This research was supported by Creative Materials Discovery Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT and Future Planning (NRF-2017M3D1A1039421) and by Marine Biotechnology Program funded by the Ministry of Oceans and Fisheries, Republic of Korea and a grant (20150220).

All experimental procedures including animal handling, sacrifice, and organ isolation were performed in strict accordance with the rules of the International Animal Care and Use Committee at Shanghai Public Health Clinical Center and Asan Medical Center. The study protocol was approved by the respective committees on the Ethics of Animal Experiments at Shanghai Public Health Clinical Center (Mouse Protocol Number: SYXK-2010-0098) and Asan Medical Center (2016-02-168).
1. Preparation of Material
NOTE: A nano-adjuvant, INP, comprised of a self-assembled lipid-DNA carrier, namely U4T, and CpG-motif containing oligonucleotide, eCpG, was employed in the current study to test the appropriate injection route for immunotherapy and vaccination in mice2. Importantly, other potential therapeutic molecules such as antigens, antibodies, and adjuvant agents can be substituted for INP and tested using the same methodology described below.
<ol>
	<li>INP formulation2,4,7
	<ol>
		<li>Anneal U4T (160 &#181;M, carrier) with eCpG (80 &#181;M, containing a TLR9 agonist sequence) in the presence of 1x phosphate-buffered saline (PBS, pH 7.4) and MgCl2 (2 mM). The standard volume for annealing is between 50 and 110 &#181;L in a PCR tube. Heat the mixture to 95 &#176;C for 10 min and then slowly cool (1 &#176;C/16 min) to 25 &#176;C using a thermocycler. The annealing protocol requires approximately 2 h. 
		CAUTION: The U4T-containing liquid is very foamy and must be carefully handled.</li>
		<li>Make 50 &#181;L aliquots of the INP preparation; use 50 &#181;L of the material for each injection. 
		NOTE: In the case of 100 &#181;L per injection, repeat step 1.1 with proper calculation of ingredients.</li>
	</ol>
	</li>
</ol>
2. General Animal Procedures
Note: Alternative methods for handling the mice can be used depending on the laboratory requirements and approved animal protocols8. The type of mice used is 6-week-old C57BL/6 and female mice.
<ol>
	<li>List of materials
	<ol>
		<li>Prepare the liquid anesthetic (isoflurane), anesthetic machine, carbon dioxide (CO2)&#160;inhalation chamber, sterilized gauze, forceps, mouse restrainer, gloves, hooked forceps, 1-inch dissecting scissors, 2-inch dissecting scissors, alcohol for sterilization, and insulin syringe.</li>
	</ol>
	</li>
	<li>Inhalant anesthesia 
	NOTE: Anesthesia is mandatory for intranasal, subcutaneous, footpad, and intravenous injections of mice9. An induction chamber with a vaporizer machine and oxygen tank connected is used for this procedure.
	<ol>
		<li>Place the animal in the induction chamber and make sure to tightly close the lid.</li>
		<li>Flow the oxygen at 1 L/min and set the vaporizer setting to an induction level of 4% for isoflurane.</li>
		<li>Vary the exposure time of animals in the chamber depending on animal weight; however, more than 10 s of exposure and monitoring every 5 min is necessary for anesthesia10. 
		NOTE: No pedal reflex indicates successful anesthesia.</li>
		<li>Turn off the isoflurane inductor and flow 1 L/min of oxygen to prevent personnel exposure to isoflurane gas.</li>
	</ol>
	</li>
	<li>Sacrifice
	<ol>
		<li>Prepare a chamber for CO2 inhalation sacrifice in accordance with the NIH &#34;Guidelines for Euthanasia of Rodents Using Carbon Dioxide&#34;11.</li>
	</ol>
	</li>
	<li>Test gag response to confirm that the animal is fully sacrificed.
	<ol>
		<li>Verify that the mouse is properly sacrificed by pinching the tip of its foot and confirming no gag response. If there is a response, repeat step 2.2.</li>
	</ol>
	</li>
	<li>Disinfection
	<ol>
		<li>Disinfect all surface areas used for injections or dissections with alcohol spray and sterilized gauze.</li>
	</ol>
	</li>
	<li>Organ storage
	<ol>
		<li>Use forceps to remove all fat and blood surrounding the harvested organs. Store these harvested tissues individually in a Petri dish filled with 3 mL of PBS. The container and media may differ depending on the subsequent procedure requirements.</li>
	</ol>
	</li>
</ol>
3. Injection Routes
<ol>
	<li>Use the six injection methods to investigate injection-dependent immune responses: oral, intranasal, subcutaneous, footpad, intraperitoneal, and intravenous8,12,13.</li>
</ol>
4. Isolation of Lymph Nodes and Spleen
Note: Use young and healthy lean mice (6 weeks old) for these procedures because the fats that typically build up around the lymph nodes in older mice are difficult to remove and can prevent proper visualization of the organ.
<ol>
	<li>Pre-harvesting steps 
	NOTE: This section describes the post-injection harvesting of the iLN, spleen, and mLN from mice for analyzing immune stimulation. Refer to steps 2.3-2.5 for sacrifice, gag response, and disinfection methods.
	<ol>
