Name: isolation and adoptive transfer of high salt treated antigen-presenting dendritic cells
Uploaded: 2019-03-05T13:30:00.000+00:00
Duration: 9 min 29 s
Description: Watch this Scientific Journal Video about Isolation and Adoptive Transfer of High Salt Treated Antigen-presenting Dendritic Cells at JoVE.com

This work was supported by American Heart Association grants POST290900 to N.R.B., 17SDG33670829 to L.X. and National Institutes of Health grant K01HL130497 to A.K.

Vanderbilt University&#39;s Institutional Animal Care and Use Committee have approved the procedures described herein. Mice are housed and cared for in accordance with the Guide for the Care and Use of Laboratory Animals (National Academies Press. Revised 2010).
1. Isolation of Spleens from Mice
<ol>
	<li>Prepare 1640 RPMI: 10% FBS, 0.10 mM HEPES, 1 mM sodium pyruvate, 50 &#181;M &#946;-mercaptoethanol and 1% penicillin/streptomycin.</li>
	<li>Euthanize 10&#8211;12 week-old C57bl/6 male mice by CO2 inhalation. Spray the chest and sides of the mouse with 70% ethanol. On the left side, carefully open the skin and the peritoneal cavity to expose the spleen.</li>
	<li>Using small forceps, cautiously move the intestines and liver to the left side of the mouse, and using fine scissors gently excise the spleen. 
	NOTE: This step must be done very carefully as damage to the gastrointestinal tract can induce contamination.</li>
	<li>Place each excised spleen in labeled 15 mL conical tube containing 3 mL of RPMI 1640 medium.</li>
</ol>
2. Generation of Single Cell Suspensions from Spleens
NOTE: The spleen can be dissociated into a single cell suspension by combining mechanical dissociation plus enzymatic digestion.
<ol>
	<li>Prepare the spleen digestion solution by adding collagenase D (1 mg/mL; 0.29 U/mg lyophilized) and DNase I (0.1 mg/mL) to prepared RPMI 1640 medium (see step 1.1)</li>
	<li>Use forceps to transfer the spleen into a C tube (dissociation tube) containing 3 mL of spleen digestion solution.</li>
	<li>Perform the mechanical dissociation using a semi-automated homogenizer according to the manufacturer&#39;s instructions.
	<ol>
		<li>Place the C tube onto the semi-automated homogenizer, ensuring that the C tube is tightly placed on the rotating arm. Once the C tube is placed, press the start button on the semi-automated homogenizer to run the Spleen 04.01 protocol for 60 s. 
		NOTE: Mechanical dissociation can also be performed by placing a 40 &#956;m filter on top of a 50 mL conical tube and gently grinding the spleen through the filter. After grinding the spleen, through rinse the 40 &#181;m filter with 10 mL of RPMI media.</li>
	</ol>
	</li>
	<li>After mechanical dissociation, detach the tube from the homogenizer and perform the enzymatic digestion. Incubate the samples for 15 min at 37 &#176;C under continuous rotation (20 rpm).</li>
	<li>Transfer the solution by pipetting into a 50 mL conical tube after filtering through a 40 &#181;m strainer. Wash the 40 &#181;m strainer with 10 mL of Dulbecco&#39;s PBS (dPBS). Centrifuge the cells at 300 x g for 10 min at 4 &#176;C.</li>
	<li>After centrifugation, aspirate the supernatant completely and wash the pellet by resuspending in 10 mL of dPBS. Centrifuge at 300 x g for 10 min at 4 &#176;C.</li>
</ol>
3. Isolation of CD11c+ DCs from Splenic Single Cell Suspension
<ol>
	<li>Resuspend the cell pellet in 5 mL of dPBS and count the number of cells using a hemocytometer and trypan blue exclusion.</li>
	<li>Pellet the cells by centrifuging at 300 x g, for 10 min at 4 &#176;C.</li>
	<li>Aspirate the supernatant completely and resuspend pellet in 400 &#181;L of magnetically activated cell-sorting (MACs) buffer.</li>
	<li>Add 100 &#181;L of CD11c microbeads (see Table of Materials) per 1 x 108 cells.</li>
	<li>Vortex the cell suspension and incubate for 10 min in the refrigerator at 4 &#176;C.</li>
	<li>Add 10 mL of MACs buffer to wash cells. Centrifuge at 300 x g for 10 min at 4 &#176;C.</li>
	<li>Aspirate the supernatant completely and resuspend in 500 &#181;L of MACs buffer.</li>
</ol>
4. Magnetic Separation of CD11c+ DCs
NOTE: Magnetic separation is done manually with a magnetic separator (see Table of Materials) outfitted with LS Columns. Magnetic separation can also be done automatically with an automated magnetic separator. (see Table of Materials).
