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 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.
<ol>
	<li>Mouse Bone Marrow-derived DCs Differentiation
	<ol>
		<li>Dissect tibias and femurs from one C57/BL6 mouse 22 and transfer into a plastic dish with 10 ml of RPMI medium.</li>
		<li>Cut each epiphysis off, with sterile scissors and expose bone marrow, which has a characteristic bright red color.</li>
		<li>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.</li>
		<li>Collect the cell suspension and centrifuge in a tube at 600 x g in a refrigerated centrifuge (4 &#176;C).</li>
		<li>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.</li>
		<li>Add 10 ml of RPMI 1640 medium with 10% fetal bovine serum (FBS) and filter through a cell strainer (70 &#956;m) to remove bone fragments and cell clumps before centrifuging at the same speed (600 xg) because clumps were eliminated by filtering.</li>
		<li>Count the cells and adjust to 5 x 105 cells/ml with RPMI 10% FBS (containing 50 &#956;M 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.</li>
		<li>Add this suspension to a sterile, microbiological quality, 15 cm Petri dish, and culture in a CO2 incubator (37 &#176;C, 5% CO2).</li>
		<li>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.</li>
	</ol>
	</li>
	<li>Maturation of DCs and Antigen Loading
	<ol>
		<li>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.</li>
		<li>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.
		<ol>
			<li>Incubate DCs (5 x 105 cells/sample) with an anti-mouse-CD16/CD32 monoclonal antibody at 2.5 &#956;g/ml of final concentration in 50 &#956;l PBS/BSA/EDTA buffer per sample. Afterwards, add 50 &#956;l 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.</li>
			<li>Finally, wash DCs with PBS/BSA/EDTA buffer and analyze by flow cytometry according to manufacturer&#39;s protocol. 
			Note: If the cells are well differentiated, cells are ready for antigen loading.</li>
		</ol>
		</li>
		<li>Wash DCs with RPMI 10% FBS (without antibiotics) and incubate them with 10 &#956;g/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 &#186;C. As a negative control, leave DCs without incubating with OVAp.</li>
	</ol>
	</li>
	<li>Infection of DCs
	<ol>
		<li>Infect DCs at a multiplicity of infection (MOI) of 10 bacteria (10 bacteria per DC) for 1 hr in a CO2 incubator (37 &#176;C, 5% CO2).</li>
		<li>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.</li>
	</ol>
	</li>
</ol>
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.
<ol>
	<li>Make Single-cell Suspension of Lymph Nodes
	<ol>
		<li>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.</li>
		<li>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.</li>
		<li>Wash single cell suspension in PBS/BSA/EDTA buffer.</li>
		<li>Filter the cells though a cell strainer (70 &#956;m) to remove connective tissue and wash with PBS/BSA/EDTA buffer.</li>
		<li>Resuspend the cells in 1 ml of ACK lysis buffer and incubate for 1 min at RT in order to eliminate red blood cells.</li>
	</ol>
	</li>
	<li>Isolation of CD4+ T Cells
	<ol>
		<li>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.</li>
		<li>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&#39;s protocol for 15 min on ice.</li>
		<li>Wash cells in PBS/BSA/EDTA buffer and filter them though a cell strainer (30 &#956;m) before CD4+ T cell isolation.</li>
		<li>Isolate CD4+ T cells by negative selection using a magnetic cell separation machine according to manufacturer&#39;s protocol.</li>
		<li>Count the cells and adjust to 4 x 106 cells/ml with RPMI 10% FBS medium (without antibiotics).</li>
		<li>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.</li>
	</ol>
	</li>
</ol>
3. T Cell Transinfection Measurement by Gentamicin Protection Assay
Note: Figure 2 summarizes this third step of the protocol.
<ol>
	<li>T cell Transinfection
	<ol>
		<li>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 &#176;C, 5% CO2).</li>
		<li>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.</li>
		<li>Include additional controls separating infected DCs from T cells using a polycarbonate transwell barrier (with 3 &#956;m 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).</li>
		<li>Finally, complete with RMPI medium until there is 0.6 ml in each well to make equal volumes in all samples.</li>
		<li>After 30 min of immune synapse formation, add 100 &#956;g/ ml of gentamicin and incubate for 1 hr before collecting cells in a CO2 incubator (37&#176;C, 5% CO2).</li>
	</ol>
	</li>
	<li>Re-isolation of CD4+ T Cells
	<ol>
		<li>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 &#181;l of cell supernatant as control to show that gentamicin has worked well.</li>
		<li>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.</li>
	</ol>
	</li>
	<li>Lyse T Cells and Seed Them on Bacteria Medium-agar Plates.
	<ol>
		<li>Wash CD4+ T cells twice with PBS.</li>
		<li>Count the cells and resuspend them at 2 x 106 cells/ml in PBS.</li>
		<li>Add 500 &#956;l of 0.1% Triton X-100 in 500 &#956;l of T cells to lyse them at 1 x 106 cells/ml, releasing the intracellular bacteria out of the T cells.</li>
		<li>Make 3 serial decimal dilutions of lysed cells and seed 50 &#956;l 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.
		<ol>
			<li>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 (&#60;2%), subtract the colony forming units (CFUs) corresponding to low DC contamination.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
4. Quantification of T Cell Transinfection by Flow Cytometry
Note: Figure 2 summarizes this step.
<ol>
	<li>T Cell Transinfection
	<ol>
		<li>Infect BMDCs as in step 1.3, but use GFP expressing bacteria in this case.</li>
		<li>Stain CD4+ T cells (isolated from lymph nodes in step 2.2) with a cell tracker chloromethyl aminocoumarin (CMAC) for 30 min at 37 &#186;C according to manufacturer&#39;s protocol.</li>
		<li>Wash labelled CD4+ T cells twice with RMPI 10% FBS (without antibiotics).</li>
		<li>Incubate infected DCs (after 1 hr incubation with bacteria) with labelled CD4+ T cells for 30 min to allow immune synapse formation at 37 &#186;C including all the controls explaining in step 3.1.1.</li>
	</ol>
	</li>
	<li>Staining of Infected CD4+ T Cells
	<ol>
		<li>After 30 min of DC-T immune synapse formation, collect cells and wash them in PBS/BSA/EDTA.</li>
		<li>After two washes with PBS/BSA/EDTA buffer, separate aggregated cells by gently vortexing the tubes.</li>
		<li>Fix the samples in 0.2 ml/tube of PBS containing 3% PFA for 15 min at RT. Then wash them in PBS.</li>
		<li>Incubate with anti-mouse-CD16/CD32 monoclonal antibody (2.5 &#956;g/ml) in 50 &#956;l/sample of PBS/BSA/EDTA for 15 min on ice to block Fc receptors of dendritic cells.</li>
		<li>Add a rabbit anti-bacteria antibody (10 &#956;g/ml as final concentration) in 50 &#956;l/sample of PBS/BSA/EDTA for 30 min on ice to stain extracellular bacteria.</li>
		<li>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 &#956;l/sample of PBS/BSA/EDTA for 30 min on ice.</li>
		<li>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.</li>
	</ol>
	</li>
</ol>

