We thank Dr. Simon Bartlett for English editing. This study was supported by grants from Instituto de Salud Carlos III (ISCIII) (PI14/00526; PI17/01395; CP11/00145; CPII16/00022), the Miguel Servet Program and Fundaci&#243;n Ram&#243;n Areces; with co-funding from the Fondo Europeo de Desarrollo Regional (FEDER). The CNIC is supported by the Ministry of Economy, Industry and Competitiveness (MEIC), and the Pro CNIC Foundation and is a Severo Ochoa Center of Excellence (SEV-2015-0505). RTF is founded by Fundaci&#243;n Ram&#243;n Areces and CNIC, VZG by ISCIII, BHF by Instituto de Investigaci&#243;n Sanitaria Hospital 12 de Octubre (imas12) and JMG-G by the ISCIII Miguel Servet Program and imas12.

Experimental procedures were approved by the Fundaci&#243;n Centro Nacional de Investigaciones Cardiovasculares Carlos III (CNIC) and the Comunidad Aut&#243;noma de Madrid in accordance with Spanish and European guidelines. Mice were bred in specific pathogen free (SPF) conditions and were euthanized by carbon dioxide (CO2) inhalation.
1. Isolation of Mouse Bone Marrow Cells from Tibias and Femurs
NOTE: The C57BL/6 congenic mouse strain carries the differential leukocyte marker Ptprca, generally recognized as CD45.1 or Ly5.1, whereas wild-type C57BL strains carry the Ptprcb allele, known as CD45.2 or Ly5.2. CD45.1 and CD45.2 variants can be distinguished by flow cytometry using antibodies. CD45.1, CD45.2, and CD45.1/CD45.2 mice can be used as cell sources or as recipients for adoptive transfer, permitting tracing of the distinct cell populations by flow cytometry. Preferentially use age-and sex-matched male or female mice below 12 weeks of age.
<ol>
	<li>Preparation of Femurs and Tibias
	<ol>
		<li>Euthanize mice using the protocol approved by the institutional animal care committee.</li>
		<li>Disinfect the hind limbs by spraying the animal surface with 70% ethanol.</li>
		<li>Use sterile scissors, forceps and scalpels. With a scalpel, make a cut in the skin and remove the skin from the distal part of the mouse including the skin covering the posterior extremities. Peel the skin around the lower calf muscle and remove the skin from the legs entirely (Figure 2A, 2B).</li>
		<li>Separate the quadriceps muscle from the femur using a scalpel. Disarticulate the hip joint without breaking the femur head. Remove the muscles from the tibia using a scalpel (Figure 2C, 2D). Separate the femur from the tibia without breaking the bone ends.</li>
		<li>Keep the bones in a Petri dish containing 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin in ice-cold 1x Roswell Park Memorial Institute (RPMI) 1640 medium.</li>
	</ol>
	</li>
	<li>Cell Isolation 
	NOTE: All subsequent steps must be performed under a culture hood and with sterile material to avoid contamination.
	<ol>
		<li>In a sterile Petri dish, carefully cut off the proximal and distal ends of each bone with a scalpel.</li>
		<li>Flush the bones repeatedly with a total volume of 10 mL of warm complete RPMI medium (RPMI + 10% FBS, 2 mM EDTA, 1% penicillin/streptomycin, 20 mM HEPES, 55 &#181;M 2-mercaptoethanol, 1 mM sodium pyruvate, and 2 mM L-glutamine). Flush the bones from both ends using a 25 G needle attached to a 1 mL syringe.</li>
		<li>Transfer the effluate to a 50 mL conical tube fitted with a 70 &#181;m nylon web filter. Carefully dislodge debris and cell conglomerates by gentle stirring and pipetting.</li>
		<li>Centrifuge the cell suspension at 250 x g for 10 min at room temperature (RT).</li>
		<li>Resuspend the cell pellet in 1 mL of cold red-blood-cell lysis buffer (0.15 M NH4Cl + 1 mM KHCO3 + 0.1 mM EDTA, adjusted pH to 7.2 with 1 N HCl, 0.4 &#181;m sterile filtered, and stored at 4 &#176;C). Maintain on ice for 5 min with the manual shaking.</li>
		<li>Add 10 mL of cold buffer (1x phosphate buffered saline (PBS) + 2 mM EDTA + 2% bovine serum albumin (BSA)) to inactivate and wash out the red-blood-cell lysis buffer.</li>
		<li>Centrifuge the cell suspension at 250 x g for 5 min at 4 &#176;C.</li>
		<li>Wash cells with 25 mL of complete RPMI medium and centrifuge at 250 x g for 5 min at 4 &#176;C.</li>
		<li>Resuspend the cells in 5 mL of complete RPMI medium. Mix 50 &#181;L of the cell suspension 1:1 with trypan blue. Determine the cell number in a counting chamber under a microscope. Calculate the cell number using the formula: Cells/mL = [(non-stained cell count/4) x 2 x 10,000]12. 
		NOTE: This method yields 40 x 106 to 60 x 106 bone marrow cells per uninfected 8 to 12 week-old C57BL/6 mouse.</li>
		<li>Centrifuge the cell suspension at 250 x g for 5 min at 4 &#176;C.</li>
	</ol>
	</li>
</ol>
2. DC Differentiation, Maturation and Antigen Loading
<ol>
	<li>BM Cell Harvesting
	<ol>
		<li>Seed the complete culture on 6-well ultra-low-attachment surface plates at 106 cells per mL of medium, typically in 5 mL per well. Differentiated macrophages will attach to the culture-plates. After 12 h, transfer the supernatant, which mainly contains monocytes, to clean 6-well plates, discarding the old 6-well plates with the adhered macrophages.</li>
