T Cells Capture Bacteria by Transinfection from Dendritic Cells

Summary

Here a protocol is presented to measure bacterial capture by CD4⁺ T cells which occurs during antigen presentation via transinfection from pre-infected dendritic cells (DC). We show how to perform the necessary steps: isolation of primary cells, infection of DC, DC/T cell conjugate formation, and measurement of bacterial T cell transfection.

Abstract

Recently, we have shown, contrary to what is described, that CD4⁺ T cells, the paradigm of adaptive immune cells, capture bacteria from infected dendritic cells (DCs) by a process called transinfection. Here, we describe the analysis of the transinfection process, which occurs during the course of antigen presentation. This process was unveiled by using CD4⁺ T cells from transgenic OTII mice, which bear a T cell receptor (TCR) specific for a peptide of ovoalbumin (OVAp), which therefore can form stable immune complexes with infected dendritic cells loaded with this specific OVAp. The dynamics of green fluorescent protein (GFP)-expressing bacteria during DC-T cell transmission can be monitored by live-cell imaging and the quantification of bacterial transinfection can be performed by flow cytometry. In addition, transinfection can be quantified by a more sensitive method based in the use of gentamicin, a non-permeable aminoglycoside antibiotic killing extracellular bacteria but not intracellular ones. This classical method has been used previously in microbiology to study the efficiency of bacterial infections. We hereby explain the protocol of the complete process, from the isolation of the primary cells to the quantification of transinfection.

Introduction

When a pathogen infects its host, there is usually an activation of the innate and adaptive immune responses, necessary for bacterial clearance. Innate immunity is the first line of defense that prevents most infections. Innate immunity distinguish in a precise way elements that are conserved among broad groups of microorganisms (pathogen-associated molecular patterns, PAMPS)¹. The mechanisms of innate immunity include physical barriers such as skin, chemicals barriers (antimicrobial peptides, lysozyme) and the innate leukocytes, which include the phagocytes (macrophages, neutrophils, and dendritic cells), mast cells, eosinophils, basophils, and natural kil....

Protocol

Note: Experimental procedures were approved by the Committee for Research Ethics of the Universidad Autònoma de Madrid and conducted under the supervision of the Universidad Autònoma de Madrid Head of Animal Welfare and Health in accordance with Spanish and European guidelines. Mice were bred in specific pathogen free (SPF) housing and they were euthanized by trained and qualified personnel using carbon dioxide (CO₂) inhalation method.

1. Mouse Bone Marrow-derived DCs Differentiation and Infection

Note: Figure 1 summarizes this first step. All procedures should be carried out i....

Results

Herein we described how to perform murine T cell bacterial transinfection from infected bone marrow derived-DCs and how to measure bacterial transinfection via two different approaches: flow cytometry and gentamicin survival assay. Figure 1 summarizes the procedure to obtain the cells. DCs are generated by incubation of bone marrow cells with GM-CSF for 9 days. Then, DCs are maturated with LPS to increase MHCII on its membrane to load them later on with a specific peptide.......

Discussion

T cells or T lymphocytes are a type of leukocytes that play a central role in cell-mediated immunity and belong to the adaptive immune response²⁶. T cells are refractory to being infected in vitro but some reports indicate that they can be infected in vivo^14,15. The intimate contacts of APC and T cells during immune synapse serve as platforms for exchanging biological material¹³, including some viruses such as HIV¹¹. It was recently shown that contrary to the .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
RPMI	Fisher Scientific	SH3025501
r-GMCSF	Peprotech	315-03
LPS	SIGMA	L2630-10mg
Na Pyruvate	Thermo Scientific	SH3023901
2-ME	Gibco	31350-010
OVAp OTII (323–339)	GenScript
Cell Strainer 70uM	BD	352350
30 uM Syringe Filcons Sterile	BD	340598
AutoMacs Classic	Miltenyi Biotec	130-088-887
Gentamicin	Normon	624601.6
Transwell	Costar	3415
LB	Pronadisa	1231
Agar	Pronadisa	1800
Paraformaldehyde 16%	Electron Microscopy Sciences	15710
Triton X-100
CD8 biot	BD Biosciences	553029
IgM Biot	ImmunoStep	Clone RMM-1
B220 Biot	BD Biosciences	553086
CD19 biot	BD Biosciences	553784
MHC-II Biot (I-A/I-E)	BD Biosciences	553622
CD11b biot	Immunostep	11BB-01mg
CD11c biot	Immunostep	11CB3-01mg
DX5 biot	BD Biosciences	553856
Gr-1 biot	BD Biosciences	553125
CD16/CD32	ImmunoStep	M16PU-05MG
anti Salmonella	ABD Serotec	8209-4006
CD11cPE	BD Biosciences	553802
CD4-APC	Tonbo Biosciences	20-0041-U100
Gr-1 APC	BD Biosciences	553129
MHC-II (I-A/I-E) FITC	BD Biosciences	553623
Alexa-Fluor 647 Goat Anti-Rabbit IgG (H+L) Antibody, highly cross-adsorbed	Invitrogen	A-21245
CMAC (7-amino-4-chloromethylcoumarin)	Life technologies	C2110
BSA	SIGMA	A7030-100G
Streptavidin MicroBeads	Miltenyi Biotec	130-048-101
BD FACSCanto II	BD Biosciences