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EVERY STEP OF THE WAY

RESEARCH MODELS

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DISCOVERY

Complex Biology In Vitro Assays: Immunology Plasmacytoid Dendritic Cells Activation AssayPlasmacytoid dendritic cells (pDCs) are a rare subset of dendritic cells that primarily regulate immune responses to

viral infections through a massive release of type I interferons. Viral RNA or DNA nucleic acids are recognized by

specific pattern recognition receptors (PRRs), such as toll-like receptor 7 and 9 (TLR7/9)1-6, expressed on pDCs. The

engagement of these receptors results in the activation of downstream molecules like MyD88, NF-κB, MAPK, and IRF-

7/5, which initiates a cascade of responses leading to pro-inflammatory cytokine production and upregulation of specific

surface molecules2,6. All these events contribute to the final pDCs effector function, which is essential for viral clearance.

Phenotypically, pDCs are characterized by the expression of surface molecules CD303, CD304, and CD123, but lack

the expression of CD11c. Contrarily to cDCs (conventional DCs), pDCs have a rounded-shape and lack dendrites. pDCs

have mainly anti-viral functions with a limited antigen presentation capacity, and act as poor, naïve T cell activators1,2,8-11.

In vitro pDC activation can be achieved using short, single-stranded oligodeoxynucleotides (ODNs), such as imiquimod

and CpG, which are synthetic ligands for TLR7 and 9, respectively. The binding of ODNs to TLR7/9 results in a massive

release of type I IFNs, such as IFN-α2a6,12.

pDCs have also been found to play a role in the pathogenesis of both autoimmune diseases and cancer5-7.

Overproduction of pDC-derived cytokines induced the activation of cDCs, triggering autoreactive CD8+ T cells in

patients with psoriasis, dermatomyositis, and systemic lupus erythematosus13. Targeting type I interferon pathways

has thus been suggested to be a compelling approach for the treatment of some autoimmune diseases14. By contrast,

the role of pDCs in cancer has not been fully defined, yet. Glioma-bearing mice vaccinated with pDCs did not show

a robust anti-tumor response, when compared to mice vaccinate with mDCs15; by contrast, CpG-ODN-mediated

activation of pDCs demonstrated to enhance NK and CD8+ T cell tumor killing functions16. As a result, modulating

pDC functions could be an effective strategy for the induction of antitumor immune responses.

SummaryPowerful novel immunological in

vitro assays provide a translational

tool to study the potency of

biologics or small molecules

as modulators of immune

responses. We have developed a

cell-based assay mimicking the

in vitro activation of plasmacytoid

dendritic cells isolated from the

peripheral blood of healthy donors,

to assess the complex biology

of plasmacytoid dendritic cells in

modulating immune responses.

Click to learn more

Need a custom version of this assay? Visit criver.com/ds-vitro-assay.

Complex Biology In Vitro Assays: Immunology Plasmacytoid Dendritic Cells Activation Assay

Cultures of human pDCs can be performed either by differentiating CD34+ progenitor cells derived from human

umbilical cord blood17,18 or by isolating already differentiated pDCs from peripheral blood mononuclear cells (PBMCs).

The first approach is more laborious, with the latter demonstrated to be a faster and reliable method suitable for in vitro

compound screening.

Here, we have developed and validated an in vitro immunology ‘pDCs activation assay’ using human primary pDCs isolated

from blood of healthy donors to study the effects of compounds on pDCs function. Our in vitro pDCs activation assay offers a

unique approach for the identification of candidate drugs in translational and clinical research.

Assay Principle Human primary pDCs were purified from peripheral blood mononuclear cells (PBMCs) by depletion of non-pDCs using

a negative magnetic beads-based procedure. Purity of ‘untouched’ pDCs was verified by multicolor flow cytometry and

pDCs were seeded in a 96-well plate in culture medium containing interleukin-3 (IL-3), a pDCs survival factor. Due to their

relatively short life-span19, 24 hours after the isolation, pDCs were triggered with CpG ODN 2216 (TLR-9 agonist) and

compounds added to the cells. 24 hours later, cell supernatant was collected and pDCs activation is assessed by measuring

IFN-α2a release using a MSD platform.

Assay Workflow

Assay Setup

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Cells Untouched primary pDCs isolated from healthy human blood donors

Cells QC pDCs surface markers by FACS (at day 0)

Seeding density 2.5 x 104 cells/well in 96-well plate

Culture medium Complete medium + IL-3 (20ng/ml)

Trigger CpG ODN 2216 (3 µM) at day 1

Inhibitor IKK inhibitor VII at 8-point CRC (0.032 - 100 µM) at day 1

Compounds 8-point concentration response curves

Controls Vehicle (0.1% DMSO), vehicle (0.1% DMSO) + trigger, culture medium

Time point assay 24 hours (post-trigger addition)

Replicates Biological duplicates

Readout IFN-α2a secretion (by MSD)

askcharlesriver@crl.com • www.criver.com

Figure 1. Quality check of isolated primary human pDCs. A. Representative flow cytometry histograms showed the

expression of pDC markers on cells isolated from PBMCs of one healthy blood donor. Isotype controls showed the lack

of non-specific binding. B. Flow cytometry-dot plot represented the expression of CD304 (Y-axis) versus CD123 (X-axis)

gated on CD11c- population. The double positive population appeared in the upper right quadrant and confirmed the high

level of purity (~91%) for the freshly isolated pDCs population (CD11c-, CD123+, CD304+ cells). C. Percentages of cells

expressing pDC- markers are shown for five representatives human PBMCs donors. Every donor is represented by a dot.