<ol>
	<li>Place the LS column in the magnetic field of MACs separator and a 15 mL conical tube under each column to collect the flow-through.</li>
	<li>Rinse the LS column with 3 mL of buffer.</li>
	<li>Place the cell suspension onto each LS column and collect the flow-through containing unlabeled cells.</li>
	<li>Wash the LS column with 3 mL of MACs buffer collecting the unlabeled cells that pass through. Wash the LS column 2 more times.</li>
	<li>Remove the LS column from the magnetic separator. Add 5 mL of MACs buffer to each column and immediately flush out the magnetically labeled cells by firmly plunging the LS column into a clean 15 mL conical tube. 
	NOTE: It is important to perform a double isolation of CD11c cells to obtain a high purity cell suspension. Therefore, repeat steps 3.1 through 4.5. and reference Figure 4 for purity standards. This step improves the purity of CD11c+ cells to 90&#8211;95%.</li>
	<li>Determine CD11c+ DC number on a hemocytometer using trypan blue exclusion. Dilute the cell sample at a 1:1 dilution in 0.4% trypan blue solution. 
	NOTE: Non-viable cells will be stained blue, while viable cells will remain unstained.
	<ol>
		<li>To do this, carefully fill a hemocytometer with 10 &#181;L of the cell sample dilution and incubate for 1 minute.</li>
		<li>Count cells in 4 squares of 1 x 1 mm2 in the chamber that has been loaded to determine the viable cell number.</li>
	</ol>
	</li>
</ol>
5. In Vitro High Salt Treatment of CD11c+ DCs
<ol>
	<li>Prepare DC culture medium by adding 10% FBS, 0.1 mM HEPES, 1 mM sodium pyruvate, 50 &#181;M &#946;-mercaptoethanol and 1% penicillin/streptomycin to 500 mL 1640 RPMI.</li>
	<li>Prepare 10x DC high salt cell culture media (400 mM) by weighing out 1.17 g of NaCl and placing it into a sterile 50 mL falcon tube. Add 50 mL of sterile DC cell culture media (prepared in step 5.1) to the 50 mL falcon tube and vortex until the NaCl is in solution.</li>
	<li>Immediately filter the high salt DC culture media using vacuum filtration through a 0.2 &#956;m filter in a cell culture hood.</li>
	<li>Centrifuge each cell suspension from the spleens at 300 x g for 10 min at 4&#186;C. Resuspend each cell pellet in DC culture media at a concentration of 1 x 106 cells/mL.</li>
	<li>Pipette 900 &#956;L (approximately 1 x 106 cells) of the DC suspension in a 24-well flat bottom Falcon plate. Add 100 &#181;L of high salt DC culture media to each well for a final sodium concentration of 190 mM. Label the plate and place in a 37 &#176;C humified CO2 incubator for 48 h.</li>
</ol>
6. Adoptive Transfer of High Salt Treated CD11c+ DCs
<ol>
	<li>After in vitro stimulation for 48 h, remove the plate from the 37 &#176;C humified CO2 incubator.</li>
	<li>Pipette the cell culture media up and down to resuspend the CD11c+ DCs. Pipette all the CD11c+ DC suspension into a corresponding labeled 1.6 mL tube. Repeat this step for each individual well plated.</li>
	<li>Centrifuge each CD11c+ DC suspension at 300 x g for 10 min at 4 &#176;C. Aspirate the supernatant. Resuspend the DC pellet with 100 &#181;L of sterile dPBS.</li>
	<li>Draw DC suspension into a 1 mL syringe equipped with a 27 G &#189; inch needle.</li>
	<li>Place the na&#239;ve male 10 week-old C57bl/6 mice under 2% isoflurane to achieve a stable surgical plane. Check the level of anesthesia with the lack of pedal reflex. Once a stable surgical plane is achieved, slowly and carefully introduce the needle into the retro-orbital space at an angle of approximately 30&#176;. 
	NOTE: The bevel of the 27 G needle should be pointing down, away from the eye, to prevent ocular damage.</li>
	<li>Slowly and smoothly inject the CD11c+ DC suspension (approximately 1 x 106 cells). Once the injection is complete, carefully remove the needle for the retro-orbital space being conscious to not damage the eye. 
	NOTE: After the injection, there should be little or no bleeding. Adoptive transfer of CD11c+ DCs can also be performed using an I.V. method of injection (e.g. Tail vein). The control mice receive CD11c+ DCs that were cultured in normal salt media.</li>
	<li>Remove the mice from the nose cone and monitor them for approximately 30 min during the recovery from anesthesia.</li>
</ol>
7. Preparation of 14 Day Low-dose Angiotensin II (140 ng/kg/min)
<ol>
