Name: immunostimulatory agent evaluation: lymphoid tissue extraction and injection route-dependent dendritic cell activation
Uploaded: 2018-09-16T14:00:00
Description: Watch this Scientific Journal Video about Immunostimulatory Agent Evaluation: Lymphoid Tissue Extraction and Injection Route-Dependent Dendritic Cell Activation at JoVE.com

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<...

<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. Lab Animal Research. Compound Administration I.</a> Journal of Visualized Experiments. , (2018).</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+III.">JoVE Science Education Database. Lab Animal Research. Compound Administration III.</a> Journal of Visualized Experiments. , (2018).</li><li>Mebius, R. E., Kraal, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+and+function+of+the+spleen.">Structure and function of the spleen.</a> Nature Review Immunology. 5 (8), 606-616 (2005).</li></ol>

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.

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	<tbody>
		<tr height="22">
			<td height="22" style="height:22px;width:325px;">Material</td>
			<td style="width:96px;"></td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">phosphate buffer saline</td>
			<td rowspan="2" style="width:96px;">Corning</td>
			<td rowspan="2" style="width:119px;">21-040-CVR</td>
			<td rowspan="2">Washing organs</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">(PBS, pH 7.4)</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;">isoflurane solution&#160;</td>
			<td style="width:96px;">Aesica Queenborough limited</td>
			<td style="width:119px;">26675-46-7</td>
			<td>Anesthesia process</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tuberculin 1mL syringe -</td>
			<td style="width:96px;">Junglim</td>
			<td style="width:119px;">N/A</td>
			<td>Injection</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">50 mL conical tube&#160;</td>
			<td style="width:96px;">S.P.L</td>
			<td style="width:119px;">50050</td>
			<td>Anesthesia process</td>
		</tr>
		<tr height="67">
			<td height="67" style="height:67px;">1mL Insulin Syringe&#160;</td>
			<td style="width:96px;">(BD Ultra-FineTM&#173;II)_short needle</td>
			<td style="width:119px;">324826</td>
			<td>Intramuscular Injection</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DMEM High Glucose</td>
			<td style="width:96px;">Hyclone</td>
			<td style="width:119px;">SH30081.01</td>
			<td>Storing organs</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Histopaque</td>
			<td style="width:96px;">&#160;Sigma-Aldrich</td>
			<td style="width:119px;">10771</td>
			<td>FACS analysis</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Ethyl alcohol anhydrous 99.5 %&#160;</td>
			<td style="width:96px;">Daejung</td>
			<td style="width:119px;">4022-4110</td>
			<td>Disinfectant</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:325px;">Equipments</td>
			<td style="width:96px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:325px;">FineCycler C100 (Thermocycler)</td>
			<td style="width:96px;">Ssufine</td>
			<td style="width:119px;">-</td>
			<td>Anealing</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:325px;">Centrifuge</td>
			<td style="width:96px;"></td>
			<td style="width:119px;"></td>
			<td>Centrifuge</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">FACS tube&#160;</td>
			<td style="width:96px;">FALCON</td>
			<td style="width:119px;">2052</td>
			<td>FACS analysis</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:325px;">Automated High-performance Flow Cytometer</td>
			<td style="width:96px;">BD (USA), FACSVerse</td>
			<td style="width:119px;">-</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 ...

