression and the level of cytokines secreted by DCs were significantly increased in RA patients and greater stimulating capacity of DCs in MLR was demonstrated in RA patients. T lymphocytes in MLR secreted higher levels of proinflammatory cytokines (IL-2, IL-17, TNF-α and INF-γ) and lower level of anti-inflammation cytokine (IL-10). The function of DCs was markedly weakened and the level of hs-CRP and low-density lipoprotein was substantially lowered in group R+S in comparison to group R. The CD86 expression was positively correlated with hs-CRP. It was concluded that DCs in RA are highly activated and DC-initiated immune reaction may play an important role in the pathogenesis of RA. Simvastatin administration can significantly inhibit the DCs function and reduce the level of hs-CRP, indicating the suppression on inflammatory reaction may be one of the mechanisms by which simvastatin exerts its effect in treating RA.

Serum-free generation and quantification of functionally active Leukemia-derived DC is possible from malignant blasts in acute myeloid leukemia and myelodysplastic syndromes.

Functional dendritic cells (DC) are professional antigen presenting cells (APC) and can be generated in vitro from leukemic cells from acute myeloid leukemia AML patients, giving rise to APC of leukemic origin presenting leukemic antigens (DC(leu)). We have already shown that DC can be successfully generated from AML and myeloplastic syndromes (MDS) cells in serum-free 'standard' medium (X-vivo + GM-CSF + IL-4 +TNFalpha + FL) in 10-14 days. In this study, we present that DC counts generated from mononuclear cells (MNC) varied between 20% (from 55 MDS samples), 34% (from 100 AML samples) and 25% (from 38 healthy MNC samples) medium. Between 53% and 58% of DC are mature CD83+ DC. DC harvests were highest in monocytoid FAB types (AML-M4/M5, MDS-CMML) and independent from cytogenetic risk groups, demonstrating that DC-based strategies can be applied for patients with all cytogenetic risk groups. Proof of the clonal derivation of DC generated was obtained in five AML and four MDS cases with a combined FISH/immunophenotype analysis (FISH-IPA): The clonal numerical chromosome aberrations of the diseases were regularly codetectable with DC markers; however, not with all clonal cells being convertible to leukemia-derived DC(leu) (on average, 53% of blasts in AML or MDS). To the contrary, not all DC generated carried the clonal aberration (on average, 51% of DC). In 41 AML and 13 MDS cases with a suitable antigen expression, we could confirm FISH-IPA data by Flow cytometry: although DC(leu) are regularly detectable, on average only 57% of blasts in AML and 64% of blasts in MDS were converted to DC(leu). After coculture with DC in mixed lymphocyte reactions (MLR), autologous T cells from AML and MDS patients proliferate and upregulate costimulatory receptors. The specific lysis of leukemic cells by autologous T cells could be demonstrated in three cases with AML in a Fluorolysis assay. In six cases with only few DC(leu) or few vital T cells available after the DC/MLR procedure, no lysis of allogeneic or autologous leukemic cells was seen, pointing to the crucial role of both partners in the lysis process. We conclude: (1) the generation of DC is regularly possible in AML and also in MDS under serum-free conditions. (2) Clonal/leukemia-derived DC(leu) can be regularly generated from MDS and AML-MNC; however, not with all blasts being converted to DC(leu) and not all DC generated carrying leukemic markers. We recommend to select DC(leu) for vaccinations or ex vivo T-cell activations to avoid contaminations with non-converted blasts and non-leukemia-derived DC and to improve the harvest of specific, anti-leukemic T cells. DC and DC-primed T cells could provide a practical strategy for the immunotherapy of AML and MDS.

Generation of Dendritic Cells Using Cell Culture Bags--Description of a Method and Review of Literature

Anticancer immunotherapy using dendritic cell-based vaccines is a strategy aimed at the induction and maintenance of immune responses against cancer cells. Clinical applications of dendritic cells (DCs) require stringent adherence to Good Manufacturing Practice (GMP) methods and rigorous standardization of DC-based vaccine preparation. Recently, closed systems for DC culture have been developed with a goal to minimize the risk of contamination. Here, we compare the yield, immunophenotype, and functional properties of DCs generated in Lifecell X-Fold culture bags and in plastic wells, both from adherence-selected monocytes, and review the current literature on closed systems for DC generation. We found that both the overall yield and the yield of CD83+ cells in cell culture bags was lower than in the standard culture method. No statistically significant differences were observed in the expression of DC immunophenotypic markers. The capability of DCs cultured in bags and in wells to induce the proliferation of allogeneic mononuclear cells were equivalent. The performance of DCs in mixed lymphocyte reaction correlated significantly (p=0.005) with the CD83 expression but not with the CD80, CD86, HLA-DR, CD1a, and CD1c expression. We conclude that the immunophenotype and stimulatory properties of DCs cultured in closed cell culture bags are similar to those generated by conventional method using cell culture wells.

