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><tbody><tr><td>RPMI</td><td>Fisher Scientific</td><td>SH3025501</td><td></td></tr><tr><td>r-GMCSF</td><td>Peprotech</td><td>315-03</td><td></td></tr><tr><td>LPS</td><td>SIGMA</td><td>L2630-10mg</td><td></td></tr><tr><td>Na Pyruvate</td><td>Thermo Scientific</td><td>SH3023901</td><td></td></tr><tr><td>2-ME</td><td>Gibco</td><td>31350-010</td><td></td></tr><tr><td>OVAp OTII (323&amp;ndash;339)</td><td>GenScript</td><td></td><td></td></tr><tr><td>Cell Strainer 70uM</td><td>BD</td><td>352350</td><td></td></tr><tr><td>&amp;nbsp;30 uM Syringe Filcons Sterile</td><td>BD</td><td>340598</td><td></td></tr><tr><td>AutoMacs Classic</td><td>Miltenyi Biotec</td><td>130-088-887</td><td></td></tr><tr><td>Gentamicin</td><td>Normon</td><td>624601.6</td><td></td></tr><tr><td>Transwell</td><td>Costar</td><td>3415</td><td></td></tr><tr><td>LB</td><td>Pronadisa</td><td>1231</td><td></td></tr><tr><td>Agar</td><td>Pronadisa</td><td>1800</td><td></td></tr><tr><td>Paraformaldehyde 16%</td><td>Electron Microscopy Sciences</td><td>15710</td><td></td></tr><tr><td>Triton X-100</td><td></td><td></td><td></td></tr><tr><td>CD8 biot</td><td>BD Biosciences</td><td>553029</td><td></td></tr><tr><td>IgM Biot</td><td>ImmunoStep</td><td>Clone RMM-1</td><td></td></tr><tr><td>B220 Biot</td><td>BD Biosciences</td><td>553086</td><td></td></tr><tr><td>CD19 biot</td><td>BD Biosciences</td><td>553784</td><td></td></tr><tr><td>MHC-II Biot (I-A/I-E)</td><td>BD Biosciences</td><td>553622</td><td></td></tr><tr><td>CD11b biot</td><td>Immunostep</td><td>11BB-01mg</td><td></td></tr><tr><td>CD11c biot</td><td>Immunostep</td><td>11CB3-01mg</td><td></td></tr><tr><td>DX5 biot</td><td>BD Biosciences</td><td>553856</td><td></td></tr><tr><td>Gr-1 biot</td><td>BD Biosciences</td><td>553125</td><td></td></tr><tr><td>CD16/CD32</td><td>ImmunoStep</td><td>M16PU-05MG</td><td></td></tr><tr><td>anti Salmonella</td><td>ABD Serotec</td><td>8209-4006</td><td></td></tr><tr><td>CD11cPE</td><td>BD Biosciences</td><td>553802</td><td></td></tr><tr><td>CD4-APC</td><td>Tonbo Biosciences</td><td>20-0041-U100</td><td></td></tr><tr><td>Gr-1 APC</td><td>BD Biosciences</td><td>553129</td><td></td></tr><tr><td>MHC-II (I-A/I-E) FITC</td><td>BD Biosciences</td><td>553623</td><td></td></tr><tr><td>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><td>CMAC (7-amino-4-chloromethylcoumarin)</td><td>Life technologies</td><td>C2110</td><td></td></tr><tr><td>BSA</td><td>SIGMA</td><td>A7030-100G</td><td></td></tr><tr><td>Streptavidin MicroBeads</td><td>Miltenyi Biotec</td><td>130-048-101</td><td></td></tr><tr><td>BD FACSCanto II</td><td>BD Biosciences</td><td></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.bdigitaluss.remotexs.co/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.bdigitaluss.remotexs.co/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.bdigitaluss.remotexs.co/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.bdigitaluss.remotexs.co/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.bdigitaluss.remotexs.co/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

Universidad San Sebastian

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

Transnuclear Mice with Pre-defined T Cell Receptor Specificities Against Toxoplasma gondii Obtained Via SCNT

