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 killer cells². These cells identify and eliminate pathogens, either by attacking them through contact or via phagocytosis, which includes pathogen engulfing and killing. This system does not allow lifelong defense, in contrast to adaptive immunity, which confer immunological memory against pathogens. The adaptive immune system is the second line of defense and is able to recognize and react to specific antigens of multiple microbial and non-microbial substances³. The main components of the adaptive immune system are the lymphocytes, which include B and T cells. B cells are involved in the humoral response, secreting antibodies against pathogens or exogenous proteins. However, T cells represent the cell-mediated immunity, modulating the immune response with cytokines secretion or killing pathogen-infected cells⁴.

Antigen presenting cells (APCs) including dendritic cells or macrophages, constituents of the innate immune system, can recognize phagocytose pathogens and process bacterial components into antigens, which are presented at the cell surface by the Major Histocompatibility Complex (MHC)^5-7. After APCs have phagocytized pathogens, they usually migrate to the draining lymph nodes, where they interact with T cells. T lymphocytes can recognize specific peptide-MHC complexes by their T cell receptors. The immune synapse (IS) occurs in the interface between an antigen-loaded APC and a lymphocyte during antigen presentation^8,9. Some bacteria can survive phagocytosis and disseminate systematically within APCs. In this view, infected APCs serve as bacterial reservoirs or "Trojan horses" that facilitate bacterial spread¹⁰. The intimate contact between APCs and lymphocytes that take place during the course of IS formation also function as a platform for exchange of part of membranes, genetic material and exosomes and can be hijacked for some viruses to infect T cells; this process is called transinfection^11-13.

Some pathogenic bacteria (Listeria monocytogenes, Salmonella enterica and Shigella flexneri) are able to invade T lymphocytes in vivo and modify their behaviour^14-16 . We have recently described that T lymphocytes are also able to capture bacteria by transinfection from previously infected dendritic cells (DCs) during the course of antigen presentation¹⁶. T cell bacterial capture by transinfection exceedingly more effective (1,000-4,000x) than direct infections. T cells capture pathogen and non-pathogen bacteria indicating than transinfection is a process driven by T cells. Strikingly, transinfected T (tiT) cells rapidly killed the captured bacteria and did so more efficiently than professional phagocytes¹⁶. These results, which break a dogma of immunology, show that the cells of adaptive immunity can perform functions that were supposedly exclusive of the innate immunity. In addition, we showed that tiT cells secrete large amounts of pro-inflammatory cytokines and protect from bacterial infections in vivo.

Here we present the different protocols used to study the bacterial transinfection process in a mouse model. This model is based on the use of CD4⁺ T cells from transgenic OTII mice, which bear a TCR specific for peptide 323-339 of OVA (OVAp) in the context of I-Ab¹⁷ that interact specifically with bacterial-infected bone marrow-derived DCs (BMDCs)^18,19 loaded with OVAp, forming stable immune synapses.

T cell transinfection can be visualized and tracked using fluorescence microscopy. Additionally, flow cytometry can be used for detecting infected cells by taking advantage of the fluorescence emitted by bacteria expressing green fluorescent protein (GFP)^16,20. Moreover, T cell transinfection can be quantified by a more sensitive approach, the gentamicin survival assay that allows measurement of a large number of events. Gentamicin is an antibiotic that cannot penetrate eukaryotic cells. Therefore, using this antibiotic allows differentiation of intracellular bacteria that survived the antibiotic addition from extracellular ones that were killed²¹.

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 in the hood from this point on, using only sterile media, instruments, pipette tips and culture dishes.

