The authors would like to thank Andres Salazar (Oncovir, Inc.) for the gift of the Poly-ICLC.

1. Isolation and Cryopreservation of PBMCs 4
<ol>
	<li>Aseptically spike one of the access ports in the leukapheresis bag using a plasma transfer set. Using a 60 ml syringe, transfer the leukapheresis obtained from patients into a sterile 500 ml bottle.</li>
	<li>Adjust the volume of the leukapheresis to 2x its original volume using room temperature RPMI. Mix thoroughly.</li>
	<li>Gently mix the bottle of Ficoll-Paque PLUS. Add 12 ml Ficoll-Paque PLUS into sterile 50 ml conical tube.</li>
	<li>Gently layer 30 ml of the diluted leukapheresis product to each sterile 50 ml conical tube containing Ficoll. Be careful not to disturb the interface between the Ficoll and cell suspension.</li>
	<li>Repeat steps 1.3 and 1.4 until all of the leukapheresis has been layered.</li>
	<li>Centrifuge the 50 ml conical tubes at 1,000 &times; g for 20 min at room temperature with no brake.</li>
	<li>Carefully harvest the cloudy layer of PBMCs from each tube and transfer to new sterile 50 ml tubes.</li>
	<li>Add RPMI to each tube to a final volume of 50 ml. Mix gently by inversion.</li>
	<li>Centrifuge the cells at 500 &times; g for 10 min at 4 &deg;C with full brake.</li>
	<li>Remove the supernatants from each tube. Resuspend the cell pellets from each tube and add RPMI to a final volume of 50 ml. Mix gently by inversion.</li>
	<li>Centrifuge the cells at 500 &times; g for 6 min at 4 &deg;C.</li>
	<li>Remove the supernatants from each tube. Resuspend and pool together the cell suspensions into one 50 ml conical tube.</li>
	<li>Bring the volume of the pooled PBMCs up to 50 ml with more RPMI. Mix gently by inversion.</li>
	<li>Centrifuge the cells at 300 &times; g for 6 min at 4 &deg;C.</li>
	<li>Remove the supernatant. Resuspend the cell pellet in 50 ml RPMI. Mix gently to ensure uniform cell suspension.</li>
	<li>Count the number of cells and determine cell viability. Calculate the total number of viable cells.</li>
	<li>Centrifuge the cells at 300 &times; g for 6 min at 4 &deg;C. Prepare the freezing media. Freezing media consists of 10% Dimethyl sulfoxide (DMSO; Miltenyi Biotec) in human AB serum (Valley Biomedical).</li>
	<li>Remove the supernatant. Resuspend the cells at a final concentration of 2 x 108 cells/ml in cold freezing media.</li>
	<li>Make 1 ml aliquots in 1.8 ml cryovials.</li>
	<li>Transfer the cryovials into the controlled-rate freezer and begin the freezing run, Program 1. At the end of the run, transfer the frozen cryovials immediately into the vapor phase of the liquid nitrogen freezer.</li>
</ol>
2. Day 0: Differentiation of Dendritic Cells from Monocytes
<ol>
	<li>Add 30 ml of RPMI/1% autologous plasma to a sterile 50 ml conical tube.</li>
	<li>Thaw aliquots of frozen PBMCs by gentle agitation in the 37 &deg;C water bath. When completely thawed, transfer the contents of the vials to the sterile 50 ml conical tube. Mix thoroughly.</li>
	<li>Centrifuge the cells for 6 min at 500 &times; g at room temperature. Remove the supernatant and resuspend the pelleted cells in a small volume of RPMI/1% autologous plasma. Then add more RPMI/1% autologous plasma to a final volume of 50 ml. Mix thoroughly.</li>