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The authors have nothing to disclose.

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 dogma, T cells, the paradigm of cells of adaptive immunity, are also able to efficiently capture bacteria by transinfection from infected DCs11,16. Herein we show he detailed protocols to quantify T cell bacterial transinfection in vitro from infected BMDCs.
T cell bacterial transinfection was enhanced by antigen recognition16. We showed that by using CD4+ T cells isolated from OTII transgenic mice recognizing a specific OVAp, BMDCs loaded with OVAp and mice infected with different bacteria (here data is presented from Salmonella enterica). CD4+ T cell isolated from OTII mice are incubated with infected DCs for 30 min to allow immune synapse formation. The data presented here correspond to the bacteria that T cells can capture from DCs in this short period of time. We know that longer exposures of T cells to infected DCs resulted in larger bacterial captures (not shown), but as T cells also destroy the uptaken bacteria very quickly, quantification of bacterial transinfection occurring during longer DC/T cell time contacts proved to be difficult. Gentamicin survival assay is a very sensitive method to quantify T cell transinfection because it is able to detect one single infecting bacterium21. After incubation with gentamicin that kill just extracellular bacteria, T cells are isolated and lysed to seed on bacteria medium agar plates. The isolation of T cells after forming conjugates with DCs is a critical step within the protocol. Only experiments with less than 2% of contaminants should be taken into account. DC contamination can be measured by flow cytometry and CFUs corresponding to low DC contamination should be subtracted. Alternatively, CD4+ T cells purity can be improved by using cell sorting.
Another approach to quantify T cell transinfection is by flow cytometry, using GFP expressing bacteria and labelling the extracellular bacteria with a compatible fluorophore. One limitation of this approach is that bacteria have to express a GFP that is sufficiently bright for detecting infected cells against the autofluorescence background. Alternatively, bacteria could be stained before infecting DCs with a cell tracker. Another approach would be staining the extracellular bacteria, permeabilizing the T cells, then labelling all the bacteria (intracellular + extracellular) with a different fluorochrome.
Regarding future directions of this protocol, we are trying to set up a protocol to quantify the number of T cells that captured bacteria after longer exposition to infected DCs, by using bacteria decorated with magnetic particles. T cells capturing bacteria would retain the magnetic particles and therefore it should be easy to isolate them by using magnets.

