Name: A Simple and Efficient Method for Testing Immunomodulatory Agents for Generation of Tolerogenic Dendritic Cells from Human CD14+ Monocytes (Video) | JoVE Journal
Uploaded: 2025-04-11T14:50:00.000+00:00
Duration: 11 min 34 s
Description: Learn a method to generate tolerogenic dendritic cells from human CD14+ monocytes using immunomodulatory agents. This technique enables regulatory T cell induction for autoimmune and transplantation research.

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5 minutes

We would like to thank the University of Pennsylvania&#39;s Human Immunology Core (HIC) for providing fresh human PBMCs from donors. The HIC is supported in part by NIH P30 AI045008 and P30 CA016520.

All human peripheral blood mononuclear cell (PBMC) samples were obtained from the University of Pennsylvania&#39;s Human Immunology Core from deidentified donors with prior approval from the University of Pennsylvania&#39;s Institutional Review Board (IRB) with patient consent.
Optional: While in this method, we used freshly isolated PBMCs obtained from an academic laboratory, PBMCs can be isolated from either whole blood or leukapheresis-enriched blood products. We recommend using the density gradient centrifugation method, as this is a well-established and reliable method that is described elsewhere23.
1. Isolation of monocytes/T cells and moDC differentiation
<ol>
	<li>Isolation of monocytes and T cells from PBMCs-Day 1
	<ol>
		<li>Obtain 200 million human PBMCs from a healthy donor. Keep the cells in a 15 mL tube and transfer them on ice.</li>
		<li>Aliquot 50 mL of Separation Buffer (provided in commercially available separation kits) in a conical tube. 
		NOTE: Separation buffer can also be replaced with DPBS with 2% fetal bovine serum (FBS) and 1 mM Ethylenediaminetetraacetic acid (EDTA).</li>
		<li>Remove the PBMC tube from ice and fill it with Separation Buffer. Centrifuge at 300 x g for 5 minutes.</li>
		<li>Aspirate the supernatant using a 10 mL serological pipette. Resuspend the cell pellet in 4 mL of Recommended Medium using a serological pipette.</li>
		<li>Divide the suspension into two different 15 mL tubes, adding 2 mL per tube (5 &#215; 107 cells/mL per tube). Label one tube &#34;T&#34; and the other &#34;Monocytes.&#34;</li>
		<li>Retrieve the Human Monocyte Isolation Kit and follow the manufacturer&#39;s protocol to isolate monocytes from the monocyte-labeled tube.</li>
		<li>Retrieve the Human T Cell Isolation Kit and follow the manufacturer&#39;s protocol for the second tube to isolate T cells.</li>
	</ol>
	</li>
	<li>Plating of monocytes for differentiation and freezing of T cells-Day 1
	<ol>
		<li>Preheat 50 mL of moDC Culture Medium (Table 1) at 37 &#176;C for at least 10 min.</li>
		<li>Centrifuge the 15 mL tubes with enriched monocytes and the 15 mL tube with T cells from step 1.1 at 250 &#215; g for 5 min.</li>
		<li>Aspirate the supernatant. Resuspend monocytes in 1 mL of moDC Culture Medium and T cells in T Cell Culture Medium (Table 1) by pipetting up and down. Ensure the pellet is properly dispersed.</li>
		<li>Count the cells using an automated cell counter or manual hemacytometer using Trypan Blue staining. 
		NOTE: For the representative data, 10 million T cells and 3 million monocytes were isolated.</li>
		<li>Take the tube with monocytes and add 9 mL of warm moDC Culture Medium to the suspension for a final volume of 10 mL. Then, add 100 &#181;L each of 10 &#181;g/mL GM-CSF and 10 &#181;g/mL IL-4 stock for a final concentration of 100 ng/mL of both GM-CSF and IL-4 in the media. Transfer the suspension to a Petri dish and label as &#34;moDC&#34; (Day 1) and incubate at 37 &#176;C with 5% CO2.</li>
		<li>Take the T cell tube and mix 1 mL of T Cell Freeze Medium (Table 1) with 1 mL of T cell suspension. Divide into two 2 mL cryovials (1 mL/vial); seal tightly (final concentration of 5 million cells per vial). Place the cryovials in a freezing chamber with balancing tubes in unused wells. Store the chamber at &#8722;80 &#176;C overnight, then transfer the cryovials to a cryotank.</li>
		<li>On Day 4, refresh the medium with the addition of 5 mL of fresh moDC Culture Medium and add 100 &#181;L each of GM-CSF and IL-4 stock. Differentiated moDCs will be ready on Day 7.</li>
	</ol>
	</li>
</ol>
2. Adding immunomodulatory drugs for generating tolerogenic moDCs
<ol>
	<li>Set up the moDC plate.
	<ol>
		<li>On Day 7, retrieve the differentiated moDCs from the incubator and transfer the moDC cell suspension into a 50 mL tube.</li>
		<li>Centrifuge the suspension at 250 &#215; g for 5 min. Aspirate the supernatant and resuspend the cell pellet in 1 mL of warm moDC culture media by pipetting up and down. Count the cells with hemacytometer and dilute the cells to a concentration of 3 &#215; 105 cells/mL in warm moDC culture medium containing 100 ng/mL GM-CSF and IL-4.</li>
		<li>Add 3 &#215; 104 cells/well in 100 &#181;L to the desired number of wells in a flat bottom 96-well tissue culture plate. 
		NOTE: For representative results, we used a total of 48 wells (four conditions done in triplicate prepared for four different analyses in sections 3 and 4), but this can be adjusted for up to 60 wells. Fill all remaining wells with 100 &#181;L/well PBS to prevent drying, especially outer wells. We recommend preparing different plates for each method of analysis.</li>
		<li>Add 1 &#181;L/well of immunomodulatory drugs to the desired wells (here 10 ng/mL rapa). Incubate the plate overnight at 37 &#176;C with 5% CO2. 
