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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Oncol. 127, 1-2 (2001).</li><li>Jaatinen, T., Laine, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+mononuclear+cells+from+human+cord+blood+by+Ficoll-Paque+density+gradient.">Isolation of mononuclear cells from human cord blood by Ficoll-Paque density gradient.</a> Curr. Protoc. Stem Cell Biol. 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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 gener...

<table border="1">
<tbody>
 <tr>
 <td fo:width="2in">Reagent/Supplies/Equipment</td>
 <td>Manufacturer</td>
 <td>Catalog No.</td>
 <td></td>
 </tr>
 <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 a...

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 ...

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Research

JoVE Journal

Immunology and Infection

18.1K 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

Murine Model of CD40-activation of B cells

Research on B cells has shown that CD40 activation improves their antigen presentation capacity. When stimulated with interleukin-4 and CD40 ligand (CD40L), human B cells can be expanded without difficulties from small amounts of peripheral blood within 14 days to very large amounts of highly-pure CD40-B cells (&gt;109 cells per patient) from healthy donors as well as cancer patients1-4. CD40-B cells express important lymph node homing molecules and can attract T cells in vitro5. Furthermore they efficiently take up, process and present antigens to T cells6,7. CD40-B cells were shown to not only prime na&iacute;ve, but also expand memory T cells8,9. Therefore CD40-activated B cells (CD40-B cells) have been studied as an alternative source of immuno-stimulatory antigen-presenting cells (APC) for cell-based immunotherapy1,5,10. In order to further study whether CD40-B cells induce effective T cell responses in vivo and to study the underlying mechanism we established a cell culture system for the generation of murine CD40-activated B cells. Using splenocytes or purified B cells from C57BL/6 mice for CD40-activation, optimal conditions were identified as follows: Starting from splenocytes of C57BL/6 mice (haplotype H-2b) lymphocytes are purified by density gradient centrifugation and co-cultured with HeLa cells expressing recombinant murine CD40 ligand (tmuCD40L HeLa)11. Cells are recultured every 3-4 days and key components such as CD40L, interleukin-4, -Mercaptoethanol and cyclosporin A are replenished. In this protocol we demonstrate how to obtain fully activated murine CD40-B cells (mCD40B) with similar APC-phenotype to human CD40-B cells (Fig 1a,b). CD40-stimulation leads to a rapid outgrowth and expansion of highly pure (&gt;90%) CD19+ B cells within 14 days of cell culture (Fig 1c,d). To avoid contamination with non-transfected cells, expression of the murine CD40 ligand on the transfectants has to be controlled regularly (Fig 2). Murine CD40-activated B cells can be used to study B-cell activation and differentiation as well as to investigate their potential to function as APC in vitro and in vivo. Moreover, they represent a promising tool for establishing therapeutic or preventive vaccination against tumors and will help to answer questions regarding safety and immunogenicity of this approach12.

In this video, we demonstrate the procedure of CD40-activation and expansion of murine B cells from splenocytes of C57BL/6 mice, which can be used as a model antigen-presenting cell (APC) to study induction of immunity.

Research on B cells has shown that CD40 activation improves their antigen presentation capacity. When stimulated with interleukin-4 and CD40 ligand ...

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 of Mouse Lung Dendritic Cells

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

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

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

Isolation of Myeloid Dendritic Cells and Epithelial Cells from Human Thymus

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

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

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

Generation of CAR T Cells for Adoptive Therapy in the Context of Glioblastoma Standard of Care

Adoptive T cell immunotherapy offers a promising strategy for specifically targeting and eliminating malignant gliomas. T cells can be engineered ex vivo to express chimeric antigen receptors specific for glioma antigens (CAR T cells). The expansion and function of adoptively transferred CAR T cells can be potentiated by the lymphodepletive and tumoricidal effects of standard of care chemotherapy and radiotherapy. We describe a method for generating CAR T cells targeting EGFRvIII, a glioma-specific antigen, and evaluating their efficacy when combined with a murine model of glioblastoma standard of care. T cells are engineered by transduction with a retroviral vector containing the anti-EGFRvIII CAR gene. Tumor-bearing animals are subjected to host conditioning by a course of temozolomide and whole brain irradiation at dose regimens designed to model clinical standard of care. CAR T cells are then delivered intravenously to primed hosts. This method can be used to evaluate the antitumor efficacy of CAR T cells in the context of standard of care.

The lymphodepletive and immunomodulatory effects of chemotherapy and radiation standard of care can be leveraged to enhance the antitumor efficacy of T cell immunotherapy. We outline a method for generating EGFRvIII-specific chimeric antigen receptor (CAR) T cells and administering them in the context of glioblastoma standard of care.

Adoptive T cell immunotherapy offers a promising strategy for specifically targeting and eliminating malignant gliomas. T cells can be engineered ex vivo ...

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. ...