	<li>Prepare the angiotensin II buffer by adding 500 &#956;L of 5 M NaCl and 300 &#956;L of acetic acid. Quantum Satis (QS) up to 30 mL of deionized H20.</li>
	<li>Dissolve angiotensin II to 5 mg/mL stock solution with angiotensin II buffer. Dilute angiotensin II stock solution with additional buffer to achieve a final concentration such that the osmotic minipump will deliver angiotensin II in a rate of 140 ng/kg/min according to the body weight of each mouse. 
	NOTE: Essential Angiotensin II (mg) = (mouse weight (g) x 0.2 mg/day x 14 days)/1,000 
	Prepared Angiotensin II (mg) = (Essential angiotensin II x 260 &#181;L) / pump rate (0.25 &#956;L/hr) 
	Angiotensin II Stock Volume (&#956;L) = Prepared angiotensin II / stock concentration (5 mg/mL) x 1,000 
	Dilute volume (&#956;L) = 270 - angiotensin II stock volume</li>
	<li>Add angiotensin II stock volume to the dilute (angiotensin II buffer) based on the volumes calculated in step 7.2.</li>
	<li>Under a sterile cell culture hood, open osmotic minipump.</li>
	<li>Add diluted angiotensin II solution and expel any air from the syringe and needle. Insert the supplied needle into the bottom of the osmotic minipump and slowly inject angiotensin II solution while slowly and carefully retracting the needle from the bladder.</li>
	<li>Insert the osmotic minipump head into the osmotic bladder fully, paying attention not to get any air into the bladder.</li>
</ol>
8. Implantation of 14 Day Low-dose Angiotensin II Osmotic Minipumps
<ol>
	<li>Ten days after adoptive transfer of CD11c+ DCs, place mice in an isoflurane chamber to initiate anesthesia and then move mice to nose cone to continuously receive 2% isoflurane to achieve and maintain an appropriate surgical plane.</li>
	<li>Remove the fur along the dorsal side of the neck with clippers to prepare surgical site. Clean the shaved area with 70% ethanol followed by 7.5% betadine prior to the start of surgery.</li>
	<li>Create a small incision (approximately 1.5 cm) in the skin. Insert hemostats into the incision to create a subcutaneous pocket for placement of the osmotic minipump.</li>
	<li>Insert the minipump into the subcutaneous pocket. Close the skin wound with 2-3 surgical staples and apply another application of 7.5% betadine to the wound and staples to prevent infection.</li>
	<li>Monitor the mice for 30&#8211;60 min during recovery from anesthesia before returning them to their cages. 
	NOTE: Mice need to be monitored up to 3&#8211;4 days after implantation of the osmotic minipump.</li>
</ol>
9. Isolation of Spleens of Recipient Mice for Flow Cytometric Analysis
<ol>
	<li>After 14 days of low-dose angiotensin II infusion, isolate spleens from mice receiving in vitro high salt treated DCs by adoptive transfer. Follow the precise steps listed above (Steps 1 and 2).</li>
</ol>
10. Surface Staining
<ol>
	<li>Transfer splenocytes that are resuspended in 1x PBS into a polystyrene FACS tube and centrifuge at 350 x g for 8 min at 4 &#176;C. Aspirate supernatant after centrifugation. Wash twice with 1 mL of MACs Buffer and centrifugation (350 x g, 8 min, 4 &#176;C).
	<ol>
		<li>Divide the splenocytes equally into different polystyrene FACS tubes based on the number of flow cytometric panels desired.</li>
	</ol>
	</li>
	<li>To perform the Live/Dead Cell Viability Staining, wash the cells with 1x PBS and centrifuge (350 x g, 8 min, 4 &#176;C). Resuspend in 1 mL of 1x PBS and add 1 &#181;L of a cell-viability stain (see materials table). Incubate for 15 min at 4 &#176;C (refrigerator or on ice) covered in foil to protect from light.</li>
	<li>During incubation of the cell viability stain, prepare the cocktail of antibodies for the cell surface staining in an appropriate volume of MACS Buffer.</li>
	<li>Wash the cells with 1 mL of MACS Buffer, centrifuge (350 x g, 8 min, 4 &#176;C), and resuspend each cell pellet with 100 &#181;L of the antibody cocktail. Incubate protected from light for 30 min at 4 &#176;C. 
	NOTE: Each flow cytometry antibody is unique, and it is highly useful and recommended to determine the optimal amount of antibody needed by performing a dose titration curve for each specific antibody.</li>
	<li>Wash the cells twice with 1 mL of MACS buffer and resuspend the splenocytes in an appropriate amount of MACS Buffer for flow cytometric analysis.</li>
</ol>