	<li>Centrifuge the cells again for 6 min at 500 &times; g at room temperature. Aspirate the supernatant and resuspend the pellet in 50 ml RPMI/1% autologous plasma. Mix thoroughly.</li>
	<li>Count the number of cells and determine cell viability. Calculate the total number of viable cells.</li>
	<li>Plate 1.40 x 108 cells in 40 ml of RPMI/1% autologous plasma onto 225 cm2 EasyFlask. Repeat until all cells have been plated.</li>
	<li>Incubate the EasyFlasks flat on its side for 1 - 2 hr at 37 &deg;C in the tissue culture incubator containing 5% CO2 to allow the monocytes to adhere to the flask.</li>
	<li>When the incubation is complete, remove the EasyFlask from the incubator and wash twice to remove the non-adherent cells using 30 ml of prewarmed RPMI.</li>
	<li>After the second wash, add 40 ml of RPMI/1% autologous plasma with 400-1,000 IU/ml IL-4 and 100-1,000 IU/ml GM-CSF 5-8. For this protocol, we are using 400 IU/ml IL-4 and 100 IU/ml GM-CSF.</li>
	<li>Incubate the EasyFlasks for 2 days at 37 &deg;C in the tissue culture incubator containing 5% CO2.</li>
</ol>
3. Day 2: Feeding of Dendritic Cells with IL-4 and GM-CSF
<ol>
	<li>Add 4 ml of RPMI/1% autologous plasma with 400-1,000 IU/ml IL-4 and 100-1,000 IU/ml GM-CSF to each EasyFlask. Mix by swirling and rocking on its side.</li>
	<li>Incubate the EasyFlasks for 3 more days (until Day 5) at 37 &deg;C in the tissue culture incubator containing 5% CO2.</li>
</ol>
4. Day 5: Harvesting of Immature Dendritic Cells
<ol>
	<li>Harvest the cultures by vigorously swirling the flasks and by pipetting up and down to resuspend non-adherent and loosely adherent cells. Transfer the harvested cells to new 50 ml conical tubes.</li>
	<li>Centrifuge the tubes at 500 &times; g for 6 min at room temperature. Remove the supernatants and resuspend the cell pellet in 50 ml RPMI/1% autologous plasma. Mix thoroughly.</li>
	<li>Count the number of cells and determine cell viability. Calculate the total number of viable cells.</li>
	<li>Centrifuge the tubes at 500 &times; g for 6 min at room temperature. Plate 1-2 x 106 cells per well in 3 ml RPMI/1% autologous plasma with 400 IU/ml IL-4 and 100 IU/ml GM-CSF in 6-well tissue culture plates.</li>
	<li>Incubate the plates at 37 &deg;C in the tissue culture incubator containing 5% CO2 overnight.</li>
</ol>
5. Day 6: Maturation and Antigen Loading of Dendritic Cells
<ol>
	<li>Add 10 &mu;g/ml KLH to 1/3 of the wells in the 6-well culture plates. Swirl the plates to mix. KLH is only added to 1/3 of the wells because KLH is only used for the first DC vaccine. Subsequent DC vaccines contain only the tumor antigen peptides.</li>
	<li>DCs can be stimulated to mature using different stimuli. The most common maturation stimuli that has been used in clinical studies has been a cocktail of cytokines (IL-1 &beta;, TNF&alpha;, and IL-6) and prostaglandin E2 (PGE2) 9,10. More recently, the use of Toll-like receptor (TLR) agonists have gained popularity 11,12. In this protocol, add 2 mg/ml Polyinosinic-Polycytidylic Acid-poly-L-lysine Carboxymethylcellulose (Poly-ICLC) to the wells and mix well. Poly-ICLC is the clinical-grade formulation of Poly-IC that is stabilized with poly-L-*-lysine and carboxymethylcellulose (manufactured by Oncovir, Inc.).</li>