		NOTE: If including immunostimulation on day 8, prepare wells for the immunomodulatory drug both with and without immunostimulation. Use of immunostimulation to mature moDCs is optional.</li>
	</ol>
	</li>
	<li>Rethaw T cells.
	<ol>
		<li>On Day 8, retrieve two cryovials from the cryotank and thaw the cryovials in a hot bead bath or water bath at 37 &#176;C until the contents start melting (in &#60;3 min).</li>
		<li>As the contents start to melt, transfer the vials to a cell culture hood and pipet 1 mL of warm T cell culture media into the vial to rapidly thaw. Transfer the entire contents of the vial into a 50 mL tube. Rinse the cryovials with 1 mL of T cell culture medium to ensure all cells are transferred into the tube.</li>
		<li>Top up the suspension with Flow Staining Solution (Table 1) to 15 mL. Centrifuge at 200 &#215; g for 10 min and aspirate the supernatant. Then, resuspend the pellet in 5 mL of Flow Staining Solution and centrifuge again.</li>
		<li>Aspirate the supernatant and resuspend the cells in 1 mL of warm T cell culture medium. Count the cells using a hemacytometer. 
		NOTE: Viability should be &#62;70% at this stage; if large amounts of dead cells remain, transfer to a fresh tube, add 10 mL of cell stain solution, centrifuge at 200 &#215; g for 5 min, resuspend in 1 mL of T cell media, and recount.</li>
		<li>Add 9 mL of warm T cell culture medium to make a final volume of 10 mL. Transfer the suspension into a Petri dish. Label it as &#34;Rethawed T Cells&#34; and incubate overnight at 37 &#176;C with 5% CO2 to let the cells rest.</li>
	</ol>
	</li>
	<li>Wash the moDC plate and add the immunostimulator if desired.
	<ol>
		<li>On Day 8, centrifuge the moDC plate (from Day 7) at 300 &#215; g for 5 min.</li>
		<li>Aspirate the supernatant, wash with warm HBSS, repeat centrifugation, aspirate again, and resuspend the cells in warm moDC culture medium containing GM-CSF and IL-4 (100 &#181;L/well). 
		NOTE: Be careful not to disturb the cells. Tilt the plate during aspiration. Approximately 10 &#181;L can remain in the well.</li>
		<li>If using immunostimulation, add 1 &#181;L/well of 100x stock LPS (or other immunostimulatory agent) to the desired wells. Incubate the plate overnight at 37 &#176;C with 5% CO2.</li>
	</ol>
	</li>
</ol>
3. Flow analysis for moDC (Validation + Tolerance)
<ol>
	<li>Flow analysis for validation of moDC
	<ol>
		<li>On day 8, prepare antibody cocktails for the Validation Panel, targeting the following markers: HLA-DR, CD14, dendritic cell-specific C-type lectin (DC-SIGN), CD1c, and CD40 as described in Table 2. Dilute all antibodies with flow stain solution. 
		NOTE: The panel was designed for a 7 channel, Red/Blue laser flow cytometer.</li>
		<li>Retrieve the moDC plate from the incubator. Centrifuge the plate at 300 &#215; g for 5 min. Remove the supernatants. Optional: Retain the supernatants for ELISA analysis for cytokines IL-10 and TNF&#945; using commercially available kits according to the manufacturers&#39; instructions.</li>
		<li>Add 200 &#181;L/well of Flow Staining Solution. Centrifuge the plate at 300 &#215; g for 5 min.</li>
		<li>Aspirate the supernatant and add 50 &#181;L/well of Fc receptor binding inhibitor antibody diluted 1:200 in flow staining solution to block unspecific binding. Incubate at room temperature (RT) for 30 min.</li>
		<li>Add 50 &#181;L/well of the prepared Validation Panel antibody cocktail.</li>
		<li>Prepare compensation controls in the plate or in 1.5 mL tubes by mixing 50 &#181;L of compensation beads with 50 &#181;L of each diluted antibody.Incubate the plate at 4 &#176;C for 1 h. 
		NOTE: Compensation beads are used here rather than cells for compensation due to potentially low expression of markers on moDCs.</li>
		<li>Wash by centrifuging at 300 &#215; g for 5 min, removing the supernatant, and resuspending in 200 &#181;L of Flow Staining Solution. Perform two washes.</li>
		<li>After the final wash, centrifuge again and resuspend the cells in 110 &#181;L/well of Flow Staining Solution. The samples are now ready for flow cytometry analysis.</li>
	</ol>
	</li>
	<li>Flow analysis for tolerogenic moDC (Tolerance Panel)
	<ol>
		<li>Similar to step 3.1 on day 8, prepare antibody cocktails for the Tolerance Panel, targeting the following markers: CD86, PD-L1, DC-SIGN, CD1c, BTLA, and CD40 (Table 2).</li>
		<li>Repeat steps 3.1.2-3.1.8, substituting the antibody cocktail with the Tolerance Panel cocktail.</li>
	</ol>
	</li>
</ol>
4. Flow analysis of T cells
<ol>
	<li>Combine T cells with treated Tolerogenic moDCs.
	<ol>
		<li>On day 9, retrieve the rethawed T cell Petri dish from the incubator and transfer all T cells into a 50 mL tube. Top up with Flow Staining Solution.</li>
		<li>Optional: If performing T cell proliferation analysis, divide T cells into two tubes.
		<ol>
			<li>With the first tube, spin down at 250 &#215; g for 5 min and remove the supernatant. Stain this tube with cell proliferation dye according to the manufacturer&#39;s instructions. 
			NOTE: We used Fixable Viability Dye eFluor 670, which requires a 30 min incubation step, followed by two washing steps. We suggest using 50 mL conical tubes to prevent cell loss and using HBSS with 10% HIFBS to wash after staining.</li>
			<li>Use the second tube containingunstained T cells for Treg analysis. Similarly spin down at 250 &#215; g for 5 min, remove the supernatant, resuspend in warm cell culture media, and keep in the incubator during the staining process. If no T cell proliferation is required, spin down all T cells and replace them with warm T cell culture media.</li>