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S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Definitions+and+characteristics+of+sodium+sensitivity+and+blood+pressure+resistance.">Definitions and characteristics of sodium sensitivity and blood pressure resistance.</a> Hypertension. 8, 127-134 (1986).</li><li>Morimoto, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sodium+sensitivity+and+cardiovascular+events+in+patients+with+essential+hypertension.">Sodium sensitivity and cardiovascular events in patients with essential hypertension.</a> Lancet. 350, 1734-1737 (1997).</li><li>Weinberger, M. H., Fineberg, N. S., Fineberg, S. 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The authors have nothing to disclose.

In the current protocol, we have optimized procedures to isolate CD11c+ DCs from spleens of mice and adoptively transfer them into na&#239;ve animals to study the role of DCs in salt-induced hypertension. This protocol can be adapted to isolate and adoptively transfer other immune cell subsets including macrophages, monocytes, and adaptive immune cells including T and B lymphocytes. We have optimized the splenic digestion process to achieve adequate cell survival and stability of DC surface expression markers....

Excess dietary salt is a major risk factor for hypertension.1,2 The American Heart Association recommends a maximum of 2,300 milligrams (mg) of sodium (Na+) intake per day, however; less than 10% of the U.S. population observes this recommendation.3,4 Modest reductions in Na+ intake lower blood pressure and reduce the annual new cases of coronary heart disease and stroke in the U.S. by 20%.5 A major problem with excess salt consumption is that 50% of the hypertensive population exhibits salt-sensitivity, defined as a 10 mmHg increase in the blood pressure following Na+ loading or a similar drop in blood pressure after Na+ restriction and diuresis.6 Salt-sensitivity also occurs in 25% of normotensive individuals, and is an independent predictor of death and cardiovascular events.7,8 Salt-sensing mechanisms in hypertension involving the kidney have been well studied; however, recent studies suggest that immune cells can sense Na+.9,10
Recent evidence suggests that changes in extra-renal Na+ handling can cause accumulation of Na+ in the interstitium and promote inflammation.11,12 Our laboratory and others have shown that cells of both the innate and adaptive immune system contribute to the exacerbation of hypertension.9,13,14,15 Various hypertensive stimuli, including angiotensin II, norepinephrine, and salt cause macrophages, monocytes and T lymphocytes to infiltrate the kidney and vasculature and promote Na+ retention, vasoconstriction, blood pressure elevation and end-organ damage.9,16,17,18,19,20 In prior studies, we found that DCs accumulate isolevuglandin (IsoLG)-protein adducts in response to various hypertensive stimuli including angiotensin II and DOCA-salt hypertension.14 IsoLGs are highly reactive products of lipid peroxidation that rapidly and covalently adduct to lysines on proteins and their accumulation is associated with DC activation.14 We have recently established that elevated Na+ is a potent stimulus for IsoLG-protein adduct formation in murine DCs.9 Na+ entry into DCs is mediated through amiloride sensitive transporters. Na+ is then exchanged for calcium (Ca2+) via the Na+/Ca2+ exchanger. Ca2+ activates protein kinase C (PKC) which activates the NADPH oxidase leading to increased superoxide (O2&#183;-) and IsoLG-protein adduct formation.9 Adoptive transfer of salt-exposed DCs primes hypertension in response to a sub-pressor dose of angiotensin II.9
Identification of CD11c+ DCs from tissues has been previously limited to immunohistochemistry and RT-PCR, and isolation of DCs has been limited to cell sorting by flow cytometry. Although flow cytometry cell sorting is a powerful method for the isolation of immune cells, it is costly, time-consuming, and leads to a low yield of viable cells. Therefore, we have optimized a step by step protocol for tissue digestion, in vitro stimulation, and adoptive transfer of CD11c+ DCs to study hypertension.