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>
</table>

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 of OVA (OVAp). As a control, some DCs are not loaded with OVAp. Both DCs (OVAp-loaded and non-loaded) are infected at a multiplicity of infection (MOI) of 10 bacteria. On the other hand, CD4+ T cells are isolated from lymph nodes of OTII transgenic mice, which recognize the specific OVAp. Figure 2 depicts the procedure to perform and quantify T cell transinfection including the appropriate negative controls. As described before, some DCs are not loaded with OVAp. In this case, there is an interaction between DCs and T cells, but not immune synapse formation due to the absence of OVAp. An additional control is included, separating DCs and T cells with a polycarbonate transwell barrier (with 3 &#956;m of pore size), which impedes DC-T physical contact. DCs are included inside the transwell; as a result they are not able to pass through this pore size. Another negative control is also incorporated, the direct T cell infection, by incubating T cells directly at a MOI of 10 bacteria. T cell transinfection can be quantified by two approaches: flow cytometry or gentamicin survival assay. To measure by flow cytometry, DCs should have been infected previously with bacteria-GFP and CD4+ T cells stain with a cell tracker (T cells-blue). After conjugate formation, the cells should be stained with antibodies against DCs (CD11c-PE). The extracellular bacteria should also be stained in order to quantify the percentage of infected CD4+ T cells by flow cytometry later on. To measure transinfection by gentamicin survival assay, cells are incubated with gentamicin for 1 hr to kill extracellular bacteria, then T cells are re-isolated to seed on agar plate. Decimal serial dilutions are made to seed 50 &#956;l of each dilution on agar plate and facilitate the counting of CFUs.
As shown in Figure 3, DC differentiation has to be checked by flow cytometry (Figure 3A and 3B). DCs should express CD11c and MHCII (Figure 3A) and not Gr-1, which is a marker of neutrophils. As presented in Figure 3B, some neutrophils (3.27% of Gr-1+, MHC-) were presented in the culture, but DCs culture with more than 5% of neutrophils contamination should not be used. CD4+ T cells purity should be checked after isolation from lymph nodes (Figure 3C) and after separation from DCs conjugates formation (Figure 3D). Take into account the experiments with less than 2% of contaminants after conjugates formation.
Figure 4 shows an example of T transinfection experiment using Salmonella. As illustrated in Figure 4A, plates are divided in four portions to seed the serial dilutions in each part. Salmonella have grown on LB agar plates and colony-forming units can be counted. As shown in Figure 4A, Salmonella was captured by T cells from infected DCs and this process was clearly enhanced with OVAp recognition (plates on the left of Figure 4A and Figure 4B). However, when there was a physical barrier impeding the contact between DCs and T cells, there was virtually no transinfection, as in the case of doing a direct T cell infection (plates on the right of Figure 4A and Figure 4B).
As shown in Figure 5, T cell transinfection can be quantified by flow cytometry using bacteria expressing a bright GFP. DCs and T cells can be differentiated by size and complexity in a forward and side scatter plot, but different markers can be used to well distinguish them. CD4+ T cells were labelled with a cell tracker (CMAC) and DCs with an antibody against CD11c. Extracellular bacteria were stained with a rabbit antibody against Salmonella and secondary antibody anti-rabbit conjugated with Alexa Fluor 647. Therefore, the percentage of infected CD4+ T cells can be quantified because they are labelled with CMAC (not with CD11c-PE) and expressed GFP (bacteria-GFP), but they were not stained with the antibody against extracellular bacteria (Alexa Fluor 647).
<img alt="Figure 1" src="/files/ftp_upload/52976/52976fig1.jpg" /> 