		<li>Pin the limbs of the mouse to the surgical foam board and sterilize the ventral side of the mouse with 70% ethanol.</li>
		<li>Pick a location 2 cm above the genital area and use hooked forceps to pull up the outer skin cover like a tent. Make an approximately 1-cm cut at this location and slide the scissors (2-inch) underneath the skin, making further dissection cuts.</li>
		<li>For the integument, cut vertically through the outer skin with both arms of the scissors (2-inch). Expose the internal organs covered with peritoneum.</li>
		<li>Pin the outer layer of the skin.</li>
	</ol>
	</li>
	<li>Isolation of the inguinal lymph node (iLN)
	<ol>
		<li>Locate the iLN at the left side of the leg following the conjunction of the blood vessel going down the left leg, which appears as a tilted letter &#39;Y&#39;. 
		NOTE: For subcutaneously injected mice, choose an iLN on the same side of the injection location.</li>
		<li>Uncover the layer with two forceps to rip the covered lipid layer and harvest the pale yellow iLN (bean shape, 2 mm). For subsequent treatment and storage of the tissue, see step 2.6.</li>
	</ol>
	</li>
	<li>Isolation of the spleen14
	<ol>
		<li>Make a tent at the center of the peritoneum using hooked forceps in one hand and use small thin scissors (1 inch) in the other hand to cut the peritoneum.</li>
		<li>Use hooked forceps in the left hand to grasp the intestine and flip it over to locate the red bean-like spleen (length of approximately 14 mm) attached to the left upper side of the mouse abdomen.</li>
		<li>Gently pull out the spleen with another set of forceps in the right hand while detaching other organs with the hooked forceps. Follow step 2.6 to store the spleen. 
		NOTE: Care is required when harvesting the spleen, as it is an easily damaged soft organ.</li>
	</ol>
	</li>
	<li>Isolation of the mediastinal lymph node (mLN) 
	NOTE: The mLN is located approximately 1 cm beneath the ribs and is covered by other organs such as the heart and lungs. Because of its location and small size, it is important to carefully expose the surrounding area using forceps and scissors (1-inch).
	<ol>
		<li>Cut the diaphragm and detach it from the ribs. Hold the body of the ribs with forceps and then cut the left and right side of the ribs with scissors (1-inch). Pin the cutoff ribs over the mouse&#39;s shoulder on the right to expose the heart and lungs. 
		NOTE: Take care not to cut any of the blood vessels around the heart and lungs, as leaking blood makes it very difficult to locate the tiny translucent mLN. If any blood vessels are cut, gently absorb the blood in the area with gauze.</li>
		<li>Using hooked forceps in the right hand, flip the heart and lungs gently to the right until the backbone is exposed. Use a pair of regular forceps in the left hand to remove the iLN with the hooked forceps in the right hand to assist with this harvesting.</li>
		<li>Grab the lungs and flip them until the backbone is widely exposed. The mLN (a translucent bean-shaped structure of approximately 2 mm in size) is located between the lungs and the spine.</li>
		<li>Utilize the hooked forceps to expose the area around the mLN and the other forceps to extract the tissue. 
		NOTE: The mLN is surrounded by numerous other tissues which must be removed carefully with forceps before harvest.</li>
		<li>See step 2.7 for instructions on storing the harvested mLN.</li>
	</ol>
	</li>
</ol>
5. Preparation of samples for flow cytometry
Note: To assay the activation of DCs in the harvested iLN, spleen, and mLN, single-cell suspensions of these tissues are stained with fluorescence-conjugated antibodies as DC-specific markers, co-stimulatory molecules, and major histocompatibility complex molecules and analyzed by flow cytometry.
<ol>
	<li>Preparation of single-cell suspensions: spleen
	<ol>
		<li>Add 5 mL of fresh culture medium (CM) into 6-mm culture dish and place the samples on ice.</li>
		<li>Remove surrounding blood and fat tissues and place the tissue in the lid of the culture dish.</li>
		<li>Cut the tissues into small fragments (&#60;1 mm) with curved scissors. Suspend the fragments in PBS followed by spinning down the tissue using a centrifuge at 646 &#215; g for 5 min and removing the supernatant.</li>
		<li>Suspend the samples again in 3 mL of CM and digest the fragments with 200 &#181;L of solution of 2% fetal bovine serum (FBS) supplemented with collagenase IV for 20 min.</li>
		<li>Gently shake and incubate the sample at 37 &#176;C for 1 h. Filter the digested tissues through nylon mesh (100 nm) and spin them down. Wash the single cells in 5 mL of PBS and remove the supernatant by centrifugation.</li>
		<li>Re-suspend the cells in 5 mL of cell isolating solution. Re-load another layer of 5 mL of clear cell isolation solution to form an aqueous layer-by-layer stack.</li>
		<li>Centrifuge the cell preparation at 2012 &#215; g for 10 min. Obtain the supernatant, which is the lighter density fraction (&#60;1.077 g/cm3), for subsequent flow cytometry analysis.</li>
