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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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 &amp;mu;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&amp;alpha; 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&amp;trade; 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 NaÃ¯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

19.4K 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

Murine Model of CD40-activation of B cells

Research on B cells has shown that CD40 activation improves their antigen presentation capacity. When stimulated with interleukin-4 and CD40 ligand (CD40L), human B cells can be expanded without difficulties from small amounts of peripheral blood within 14 days to very large amounts of highly-pure CD40-B cells (&gt;109 cells per patient) from healthy donors as well as cancer patients1-4. CD40-B cells express important lymph node homing molecules and can attract T cells in vitro5. Furthermore they efficiently take up, process and present antigens to T cells6,7. CD40-B cells were shown to not only prime na&iacute;ve, but also expand memory T cells8,9. Therefore CD40-activated B cells (CD40-B cells) have been studied as an alternative source of immuno-stimulatory antigen-presenting cells (APC) for cell-based immunotherapy1,5,10. In order to further study whether CD40-B cells induce effective T cell responses in vivo and to study the underlying mechanism we established a cell culture system for the generation of murine CD40-activated B cells. Using splenocytes or purified B cells from C57BL/6 mice for CD40-activation, optimal conditions were identified as follows: Starting from splenocytes of C57BL/6 mice (haplotype H-2b) lymphocytes are purified by density gradient centrifugation and co-cultured with HeLa cells expressing recombinant murine CD40 ligand (tmuCD40L HeLa)11. Cells are recultured every 3-4 days and key components such as CD40L, interleukin-4, -Mercaptoethanol and cyclosporin A are replenished. In this protocol we demonstrate how to obtain fully activated murine CD40-B cells (mCD40B) with similar APC-phenotype to human CD40-B cells (Fig 1a,b). CD40-stimulation leads to a rapid outgrowth and expansion of highly pure (&gt;90%) CD19+ B cells within 14 days of cell culture (Fig 1c,d). To avoid contamination with non-transfected cells, expression of the murine CD40 ligand on the transfectants has to be controlled regularly (Fig 2). Murine CD40-activated B cells can be used to study B-cell activation and differentiation as well as to investigate their potential to function as APC in vitro and in vivo. Moreover, they represent a promising tool for establishing therapeutic or preventive vaccination against tumors and will help to answer questions regarding safety and immunogenicity of this approach12.

In this video, we demonstrate the procedure of CD40-activation and expansion of murine B cells from splenocytes of C57BL/6 mice, which can be used as a model antigen-presenting cell (APC) to study induction of immunity.

Research on B cells has shown that CD40 activation improves their antigen presentation capacity. When stimulated with interleukin-4 and CD40 ligand ...

Induction of Graft-versus-host Disease and In Vivo T Cell Monitoring Using an MHC-matched Murine Model

Graft-versus-host disease (GVHD) is the limiting barrier to the broad use of bone marrow transplant as a curative therapy for a variety of hematological deficiencies. GVHD is caused by mature alloreactive T cells present in the bone marrow graft that are infused into the recipient and cause damage to host organs. However, in mice, T cells must be added to the bone marrow inoculum to cause GVHD. Although extensive work has been done to characterize T cell responses post transplant, bioluminescent imaging technology is a non-invasive method to monitor T cell trafficking patterns in vivo. 
Following lethal irradiation, recipient mice are transplanted with bone marrow cells and splenocytes from donor mice. T cell subsets from L2G85.B6 (transgenic mice that constitutively express luciferase) are included in the transplant. By only transplanting certain T cell subsets, one is able to track specific T cell subsets in vivo, and based on their location, develop hypotheses regarding the role of specific T cell subsets in promoting GVHD at various time points. At predetermined intervals post transplant, recipient mice are imaged using a Xenogen IVIS CCD camera. Light intensity can be quantified using Living Image software to generate a pseudo-color image based on photon intensity (red = high intensity, violet = low intensity). 
Between 4-7 days post transplant, recipient mice begin to show clinical signs of GVHD. Cooke et al.1 developed a scoring system to quantitate disease progression based on the recipient mice fur texture, skin integrity, activity, weight loss, and posture. Mice are scored daily, and euthanized when they become moribund. Recipient mice generally become moribund 20-30 days post transplant. 
Murine models are valuable tools for studying the immunology of GVHD. Selectively transplanting particular T cell subsets allows for careful identification of the roles each subset plays. Non-invasively tracking T cell responses in vivo adds another layer of value to murine GVHD models.