		<li>To promote cell differentiation, culture the non-adherent cells at 5 x 105 cells per mL in typically 5 mL per well of complete RPMI medium containing 20 ng/mL commercially obtained recombinant murine GM-CSF at 37 &#176;C and 5% carbon dioxide (CO2) and observe daily.</li>
		<li>Every 3 days, spin down suspended cells and collect the adherent cells with 5 mM EDTA in PBS and spin down. Combine and resuspend at 5 x 105 cells/mL in fresh medium containing 20 ng/mL rm-GM-CSF.</li>
		<li>On the day 8, collect adherent and detached cells as above and resuspend at 5 x 105 cells/mL in medium containing 20 ng/mL GM-CSF and 20 ng/mL lipopolysaccharide (LPS) for 24 h in a non-tissue culture-treated sterile Petri dish, to induce high MHC-II, CD80 and CD86 expression.</li>
		<li>Check DC differentiation by flow cytometry as indicated in step 2.2.</li>
	</ol>
	</li>
	<li>Flow Cytometry Differentiation and Maturation Analysis.
	<ol>
		<li>Centrifuge the cell suspension at 250 x g for 5 min at 4 &#176;C. Repeat this step twice.</li>
		<li>Block Fc receptors by incubating the resuspended cell pellet in mouse Fc receptor blocker (purified anti mouse CD16/CD32 IgG2b antibody) for 15 min at 4 &#176;C according to the manufacturer&#39;s instructions.</li>
		<li>Centrifuge the cell suspension at 250 x g for 5 min at 4 &#176;C.</li>
		<li>For each probe, resuspend 250,000 cells in 300 &#181;L of flow cytometry buffer (1x PBS + 2 mM EDTA + 2% BSA) at 4 &#176;C.</li>
		<li>Transfer 30 &#181;L of cell solution per well to a 96-well-plate.</li>
		<li>Centrifuge the cell suspension at 250 x g for 5 min at 4 &#176;C.</li>
		<li>Discard the supernatant and add 30 &#181;L of antibody-containing buffer to each well following the manufacturer&#39;s indications. Incubate the cells with antibodies for 20 min at 4 &#176;C. 
		NOTE: Usually employed markers for DCs, monocytes, and macrophages are CD11b, CD64, CD115, CD11c, MHCII, CD80, and CD86 (Figure 3).</li>
		<li>Centrifuge the samples at 250 x g for 5 min at 4 &#176;C and resuspend in 200 &#181;L per well of cold flow cytometry buffer containing a marker to exclude dead cells (e.g., propidium iodide, 4&#8242;,6-diamidino-2-phenylindole (DAPI) or an amine-reactive dye) at the manufacturer&#39;s recommended concentration13.</li>
		<li>Monitor cell differentiation by transferring the samples to flow cytometry tubes and performing the flow cytometry analysis excluding dead cells on days 0, 3, 6, and 9.</li>
	</ol>
	</li>
	<li>DC Antigen loading.
	<ol>
		<li>On day 8, centrifuge the samples at 250 x g for 5 min at 4 &#176;C and resuspend the pellets in 200 &#181;L medium (RPMI + 10% FBS without antibiotics). Repeat this step twice.</li>
		<li>Incubate the DCs with 10 &#181;g/mL OVAp (323-339; ISQAVHAAHAEINEAGR) per 1 x 107 DCs in 1 mL of medium (RMPI + 1% FBS) in a plastic tube for at least 30 min at 37 &#176;C. Use non OVAp-loaded DCs as a negative control condition.</li>
		<li>Centrifuge the samples at 250 x g for 5 min at 4 &#176;C and resuspend the pellets in 200 &#181;L complete RPMI medium. Repeat this step twice.</li>
		<li>Centrifuge the samples at 250 x g for 5 min at 4 &#176;C and resuspend the pellets in 200 &#181;L medium (RPMI + 10% FBS) at 2 x 106 cells/mL.</li>
	</ol>
	</li>
</ol>
3. Isolation of Na&#239;ve CD4 T Cells from OTII Mice
NOTE: This method yields approximately 100 x 106 spleenocytes per uninfected 8 to 12 week-old C57BL/6 mouse. Both males and females can be used. CD4 T cells can be isolated from spleen or lymph nodes; CD4 T cells account for approximately 25% and 50% of cells in these organs, respectively. Perform the following steps in sterile conditions.
<ol>
	<li>Isolate inguinal, axillary, brachial, cervical, and mesenteric lymph nodes and the spleen from OTII transgenic mice (Figure 4) and transfer them to a plastic Petri dish containing 10 mL of complete RPMI medium.</li>
	<li>Rinse the cell strainer with 2 mL of complete RPMI medium and remove it from the 50 mL tube. Transfer lymph nodes and spleen to a 70 &#181;m cell strainer placed in a 50 mL tube. Homogenize using a syringe plunger (Figure 5) and add medium up to 15 mL.</li>
	<li>Centrifuge the samples at 250 x g for 5 min at 4 &#176;C and resuspend the pellet in 15 mL of complete RPMI medium. Repeat this step twice.</li>
	<li>For spleen cell suspension, resuspend the cell pellet and lyse the red blood cells as indicated in step 1.2.5.</li>
	<li>Centrifuge the cell suspension at 250 x g for 5 min at 4 &#176;C.</li>
	<li>Wash cells with 25 mL of complete RPMI medium and centrifuge at 250 x g for 5 min at RT.</li>
	<li>Resuspend the cells in 5 mL of medium. Mix 50 &#181;L of the cell suspension 1:1 with Trypan blue. Determine the cell number using a counting chamber under a microscope. Calculate the cell number using the formula: cells/mL = [(non-stained cell count/4) x 2 x 10,000]12.</li>
	<li>Repeat step 2.2.2.</li>
	<li>Following the manufacturer&#39;s instructions, incubate the cells with 1:800 dilutions of biotinylated antibodies to CD8&#945;, IgM, B220, CD19, MHCII (I-Ab), CD11b, CD11c, CD44, CD25, and DX5 for 30 min on ice for later negative selection of CD4 T cells.</li>