Lymphocytes, such as T cells, undergo genetic V(D)J recombination, to generate a receptor with a certain specificity1. Mice transgenic for a rearranged antigen-specific T cell receptor (TCR) have been an indispensable tool to study T cell development and function. However, such TCRs are usually isolated from the relevant T cells after long-term culture often following repeated antigen stimulation, which unavoidably selects for T cells with high affinity. Random genomic integration of the TCR &alpha;- and &beta;-chain and expression from non-endogenous promoters can lead to variations in expression level and kinetics.
Epigenetic reprogramming via somatic cell nuclear transfer provides a tool to generate embryonic stem cells and mice from any cell of interest. Consequently, when SCNT is applied to T cells of known specificity, these genetic V(D)J rearrangements are transferred to the SCNT-embryonic stem cells (ESCs) and the mice derived from them, while epigenetic marks are reset. We have demonstrated that T cells with pre-defined specificities against Toxoplasma gondii can be used to generate mouse models that express the specific TCR from their endogenous loci, without experimentally introduced genetic modification. The relative ease and speed with which such transnuclear models can be obtained holds promise for the construction of other disease models.

Transnuclear Mice with Pre-defined T Cell Receptor Specificities Against Toxoplasma gondii Obtained Via SCNT

We demonstrate here that epigenetic reprogramming via Somatic Cell Nuclear Transfer (SCNT) can be used as a tool to generate mouse models with pre-defined T cell receptor (TCR) specificities. These transnuclear mice express the corresponding TCR from their endogenous locus under the control of the endogenous promoter.

Lymphocytes, such as T cells, undergo genetic V(D)J recombination, to generate a receptor with a certain specificity1. Mice transgenic for a rearranged ...

Retroviral Transduction of T-cell Receptors in Mouse T-cells

T-cell receptors (TCRs) play a central role in the immune system. TCRs on T-cell surfaces can specifically recognize peptide antigens presented by antigen presenting cells (APCs)1. This recognition leads to the activation of T-cells and a series of functional outcomes (e.g. cytokine production, killing of the target cells). Understanding the functional role of TCRs is critical to harness the power of the immune system to treat a variety of immunology related diseases (e.g. cancer or autoimmunity).
It is convenient to study TCRs in mouse models, which can be accomplished in several ways. Making TCR transgenic mouse models is costly and time-consuming and currently there are only a limited number of them available2-4. Alternatively, mice with antigen-specific T-cells can be generated by bone marrow chimera. This method also takes several weeks and requires expertise5. Retroviral transduction of TCRs into in vitro activated mouse T-cells is a quick and relatively easy method to obtain T-cells of desired peptide-MHC specificity. Antigen-specific T-cells can be generated in one week and used in any downstream applications. Studying transduced T-cells also has direct application to human immunotherapy, as adoptive transfer of human T-cells transduced with antigen-specific TCRs is an emerging strategy for cancer treatment6.
Here we present a protocol to retrovirally transduce TCRs into in vitro activated mouse T-cells. Both human and mouse TCR genes can be used. Retroviruses carrying specific TCR genes are generated and used to infect mouse T-cells activated with anti-CD3 and anti-CD28 antibodies. After in vitro expansion, transduced T-cells are analyzed by flow cytometry.

We present a protocol to produce antigen-specific mouse T-cells using retroviral transduction

T-cell receptors (TCRs) play a central role in the immune system. TCRs on T-cell surfaces can specifically recognize peptide antigens presented by antigen ...