1. Mouse Bone Marrow-derived DCs Differentiation
   1. Dissect tibias and femurs from one C57/BL6 mouse ²² and transfer into a plastic dish with 10 ml of RPMI medium.
   2. Cut each epiphysis off, with sterile scissors and expose bone marrow, which has a characteristic bright red color.
   3. Infuse the inside of the bone with 10 ml of phosphate-buffered saline (PBS) containing 0.5% bovine serum albumin (BSA) and 5 mM ethylenediaminetetraacetic acid (EDTA) (PBS/BSA/EDTA buffer) using a sterile syringe on a sterile Petri dish.
   4. Collect the cell suspension and centrifuge in a tube at 600 x g in a refrigerated centrifuge (4 °C).
   5. Resuspend the cells in 1 ml of Ammonium-Chloride-Potassium (ACK) lysing buffer (0.15 M NH₄Cl, 10 mM KHCO₃ and 0.1 mM EDTA) and incubate for 30 sec at RT in order to eliminate red blood cells.
   6. Add 10 ml of RPMI 1640 medium with 10% fetal bovine serum (FBS) and filter through a cell strainer (70 μm) to remove bone fragments and cell clumps before centrifuging at the same speed (600 xg) because clumps were eliminated by filtering.
   7. Count the cells and adjust to 5 x 10⁵ cells/ml with RPMI 10% FBS (containing 50 μM B-mercaptoethanol, 1 mM sodium pyruvate) and add 20 ng/ml recombinant murine granulocyte-macrophage colony- stimulating factor (rm-GM-CSF) as a final concentration.
   8. Add this suspension to a sterile, microbiological quality, 15 cm Petri dish, and culture in a CO₂ incubator (37 °C, 5% CO₂).
   9. Every three days, spin down both non-adherent cells and detached cells with 5 mM EDTA in PBS and resuspend at 5 x 10⁵ cells/ml of cell density, with fresh medium containing 20 ng/ml rm-GM-CSF.
2. Maturation of DCs and Antigen Loading
   1. At day 9, resuspend the cells at 5 x 10⁵ cells/ml of density in medium containing 20 ng/ml rm-GM-CSF. Then add 20 ng/ml of lipopolysaccharide (LPS) to allow high MHC-II expression on cells and incubate in a non-tissue culture-treated sterile 15 cm Petri dish for 24 hr.
   2. After one day of LPS-stimulation, stain some DC cells with antibodies against mouse CD11c, MHCII and Gr-1 to check DCs differentiation by flow cytometry. An example of flow cytometry analysis of DCs is shown in Figure 3A and 3B.
      1. Incubate DCs (5 x 10⁵ cells/sample) with an anti-mouse-CD16/CD32 monoclonal antibody at 2.5 μg/ml of final concentration in 50 μl PBS/BSA/EDTA buffer per sample. Afterwards, add 50 μl of the antibodies against MHCII (I-A/I-E), CD11c and Gr-1 conjugated with different fluorochromes at 1:100, 1:200 and 1:500 in PBS/BSA/EDTA buffer respectively.
      2. Finally, wash DCs with PBS/BSA/EDTA buffer and analyze by flow cytometry according to manufacturer's protocol.
         Note: If the cells are well differentiated, cells are ready for antigen loading.
   3. Wash DCs with RPMI 10% FBS (without antibiotics) and incubate them with 10 μg/ml OTII OVAp (OVA 323-339; I S Q A V H A A H A E I N E A G R) on a plastic tube in 1 ml of RMPI 10% FBS medium per 5 x 10⁶ DCs for minimum 1 hr at 37 ºC. As a negative control, leave DCs without incubating with OVAp.
3. Infection of DCs
   1. Infect DCs at a multiplicity of infection (MOI) of 10 bacteria (10 bacteria per DC) for 1 hr in a CO₂ incubator (37 °C, 5% CO₂).
   2. Wash DC cells 3x in PBS and 1x in RMPI 10% FBS and centrifuge at 450 x g in order to wash most of extracellular bacteria. Finally, resuspend cells in RMPI 10% FBS medium (without antibiotics) at 20 x 10⁶ cells/ml.

2. Isolation of CD4 ⁺ T Lymphocytes from OTII Transgenic Mice

Note: Figure 1 summarizes this second step. Lymph nodes should be used instead of spleen to isolate CD4⁺ T cells, because the proportion of CD4⁺ lymphocytes in lymph nodes (~50%) is larger than in spleen (~25%) and therefore the purification would be more effective.

1. Make Single-cell Suspension of Lymph Nodes
   1. Remove inguinal, axillary, brachial, cervical and mesenteric lymph nodes ²³ from OTII transgenic mice and transfer into a plastic dish with 10 ml of RPMI 10% FBS medium.
   2. Grind lymph nodes under a sterile hood using two frosted microscope slides. Place lymph nodes on a frosted side of one microscope slide, and rub with frosted side of the second slide until organs have been ground.
   3. Wash single cell suspension in PBS/BSA/EDTA buffer.
   4. Filter the cells though a cell strainer (70 μm) to remove connective tissue and wash with PBS/BSA/EDTA buffer.
   5. Resuspend the cells in 1 ml of ACK lysis buffer and incubate for 1 min at RT in order to eliminate red blood cells.
2. Isolation of CD4⁺ T Cells
   1. Wash again as in step 2.1.3 and count cells to resuspend them at 100 x 10⁶ cells /ml in PBS/BSA/EDTA buffer. Add biotinylated antibodies against CD8, IgM, B220, CD19, MHC class II (I-Ab), CD11b, CD11c and DX5 at 1:250 for 30 min on ice for later negative selection of CD4⁺ T cells.
   2. Wash cells in PBS/BSA/EDTA buffer and incubate the cells (100 x 10⁶ cells/ml) with streptavidin microbeads at the concentration recommended in manufacturer's protocol for 15 min on ice.
   3. Wash cells in PBS/BSA/EDTA buffer and filter them though a cell strainer (30 μm) before CD4⁺ T cell isolation.
   4. Isolate CD4⁺ T cells by negative selection using a magnetic cell separation machine according to manufacturer's protocol.
   5. Count the cells and adjust to 4 x 10⁶ cells/ml with RPMI 10% FBS medium (without antibiotics).
   6. Fix and keep on ice isolated CD4⁺ T cells (3 x 10⁵ cells) to check the purity by flow cytometer as described²⁴. An example of CD4⁺ T cells purification is shown in Figure 3C.

3. T Cell Transinfection Measurement by Gentamicin Protection Assay

Note: Figure 2 summarizes this third step of the protocol.