	<li>Following the manufacturer&#39;s instructions and based on the number of cells and the capacity of the beads, calculate the required amount of streptavidin-coated magnetic microbeads and isolate CD4 T cells using a magnetic cell separator and the appropriate separation columns.</li>
	<li>Resuspend the cells in 5 mL of complete RPMI medium and keep them on ice while counting live cells as described in step 3.7.</li>
	<li>Extract 2 x 105 cells and check the purity of the collected cells using anti CD4, CD3, B220 and CD8 fluorescent antibodies in a flow cytometer using manufacturer-recommending antibody dilutions.</li>
	<li>To stain isolated na&#239;ve CD4/OTII T cells, resuspend 106 cells in 500 &#181;L buffer (1x PBS + 2 mM EDTA + 2% BSA). Prepare 500 &#181;L buffer (1x PBS + 2 mM EDTA + 2% BSA) containing double the final manufacturer-recommended concentration of vital cell tracer dye. Mix both solutions by slowly adding the cell tracer solution to the cell suspension. Incubate the cells for 5 min at 37 &#176;C.</li>
	<li>Wash the cells with 5 mL of complete RPMI medium and centrifuge at 250 x g for 5 min at 4 &#176;C. Repeat this step twice. Resuspend the cells in 1 mL of complete RPMI medium at 1 x 107cells/mL.</li>
</ol>
4. In Vivo Activation, Proliferation and Th1 Differentiation Assay
NOTE: The vital cell tracer dye carboxyfluorescein diacetate succinimidyl ester (CFSE) and other succinimidyl ester-based dyes, which are excited and emit fluorescence at different wavelengths, are widely used to assess lymphocyte proliferation by flow cytometry due to their high fluorescence intensity, long-life, low variability, and low toxicity14.
<ol>
	<li>Adoptively transfer intravenously (i.v.) 1 x 106 live vital cell tracer dye-stained na&#239;ve CD4/OTII T cells in 100 &#181;L of PBS per mouse. Labeled T cells can be adoptively transferred by tail vein or retro-orbital injection15; in both cases, the cells will home to lymphoid organs (Figure 6A).</li>
	<li>Challenge the recipient mice one day later by adoptively transferring 100,000 LPS-matured OVAp-loaded DCs by subcutaneous injection (s.i.) in the footpads (Figure 6B).</li>
	<li>Harvest popliteal lymph nodes from recipient mice and process them until the obtainment of single cell suspensions following the same steps as previously described in 3.1 and 3.2. Do this on day 2 post-immunization to measure CD4/OTII T cell activation, and on day 5 to measure proliferation plus Th1 differentiation.</li>
	<li>To analyze T cell activation on day 2, stain cells with fluorescent antibodies against CD4, CD69, and CD25 (optionally CD45.1 and CD45.2) and determine the percentage of C69+CD25+ T cells in the CD4 population. Analyze by flow cytometry (Figure 7).</li>
	<li>To check T cell proliferation on day 5, stain cells using appropriate fluorescent antibodies against CD4 and optionally CD45.1 and CD45.2. Analyze the decay of the vital cell tracer dye signal in the CD4 population by flow cytometry (Figure 8). Several parameters can be determined in these studies, such as the number of cell divisions undergone by cells labelled with the vital cell tracer dye, the percentage of dividing cells in each fluorescence peak, and the percentage of dividing cells in the total cell population.</li>
	<li>To determine Th1 differentiation at day 5, plate 400,000 cells per well of a 96-well plate in 200 &#181;L of complete RPMI medium containing 1 &#181;M ionomycin and 10 ng/mL phorbol 12 myristate 13 acetate (PMA) for 6 h at 37 &#176;C in the incubator. For the last 4 h, add 3&#8211;10 &#181;g/mL brefeldin A to block cytokine secretion to the medium.</li>
	<li>Centrifuge the plates for 5 min at 250 x g at 4 &#176;C. Discard the supernatant by rapid inversion of the 96-well plate.</li>
	<li>Repeat step 2.2.2.</li>
	<li>Stain cell membranes by incubating for 15 min at 4 &#176;C in 30 &#181;L of buffer (1x PBS + 2 mM EDTA + 2% BSA) containing antibodies to CD4 and optionally to CD45.1 and CD45.2 at manufacturer-recommended dilutions. Wash the cells by adding 150 &#181;L of buffer (1x PBS + 2 mM EDTA + 2% BSA) and centrifuge the plates for 5 min at 250 x g at 4 &#176;C and discard supernatant.</li>
	<li>Fix the cells by adding 100 &#181;L of 2% paraformaldehyde/1% sucrose for 20 min at RT. Centrifuge the plates for 5 min at 1,000 x g and 4 &#176;C. Discard the supernatant.</li>
	<li>Permeabilize the cells by adding 50 &#181;L of intracellular permeabilization buffer (1x PBS + 0.1% BSA, 0.01 M HEPES, 0.3% saponin) and incubate for 30 min at 4 &#176;C.</li>
	<li>Add 150 &#181;L of PBS and centrifuge for 5 min at 1,000 x g at RT. Discard the supernatant.</li>
	<li>Stain intracellular cytokines by adding 30 &#181;L of fluorophore-conjugated anti IFN&#947; antibody in buffer (PBS + 0.1% saponin) and incubate for 30 min at RT.</li>
	<li>Wash the cells by adding 150 &#181;L of buffer (PBS + 0.1% saponin). Centrifuge the plates for 5 min at 1,000 x g at RT and discard the supernatant. Repeat this step twice.</li>
	<li>Analyze cell populations by flow cytometry (Figure 8).</li>
</ol>