		</ol>
		</li>
		<li>Count the cells and obtain at least 4 million (for just Treg analysis) or 8 million (for Treg and T proliferation analysis).</li>
		<li>Retrieve the moDC plate from incubator with the remaining wells, centrifuge at 300 &#215; g for 5 min, and apirate the supernatants. Add 200 &#181;L/well T cells at 1.6 &#215; 105 cells/well. Add unstained T cells for Treg analysis plates and stained T cells for T cell proliferation plates. 
		NOTE: This will maintain a T cell to moDC ratio of 5:1, as the initial 3 &#215;104 moDCs typically expand slightly.</li>
		<li>Add 25 &#181;L/mL of anti-CD3/CD28 antibody cocktail to all groups except negative control wells to stimulate T cells. Incubate the plate for 72 h at 37 &#176;C with 5% CO2 until day 12. 
		NOTE: Be sure to use stained T cells for proliferation analysis and unstained for Treg analysis.</li>
	</ol>
	</li>
	<li>Treg flow analysis
	<ol>
		<li>Surface marker staining
		<ol>
			<li>On day 12, prepare antibody cocktails for the Treg Panel (Table 2).</li>
			<li>Transfer all cell suspensions from the 96-well culture plate into a V-bottom plate with Flow Staining Solution. Retain the supernatants for cytokine analysis.</li>
			<li>Wash the cells 2x by centrifugation (200 &#215; g for 5 min followed by 200 &#181;L of Flow stain solution). Set aside some cells for compensation controls. Set one aside for FOXP3 staining and for unstained controls. 
			NOTE: We suggest combing 10 &#181;L of all samples prior to the final spin in step 4.2.1.3 to obtain necessary compensation controls. This provides a representative sample of all surface level expression of each marker on the cells.</li>
			<li>After the second wash, centrifuge at 200 x g for 5 min at room temperature and aspirate, then add 50 &#181;L/well Fc receptor binding inhibitor antibody (diluted 1:200 in Flow Staining Solution). Incubate at room temperature for 10 min.</li>
			<li>Add 50 &#181;L/well of the prepared Treg Panel antibody cocktail.</li>
			<li>For compensation controls, mix 50 &#181;L of unstained compensation cells with 50 &#181;L of each single stained antibody.</li>
			<li>Incubate the plate and compensation controls at 4 &#176;C for 1 h. Wash the plate once with Flow Stain Solution and centrifuge at 300 &#215; g for 5 min.</li>
			<li>Stain with Live/Dead Near-IR dye diluted in HBSS (1:1,000) at 200 &#181;L/well. Incubate at 4 &#176;C for 30 min.</li>
			<li>Wash the plate once with Flow Staining Solution (200 &#181;L/well) at 300 &#215; g for 5 min.</li>
		</ol>
		</li>
		<li>Stain with FoxP3 according to the manufacturer&#39;s instructions, which will require an overnight incubation. A brief overview of the FoxP3 staining protocol is described below .
		<ol>
			<li>Prepare the FoxP3 Fixation/Permeabilization Working Solution according to the manufacturer&#39;s instructions. Mix 1 part of FoxP3 Fixation/Permeabilization Concentrate with 3 parts of FoxP3 Fixation/Permeabilization Diluent. Prepare 200 &#181;L of the working solution per sample.</li>
			<li>Incubate all samples in 200 &#181;L/well of the working Fixation/Permeabilization solution. Leave at 4 &#176;C for 1 h.</li>
			<li>Prepare 1x Permeabilization Buffer: Mix 1 part of 10x Permeabilization Buffer (obtained from commercial kit) with 9 parts of distilled water.</li>
			<li>Wash the plate 2x with 1x Permeabilization Buffer (200 &#181;L/well) at 300 &#215; g for 5 min.</li>
			<li>Stain with FoxP3 antibody (PE-Cy5.5, 1:300 dilution in 1x Permeabilization Buffer): Add 100 &#181;L/well of the diluted FoxP3 antibody and incubate at 4 &#176;C overnight.</li>
			<li>Wash the plate 2x with 1x Permeabilization Buffer (200 &#181;L/well) at 300 &#215; g for 5 min.</li>
			<li>Resuspend the cells in 110 &#181;L/well of Flow Staining Solution.</li>
			<li>Perform flow cytometry analysis.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>T cell proliferation analysis
	<ol>
		<li>Prepare antibody cocktails for the T Proliferation Panel (Table 2).</li>
		<li>Transfer all cell suspensions from the 96-well culture plate into a V-bottom plate with Flow Staining Solution. Retain supernatants for cytokine analysis.</li>
		<li>Wash the cells 2x by centrifugation (200 &#215; g for 5 min followed by 200 &#181;L of Flow stain solution). Set aside some cells for compensation controls. Set one aside for FOXP3 staining and for unstained controls. 
		NOTE: We suggest combing 10 &#181;L of all samples prior to the final spin in step 4.3.3 to obtain the necessary compensation controls. This provides a representative sample of all surface level expression of each marker on the cells.</li>
		<li>After the second wash, centrifuge (200 &#215; g for 5 min at room temperature) and aspirate, then add 50 &#181;L/well Fc receptor binding inhibitor antibody (diluted 1:200 in Flow Staining Solution). Incubate at room temperature for 10 min.</li>
		<li>Add 50 &#181;L/well of the prepared T Proliferation Panel antibody cocktail.</li>
		<li>For compensation controls, mix 50 &#181;L of unstained compensation cells with 50 &#181;L of each single stained antibody.</li>
		<li>Incubate the plate and compensation controls at 4 &#176;C for 1 h. Wash the plate once with Flow Stain Solution and centrifuge at 300 &#215; g for 5 min.</li>
		<li>Stain with Live/Dead Near-IR dye diluted in HBSS (1:1,000) at 200 &#181;L/well. Incubate at 4 &#176;C for 30 min.</li>
		<li>Wash the plate once with Flow Staining Solution (200 &#181;L/well) at 300 &#215; g for 5 min.</li>
		<li>Perform flow cytometry analysis.</li>
	</ol>
	</li>
</ol>