The overall goal of this procedure is to familiarize the unique technique required in isolating mediastinal lymph node from mice. The operation was conducted after injecting immunostimulant material on six different routes in mice. In this study, we used a new class of material forming nano-sized structures.Surfactants consist of hydrophilic head group and hydrophobic tail. Those form spherical micelle or other secondary structure due to microphage separation. We utilized this property to a bio macromolecule that is DNA.DNA amphiphile, shown here, as regular nucleotides such as A, C, G, T, has hydrophilic blocks while lipid modified neuro-cell nucleobases act as the hydrophobic units. The modified oligo was synthesized by DNA synthesizer in large scale octograms. In equi-media the amphiphilic lipid DNAs spontaneously form nano-sized spherical micelles.When micelle is formed, the DNA remains single-stranded as an empty carrier with about 10 nanometer in diameter. To equip the carrier with immune-stimulant, it is simple matter of extending the DNA sequence with toll-like receptor nine adjuvant namely eCpG. Lipid DNA and eCpG base here therefore the outer surface of micelle is degraded with CpG sequence.We then name this Immune-Stimulating Nano particle as the INP. To determine the proper way of INPs for immune response we injected INPs to mice in 64 injection routes and consequently a proper lymph node tissue were analyzed. For more detailed injection instruction please find a JOB article published by Charles River in 2012.The main advantage of this video is visually showing how to obtain mediastinal lymph node. The video explicitly shows the location of mediastinal lymph node which a lot of people find a difficulty in isolating the organs. All experiments were carried out under the guidelines of the Institutional Animal Care and Use Committee at the Shanghai Public Health Clinical Center, or University of Ulsan College of Medicine.This part will show the specific location of organs or tissues, which are crucial for immunological analysis. Spleen and two lymph nodes will be isolated. First of all, mice were sacrificed by the carbon dioxide inhalation euthanasia and all efforts were made to minimize suffering as following the guidance of Institutional Animal Care and Use Committee.From now on let's find the organs and isolate them. First, properly fix the mouse on the operating desk with four pins on the palm of the mouse. Sterilize the dissection area with alcohol.Dissect the outer cover the bottom of the mouse abdomen with a big-sized scissor. Use forceps to open the both sides of attachment and fix it with pins on the operating table. The location of inguinal lymph node is exposed by finding the conjunction of vessel going down toward the left leg.The shape of the vessel looks like tilted alphabet letter Y.Uncover the layer with the forceps and pull it from the skin. Cut the peritoneum with smaller scissor to expose internal organs. Move intestine to the right by using forceps then wrap in shaped spleen, which show on the left, upper left side of the abdomen.Additional attention is required during the procedure since spleen is easy to disrupt. Finally the key point of this video:harvesting mediastinal lymph node. It is located underneath the upper side of the lung, which is hard to see due to its small size and somewhat hidden location.Carefully expose the area using small scissors and forceps. Cut both sides of the ribs and the diaphragm as well. Clip the cut off ribs above the chest.During the procedure avoid damaging heart and blood vessels since it might blind the site, finding the translucent mediastinal lymph node. If by accident the blood vessel burst, gently wipe out the blood with gauze. Grab the left side of the lung and softly turn it over to the other side to see underneath the lung.Pull up the mediastinal lymph node using forceps. After harvesting the mediastinal lymph node, make sure to perfectly remove all the fat and blood surrounding the lymph node since undesired tissue may influence the analysis. Finally, all the isolated organs on a petri dish filled with media.Total 0.1 times 10 to the sixth cells were analyzed on this flow cytometry. Based on force getter and size getter leukocyte population was gated and that cells were excluded by dark staining. 24 hours after INP injection by 64 injection routes, spleen, inguinal lymph node, and mediastinal lymph node were harvested and analyzed DC activation by flow cytometry.Based on the strategy of flow cytometry DC population was defined as CD11C positive and lineage negative cells in the live leukocyte. Notably, intranasal injection of INP promoted significant increases in the CD40, 80, 86 expression in the mediastinal lymph node compared to PBS for the control. Therefore, this data suggested that intranasal injection of INP can be used as a mucosal adjuvant for enhancement of immunity in the lung.

immunostimulatory-agent-evaluation-lymphoid-tissue-extraction

Research

JoVE Journal

Immunology and Infection

Optimized Protocol for Efficient Transfection of Dendritic Cells without Cell Maturation