Figure 1. Flow chart of DC generation and CD4+ T cells isolation. On the left hand side of the figure, the process to differentiate dendritic cells (DCs) from bone marrow cells is detailed. After dissecting femurs and tibias, bone marrow cells are extracted from the interior of the bones. Afterwards, red blood cells (RBC) are lysed and bone marrow cells are cultured with recombinant murine granulocyte-macrophage colony- stimulating factor (GM-CSF) for 9 days. Once they are well differentiated, DCs are incubated with lipopolysaccharide (LPS) to increase Major Histocompatibility Complex II (MHCII) expression. Then, DCs are loaded with a specific ovoalbumin peptide (OVAp) and infected with a multiplicity of infection (MOI) of 10 bacteria. Some of them are not loaded with OVAp as a control. On the right hand side of the figure, how to isolate CD4+ T cells from OTII transgenic mice is represented. Lymph nodes (LNs) are removed and ground, obtaining a single-cell suspension. Finally, CD4+ T cells are isolated by negative selection using a magnetic cell separation machine. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52976/52976fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/52976/52976fig2.jpg" /> 
Figure 2. Flow chart of T cell transinfection procedure. Infected (bacteria are depicted in green) and OVAp loaded-DCs (red cells) are incubated with CD4+ T cells (blue cells) to allow immune synapse formation. Infected DCs (but not loaded with OVAp) are also incubated with T cells, which allows interaction between both cells but without immune synapse formation. An additional control is included, separating CD4+ T cells and DCs with a polycarbonate transwell barrier (+TW), which impedes DC-T cell contact. Direct T cell infection is also incorporated as negative control. Finally, T cell transinfection can be quantified by two methods, by flow cytometry (left side of the figure), which indicates the number of T cells capturing bacteria, and by gentamicin survival assay (right side of the figure) a very sensitive method (one infecting bacterium can be detected) indicating the bacterial infection rate. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52976/52976fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/52976/52976fig3.jpg" /> 
Figure 3. Flow cytometer analysis of bone marrow derived-DCs and isolated CD4+ T cells. Representative dot plots of bone marrow derived-DCs analysed by flow cytometry. (A) DCs expressed MHCII and CD11c (85.8%; upper right quadrant) (B) DCs expressed MHCII but not Gr-1 (85%; lower right quadrant). However, there was a 3.27% of cells that expressed Gr-1 but not MHCII cells (upper left quadrant). These cells were slight contaminated neutrophils. (C and D) Histograms representing the purity of CD4+ T cells isolated from lymph nodes (C), and after conjugate formation with dendritic cells (D). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52976/52976fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" src="/files/ftp_upload/52976/52976fig4.jpg" /> 
Figure 4. T cell transinfection quantified by gentamicin survival assay. LB agar plates were divided into 4 portions corresponding to decimal serial dilutions of lysed CD4+ T cells. (A) Colony forming units of Salmonella enterica grew on LB agar plates corresponding to T cell transinfection in the presence (+) or absence (-) of OVAp on the surface of infected DC, and in conditions allowing DC/T cell contact (-TW) or in the presence of a physical barrier impeding such contacts (+TW). An empty plate corresponding to direct T cell infection (negative control) is also shown. There was almost no transinfection when DC/T cell contact was impeded (+TW). (B) Quantification of Colony Forming Units (CFUs) from several experiments showing the rate of bacterial captured by T cells from infected DCs. Direct T cell infection, and conditions impeding DC/T cell contact produce little to no levels of T cell infection. When DC/T cell contacts were permitted, there was T cell transinfection, and it was enhanced by antigen recognition (+OVAp). Column bars represent the mean of 3 independent experiments. Error bars indicate the SD. Significant differences are represented by ***, corresponding to p &#60;0,0001. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52976/52976fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" src="/files/ftp_upload/52976/52976fig5.jpg" /> 
Figure 5. T cell transinfection quantified by flow cytometry. Representative dot plot showing DCs and T cell conjugates after transinfection process. CD4+ T cells are small and round as forward and side scatter (FCS - SSC) dot blot shows, and in order to be well differentiated, they were stained with a cell tracker (CMAC). DCs expressing CD11c are not labelled with CMAC and are larger than T CD4+ T cells with irregular shape. Infected CD4+ T cells containing intracellular bacteria-GFP but negative for extracellular bacteria (6.19%) are shown in the lower right quadrant. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52976/52976fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about T Cells Capture Bacteria by Transinfection from Dendritic Cells at JoVE.com

