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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Disclosures
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

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.

Abstract

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.

Introduction

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....

Protocol

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.......

Representative Results

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 .......

Discussion

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.......

Disclosures

The authors have nothing to disclose

Acknowledgements

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....

Materials

NameCompanyCatalog NumberComments
EthanolVWR Chemicals20,824,3655 L
ScissorsFine Science Tools (F.S.T.)14001-12
ForcepsFine Science Tools (F.S.T.)11000-13
Fine ForcepsFine Science Tools (F.S.T.)11253-20
ScalpelFine Science Tools (F.S.T.)10020-00Box of 100 blades
Fetal Bovine SerumSIGMAF7524
Penicillin/streptomycinLONZADE17-602E
Roswell Park Memorial Institute medium (RPMI)GIBCO21875-034
Sterile Petri dishesFalcon353003
25-gauge needleBD Microlance 3300600
1 ml syringeNovicoN15663
15 ml conical tubesFalcon352096
50 ml conical tubesFalcon352098
70 μm nylon web filterBD Falcon352350
Red blood lysis bufferSIGMAR7757100 Ml
EDTASIGMAED2SS-250
Bovine Serum AlbuminSIGMAA7906100 g
Trypan blueSIGMA302643-25G
Culture-platesFalcon353003
4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES)CambrexBE17-737E
Beta-mercaptoethanolMerck8-05740-0250
Sodium pyruvateLONZABE13-115E
L-glutamineLONZABE17-605E
Recombinant murine Granulocyte Macrophage colony-stimulating factor (GM-CSF)Prepotech315-03
lipopolysaccharide (LPS)SIGMA-ALDRICHL2018
96-well-plateCostar3799
v450 anti-mouse CD11b antibodyBD Biosciences560455
PE anti-mouse CD64 antibodyBioLegend139303
PE anti-mouse CD115 antibodyBioLegend135505
FITC anti-mouse CD11c antibodyBioLegend117305
FITC anti-mouse MHCII antibodyBioLegend125507
APC anti-mouse CD86 antibodyBioLegend105011
APC anti-mouse CD80 antibodyBioLegend104713
Flow Cytometry tubesZelian300800-1PS 12X75 5mL
OTII Ovoalbumine peptideInvivoGen323-339
anti-mouse biotinylated CD8α antibodyTonbo Biosciences30-0081-U500
anti-mouse biotinylated IgM antibodyBioLegend406503
anti-mouse biotinylated B220 antibodyTonbo Biosciences30-0452-U500
anti-mouse biotinylated CD19 antibodyTonbo Biosciences30-0193-U500
anti-mouse biotinylated MHCII (I-Ab) antibodyBioLegend115302
anti-mouse biotinylated CD11b antibodyTonbo Biosciences30-0112-U500
anti-mouse biotinylated CD11c antibodyBioLegend117303
anti-mouse biotinylated CD44 antibodyBioLegend103003
anti-mouse biotinylated CD25 antibodyTonbo Biosciences30-0251-U100
anti-mouse biotinylated DX5 antibodyBioLegend108903
streptavidin-coated magnetic microbeadsMACS Miltenyi Biotec130-048-101
Magnetic cell separatorMACS Miltenyi Biotec130-090-976QuadroMACS Separator
Separation columnsMACS Miltenyi Biotec130-042-401
PE anti-mouse CD4 antibodyBioLegend100408
APC anti-mouse CD3 antibodyBioLegend100235
FITC anti-mouse CD8 antibodyTonbo Biosciences35-0081-U025
Cell Violet TracerThermofisherC34557
APC anti-mouse CD25 antibodyTonbo Biosciences20-0251-U100
Alexa647 anti-mouse CD69 antibodyBioLegend104518
PerCPCY5.5 anti-mouse CD45.1 antibodyTonbo Biosciences65-0453
APC anti-mouse CD45.1 antibodyTonbo Biosciences20-0453
PECY7 anti-mouse CD45.1 antibodyTonbo Biosciences60-0453
FITC anti-mouse CD45.2 antibodyTonbo Biosciences35-0454
IonomycinSIGMA-ALDRICHI0634
Phorbol 12 Myristate 13 Acetate (PMA)SIGMAP8139
Brefeldin A (BD GolgiPlug)BD555029
ParaformaldehydeMillipore8-18715-02100
Intracellular permeabilization buffereBioscience00-8333
APC anti-mouse IFNg antibodyTonbo Biosciences20-7311-U100
Fc-block (anti-mouse CD16/CD32)Tonbo Biosciences70-0161-U100
B6.SJL CD45.1 miceThe Jackson Laboratory002014
BD LSRFortessa™ Cell AnalyzerBD Biosciences649225
DAPI SolutionThermofisher62248

References

  1. Zhu, J., Yamane, H., Paul, W. E. Differentiation of effector CD4 T cell populations (*). Annual Review of Immunology. 28, 445-489 (2010).
  2. Bustos-Moran, E., Blas-Rus, N., Martin-Cofreces, N. B., Sanchez-Madrid, F. Orchestrat....

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