		<li>Count the number of cells with a hemocytometer.</li>
	</ol>
	</li>
	<li>Preparation of single-cell suspensions: lymph node
	<ol>
		<li>Follow step 5.1.1-5.1.2.</li>
		<li>Add 5 mL of CM using a 10-mL pipet to suspend the cells by pipetting 3-4 times. After thoroughly suspending the cells, use sterile nylon mesh to filter the cells for collection in a 15-mL conical tube.</li>
		<li>Spin the cells down at 646 &#215;g for 7 min at 4 &#176;C and remove the supernatant.</li>
		<li>Add an additional 5 mL of CM into 10-mL pipet to better suspend the cells by pipetting.</li>
		<li>Wash with PBS and count the number of cells.</li>
	</ol>
	</li>
	<li>Flow cytometry analysis
	<ol>
		<li>Aliquot 0.5-1 &#215; 106 cells into each fluorescence-activated cell sorting (FACS) tube.</li>
		<li>Centrifuge at 646 &#215; g at 4 &#176;C for 5 min and aspirate the supernatant.</li>
		<li>Add 5 &#181;L of each diluted fluorescence-conjugated antibody preparation to the cells to re-suspend the cell pellet and vortex. 
		NOTE: It is important to shield the cell pellet from light exposure as much as possible because fluorescently labeled antibodies are light-sensitive. Generate an antibody master mix and always add Fc block.</li>
		<li>Prepare FACS buffer using 100 &#181;L of PBS, 1% of heat-inactivated regular FBS, and 0.1% sodium azide.</li>
		<li>Place the sample in the tube at 4 &#176;C for 30 min. Rinse the cells with 2-4 mL of FACS buffer and centrifuge the sample at 1700 rpm for 7 min at 4 &#176;C.</li>
		<li>Re-suspend the cells in 500 &#181;L of FACS buffer in the dark. Analyze the cells by flow cytometry.</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Park, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Preparation of dodecynyl modified DNA nanoparticles with unmethylated CpG adjuvant and ovalbumin for immunostimulation. , (2016).</li><li>Jin, J. O., Park, H., Zhang, W., de Vries, J. W., Gruszka, A., Lee, M. W., Ahn, D. R., Herrmann, A., Kwak, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modular+delivery+of+CpG-incorporated+lipid-DNA+nanoparticles+for+spleen+DC+activation.">Modular delivery of CpG-incorporated lipid-DNA nanoparticles for spleen DC activation.</a> Biomaterials. 115, 81-89 (2017).</li><li>Pal, I., Ramsey, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+the+lymphatic+system+in+vaccine+trafficking+and+immune+response.">The role of the lymphatic system in vaccine trafficking and immune response.</a> Advanced Drug Delivery Reviews. 63 (10-11), 909-922 (2011).</li><li>Jin, J. O., Kwak, M., Xu, L., Kim, H., Lee, T. H., Kim, J. O., Liu, Q., Herrmann, A., Lee, P. C. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Administration+of+soft+matter+lipid-DNA+nanoparticle+as+the+immunostimulant+via+multiple+routes+of+injection+in+vivo.">Administration of soft matter lipid-DNA nanoparticle as the immunostimulant via multiple routes of injection in vivo.</a> ACS Biomaterial Science &amp; Engineering. 3 (9), 2054-2058 (2017).</li><li>Worbs, T., Hammerschmidt, S. I., F&#246;rster, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dendritic+cell+migration+in+health+and+disease.">Dendritic cell migration in health and disease.</a> Nature Review Immunology. 17 (1), 30-48 (2017).</li><li>Milling, S. W. F., Jenkins, C., MacPherson, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Collection+of+lymph-borne+dendritic+cells+in+the+rat.">Collection of lymph-borne dendritic cells in the rat.</a> Nature Protocols. 1 (5), 2263-2270 (2006).</li><li>Desormeaux, A., Bergeron, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lymphoid+tissue+targeting+of+anti-HIV+drugs+using+liposomes.">Lymphoid tissue targeting of anti-HIV drugs using liposomes.</a> Methods in Enzymology. 391, 330-351 (2005).</li><li>Machholz, E., Mulder, G., Ruiz, C., Corning, B. F., Pritchett-Corning, K. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Manual+restraint+and+common+compound+administration+routes+in+mice+and+rats.">Manual restraint and common compound administration routes in mice and rats.</a> Journal of Visualized Experiments. (67), (2012).</li><li>Turner, P. V., Brabb, T., Pekow, C., Vasbinder, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Administration+of+substances+to+laboratory+animals:+routes+of+administration+and+factors+to+consider.">Administration of substances to laboratory animals: routes of administration and factors to consider.</a> Journal of the American Association for Laboratory Animal Science. 50 (5), 600-613 (2011).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> General anesthesia of mice and rats. , (2016).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Guidelines for the Euthanasia of Animals. , (2013).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=JoVE+Science+Education+Database.+Lab+Animal+Research.+Compound+Administration+I.">JoVE Science Education Database. 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The authors have nothing to disclose.

