This work was supported by grants BFU2011-29450, BFU2008-04342/BMC from the Spanish Ministry of Science and Innovation and PIES201020I046 from Consejo Superior de Investigaciones Cient&#236;ficas (CSIC).

Note: Experimental procedures were approved by the Committee for Research Ethics of the Universidad Aut&#242;noma de Madrid and conducted under the supervision of the Universidad Aut&#242;noma 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 Differ...

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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 vivo14,15. 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 ...

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 presentation8,9. Some bacteria can survive phagocytosis and disseminate systematically within APCs. In this view, infected APCs serve as bacterial reservoirs or &#34;Trojan horses&#34; 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 transinfection11-13.
Some pathogenic bacteria (Listeria monocytogenes, Salmonella enterica and Shigella flexneri) are able to invade T lymphocytes in vivo and modify their behaviour14-16 . 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.

<table border="0" cellpadding="0" cellspacing="0" width="743">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:341px;">RPMI</td>
			<td style="width:131px;">Fisher Scientific</td>
			<td style="width:104px;">SH3025501</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:341px;">r-GMCSF</td>
			<td style="width:131px;">Peprotech</td>
			<td style="width:104px;">315-03</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:341px;">LPS</td>
			<td style="width:131px;">SIGMA</td>
			<td style="width:104px;">L2630-10mg</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:341px;">Na Pyruvate</td>
			<td style="width:131px;">Thermo Scientific</td>
			<td style="width:104px;">SH3023901</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:341px;">2-ME</td>
			<td style="width:131px;">Gibco</td>
			<td style="width:104px;">31350-010</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:341px;">OVAp OTII (323&#8211;339)</td>
			<td style="width:131px;">GenScript</td>
			<td style="width:104px;">---</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:341px;">Cell Strainer 70uM</td>
			<td style="width:131px;">BD</td>
			<td align="right" style="width:104px;">352350</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">&#160;30uM Syringe Filcons Sterile</td>
			<td>BD</td>
			<td align="right">340598</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:341px;">AutoMacs Classic</td>
			<td style="width:131px;">Miltenyi Biotec</td>
			<td style="width:104px;">130-088-887</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:341px;">Gentamicin</td>
			<td style="width:131px;">Normon</td>
			<td style="width:104px;">624601.6</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:341px;">Transwell</td>
			<td style="width:131px;">Costar</td>
			<td align="right" style="width:104px;">3415</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:341px;">LB</td>
			<td style="width:131px;">Pronadisa</td>
			<td align="right">1231</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:341px;">Agar</td>
			<td style="width:131px;">Pronadisa</td>
			<td align="right">1800</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:341px;">Paraformaldehyde 16%</td>
			<td style="width:131px;">Electron Microscopy Sciences</td>
			<td align="right" style="width:104px;">15710</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:341px;">Triton X-100</td>
			<td style="width:131px;"></td>
			<td style="width:104px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:341px;">CD8 biot</td>
			<td style="width:131px;">BD Biosciences</td>
			<td align="right" style="width:104px;">553029</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:341px;">IgM Biot</td>
			<td style="width:131px;">ImmunoStep</td>
			<td style="width:104px;">Clone RMM-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:341px;">B220 Biot</td>
			<td style="width:131px;">BD Biosciences</td>
			<td align="right" style="width:104px;">553086</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:341px;">CD19 biot</td>
			<td style="width:131px;">BD Biosciences</td>
			<td align="right">553784</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:341px;">MHC-II Biot (I-A/I-E)</td>
			<td style="width:131px;">BD Biosciences</td>
			<td align="right">553622</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:341px;">CD11b biot</td>
			<td style="width:131px;">Immunostep</td>
			<td style="width:104px;">11BB-01mg</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:341px;">CD11c biot</td>
			<td style="width:131px;">Immunostep</td>
			<td>11CB3-01mg</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:341px;">DX5 biot</td>
			<td style="width:131px;">BD Biosciences</td>
			<td align="right" style="width:104px;">553856</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:341px;">Gr-1 biot</td>
			<td style="width:131px;">BD Biosciences</td>
			<td align="right" style="width:104px;">553125</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:341px;">CD16/CD32</td>
			<td style="width:131px;">ImmunoStep</td>
			<td>M16PU-05MG</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:341px;">anti Salmonella</td>
			<td style="width:131px;">ABD Serotec</td>
			<td style="width:104px;">8209-4006</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:341px;">CD11cPE</td>
			<td style="width:131px;">BD Biosciences</td>
			<td align="right" style="width:104px;">553802</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:341px;">CD4-APC</td>
			<td style="width:131px;">Tonbo Biosciences</td>
			<td style="width:104px;">20-0041-U100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Gr-1 APC</td>
			<td style="width:131px;">BD Biosciences</td>
			<td align="right">553129</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MHC-II (I-A/I-E) FITC</td>
			<td style="width:131px;">BD Biosciences</td>
			<td align="right">553623</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Alexa-Fluor 647 Goat Anti-Rabbit IgG (H+L) Antibody, highly cross-adsorbed</td>
			<td>Invitrogen</td>
			<td>A-21245</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:341px;">CMAC (7-amino-4-chloromethylcoumarin)</td>
			<td style="width:131px;">Life technologies</td>
			<td>C2110</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:341px;">BSA</td>
			<td style="width:131px;">SIGMA</td>
			<td style="width:104px;">A7030-100G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:341px;">Streptavidin MicroBeads</td>
			<td style="width:131px;">Miltenyi Biotec</td>
			<td style="width:104px;">130-048-101</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:341px;">BD FACSCanto II</td>
			<td style="width:131px;">BD Biosciences</td>
			<td style="width:104px;">---</td>
			<td></td>
		</tr>
	</tbody>
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t cells capture bacteria by transinfection from dendritic cells