<table><tbody><tr><td>APC/Cy7 anti-mouse CD11c</td><td>Biolegend</td><td>117324</td><td></td></tr><tr><td>autoMACS Running Buffer&amp;nbsp;</td><td>Miltenyi Biotec</td><td>130-091-221</td><td></td></tr><tr><td>CD11c MicroBeads Ultrapure&amp;nbsp;</td><td>Miltenyi Biotec</td><td>130-108-338</td><td></td></tr><tr><td>Collagenase D</td><td>Roche</td><td>11088866001</td><td></td></tr><tr><td>DNase I</td><td>Roche</td><td>10104159001</td><td></td></tr><tr><td>DPBS without calcium and magnesium</td><td>Corning</td><td>21-031-CV</td><td></td></tr><tr><td>FcR Blocking Reagent</td><td>Miltenyi Biotec</td><td>&amp;nbsp;130-092-575</td><td></td></tr><tr><td>FITC anti-mouse CD45</td><td>Biolegend</td><td>103108</td><td></td></tr><tr><td>GentleMACS C tube</td><td>Miltenyi Biotec</td><td>130-096-334</td><td></td></tr><tr><td>GentleMACS dissociator device</td><td>Miltenyi Biotec</td><td>130-093-235</td><td>Use protocol: Spleen 04.01</td></tr><tr><td>LIVE/DEAD fixable violet dead cell stain kit</td><td>Invitrogen</td><td>L34964</td><td></td></tr><tr><td>LS Columns</td><td>Miltenyi Biotec</td><td>130-042-401</td><td></td></tr><tr><td>QuadroMACs Seperator&amp;nbsp;</td><td>Miltenyi Biotec</td><td>130-090-976</td><td></td></tr><tr><td>RPMI 1640 medium&amp;nbsp;</td><td>Gibco</td><td>11835-030</td><td></td></tr></tbody></table>

isolation and adoptive transfer of high salt treated antigen-presenting dendritic cells

Excess dietary salt intake contributes to inflammation and plays a vital role in the development of hypertension. We previously found that antigen-presenting dendritic cells (DCs) can sense elevated extracellular sodium leading to the activation of the NADPH oxidase and formation of isolevuglandin (IsoLG)-protein adducts. These IsoLG-protein adducts react with self-proteins and promote an autoimmune-like state and hypertension. We have developed and optimized state-of-the-art methods to study DC function in hypertension. Here, we provide a detailed protocol for isolation, in vitro treatment with elevated sodium, and adoptive transfer of murine splenic CD11c+ cells into recipient mice to study their role in hypertension.

Figure 1 represents a schematic of the described steps above. Isolated murine spleens are sorted for CD11c+ DCs by magnetic cell sorting and plated in either normal salt media (NS; 150 mmol NaCl) or high salt media (HS; 190 mmol NaCl) for 48 h. CD11c+ DCs are then adoptively transferred by retro-orbital injection to na&#239;ve recipient mice. Ten days later, mice are implanted with osmotic minipumps for low-dose angiotensin II (140 ng/kg...

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Isolation and Adoptive Transfer of High Salt Treated Antigen-presenting Dendritic Cells

Here, we present a protocol to isolate dendritic cells from murine spleens by magnetic cell sorting and subsequent adoptive transfer into na&#239;ve mice. The model of high-salt activated dendritic cells was chosen to explain the step-by-step procedures of adoptive transfer and flow cytometry.

Excess dietary salt intake contributes to inflammation and plays a vital role in the development of hypertension. We previously found that ...

isolation-adoptive-transfer-high-salt-treated-antigen-presenting

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Immunology and Infection

5.6K Views.  Vanderbilt University Medical Center. Deze methode kan antwoord geven op belangrijke vragen op het gebied van hypertensie en hart- en vaatziekten, waaronder de studie van CD11c positieve dendritische cellen en hoe ze de rol van hypertensieve letsel versterken. Het belangrijkste voordeel van deze techniek is de isolatie van CD11c positieve dendritische cellen van vaste organen en de studie van hun fysiologische rol in verschillende ziektetoestanden. Dit proces kan dus worden toegepast op meerdere immuunceltypen, waaronder B-lymfoc...