piggyBac Transposon System Modification of Primary Human T Cells

The piggyBac transposon system is naturally active, originally derived from the cabbage looper moth1,2. This non-viral system is plasmid based, most commonly utilizing two plasmids with one expressing the piggyBac transposase enzyme and a transposon plasmid harboring the gene(s) of interest between inverted repeat elements which are required for gene transfer activity. PiggyBac mediates gene transfer through a "cut and paste" mechanism whereby the transposase integrates the transposon segment into the genome of the target cell(s) of interest. PiggyBac has demonstrated efficient gene delivery activity in a wide variety of insect1,2, mammalian3-5, and human cells6 including primary human T cells7,8. Recently, a hyperactive piggyBac transposase was generated improving gene transfer efficiency9,10.
Human T lymphocytes are of clinical interest for adoptive immunotherapy of cancer11. Of note, the first clinical trial involving transposon modification of human T cells using the Sleeping beauty transposon system has been approved12. We have previously evaluated the utility of piggyBac as a non-viral methodology for genetic modification of human T cells. We found piggyBac to be efficient in genetic modification of human T cells with a reporter gene and a non-immunogenic inducible suicide gene7. Analysis of genomic integration sites revealed a lack of preference for integration into or near known proto-oncogenes13. We used piggyBac to gene-modify cytotoxic T lymphocytes to carry a chimeric antigen receptor directed against the tumor antigen HER2, and found that gene-modified T cells mediated targeted killing of HER2-positive tumor cells in vitro and in vivo in an orthotopic mouse model14. We have also used piggyBac to generate human T cells resistant to rapamycin, which should be useful in cancer therapies where rapamycin is utilized15.
Herein, we describe a method for using piggyBac to genetically modify primary human T cells. This includes isolation of peripheral blood mononuclear cells (PBMCs) from human blood followed by culture, gene modification, and activation of T cells. For the purpose of this report, T cells were modified with a reporter gene (eGFP) for analysis and quantification of gene expression by flow cytometry.
PiggyBac can be used to modify human T cells with a variety of genes of interest. Although we have used piggyBac to direct T cells to tumor antigens14, we have also used piggyBac to add an inducible safety switch in order to eliminate gene modified cells if needed7. The large cargo capacity of piggyBac has also enabled gene transfer of a large rapamycin resistant mTOR molecule (15 kb)15. Therefore, we present a non-viral methodology for stable gene-modification of primary human T cells for a wide variety of purposes.

piggyBac Transposon System Modification of Primary Human T Cells

We describe a method to genetically modify primary human T cells with a transgene using the non-viral piggyBac transposon system. T cells modified to using the piggyBac transposon system exhibit stable transgene expression.

The piggyBac transposon system is naturally active, originally derived from the cabbage looper moth1,2. This non-viral system is plasmid based, most ...

Adenoviral Transduction of Naive CD4 T Cells to Study Treg Differentiation

Regulatory T cells (Tregs) are essential to provide immune tolerance to self as well as to certain foreign antigens. Tregs can be generated from naive CD4 T cells in vitro with TCR- and co-stimulation in the presence of TGF&beta; and IL-2. This bears enormous potential for future therapies, however, the molecules and signaling pathways that control differentiation are largely unknown.
Primary T cells can be manipulated through ectopic gene expression, but common methods fail to target the most important naive state of the T cell prior to primary antigen recognition. Here, we provide a protocol to express ectopic genes in naive CD4 T cells in vitro before inducing Treg differentiation. It applies transduction with the replication-deficient adenovirus and explains its generation and production. The adenovirus can take up large inserts (up to 7 kb) and can be equipped with promoters to achieve high and transient overexpression in T cells. It effectively transduces naive mouse T cells if they express a transgenic Coxsackie adenovirus receptor (CAR). Importantly, after infection the T cells remain naive (CD44low, CD62Lhigh) and resting (CD25-, CD69-) and can be activated and differentiated into Tregs similar to non-infected cells. Thus, this method enables manipulation of CD4 T cell differentiation from its very beginning. It ensures that ectopic gene expression is already in place when early signaling events of the initial TCR stimulation induces cellular changes that eventually lead into Treg differentiation.

Adenoviral gene transfer into naive CD4 T cells with transgenic expression of the Coxsackie adenovirus receptor enables the molecular analysis of regulatory T cell differentiation in vitro.

Regulatory T cells (Tregs) are essential to provide immune tolerance to self as well as to certain foreign antigens. Tregs can be generated from naive CD4 ...