Induction of Graft-versus-host Disease and In Vivo T Cell Monitoring Using an MHC-matched Murine Model

Murine bone marrow transplantation is a widely used technique to study immunological mechanisms governing graft-versus-host disease in humans. The ability to monitor T cell trafficking patterns in vivo allows for detailed analysis of the development and perpetuation of T cell responses during graft-versus-host disease.

Graft-versus-host disease (GVHD) is the limiting barrier to the broad use of bone marrow transplant as a curative therapy for a variety of hematological ...

Culture of myeloid dendritic cells from bone marrow precursors

Myeloid dendritic cells (DCs) are frequently used to study the interactions between innate and adaptive immune mechanisms and the early response to infection. Because these are the most potent antigen presenting cells, DCs are being increasingly used as a vaccine vector to study the induction of antigen-specific immune responses. In this video, we demonstrate the procedure for harvesting tibias and femurs from a donor mouse, processing the bone marrow and differentiating DCs in vitro. The properties of DCs change following stimulation: immature dendritic cells are potent phagocytes, whereas mature DCs are capable of antigen presentation and interaction with CD4+ and CD8+ T cells. This change in functional activity corresponds with the upregulation of cell surface markers and cytokine production. Many agents can be used to mature DCs, including cytokines and toll-like receptor ligands. In this video, we demonstrate flow cytometric comparisons of expression of two co-stimulatory molecules, CD86 and CD40, and the cytokine, IL-12, following overnight stimulation with CpG or mock treatment. After differentiation, DCs can be further manipulated for use as a vaccine vector or to generate antigen-specific immune responses by in vitro pulsing using peptides or proteins, or transduced using recombinant viral vectors. 

This video demonstrates the procedure for differentiating myeloid dendritic cells from mouse bone marrow. Isolation of mouse tibia and femur, and processing of bone marrow are demonstrated. Pictures demonstrating cell morphology before and after differentiation, and figures depicting cell phenotype and IL-12 production following maturation using CpG are shown.

Myeloid dendritic cells (DCs) are frequently used to study the interactions between innate and adaptive immune mechanisms and the early response to ...

Biology

Generation of Bone Marrow Derived Murine Dendritic Cells for Use in 2-photon Imaging

Several methods for the preparation of murine dendritic cells can be found in the literature. Here, we present a method that produces greater than 85% CD11c high dendritic cells in culture that home to the draining lymph node after subcutaneous injection and present antigen to antigen specific T cells (see video). Additionally, we use Essen Instruments Incucyte to track dendritic cell maturation, where, at day 10, the morphology of the cultured cells is typical of a mature dendritic cell and &lt;85% of cells are CD11chigh. The study of antigen presentation in peripheral lymph nodes by 2-photon imaging revealed that there are three distinct phases of dendritic cell and T cell interaction1, 2. Phase I consists of brief serial contacts between highly motile antigen specific T cells and antigen carrying dendritic cells1, 2. Phase two is marked by prolonged contacts between antigen-specific T cell and antigen bearing dendritic cells1, 2. Finally, phase III is characterized by T cells detaching from dendritic cells, regaining motility and beginning to divide1, 2. This is one example of the type of antigen-specific interactions that can be analyzed by two-photon imaging of antigen-loaded cell tracker dye-labeled dendritic cells.

Antigen presentation in secondary lymphoid organs by dendritic cells is crucial for the initiation of the T cell mediated adaptive immune response. Here we demonstrate the culture of bone marrow derived murine dendritic cells, activation, and labeling for 2-photon imaging.

Several methods for the preparation of murine dendritic cells can be found in the literature. Here, we present a method that produces greater than 85% ...

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 ...

Tailoring In Vivo Cytotoxicity Assays to Study Immunodominance in Tumor-specific CD8+ T Cell Responses