Video: Isolation and Adoptive Transfer of High Salt Treated Antigen-presenting Dendritic Cells

HLA-Ig Based Artificial Antigen Presenting Cells for Efficient ex vivo Expansion of Human CTL

CTL with optimal effector function play critical roles in mediating protection against various intracellular infections and cancer. However, individuals may exhibit suppressive immune microenvironment and, in contrast to activating CTL, their autologous antigen presenting cells may tend to tolerize or anergize antigen specific CTL. As a result, although still in the experimental phase, CTL-based adoptive immunotherapy has evolved to become a promising treatment for various diseases such as cancer and virus infections. In initial experiments ex vivo expanded CMV (cytomegalovirus) specific CTL have been used for treatment of CMV infection in immunocompromised allogeneic bone marrow transplant patients. While it is common to have life-threatening CMV viremia in these patients, none of the patients receiving expanded CTL develop CMV related illness, implying the anti-CMV immunity is established by the adoptively transferred CTL1. Promising results have also been observed for melanoma and may be extended to other types of cancer2. 
While there are many ways to ex vivo stimulate and expand human CTL, current approaches are restricted by the cost and technical limitations. For example, the current gold standard is based on the use of autologous DC. This requires each patient to donate a significant number of leukocytes and is also very expensive and laborious. Moreover, detailed in vitro characterization of DC expanded CTL has revealed that these have only suboptimal effector function 3. 
Here we present a highly efficient aAPC based system for ex vivo expansion of human CMV specific CTL for adoptive immunotherapy (Figure 1). The aAPC were made by coupling cell sized magnetic beads with human HLA-A2-Ig dimer and anti-CD28mAb4. Once aAPC are made, they can be loaded with various peptides of interest, and remain functional for months. In this report, aAPC were loaded with a dominant peptide from CMV, pp65 (NLVPMVATV). After culturing purified human CD8+ CTL from a healthy donor with aAPC for one week, CMV specific CTL can be increased dramatically in specificity up to 98% (Figure 2) and amplified more than 10,000 fold. If more CMV-specific CTL are required, further expansion can be easily achieved by repetitive stimulation with aAPC. Phenotypic and functional characterization shows these expanded cells have an effector-memory phenotype and make significant amounts of both TNF&alpha; and IFN&gamma; (Figure 3).

HLA-Ig Based Artificial Antigen Presenting Cells for Efficient ex vivo Expansion of Human CTL

A new DC independent method for induction and expansion of antigen-specific T cells is described. HLA A2-Ig based artificial Antigen Presenting Cells (aAPC) are loaded with HLA-A2 restricted peptides to efficiently expand CTL of diverse antigen specificity. This technology holds great potential for CTL-based adoptive immunotherapy.

CTL with optimal effector function play critical roles in mediating protection against various intracellular infections and cancer. However, individuals ...

Isolation Protocol of Mouse Monocyte-derived Dendritic Cells and Their Subsequent In Vitro Activation with Tumor Immune Complexes

Dendritic cells (DC) are heterogeneous cell populations that differ in their cell membrane markers, migration patterns and distribution, and in their antigen presentation and T cell activation capacities. Since most vaccinations of experimental tumor models require millions of DC, they are widely isolated from the bone marrow or spleen. However, these DC significantly differ from blood and tumor DC in their responses to immune complexes (IC), and presumably to other Syk-coupled lectin receptors. Importantly, given the sensitivity of DC to danger-associated molecules, the presence of endotoxins or antibodies that crosslink activation receptors in one of the isolating steps could result in the priming of DC and thus affect the parameters, or at least the dosage, required to activate them. Therefore, here we describe a detailed protocol for isolating MoDC from blood and tumors while avoiding their premature activation. In addition, a protocol is provided for MoDC activation with tumor IC, and their subsequent analyses.

Isolation Protocol of Mouse Monocyte-derived Dendritic Cells and Their Subsequent In Vitro Activation with Tumor Immune Complexes

Monocyte-derived DC (MoDC) can sense minor amounts of danger-associated molecules and are therefore easily primed. We provide a detailed protocol for the isolation of MoDC from blood and tumors and their activation with immune complexes while highlighting key precautions that should be considered in order to avoid their premature activation.

Dendritic cells (DC) are heterogeneous cell populations that differ in their cell membrane markers, migration patterns and distribution, and in their ...