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<li>Jia, S., Kim, J., Esser-Kahn, A. P., Deak, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((High-throughput+screening+identification+of+novel+immunomodulatory+combinations+for+the+generation+of+tolerogenic+dendritic+cells%5BTitle%5D)+AND+%22Front+Med%22%5BJournal%5D)">High-throughput screening identification of novel immunomodulatory combinations for the generation of tolerogenic dendritic cells.</a> Front Med. 10, 1298424(2024).</li>
<li>Dinh, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Isolation+and+cryopreservation+of+highly+viable+human+peripheral+blood+mononuclear+cells+from+whole+blood%3A+A+guide+for+beginners%5BTitle%5D)+AND+%22J+Vis+Exp%22%5BJournal%5D)">Isolation and cryopreservation of highly viable human peripheral blood mononuclear cells from whole blood: A guide for beginners.</a> J Vis Exp. (212), e66794(2024).</li>
<li>Akkaya, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Regulatory+T+cells+mediate+specific+suppression+by+depleting+peptide-MHC+class+II+from+dendritic+cells%5BTitle%5D)+AND+%22Nat+Immunol%22%5BJournal%5D)">Regulatory T cells mediate specific suppression by depleting peptide-MHC class II from dendritic cells.</a> Nat Immunol. 20 (2), 218-231 (2019).</li>
<li>Stallone, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((mTOR+inhibitors+effects+on+regulatory+T+cells+and+on+dendritic+cells%5BTitle%5D)+AND+%22J+Trans+Med%22%5BJournal%5D)">mTOR inhibitors effects on regulatory T cells and on dendritic cells.</a> J Trans Med. 14 (1), 152(2016).</li>
<li>Dahlqvist, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Modulatory+effect+of+rapamycin+and+tacrolimus+on+monocyte-derived+dendritic+cells+phenotype+and+function%5BTitle%5D)+AND+%22Immunobiology%22%5BJournal%5D)">Modulatory effect of rapamycin and tacrolimus on monocyte-derived dendritic cells phenotype and function.</a> Immunobiology. 226 (1), 152031(2021).</li>
<li>Cimen Bozkus, C., Blazquez, A. B., Enokida, T., Bhardwaj, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+T-cell-based+immunogenicity+protocol+for+evaluating+human+antigen-specific+responses%5BTitle%5D)+AND+%22STAR+Protoc%22%5BJournal%5D)">A T-cell-based immunogenicity protocol for evaluating human antigen-specific responses.</a> STAR Protoc. 2 (3), 100758(2021).</li>
<li>Nakayama, M., Michels, A. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Determining+antigen+specificity+of+human+islet+infiltrating+T+cells+in+type+1+diabetes%5BTitle%5D)+AND+%22Front+Immunol%22%5BJournal%5D)">Determining antigen specificity of human islet infiltrating T cells in type 1 diabetes.</a> Front Immunol. 10, 365(2019).</li>
<li>Gregori, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Differentiation+of+type+1+T+regulatory+cells+%28Tr1%29+by+tolerogenic+DC-10+requires+the+IL-10-dependent+ILT4%2FHLA-G+pathway%5BTitle%5D)+AND+%22Blood%22%5BJournal%5D)">Differentiation of type 1 T regulatory cells (Tr1) by tolerogenic DC-10 requires the IL-10-dependent ILT4/HLA-G pathway.</a> Blood. 116 (6), 935-944 (2010).</li>
<li>Taner, T., Hackstein, H., Wang, Z., Morelli, A. E., Thomson, A. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Rapamycin-treated%2C+alloantigen-pulsed+host+dendritic+cells+induce+Ag-specific+T+cell+regulation+and+prolong+graft+survival%5BTitle%5D)+AND+%22Am+J+Transpl%22%5BJournal%5D)">Rapamycin-treated, alloantigen-pulsed host dendritic cells induce Ag-specific T cell regulation and prolong graft survival.</a> Am J Transpl. 5 (2), 228-236 (2005).</li>
<li>Baroja-Mazo, A., Revilla-Nuin, B., Ramírez, P., Pons, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Immunosuppressive+potency+of+mechanistic+target+of+rapamycin+inhibitors+in+solid-organ+transplantation%5BTitle%5D)+AND+%22World+J+Transplant%22%5BJournal%5D)">Immunosuppressive potency of mechanistic target of rapamycin inhibitors in solid-organ transplantation.</a> World J Transplant. 6 (1), 183-192 (2016).</li>
<li>Phillips, B. E., Garciafigueroa, Y., Trucco, M., Giannoukakis, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Clinical+tolerogenic+dendritic+cells%3A+Exploring+therapeutic+impact+on+human+autoimmune+disease%5BTitle%5D)+AND+%22Front+Immunol%22%5BJournal%5D)">Clinical tolerogenic dendritic cells: Exploring therapeutic impact on human autoimmune disease.</a> Front Immunol. 8, 1279(2017).</li>
</ol>