	<li>Add 100 &mu;g/ml long peptide antigens (NY-ESO-1 and/or Melan-A/MART-1) to their designated wells, each well receiving only a single peptide to avoid cross-competition 13.</li>
	<li>Incubate the plates at 37 &deg;C in the tissue culture incubator containing 5% CO2 O/N.</li>
</ol>
6. Day 7: Cryopreservation of Dendritic Cells
<ol>
	<li>Prepare DC Freezing Solution using autologous plasma and 10% DMSO. Centrifuge the DC Freezing Solution at 2,000 &times; g for 20 min at 4 &deg;C to remove any particulate material. Transfer supernatant to a new labeled 15 ml conical tube. Store at 4 &deg;C until use.</li>
	<li>When the overnight incubation is finished, harvest and pool all the wells containing DCs pulsed with the peptides into 50 ml conical tubes.</li>
	<li>Centrifuge the tubes at 500 &times; g for 6 min. Remove the supernatants. Resuspend the cell pellet in 5 ml of RPMI/1% autologous plasma. Bring the total volume up to 50 ml with RPMI/1% autologous plasma. Mix thoroughly.</li>
	<li>Count the number of cells and determine cell viability. Calculate total number of viable cells.</li>
	<li>Centrifuge the tubes at 500 &times; g for 6 min at room temp. Remove the supernatants and resuspend each pellet in 5 ml sterile 0.9% NaCl, USP and transfer to new 15 ml conical tubes. Bring each cell suspension to 14 ml with more sterile 0.9% NaCl, USP. Cap and mix by inversion.</li>
	<li>Repeat previous step twice. After the last centrifugation, remove the supernatants, getting as close as possible to the cell pellets without disturbing the pellets.</li>
	<li>Resuspend the cell pellets in DC Freezing Solution at 4 - 20 x 106 cells/ml and aliquot 1 ml to each 1.8 ml CryoTube vial.</li>
	<li>Use the controlled-rate freezer, Program 1, to freeze down the aliquots of DC vaccines and transfer immediately into the vapor phase of a liquid nitrogen freezer for long-term storage.</li>
</ol>
7. Lot Release Testing
<ol>
	<li>Each batch of DC vaccine must be evaluated and meet specific lot release criteria (Table 1) before it is released for injection into patients in the study, typically 5 to 6 weeks after vaccine preparation.</li>
	<li>Viability: Evaluate viability of the DC vaccine using an automated cell counter. The minimum acceptable viability specification is &gt;70%.</li>
	<li>Identity: Evaluate the identity of the mature DCs (CD11c+CD14-CD83+) by flow cytometry. The percentage of CD11c+ cells should be &gt;50%. The percentage of CD11c+ and CD14+ cells should be &lt;30% and the percentage of CD11c+, CD14-, and CD83+ cells should be &gt;50%.</li>
	<li>Sterility: Test the DC vaccine for the presence of anaerobic and aerobic bacteria and fungus by direct culture and Gram stains. Evaluate for the presence of mycoplasma using a direct culture system and DNA fluorochrome Staining Assay with Indicator Cell Line. For these tests, the minimum specification set is that no growth is observed from all the cultures.</li>
	<li>Endotoxin: Evaluate the endotoxin content using the Kinetic-QCL kinetic chromogenic LAL assay and it must be &lt; 50 EU/ml to meet the minimum criteria.</li>
	<li>Function: Evaluate DC function in a mixed lymphocyte reaction by incubation with allogeneic T cells. The presence of proliferation compared to unstimulated DCs is reported (Figure 3).</li>
</ol>