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 ...

t-cells-capture-bacteria-by-transinfection-from-dendritic-cells

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

11.8K Views.  Spanish National Biotechnology Centre (CNB-CSIC). 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.

Video: T Cells Capture Bacteria by Transinfection from Dendritic Cells

Following Cell-fate in E. coli After Infection by Phage Lambda

The system comprising bacteriophage (phage) lambda and the bacterium E. coli has long served as a paradigm for cell-fate determination1,2. Following the simultaneous infection of the cell by a number of phages, one of two pathways is chosen: lytic (virulent) or lysogenic (dormant)3,4. We recently developed a method for fluorescently labeling individual phages, and were able to examine the post-infection decision in real-time under the microscope, at the level of individual phages and cells5. Here, we describe the full procedure for performing the infection experiments described in our earlier work5. This includes the creation of fluorescent phages, infection of the cells, imaging under the microscope and data analysis. The fluorescent phage is a "hybrid", co-expressing wild- type and YFP-fusion versions of the capsid gpD protein. A crude phage lysate is first obtained by inducing a lysogen of the gpD-EYFP (Enhanced Yellow Fluorescent Protein) phage, harboring a plasmid expressing wild type gpD. A series of purification steps are then performed, followed by DAPI-labeling and imaging under the microscope. This is done in order to verify the uniformity, DNA packaging efficiency, fluorescence signal and structural stability of the phage stock. The initial adsorption of phages to bacteria is performed on ice, then followed by a short incubation at 35&deg;C to trigger viral DNA injection6. The phage/bacteria mixture is then moved to the surface of a thin nutrient agar slab, covered with a coverslip and imaged under an epifluorescence microscope. The post-infection process is followed for 4 hr, at 10 min interval. Multiple stage positions are tracked such that ~100 cell infections can be traced in a single experiment. At each position and time point, images are acquired in the phase-contrast and red and green fluorescent channels. The phase-contrast image is used later for automated cell recognition while the fluorescent channels are used to characterize the infection outcome: production of new fluorescent phages (green) followed by cell lysis, or expression of lysogeny factors (red) followed by resumed cell growth and division. The acquired time-lapse movies are processed using a combination of manual and automated methods. Data analysis results in the identification of infection parameters for each infection event (e.g. number and positions of infecting phages) as well as infection outcome (lysis/lysogeny). Additional parameters can be extracted if desired.

Following Cell-fate in E. coli After Infection by Phage Lambda

This article describes the procedure for preparing a fluorescently-labeled version of bacteriophage lambda, infection of E. coli bacteria, following the infection outcome under the microscope, and analysis of infection results.

The system comprising bacteriophage (phage) lambda and the bacterium E. coli has long served as a paradigm for cell-fate determination1,2. Following the ...

Isolation of Mouse Lung Dendritic Cells

Lung dendritic cells (DC) play a fundamental role in sensing invading pathogens 1,2 as well as in the control of tolerogenic responses 3 in the respiratory tract. At least three main subsets of lung dendritic cells have been described in mice: conventional DC (cDC) 4, plasmacytoid DC (pDC) 5 and the IFN-producing killer DC (IKDC) 6,7. The cDC subset is the most prominent DC subset in the lung 8. 
The common marker known to identify DC subsets is CD11c, a type I transmembrane integrin (&beta;2) that is also expressed on monocytes, macrophages, neutrophils and some B cells 9. In some tissues, using CD11c as a marker to identify mouse DC is valid, as in spleen, where most CD11c+ cells represent the cDC subset which expresses high levels of the major histocompatibility complex class II (MHC-II). However, the lung is a more heterogeneous tissue where beside DC subsets, there is a high percentage of a distinct cell population that expresses high levels of CD11c bout low levels of MHC-II. Based on its characterization and mostly on its expression of F4&#47;80, an splenic macrophage marker, the CD11chiMHC-IIlo lung cell population has been identified as pulmonary macrophages 10 and more recently, as a potential DC precursor 11. 
In contrast to mouse pDC, the study of the specific role of cDC in the pulmonary immune response has been limited due to the lack of a specific marker that could help in the isolation of these cells. Therefore, in this work, we describe a procedure to isolate highly purified mouse lung cDC. The isolation of pulmonary DC subsets represents a very useful tool to gain insights into the function of these cells in response to respiratory pathogens as well as environmental factors that can trigger the host immune response in the lung.

A highly purified preparation of mouse lung dendritic cells is described. Specific emphasis is given to the isolation of conventional dendritic cell subset.

Lung dendritic cells (DC) play a fundamental role in sensing invading pathogens 1,2 as well as in the control of tolerogenic responses 3 in the ...

Generation of Induced Regulatory T Cells from Primary Human Na&iuml;ve and Memory T Cells

