Antibodies for human plasmacytoïd dendritic cells studiesdev.dendritics.net/files/documentation/PDC_report.pdf · Human plasmacytoid dendritic cells. DENDRITICS- Immeuble Laennec-60

DENDRITICS- Immeuble Laennec-60 Avenue Rockefeller-F-69008 Lyon. Tél : +33 (0)4 72 71 74 03 SAS au capital de 46300€-RCS LYON 484 431 085 00048-APE 2120Z n° TVA INTRA : FR79484431085

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Antibodies for human plasmacytoïd dendritic cells studies Dendritics SAS, 60 avenue Rockefeller, F-69008 Lyon

www.dendritics.net

Human plasmacytoïd dendritic cells (PDCs) are considered the main sentinels against viral infections and key effectors in host innate immunity. Plasmacytoïd dendritic cell precursors are the major producers of type I interferon and have the unique ability to link innate and adaptive immunity. After producing large amounts of type I IFN in response to pathogen stimulation, they can differentiate into DC capable of stimulating naive T cells and modulate the adaptive immune response.

Dendritics, internal data

PDC constitute <0.4% of peripheral mononuclear blood cells. As professional interferon producing cells (IPC), PDC regulate the function of other immune cells as well as the pathogenesis of human diseases.

Liu YJ, Annu.Rev.Immunol, 2005, 23;275-306

Human PDCs express the surface markers CD123, CD303 and neuropilin-1(CD304), but do not express CD11c (conventional dendritic cells) or CD14 (monocytes). PDCs orchestrate adaptive immune responses and play a major role in immune responses against pathogens through the recognition of pathogen associated molecular patterns (PAMPs) by two intracellular Toll-like receptors TLR7 and TLR9, which recognize ssRNA and CpG DNA motifs, respectively. TLR-triggering induces the secretion of high levels of type I IFN (mainly IFN and IFN), resulting from a signaling pathway involving in particular Myd88 and IRAK4 molecules.

Immature Mature

Human plasmacytoid dendritic cells


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Dendritics provides a panel of antibodies for human plasmacytoïd dendritic cells studies.

CD304 ( neuropilin-1):DDX0440 We have generated a mAb (211H6) that identifies human PDCs ex vivo after mice immunization with CD34+-derived DCs. In blood, the 211H6 antigen is highly expressed on IL3R+ + CD11c- PDCs (A). In situ, 211H6 recognizes some isolated cells in T cell zones of tonsils (B), lymphatic vessels and high endothelial venules (C).

The 211H6 antigen has been identified as Neuropilin-1, a membrane-bound co-receptor to a tyrosine kinase receptor for both vascular endothelial growth factor (VEGF) and semaphorin (SEMA3A) family members (Dzionek A and al., 2000 J. Immunol., 165: 6037-6046; Dzionek A. et al.,2002, Human Immunol, 63,1133-48). 211H6 does not stain lymphocytes, monocytes, basophils and granulocytes. Interestingly, despite overlapping stainings, 211H6 and display slightly different expression profiles as witnessed by the double-staining presented below:

BDCA-4-FITC and 211H6-Alexa546 staining of

CD3, CD19 and CD56-depleted PBMC

The double-staining revealed a BDCA4-211H6+ subset, suggesting that 211H6 might recognize a neuropilin-1 isoform, selectively expressed on PDCs, and which is different from the primarily identified BDCA4 (Dzionek A and al., 2000 J. Immunol., 165: 6037-6046).

CD303 : several clones We have generated a panel of anti-human CD303 monoclonal antibodies using mice immunization with the recombinant human CD303 protein. CD303 is a calcium dependent type II lectin also known as Blood-Dendritic-Cell-Antigen-2, specifically expressed by human PDCs. CD303 is a 25kDa type II membrane glycoprotein with single carbohydrate recognition domain (CRD) predicting mannose-type specificity. CD303 protein expression is restricted to immature human PDCs subset. CD303 was described to be involved in ligand internalization, processing and presentation as well as inhibition of IFN- synthesis (Dzionek A. et al, 2000, J.Immunol., 165, 6037; Dzionek et al, 2001, J.Exp.Med., 194,

211H6

IL3Ra

-PE

211H6-FITC

211H6

A B C


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1823-1834). These antibodies were used in immunohistochemistry assays to evaluate their ability to stain CD303-expressing cells in situ. Interestingly, anti-CD303 demonstrated that infiltration by PDCs of primary localized breast tumors correlates with an adverse outcome (Treilleux I. et al, 2004, Clin. Canc.Res., 10:7466).