A Tetracycline-regulated Cell Line Produces High-titer Lentiviral Vectors that Specifically Target Dendritic Cells

Lentiviral vectors (LVs) are a powerful means of delivering genetic material to many types of cells. Because of safety concerns associated with these HIV-1 derived vectors, producing large quantities of LVs is challenging. In this paper, we report a method for producing high titers of self-inactivating LVs. We retrovirally transduce the tet-off stable producer cell line GPR to generate a cell line, GPRS, which can express all the viral components, including a dendritic cell-specific glycoprotein, SVGmu. Then, we use concatemeric DNA transfection to transfect the LV transfer plasmid encoding a reporter gene GFP in combination with a selectable marker. Several of the resulting clones can produce LV at a titer 10-fold greater than what we achieve with transient transfection. Plus, these viruses efficiently transduce dendritic cells in vitro and generate a strong T cell immune response to our reporter antigen. This method may be a good option for producing strong LV-based vaccines for clinical studies of cancer or infectious diseases.

Here, we use retroviral transduction and concatemeric transfection to create a cell line that can express the components of a lentiviral vector (LV) in the absence of tetracycline. This LV encodes GFP and is pseudotyped with a glycoprotein, SVGmu, which is specific for a receptor on dendritic cells.

Lentiviral  vectors (LVs) are a powerful means of delivering genetic material to many types  of cells. Because of safety concerns associated with these ...

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 ...

Induction of Murine Intestinal Inflammation by Adoptive Transfer of Effector CD4+CD45RBhigh T Cells into Immunodeficient Mice

There are many different animal models available for studying the pathogenesis of human inflammatory bowel diseases (IBD), each with its own advantages and disadvantages. We describe here an experimental colitis model that is initiated by adoptive transfer of syngeneic splenic CD4+CD45RBhigh T cells into T and B cell deficient recipient mice. The CD4+CD45RBhigh T cell population that largely consists of na&#239;ve effector cells is capable of inducing chronic intestinal inflammation, closely resembling key aspects of human IBD. This method can be manipulated to study aspects of disease onset and progression. Additionally it can be used to study the function of innate, adaptive, and regulatory immune cell populations, and the role of environmental exposures, i.e., the microbiota, in intestinal inflammation. In this article we illustrate the methodology for inducing colitis with a step-by-step protocol. This includes a video demonstration of key technical aspects required to successfully develop this murine model of experimental colitis for research purposes.

Induction of Murine Intestinal Inflammation by Adoptive Transfer of Effector CD4+CD45RBhigh T Cells into Immunodeficient Mice

Here, we present a protocol to induce colonic inflammation in mice by adoptive transfer of syngeneic CD4+CD45RBhigh T cells into T and B cell deficient recipients. Clinical and histopathological features mimic human inflammatory bowel diseases. This method allows the study of the initiation of colonic inflammation and progression of disease.

There are many different animal models available for studying the pathogenesis of human inflammatory bowel diseases (IBD), each with its own advantages ...

Generation of Human Monocyte-derived Dendritic Cells from Whole Blood

Dendritic cells (DCs) recognize foreign structures of different pathogens, such as viruses, bacteria, and fungi, via a variety of pattern recognition receptors (PRRs) expressed on their cell surface and thereby activate and regulate immunity. 
The major function of DCs is the induction of adaptive immunity in the lymph nodes by presenting antigens via MHC I and MHC II molecules to na&#239;ve T lymphocytes. Therefore, DCs have to migrate from the periphery to the lymph nodes after the recognition of pathogens at the sites of infection. For in vitro experiments or DC vaccination strategies, monocyte-derived DCs are routinely used. These cells show similarities in physiology, morphology, and function to conventional myeloid dendritic cells. They are generated by interleukin 4 (IL-4) and granulocyte-macrophage colony-stimulating factor (GM-CSF) stimulation of monocytes isolated from healthy donors. Here, we demonstrate how monocytes are isolated and stimulated from anti-coagulated human blood after peripheral blood mononuclear cell (PBMC) enrichment by density gradient centrifugation. Human monocytes are differentiated into immature DCs and are ready for experimental procedures in a non-clinical setting after 5 days of incubation.

Here, we demonstrate how monocytes are isolated by magnetic bead separation from peripheral blood mononuclear cells after density gradient centrifugation of human anti-coagulated blood. Following incubation for 5 days, human monocytes are differentiated into immature dendritic cells and are ready for experimental procedures in a non-clinical setting.