Cancer Research

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

Preparation of Tumor Antigen-loaded Mature Dendritic Cells for Immunotherapy

While clinical studies have established that antigen-loaded DC vaccines are safe and promising therapy for tumors 1, their clinical efficacy remains to be established. The method described below, prepared in accordance with Good Manufacturing Process (GMP) guidelines, is an optimization of the most common ex vivo preparation method for generating large numbers of DCs for clinical studies 2.
Our method utilizes the synthetic TLR 3 agonist Polyinosinic-Polycytidylic Acid-poly-L-lysine Carboxymethylcellulose (Poly-ICLC) to stimulate the DCs. Our previous study established that Poly-ICLC is the most potent individual maturation stimulus for human DCs as assessed by an upregulation of CD83 and CD86, induction of interleukin-12 (IL-12), tumor necrosis factor (TNF), interferon gamma-induced protein 10 (IP-10), interleukmin 1 (IL-1), and type I interferons (IFN), and minimal interleukin 10 (IL-10) production.
DCs are differentiated from frozen peripheral blood mononuclear cells (PBMCs) obtained by leukapheresis. PBMCs are isolated by Ficoll gradient centrifugation and frozen in aliquots. On Day 1, PBMCs are thawed and plated onto tissue culture flasks to select for monocytes which adhere to the plastic surface after 1-2 hr incubation at 37 &deg;C in the tissue culture incubator. After incubation, the lymphocytes are washed off and the adherent monocytes are cultured for 5 days in the presence of interleukin-4 (IL-4) and granulocyte macrophage-colony stimulating factor (GM-CSF) to differentiate to immature DCs. On Day 6, immature DCs are pulsed with the keyhole limpet hemocyanin (KLH) protein which serves as a control for the quality of the vaccine and may boost the immunogenicity of the vaccine 3. The DCs are stimulated to mature, loaded with peptide antigens, and incubated overnight. On Day 7, the cells are washed, and frozen in 1 ml aliquots containing 4 - 20 x 106 cells using a controlled-rate freezer. Lot release testing for the batches of DCs is performed and must meet minimum specifications before they are injected into patients.

The most commonly used method for generating large numbers of autologous dendritic cells (DCs) for use in tumor immunotherapy is described. The method uses IL-4 and GM-CSF to differentiate DCs from monocytes. The immature DCs are stimulated to mature and then loaded with antigens before they are injected back into the patient.

While clinical studies have established that antigen-loaded DC vaccines are safe and promising therapy for tumors 1, their clinical efficacy remains to be ...

Isolation of Myeloid Dendritic Cells and Epithelial Cells from Human Thymus

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

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

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

An Efficient and High Yield Method for Isolation of Mouse Dendritic Cell Subsets

Dendritic cells (DCs) are professional antigen-presenting cells primarily responsible for acquiring, processing and presenting antigens on antigen presenting molecules to initiate T-cell-mediated immunity. Dendritic cells can be separated into several phenotypically and functionally heterogeneous subsets. Three important subsets of splenic dendritic cells are plasmacytoid, CD8&#945;Pos and CD8&#945;Neg cells. The plasmacytoid DCs are natural producers of type I interferon and are important for anti-viral T cell immunity. The CD8&#945;Neg DC subset is specialized for MHC class II antigen presentation and is centrally involved in priming CD4 T cells. The CD8&#945;Pos DCs are primarily responsible for cross-presentation of exogenous antigens and CD8 T cell priming. The CD8&#945;Pos DCs have been demonstrated to be most efficient at the presentation of glycolipid antigens by CD1d molecules to a specialized T cell population known as invariant natural killer T (iNKT) cells. Administration of Flt-3 ligand increases the frequency of migration of dendritic cell progenitors from bone marrow, ultimately resulting in expansion of dendritic cells in peripheral lymphoid organs in murine models. We have adapted this model to purify large numbers of functional dendritic cells for use in cell transfer experiments to compare in vivo proficiency of different DC subsets.

Distinct dendritic cell subsets exist as rare populations in lymphoid organs, and therefore are challenging to isolate in sufficient numbers and purity for immunological experiments. Here we describe a high efficiency, high yield method for isolation of all of the currently known major subsets of mouse splenic dendritic cells.

Dendritic cells (DCs) are professional antigen-presenting cells primarily responsible for acquiring, processing and presenting antigens on antigen ...