IHC staining with anti-CD303 antibodies

These antibodies were characterized by flow cytometry for their ability to stain CD303-transfected COP5 cells and monocytes-derived dendritic cells, both at the surface and the intracytoplasmic level.

FACS staining with anti-CD303 antibodies

Human spleen frozen section stained with 108H10

Paraffin-embedded human Kikuchi’sdisease lymph node stained with

104C12

Formol-paraffin-embedded human lymph node section stained with 124B3.13

Human tonsil frozen section stainedwith 109B8

Isotype control 124B3

Surf

ace

Intr

acel

lula

r

FACS staining of human monocyte-derived DCs with 124B3

Isotype control 104C12

Surface FACS staining of CD303-transfected COP5 cells

Isotype control 108H10

Intracellular FACS staining of monocyte-derived (GM+IL4) Dcs


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The panel of anti-CD303 antibodies was included in biological assays aiming at measuring their influence on the amount of IFN secretion by CpG2216-treated pDCs. Antibodies were added in a soluble form. In addition, the capacity of the antibody to induce apoptosis was measured through the determination of cell viability.

Effects of anti-CD303 monoclonal antibodies CpG-treated PDCs

These biological assays revealed remarkable properties of some anti-CD303 clones: DDX0040, DDX0042 are able to induce the apoptosis of CpG-treated PDC. DDX0041, DDX0044, DDX0047, DDX0048, and DDX1051 inhibit the secretion of IFN by CpG-treated PDC, without affecting cell viability. Increasing number of reports described the leukemic counterpart of PDC, BPDCN (blastic plasmacytoïd dendritic cells neoplasm), as well as the infiltration of solid tumors by PDC. These newly generated anti-CD303 antibodies could be powerful tools for studies aiming at understanding the physiopathology of PDC-related diseases.

The overall data are summarized in the table below:

0102030405060708090

100

cont

rol a

ntib

ody

BDCA

2 (M

ilten

yi)