Altered phenotype and function of blood dendritic cells in multiple sclerosis are modulated by IFN-beta and IL-10.

Multiple sclerosis (MS) is assumed to result from autoaggressive T cell-mediated immune responses, in which T helper type 1 (Th1) cells producing cytokines, e.g. IFN-gamma and lymphotoxin promote damage of oligodendrocyte-myelin units. Dendritic cells (DCs) as potent antigen presenting cells initiate and orchestrate immune responses. Whether phenotype and function of DCs with respect to Th1 cell promotion are altered in MS, are not known. This study revealed that blood-derived DCs from MS patients expressed low levels of the costimulatory molecule CD86. In addition, production of IFN-gamma by blood mononuclear cells (MNCs) was strongly enhanced by DCs derived from MS patients. IFN-beta and IL-10 inhibited the costimulatory capacity of DCs in mixed lymphocyte reaction (MLR) and showed additive effects on suppression of IL-12 production by DCs. Correspondingly, DCs pretreated with IFN-beta and IL-10 significantly suppressed IFN-gamma production by MNCs. IFN-beta in vitro also upregulated CD80 and, in particular, CD86 expression on DCs. In vitro, anti-CD80 antibody remarkably increased, while anti-CD86 antibody inhibited DC-induced IL-4 production in MLR. We conclude that DC phenotype and function are altered in MS, implying Th1-biased responses with enhanced capacity to induce Th1 cytokine production. In vitro modification of MS patients' DCs by IFN-beta and IL-10 could represent a novel way of immunomodulation and of possible usefulness for future immunotherapy of MS.

Differentiation and expansion of lentivirus vector-marked dendritic cells derived from human CD34(+) cells.

The in vitro genetic manipulation of dendritic cells (DCs) for the expression of foreign proteins or peptides will assist in the development of immunotherapeutic approaches to treat cancer, immunological disorders, and/or infectious diseases. Reports have shown the expansion and differentiation of CD34(+) progenitor cells into mature DCs. In this article we describe the differentiation and expansion of lentivirus vector-marked DCs from umbilical cord blood, bone marrow, and cytokine-mobilized peripheral blood CD34(+) cells in the presence of GM-CSF, TNF-alpha, SCF, Flt-3, and IL-4. Lentivirus-marked DCs expressed high levels of enhanced green fluorescent protein and the characteristic DC surface markers CD1a, CD83, HLA-DR, and CD80. Transduced DCs activated allogeneic CD3(+) T cells as efficiently as control (nontransduced) DCs in mixed lymphocyte reactions. These results demonstrate the potential utility of lentivirus-transduced DCs in future immunotherapy protocols.

Physiologic doses of urocanic acid do not alter the allostimulatory function or the development of murine dendritic cells in vitro.

Exposure to UVB results in the isomerization of trans-urocanic acid (UCA), localized in the stratum corneum, to cis-UCA. Cis-UCA can mediate at least some of the immunosuppressive effects of UVB, though the mechanism of cis-UCA action remains incompletely defined. Alterations in Langerhans cells, and other dendritic antigen presenting cell populations in the skin, may contribute to the loss of skin immune function following UVB exposure. Hence, this study was designed to investigate whether cis-UCA directly can induce changes in the immunostimulatory capacity of dendritic cells (DC) and the development of DC from precursor cells. Murine DC were generated from C57BL/6 bone marrow (BM) using granulocyte-macrophage colony-stimulating factor (GM-CSF), and were used as stimulator cells in mixed lymphocyte reactions (MLR) using BALB/c lymph node cells (LNC) as responders. The addition of cis- and trans-UCA at concentrations ranging from 0.1-500 micrograms/ml to the MLR did not affect proliferative responses. Cis- or trans-UCA (100 micrograms/ml) was added to GM-CSF stimulated mouse BM cells on day 0, day 3 or day 5 of culture, and the phenotype and allo-stimulatory function of the DC were analysed on day 7. Treatment with cis- or trans-UCA did not affect the numbers or the viability of cells in the BM cultures. In addition, the expression on DC of Iab, CD11c or the costimulatory molecules ICAM-1, B7-1, B7-2 and CD40 was not altered by the addition of cis-UCA to BM cultures. The inability of cis-UCA to alter the development of DC in vitro was confirmed by analysing the functional capacity of DC in MLR. DC generated in the presence of cis-UCA were equally efficient in the induction of allo-stimulation, when compared with control DC. These results suggest that cis-UCA does not exert its immunosuppressive activity through direct effects on DC. Such activity may be independent of DC, or alternatively, cis-UCA may influence DC function indirectly, through the induction of secondary mediators.