The development and maintenance of immunosuppressive CD4+ regulatory T cells (Tregs) contribute to the peripheral tolerance needed to remain in immunologic homeostasis with the vast amount of self and commensal antigens in and on the human body. Perturbations in the balance between Tregs and inflammatory conventional T cells can result in immunopathology or cancer. Although therapeutic injection of Tregs has been shown to be efficacious in murine models of colitis1 , type I diabetes2 , rheumatoid arthritis and graft versus host disease,4 several fundamental differences in human versus mouse Treg biology5 has thus far precluded clinical use. The lack of sufficient number, purity, stability and homing specificity of therapeutic Tregs necessitated a dynamic platform of human Treg development on which to optimize conditions for their ex vivo expansion6.
Here we describe a method for the differentiation of induced Tregs (iTregs) from a single human peripheral blood donor which can be broken down into four stages: isolation of peripheral blood mononuclear cells, magnetic selection of CD4+ T cells, in vitro cell culture and fluorescence activated cell sorting (FACS) of T cell subsets. Since the Treg signature transcription factor forkhead box P3 (FoxP3) is an activation-induced transcription factor in humans7 and no other unique marker exists, a combinatorial panel of markers must be used to identify T cells with suppressor activity. After six days in culture, cells in our system can be demarcated into na&iuml;ve T cells, memory T cells or iTregs based on their relative expression of CD25 and CD45RA. As memory and na&iuml;ve T cells have different reported polarization requirements and plasticities8 , pre-sorting of the initial T cell population into CD45RA+ and CD45RO+ subsets can be used to examine these discrepancies. Consistent with others, our CD25HiCD45RA- iTregs express high levels of FoxP39 , GITR and CTLA-411 and low levels of CD12712 . Following FACS of each population, resultant cells can be used in a suppressor assay which evaluates the relative ability to retard the proliferation of carboxyfluorescein succinimidyl ester (CFSE)-labeled autologous T cells.

Generation of Induced Regulatory T Cells from Primary Human Na&#239;ve and Memory T Cells

We describe a method for generating regulatory, memory and na&#239;ve T cells from a single human blood donor. Polarized Tregs can be then compared to other subsets in a variety of genetic and functional applications with genetic homogeneity, including a suppression assay also detailed here.

The development and maintenance of immunosuppressive CD4+ regulatory T cells (Tregs) contribute to the peripheral tolerance needed to remain in ...

Isolation and Characterization of Dendritic Cells and Macrophages from the Mouse Intestine

Within the intestine reside unique populations of innate and adaptive immune cells that are involved in promoting tolerance towards commensal flora and food antigens while concomitantly remaining poised to mount inflammatory responses toward invasive pathogens1,2. Antigen presenting cells, particularly DCs and macrophages, play critical roles in maintaining intestinal immune homeostasis via their ability to sense and appropriately respond to the microbiota3-14. Efficient isolation of intestinal DCs and macrophages is a critical step in characterizing the phenotype and function of these cells. While many effective methods of isolating intestinal immune cells, including DCs and macrophages, have been described6,10,15-24, many rely upon long digestions times that may negatively influence cell surface antigen expression, cell viability, and/or cell yield. Here, we detail a methodology for the rapid isolation of large numbers of viable, intestinal DCs and macrophages. Phenotypic characterization of intestinal DCs and macrophages is carried out by directly staining isolated intestinal cells with specific fluorescence-labeled monoclonal antibodies for multi-color flow cytometric analysis. Furthermore, highly pure DC and macrophage populations are isolated for functional studies utilizing CD11c and CD11b magnetic-activated cell sorting beads followed by cell sorting.

Here, we detail a methodology for the rapid isolation of mouse intestinal dendritic cells (DCs) and macrophages. Phenotypic characterization of intestinal DCs and macrophages is performed using multi-color flow cytometric analysis while magnetic bead enrichment followed by cell sorting is used to yield highly pure populations for functional studies.

Within the intestine reside unique populations of innate and adaptive immune cells that are involved in promoting tolerance towards commensal flora and ...

Isolating And Immunostaining Lymphocytes and Dendritic Cells from Murine Peyer's Patches

Peyer's patches (PPs) are integral components of the gut-associated lymphoid tissues (GALT) and play a central role in intestinal immunosurveillance and homeostasis. Particulate antigens and microbes in the intestinal lumen are continuously sampled by PP M cells in the follicle-associated epithelium (FAE) and transported to an underlying network of dendritic cells (DCs), macrophages, and lymphocytes. In this article, we describe protocols in which murine PPs are (i) dissociated into single cell suspensions and subjected to flow cytometry and (ii) prepared for cryosectioning and immunostaining. For flow cytometry, PPs are mechanically dissociated and then filtered through 70 &mu;m membranes to generate single cell suspensions free of epithelial cells and large debris. Starting with 20-25 PPs (from four mice), this quick and reproducible method yields a population of &gt;2.5 x 106 cells with &gt;90% cell viability. For cryosectioning, freshly isolated PPs are immersed in Optimal Cutting Temperature (OCT) medium, snap-frozen in liquid nitrogen, and then sectioned using a cryomicrotome. Tissue sections (5-12 &mu;m) are air-dried, fixed with acetone or methanol, and then subjected to immunolabeling.

There is an increasing interest in understanding the immunological functions of specific subpopulations of cells in Peyer's patches (PPs), the primary inductive sites of gut-associated lymphoid tissues. Here we outline parallel protocols for preparing PP single cell preparations for flow cytometric analysis and PP cryosections for immunostaining.