DD

X004

0

DD

X004

1

DD

X004

2

DD

X004

3

DD

X004

4

DD

X004

5

DD

X004

6

DD

X004

7

DD

X004

8

DD

X105

0

DD

X105

1

% viable cells

% inhibition of IFN secretion

reference clone IsotypeCOP5-CD303

surface protein

COP5-CD303 intracyto

MonoDC GMCSF-IL-4

intracyto

MonoDC GMCSF-IL-4

surface

Inhibits IFN a

Increases IFN a

Apoptosis frozen section

paraffin section

DDX0040 108H10.03 IgM +++ + ++ - +++ - +++ +++ +

DDX0041 104C12.08 IGg1 ++ + - +/- - - - ++ +++

DDX0042 110H7.05 IGg1 +++ +++ +++ + +++ - +++ +++ +

DDX0043 124B3.13 IGg1 +/- +++ - +/- - - - +++ +++

DDX0044 102G7.04 IgG2K ++ ++ ND ND - +++ - ++ -

DDX0045 109B8.10 IGg1 +++ ++ ND ND +++ - - ++ ND

DDX0046 101C9.01 IgG1K +++ ++ ND ND - - - +++ -

DDX0047 113H3.01 IgG1K +++ ++ ND ND - +++ - ++ ND

DDX0048 122A2.01 IgG1K +++ ++ ND ND +++ - - ++ ND

DDX1050 101C11.31 IgG1K +++ ++ ND ND - - - ++ ND

DDX1051 104D7 IgG1K +++ ++ ND ND +++ - - ++ ND

Flow cytometry Biological effects Immunohistochemistry


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CD123 (IL3R): DDX0300 IL-3 exerts its biologic activity (eg survival and proliferation of multipotent hematopoietic cells, development of pDCs) through its interaction with a cell surface receptor that consists of two subunits. The subunit (CD123) specifically binds IL-3, whereas the subunit is required for signaling and is common to the GMCSF-R and IL-5-R. Interplay of α and chains leads to the formation of a high-affinity receptor complex able to transduce proliferative, apoptotic and differentiative signals. Plasmacytoid DCs express IL3-R and differentiate into functional DCs with IL-3. The leukemic pDC counterpart also expresses high levels of IL3-Rα. Upon IL-3 treatment in vitro, leukemic cells become inducers of T-cell proliferation, as reported for normal pDCs. The role of IL3-R in oncogenesis has been explored in several animal models. Over expression of the IL3-R chain may represent one of the mechanisms contributing to the development of a highly malignant leukemic phenotype. (Treilleux I et al., 2004, Clinical Cancer Research, 10: 7466-7474). A monoclonal antibody anti-IL3R (107D2.08) was obtained after mice immunization with sorted human PDCs.

This antibody revealed the IL3R expression on freshly isolated human PDCs by flow cytometry, the presence of IL3R+ PDCs in human breast tumours and in human tonsil cryosection by immunochemistry.

FACS and IHC staining with 107D2.08

CD287 (TLR7): DDX0500 TLR7 is expressed on plasmacytoid DCs, B and T lymphocytes, neutrophils, eosinophils, NK cells, and lung and placenta epithelial cells. TLR7 is localized to endosomal/lysosomal compartments and is reported to respond to single-stranded RNA. Plasmacytoid DCs express TLR7 and respond to activation by producing high amounts of IFN-TLR7 ligands are R848, Imiquimod (Imidazoquinolin family) and loxoribine (guanosin analog). (Diebold S.S.et al, Science 2004, 303, 1529; Barchet W.A. et al, 2005, Eur J Immunol 3:236; Lund J.M. et al., 2004, PNAS 5598 ;Hell F. et al., 2004 Science, 303, 1526 ; Gilliet M et al.,2008, Natur Rev Immunol.8, 594). We have generated a monoclonal antibody (66H3) after mice immunization with human TLR7-transfected eukaryotic cells.

blood tonsil

Bouin-paraffin section of human invasive breast tumor double-stained with anti-CD123 (blue)

anti-CD303 (red)

Human tonsil cryosectionHuman sorted PDCs


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Intracellular FACS staining of human lymphocytes with 66H3

Immunocytochemistry and immunohistochemistry staining with 66H3

Myd88: DDX1400 and DDX1401 MyD88 (myeloid differentiation primary response gene 88) is a universal adapter protein of 296 aa used by all TLRs (except TLR3) and by IL1-R to activate the transcription factor NF-B. Myd88 binds IRAK1, IRAK2 and TRAF6, leading to NF-B activation, cytokine secretion and inflammatory response. Myd88 increases IL8 transcription, and is involved in IL18 signaling pathway. Two monoclonal antibodies, 603E10.05 and 603E10.06 were obtained after mice immunization with human Myd88-transfected 293T cells. These antibodies specifically recognize endogenously expressed Myd88 and do not cross-react with the mouse Myd88.

Flow cytometry and immunofluorescent staining with anti-Myd88 monoclonal antibodies

PBMC

isotype control 66H3

IHC staining of tonsil cryosection with 66H3

TLR7-transfected 293 cells stained with 66H3

100 101 102 103 104FL1-H

Isotype controlsiRNA Myd88siRNA scramble

Endogenous MyD88 expression revealed by intracytoplasmic FACS staining of HCT116 cells with

603E10.05

Endogenous MyD88 expression revealed by IF staining of Hela cells with 603E10.06


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IRAK4: DDX0340 and DDX0341 IL-1R associated kinases (IRAKs) are important mediators in the signal transduction of Toll / IL-1R (TIR) family members. The mammalian family of IRAK molecules contains four members (IRAK1, IRAK2, IRAK-M and IRAK4). IL-1 stimulation leads to the recruitment of the IL-1R associated kinase (IRAK) to the IL-1 Receptor where IRAK is phosphorylated, ubiquinated, and eventually degraded. Endogenous IRAK-4 interacts with IRAK-1 and TRAF6 in an IL-1 dependent manner. IRAK-4 is able to phosphorylate IRAK-1 (Cao S. et al, 1996; Science, 271, 1128-1131 ; Li S. et al, 2002; PNAS, 99, 5567-5572). Two monoclonal antibodies (6F8 and 7D8) were obtained after mice immunization with human IRAK4-transfected 293T cells. The 6F8 clone revealed IRAK4 expression in several cell types among which Gen 2.2 (a human PDC cell line) by western-blot whereas the 7D8 clone revealed the presence of IRAK4+ cells by in situ staining.