The authors have no conflicts of interest to disclose.

Here we describe a reliable and versatile method to assess the functionality of immunomodulatory agents to induce tolDCs from moDCs and validate their functionality to generate Tregs from autogenic T cells ex vivo. There are several critical steps in this protocol. First, monocytes are notoriously sensitive cells and must be obtained from fresh, not previously frozen PBMCs for the best results. Monocytes should be isolated as soon as possible and placed in the differentiation cocktail. Typically, poor monocyte y...

Dendritic cells (DCs) are critical mediators between innate and adaptive immunity. DCs, which mainly reside in mucosal membranes, skin and lymphoid tissue, are the primary antigen presenting cells (APCs)1. DCs uptake foreign proteins and process and present them on major histocompatibility (MHC) proteins to na&#239;ve T cells. DCs specifically express MHC class II proteins, such as human leukocyte antigen-DR (HLA-DR) in humans. The activation state of the DCs upon antigen exposure is critical for the downstream T cell response2. Immature DCs express various pattern recognition receptors (PRRs) that recognize classes of molecules call pathogen associated molecular patterns (PAMPs), such as the bacterial wall component, lipopolysaccharide (LPS)3. Upon PRR stimulation, DCs become matured DCs and upregulate important T cell co-stimulatory proteins, such as CD80, CD86, and CD40, and secrete pro-inflammatory cytokines such as tumor necrosis factor-alpha (TNF&#945;), facilitating the differentiation of na&#239;ve T cells into conventional effector or helper T cells2. On the contrary, if DC maturation is interrupted or if DCs develop in a tolerogenic environment, DCs can generate a tolerogenic DC state (tolDCs)4. TolDCs downregulate classical T cell co-stimulatory receptors and instead upregulate tolerance receptors such as programmed cell death ligand 1 (PD-L1) and B- and T-lymphocyte attenuator (BTLA) and generate suppressive cytokines such as interleukin 10 (IL-10) and transforming growth factor beta (TGF-&#946;)4. This is not a comprehensive list of tolerance markers and, in fact, there is limited consensus as to which tolDC markers are appropriate to define the tolDC state5. Considering this, we propose regulatory T cell (Treg) generation as a functional marker that ought to be used to compare the effectiveness of various tolDC induction agents.
In addition to tolDC/matured DC activation states, DCs can also be categorized based on their lineage or tissue location, with each subset displaying slightly different functionality. While the tolDC/matured DC division is less definitive and exists more as a continuum, lineage divisions have well-defined markers in both human and mice. DC precursors are formed in the bone marrow, but there are two main subtypes of DCs based on their lineage: 1) plasmacytoid dendritic cell (pDCs), which derive from lymphoid lineages and 2) conventional dendritic cells (cDCs), which derive from myeloid lineages. In humans, pDCs are matured in lymphoid organs, express CD303, and are highly responsive in viral infections6. CD11c expressing cDCs, meanwhile, are matured in peripheral tissues and exist in two distinct subtypes, CD1c+ cDC1s and CD141+ cDC2, which each generate distinct T cell responses7. Furthermore, all cDCs can exist in either tissue-resident (CD103-) or migratory (CD103+) substates8. Finally, under certain conditions, cells from monocyte lineages (CD14+) can be induced toward a dendritic cell phenotype and are identified as CD14-, CD141+, CD1c+ 9. These cells, known as monocyte-derived DCs (moDCs), are the most commonly used for ex vivo analysis in humans as monocytes constitute approximately 10-30% of human peripheral blood mononuclear cells (PBMCs), whereas pDCs constitute only 1-3%10. This makes moDCs an attractive choice, but it is also known that moDCs are more inflammatory that typical cDCs isolated from primary tissue9.
There are currently two broad categories of efforts to employ tolDCs to generate clinical tolerance. First, tolDCs are generated from monocytes for use as a cell therapy. In this paradigm, moDCs are typically differentiated using IL-4/GM-CSF concurrently with immunomodulators such as vitamin D3, rapamycin (rapa), IL-10, dexamethasone, or combinations of these11,12. These tolDCs have been explored as autologous cell therapies for autoimmunity and transplants13. The other use of tolDCs is to reprogram endogenous DCs towards tolDCs using free drugs or nanocarriers to deliver both immunomodulator and antigen of interest14,15,16. Induction of already differentiated DCs is more challenging, however, due to the development of robust metabolic phenotypes of DCs that are typically in contrast with tolDC metabolism17,18. This is a high bar for most pharmacological immunomodulators; for this reason, most endogenous DC reprogramming studies report effective DC suppression and often some Treg induction, but lack clinical success, often due to lack of T cell persistence15,19,20. This highlights the need for strategies to identify potential tolDC induction agents from existing DCs.
Here, we present a method for in vitro evaluation of immunomodulatory agents against differentiated moDCs with the end metric of autologous Treg induction. This protocol is designed to assess the effectiveness of immunomodulatory agents to reprogram already-differentiated human moDCs towards tolerance. Furthermore, this protocol validates the functionality of reprogrammed tolDCs to generate Tregs against autologous T cells isolated from the same PBMC sample. This is in contrast to other protocols that induce tolerance during differentiation and/or challenge tolDCs with T cells from allogenic donors21. In this protocol, we use the common tolerizing agent rapa as an example but also demonstrate the limited effectiveness of rapa-treated moDCs to generate Tregs. In our representative results, we also show the efficacy of other common immunomodulatory treatments such as IL-10, dexamethasone, and vitamin D3. We envision this protocol being used to screen potentially more effective tolDC-inducting agents against already established moDCs22.

