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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....
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.......
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 .......
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.......
The authors have nothing to disclose
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....
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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