Many advances in nanotechnology and immunology have been achieved through therapeutic research of drug delivery and immunostimulation. Careful selection of the injection method is known to be important for immunostimulation, which was the focus of the present study.
Different injection routes were evaluated for a naturally non-toxic and biodegradable DNA-based material, INP (immunostimulatory nanoparticle), to determine which route yielded the best outcome. This approach is also relevant for d...

Advances in immunology and nanomaterials have led to an abundance of potential new therapeutic strategies for applications in biomedicine, including drug delivery and immunostimulation. Optimization of the administration route is a vital aspect affecting the efficacy of immunostimulatory agents. An immunostimulatory nanoparticle (INP) consisting of DNA is a newly developed nano-immune adjuvant self-assembled by microphase separation because of the amphiphilic structure of lipid-DNA1. Therefore, protocols for INP involving administration of the material1&#160;in vivo via different routes, and three procedures for harvesting appropriate tissues such as the inguinal lymph node (iLN), mediastinal LN (mLN), and spleen, are described. Finally, these tissues were analyzed for dendritic cell (DC) activation, the most powerful antigen-presenting cells in the immune system. This protocol can also be applied for evaluating antigens, antibodies, or other immune adjuvants2.
We tested the INP formulation because it is an agent that has shown great promise. INP is a Toll-like receptor 9 (TLR9) adjuvant material that contains nucleic acids, for which assessment of immunostimulation efficacy is required to test different injection methods3. In this context, the stimulation of DCs is a potent endpoint for in vivo evaluation. After antigen or immunostimulatory molecules are phagocytosed by DCs in the peripheral tissues or blood, these cells migrate to lymphoid organs such as the spleen and LNs4,5. Thus, DC activation was analyzed in the spleen, iLN, and mLN of the injected animals. Properly harvesting of these tissues is therefore also crucial for evaluating the immune response to a novel adjuvant or pathogens5. Such tissue harvesting is also important for developing a novel immunological methodology as a cancer therapy. Furthermore, this protocol can be used to verify the efficiency of other drugs, such as anti-human immunodeficiency virus therapeutics6.

