Dataset Information

Simplified Approaches for the Production of Monocyte-Derived Dendritic Cells and Study of Antigen Presentation in Bovine.

ABSTRACT: Dendritic cells are sentinels of the immune system responsible for the initiation of adaptive immune mechanisms. In that respect, the study of these cells is essential for a full understanding of host response to infectious agents and vaccines. In ruminants, the large blood volume facilitates the isolation of abundant monocytes and their derivation to other antigen-presenting cells such as dendritic cells and macrophages. However, the available protocols for the production of bovine monocyte-derived dendritic cells (moDCs) rely mostly on time-consuming and costly techniques such as density gradient centrifugation and magnetic sorting of cells. In this study, we describe a simplified protocol for the production of bovine moDC using conventional and serum-free media. We also employ moDC produced by this approach to carry out a flow cytometry-based antigen presentation assay adapted to blood fresh or frozen cells. The experimental strategies described here might enable the setup of studies involving a large number of individuals, requiring a large number of dendritic cells, or relying on the utilization of cryopreserved blood cells. These simplified protocols might contribute to the elucidation of cell-mediated immune responses in bovine.

SUBMITTER: Cunha P

PROVIDER: S-EPMC9223769 | biostudies-literature | 2022

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Simplified Approaches for the Production of Monocyte-Derived Dendritic Cells and Study of Antigen Presentation in Bovine.

Cunha Patricia P Gilbert Florence B FB Bodin Jennifer J Godry Lise L Germon Pierre P Holbert Sebastien S Martins Rodrigo Prado RP

Frontiers in veterinary science 20220609

Dendritic cells are sentinels of the immune system responsible for the initiation of adaptive immune mechanisms. In that respect, the study of these cells is essential for a full understanding of host response to infectious agents and vaccines. In ruminants, the large blood volume facilitates the isolation of abundant monocytes and their derivation to other antigen-presenting cells such as dendritic cells and macrophages. However, the available protocols for the production of bovine monocyte-der ...[more]

PMID: 35754538

Similar Datasets

Project description:UnlabelledMonocyte-derived dendritic cells (MDDC) stimulate CD8 cytotoxic T lymphocytes (CTL) by presenting endogenous and exogenous viral peptides via major histocompatibility complex class I (MHC-I) molecules. MDDC are poorly susceptible to HIV-1, in part due to the presence of SAMHD1, a cellular enzyme that depletes intracellular deoxynucleoside triphosphates (dNTPs) and degrades viral RNA. Vpx, an HIV-2/SIVsm protein absent from HIV-1, antagonizes SAMHD1 by inducing its degradation. The impact of SAMHD1 on the adaptive cellular immune response remains poorly characterized. Here, we asked whether SAMHD1 modulates MHC-I-restricted HIV-1 antigen presentation. Untreated MDDC or MDDC pretreated with Vpx were exposed to HIV-1, and antigen presentation was examined by monitoring the activation of an HIV-1 Gag-specific CTL clone. SAMHD1 depletion strongly enhanced productive infection of MDDC as well as endogenous HIV-1 antigen presentation. Time-lapse microscopy analysis demonstrated that in the absence of SAMHD1, the CTL rapidly killed infected MDDC. We also report that various transmitted/founder (T/F) HIV-1 strains poorly infected MDDC and, as a consequence, did not stimulate CTL. Vesicular stomatitis virus glycoprotein (VSV-G) pseudotyping of T/F alleviated a block in viral entry and induced antigen presentation only in the absence of SAMHD1. Furthermore, by using another CTL clone that mostly recognizes incoming HIV-1 antigens, we demonstrate that SAMHD1 does not influence exogenous viral antigen presentation. Altogether, our results demonstrate that the antiviral activity of SAMHD1 impacts antigen presentation by DC, highlighting the link that exists between restriction factors and adaptive immune responses.ImportanceUpon viral infection, DC may present antigens derived from incoming viral material in the absence of productive infection of DC or from newly synthesized viral proteins. In the case of HIV, productive infection of DC is blocked at an early postentry step. This is due to the presence of SAMHD1, a cellular enzyme that depletes intracellular levels of dNTPs and inhibits viral reverse transcription. We show that the depletion of SAMHD1 in DCs strongly stimulates the presentation of viral antigens derived from newly produced viral proteins, leading to the activation of HIV-1-specific cytotoxic T lymphocytes (CTL). We further show in real time that the enhanced activation of CTL leads to killing of infected DCs. Our results indicate that the antiviral activity of SAMHD1 not only impacts HIV replication but also impacts antigen presentation by DC. They highlight the link that exists between restriction factors and adaptive immune responses.

Project description:Advances in fundamental and applied immunology research often originate from pilot studies utilizing animal models. While cattle represent an ideal model for disease pathogenesis and vaccinology research for a number of human disease, optimized bovine culture models have yet to be fully established. Monocyte-derived dendritic cells (MoDC) are critical in activating adaptive immunity and are an attractive subset for experimental and clinical applications. The use of serum-supplemented culture medium in this ex vivo approach is undesirable as serum contains unknown quantities of immune-modulating components and may induce unwanted immune responses if not autologous. Here, we describe a standardized protocol for generating bovine MoDC in serum-free medium (AIM-V) and detail the MoDC phenotype, cytokine profile, and metabolic signature achieved using this culture methodology. MoDC generated from adult, barren cattle were used for a series of experiments that evaluated the following culture conditions: medium type, method of monocyte enrichment, culture duration, and concentration of differentiation additives. Viability and yield were assessed using flow cytometric propidium iodide staining and manual hemocytometer counting, respectively. MoDC phenotype and T cell activation and proliferation were assessed by flow cytometric analysis of surface markers (MHC class II, CD86, CD14, and CD205), and CD25 and CFSE respectively. Cytokine secretion was quantified using a multiplex bovine cytokine panel (IL-1α, IL-1β, IL-8, IL-10, IL-17A, IFN-γ, MIP-1α, TNF-α, and IL-4). Changes in cell metabolism following stimulation were analyzed using an Extracellular Flux (XFe96) Seahorse Analyzer. Data were analyzed using paired t-tests and repeated measures ANOVA. Immature MoDC generated in serum-free medium using magnetic-activated cell sorting with plate adhesion to enrich monocytes and cultured for 4 days have the following phenotypic profile: MHC class II+++, CD86+, CD205++, and CD14-. These MoDC can be matured with PMA and ionomycin as noted by increased CD86 and CD40 expression, increased cytokine secretion (IL-1α, IL-10, MIP-1α, and IL-17A), a metabolic switch to aerobic glycolysis, and induction of T cell activation and proliferation following maturation. Cultivation of bovine MoDC utilizing our well-defined culture protocol offers a serum-free approach to mechanistically investigate mechanisms of diseases and the safety and efficacy of novel therapeutics for both humans and cattle alike.

Dataset Information

Simplified Approaches for the Production of Monocyte-Derived Dendritic Cells and Study of Antigen Presentation in Bovine.

Publications

Simplified Approaches for the Production of Monocyte-Derived Dendritic Cells and Study of Antigen Presentation in Bovine.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets