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

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1 Antibodies for human plasmacytoïd dendritic cells studies Dendritics SAS, 60 avenue Rockefeller, F Lyon 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. Human plasmacytoid dendritic cells Immature Mature 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; 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. Page 1

2 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). A B C 211H6 211H6 IL3Ra-PE 211H6-FITC 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: ; Dzionek A. et al.,2002, Human Immunol, 63, ). 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: ). 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, Page 2

3 ). 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 Formol-paraffin-embedded human lymph node section stained with 124B3.13 Human spleen frozen section stained with 108H10 Human tonsil frozen section stained with 109B8 Paraffin-embeddedhuman Kikuchi s diseaselymphnode stainedwith 104C12 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 Isotype control 124B3 Isotype control 104C12 Intracellular Surface Surface FACS staining of CD303-transfected COP5 cells FACS staining of human monocyte-deriveddcs with 124B3 Isotype control 108H10 Intracellular FACS staining of monocyte-derived (GM+IL4) Dcs Page 3

4 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 control antibody BDCA2 (Miltenyi) DDX0040 DDX0041 DDX0042 DDX0043 DDX0044 DDX0045 DDX0046 DDX0047 DDX0048 DDX1050 DDX1051 % viable cells % inhibition of IFN secretion 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: Flow cytometry Biological effects Immunohistochemistry reference clone Isotype COP5-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 DDX H10.03 IgM DDX C12.08 IGg / DDX H7.05 IGg DDX B3.13 IGg1 +/ / DDX G7.04 IgG2K ND ND DDX B8.10 IGg ND ND ND DDX C9.01 IgG1K ND ND DDX H3.01 IgG1K ND ND ND DDX A2.01 IgG1K ND ND ND DDX C11.31 IgG1K ND ND ND DDX D7 IgG1K ND ND ND Page 4

5 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: ). 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 blood tonsil Human sorted PDCs Human tonsil cryosection Bouin-paraffin section of human invasive breast tumor doublestained with anti-cd123 (blue) anti-cd303 (red) 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. Page 5

6 Intracellular FACS staining of human lymphocytes with 66H3 PBMC isotype control 66H3 Immunocytochemistry and immunohistochemistry staining with 66H3 TLR7-transfected 293 cells stained with 66H3 IHC staining of tonsil cryosection 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 Myd88. Flow cytometry and immunofluorescent staining with anti-myd88 monoclonal antibodies Isotype control sirna Myd88 sirna scramble FL1-H 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 Page 6

7 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, ; Li S. et al, 2002; PNAS, 99, ). 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) PBMC RPMI8226 HEK KDa 75KDa IRAK4 55KDa 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. Page 7

8 List of monoclonal antibodies targeting human plasmacytoïd cells provided by Dendritics CD304 (neuropilin1) DDX0440 DDX0440A488 Neuropilin1 211H6.01 IgG1 Flow cytometry, IHC DDX0440A546 CD304 DDX0440A647 DDX0440B CD303 CD H10.03 IgG1 104C12.08 IgG1 110H7.05 IgG1 124B3.13 IgG1 102G B C9.01 IgG1K 113H3.01 IgG2K IGg1 IgG1K 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 DDX0040 DDX0040A488 DDX0040A546 DDX0040A647 DDX0040B DDX0041 DDX0041A488 DDX0041A546 DDX0041A647 DDX0041B DDX0042 DDX0042A488 DDX0042A546 DDX0042A647 DDX0042B DDX0043 DDX0043A488 DDX0043A546 DDX0043A647 DDX0043B DDX0044 DDX0044A488 DDX0044A546 DDX0044A647 DDX0044B DDX0045 DDX0045A488 DDX0045A546 DDX0045A647 DDX0045B DDX0046 DDX0046A488 DDX0046A546 DDX0046A647 DDX0046B DDX0047 DDX0047A488 DDX0047A546 DDX0047A647 DDX0047B Page 8

9 CD A C D7 IgG1K IgG1K IgG1K Surface flow cytometry, IHC, Inhibition of IFN secretion, IF Flow cytometry, IHC Surface flow cytometry, IHC, Inhibition of IFN secretion, IF DDX0048 DDX0048A488 DDX0048A546 DDX0048A647 DDX0048B DDX1050 DDX1050A488 DDX1050A546 DDX1050A647 DDX1050B DDX1051 DDX1051A488 DDX1051A546 DDX1051A647 DDX1051B CD123 (IL3R ) IL3R α/ CD D2.08 IgG1 CD287 (TLR7) Bouin-paraffin IHC, IP, IF, Flow cytometry DDX0300 DDX0300A488 DDX0300A546 DDX0300A647 DDX0300B DDX0500 DDX0500A488 TLR7/CD287 66H3 IgG1 Intracellular flow cytometry, IHC DDX0500A546 DDX0500A647 DDX0500B Myd88 Myd88 603E10.05 IgG2b 603E10.06 IgG1 IRAK-4 Intracellular flow cytometry, WB on Myd88-transfected cells IF, WB on Myd88-transfected cells DDX1400 DDX1400A488 DDX1401 DDX1401A488 6F8 IgG1 WB DDX0340 IRAK-4 DDX0341 7D8 IgG1 IHC, IF DDX0341A488 DDX0341A546 For technical informations: contact@dendritics.net Ordering informations: find your distributor at Page 9