Western-blot analysis with 6F8 monoclonal antibody (Innate sensors plateform, CLARA, LYON)

Frozen section of human tonsil stained with Alexa546-coupled 7D8 antibody

Concluding remarks:

Altogether, Dendritics has developed a series of monoclonal antibodies allowing the isolation as well as the study of human plasmacytoïd dendritic cells. The emerging data reporting the involvement of PDC in human diseases (infiltration in breast cancer, PDC lymphomas) strongly suggested that these antibodies should be further characterized in order to evaluate their prognostic as well as therapeutic values, so that their use will not be limited only to research and diagnosis areas.

55KDa

75KDa

100KDa

IRAK4

PBM

C

RPM

I822

6

HEK

293


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List of monoclonal antibodies targeting human plasmacytoïd cells provided by Dendritics

CD304 (neuropilin1)

CD303

Antigen Clone Format Isotype Source / Host Applications Product No.purified DDX0440

Alexa-fluor®-488 coupled DDX0440A488 Alexa-fluor®-647 coupled DDX0440A546 Alexa-fluor®-546 coupled DDX0440A647

biotin-coupled DDX0440B

Neuropilin1 CD304 211H6.01 IgG1 mouse Flow cytometry, IHC




purified DDX0041



purified DDX0042



purified DDX0043



purified DDX0044



purified DDX0045



purified DDX0046



purified DDX0047



CD303

108H10.03 IgG1 mouse

IGg1109B8.10

101C9.01 IgG1K

IgG1K113H3.01

mouse

124B3.13 IgG1 mouse

IgG2K102G7.04

104C12.08 IgG1 mouse

110H7.05 IgG1 mouse

mouse

mouse

mouse

IHC Bouin-paraffin, Inhibition of IFN secretion, IF, Flow cytometry

Formol, Bouin-paraffin IHC, IF, Flow cytometry

Flow cytometry, Bouin-paraffin IHC, IF, Inhibition of IFN

secretion, Apoptosis

Bouin, Formol-paraffin IHC, IF, Intracellular flow cytometry

Flow cytometry, Increase IFN secretion

Surface flow cytometry, IHC, Inhibition of IFN secretion, IF

Flow cytometry, IHC

Flow cytometry, Increase IFN secretion, IHC


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CD123 (IL3R)

CD287 (TLR7)

Myd88

IRAK-4

For technical informations: [email protected] Ordering informations: find your distributor at www.dendritics.net


Alexa-fluor®-488 coupled DDX0300A488




IL3R α/ CD123 107D2.08 IgG1 mouseBouin-paraffin IHC, IP, IF, Flow

cytometry






IgG1 mouse66H3TLR7/CD287 Intracellular flow cytometry, IHC



purified DDX1401


IF, WB on Myd88-transfected cells

Intracellular flow cytometry, WB on Myd88-transfected cells

Myd88603E10.05 IgG2b mouse

603E10.06 IgG1 mouse

Antigen Clone Format Isotype Source / Host Applications Product No.6F8 purified IgG1 mouse WB DDX0340

purified DDX0341



IHC, IFIRAK-4

7D8 IgG1 mouse




purified DDX1050



purified DDX1051



CD303

mouse

mouse

mouse

122A2.01

101C11.31

104D7 IgG1K

IgG1K

IgG1K Flow cytometry, IHC



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Antibodies for human plasmacytoïd dendritic cells studiesdev.dendritics.net/files/documentation/PDC_report.pdf · Human plasmacytoid dendritic cells. DENDRITICS- Immeuble Laennec-60