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The authors have received financial support from the following: NIH (AI061684, AI071078, AI044628, and K08 AI084578 (Miller)), the Bill and Melinda Gates Foundation, Doris Duke Charitable Foundation, Cancer Research Institute, Alliance for Lupus Research, and the Emerald Foundation. Nina Bhardwaj is a coinventor on patents relating to the preparation and use of dendritic cells to manipulate immunity. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.

Phase I and II clinical trials of monocyte-derived DCs have shown that they induce immune responses in patients however clinical success has been limited 1. This may be partly due to the lack of consensus on how to generate the optimal DCs for tumor immunotherapeutic use. Although there are numerous ways to generate clinical-grade DCs, these methods vary in terms of the use of cytokines used to differentiate the monocytes, stimuli used to induce maturation, and methods of antigen loading. The formula for generating the optimal DCs still remains to be defined 2.
Recent in vitro studies have shown that the DCs matured with a cocktail of proinflammatory cytokines 10 which has been used in the majority of clinical trials, in particular the presence of PGE2, may induce the differentiation of regulatory T cells and Th2 responses 17, express IDO 18, and are deficient in IL-12p70 production 19. These effects significantly undermine the vaccine's ability to induce immune responses and therefore support the need to evaluate alternative methods of maturing DCs in order to optimize their effects in vivo.
The use of TLR agonists, in particular the TLR 3 agonist Poly-IC, to mature the DCs may improve the clinical efficacy of DCs. In vitro studies have shown that Poly-IC-matured DCs retained stable high expression of MHC molecules and CD83 and costimulatory molecules CD40, CD80, and CD86 14,15,20. Additionally, Poly-IC-matured DCs produced high levels of IL-12 14,16,20, an important cytokine for the generation of anti-tumor response 21, as well as other proinflammatory cytokines such as TNF-&alpha;, IL-6, IL-1&beta;, IP-10, and type I IFNs 14. More importantly, as has been shown in murine tumor models, DCs pulsed with Human Papilloma Virus (HPV) antigens and matured with Poly-IC-primed cytotoxic T cell responses were capable of eradicating established HPV16-expressing tumors 22. As the maturation status of DCs and the presence of IL-12 appear to correlate with efficacy in clinical trials 23,24, the use of Poly-IC to mature DCs may be an important step towards attaining the goal of clinical success.

<table><tbody><tr><td>RPMI-1640 medium with L-glutamine</td><td>BioWhittaker</td><td>12-702F</td><td></td></tr><tr><td>1M HEPES buffered saline</td><td>BioWhittaker</td><td>17-737E</td><td></td></tr><tr><td>Phosphate buffered saline (PBS)</td><td>BioWhittaker</td><td>17-516F</td><td></td></tr><tr><td>Human albumin, 25% solution USP</td><td>Aventis Behring</td><td></td><td></td></tr><tr><td>Ficoll-Hypaque PREMIUM</td><td>GE Healthcare</td><td>17-5442-03</td><td></td></tr><tr><td>Human AB serum</td><td>Valley Biomedical</td><td>HP1022</td><td></td></tr><tr><td>Sterile saline USP</td><td>Hospira</td><td></td><td></td></tr><tr><td>CryoMACS DMSO </td><td>Miltenyi Biotec</td><td>170-076-303</td><td></td></tr><tr><td>Leukine GM-CSF, 0.5 mg/ml</td><td>Berlex</td><td>A02266</td><td></td></tr><tr><td>MACS GMP IL-4</td><td>Miltenyi Biotec</td><td>170-076-101</td><td></td></tr><tr><td>Hiltonol, Poly-ICLC, 2 mg/ml</td><td>Oncovir</td><td>NA</td><td></td></tr><tr><td>VACMUNE KLH</td><td>Biosyn</td><td></td><td></td></tr><tr><td>225 sq cm EasyFlasks</td><td>Nalgene Nunc</td><td>159934</td><td></td></tr><tr><td>Falcon 6-well tissue culture plates</td><td>Becton Dickinson</td><td>353046</td><td></td></tr><tr><td>1.8 ml CryoTube vials</td><td>Nalgene Nunc</td><td>377267</td><td></td></tr><tr><td>Controlled Rate Freezer</td><td>Thermo</td><td>CryoMed</td><td></td></tr></tbody></table>

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.