<table><tbody><tr><td>0.1-10 &amp;micro;L Filtered Pipet tips</td><td>VWR</td><td>76322-158</td><td>General Cell Culture</td></tr><tr><td>1.5 mL Centrifuge Tube</td><td>VWR</td><td>77508-358</td><td>General Cell Culture</td></tr><tr><td>10 mL Serological Pipets</td><td>VWR</td><td>414004-267</td><td>General Cell Culture</td></tr><tr><td>100-1000 &amp;micro;L Filtered Pipet tips</td><td>VWR</td><td>76322-164</td><td>General Cell Culture</td></tr><tr><td>15 mL Conical Tube</td><td>VWR</td><td>77508-212</td><td>General Cell Culture</td></tr><tr><td>20-200 &amp;micro;L Filtered Pipet tips</td><td>VWR</td><td>76322-160</td><td>General Cell Culture</td></tr><tr><td>2-Mercaptoethanol</td><td>MP Biomedical</td><td>194834</td><td>T Cell Culture</td></tr><tr><td>50 mL Conical Tube</td><td>VWR</td><td>21008-736</td><td>General Cell Culture</td></tr><tr><td>60 x 15 mm Dish, Nunclon Delta</td><td>Thermo Fischer</td><td>150326</td><td>General Cell Culture</td></tr><tr><td>96 Well Conical (V) Bottom Plate, Non-Treated Surface</td><td>Thermo Fischer</td><td>277143</td><td>General Cell Culture</td></tr><tr><td>96 well Flat Bottom Plate</td><td>Thermo Fischer</td><td>161093</td><td>General Cell Culture</td></tr><tr><td>APC/Cyanine7 anti-human CD272 (BTLA) Antibody</td><td>Biolegend</td><td>344518</td><td>Flow Cytometry</td></tr><tr><td>Attune NxT (Red/Blue Laser, 7 Channel)</td><td>Thermo Fischer</td><td>A24863</td><td>Flow Cytometry</td></tr><tr><td>BSA</td><td>Thermo Fischer</td><td>15260-037</td><td>General Cell Culture</td></tr><tr><td>CD14 Monoclonal Antibody (61D3), PE</td><td>Thermo Fischer</td><td>12-0149-42</td><td>Flow Cytometry</td></tr><tr><td>CD1c Monoclonal Antibody (L161), PE-Cyanine7</td><td>Thermo Fischer</td><td>25-0015-42</td><td>Flow Cytometry</td></tr><tr><td>CD209 (DC-SIGN) Monoclonal Antibody (eB-h209), PerCP-Cyanine5.5</td><td>Thermo Fischer</td><td>45-2099-42</td><td>Flow Cytometry</td></tr><tr><td>CD25 Monoclonal Antibody (CD25-4E3), APC</td><td>Thermo Fischer</td><td>17-0257-42</td><td>Flow Cytometry</td></tr><tr><td>CD274 (PD-L1, B7-H1) Monoclonal Antibody (MIH1), PE</td><td>Thermo Fischer</td><td>12-5983-42</td><td>Flow Cytometry</td></tr><tr><td>CD4 Monoclonal Antibody (RPA-T4), Alexa Fluor 488</td><td>Thermo Fischer</td><td>53-0049-42</td><td>Flow Cytometry</td></tr><tr><td>CD40 Monoclonal Antibody (5C3), APC</td><td>Thermo Fischer</td><td>17-0409-42</td><td>Flow Cytometry</td></tr><tr><td>CD40 Monoclonal Antibody (5C3), APC-eFluor 780</td><td>Thermo Fischer</td><td>47-0409-42</td><td>Flow Cytometry</td></tr><tr><td>CD69 Monoclonal Antibody (FN50), PE</td><td>Thermo Fischer</td><td>MA1-10276</td><td>Flow Cytometry</td></tr><tr><td>CD86 Monoclonal Antibody (BU63), FITC</td><td>Thermo Fischer</td><td>MHCD8601</td><td>Flow Cytometry</td></tr><tr><td>CD8a Monoclonal Antibody (RPA-T8), PE-Cyanine7</td><td>Thermo Fischer</td><td>25-0088-42</td><td>Flow Cytometry</td></tr><tr><td>Conical Bottom (V-well) 96 Well Plate</td><td>Thermo Fischer</td><td>2605</td><td>Flow Cytometry</td></tr><tr><td>Cryogenic Vials, 2 mL</td><td>Thermo Fischer</td><td>430488</td><td>T Cell Culture</td></tr><tr><td>Dimethylsulfoxide (DMSO), Sequencing Grade</td><td>Thermo Fischer</td><td>20688</td><td>General Cell Culture</td></tr><tr><td>DPBS</td><td>Thermo Fischer</td><td>14200166</td><td>General Cell Culture</td></tr><tr><td>EasySep Human Monocyte Isolation Kit</td><td>Stem Cell Technologies</td><td>19359</td><td>Cell Separation</td></tr><tr><td>EasySep