<table><tbody><tr><td>&lt;strong&gt;Material&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>phosphate buffer saline</td><td>Corning</td><td>21-040-CVR</td><td>Washing organs</td></tr><tr><td>(PBS, pH 7.4)</td><td></td><td></td><td></td></tr><tr><td>isoflurane solution&amp;nbsp;</td><td>Aesica Queenborough limited</td><td>26675-46-7</td><td>Anesthesia process</td></tr><tr><td>Tuberculin 1mL syringe -</td><td>Junglim</td><td>N/A</td><td>Injection</td></tr><tr><td>50 mL conical tube&amp;nbsp;</td><td>S.P.L</td><td>50050</td><td>Anesthesia process</td></tr><tr><td>1mL Insulin Syringe&amp;nbsp;</td><td>(BD Ultra-Fine&lt;sup&gt;TM&lt;/sup&gt;&amp;shy;II)_short needle</td><td>324826</td><td>Intramuscular Injection</td></tr><tr><td>DMEM High Glucose</td><td>Hyclone</td><td>SH30081.01</td><td>Storing organs</td></tr><tr><td>Histopaque</td><td>&amp;nbsp;Sigma-Aldrich</td><td>10771</td><td>FACS analysis</td></tr><tr><td>Ethyl alcohol anhydrous 99.5 %&amp;nbsp;</td><td>Daejung</td><td>4022-4110</td><td>Disinfectant</td></tr><tr><td>&lt;strong&gt;Equipments&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>FineCycler C100 (Thermocycler)</td><td>Ssufine</td><td>-</td><td>Anealing</td></tr><tr><td>Centrifuge</td><td></td><td></td><td>Centrifuge</td></tr><tr><td>FACS tube&amp;nbsp;</td><td>FALCON</td><td>2052</td><td>FACS analysis</td></tr><tr><td>Automated High-performance Flow Cytometer</td><td>BD (USA), FACSVerse</td><td>-</td><td>FACS analysis</td></tr></tbody></table>

immunostimulatory agent evaluation: lymphoid tissue extraction and injection route-dependent dendritic cell activation

For evaluation of a new therapeutic agent for immunotherapy or vaccination, analysis of immune cell activation in lymphatic tissues is essential. Here, we investigated immunological effects of a novel lipid-DNA immunostimulant in nanoparticle form from different administration routes in the mouse: oral, intranasal, subcutaneous, footpad, intraperitoneal, and intravenous. These injections will directly influence the immune response, and harvesting lymphatic tissues and analysis of dendritic cell (DC) activation in the tissues are crucial parts of these evaluations. The extraction of mediastinal lymph nodes (mLNs) is important but quite complex because of the size and location of this organ. A stepwise procedure for harvesting the inguinal lymph node (iLN), mLN, and spleen and analyzing DC activation by flow cytometry is described.

To evaluate appropriate injection routes of INP for lymphatic tissue DC activation, the DC population as lineage was defined as -CD11c+ cells in the spleen, iLN, and mLN and analyzed the expression levels of co-stimulatory molecules. Treatment of INP by subcutaneous (s.c.) and intravenous (i.v.) injection promoted substantial increases in CD40, CD80, and CD86 expression in the spleen and iLN DCs (Figure 2B and 2...

Watch this Scientific Journal Video about Immunostimulatory Agent Evaluation: Lymphoid Tissue Extraction and Injection Route-Dependent Dendritic Cell Activation at JoVE.com

Immunostimulatory Agent Evaluation: Lymphoid Tissue Extraction and Injection Route-Dependent Dendritic Cell Activation

Experimental procedures for the subsequent extraction of lymphatic tissues to test lymphoid dendritic cell activation are described after treatment of an immunostimulating nanomaterial.

