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>

<ol>
	<li>Kearney, P. M., 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=Global+burden+of+hypertension:+analysis+of+worldwide+data.">Global burden of hypertension: analysis of worldwide data.</a> Lancet. 365, 217-223 (2005).</li><li>Murray, C. J., Lopez, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measuring+the+global+burden+of+disease.">Measuring the global burden of disease.</a> N Engl J Med. 369, 448-457 (2013).</li><li>Lev-Ran, A., Porta, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Salt+and+hypertension:+a+phylogenetic+perspective.">Salt and hypertension: a phylogenetic perspective.</a> Diabetes/metabolism research and reviews. 21, 118-131 (2005).</li><li>Frisoli, T. M., Schmieder, R. E., Grodzicki, T., Messerli, F. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Salt+and+hypertension:+is+salt+dietary+reduction+worth+the+effort.">Salt and hypertension: is salt dietary reduction worth the effort.</a> The American journal of medicine. 125, 433-439 (2012).</li><li>He, F. J., Li, J., Macgregor, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+longer-term+modest+salt+reduction+on+blood+pressure.">Effect of longer-term modest salt reduction on blood pressure.</a> Cochrane Database Syst Rev. 4, 004937 (2013).</li><li>Weinberger, M. H., Miller, J. Z., Luft, F. C., Grim, C. E., Fineberg, N. 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 border="0" cellpadding="0" cellspacing="0" style="width:824px;" width="823">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:341px;">APC/Cy7 anti-mouse CD11c</td>
			<td style="width:160px;">Biolegend</td>
			<td align="right" style="width:140px;">117324</td>
			<td style="width:183px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">autoMACS Running Buffer&#160;</td>
			<td>Miltenyi Biotec</td>
			<td>130-091-221</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CD11c MicroBeads Ultrapure&#160;</td>
			<td>Miltenyi Biotec</td>
			<td>130-108-338</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Collagenase D</td>
			<td>Roche</td>
			<td align="right">11088866001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DNase I</td>
			<td>Roche</td>
			<td>10104159001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DPBS without calcium and magnesium</td>
			<td>Corning</td>
			<td>21-031-CV</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">FcR Blocking Reagent</td>
			<td>Miltenyi Biotec</td>
			<td>&#160;130-092-575</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:341px;">FITC anti-mouse CD45</td>
			<td style="width:160px;">Biolegend</td>
			<td align="right" style="width:140px;">103108</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">GentleMACS C tube</td>
			<td>Miltenyi Biotec</td>
			<td>130-096-334</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">GentleMACS dissociator device</td>
			<td>Miltenyi Biotec</td>
			<td>130-093-235</td>
			<td>Use protocol: Spleen 04.01</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">LIVE/DEAD fixable violet dead cell stain kit</td>
			<td>Invitrogen</td>
			<td>L34964</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">LS Columns</td>
			<td>Miltenyi Biotec</td>
			<td>130-042-401</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">QuadroMACs Seperator&#160;</td>
			<td>Miltenyi Biotec</td>
			<td>130-090-976</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">RPMI 1640 medium&#160;</td>
			<td style="width:160px;">Gibco</td>
			<td style="width:140px;">11835-030</td>
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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...

Watch this Scientific Journal Video about Isolation and Adoptive Transfer of High Salt Treated Antigen-presenting Dendritic Cells at JoVE.com

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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JoVE Journal

Immunology and Infection

5.6K Views.  Vanderbilt University Medical Center. Here, we present a protocol to isolate dendritic cells from murine spleens by magnetic cell sorting and subsequent adoptive transfer into naïve mice. The model of high-salt activated dendritic cells was chosen to explain the step-by-step procedures of adoptive transfer and flow cytometry.

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

Experimental Metastasis and CTL Adoptive Transfer Immunotherapy Mouse Model

Experimental metastasis mouse model is a simple and yet physiologically relevant metastasis model. The tumor cells are injected intravenously (i.v) into mouse tail veins and colonize in the lungs, thereby, resembling the last steps of tumor cell spontaneous metastasis: survival in the circulation, extravasation and colonization in the distal organs. From a therapeutic point of view, the experimental metastasis model is the simplest and ideal model since the target of therapies is often the end point of metastasis: established metastatic tumor in the distal organ. In this model, tumor cells are injected i.v into mouse tail veins and allowed to colonize and grow in the lungs. Tumor-specific CTLs are then injected i.v into the metastases-bearing mouse. The number and size of the lung metastases can be controlled by the number of tumor cells to be injected and the time of tumor growth. Therefore, various stages of metastasis, from minimal metastasis to extensive metastasis, can be modeled. Lung metastases are analyzed by inflation with ink, thus allowing easier visual observation and quantification.

An experimental lung metastasis and CTL immunotherapy mouse model for analysis of tumor cells-T cell interaction in vivo.