A CFSE-based Assay to Study the Migration of Murine Skin Dendritic Cells into Draining Lymph Nodes During Infection with Mycobacterium bovis Bacille Calmette-Gu&#233;rin

Dendritic cells (DCs) are important for initiating immune responses, in part through their ability to acquire and shuttle antigen to the draining lymph node (DLN). The mobilization of DCs to the DLN is complex and remains to be fully elucidated during infection. Herein described is the use of an innovative, simple assay that relies on the fluorochrome 5- and 6-carboxyfluorescein diacetate succinimidyl ester (CFSE) to track the migration of DCs during footpad infection with Mycobacterium bovis Bacille Calmette-Gu&#233;rin (BCG) in C57BL/6 mice. This assay enables the characterization of skin DC sub-populations that actively relocate to the draining, popliteal LN in response to BCG. This protocol originates from a BCG model where migratory skin DCs were identified by flow cytometry. The assay is amiable to the study and identification of DCs or other cells that home to the popliteal LN after inoculation of microbes, their metabolites or other inflammatory stimuli in the footpad, and consequently to study factors that regulate the migration of these cells.

A CFSE-based Assay to Study the Migration of Murine Skin Dendritic Cells into Draining Lymph Nodes During Infection with &lt;em&gt;Mycobacterium bovis&lt;/em&gt; Bacille Calmette-Gu&amp;#233;rin

An assay in mice to track cell migration from skin to draining lymph node is described which enables the characterization of skin Dendritic cells mobilized to the lymph node after footpad infection with Bacille Calmette-Gu&#233;rin.

Dendritic cells (DCs) are important for initiating immune responses, in part through their ability to acquire and shuttle antigen to the draining lymph ...

Generation of Human Monocyte-derived Dendritic Cells from Whole Blood

Dendritic cells (DCs) recognize foreign structures of different pathogens, such as viruses, bacteria, and fungi, via a variety of pattern recognition receptors (PRRs) expressed on their cell surface and thereby activate and regulate immunity. 
The major function of DCs is the induction of adaptive immunity in the lymph nodes by presenting antigens via MHC I and MHC II molecules to na&#239;ve T lymphocytes. Therefore, DCs have to migrate from the periphery to the lymph nodes after the recognition of pathogens at the sites of infection. For in vitro experiments or DC vaccination strategies, monocyte-derived DCs are routinely used. These cells show similarities in physiology, morphology, and function to conventional myeloid dendritic cells. They are generated by interleukin 4 (IL-4) and granulocyte-macrophage colony-stimulating factor (GM-CSF) stimulation of monocytes isolated from healthy donors. Here, we demonstrate how monocytes are isolated and stimulated from anti-coagulated human blood after peripheral blood mononuclear cell (PBMC) enrichment by density gradient centrifugation. Human monocytes are differentiated into immature DCs and are ready for experimental procedures in a non-clinical setting after 5 days of incubation.

Here, we demonstrate how monocytes are isolated by magnetic bead separation from peripheral blood mononuclear cells after density gradient centrifugation of human anti-coagulated blood. Following incubation for 5 days, human monocytes are differentiated into immature dendritic cells and are ready for experimental procedures in a non-clinical setting.

Dendritic cells (DCs) recognize foreign structures of different pathogens, such as viruses, bacteria, and fungi, via a variety of pattern recognition ...

Development and Validation of an Ultrasensitive Single Molecule Array Digital Enzyme-linked Immunosorbent Assay for Human Interferon-&#945;

The main aim of this protocol is to describe the development and validation of an interferon (IFN)-&#945; single molecule array digital Enzyme-Linked ImmunoSorbent Assay (ELISA) assay. This system enables the quantification of human IFN-&#945; protein with unprecedented sensitivity, and with no cross-reactivity for other species of IFN.
The first key step of the protocol is the choice of the antibody pair, followed by the conjugation of the capture antibody to paramagnetic beads, and biotinylation of the detection antibody. Following this step, different parameters such as assay configuration, detector antibody concentration, and buffer composition can be modified until optimum sensitivity is achieved. Finally, specificity and reproducibility of the method are assessed to ensure confidence in the results. Here, we developed an IFN-&#945; single molecule array assay with a limit of detection of 0.69 fg/mL using high-affinity autoantibodies isolated from patients with biallelic mutations in the autoimmune regulator (AIRE) protein causing autoimmune polyendocrinopathy syndrome type 1/autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APS1/APECED). Importantly, these antibodies enabled detection of all 13 IFN-&#945; subtypes.
This new methodology allows the detection and quantification of IFN-&#945; protein in human biological samples at attomolar concentrations for the first time. Such a tool will be highly useful in monitoring the levels of this cytokine in human health and disease states, most particularly infection, autoimmunity, and autoinflammation.

Development and Validation of an Ultrasensitive Single Molecule Array Digital Enzyme-linked Immunosorbent Assay for Human Interferon-&amp;#945;

Here we present a protocol to describe the development and validation of a single molecule array digital ELISA assay, which enables the ultra-sensitive detection of all IFN-&#945; subtypes in human samples.

The main aim of this protocol is to describe the development and validation of an interferon (IFN)-&#945; single molecule array digital Enzyme-Linked ...

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 ...