Leukemic cells exert immunosuppressive effects that interfere with dendritic cell (DC) function and hamper effective antileukemic immune responses. Here, we sought to enhance the immunogenicity of ...leukemic cells by loading them with the double-stranded (ds) RNA Toll-like receptor 3 (TLR3) ligand polyriboinosinic polyribocytidylic acid (poly(I:C)), mimicking viral infection of the tumor cells. Given the responsiveness of DC to TLR ligands, we hypothesized that the uptake of poly(I:C)-loaded leukemic cells by immature DC (iDC) would lead to DC activation. Primary acute myeloid leukemia (AML) cells and AML cell lines markedly responded to poly(I:C) electroporation by apoptosis, upregulation of TLR3 expression, enhanced expression of major histocompatibility complex (MHC) and costimulatory molecules and by production of type I interferons (IFN). Upon phagocytosis of poly(I:C)-electroporated AML cells, DC maturation and activation were induced as judged by an increased expression of MHC and costimulatory molecules, production of proinflammatory cytokines and an increase of T helper 1 (T(H)1)-polarizing capacity. These immune effects were suboptimal when AML cells were passively pulsed with poly(I:C), indicating the superiority of poly(I:C) transfection over pulsing. Our results demonstrate that poly(I:C) electroporation is a promising strategy to increase the immunogenicity of AML cells and to convert iDC into activated mature DC following the phagocytosis of AML cells.
Background
Intestinal dendritic cells (DCs) maintain immune homeostasis, only initiating an active immune response against invading pathogens. However, little information is available on the reaction ...of mononuclear phagocytes (MNP) to intestinal trematode infection, a reaction equally important in helminth‐based therapies. The CD11c+ CX3CR1+ F4/80− DCs in the ileal lamina propria (LP) of the mouse were proven to migrate to the mesenteric lymph nodes (MLNs). We analyzed all MNP subsets present in the mouse LP and MLNs, under steady‐state conditions and during acute Schistosoma mansoni‐induced inflammation. Furthermore, we studied the uptake of schistosomal antigens by MNP in vivo in the LP and MLNs.
Methods
Using a combination of immunohistochemistry and multiparametric flow cytometry, we investigated distributional changes of the MNP during acute intestinal schistosomiasis. Next, S. mansoni‐derived products, i.e., S. mansoni soluble worm proteins (SmSWP) and S. mansoni soluble egg antigens (SmSEA) were intraperitoneally injected into CX3CR1+/GFP C57BL/6 mice and antigen uptake was analyzed using confocal microscopy.
Key Results
The CD11c+ CX3CR1+ F4/80− DCs significantly increased during intestinal schistosomiasis in the LP and MLNs. Only CX3CR1‐expressing DC and MФ subsets, but not other LP DCs, are involved in both SmSWP and SmSEA antigen uptake and processing.
Conclusions & inferences
The significant upregulation of CD11c+ CX3CR1+ F4/80− DCs during intestinal schistosomiasis and the restriction of phagocytosis of parasitic antigens to CX3CR1‐expresssing MNP indicate a crucial role for this immune cell niche in response to trematodiasis. These findings add insight into the functional specialization of LP immune cells and add to the understanding of cellular mechanisms behind helminth‐based therapies.
The significant upregulation of CD11c+ CX3CR1+ F4/80− DCs during intestinal schistosomiasis and the restriction of phagocytosis of parasitic antigens to CX3CR1‐expressing MNP in the mouse intestine indicate a crucial role for this immune cell niche in response to trematodiasis. These findings add insight into the functional specialization of LP immune cells and add to the understanding of cellular mechanisms behind helminth‐based therapies.
To date, the number of immunotherapy vaccines in clinical trials increases exponentially. To evaluate the efficacy of these clinical vaccination trials, we fervently need cellular immunomonitoring ...tools. In this study, we present a newly developed short-time assay which allows direct ex vivo analysis of multi-epitope antigen-specific T cell immune responses. This novel method is based on mRNA electroporation of isolated peripheral blood mononuclear cells (PBMC).
Fresh and cryopreserved PBMC of both healthy volunteers as well as of allogeneic stem cell transplanted patients enrolled in a cytomegalovirus (CMV) dendritic cell vaccination trial were electroporated with CMV pp65-encoding mRNA. Using a direct IFN-γ EliSPOT and intracellular cytokine flow cytometry we detected significantly higher numbers of CMV pp65-specific IFN-γ-secreting T cells as compared to the assay with non-treated PBMC and as compared to PBMC electroporated with mRNA coding for an irrelevant protein.
Compared to conventional methods to evaluate T cell-mediated immune responses, this method is time-saving and less labor-intensive because it obviates the need for in vitro cultured antigen-presenting cells and because an overnight incubation is sufficient for activation of T cells. Moreover, the use of CMV pp65-encoding mRNA will broaden the immune response because it covers every epitope with potential relevance. This is a major advantage compared to the recognition of a single epitope covered by a HLA-restricted peptides.
In summary, we developed a highly efficient mRNA electroporation protocol for fresh and cryopreserved PBMC. This novel method is a rapid and elegant tool and will be convenient for monitoring the cellular immune status of patients in a clinical vaccination setting.