Experimental metastasis mouse model is a simple and yet physiologically relevant metastasis model. The tumor cells are injected intravenously (i.v) into ...

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 of Mouse Lung Dendritic Cells

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

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

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

Artificial Antigen Presenting Cell (aAPC) Mediated Activation and Expansion of Natural Killer T Cells

Natural killer T (NKT) cells are a unique subset of T cells that display markers characteristic of both natural killer (NK) cells and T cells1. Unlike classical T cells, NKT cells recognize lipid antigen in the context of CD1 molecules2. NKT cells express an invariant TCR&alpha; chain rearrangement: V&alpha;14J&alpha;18 in mice and V&alpha;24J&alpha;18 in humans, which is associated with V&beta; chains of limited diversity3-6, and are referred to as canonical or invariant NKT (iNKT) cells. Similar to conventional T cells, NKT cells develop from CD4-CD8- thymic precursor T cells following the appropriate signaling by CD1d 7. The potential to utilize NKT cells for therapeutic purposes has significantly increased with the ability to stimulate and expand human NKT cells with &alpha;-Galactosylceramide (&alpha;-GalCer) and a variety of cytokines8. Importantly, these cells retained their original phenotype, secreted cytokines, and displayed cytotoxic function against tumor cell lines. Thus, ex vivo expanded NKT cells remain functional and can be used for adoptive immunotherapy. However, NKT cell based-immunotherapy has been limited by the use of autologous antigen presenting cells and the quantity and quality of these stimulator cells can vary substantially. Monocyte-derived DC from cancer patients have been reported to express reduced levels of costimulatory molecules and produce less inflammatory cytokines9,10. In fact, murine DC rather than autologous APC have been used to test the function of NKT cells from CML patients11. However, this system can only be used for in vitro testing since NKT cells cannot be expanded by murine DC and then used for adoptive immunotherapy. Thus, a standardized system that relies on artificial Antigen Presenting Cells (aAPC) could produce the stimulating effects of DC without the pitfalls of allo- or xenogeneic cells12, 13. Herein, we describe a method for generating CD1d-based aAPC. Since the engagement of the T cell receptor (TCR) by CD1d-antigen complexes is a fundamental requirement of NKT cell activation, antigen: CD1d-Ig complexes provide a reliable method to isolate, activate, and expand effector NKT cell populations.

Artificial Antigen Presenting Cell aAPC Mediated Activation and Expansion of Natural Killer T Cells

Here we describe a method for activating and expanding human NKT cells from bulk T cell populations using artificial antigen presenting cells (aAPC). The use of CD1d-based aAPC provides a standardized method for generating high numbers of functional NKT cells.

Natural killer T (NKT) cells are a unique subset of T cells that display markers characteristic of both natural killer (NK) cells and T cells1. Unlike ...

Clinical Application of Sleeping Beauty and Artificial Antigen Presenting Cells to Genetically Modify T Cells from Peripheral and Umbilical Cord Blood

The potency of clinical-grade T cells can be improved by combining gene therapy with immunotherapy to engineer a biologic product with the potential for superior (i) recognition of tumor-associated antigens (TAAs), (ii) persistence after infusion, (iii) potential for migration to tumor sites, and (iv) ability to recycle effector functions within the tumor microenvironment. Most approaches to genetic manipulation of T cells engineered for human application have used retrovirus and lentivirus for the stable expression of CAR1-3. This approach, although compliant with current good manufacturing practice (GMP), can be expensive as it relies on the manufacture and release of clinical-grade recombinant virus from a limited number of production facilities. The electro-transfer of nonviral plasmids is an appealing alternative to transduction since DNA species can be produced to clinical grade at approximately 1/10th the cost of recombinant GMP-grade virus. To improve the efficiency of integration we adapted Sleeping Beauty (SB) transposon and transposase for human application4-8. Our SB system uses two DNA plasmids that consist of a transposon coding for a gene of interest (e.g. 2nd generation CD19-specific CAR transgene, designated CD19RCD28) and a transposase (e.g. SB11) which inserts the transgene into TA dinucleotide repeats9-11. To generate clinically-sufficient numbers of genetically modified T cells we use K562-derived artificial antigen presenting cells (aAPC) (clone #4) modified to express a TAA (e.g. CD19) as well as the T cell costimulatory molecules CD86, CD137L, a membrane-bound version of interleukin (IL)-15 (peptide fused to modified IgG4 Fc region) and CD64 (Fc-&gamma; receptor 1) for the loading of monoclonal antibodies (mAb)12. In this report, we demonstrate the procedures that can be undertaken in compliance with cGMP to generate CD19-specific CAR+ T cells suitable for human application. This was achieved by the synchronous electro-transfer of two DNA plasmids, a SB transposon (CD19RCD28) and a SB transposase (SB11) followed by retrieval of stable integrants by the every-7-day additions (stimulation cycle) of &gamma;-irradiated aAPC (clone #4) in the presence of soluble recombinant human IL-2 and IL-2113. Typically 4 cycles (28 days of continuous culture) are undertaken to generate clinically-appealing numbers of T cells that stably express the CAR. This methodology to manufacturing clinical-grade CD19-specific T cells can be applied to T cells derived from peripheral blood (PB) or umbilical cord blood (UCB). Furthermore, this approach can be harnessed to generate T cells to diverse tumor types by pairing the specificity of the introduced CAR with expression of the TAA, recognized by the CAR, on the aAPC.