For evaluation of a new therapeutic agent for immunotherapy or vaccination, analysis of immune cell activation in lymphatic tissues is essential. Here, we ...

immunostimulatory-agent-evaluation-lymphoid-tissue-extraction

Research

JoVE Journal

Immunology and Infection

10.0K Views.  Fudan University. Het algemene doel van deze procedure is om de unieke techniek die nodig is bij het isoleren van mediastinale lymfeklier van muizen vertrouwd te maken. De operatie werd uitgevoerd na het injecteren van immunostimulant materiaal op zes verschillende routes bij muizen. In deze studie gebruikten we een nieuwe klasse van materiaalvormende nano-sized structuren.Oppervlakteactieve stoffen bestaan uit hydrofiele kopgroep en hydrofobe staart. Die vormen bolvormige micelle of andere secundaire s...

Video: Immunostimulatory Agent Evaluation: Lymphoid Tissue Extraction and Injection Route-Dependent Dendritic Cell Activation

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

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 ...

Intralymphatic Immunotherapy and Vaccination in Mice

Vaccines are typically injected subcutaneously or intramuscularly for stimulation of immune responses. The success of this requires efficient drainage of vaccine to lymph nodes where antigen presenting cells can interact with lymphocytes for generation of the wanted immune responses. The strength and the type of immune responses induced also depend on the density or frequency of interactions as well as the microenvironment, especially the content of cytokines. As only a minute fraction of peripherally injected vaccines reaches the lymph nodes, vaccinations of mice and humans were performed by direct injection of vaccine into inguinal lymph nodes, i.e.&#160;intralymphatic injection. In man, the procedure is guided by ultrasound. In mice, a small (5-10 mm) incision is made in the inguinal region of anesthetized animals, the lymph node is localized and immobilized with forceps, and a volume of 10-20 &#956;l of the vaccine is injected under visual control. The incision is closed with a single stitch using surgical sutures. Mice were vaccinated with plasmid DNA, RNA, peptide, protein, particles, and bacteria as well as adjuvants, and strong improvement of immune responses against all type of vaccines was observed. The intralymphatic method of vaccination is especially appropriate in situations where conventional vaccination produces insufficient immunity or where the amount of available vaccine is limited.

Prophylactic and therapeutic vaccination often fails to stimulate strong immune responses due to week drainage of the vaccine to lymph nodes and consequently poor involvement of immune cells. By direct injection of vaccine to lymph nodes, so-called intralymphatic injection, vaccine efficacy can be strongly improved and vaccine doses can be reduced.

Vaccines are typically injected subcutaneously or intramuscularly for stimulation of immune responses. The success of this requires efficient drainage of ...

Generation of Immature, Mature and Tolerogenic Dendritic Cells with Differing Metabolic Phenotypes

Immune response results from a complex interplay between the antigen non-specific innate immune system and the antigen specific adaptive immune system. The immune system is a constant balance in maintaining tolerance to self-molecules and reacting rapidly to pathogens. Dendritic cells (DCs) are powerful professional antigen presenting cells that link the innate immune system to the adaptive immune system and balance the adaptive response between self and non-self. Depending on the maturation signals, immature dendritic cells can be selectively stimulated to differentiate into immunogenic or tolerogenic DCs. Immunogenic dendritic cells provide proliferation signals to antigen-specific T cells for clonal expansion; while tolerogenic dendritic cells regulate tolerance by antigen-specific T-cell deletion or clonal expansion of regulatory T-cells. Due to this unique property, dendritic cells are highly sought after as therapeutic agents for cancer and autoimmune diseases. Dendritic cells can be loaded with specific antigens in vitro and injected into the human body to mount a specific immune response both immunogenic and tolerogenic. This work presents a means to generate in vitro from monocytes, immature monocyte derived dendritic cells (moDCs), tolerogenic and mature moDCs that differ in surface marker expression, function and metabolic phenotypes.

Immature dendritic cells can be selectively differentiated into tolerogenic or mature dendritic cells to regulate the balance between immunity and tolerance. This work presents a means to generate from immature monocyte derived dendritic cells (moDCs), in vitro tolerogenic and mature moDCs that differ in metabolic phenotypes.

Immune response results from a complex interplay between the antigen non-specific innate immune system and the antigen specific adaptive immune system. ...