Human T Cell Isolation Kit</td><td>Stem Cell Technologies</td><td>17951</td><td>Cell Separation</td></tr><tr><td>EasySep Magnet</td><td>Stem Cell Technologies</td><td>18000</td><td>Cell Separation</td></tr><tr><td>EDTA</td><td>Thermo Fischer</td><td>AIM9260G</td><td>General Cell Culture</td></tr><tr><td>Falcon Round-Bottom Polystyrene Tubes, 5 mL</td><td>Stem Cell Technologies</td><td>38025</td><td>Cell Separation</td></tr><tr><td>Fc Receptor Binding Inhibitor Polyclonal Antibody</td><td>Thermo Fischer</td><td>14-9161-73</td><td>Flow Cytometry</td></tr><tr><td>Fetal Bovine Serum</td><td>Thermo Fischer</td><td>A5670701</td><td>General Cell Culture</td></tr><tr><td>Fixable Viability Dye eFluor 780</td><td>Thermo Fischer</td><td>65-0865-18</td><td>Flow Cytometry</td></tr><tr><td>Foxp3 / Transcription Factor Staining Buffer Set</td><td>Thermo Fischer</td><td>00-5523-00</td><td>Flow Cytometry</td></tr><tr><td>FOXP3 Monoclonal Antibody (PCH101), PE-Cyanine5.5</td><td>Thermo Fischer</td><td>35-4776-42</td><td>Flow Cytometry</td></tr><tr><td>HBSS</td><td>Thermo Fischer</td><td>14170-112</td><td>General Cell Culture</td></tr><tr><td>Heat Inactivated Fetal Bovine Serum</td><td>Thermo Fischer</td><td>A5670801</td><td>General Cell Culture</td></tr><tr><td>HEPES (1 M)</td><td>Thermo Fischer</td><td>15630106</td><td>moDC Cell Culture</td></tr><tr><td>HLA-DR Monoclonal Antibody (L243), Alexa Fluor 488</td><td>Thermo Fischer</td><td>A51009</td><td>Flow Cytometry</td></tr><tr><td>Human CD3/CD28/CD2 T Cell Activator</td><td>StemCell Technologies</td><td>10970</td><td>T Cell Culture</td></tr><tr><td>Human GM-CSF Recombinant Protein</td><td>Thermo Fischer</td><td>300-03</td><td>moDC Cell Culture</td></tr><tr><td>Human IL-10 ELISA Kit, High Sensitivity</td><td>Thermo Fischer</td><td>BMS215-2HS</td><td>ELISA</td></tr><tr><td>Human IL-4, Animal-Free Recombinant Protein</td><td>Thermo Fischer</td><td>AF-200-04</td><td>moDC Cell Culture</td></tr><tr><td>Human PBMC (Freshly Isolated)</td><td>UPenn HIC</td><td>N/A</td><td>Cells</td></tr><tr><td>Human TNF alpha ELISA Kit</td><td>Thermo Fischer</td><td>BMS223-4</td><td>ELISA</td></tr><tr><td>Light Microscope (DMi1)</td><td>Lucia</td><td>391240</td><td>General Cell Culture</td></tr><tr><td>Lipopolysaccaride (LPS)</td><td>Invivogen</td><td>tlrl-eblps</td><td>moDC Cell Culture</td></tr><tr><td>LIVE/DEAD Fixable Near-IR Dead Cell Stain Kit</td><td>Thermo Fischer</td><td>L34975</td><td>Flow Cytometry</td></tr><tr><td>MEM Non-Essential Amino Acids Solution (100x)</td><td>Thermo Fischer</td><td>11140050</td><td>T Cell Culture</td></tr><tr><td>Penicillin-Streptomycin (100x)</td><td>Thermo Fischer</td><td>15140122</td><td>General Cell Culture</td></tr><tr><td>Pipette Controller</td><td>VWR</td><td>77575-370</td><td>General Cell Culture</td></tr><tr><td>Rapamycin, 98+%</td><td>Thermo Fischer</td><td>J62473.MF</td><td>moDC Cell Culture</td></tr><tr><td>RPMI 1640 with Glutamax</td><td>Thermo Fischer</td><td>61870-036</td><td>General Cell Culture</td></tr><tr><td>Separation Buffer</td><td>Stem Cell Technologies</td><td>20144</td><td>Cell Separation</td></tr><tr><td>T Cell Stimulation Cocktail (500x)</td><td>Thermo Fischer</td><td>00-4970-93</td><td>T Cell Culture</td></tr><tr><td>UltraComp eBead Plus Compensation Beads</td><td>Thermo Fischer</td><td>01-3333-41</td><td>Flow Cytometry</td></tr><tr><td>Variable Pipette Set</td><td>Fischer Scientific</td><td>05-403-152</td><td>General Cell Culture</td></tr></tbody></table>