Between 10 - 20% of starting PBMCs differentiate into DCs at the end of the culture period. Mature DCs are CD11c+, CD14-, CD83+, CD40+, and CCR7+ (Figure 1). They express high levels of MHC class I and II molecules and the costimulatory molecules CD80 and CD86. Poly-ICLC also induced lower levels of PDL-1 as compared to other TLR agonists 14. Additionally, these Poly-IC-matured DCs secrete large amounts of IL-12 (Figure 2 and 15,16) and induce the proliferation of allogeneic T cells (Figure 3).
<img alt="Figure 1" fo:content-width="6in" fo:src="/files/ftp_upload/50085/50085fig1highres.jpg" src="/files/ftp_upload/50085/50085fig1.jpg" /> 
 Figure 1. Representative phenotype of fully differentiated DCs matured with Poly-IC. All the DCs were gated on the forward- and side-scatter plot. DCs were identified as CD11c+ and CD14-. Maturation of DCs in response to Poly-ICLC (bold line) was evaluated based on the expression of CD83, CD40, and CCR7 and compared to unstimulated DCs (gray shade). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/50085/50085fig1large.jpg" target="_blank">Click here to view larger figure</a>.
<img alt="Figure 2" src="/files/ftp_upload/50085/50085fig2.jpg" /> 
 Figure 2. DCs matured with Poly-IC produce high levels of cytokines. Supernatants from DCs differentiated from 3 healthy donors were collected after overnight maturation of DCs with Poly-IC. Cytokines produced were measured by Cytokine Bead Array (CBA) Human Inflammatory Cytokines Kit (BD Biosciences) which measures IL-12p70, TNF, IL-10, IL-6, IL-1&beta;, and IL-8.
<img alt="Figure 3" fo:content-width="6in" fo:src="/files/ftp_upload/50085/50085fig3highres.jpg" src="/files/ftp_upload/50085/50085fig3.jpg" /> 
 Figure 3. DCs matured with Poly-ICLC induce the proliferation of allogeneic T cells. Poly-ICLC-matured DCs from healthy donors were incubated 1:10 with Carboxyfluorescein Diacetate Succinimidyl Ester (CFSE)-labeled allogeneic T cells. After 6 days, proliferation (A) was evaluated by flow cytometry by gating on the CD3+ T cell population. X-axis shows the dilution of CFSE, as a measurement of proliferation, in the various co-culture conditions: T cells alone, unstimulated DC, Poly-ICLC stimulated DC, and phytohaemagglutinin (PHA)-stimulated T cells. Cytokine secretion (B) during the proliferation was evaluated from the supernatants in the cultures using the BD CBA Human Th1/Th2 Cytokine Kit II (BD Biosciences) which measures IFN&gamma;, TNF, IL-10, IL-6, IL-4, and IL-2. Only IFN&gamma; is shown as the other cytokines were not detected to measureable levels in the assay. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/50085/50085fig3large.jpg" target="_blank">Click here to view larger figure</a>.
<table border="1">
	<tbody>
		<tr>
			<td>Test</td>
			<td>Method</td>
			<td>Criteria</td>
		</tr>
		<tr>
			<td>Viability</td>
			<td>Guava Personal Cell Analysis with ViaCount Reagent</td>
			<td>&gt;70%</td>
		</tr>
		<tr>
			<td rowspan="3">Identity</td>
			<td>Flow Cytometry - % CD11c+ cells</td>
			<td>&gt; 50%</td>
		</tr>
		<tr>
			<td>Flow Cytometry - % CD11c+CD14+ cells</td>
			<td>&lt; 30%</td>
		</tr>
		<tr>
			<td>Flow Cytometry - % CD11c+CD14-CD83+ cells</td>
			<td>&gt; 50%</td>
		</tr>
		<tr>
			<td rowspan="2">Sterility</td>
			<td>Bacterial and Fungal Cultures</td>
			<td>Negative</td>
		</tr>
		<tr>
			<td>Mycoplasma: Direct Cell Culture</td>
			<td>Negative</td>
		</tr>
		<tr>
			<td>Endotoxin</td>
			<td>Kinetic Chromogenic LAL Assay</td>
			<td>&lt; 50 EU/ml</td>
		</tr>
		<tr>
			<td>Function</td>
			<td>Mixed Lymphocyte Reaction</td>
			<td>Report Results</td>
		</tr>
	</tbody>
</table>
Table 1. Lot Release Criteria.

Watch this Scientific Journal Video about Preparation of Tumor Antigen-loaded Mature Dendritic Cells for Immunotherapy at JoVE.com

Preparation of Tumor Antigen-loaded Mature Dendritic Cells for Immunotherapy

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 ...

preparation-tumor-antigen-loaded-mature-dendritic-cells-for

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

18.2K Views.  NYU Langone Medical Center. 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.

Video: Preparation of Tumor Antigen-loaded Mature Dendritic Cells for Immunotherapy

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 ...

