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

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 pathogens^1,2. In peripheral lymphoid organs, CD4 T cells are activated upon recognition of specific antigens presented by APCs^3,4,5. Activated CD4 T cells proliferate and differentiate into distinct specific effector Th cells that are necessary for the development of a correct adaptive immune response^6,7. 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 differential leukocyte marker Ptprc^a, generally recognized as CD45.1 or Ly5.1, whereas wild-type C57BL strains carry the Ptprc^b

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.......

Materials

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