In Vivo Augmentation of Gut-Homing Regulatory T Cell Induction

Inflammatory bowel disease (IBD) is an inflammatory chronic disease in the gastrointestinal tract (GUT). In the United States, there are approximately 1.4 million IBD patients. It is generally accepted that a dysregulated immune response to gut bacteria initiates the disease and disrupts the mucosal epithelial barrier. We recently show that gut-homing regulatory T (Treg) cells are a promising therapy for IBD. Accordingly, this article presents a protocol for in vivo augmentation of gut-homing Treg cell induction. In this protocol, dendritic cells are engineered to produce locally high concentrations of two molecules de novo, active vitamin D (1,25-dihydroxyvitamin D or 1,25[OH]2D) and active vitamin A (retinoic acid or RA). We chose 1,25(OH)2D and RA based on previous findings showing that 1,25(OH)2D can induce the expression of regulatory molecules (e.g., forkhead box P3 and interleukin-10) and that RA can stimulate the expression of gut-homing receptors in T cells. To generate such engineered dendritic cells, we use a lentiviral vector to transduce dendritic cells to overexpress two genes. One gene is the cytochrome P450 family 27 subfamily B member 1 that encodes 25-hydroxyvitamin D 1&#945;-hydroxylase, which physiologically catalyzes the synthesis of 1,25(OH)2D. The other gene is the aldehyde dehydrogenase 1 family member A2 that encodes retinaldehyde dehydrogenase 2, which physiologically catalyzes the synthesis of RA. This protocol can be used for future investigation of gut-homing Treg cells in vivo.

Here we present a protocol for in vivo augmentation of gut-homing regulatory T cell induction. In this protocol, dendritic cells are engineered to locally produce high concentrations of the active vitamin D (1,25-dihydroxyvitamin D or 1,25[OH]2D) and the active vitamin A (retinoic acid or RA) de novo.

Inflammatory bowel disease (IBD) is an inflammatory chronic disease in the gastrointestinal tract (GUT). In the United States, there are approximately 1.4 ...

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

Isolation And Dendritic Cell-Uptake of Small Extracellular Vesicles from Echinococcus granulosus

The secretion of extracellular vesicles by cestodes is crucial for enabling cellular communication not only among parasites but also with host tissues. In particular, small extracellular vesicles (sEVs) act as nano-carriers transferring natural antigens, which are critical in host immunomodulation and parasite survival. This article presents a step-by-step protocol to isolate sEVs from larval stage cultures of Echinococcus granulosus and analyzes their uptake by dendritic cells obtained from murine bone marrow, which acquire adhesion and antigen presentation capacity during their maturation after one week of in vitro culture. This article provides comprehensive information for generating, purifying, and quantifying sEVs using ultracentrifugation alongside parallel analyses of dynamic light scattering and transmission electron microscopy. Additionally, a detailed experimental protocol is outlined for isolating and cultivating mouse bone marrow cells and driving their differentiation into dendritic cells using Flt3L. These dendritic cells can present antigens to na&#239;ve T cells, thereby modulating the type of immune response in vivo. Thus, alternative protocols, including confocal microscopy and flow cytometry analysis, are proposed to check the acquired maturational phenotype of dendritic cells previously exposed to parasitic sEVs. Finally, it is worth noting that the described protocol can be applied as a whole or in individual parts to carry out parasite in vitro culture, isolate extracellular vesicles, generate bone marrow-derived dendritic cell cultures, and perform uptake assays with these cells.

Here, we describe in vitro culture conditions, isolation, and increased generation of extracellular vesicles (EVs) from Echinococcus granulosus. The small EVs were characterized by dynamic light scattering and transmission electron microscopy. The uptake by bone marrow-derived dendritic cells and their phenotypic modulation were studied using confocal microscopy and flow cytometry.

The secretion of extracellular vesicles by cestodes is crucial for enabling cellular communication not only among parasites but also with host tissues. In ...

Effect of an Immunomodulatory Pharmacological Agent on the Interaction Between Dendritic Cells and CD4+ T Cells

Source: Wei, H., et al. <a href="https://app.jove.com/v/57637">Development and Functional Characterization of Murine Tolerogenic Dendritic Cells.</a> J. Vis. Exp. (135), (2018).
This video demonstrates a method of converting dendritic cells (DCs) to tolerogenic DCs (TolDCs) using an immunomodulatory pharmacological agent. This conversion is confirmed by evaluating the TolDC function through their immunosuppressive effect on CD4+ T cells.

1. Evaluate the Function of TolDCs In Vitro and In Vivo

	T-cell Syngeneic Proliferation Assay
	
		Sterilize all the surgical instruments via autoclaving ...

Isolation and Adoptive Transfer of High Salt Treated Antigen-presenting Dendritic Cells

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