Novel Protocol for Generating Physiologic Immunogenic Dendritic Cells

Extracorporeal photochemotherapy (ECP) is a widely used cancer immunotherapy for cutaneous T cell lymphoma (CTCL), operative in over 350 university centers worldwide. While ECP&#8217;s clinical efficacy and exemplary safety profile have driven its widespread use, elucidation of the underlying mechanisms has remained a challenge, partly owing to lack of a laboratory ECP model. To overcome this obstacle and create a simple, user-friendly platform for ECP research, we developed a scaled-down version of the clinical ECP leukocyte-processing device, suitable for work with both mouse models, and small human blood samples. This device is termed the Transimmunization (TI) chamber, or plate. In a series of landmark experiments, the miniaturized device was used to produce a cellular vaccine that regularly initiated therapeutic anti-cancer immunity in several syngeneic mouse tumor models. By removing individual factors from the experimental system and ascertaining their contribution to the in vivo anti-tumor response, we then elucidated key mechanistic drivers of ECP immunizing potential. Collectively, our results revealed that anti-tumor effects of ECP are initiated by dendritic cells (DC), physiologically generated through blood monocyte interaction with platelets in the TI plate, and loaded with antigens from tumor cells whose apoptotic cell death is finely titrated by exposure to the photoactivatable DNA cross-linking agent 8-methoxypsoralen and UVA light (8-MOPA). When returned to the mouse, this cellular vaccine leads to specific and transferable anti-tumor T cell immunity. We verified that the TI chamber is also suitable for human blood processing, producing human DCs fully comparable in activation state and profile to those derived from the clinical ECP chamber. The protocols presented here are intended for ECP studies in mouse and man, controlled generation of apoptotic tumor cells with 8-MOPA, and rapid production of physiologic human and mouse monocyte-derived DCs for a variety of applications.

The transimmunization (TI) device or plate and related protocols have been developed to replicate the key features of extracorporeal photochemotherapy (ECP), in an experimental setting, allowing for production of physiologically activated, tunable dendritic cells (DCs) for cancer immunotherapy.

Extracorporeal photochemotherapy (ECP) is a widely used cancer immunotherapy for cutaneous T cell lymphoma (CTCL), operative in over 350 university ...

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 ...

In Vitro Cellular Activity Evaluation of the Nanoemulsion Vaccine Adjuvant Ophiopogonin D

As a principal ingredient of vaccines, adjuvants can directly induce or enhance the powerful, widespread, innate, and adaptive immune responses associated with antigens. Ophiopogonin D (OP-D), a purified component extracted from the plant Ophiopogon japonicus, has been found to be useful as a vaccine adjuvant. The problems of the low solubility and toxicity of OP-D can be effectively overcome by using a low-energy emulsification method to prepare nanoemulsion ophiopogonin D (NOD). In this article, a series of in vitro protocols for cellular activity evaluation are examined. The cytotoxic effects of L929 were determined using a cell counting kit-8 assay. Then, the secreted cytokine levels and corresponding immune cell numbers after the stimulation and culture of splenocytes from immunized mice were detected by ELISA and ELISpot methods. In addition, the antigen uptake ability in bone marrow-derived dendritic cells (BMDCs), which were isolated from C57BL/6 mice and matured after incubation with GM-CSF plus IL-4, was observed by laser scanning confocal microscopy (CLSM). Importantly, macrophage activation was confirmed by measuring the levels of IL-1&#946;, IL-6, and tumor necrosis factor alpha (TNF-&#945;) cytokines by ELISA kits after coculturing peritoneal macrophages (PMs) from blank mice with the adjuvant for 24 h. It is hoped that this protocol will provide other researchers with direct and effective experimental approaches to evaluate the cellular response efficacies of novel vaccine adjuvants.

In Vitro Cellular Activity Evaluation of the Nanoemulsion Vaccine Adjuvant Ophiopogonin D

The protocol presents detailed methods for evaluating whether the nanoemulsion ophiopogonin D adjuvant promotes effective cellular immune responses.

As a principal ingredient of vaccines, adjuvants can directly induce or enhance the powerful, widespread, innate, and adaptive immune responses associated ...