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	<li>Zhu, J., Yamane, H., Paul, W. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differentiation+of+effector+CD4+T+cell+populations+(*).">Differentiation of effector CD4 T cell populations (*).</a> Annual Review of Immunology. 28, 445-489 (2010).</li><li>Bustos-Moran, E., Blas-Rus, N., Martin-Cofreces, N. B., Sanchez-Madrid, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Orchestrating+Lymphocyte+Polarity+in+Cognate+Immune+Cell-Cell+Interactions.">Orchestrating Lymphocyte Polarity in Cognate Immune Cell-Cell Interactions.</a> International Review of Cell and Molecular Biology. 327, 195-261 (2016).</li><li>Gonzalez-Granado, J. 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The authors have nothing to disclose

This protocol allows for the characterization of the capacity of BM-derived DCs to modulate the activation, proliferation, and differentiation of na&#239;ve CD4 T cells. Moreover, it can also be used to assess the susceptibility of CD4 T cells to modulation by BM-derived DCs. With this protocol, changes in these events can be measured in vivo.
Depending on the hypothesis under investigation, several combinations of T cells and DCs can be used. For example, one can analyze the conseque...

CD4 T cells and antigen presenting cells (APCs) such as DCs are required mediators of immunity to microbial pathogens1,2. In peripheral lymphoid organs, CD4 T cells are activated upon recognition of specific antigens presented by APCs3,4,5. Activated CD4 T cells proliferate and differentiate into distinct specific effector Th cells that are necessary for the development of a correct adaptive immune response6,7. Control of these processes is critical for producing an adequate adaptive defense that kills the pathogen without producing harmful tissue damage8. Th cells are defined according to the expression or production of surface molecules, transcription factors, and effector cytokines and perform essential and precise functions in response to pathogens1. Cells of the Th1 cell subset express the transcription factor T-bet and the cytokine interferon &#947; (IFN&#947;) and participate in host defense against intracellular pathogens1. Quantification of na&#239;ve CD4 T cell activation, proliferation, and Th1 differentiation is a useful means of assessing the role T cells play in an immune response.
This protocol enables in vivo analysis of the capacity of in vitro-generated BM-derived DCs to modulate the activation, proliferation, and Th1 differentiation of na&#239;ve CD4 T cells. The protocol also serves to assess the capacity of na&#239;ve CD4 T cells to be activated, induced to proliferate, and Th1 differentiated (Figure 1). This versatile protocol circumvents the inability to specifically manipulate or select the studied cell population in purely in vivo protocols. The effects of diverse molecules and treatments on DCs can be studied by using BM from genetically modified mice5 or by treating or genetically manipulating isolated BM cells9. Similarly, T cell responses can be explored by obtaining T cells for adoptive transfer from different sources or after several manipulations3,8,10.
The main advantages of this protocol are twofold. T cell activation, proliferation, and Th1 differentiation are analyzed with a flow cytometry approach; and this is combined with in vivo studies, thus averting alterations that may occur in vitro and including cell types and other factors only found in intact organs11.
The use of vital dyes is a widely used technique to track cell proliferation while avoiding the use of radioactivity. The measurement of proliferation with these reagents is based on dye dilution after cell division. Moreover, these dyes can be detected at multiple wavelengths and are easily analyzed by flow cytometry in combination with multiple fluorescent antibodies or markers. We highlight the utility of this protocol by showing how T cell activation, proliferation, and Th1 differentiation can be analyzed by flow cytometry.