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.

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...

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T Cells Capture Bacteria by Transinfection from Dendritic Cells

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.

Recently, we have shown, contrary to what is described, that CD4+ T cells, the paradigm of adaptive immune cells, capture bacteria from infected dendritic ...

The overall goal of this procedure is to quantify bacterial CD4+T cell transinfection, using a mouse cell model. In vivo, some pathogenic bacteria such as Listeria monocytogenes and Salmonella enteritidis are able to infect T lymphocytes and modify their behavior. However, in vitro, T cells are not easy to infect.Based on this observation, we investigated whether T cells can capture bacteria from infected dendritic cells during antigen presentation as occurs in viral infections. We found that T cells capture bacteria and then kill them. Actually, we found that T cells do this more efficiently than professional phagocytes.These results contradict a central tenet of immunology, showing that cells from adaptive immunity can perform functions supposedly exclusive to innate immunity. Today we present the protocol used to study the bacteria transinfection process in a mouse model. Esteban Veiga and I will be demonstrating this procedure along with Guillermo Ramirez-Santiago, Raquelle Garcia-Ferreras, two graduate students, and Monica Torres-Torresano, a technician.Note that all the procedures described in this video should be carried out in the hood, using only sterile media, instruments, pipette tips, and culture dishes. In a non-tissue culture treated sterile 15 centimeter Petri dish, add one microliter of one milligram per milliliter lipopolysaccharide to the bone marrow derived dendritic cells from a single mouse. Incubate the cells for 24 hours to increase major histocompatibility complex II, or MHC II, expression.Following the incubation, perform flow cytometry to confirm MHC II expression and differentiation. After MHC II expression has been confirmed, as shown here, transfer the cells to a sterile 50 milliliter polypropylene tube. Add 10 milliliters of PBS containing five millimolar EDTA, then centrifuge the cells at 600 x g.Once the spin is complete, perform a wash by resuspending the cells in RPMI with 10%FBS, then centrifuging again. Then, after the wash, add one milliliter of RPMI 10%FBS for every five times 10 to the sixth dendritic cells, and pipette up and down to resuspend them. Next, separate the resuspended dendritic cells into two tubes of equal volume.To one tube, add 10 micrograms per milliliter OT-II OVAp. Leave the other tube untreated. It will serve as a negative control for antigen loading.Incubate the tubes for at least one hour to load the MHC II molecules of dendritic cells with OVA peptide. Meanwhile, prepare the Salmonella enteritidis for infection by washing them with a PBS solution three times. Centrifuge between each wash at 1800 x g to eliminate the LB medium and any secreted toxins.Then, resuspend the bacteria in RPMI. Following the incubation, infect each tube of dendritic cells at a multiplicity of infection, or MOI, of 10, and then incubate the cells for one hour. Once the cells have been infected, wash them three times with PBS, and then once with RPMI 10%FBS.Centrifuge between washes at 450 x g to wash away most of the extracellular bacteria in the soluble OVAp. After the last centrifugation, resuspend the cells in RPMI 10%FBS at 20 times 10 to the sixth cells per milliliter. To a 24-well culture plate, add two million CD4+T cells in a volume of 0.5 milliliters to each of five wells.Next, to control for physical contact, place polycarbonate trans-well barriers with a three micrometer pore size into the third and fourth wells. To the first well, add 0.1 milliliters of the infected overloaded dendritic cells. To the second well, add 0.1 milliliters of the infected non-loaded dendritic cells.Add 0.1 milliliters of the infected overloaded dendritic cells to one well, and 0.1 milliliters of the infected non-loaded dendritic cells into the other well. Finally, as a negative control, directly infect the fifth well of CD4+T cells at an MOI of 10. Then, bring the total volume of the well up to 0.6 milliliters with RPMI.Incubate the plate for 30 minutes to allow immune synapses to form. After the incubation, add 100 micrograms of Gentamicin per milliliter of medium to each well, and incubate for one hour. Gentamicin is an aminoglycoside antibiotic that is not able to penetrate eukaryotic cells.Therefore, in this step, aminoglycoside will kill extracellular bacteria but not intracellular bacteria. Following the incubation, collect the non-adherent cells in 15 milliliter tubes, and resuspend them in 10 