Peyer's  patches (PPs) are integral components of the gut-associated lymphoid tissues  (GALT) and play a central role in intestinal immunosurveillance and ...

Isolation of Myeloid Dendritic Cells and Epithelial Cells from Human Thymus

In this protocol we provide a method to isolate dendritic cells (DC) and epithelial cells (TEC) from the human thymus. DC and TEC are the major antigen presenting cell (APC) types found in a normal thymus and it is well established that they play distinct roles during thymic selection. These cells are localized in distinct microenvironments in the thymus and each APC type makes up only a minor population of cells. To further understand the biology of these cell types, characterization of these cell populations is highly desirable but due to their low frequency, isolation of any of these cell types requires an efficient and reproducible procedure. This protocol details a method to obtain cells suitable for characterization of diverse cellular properties. Thymic tissue is mechanically disrupted and after different steps of enzymatic digestion, the resulting cell suspension is enriched using a Percoll density centrifugation step. For isolation of myeloid DC (CD11c+), cells from the low-density fraction (LDF) are immunoselected by magnetic cell sorting. Enrichment of TEC populations (mTEC, cTEC) is achieved by depletion of hematopoietic (CD45hi) cells from the low-density Percoll cell fraction allowing their subsequent isolation via fluorescence activated cell sorting (FACS) using specific cell markers. The isolated cells can be used for different downstream applications.

This protocol details a method to isolate antigen presenting cells from human thymus via different steps of enzymatic digestion of the tissue followed by density centrifugation of the single cell suspension and finally magnetic and/or FACS sorting of the cell populations of interest.

In this protocol we provide a method to isolate dendritic cells (DC) and epithelial cells (TEC) from the human thymus. DC and TEC are the major antigen ...

Isolation of Double Negative &#945;&#946; T Cells from the Kidney

There is currently no standard protocol for the isolation of DN T cells from the non-lymphoid tissues despite their increasingly reported involvement in various immune responses. DN T cells are a unique immune cell type that has been implicated in regulating immune and autoimmune responses and tolerance to allotransplants1-6. DN T cells are, however, rare in peripheral blood and secondary lymphoid organs (spleen and lymph nodes), but are major residents of the normal kidney. Very little is known about their pathophysiologic function7 due to their paucity in the periphery. We recently described a comprehensive phenotypic and functional analysis of this population in the kidney8 in steady state and during ischemia reperfusion injury. Analysis of DN T cell function will be greatly enhanced by developing a protocol for their isolation from the kidney.
Here, we describe a novel protocol that allows isolation of highly pure ab CD4+ CD8+ T cells and DN T cells from the murine kidney. Briefly, we digest kidney tissue using collagenase and isolate kidney mononuclear cells (KMNC) by density gradient. This is followed by two steps to enrich hematopoietic T cells from 3% to 70% from KMNC. The first step consists of a positive selection of hematopoietic cells using a CD45+ isolation kit. In the second step, DN T cells are negatively isolated by removal of non-desired cells using CD4, CD8, and MHC class II monoclonal antibodies and CD1d &#945;-galcer tetramer. This strategy leads to a population of more than 90% pure DN T cells. Surface staining with the above mentioned antibodies followed by FACs analysis is used to confirm purity.

DNabT cells are rare among peripheral T cells; however, they are abundant in certain non-lymphoid tissues. Difficulty of isolating DN T cells from non-lymphoid tissue hinders their functional analysis despite increasing recognized pathophysiologic significance. We describe a novel protocol for isolation of highly purified DN T cells from murine kidney.

There is currently no standard protocol for the isolation of DN T cells from the non-lymphoid tissues despite their increasingly reported involvement in ...

Assessing the Innate Sensing of HIV-1 Infected CD4+ T Cells by Plasmacytoid Dendritic Cells Using an Ex vivo Co-culture System.

HIV-1 innate sensing requires direct contact of infected CD4+ T cells with plasmacytoid dendritic cells (pDCs). In order to study this process, the protocols described here use freshly isolated human peripheral blood mononuclear cells (PBMCs) or plasmacytoid dendritic cells (pDCs) to sense infections in either T cell line (MT4) or heterologous primary CD4+ T cells. In order to ensure proper sensing, it is essential that PBMC are isolated immediately after blood collection and that optimal percentage of infected T cells are used. Furthermore, multi-parametric flow cytometric staining can be used to confirm that PBMC samples contain the different cell lineages at physiological ratios. A number of controls can also be included to evaluate viability and functionality of pDCs. These include, the presence of specific surface markers, assessing cellular responses to known agonist of Toll-Like Receptors (TLR) pathways, and confirming a lack of spontaneous type-I interferon (IFN) production. In this system, freshly isolated PBMCs or pDCs are co-cultured with HIV-1 infected cells in 96 well plates for 18-22 hr. Supernatants from these co-cultures are then used to determine the levels of bioactive type-I IFNs by monitoring the activation of the ISGF3 pathway in HEK-Blue IFN-&#945;/&#946; cells. Prior and during co-culture conditions, target cells can be subjected to flow cytometric analysis to determine a number of parameters, including the percentage of infected cells, levels of specific surface markers, and differential killing of infected cells. Although, these protocols were initially developed to follow type-I IFN production, they could potentially be used to study other imuno-modulatory molecules released from pDCs and to gain further insight into the molecular mechanisms governing HIV-1 innate sensing.