<table>
	<tbody>
		<tr>
			<td>Ethanol</td>
			<td>VWR Chemicals</td>
			<td>20,824,365</td>
			<td>5 L</td>
		</tr>
		<tr>
			<td>Scissors</td>
			<td>Fine Science Tools (F.S.T.)</td>
			<td>14001-12</td>
			<td></td>
		</tr>
		<tr>
			<td>Forceps</td>
			<td>Fine Science Tools (F.S.T.)</td>
			<td>11000-13</td>
			<td></td>
		</tr>
		<tr>
			<td>Fine Forceps</td>
			<td>Fine Science Tools (F.S.T.)</td>
			<td>11253-20</td>
			<td></td>
		</tr>
		<tr>
			<td>Scalpel</td>
			<td>Fine Science Tools (F.S.T.)</td>
			<td>10020-00</td>
			<td>Box of 100 blades</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>SIGMA</td>
			<td>F7524</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/streptomycin</td>
			<td>LONZA</td>
			<td>DE17-602E</td>
			<td></td>
		</tr>
		<tr>
			<td>Roswell Park Memorial Institute medium (RPMI)</td>
			<td>GIBCO</td>
			<td>21875-034</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile Petri dishes</td>
			<td>Falcon</td>
			<td>353003</td>
			<td></td>
		</tr>
		<tr>
			<td>25-gauge needle</td>
			<td>BD Microlance 3</td>
			<td>300600</td>
			<td></td>
		</tr>
		<tr>
			<td>1 ml syringe</td>
			<td>Novico</td>
			<td>N15663</td>
			<td></td>
		</tr>
		<tr>
			<td>15 ml conical tubes</td>
			<td>Falcon</td>
			<td>352096</td>
			<td></td>
		</tr>
		<tr>
			<td>50 ml conical tubes</td>
			<td>Falcon</td>
			<td>352098</td>
			<td></td>
		</tr>
		<tr>
			<td>70 &#956;m nylon web filter</td>
			<td>BD Falcon</td>
			<td>352350</td>
			<td></td>
		</tr>
		<tr>
			<td>Red blood lysis buffer</td>
			<td>SIGMA</td>
			<td>R7757</td>
			<td>100 Ml</td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>SIGMA</td>
			<td>ED2SS-250</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin</td>
			<td>SIGMA</td>
			<td>A7906</td>
			<td>100 g</td>
		</tr>
		<tr>
			<td>Trypan blue</td>
			<td>SIGMA</td>
			<td>302643-25G</td>
			<td></td>
		</tr>
		<tr>
			<td>Culture-plates</td>
			<td>Falcon</td>
			<td>353003</td>
			<td></td>
		</tr>
		<tr>
			<td>4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES)</td>
			<td>Cambrex</td>
			<td>BE17-737E</td>
			<td></td>
		</tr>
		<tr>
			<td>Beta-mercaptoethanol</td>
			<td>Merck</td>
			<td>8-05740-0250</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium pyruvate</td>
			<td>LONZA</td>
			<td>BE13-115E</td>
			<td></td>
		</tr>
		<tr>
			<td>L-glutamine</td>
			<td>LONZA</td>
			<td>BE17-605E</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant murine Granulocyte Macrophage colony-stimulating factor (GM-CSF)</td>
			<td>Prepotech</td>
			<td>315-03</td>
			<td></td>
		</tr>
		<tr>
			<td>lipopolysaccharide (LPS)</td>
			<td>SIGMA-ALDRICH</td>
			<td>L2018</td>
			<td></td>
		</tr>
		<tr>
			<td>96-well-plate</td>
			<td>Costar</td>
			<td>3799</td>
			<td></td>
		</tr>
		<tr>
			<td>v450 anti-mouse CD11b antibody</td>
			<td>BD Biosciences</td>
			<td>560455</td>
			<td></td>
		</tr>
		<tr>
			<td>PE anti-mouse CD64 antibody</td>
			<td>BioLegend</td>
			<td>139303</td>
			<td></td>
		</tr>
		<tr>
			<td>PE anti-mouse CD115 antibody</td>
			<td>BioLegend</td>
			<td>135505</td>
			<td></td>
		</tr>
		<tr>
			<td>FITC anti-mouse CD11c antibody</td>
			<td>BioLegend</td>
			<td>117305</td>
			<td></td>
		</tr>
		<tr>
			<td>FITC anti-mouse MHCII antibody</td>
			<td>BioLegend</td>
			<td>125507</td>
			<td></td>
		</tr>
		<tr>
			<td>APC anti-mouse CD86 antibody</td>
			<td>BioLegend</td>
			<td>105011</td>
			<td></td>
		</tr>
		<tr>
			<td>APC anti-mouse CD80 antibody</td>
			<td>BioLegend</td>
			<td>104713</td>
			<td></td>
		</tr>
		<tr>
			<td>Flow Cytometry tubes</td>
			<td>Zelian</td>
			<td>300800-1</td>
			<td>PS 12X75 5mL</td>
		</tr>
		<tr>
			<td>OTII Ovoalbumine peptide</td>
			<td>InvivoGen</td>
			<td>323-339</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-mouse biotinylated CD8&#945; antibody</td>
			<td>Tonbo Biosciences</td>
			<td>30-0081-U500</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-mouse biotinylated IgM antibody</td>
			<td>BioLegend</td>
			<td>406503</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-mouse biotinylated B220 antibody</td>
			<td>Tonbo Biosciences</td>
			<td>30-0452-U500</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-mouse biotinylated CD19 antibody</td>
			<td>Tonbo Biosciences</td>
			<td>30-0193-U500</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-mouse biotinylated MHCII (I-Ab) antibody</td>
			<td>BioLegend</td>
			<td>115302</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-mouse biotinylated CD11b antibody</td>
			<td>Tonbo Biosciences</td>
			<td>30-0112-U500</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-mouse biotinylated CD11c antibody</td>
			<td>BioLegend</td>
			<td>117303</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-mouse biotinylated CD44 antibody</td>
			<td>BioLegend</td>
			<td>103003</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-mouse biotinylated CD25 antibody</td>
			<td>Tonbo Biosciences</td>
			<td>30-0251-U100</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-mouse biotinylated DX5 antibody</td>
			<td>BioLegend</td>
			<td>108903</td>
			<td></td>
		</tr>
		<tr>
			<td>streptavidin-coated magnetic microbeads</td>
			<td>MACS Miltenyi Biotec</td>
			<td>130-048-101</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnetic cell separator</td>
			<td>MACS Miltenyi Biotec</td>
			<td>130-090-976</td>
			<td>QuadroMACS Separator</td>
		</tr>
		<tr>
			<td>Separation columns</td>
			<td>MACS Miltenyi Biotec</td>
			<td>130-042-401</td>
			<td></td>
		</tr>
		<tr>
			<td>PE anti-mouse CD4 antibody</td>
			<td>BioLegend</td>
			<td>100408</td>
			<td></td>
		</tr>
		<tr>
			<td>APC anti-mouse CD3 antibody</td>
			<td>BioLegend</td>
			<td>100235</td>
			<td></td>
		</tr>
		<tr>
			<td>FITC anti-mouse CD8 antibody</td>
			<td>Tonbo Biosciences</td>
			<td>35-0081-U025</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell Violet Tracer</td>
			<td>Thermofisher</td>
			<td>C34557</td>
			<td></td>
		</tr>
		<tr>
			<td>APC anti-mouse CD25 antibody</td>
			<td>Tonbo Biosciences</td>
			<td>20-0251-U100</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa647 anti-mouse CD69 antibody</td>
			<td>BioLegend</td>
			<td>104518</td>
			<td></td>
		</tr>
		<tr>
			<td>PerCPCY5.5 anti-mouse CD45.1 antibody</td>
			<td>Tonbo Biosciences</td>
			<td>65-0453</td>
			<td></td>
		</tr>
		<tr>
			<td>APC anti-mouse CD45.1 antibody</td>
			<td>Tonbo Biosciences</td>
			<td>20-0453</td>
			<td></td>
		</tr>
		<tr>
			<td>PECY7 anti-mouse CD45.1 antibody</td>
			<td>Tonbo Biosciences</td>
			<td>60-0453</td>
			<td></td>
		</tr>
		<tr>
			<td>FITC anti-mouse CD45.2 antibody</td>
			<td>Tonbo Biosciences</td>
			<td>35-0454</td>
			<td></td>
		</tr>
		<tr>
			<td>Ionomycin</td>
			<td>SIGMA-ALDRICH</td>
			<td>I0634</td>
			<td></td>
		</tr>
		<tr>
			<td>Phorbol 12 Myristate 13 Acetate (PMA)</td>
			<td>SIGMA</td>
			<td>P8139</td>
			<td></td>
		</tr>
		<tr>
			<td>Brefeldin A (BD GolgiPlug)</td>
			<td>BD</td>
			<td>555029</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Millipore</td>
			<td>8-18715-02100</td>
			<td></td>
		</tr>
		<tr>
			<td>Intracellular permeabilization buffer</td>
			<td>eBioscience</td>
			<td>00-8333</td>
			<td></td>
		</tr>
		<tr>
			<td>APC anti-mouse IFNg antibody</td>
			<td>Tonbo Biosciences</td>
			<td>20-7311-U100</td>
			<td></td>
		</tr>
		<tr>
			<td>Fc-block (anti-mouse CD16/CD32)</td>
			<td>Tonbo Biosciences</td>
			<td>70-0161-U100</td>
			<td></td>
		</tr>
		<tr>
			<td>B6.SJL CD45.1 mice</td>
			<td>The Jackson Laboratory</td>
			<td>002014</td>
			<td></td>
		</tr>
		<tr>
			<td>BD LSRFortessa&#8482; Cell Analyzer</td>
			<td>BD Biosciences</td>
			<td>649225</td>
			<td></td>
		</tr>
		<tr>
			<td>DAPI Solution</td>
			<td>Thermofisher</td>
			<td>62248</td>
			<td></td>
		</tr>
	</tbody>
</table>