Dendritic cells (DCs) can be considered sentinels of the immune system which play a critical role in its initiation and response to infection1. Detection of pathogenic antigen by na&iuml;ve DCs is through pattern recognition receptors (PRRs) which are able to recognize specific conserved structures referred to as pathogen-associated molecular patterns (PAMPS). Detection of PAMPs by DCs triggers an intracellular signaling cascade resulting in their activation and transformation to mature DCs. This process is typically characterized by production of type 1 interferon along with other proinflammatory cytokines, upregulation of cell surface markers such as MHCII and CD86 and migration of the mature DC to draining lymph nodes, where interaction with T cells initiates the adaptive immune response2,3. Thus, DCs link the innate and adaptive immune systems. 
The ability to dissect the molecular networks underlying DC response to various pathogens is crucial to a better understanding of the regulation of these signaling pathways and their induced genes. It should also help facilitate the development of DC-based vaccines against infectious diseases and tumors. However, this line of research has been severely impeded by the difficulty of transfecting primary DCs4.
Virus transduction methods, such as the lentiviral system, are typically used, but carry many limitations such as complexity and bio-hazardous risk (with the associated costs)5,6,7,8. Additionally, the delivery of viral gene products increases the immunogenicity of those transduced DCs9,10,11,12. Electroporation has been used with mixed results13,14,15, but we are the first to report the use of a high-throughput transfection protocol and conclusively demonstrate its utility.
In this report we summarize an optimized commercial protocol for high-throughput transfection of human primary DCs, with limited cell toxicity and an absence of DC maturation16. Transfection efficiency (of GFP plasmid) and cell viability were more than 50% and 70% respectively. FACS analysis established the absence of increase in expression of the maturation markers CD86 and MHCII in transfected cells, while qRT-PCR demonstrated no upregulation of IFN&beta;. Using this electroporation protocol, we provide evidence for successful transfection of DCs with siRNA and effective knock down of targeted gene RIG-I, a key viral recognition receptor16,17, at both the mRNA and protein levels.

We present our optimized high-throughput nucleofection protocol as an efficient way of transfecting primary human monocyte-derived dendritic cells with either plasmid DNA or siRNA without causing cell maturation. We further provide evidence for successful siRNA silencing of targeted gene RIG-I at both the mRNA and protein levels.

Dendritic cells (DCs) can be considered sentinels of the immune system which play a critical role in its initiation and response to infection1. Detection ...

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

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

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

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

Analysis of Pulmonary Dendritic Cell Maturation and Migration during Allergic Airway Inflammation

Dendritic cells (DCs) are the key players involved in initiation of adaptive immune response by activating antigen-specific T cells. DCs are present in peripheral tissues in steady state; however in response to antigen stimulation, DCs take up the antigen and rapidly migrate to the draining lymph nodes where they initiate T cell response against the antigen1,2. Additionally, DCs also play a key role in initiating autoimmune as well as allergic immune response3.
DCs play an essential role in both initiation of immune response and induction of tolerance in the setting of lung environment4. Lung environment is largely tolerogenic, owing to the exposure to vast array of environmental antigens5. However, in some individuals there is a break in tolerance, which leads to induction of allergy and asthma. In this study, we describe a strategy, which can be used to monitor airway DC maturation and migration in response to the antigen used for sensitization. The measurement of airway DC maturation and migration allows for assessment of the kinetics of immune response during airway allergic inflammation and also assists in understanding the magnitude of the subsequent immune response along with the underlying mechanisms.
Our strategy is based on the use of ovalbumin as a sensitizing agent. Ovalbumin-induced allergic asthma is a widely used model to reproduce the airway eosinophilia, pulmonary inflammation and elevated IgE levels found during asthma6,7. After sensitization, mice are challenged by intranasal delivery of FITC labeled ovalbumin, which allows for specific labeling of airway DCs which uptake ovalbumin. Next, using several DC specific markers, we can assess the maturation of these DCs and can also assess their migration to the draining lymph nodes by employing flow cytometry.

We describe a strategy to monitor maturation and migration of pulmonary dendritic cells in response to ovalbumin in the setting of ovalbumin induced allergic airway inflammation. This strategy can be modified to assess migration of pulmonary dendritic cells in settings of infection.

Dendritic cells (DCs) are the key players involved in initiation of adaptive immune response by activating antigen-specific T cells. DCs are present in ...

