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Abstract

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Protocol

Representative Results

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Materials

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Immunology and Infection

An In Vivo Mouse Model to Measure Naïve CD4 T Cell Activation, Proliferation and Th1 Differentiation Induced by Bone Marrow-derived Dendritic Cells

Published: August 22nd, 2018

DOI:

10.3791/58118

1LamImSys Lab, Centro Nacional de Investigaciones Cardiovasculares Carlos III (CNIC), 2LamImSys Lab, Instituto de Investigación Sanitaria Hospital 12 de Octubre (imas12), 3CIBER de Enfermedades Cardiovasculares

Here, we present a protocol for the in vivo determination of naïve CD4 T cell (T cell) activation, proliferation, and Th1 differentiation induced by GM-CSF bone marrow (BM)-derived dendritic cells (DCs). In addition, this protocol describes BM and T-cell isolation, DC generation, and DC and T-cell adoptive transfer.

Quantification of naïve CD4 T cell activation, proliferation, and differentiation to T helper 1 (Th1) cells is a useful way to assess the role played by T cells in an immune response. This protocol describes the in vitro differentiation of bone marrow (BM) progenitors to obtain granulocyte macrophage colony-stimulating factor (GM-CSF) derived-dendritic cells (DCs). The protocol also describes the adoptive transfer of ovalbumin peptide (OVAp)-loaded GM-CSF-derived DCs and naïve CD4 T cells from OTII transgenic mice in order to analyze the in vivo activation, proliferation, and Th1 differentiation of the transferred CD4 T cells. This protocol circumvents the limitation of purely in vivo methods imposed by the inability to specifically manipulate or select the studied cell population. Moreover, this protocol allows studies in an in vivo environment, thus avoiding alterations to functional factors that may occur in vitro and including the influence of cell types and other factors only found in intact organs. The protocol is a useful tool for generating changes in DCs and T cells that modify adaptive immune responses, potentially providing important results to understand the origin or development of numerous immune associated diseases.

CD4 T cells and antigen presenting cells (APCs) such as DCs are required mediators of immunity to microbial pathogens1,2. In peripheral lymphoid organs, CD4 T cells are activated upon recognition of specific antigens presented by APCs3,4,5. Activated CD4 T cells proliferate and differentiate into distinct specific effector Th cells that are necessary for the development of a correct adaptive immune response6,7. Control of these processes is critical for produc....

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Experimental procedures were approved by the Fundación Centro Nacional de Investigaciones Cardiovasculares Carlos III (CNIC) and the Comunidad Autónoma de Madrid in accordance with Spanish and European guidelines. Mice were bred in specific pathogen free (SPF) conditions and were euthanized by carbon dioxide (CO2) inhalation.

1. Isolation of Mouse Bone Marrow Cells from Tibias and Femurs

NOTE: The C57BL/6 congenic mouse strain carries the differen.......

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Figure 1 illustrates the steps described in this protocol. Figure 2 illustrates the isolation and culture of mouse BM cells. The addition of GM-CSF and LPS to these cultures allows the in vitro generation and maturation of DCs. Figure 3 illustrates the flow cytometry analysis of the differentiation and maturation of the obtained DCs. OVAp-loaded GM-CSF BM-derived DCs and isolated vital cell .......

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This protocol allows for the characterization of the capacity of BM-derived DCs to modulate the activation, proliferation, and differentiation of naïve CD4 T cells. Moreover, it can also be used to assess the susceptibility of CD4 T cells to modulation by BM-derived DCs. With this protocol, changes in these events can be measured in vivo.

Depending on the hypothesis under investigation, several combinations of T cells and DCs can be used. For example, one can analyze the conseque.......

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We thank Dr. Simon Bartlett for English editing. This study was supported by grants from Instituto de Salud Carlos III (ISCIII) (PI14/00526; PI17/01395; CP11/00145; CPII16/00022), the Miguel Servet Program and Fundación Ramón Areces; with co-funding from the Fondo Europeo de Desarrollo Regional (FEDER). The CNIC is supported by the Ministry of Economy, Industry and Competitiveness (MEIC), and the Pro CNIC Foundation and is a Severo Ochoa Center of Excellence (SEV-2015-0505). RTF is founded by Fundación Ramón Areces and CNIC, VZG by ISCIII, BHF by Instituto de Investigación Sanitaria Hospital 12 de Octubre (imas12) and JMG-G by the ISCIII Migue....