Carboxyfluorescein succinimidyl ester (CFSE)-based in vivo cytotoxicity assays enable sensitive and accurate quantitation of CD8+ cytolytic T lymphocyte (CTL) responses elicited against tumor- and pathogen-derived peptides. They offer several advantages over traditional killing assays. First, they permit the monitoring of CTL-mediated cytotoxicity within architecturally intact secondary lymphoid organs, typically in the spleen. Second, they allow for mechanistic studies during the priming, effector and recall phases of CTL responses. Third, they provide useful platforms for vaccine/drug efficacy testing in a truly in vivo setting. Here, we provide an optimized protocol for the examination of concomitant CTL responses against more than one peptide epitope of a model tumor antigen (Ag), namely, simian virus 40 (SV40)-encoded large T Ag (T Ag). Like most other clinically relevant tumor proteins, T Ag harbors many potentially immunogenic peptides. However, only four such peptides induce detectable CTL responses in C57BL/6 mice. These responses are consistently arranged in a hierarchical order based on their magnitude, which forms the basis for TCD8 &#8220;immunodominance&#8221; in this powerful system. Accordingly, the bulk of the T Ag-specific TCD8 response is focused against a single immunodominant epitope while the other three epitopes are recognized and responded to only weakly. Immunodominance compromises the breadth of antitumor TCD8 responses and is, as such, considered by many as an impediment to successful vaccination against cancer. Therefore, it is important to understand the cellular and molecular factors and mechanisms that dictate or shape TCD8 immunodominance. The protocol we describe here is tailored to the investigation of this phenomenon in the T Ag immunization model, but can be readily modified and extended to similar studies in other tumor models. We provide examples of how the impact of experimental immunotherapeutic interventions can be measured using in vivo cytotoxicity assays.

Tailoring In Vivo Cytotoxicity Assays to Study Immunodominance in Tumor-specific CD8+ T Cell Responses

We describe here a flow cytometry-based in vivo killing assay that enables examination of immunodominance in cytotoxic T lymphocyte (CTL) responses to a model tumor antigen. We provide examples of how this elegant assay may be employed for mechanistic studies and for drug efficacy testing.

Carboxyfluorescein succinimidyl ester (CFSE)-based in vivo cytotoxicity assays enable sensitive and accurate quantitation of CD8+ cytolytic T lymphocyte ...

Development and Functional Characterization of Murine Tolerogenic Dendritic Cells

The immune system operates by maintaining a tight balance between coordinating responses against foreign antigens and maintaining an unresponsive state against self-antigens as well as antigens derived from commensal organisms. The disruption of this immune homeostasis can lead to chronic inflammation and to the development of autoimmunity. Dendritic cells (DCs) are the professional antigen-presenting cells of the innate immune system involved in activating na&#239;ve T cells to initiate immune responses against foreign antigens. However, DCs can also be differentiated into TolDCs that act to maintain and promote T cell tolerance and to suppress effector cells contributing to the development of either autoimmune or chronic inflammation conditions. The recent advancement in our understanding of TolDCs suggests that DC tolerance can be achieved by modulating their differentiation conditions. This phenomenon has led to tremendous growth in developing TolDC therapies for numerous immune disorders caused due to break in immune tolerance. Successful studies in preclinical autoimmunity murine models have further validated the immunotherapeutic utility of TolDCs in the treatment of autoimmune disorders. Today, TolDCs have become a promising immunotherapeutic tool in the clinic for reinstating immune tolerance in various immune disorders by targeting pathogenic autoimmune responses while leaving protective immunity intact. Although an array of strategies has been proposed by multiple labs to induce TolDCs, there is no consistency in characterizing the cellular and functional phenotype of these cells. This protocol provides a step-by-step guide for the development of bone marrow-derived DCs in large numbers, a unique method used to differentiate them into TolDCs with a synthetic triterpenoid 2-cyano-3,12-dioxooleana-1,9-dien-28-oic acid-difluoro-propyl-amide (CDDO-DFPA), and the techniques used to confirm their phenotype, including analyses of essential molecular signatures of TolDCs. Finally, we show a method to assess TolDC function by testing their immunosuppressive response in vitro and in vivo in a preclinical model of multiple sclerosis.

Here, we present a protocol to develop and characterize tolerogenic dendritic cells (TolDCs) and evaluate their immunotherapeutic utility.

The immune system operates by maintaining a tight balance between coordinating responses against foreign antigens and maintaining an unresponsive state ...