Dendritic cells (DCs) recognize foreign structures of different pathogens, such as viruses, bacteria, and fungi, via a variety of pattern recognition ...

Transduction and Expansion of Primary T Cells in Nine Days with Maintenance of Central Memory Phenotype

Emerging immunotherapies to treat infectious diseases and cancers often involve transduction of cellular populations with genes encoding disease-targeting proteins. For example, chimeric antigen receptor (CAR)-T cells to treat cancers and viral infections involve the transduction of T cells with synthetic genes encoding CAR molecules. The CAR molecules make the T cells specifically recognize and kill cancer or virally infected cells. Cells can also be co-transduced with other genes of interest. For example, cells can be co-transduced with genes encoding proteins that target cells to specific locations. Here, we present a protocol to transduce primary peripheral blood mononuclear cells (PBMCs) with genes encoding a virus-specific CAR and the B cell follicle homing molecule chemokine receptor type 5 (CXCR5). This procedure takes nine days and results in transduced T cell populations that maintain a central memory phenotype. Maintenance of a central memory or less differentiated phenotype has been shown to associate with persistence of cells post-infusion. Furthermore, cells produced with this method show high levels of viability, high levels of co-expression of the two transduced genes, and large enough quantities of cells for immunotherapeutic infusion. This nine-day protocol may be broadly used for CAR-T cell and other T cell immunotherapy approaches. The methods described here are based on studies presented in our previous publications.

We outline a 9-day protocol for the transduction and expansion of rhesus macaque peripheral blood mononuclear cells which yields cells with excellent co-expression of the genes of interest in sufficient number for infusion studies of cell efficacy.

Emerging immunotherapies to treat infectious diseases and cancers often involve transduction of cellular populations with genes encoding disease-targeting ...

Bioengineering

Induction of Intestinal Inflammation by Adoptive Transfer of CBir1 TCR Transgenic CD4+ T Cells to Immunodeficient Mice

With the increase of incidence, inflammatory bowel diseases (IBD), which are chronic diseases affecting the gastrointestinal tract, impose a considerable health and financial burden on individuals and society. Therefore, it is critical to investigate the mechanisms underlying the pathogenesis and development of IBD. Here, a gut microbiota antigen-specific T cell transfer colitis model is described. CBir1 flagellin has been recognized as the immunodominant gut bacterial antigen in experimental colitis and patients with Crohn&#39;s disease. CBir1 TCR transgenic na&#970;ve CD4+ T cells, specific to CBir1 flagellin, can induce chronic colitis after adoptive transfer into immune-deficient Rag1-/- mice. The disease severity is assessed by histopathology. The CD4+ T cell phenotypes in colonic lamina propria are also determined. This model closely resembles the development of IBD, which provides an ideal murine model for investigating the mechanisms driving the pathogenesis of IBD and testing the potential drugs for treating IBD.

Induction of Intestinal Inflammation by Adoptive Transfer of CBir1 TCR Transgenic CD4+ T Cells to Immunodeficient Mice

In this protocol, a gut microbiota antigen-specific T cell adoptive transfer colitis model is described. CD4+ T cells are isolated from CBir1 TCR transgenic mice. These are specific for an immunodominant gut microbiota antigen CBir1 flagellin, which is transferred into recipient Rag1-/- mice, leading to intestinal inflammation.

With the increase of incidence, inflammatory bowel diseases (IBD), which are chronic diseases affecting the gastrointestinal tract, impose a considerable ...

T Cells Capture Bacteria by Transinfection from Dendritic Cells

Summary

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Induction of Murine Intestinal Inflammation by Adoptive Transfer of Effector CD4⁺CD45RB^high T Cells into Immunodeficient Mice

Generation of Human Monocyte-derived Dendritic Cells from Whole Blood

Transduction and Expansion of Primary T Cells in Nine Days with Maintenance of Central Memory Phenotype

Induction of Intestinal Inflammation by Adoptive Transfer of CBir1 TCR Transgenic CD4⁺ T Cells to Immunodeficient Mice