1. T cell Transinfection
   1. Incubate isolated CD4⁺ T cells with infected DC cells (1:1) (the DCs were infected for 1 hr and washed to eliminate free bacteria) for 30 min to allow immune synapse formation in a CO₂ incubator (37 °C, 5% CO₂).
   2. Add 0.5 ml of isolated CD4⁺ T cells (2 x 10⁶ cells) and 0.1 ml of infected DCs (2 x 10⁶ cells) on a 24-well culture plate. As negative control, infect directly 0.5 ml of CD4⁺ T cells at a MOI of 10 bacteria for 30 min.
   3. Include additional controls separating infected DCs from T cells using a polycarbonate transwell barrier (with 3 μm of pore size), impeding DC-T physical contact. Add 0.1 ml of DCs inside the transwell and 0.5 ml of CD4⁺ T cells into the lower compartment of the well (24-well plate).
   4. Finally, complete with RMPI medium until there is 0.6 ml in each well to make equal volumes in all samples.
   5. After 30 min of immune synapse formation, add 100 μg/ ml of gentamicin and incubate for 1 hr before collecting cells in a CO₂ incubator (37°C, 5% CO₂).
2. Re-isolation of CD4⁺ T Cells
   1. Collect non-adherent cells and resuspend them in PBS/BSA/EDTA buffer. Most of DCs remain attached to the plastic culture plate. Vortex tubes gently to ensure cell disaggregation. Keep 500 µl of cell supernatant as control to show that gentamicin has worked well.
   2. Re-isolate CD4⁺ T cells from samples that contain DCs and T cells together as in step 2.2, although most of the DCs should have attached on the plate. Keep the control samples on ice. Take into account only experiments with less than 2% contamination. An example of CD4⁺ T cells purification after transinfection is shown in Figure 3D.
3. Lyse T Cells and Seed Them on Bacteria Medium-agar Plates.
   1. Wash CD4⁺ T cells twice with PBS.
   2. Count the cells and resuspend them at 2 x 10⁶ cells/ml in PBS.
   3. Add 500 μl of 0.1% Triton X-100 in 500 μl of T cells to lyse them at 1 x 10⁶ cells/ml, releasing the intracellular bacteria out of the T cells.
   4. Make 3 serial decimal dilutions of lysed cells and seed 50 μl of each dilution on bacteria medium agar plates. Divide plates into 4 portions and seed the different dilutions of lysed cells in each part. Representative results are shown in Figure 4.
      1. As a control, also plate the stored cell supernatant of the conjugates on agar, to ensure that gentamicin has worked well. Also plate infected DCs as an additional control. In case low DCs contamination occurs (<2%), subtract the colony forming units (CFUs) corresponding to low DC contamination.

4. Quantification of T Cell Transinfection by Flow Cytometry

Note: Figure 2 summarizes this step.

1. T Cell Transinfection
   1. Infect BMDCs as in step 1.3, but use GFP expressing bacteria in this case.
   2. Stain CD4⁺ T cells (isolated from lymph nodes in step 2.2) with a cell tracker chloromethyl aminocoumarin (CMAC) for 30 min at 37 ºC according to manufacturer's protocol.
   3. Wash labelled CD4⁺ T cells twice with RMPI 10% FBS (without antibiotics).
   4. Incubate infected DCs (after 1 hr incubation with bacteria) with labelled CD4⁺ T cells for 30 min to allow immune synapse formation at 37 ºC including all the controls explaining in step 3.1.1.
2. Staining of Infected CD4⁺ T Cells
   1. After 30 min of DC-T immune synapse formation, collect cells and wash them in PBS/BSA/EDTA.
   2. After two washes with PBS/BSA/EDTA buffer, separate aggregated cells by gently vortexing the tubes.
   3. Fix the samples in 0.2 ml/tube of PBS containing 3% PFA for 15 min at RT. Then wash them in PBS.
   4. Incubate with anti-mouse-CD16/CD32 monoclonal antibody (2.5 μg/ml) in 50 μl/sample of PBS/BSA/EDTA for 15 min on ice to block Fc receptors of dendritic cells.
   5. Add a rabbit anti-bacteria antibody (10 μg/ml as final concentration) in 50 μl/sample of PBS/BSA/EDTA for 30 min on ice to stain extracellular bacteria.
   6. Wash cells with PBS/BSA/EDTA and incubate with an antibody against mouse CD11c conjugated with phycoerythrin (PE) and a secondary antibody against rabbit conjugated with other fluorochrome like Alexa-Fluor 647 at 1:200 in 100 μl/sample of PBS/BSA/EDTA for 30 min on ice.
   7. Wash cells and analyze samples by flow cytometry. Do not forget to include a sample of transinfected T cells using non-fluorescent bacteria as negative control and samples labelled with just one of each fluorochrome used in the experiment to compensate the samples and adjust the flow cytometry settings²⁵. Representative analysis is shown in Figure 5.
      Note: If bacteria do not express GFP, to distinguish intracellular and extracellular bacteria, label extracellular bacteria, fix, and permeabilize samples with Triton X-100 at 0.5% in PBS for 5 min. Afterwards stain total bacteria (intracellular + extracellular) with a different fluorochrome.

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

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