Adaptation of land use to the potentialities and constraints of local agroecologies is a key principle of sustainable land management. Farmers and land resource professionals assess the options that ...optimise the productivity and sustainable land use through different knowledge systems. Both systems have advantages and drawbacks. Through a case study in a village of northwestern Syria, an approach was developed to integrate the knowledge of both farmers and land resource experts in order to promote adoption of new land use systems. This was done by comparing a farmer-led land suitability assessment (FLSA) with the results of an expert-led land suitability assessment (ELSA) so as to evaluate respective comparative advantages and complementarities. The results of FLSA and ELSA were integrated in a geographical information system (GIS). The farmers compared the results of FLSA and ELSA and their input ELSA was upgraded to suit local circumstances.
Some striking differences came out between FLSA and ELSA, which could be explained by a participatory land evaluation. The farmers’ knowledge provided a better understanding of the impact of microclimatic variations on crop productivity. This is an important bonus of the participatory approach because detailed climatic data for long periods are rarely available in most rural communities. The FLSA procedure explained adequately the overriding weight of socio-economic constraints over biophysical opportunities. A constraint in the participatory approach is that useful and interesting indigenous knowledge is often scarce. GIS was instrumental in the correlation of indigenous and expert land units and in the farmers’ validation of land suitability. The benefits of this approach to the researchers were clear. The farmers on the other hand highly appreciated the improved communication with the scientists. The better interaction with the farmers will eventually pay off when it comes to adoption of improved management recommendations.
Dendritic cells (DC), professional antigen‐presenting cells of the immune system, exert important functions both in induction of T cell immunity, as well as tolerance. It is well established that the ...main function of immature DC (iDC) in their in vivo steady‐state condition is to maintain peripheral tolerance to self‐antigens and that these iDC mature upon encounter of so‐called danger signals and subsequently promote T cell immunity. Previously, it was believed that T cell unresponsiveness induced after stimulation with iDC is caused by the absence of inflammatory signals in steady‐state in vivo conditions and by the low expression levels of costimulatory molecules on iDC. However, a growing body of evidence now indicates that iDC can also actively maintain peripheral T cell tolerance by the induction and/or stimulation of regulatory T cell populations. Moreover, several reports indicate that traditional DC maturation can no longer be used to distinguish tolerogenic and immunogenic properties of DC. This review will focus on the complementary role of dendritic cells in inducing both tolerance and immunity, and we will discuss the clinical implications for dendritic cell‐based therapies.
Genetically modified dendritic cells (DC) are increasingly used in vitro to activate cytotoxic T lymphocyte (CTL) immune responses. Because T cell activation protocols consist of multiple ...restimulation cycles of peripheral blood lymphocytes with antigen-loaded mature DC, continuous generation of DC is needed throughout the experiment. Therefore, cryopreservation of DC loaded with antigen is a valuable alternative for weekly generation and modification of DC. Recently, we described an antigen loading method for DC based on electroporation of defined tumor antigen mRNA. In this study, we demonstrate that mRNA-electroporated DC can efficiently be prepared for cryopreservation. Using an optimized maturation and freezing protocol after mRNA electroporation, we obtained high transgene-expressing viable mature DC. In addition, we showed that these modified cryopreserved DC retain stimulatory capacity in an influenza model system. Therefore, cryopreservation of mature mRNA-electroporated DC is a useful method for continuous availability of antigen-loaded DC throughout T cell activation experiments.
In an effort to raise protective antiviral immunity, dendritic cell immunotherapy was evaluated in six adults infected with human immunodeficiency virus (HIV)-1 and stable under highly active ...antiretroviral therapy (HAART).
Autologous monocyte-derived dendritic cells electroporated with mRNA encoding Gag and a chimeric Tat-Rev-Nef protein were administered, whereas patients remained on HAART. Feasibility, safety, immunogenicity and antiviral responses were investigated.
Dendritic cell vaccine preparation and administration were successful in all patients and only mild adverse events were seen. There was a significant increase post-dendritic cell as compared to pre-dendritic cell vaccination in magnitude and breadth of HIV-1-specific interferon (IFN)-γ response, in particular to Gag, and in T-cell proliferation. Breadth of IFN-γ response and T-cell proliferation were both correlated with CD4(+) and CD8(+) polyfunctional T-cell responses. Importantly, dendritic cell vaccination induced or increased the capacity of autologous CD8(+) T cells to inhibit superinfection of CD4(+) T cells with the vaccine-related IIIB virus and some but not all other HIV-1 strains tested. This HIV-1-inhibitory activity, indicative of improved antiviral response, was correlated with magnitude and breadth of Gag-specific IFN-γ response.
Therapeutic immunization with dendritic cells was safe and successful in raising antiviral cellular immune responses, including effector CD8(+) T cells with virus inhibitory activity. The stimulation of those potent immunological and antiviral effects, which have been associated with control of HIV-1, underscores the potential of dendritic cell vaccination in the treatment of HIV-1. The incomplete nature of the response in some patients helped to identify potential targets for future improvement, that is increasing antigenic spectrum and enhancing T-cell response.
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