Monitoring Dendritic Cell Migration using 19F / 1H Magnetic Resonance Imaging

Continuous advancements in noninvasive imaging modalities such as magnetic resonance imaging (MRI) have greatly improved our ability to study physiological or pathological processes in living organisms. MRI is also proving to be a valuable tool for capturing transplanted cells in vivo. Initial cell labeling strategies for MRI made use of contrast agents that influence the MR relaxation times (T1, T2, T2*) and lead to an enhancement (T1) or depletion (T2*) of signal where labeled cells are present. T2* enhancement agents such as ultrasmall iron oxide agents (USPIO) have been employed to study cell migration and some have also been approved by the FDA for clinical application. A drawback of T2* agents is the difficulty to distinguish the signal extinction created by the labeled cells from other artifacts such as blood clots, micro bleeds or air bubbles. In this article, we describe an emerging technique for tracking cells in vivo that is based on labeling the cells with fluorine (19F)-rich particles. These particles are prepared by emulsifying perfluorocarbon (PFC) compounds and then used to label cells, which subsequently can be imaged by 19F MRI. Important advantages of PFCs for cell tracking in vivo include (i) the absence of carbon-bound 19F in vivo, which then yields background-free images and complete cell selectivityand(ii) the possibility to quantify the cell signal by 19F MR spectroscopy.

Monitoring Dendritic Cell Migration using 19F / 1H Magnetic Resonance Imaging

Tracking of cells using MRI has gained remarkable attention in the past years. This protocol describes the labeling of dendritic cells with fluorine (19F)-rich particles, the in vivo application of these cells, and monitoring the extent of their migration to the draining lymph node with 19F/1H MRI and 19F MRS.

Continuous  advancements in noninvasive imaging modalities such as magnetic resonance  imaging (MRI) have greatly improved our ability to study ...

Activation and Measurement of NLRP3 Inflammasome Activity Using IL-1&#946; in Human Monocyte-derived Dendritic Cells

Inflammatory processes resulting from the secretion of Interleukin (IL)-1 family cytokines by immune cells lead to local or systemic inflammation, tissue remodeling and repair, and virologic control1,2 . Interleukin-1&#946; is an essential element of the innate immune response and contributes to eliminate invading pathogens while preventing the establishment of persistent infection1-5.
Inflammasomes are the key signaling platform for the activation of interleukin 1 converting enzyme (ICE or Caspase-1). The NLRP3 inflammasome requires at least two signals in DCs to cause IL-1&#946; secretion6. Pro-IL-1&#946; protein expression is limited in resting cells; therefore a priming signal is required for IL-1&#946; transcription and protein expression. A second signal sensed by NLRP3 results in the formation of the multi-protein NLRP3 inflammasome. The ability of dendritic cells to respond to the signals required for IL-1&#946; secretion can be tested using a synthetic purine, R848, which is sensed by TLR8 in human monocyte derived dendritic cells&#160;(moDCs) to prime cells, followed by activation of the NLRP3 inflammasome with the bacterial toxin and potassium ionophore, nigericin.
Monocyte derived DCs&#160;are easily produced in culture and provide significantly more cells than purified human myeloid DCs. The method presented here differs from other inflammasome assays in that it uses in vitro human, instead of mouse derived, DCs thus allowing for the study of the inflammasome in human disease and infection.

Activation and Measurement of NLRP3 Inflammasome Activity Using IL-1&amp;#946; in Human Monocyte-derived Dendritic Cells

Dendritic cells (DCs)&#160;secrete IL-1&#946; in response to TLR8 recognition of synthetic purine, R848, followed by NLRP3 inflammasome activation with nigericin, therefore, IL-1&#946; can be used to measure NLRP3 inflammasome activity. Intracellular cytokine staining, immunoblotting, and ELISA are used to accurately measure NLRP3 inflammasome priming and activation via IL-1&#946; expression.

Inflammatory processes resulting from the secretion of Interleukin (IL)-1 family cytokines by immune cells lead to local or systemic inflammation, tissue ...

In Vitro Generation of Murine Plasmacytoid Dendritic Cells from Common Lymphoid Progenitors using the AC-6 Feeder System

Plasmacytoid dendritic cells (pDCs) are powerful type I interferon (IFN-I) producing cells that are activated in response to infection or during inflammatory responses. Unfortunately, study of pDC function is hindered by their low frequency in lymphoid organs, and existing methods for in vitro DC generation predominantly favor the production of cDCs over pDCs. Here we present a unique approach to efficiently generate pDCs from common lymphoid progenitors (CLPs) in vitro. Specifically, the protocol described details how to purify CLPs from bone marrow and generate pDCs by coculturing with &#947;-irradiated AC-6 feeder cells in the presence of Flt3 ligand. A unique characteristic of this culture system is that the CLPs migrate underneath the AC-6 cells and become cobblestone area-forming cells, a critical step for expanding pDCs. Morphologically distinct DCs, namely pDCs and cDCs, were generated after approximately 2 weeks with a composition of 70-90% pDCs under optimal conditions. Typically, the number of pDCs generated by this method is roughly 100-fold of the number of CLPs seeded. Therefore, this is a novel system with which to robustly generate the large numbers of pDCs required to facilitate further studies into the development and function of these cells.