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Name Company Catalog Number Comments
Ethanol VWR Chemicals 20,824,365 5 L
Scissors Fine Science Tools (F.S.T.) 14001-12
Forceps Fine Science Tools (F.S.T.) 11000-13
Fine Forceps Fine Science Tools (F.S.T.) 11253-20
Scalpel Fine Science Tools (F.S.T.) 10020-00 Box of 100 blades
Fetal Bovine Serum SIGMA F7524
Penicillin/streptomycin LONZA DE17-602E
Roswell Park Memorial Institute medium (RPMI) GIBCO 21875-034
Sterile Petri dishes Falcon 353003
25-gauge needle BD Microlance 3 300600
1 ml syringe Novico N15663
15 ml conical tubes Falcon 352096
50 ml conical tubes Falcon 352098
70 μm nylon web filter BD Falcon 352350
Red blood lysis buffer SIGMA R7757 100 Ml
EDTA SIGMA ED2SS-250
Bovine Serum Albumin SIGMA A7906 100 g
Trypan blue SIGMA 302643-25G
Culture-plates Falcon 353003
4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) Cambrex BE17-737E
Beta-mercaptoethanol Merck 8-05740-0250
Sodium pyruvate LONZA BE13-115E
L-glutamine LONZA BE17-605E
Recombinant murine Granulocyte Macrophage colony-stimulating factor (GM-CSF) Prepotech 315-03
lipopolysaccharide (LPS) SIGMA-ALDRICH L2018
96-well-plate Costar 3799
v450 anti-mouse CD11b antibody BD Biosciences 560455
PE anti-mouse CD64 antibody BioLegend 139303
PE anti-mouse CD115 antibody BioLegend 135505
FITC anti-mouse CD11c antibody BioLegend 117305
FITC anti-mouse MHCII antibody BioLegend 125507
APC anti-mouse CD86 antibody BioLegend 105011
APC anti-mouse CD80 antibody BioLegend 104713
Flow Cytometry tubes Zelian 300800-1 PS 12X75 5mL
OTII Ovoalbumine peptide InvivoGen 323-339
anti-mouse biotinylated CD8α antibody Tonbo Biosciences 30-0081-U500
anti-mouse biotinylated IgM antibody BioLegend 406503
anti-mouse biotinylated B220 antibody Tonbo Biosciences 30-0452-U500
anti-mouse biotinylated CD19 antibody Tonbo Biosciences 30-0193-U500
anti-mouse biotinylated MHCII (I-Ab) antibody BioLegend 115302
anti-mouse biotinylated CD11b antibody Tonbo Biosciences 30-0112-U500
anti-mouse biotinylated CD11c antibody BioLegend 117303
anti-mouse biotinylated CD44 antibody BioLegend 103003
anti-mouse biotinylated CD25 antibody Tonbo Biosciences 30-0251-U100
anti-mouse biotinylated DX5 antibody BioLegend 108903
streptavidin-coated magnetic microbeads MACS Miltenyi Biotec 130-048-101
Magnetic cell separator MACS Miltenyi Biotec 130-090-976 QuadroMACS Separator
Separation columns MACS Miltenyi Biotec 130-042-401
PE anti-mouse CD4 antibody BioLegend 100408
APC anti-mouse CD3 antibody BioLegend 100235
FITC anti-mouse CD8 antibody Tonbo Biosciences 35-0081-U025
Cell Violet Tracer Thermofisher C34557
APC anti-mouse CD25 antibody Tonbo Biosciences 20-0251-U100
Alexa647 anti-mouse CD69 antibody BioLegend 104518
PerCPCY5.5 anti-mouse CD45.1 antibody Tonbo Biosciences 65-0453
APC anti-mouse CD45.1 antibody Tonbo Biosciences 20-0453
PECY7 anti-mouse CD45.1 antibody Tonbo Biosciences 60-0453
FITC anti-mouse CD45.2 antibody Tonbo Biosciences 35-0454
Ionomycin SIGMA-ALDRICH I0634
Phorbol 12 Myristate 13 Acetate (PMA) SIGMA P8139
Brefeldin A (BD GolgiPlug) BD 555029
Paraformaldehyde Millipore 8-18715-02100
Intracellular permeabilization buffer eBioscience 00-8333
APC anti-mouse IFNg antibody Tonbo Biosciences 20-7311-U100
Fc-block (anti-mouse CD16/CD32) Tonbo Biosciences 70-0161-U100
B6.SJL CD45.1 mice The Jackson Laboratory 002014
BD LSRFortessa™ Cell Analyzer BD Biosciences 649225
DAPI Solution Thermofisher 62248

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