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

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

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

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

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

Cancer Research

Culture of myeloid dendritic cells from bone marrow precursors

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

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

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

Biology

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

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

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

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

Generation of a Novel Dendritic-cell Vaccine Using Melanoma and Squamous Cancer Stem Cells

We identified cancer stem cell (CSC)-enriched populations from murine melanoma D5 syngeneic to C57BL/6 mice and the squamous cancer SCC7 syngeneic to C3H mice using ALDEFLUOR/ALDH as a marker, and tested their immunogenicity using the cell lysate as a source of antigens to pulse dendritic cells (DCs). DCs pulsed with ALDHhigh CSC lysates induced significantly higher protective antitumor immunity than DCs pulsed with the lysates of unsorted whole tumor cell lysates in both models and in a lung metastasis setting and a s.c.&#160;tumor growth setting, respectively. This phenomenon was due to CSC vaccine-induced humoral as well as cellular anti-CSC responses. In particular, splenocytes isolated from the host subjected to CSC-DC vaccine produced significantly higher amount of IFN&#947; and GM-CSF than splenocytes isolated from the host subjected to unsorted tumor cell lysate pulsed-DC vaccine. These results support the efforts to develop an autologous CSC-based therapeutic vaccine for clinical use in an adjuvant setting.

Evaluated in syngeneic immunocompetent hosts, cancer stem cell (CSC) based dendritic cell (DC) vaccine demonstrated significantly higher antitumor immunity than traditional DC vaccines pulsed with heterogeneous bulk tumor cells.

We identified cancer stem cell (CSC)-enriched populations from murine melanoma D5 syngeneic to C57BL/6 mice and the squamous cancer SCC7 syngeneic to C3H ...

Experimental Melanoma Immunotherapy Model Using Tumor Vaccination with a Hematopoietic Cytokine

Fms-like tyrosine kinase 3 ligand (Flt3L) is a hematopoietic cytokine that promotes the survival and differentiation of dendritic cells (DCs). It has been used in tumor vaccines to activate innate immunity and enhance antitumor responses. This protocol demonstrates a therapeutic model using cell-based tumor vaccine consisting of Flt3L-expressing B16-F10 melanoma cells along with phenotypic and functional analysis of immune cells in the tumor microenvironment (TME). Procedures for cultured tumor cell preparation, tumor implantation, cell irradiation, tumor size measurement, intratumoral immune cell isolation, and flow cytometry analysis are described. The overall goal of this protocol is to provide a preclinical solid tumor immunotherapy model, and a research platform to study the relationship between tumor cells and infiltrating immune cells. The immunotherapy protocol described here can be combined with other therapeutic modalities, such as immune checkpoint blockade (anti-CTLA-4, anti-PD-1, anti-PD-L1 antibodies) or chemotherapy in order to improve the cancer therapeutic effect of melanoma.

The protocol presents a cancer immunotherapy model using cell-based tumor vaccination with Flt3L-expressing B16-F10 melanoma. This protocol demonstrates the procedures, including preparation of cultured tumor cells, tumor implantation, cell irradiation, measurement of tumor growth, isolation of intratumoral immune cells, and flow cytometry analysis.

Fms-like tyrosine kinase 3 ligand (Flt3L) is a hematopoietic cytokine that promotes the survival and differentiation of dendritic cells (DCs). It has been ...

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 ...

Preparation of Tumor Antigen-loaded Mature Dendritic Cells for Immunotherapy

Summary

Explore More Videos

Chapters in this video

Optimized Protocol for Efficient Transfection of Dendritic Cells without Cell Maturation

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

Culture of myeloid dendritic cells from bone marrow precursors

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

Generation of a Novel Dendritic-cell Vaccine Using Melanoma and Squamous Cancer Stem Cells

Experimental Melanoma Immunotherapy Model Using Tumor Vaccination with a Hematopoietic Cytokine

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

Novel Protocol for Generating Physiologic Immunogenic Dendritic Cells

Development and Functional Characterization of Murine Tolerogenic Dendritic Cells

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