an in vivo mouse model to measure na&#239;ve cd4 t cell activation, proliferation and th1 differentiation induced by bone marrow-derived dendritic cells

Quantification of na&#239;ve CD4 T cell activation, proliferation, and differentiation to T helper 1 (Th1) cells is a useful way to assess the role played by T cells in an immune response. This protocol describes the in vitro differentiation of bone marrow (BM) progenitors to obtain granulocyte macrophage colony-stimulating factor (GM-CSF) derived-dendritic cells (DCs). The protocol also describes the adoptive transfer of ovalbumin peptide (OVAp)-loaded GM-CSF-derived DCs and na&#239;ve CD4 T cells from OTII transgenic mice in order to analyze the in vivo activation, proliferation, and Th1 differentiation of the transferred CD4 T cells. This protocol circumvents the limitation of purely in vivo methods imposed by the inability to specifically manipulate or select the studied cell population. Moreover, this protocol allows studies in an in vivo environment, thus avoiding alterations to functional factors that may occur in vitro and including the influence of cell types and other factors only found in intact organs. The protocol is a useful tool for generating changes in DCs and T cells that modify adaptive immune responses, potentially providing important results to understand the origin or development of numerous immune associated diseases.

Figure 1 illustrates the steps described in this protocol. Figure 2 illustrates the isolation and culture of mouse BM cells. The addition of GM-CSF and LPS to these cultures allows the in vitro generation and maturation of DCs. Figure 3 illustrates the flow cytometry analysis of the differentiation and maturation of the obtained DCs. OVAp-loaded GM-CSF BM-derived DCs and isolated vital cell ...

Watch this Scientific Journal Video about An In Vivo Mouse Model to Measure NaÃƒÂ¯ve CD4 T Cell Activation, Proliferation and Th1 Differentiation Induced by Bone Marrow-derived Dendritic Cells at JoVE

An In Vivo Mouse Model to Measure Na&#239;ve CD4 T Cell Activation, Proliferation and Th1 Differentiation Induced by Bone Marrow-derived Dendritic Cells

Here, we present a protocol for the in vivo determination of na&#239;ve CD4 T cell (T cell) activation, proliferation, and Th1 differentiation induced by GM-CSF bone marrow (BM)-derived dendritic cells (DCs). In addition, this protocol describes BM and T-cell isolation, DC generation, and DC and T-cell adoptive transfer.