A

B

CD11c

CD123

CD30

4

CD123 CD304

C CD11c CD123 CD304

pDC markers

Isotype

Quality Checks

Flow cytometry was used to perform a quality check (QC) of isolated primary pDCs. After isolation from buffy coats of

healthy human donors, cells were stained with anti-CD11c, anti-CD304, and anti-CD123 antibodies and their relative isotype

controls, and cells were analyzed by flow cytometry (Fig. 1). Cells that were negative for CD11c and exhibit > 70% of

CD123 and CD304 expression were plated and used for the assay.

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Figure 2. In vitro pDCs activation. A. IFN-α2a release measured by MSD for the assay control conditions of two representative

human blood donors (Donor 01 and 02) was reported. Data are shown as mean ± SD. B. Concentration-response curves of

IKK Inhibitor VII and percentage of inhibition (PIN) for IFN-α2a secretion in two representative human blood donors (Donor 01

and 02) were reported. Data are shown as mean ± SD, full lines depict non-linear regression analysis.

A B

Complex Biology In Vitro Assays: Immunology Plasmacytoid Dendritic Cells Activation Assay

Assay Performance

Performance of the developed assay was validated using CpG (ODN 2216), as a trigger, and IKK inhibitor VII, as positive

control for inhibition of pDCs activation, together with other assay controls. Data obtained from two different human donors

are reported below (Fig. 2).

SummaryAlthough pDCs constitute only 0.1-4%19 of the PBMC population, they are one of the most important cell types involved in

the generation of anti-viral responses, through the production of type I interferons. Phenotypically, pDCs are different from

their counterpart cDCs, as they resemble plasma cells and lack dendrites, which are typical properties of cDCs. pDCs

are characterized by high expression of the interleukin-3 receptor alpha chain (CD123) and by the expression of other cell

surface markers such as CD304 and HLA-DR, but lack expression of the myeloid lineage marker CD11c.

pDCs are critical for the anti-viral responses through production of large amounts of type I IFN and cytokines via TRL7/9

signaling. However, depending on the microenvironment and their maturation state, pDCs can exert immunogenic and/

or tolerogenic functions in auto-immune disorders20-22 and cancer23,24. Understanding the mechanisms underlying pDCs

plasticity and modulating their capacity to respond to TLR-mediated signals, could lead to novel treatments or improve

existing therapeutics.

Need a custom version of this assay? Visit criver.com/ds-vitro-assay.

askcharlesriver@crl.com • www.criver.com © 2020, Charles River Laboratories International, Inc.

Here, we demonstrated an optimized in vitro pDCs activation assay by assessing CpG (ODN2216)-induced secretion of

IFN-α2a. CpG (ODN2216) at 3 µM promoted a strong activation of human primary pDCs by promoting IFN-α2a release,

which was inhibited by IKK inhibitor VII, in a dose-dependent fashion. IC50 values were consistent across different donors

(not shown). Our in vitro immunology assay is a cost-effective and robust approach to evaluate therapeutic candidates that

affect pDCs functions for the treatment of different infections, and autoimmune diseases, as well as cancer.

Therapeutic candidates can be evaluated in an eight-point CRC on pDCs derived from up to two healthy human donors in

biological duplicates for their capacity to release IFN-α2a.

Assay Reference CodepDCs Activation Assay Reference Code – OTS218 pDC Activation Assay

References1. Primer to the Immune Response, 2014, Elsevier Inc

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3. Tversky JR et al. Clinical & Experimental Allergy, 2008, 38, 781-788

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5. Mitchell D. et al. Journal of neuroimmunology, 2018, 322, 63-73

6. McKenna K et al. Journal of virology, 2005, 79, 17-27

7. Li S et al. Frontiers in immunology, 2017, 8, 1268

8. Lin YT et al. Current genomics, 2012, 13, 633-645

9. Crozat K & Beutler B, Proceedings of the National Academy of Sciences, 2004, 101, 6835-6836

10. Swiecki M & Colonna M, Nat. Rev. Immunol. 2015, 15, 471–485

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13. Klarquist J et al. Mediators of inflammation, 2016, 5045248

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16. Kawarada Y et al. J. Immunology, 2001, 167: 5247–5253

17. Fahrbach KM et al. J. Virol. 2007, 81: 6858–6868

18. Laustsen A et al. Nat Commun. 2018, 9: 3525

19. Ueda Y et al, Hum Immunol. 2003, 64,1144-51

20. Farkas L et al. L. American J. Pathol. 2001, 159:237–243

21. Jahnsen FL et al. Nasal Allergy. J. Immunol. 2000, 165:4062–4068

22. Wollenberg A et al. J. Invest Dermatol. 2002,119:1096–1102

23. Aspord C et al. Cancer Immunol Res. 2013, 6:402-415

24. Monti M et al. Cells. 2020, 9: 417

Need a custom version of this assay? Visit criver.com/ds-vitro-assay.