a simple and efficient method for testing immunomodulatory agents for generation of tolerogenic dendritic cells from human cd14+ monocytes

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

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

We have described a protocol for human PBMCs, isolate both CD3+ T cells and CD14+ monocytes using commercially available magnetic separation kits, differentiate monocytes into CD14-, HLA-DR+, CD141+, CD1c+ moDCs using GM-CSF and IL-4, treat them for 24 h, and co-culture with autologous T cells with anti-CD3/CD28 stimulation for 72 h. An experimental schematic is shown in Figure 1.
Isolation ...

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

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

a-simple-efficient-method-for-testing-immunomodulatory-agents-for

Research

JoVE Journal

Immunology and Infection

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.

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

This video demonstrates the generation of immature, mature, and tolerogenic dendritic cells, or DCs, from human monocytes. The incubation of monocytes with growth factors and cytokines leads to the production of immature DCs. In response to immune regulatory molecules, immature DCs differentiate into tolerogenic and mature DCs.

Generation of Immature, Mature, and Tolerogenic Dendritic Cells from Monocytes

This video demonstrates a method of converting dendritic cells (DCs) to tolerogenic DCs (TolDCs) using an immunomodulatory pharmacological agent. This conversion is confirmed by evaluating the TolDC function through their immunosuppressive effect on CD4+ T cells.

Effect of an Immunomodulatory Pharmacological Agent on the Interaction Between Dendritic Cells and CD4+ T Cells

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.

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

This study compares two different methods of human monocyte isolation for obtaining in vitro dendritic cells (DCs). Monocytes are selected by adherence or negatively enriched by magnetic separation. Monocyte yield and viability along with MDDC viability, proliferation and CD11c/CD14 surface marker expression will be compared between both methods.

Characterization of Human Monocyte-derived Dendritic Cells by Imaging Flow Cytometry: A Comparison between Two Monocyte Isolation Protocols

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.

Generation of Human Monocyte-derived Dendritic Cells from Whole Blood

Here, we present a protocol to develop and characterize tolerogenic dendritic cells (TolDCs) and evaluate their immunotherapeutic utility.

Development and Functional Characterization of Murine Tolerogenic Dendritic Cells

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.

Novel Protocol for Generating Physiologic Immunogenic Dendritic Cells

Here we present a protocol for in vivo augmentation of gut-homing regulatory T cell induction. In this protocol, dendritic cells are engineered to locally produce high concentrations of the active vitamin D (1,25-dihydroxyvitamin D or 1,25[OH]2D) and the active vitamin A (retinoic acid or RA) de novo.

In Vivo Augmentation of Gut-Homing Regulatory T Cell Induction

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

Determination of Vaccine Immunogenicity Using Bovine Monocyte-Derived Dendritic Cells

A unique, comprehensive protocol to generate de-sialylated human monocyte-derived dendritic cells (mo-DCs) from isolated peripheral blood mononuclear cells (PBMCs) using a sialidase treatment is presented. Further, methods to assess the phenotypic and functional characterization of mo-DCs and evaluate how sialidase treatment improves the maturation level of mo-DCs are described.

Generation of Monocyte-Derived Dendritic Cells with Differing Sialylated Phenotypes

Learn a method to generate tolerogenic dendritic cells from human CD14+ monocytes using immunomodulatory agents. This technique enables regulatory T cell induction for autoimmune and transplantation research.

A Simple and Efficient Method for Testing Immunomodulatory Agents for Generation of Tolerogenic Dendritic Cells from Human CD14+ Monocytes (Video) | JoVE Journal

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

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