Quantification of na&#239;ve CD4 T cell activation, proliferation, and differentiation to T helper 1 (Th1) cells is a useful way to assess the role played ...

an-vivo-mouse-model-to-measure-nave-cd4-t-cell-activation

Research

JoVE Journal

Immunology and Infection

19.3K Views.  Centro Nacional de Investigaciones Cardiovasculares Carlos III (CNIC). Here, we present a protocol for the in vivo determination of naïve CD4 T cell (T cell) activation, proliferation, and Th1 differentiation induced by GM-CSF bone marrow (BM)-derived dendritic cells (DCs). In addition, this protocol describes BM and T-cell isolation, DC generation, and DC and T-cell adoptive transfer.

Video: An In Vivo Mouse Model to Measure Naïve CD4 T Cell Activation, Proliferation and Th1 Differentiation Induced by Bone Marrow-derived Dendritic Cells

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 ...

Generation and Labeling of Murine Bone Marrow-derived Dendritic Cells with Qdot Nanocrystals for Tracking Studies

Dendritic cells (DCs) are professional antigen presenting cells (APCs) found in peripheral tissues and in immunological organs such as thymus, bone marrow, spleen, lymph nodes and Peyer's patches 1-3. DCs present in peripheral tissues sample the organism for the presence of antigens, which they take up, process and present in their surface in the context of major histocompatibility molecules (MHC). Then, antigen-loaded DCs migrate to immunological organs where they present the processed antigen to T lymphocytes triggering specific immune responses. One way to evaluate the migratory capabilities of DCs is to label them with fluorescent dyes 4.
Herewith we demonstrate the use of Qdot fluorescent nanocrystals to label murine bone marrow-derived DC. The advantage of this labeling is that Qdot nanocrystals possess stable and long lasting fluorescence that make them ideal for detecting labeled cells in recovered tissues. To accomplish this, first cells will be recovered from murine bone marrows and cultured for 8 days in the presence of granulocyte macrophage-colony stimulating factor in order to induce DC differentiation. These cells will be then labeled with fluorescent Qdots by short in vitro incubation. Stained cells can be visualized with a fluorescent microscopy. Cells can be injected into experimental animals at this point or can be into mature cells upon in vitro incubation with inflammatory stimuli. In our hands, DC maturation did not determine loss of fluorescent signal nor does Qdot staining affect the biological properties of DCs. Upon injection, these cells can be identified in immune organs by fluorescent microscopy following typical dissection and fixation procedures.

Dendritic cells uptake antigens and migrate towards immune organs to present processed antigens to T cells. Qdot nanocrystal labeling provides a long-lasting and stable fluorescent signal. This allows tracking of dendritic cells to different organs by fluorescent microscopy.

Dendritic cells (DCs) are professional antigen presenting cells (APCs) found in peripheral tissues and in immunological organs such as thymus, bone ...

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 ...

Isolation and Th17 Differentiation of Na&iuml;ve CD4 T Lymphocytes

Th17 cells are a distinct subset of T cells that have been found to produce interleukin 17 (IL-17), and differ in function from the other T cell subsets including Th1, Th2, and regulatory T cells. Th17 cells have emerged as a central culprit in overzealous inflammatory immune responses associated with many autoimmune disorders. In this method we purify T lymphocytes from the spleen and lymph nodes of C57BL/6 mice, and stimulate purified CD4+ T cells under control and Th17-inducing environments. The Th17-inducing environment includes stimulation in the presence of anti-CD3 and anti-CD28 antibodies, IL-6, and TGF-&#946;. After incubation for at least 72 hours and for up to five days at 37 &#176;C, cells are subsequently analyzed for the capability to produce IL-17 through flow cytometry, qPCR, and ELISAs. Th17 differentiated CD4+CD25- T cells can be utilized to further elucidate the role that Th17 cells play in the onset and progression of autoimmunity and host defense. Moreover, Th17 differentiation of CD4+CD25- lymphocytes from distinct murine knockout/disease models can contribute to our understanding of cell fate plasticity.

Isolation and Th17 Differentiation of Na&#239;ve CD4 T Lymphocytes

With effector functions distinct from other T cell subsets, Th17 cells have been centrally implicated in inflammatory autoimmunity. This in vitro Th17 differentiation protocol provides a means to determine whether na&#239;ve CD4+ T lymphocytes can differentiate into Th17 cells, and to further examine their role in autoimmunity and host response.

Th17 cells are a distinct subset of T cells that have been found to produce interleukin 17 (IL-17), and differ in function from the other T cell subsets ...

Bone Marrow-derived Macrophage Production

Macrophages are critical components of the innate and adaptive immune responses, and they are the first line of defense against foreign invaders because of their powerful microbicidal activities. Macrophages are widely distributed throughout the body and are present in the lymphoid organs, liver, lungs, gastrointestinal tract, central nervous system, bone, and skin. Because of their repartition, they participate in a wide range of physiological and pathological processes. Macrophages are highly versatile cells that are able to recognize microenvironmental alterations and to maintain tissue homeostasis. Numerous pathogens have evolved mechanisms to use macrophages as Trojan horses to survive, replicate in, and infect both humans and animals and to propagate throughout the body. The recent explosion of interest in evolutionary, genetic, and biochemical aspects of host-pathogen interactions has renewed scientific attention regarding macrophages. Here, we describe a procedure to isolate and cultivate macrophages from murine bone marrow that will provide large numbers of macrophages for studying host-pathogen interactions as well as other processes.

Macrophages have long been recognized as a critical component of the innate and adaptive immune responses. The recent explosion of knowledge pertaining to evolutionary, genetic, and biochemical aspects of the interaction between macrophages and microbes has renewed scientific attention to macrophages. This article describes a method to differentiate macrophages from mouse bone marrow.

Macrophages are critical components of the innate and adaptive immune responses, and they are the first line of defense against foreign invaders because ...

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 ...