Determination of Vaccine Immunogenicity Using Bovine Monocyte-Derived Dendritic Cells

Dendritic cells (DCs) are the most potent antigen-presenting cells (APCs) within the immune system. They patrol the organism looking for pathogens and play a unique role within the immune system by linking the innate and adaptive immune responses. These cells can phagocytize and then present captured antigens to effector immune cells, triggering a diverse range of immune responses. This paper demonstrates a standardized method for the in vitro generation of bovine monocyte-derived dendritic cells (MoDCs) isolated from cattle peripheral blood mononuclear cells (PBMCs) and their application in evaluating vaccine immunogenicity.
Magnetic-based cell sorting was used to isolate CD14+ monocytes from PBMCs, and the supplementation of complete culture medium with interleukin (IL)-4 and granulocyte-macrophage colony-stimulating factor (GM-CSF) was used to induce the differentiation of CD14+ monocytes into naive MoDCs. The generation of immature MoDCs was confirmed by detecting the expression of major histocompatibility complex II (MHC II), CD86, and CD40 cell surface markers. A commercially available rabies vaccine was used to pulse the immature MoDCs, which were subsequently co-cultured with naive lymphocytes.
The flow cytometry analysis of the antigen-pulsed MoDCs and lymphocyte co-culture revealed the stimulation of T lymphocyte proliferation through the expression of Ki-67, CD25, CD4, and CD8 markers. The analysis of the mRNA expression of IFN-&#947; and Ki-67, using quantitative PCR, showed that the MoDCs could induce the antigen-specific priming of lymphocytes in this in vitro co-culture system. Furthermore, IFN-&#947; secretion assessed using ELISA showed a significantly higher titer (**p &#60; 0.01) in the rabies vaccine-pulsed MoDC-lymphocyte co-culture than in the non-antigen-pulsed MoDC-lymphocyte co-culture. These results show the validity of this in vitro MoDC assay to measure vaccine immunogenicity, meaning this assay can be used to identify potential vaccine candidates for cattle before proceeding with in vivo trials, as well as in vaccine immunogenicity assessments of commercial vaccines.

The methodology describes the generation of bovine monocyte-derived dendritic cells (MoDCs) and their application for the in vitro evaluation of antigenic candidates during the development of potential veterinary vaccines in cattle.

Dendritic cells (DCs) are the most potent antigen-presenting cells (APCs) within the immune system. They patrol the organism looking for pathogens and ...

A Simple and Efficient Method for Testing Immunomodulatory Agents for Generation of Tolerogenic Dendritic Cells from Human CD14+ Monocytes

Tolerogenic dendritic cells (tolDCs) are a subset of dendritic cells (DCs) that are known to influence na&#239;ve T cells toward a regulatory T cell (Treg) phenotype. TolDCs are currently under investigation as therapies for autoimmunity and transplantation, both as a cell therapy and method to induce tolDCs from endogenous DCs. To date, however, the number of known agents to induce tolDCs from na&#239;ve DCs is relatively small and their potency to generate Tregs in vivo has been inconsistent, particularly therapies that induce tolDCs from endogenous DCs. This provides an opportunity to explore novel compounds to generate tolerance.
Here we describe a method to test novel immunomodulatory compounds on monocyte-derived DCs (moDCs) in vitro and validate their functionality to generate autologous Tregs. First, we obtain PBMCs and isolate CD14+ monocytes and CD3+ T cells using commercially available magnetic separation kits. Next, we differentiate monocytes into moDCs, treat them with an established immunomodulator, such as rapamycin, dexamethasone, IL-10, or vitamin D3, for 24 h and test their change in tolerogenic markers as a validation of the protocol. Finally, we co-culture the induced tolDCs with autologous T cells in the presence of anti-CD3/CD28 stimulation and observe changes in Treg populations and T cell proliferation. We envision this protocol being used to evaluate the efficacy of novel immunomodulatory agents to reprogram already differentiated DCs towards tolDC.

We describe a procedure to assess the capacity for pharmacologic agents to generate tolerogenic dendritic cells from na&#239;ve monocyte-derived dendritic cells in vitro and validate their potency by autologous regulatory T cell generation.

Tolerogenic dendritic cells (tolDCs) are a subset of dendritic cells (DCs) that are known to influence na&#239;ve T cells toward a regulatory T cell ...

Immunostimulatory Agent Evaluation: Lymphoid Tissue Extraction and Injection Route-Dependent Dendritic Cell Activation

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