Medicine

Economical and Efficient Protocol for Isolating and Culturing Bone Marrow-derived Dendritic Cells from Mice

The demand for dendritic cells (DCs) is gradually increasing as immunology research advances. However, DCs are rare in all tissues. The traditional method for isolating DCs primarily involves inducing bone marrow (BM) differentiation into DCs by injecting large doses (&#62;10 ng/mL) of granulocyte-macrophage colony-stimulating factor/interleukin-4 (GM-CSF/IL-4), making the procedure complex and expensive. In this protocol, using all BM cells cultured in 10 ng/mL GM-CSF/IL-4 medium, after 3-4 half-culture exchanges, up to 2.7 x 107 CD11c+ cells&#160;(DCs) per mouse (two femurs) were harvested with a purity of 80%-95%. After 10 days in culture, the expression of CD11c, CD80, and MHC II increased, whereas the number of cells decreased. The number of cells peaked after 7 days of culture. Moreover, this method only took 10 min to harvest all bone marrow cells, and a high number of DCs were obtained after 1 week of culture.

Here, we present an economical and efficient method to isolate and generate high-purity bone marrow-derived dendritic cells from mice after 7 days of culture with 10 ng/mL GM-CSF/IL-4.

The demand for dendritic cells (DCs) is gradually increasing as immunology research advances. However, DCs are rare in all tissues. The traditional method ...

Th17 Cell Differentiation from Naive CD4+ T-Cells Using Flow Cytometry

In Vitro Differentiation of Naive CD4+ T Cells into Pathogenic Th17 Cells in Mouse

In vitro T cell differentiation techniques are essential for both functional and mechanistic investigations of CD4+ T cells. Pathogenic Th17 cells have been linked to a wide range of diseases in recent times, including multiple sclerosis (MS), rheumatoid arthritis, acute respiratory distress syndrome (ARDS), sepsis, and other autoimmune disorders. However, the currently known in vitro differentiation protocols have difficulty achieving high purity of pathogenic Th17 cells, with the induction efficiency often below 50%, which is a key challenge in in vitro experiments. In this protocol, we propose an enhanced in vitro culture and differentiation protocol for pathogenic Th17 cells, which is used to directly differentiate naive CD4+ T cells isolated from mouse spleens into pathogenic Th17 cells. This protocol provides detailed instructions on splenocyte isolation, purification of naive CD4+ T cells, and differentiation of pathogenic Th17 cells. Through this protocol, we can achieve a differentiation purity of approximately 90% for pathogenic Th17 cells, which meets the basic needs of many cellular experiments.

Author Spotlight: Achieving High-Purity In Vitro Differentiation of Th17 Cells Using Cytokine Concentration Modulation

This protocol describes the differentiation of na&#239;ve CD4+ T cells into pathogenic Th17 cells in vitro. Specifically, when combined with a multi-parameter flow cytometry-based approach, 90% purity of pathogenic Th17 cells can be obtained from na&#239;ve CD4+ T cells using this differentiation method.

In vitro T cell differentiation techniques are essential for both functional and mechanistic investigations of CD4+ T cells. Pathogenic Th17 cells have ...

Bone Marrow-Derived Dendritic Cells Generation: A Method to Generate Dendritic Cells from Mouse Bone Marrow

Source:Nguyen, H. D., et al. <a href="https://www.jove.com/t/60083">Bone Marrow Transplantation Platform to Investigate the Role of Dendritic Cells in Graft-versus-Host Disease.</a> J. Vis. Exp.. (2020).
This video describes a protocol for the generation of dendritic cells--key antigen-presenting cells that activate T cells--from the bone marrow of an adult mouse.

Source:Nguyen, H. D., et al. Bone Marrow Transplantation Platform to Investigate the Role of Dendritic Cells in Graft-versus-Host Disease. J. Vis. Exp.. ...

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

Summary

Explore More Videos

Chapters in this video

Murine Model of CD40-activation of B cells

Induction of Graft-versus-host Disease and In Vivo T Cell Monitoring Using an MHC-matched Murine Model

Culture of myeloid dendritic cells from bone marrow precursors

Generation of Bone Marrow Derived Murine Dendritic Cells for Use in 2-photon Imaging

Mouse Naïve CD4⁺ T Cell Isolation and In vitro Differentiation into T Cell Subsets

Tailoring In Vivo Cytotoxicity Assays to Study Immunodominance in Tumor-specific CD8⁺ T Cell Responses

Development and Functional Characterization of Murine Tolerogenic Dendritic Cells

Economical and Efficient Protocol for Isolating and Culturing Bone Marrow-derived Dendritic Cells from Mice

In Vitro Differentiation of Naive CD4⁺ T Cells into Pathogenic Th17 Cells in Mouse

Bone Marrow-Derived Dendritic Cells Generation: A Method to Generate Dendritic Cells from Mouse Bone Marrow