Assessing the Innate Sensing of HIV-1 Infected CD4+ T Cells by Plasmacytoid Dendritic Cells Using an Ex vivo Co-culture System.

Unlike cell-free HIV-1 particles, infected CD4+ T cells are effectively sensed by plasmacytoid dendritic cells (pDCs). This manuscript describes a method where peripheral blood mononuclear cells (PBMCs) or isolated pDCs are co-cultured with HIV-1 infected T cells to evaluate innate sensing by pDCs as assessed by release of type-I IFN.

HIV-1 innate sensing requires direct contact of infected CD4+ T cells with plasmacytoid dendritic cells (pDCs). In order to study this process, the ...

Derivation of T Cells In Vitro from Mouse Embryonic Stem Cells

The OP9/OP9-DL1 co-culture system has become a well-established method for deriving differentiated blood cell types from embryonic and hematopoietic progenitors of both mouse and human origin. It is now used to address a growing variety of complex genetic, cellular and molecular questions related to hematopoiesis, and is at the cutting edge of efforts to translate these basic findings to therapeutic applications. The procedures are straightforward and routinely yield robust results. However, achieving successful hematopoietic differentiation in vitro requires special attention to the details of reagent and cell culture maintenance. Furthermore, the protocol features technique sensitive steps that, while not difficult, take care and practice to master. Here we focus on the procedures for differentiation of T lymphocytes from mouse embryonic stem cells (mESC). We provide a detailed protocol with discussions of the critical steps and parameters that enable reproducibly robust cellular differentiation in vitro. It is in the interest of the field to consider wider adoption of this technology, as it has the potential to reduce animal use, lower the cost and shorten the timelines of both basic and translational experimentation.

Derivation of T Cells In Vitro from Mouse Embryonic Stem Cells

Mouse embryonic stem cells can be differentiated to T cells in vitro using the OP9-DL1 co-culture system. Success in this procedure requires careful attention to reagent/cell maintenance, and key technique sensitive steps. Here we discuss these critical parameters and provide a detailed protocol to encourage adoption of this technology.

The OP9/OP9-DL1 co-culture system has become a well-established method for deriving differentiated blood cell types from embryonic and hematopoietic ...

Generation of Human Alloantigen-specific T Cells from Peripheral Blood

The study of human T lymphocyte biology often involves examination of responses to activating ligands. T cells recognize and respond to processed peptide antigens presented by MHC (human ortholog HLA) molecules through the T cell receptor (TCR) in a highly sensitive and specific manner. While the primary function of T cells is to mediate protective immune responses to foreign antigens presented by self-MHC, T cells respond robustly to antigenic differences in allogeneic tissues. T cell responses to alloantigens can be described as either direct or indirect alloreactivity. In alloreactivity, the T cell responds through highly specific recognition of both the presented peptide and the MHC molecule. The robust oligoclonal response of T cells to allogeneic stimulation reflects the large number of potentially stimulatory alloantigens present in allogeneic tissues. While the breadth of alloreactive T cell responses is an important factor in initiating and mediating the pathology associated with biologically-relevant alloreactive responses such as graft versus host disease and allograft rejection, it can preclude analysis of T cell responses to allogeneic ligands. To this end, this protocol describes a method for generating alloreactive T cells from naive human peripheral blood leukocytes (PBL) that respond to known peptide-MHC (pMHC) alloantigens. The protocol applies pMHC multimer labeling, magnetic bead enrichment and flow cytometry to single cell in vitro culture methods for the generation of alloantigen-specific T cell clones. This enables studies of the biochemistry and function of T cells responding to allogeneic stimulation.

This article describes a method for the generation and propagation of human T cell clones that specifically respond to a defined alloantigen. This protocol can be adapted for cloning human T cells specific for a variety of peptide-MHC ligands.

The study of human T lymphocyte biology often involves examination of responses to activating ligands. T cells recognize and respond to processed peptide ...