milliliters of PBS BSA EDTA buffer. Discard the plastic culture plate to which most of the dendritic cells remain attached.Next, vortex the tubes gently to ensure cell desegregation. Centrifuge the tubes at 600 x g. Then, from any tube, aspirate 500 microliters of the cell supernatant and pipette it into a new tube.This will be used as a control to show that the Gentamicin treatment was effective. Place all of the samples on ice. At this stage, there are six tubes:four tubes of test samples and two controls.Although most of the dendritic cells were discarded with the plate, the samples that were not separated by the trans-well barriers could still contain some. To reduce contamination, use magnetic cell isolation or cell sorting to re-isolate the CD4+T cells from the samples that were not separated by trans-well barriers. The final isolate should contain less than 2%dendritic cells.Keep the rest of the samples on ice. Once the CD4+T cells have been purified, it is time to save for bacteria class infection. This can be done by performing colony counts, or by flow cytometry.Here, we will show you how to perform colony counts. Wash the transinfected CD4+T cells twice with PBS. Centrifuge at 600 x g between washes.Then count the cells and resuspend them in PBS at a concentration of two times 10 to the sixth cells per milliliter. To 500 microliters of T cells, add 500 microliters of 0.1%Triton X-100 to lyse them. This releases the intracellular bacteria from the T cells.For each sample of lysed cells, make three serial dilutions. Then, divide an LB agar plate into quarters and spread 50 microliters of each dilution, including the undiluted suspension, onto each section of the plate. On a separate LB agar plate, spread the stored cell supernatant as a control for Gentamicin efficacy.Then, on another plate, spread the directly infected T cells as an additional control. Incubate overnight. The next day, count the colonies in each section of the plate and calculate the number of colony-forming units for each sample.If low-level dendritic cell contamination occurs, which is defined as less than 2%subtract the colony-forming units corresponding to low dendritic cell contamination. T cell transinfection was performed using Salmonella enteritidis and quantified by Gentamicin survival assay as described in this video. When directly infected T cells were plated, as shown on the left, growth of colonies was not observed, indicated that T cells are refractory to direct bacterial infections.When supernatant from transinfection assays was plated, as shown on the right, colonies were not observed, indicating that the Gentamicin worked properly. Results from transinfection assays performed in the presence of trans-well barriers are shown here. Only three colonies are observed in the undiluted suspension from both OVA-loaded and non-loaded samples.In contrast, numerous colonies are seen when there is direct contact of T cells with infected dendritic cells. Moreover, when dendritic cells loaded with OVAp were incubated with T cells, the uptake of bacteria was increased by about five times. Taken together, these data indicate that direct contact of infected dendritic cells with T cells facilitates bacterial uptake and that cell transinfection is enhanced during antigen recognition.After this video, you should have a good understanding of how to quantify transinfection using marine T cells. The advantage of quantifying transinfection with the Gentamicin survival is its great sensitivity. We are able to detect one single infecting bacterium.Is it is important to remember that the purification step of this procedure is critical. The purity must be very high if colony counts will be used to determine the transinfection rate. Quantification by flow cytometry does not have this limitation, because you can easily distinguish dendritic cells and T cells, but the restriction is that the GFP expressing bacteria must be bright enough to detect the infected cells against the outer fluorescence background.Don't forget that working with pathogens can be extremely hazardous and the appropriate safety precautions should always be used when performing this procedure.

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Research

JoVE Journal

Immunology and Infection

11.6K Views.  Spanish National Biotechnology Centre (CNB-CSIC). The overall goal of this procedure is to quantify bacterial CD4+T cell transinfection, using a mouse cell model. In vivo, some pathogenic bacteria such as Listeria monocytogenes and Salmonella enteritidis are able to infect T lymphocytes and modify their behavior. However, in vitro, T cells are not easy to infect.Based on this observation, we investigated whether T cells can capture bacteria from infected dendritic cells during antigen presentation as occurs in viral infections. We fou...