Clinical Application of Sleeping Beauty and Artificial Antigen Presenting Cells to Genetically Modify T Cells from Peripheral and Umbilical Cord Blood

T cells expressing a CD19-specific chimeric antigen receptor (CAR) are infused as investigational treatment of B-cell malignancies in our first-in-human gene therapy trials. We describe genetic modification of T cells using the Sleeping Beauty (SB) system to introduce CD19-specific CAR and selective propagation on designer CD19+ artificial antigen presenting cells.

The potency of clinical-grade T cells can be improved by combining gene therapy with immunotherapy to engineer  a biologic product with the potential for ...

Accelerated Type 1 Diabetes Induction in Mice by Adoptive Transfer of Diabetogenic CD4+ T Cells

The nonobese diabetic (NOD) mouse spontaneously develops autoimmune diabetes after 12 weeks of age and is the most extensively studied animal model of human Type 1 diabetes (T1D). Cell transfer studies in irradiated recipient mice have established that T cells are pivotal in T1D pathogenesis in this model. We describe herein a simple method to rapidly induce T1D by adoptive transfer of purified, primary CD4+ T cells from pre-diabetic NOD mice transgenic for the islet-specific T cell receptor (TCR) BDC2.5 into NOD.SCID recipient mice. The major advantages of this technique are that isolation and adoptive transfer of diabetogenic T cells can be completed within the same day, irradiation of the recipients is not required, and a high incidence of T1D is elicited within 2 weeks after T cell transfer. Thus, studies of pathogenesis and therapeutic interventions in T1D can proceed at a faster rate than with methods that rely on heterogenous T cell populations or clones derived from diabetic NOD mice.

We provide a reproducible method to induce type 1 diabetes (T1D) in mice within two weeks by the adoptive transfer of islet antigen-specific, primary CD4+ T cells.

The nonobese diabetic (NOD) mouse spontaneously develops autoimmune diabetes after 12 weeks of age and is the most extensively studied animal model of ...

Isolation, Purification and Labeling of Mouse Bone Marrow Neutrophils for Functional Studies and Adoptive Transfer Experiments

Neutrophils are critical effector cells of the innate immune system. They are rapidly recruited at sites of acute inflammation and exert protective or pathogenic effects depending on the inflammatory milieu. Nonetheless, despite the indispensable role of neutrophils in immunity, detailed understanding of the molecular factors that mediate neutrophils' effector and immunopathogenic effects in different infectious diseases and inflammatory conditions is still lacking, partly because of their short half life, the difficulties with handling of these cells and the lack of reliable experimental protocols for obtaining sufficient numbers of neutrophils for downstream functional studies and adoptive transfer experiments. Therefore, simple, fast, economical and reliable methods are highly desirable for harvesting sufficient numbers of mouse neutrophils for assessing functions such as phagocytosis, killing, cytokine production, degranulation and trafficking. To that end, we present a reproducible density gradient centrifugation-based protocol, which can be adapted in any laboratory to isolate large numbers of neutrophils from the bone marrow of mice with high purity and viability. Moreover, we present a simple protocol that uses CellTracker dyes to label the isolated neutrophils, which can then be adoptively transferred into recipient mice and tracked in several tissues for at least 4 hr post-transfer using flow cytometry. Using this approach, differential labeling of neutrophils from wild-type and gene-deficient mice with different CellTracker dyes can be successfully employed to perform competitive repopulation studies for evaluating the direct role of specific genes in trafficking of neutrophils from the blood into target tissues in vivo.

We describe a protocol for isolation and purification of neutrophils from mouse bone marrow by density gradient centrifugation and for neutrophil labeling using CellTracker dyes. This represents a simple, fast, reproducible and economical method for obtaining large numbers of neutrophils for downstream functional studies or adoptive transfer and tracking experiments.

Neutrophils are critical effector cells of the innate immune system. They are rapidly recruited at sites of acute inflammation and exert protective or ...

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 ...