In Vitro Generation of Murine Plasmacytoid Dendritic Cells from Common Lymphoid Progenitors using the AC-6 Feeder System

In this study we present an in vitro culture system that can efficiently generate pDCs by co-culturing common lymphoid progenitors with AC-6 feeder cells in the presence of Flt3 ligand.

Plasmacytoid dendritic cells (pDCs) are powerful type I interferon (IFN-I) producing cells that are activated in response to infection or during ...

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. ...

Ex Vivo Infection of Human Lymphoid Tissue and Female Genital Mucosa with Human Immunodeficiency Virus 1 and Histoculture

Histocultures allow studying intercellular interactions within human tissues, and they can be employed to model host-pathogen interactions under controlled laboratory conditions. Ex vivo infection of human tissues with human immunodeficiency virus (HIV), among other viruses, has been successfully used to investigate early disease pathogenesis, as well as a platform to test the efficacy and toxicity of antiviral drugs. In the present protocol, we explain how to process and infect with HIV-1 tissue explants from human tonsils and cervical mucosae, and maintain them in culture on top of gelatin sponges at the liquid-air interface for about two weeks. This non-polarized culture setting maximizes access to nutrients in culture medium and oxygen, although progressive loss of tissue integrity and functional architectures remains its main limitation. This method allows monitoring HIV-1 replication and pathogenesis using several techniques, including immunoassays, qPCR, and flow cytometry. Of importance, the physiologic variability between tissue donors, as well as between explants from different areas of the same specimen, may significantly affect experimental results. To ensure result reproducibility, it is critical to use an adequate number of explants, technical replicates, and donor-matched control conditions to normalize the results of the experimental treatments when compiling data from multiple experiments (i.e., conducted using tissue from different donors) for statistical analysis.

Ex Vivo Infection of Human Lymphoid Tissue and Female Genital Mucosa with Human Immunodeficiency Virus 1 and Histoculture

Infection of human tissues with human immunodeficiency virus (HIV) ex vivo provides a valuable 3D model of virus pathogenesis. Here, we describe a protocol to process and infect tissue specimens from human tonsils and female genital mucosae with HIV-1 and maintain them in culture at the liquid-air interface.

Histocultures allow studying intercellular interactions within human tissues, and they can be employed to model host-pathogen interactions under ...

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

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

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

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

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

Assessment of the Synaptic Interface of Primary Human T Cells from Peripheral Blood and Lymphoid Tissue

The current understanding of the dynamics and structural features of T-cell synaptic interfaces has been largely determined through the use of glass-supported planar bilayers and in vitro-derived T-cell clones or lines1,2,3,4. How these findings apply to the primary human T cells isolated from blood or lymphoid tissues is not known, partly due to significant difficulties in obtaining a sufficient number of cells for analysis5. Here we address this through the development of a technique exploiting multichannel flow slides to build planar lipid bilayers containing activating and adhesion molecules. The low height of the flow slides promotes rapid cell sedimentation in order to synchronize cell:bilayer attachment, thereby allowing researchers to study the dynamic of the synaptic interface formation and the kinetics of the granules release. We apply this approach to analyze the synaptic interface of as few as 104 to 105 primary cryopreserved T cells isolated from lymph nodes (LN) and peripheral blood (PB). The results reveal that the novel planar lipid bilayer technique enables the study of the biophysical properties of primary human T cells derived from blood and tissues in the context of health and disease.

The protocol describes a technique to study the ability of primary polyclonal human T cells to form synaptic interfaces using planar lipid bilayers. We use this technique to show the differential synapse formation capability of human primary T cells derived from lymph nodes and peripheral blood.