Isolation and Intravenous Injection of Murine Bone Marrow Derived Monocytes

As a subtype of leukocytes and progenitors of macrophages, monocytes are involved in many important processes of organisms and are often the subject of various fields in biomedical science. The method described below is a simple and effective way to isolate murine monocytes from heterogeneous bone marrow.
Bone marrow from the femur and tibia of Balb/c mice is harvested by flushing with phosphate buffered saline (PBS). Cell suspension is supplemented with macrophage-colony stimulating factor (M-CSF) and cultured on ultra-low attachment surfaces to avoid adhesion-triggered differentiation of monocytes. The properties and differentiation of monocytes are characterized at various intervals. Fluorescence activated cell sorting (FACS), with markers like CD11b, CD115, and F4/80, is used for phenotyping. At the end of cultivation, the suspension consists of 45%&#177; 12% monocytes. By removing adhesive macrophages, the purity can be raised up to 86%&#177; 6%. After the isolation, monocytes can be utilized in various ways, and one of the most effective and common methods for in vivo delivery is intravenous tail vein injection. 
This technique of isolation and application is important for mouse model studies, especially in the fields of inflammation or immunology. Monocytes can also be used therapeutically in mouse disease models.

Here we present a protocol that generates large amounts of murine monocytes from heterogeneous bone marrow for translational applications. In comparison to others, this new method helps reduce the number of sacrificed animals and lowers costs by avoiding expensive methods such as high gradient magnetic cell separation (MACS).

As a subtype of leukocytes and progenitors of macrophages, monocytes are involved in many important processes of organisms and are often the subject of ...

Mouse Na&#239;ve CD4+ T Cell Isolation and In vitro Differentiation into T Cell Subsets

Antigen inexperienced (na&#239;ve) CD4+ T cells undergo expansion and differentiation to effector subsets at the time of T cell receptor (TCR) recognition of cognate antigen presented on MHC class II. The cytokine signals present in the environment at the time of TCR activation are a major factor in determining the effector fate of a na&#239;ve CD4+ T cell. Although the cytokine environment during na&#239;ve T cell activation may be complex and involve both redundant and opposing signals in vivo, the addition of various cytokine combinations during naive CD4+ T cell activation in vitro can readily promote the establishment of effector T helper lineages with hallmark cytokine and transcription factor expression. Such differentiation experiments are commonly used as a first step for the evaluation of targets believed to promote or inhibit the development of certain CD4+ T helper subsets. The addition of mediators, such as signaling agonists, antagonists, or other cytokines, during the differentiation process can also be used to study the influence of a particular target on T cell differentiation. Here, we describe a basic protocol for the isolation of na&#239;ve T cells from mouse and the subsequent steps necessary for polarizing na&#239;ve cells to various T helper effector lineages in vitro.

Mouse Na&#239;ve CD4+ T Cell Isolation and In vitro Differentiation into T Cell Subsets

Na&#239;ve CD4+ T cells polarize to various subsets depending on the environment at the time of activation. The differentiation of na&#239;ve CD4+ T cells to various effector subsets can be achieved in vitro through the addition of T cell receptor stimuli and specific cytokine signals.

Antigen inexperienced (na&#239;ve) CD4+ T cells undergo expansion and differentiation to effector subsets at the time of T cell receptor (TCR) recognition ...

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.

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 ...

Isolation and Ex Vivo Culture of V&#948;1+CD4+&#947;&#948; T Cells, an Extrathymic &#945;&#946;T-cell Progenitor

The thymus, the primary organ for the generation of &#945;&#946; T cells and backbone of the adaptive immune system in vertebrates, has long been considered as the only source of &#945;&#946;T cells. Yet, thymic involution begins early in life leading to a drastically reduced output of na&#239;ve &#945;&#946;T cells into the periphery. Nevertheless, even centenarians can build immunity against newly acquired pathogens. Recent research suggests extrathymic &#945;&#946;T cell development, however our understanding of pathways that may compensate for thymic loss of function are still rudimental. &#947;&#948; T cells are innate lymphocytes that constitute the main T-cell subset in the tissues. We recently ascribed a so far unappreciated outstanding function to a &#947;&#948; T cell subset by showing that the scarce entity of CD4+ V&#948;1+&#947;&#948; T cells can transdifferentiate into &#945;&#946;T cells in inflammatory conditions. Here, we provide the protocol for the isolation of this progenitor from peripheral blood and its subsequent cultivation. V&#948;1 cells are positively enriched from PBMCs of healthy human donors using magnetic beads, followed by a second step wherein we target the scarce fraction of CD4+ cells with a further magnetic labeling technique. The magnetic force of the second labeling exceeds the one of the first magnetic label, and thus allows the efficient, quantitative and specific positive isolation of the population of interest. We then introduce the technique and culture condition required for cloning and efficiently expanding the cells and for identification of the generated clones by FACS analysis. Thus, we provide a detailed protocol for the purification, culture and ex vivo expansion of CD4+ V&#948;1+&#947;&#948; T cells. This knowledge is prerequisite for studies that relate to this &#945;&#946;T cell progenitor`s biology and for those who aim to identify the molecular triggers that are involved in its transdifferentiation.

Isolation and Ex Vivo Culture of V&#948;1+CD4+&#947;&#948; T Cells, an Extrathymic &#945;&#946;T-cell Progenitor

Here, we provide an optimized protocol for the isolation and cloning of the scarce T-cell entity of peripheral V&#948;1+CD4+ T cells that is, as we showed recently, an extrathymic &#945;&#946; T-cell progenitor. This technique allows to quantitatively isolate, clone and efficiently expand these cells in ex vivo culture.