Assessing the Development of Murine Plasmacytoid Dendritic Cells in Peyer's Patches Using Adoptive Transfer of Hematopoietic Progenitors

This protocol details a method to analyze the ability of purified hematopoietic progenitors to generate plasmacytoid dendritic cells (pDC) in intestinal Peyer&#39;s patch (PP). Common dendritic cell progenitors (CDPs, lin- c-kitlo CD115+ Flt3+) were purified from the bone marrow of C57BL6 mice by FACS and transferred to recipient mice that lack a significant pDC population in PP; in this case, Ifnar-/- mice were used as the transfer recipients. In some mice, overexpression of the dendritic cell growth factor Flt3 ligand (Flt3L) was enforced prior to adoptive transfer of CDPs, using hydrodynamic gene transfer (HGT) of Flt3L-encoding plasmid. Flt3L overexpression expands DC populations originating from transferred (or endogenous) hematopoietic progenitors. At 7-10 days after progenitor transfer, pDCs that arise from the adoptively transferred progenitors were distinguished from recipient cells on the basis of CD45 marker expression, with pDCs from transferred CDPs being CD45.1+ and recipients being CD45.2+. The ability of transferred CDPs to contribute to the pDC population in PP and to respond to Flt3L was evaluated by flow cytometry of PP single cell suspensions from recipient mice. This method may be used to test whether other progenitor populations are capable of generating PP pDCs. In addition, this approach could be used to examine the role of factors that are predicted to affect pDC development in PP, by transferring progenitor subsets with an appropriate knockdown, knockout or overexpression of the putative developmental factor and/or by manipulating circulating cytokines via HGT. This method may also allow analysis of how PP pDCs affect the frequency or function of other immune subsets in PPs. A unique feature of this method is the use of Ifnar-/- mice, which show severely depleted PP pDCs relative to wild type animals, thus allowing reconstitution of PP pDCs in the absence of confounding effects from lethal irradiation.

This protocol describes experimental procedures to assess the differentiation of plasmacytoid dendritic cells in Peyer&#8217;s patch from common dendritic cell progenitors, using techniques involving FACS-mediated cell isolation, hydrodynamic gene transfer, and flow analysis of immune subsets in Peyer&#8217;s patch.

This protocol details a method to analyze the ability of purified hematopoietic progenitors to generate plasmacytoid dendritic cells (pDC) in intestinal ...

Killer Artificial Antigen Presenting Cells (KaAPC) for Efficient In Vitro Depletion of Human Antigen-specific T Cells

Current treatment of T cell mediated autoimmune diseases relies mostly on strategies of global immunosuppression, which, in the long term, is accompanied by adverse side effects such as a reduced ability to control infections or malignancies. Therefore, new approaches need to be developed that target only the disease mediating cells and leave the remaining immune system intact. Over the past decade a variety of cell based immunotherapy strategies to modulate T cell mediated immune responses have been developed. Most of these approaches rely on tolerance-inducing antigen presenting cells (APC). However, in addition to being technically difficult and cumbersome, such cell-based approaches are highly sensitive to cytotoxic T cell responses, which limits their therapeutic capacity. Here we present a protocol for the generation of non-cellular killer artificial antigen presenting cells (KaAPC), which allows for the depletion of pathologic T cells while leaving the remaining immune system untouched and functional. KaAPC is an alternative solution to cellular immunotherapy which has potential for treating autoimmune diseases and allograft rejections by regulating undesirable T cell responses in an antigen specific fashion.

Killer Artificial Antigen Presenting Cells KaAPC for Efficient In Vitro Depletion of Human Antigen-specific T Cells

Guidelines are presented for the generation of killer artificial antigen presenting cells, KaAPC, an efficient tool for in vitro depletion of human antigen-specific T cells and an alternative solution to cellular immunotherapy for the treatment of T cell-mediated autoimmune diseases in an antigen-specific fashion without compromising the remaining immune system.

Current treatment of T cell mediated autoimmune diseases relies mostly on strategies of global immunosuppression, which, in the long term, is accompanied ...

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

Summary

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Chapters in this video

Experimental Metastasis and CTL Adoptive Transfer Immunotherapy Mouse Model

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

Isolation of Mouse Lung Dendritic Cells

Artificial Antigen Presenting Cell (aAPC) Mediated Activation and Expansion of Natural Killer T Cells

Clinical Application of Sleeping Beauty and Artificial Antigen Presenting Cells to Genetically Modify T Cells from Peripheral and Umbilical Cord Blood

Accelerated Type 1 Diabetes Induction in Mice by Adoptive Transfer of Diabetogenic CD4+ T Cells

Isolation, Purification and Labeling of Mouse Bone Marrow Neutrophils for Functional Studies and Adoptive Transfer Experiments

Isolation of Myeloid Dendritic Cells and Epithelial Cells from Human Thymus

Assessing the Development of Murine Plasmacytoid Dendritic Cells in Peyer's Patches Using Adoptive Transfer of Hematopoietic Progenitors

Killer Artificial Antigen Presenting Cells (KaAPC) for Efficient In Vitro Depletion of Human Antigen-specific T Cells