Effect of synthetic agonists of toll-like receptor 9 on canine lymphocyte proliferation and cytokine production in vitro Michelle Im Hof a,1, * , Lina Williamson b,1 , Artur Summerfield c , Vreni Balmer a , Virginie Dutoit b , Ekambar R. Kandimalla d , Dong Yu d , Andreas Zurbriggen a,e , Marcus G. Doherr a , John Peel b , Petra J. Roosje a,e a Department of Clinical Veterinary Medicine, Vetsuisse Faculty, University of Bern, La ¨nggassstrasse 128, CH-3012 Bern, Switzerland b Novartis Animal Health, Centre de Recherche Sante ´ Animale, 1566 St. Aubin, Switzerland c Institute of Virology and Immunoprophylaxis, Sensemattstrasse 293, 3147 Mittelha ¨usern, Switzerland d Idera Pharmaceuticals, Inc., 167 Sidney Street, Cambridge, MA 02139, USA e DermFocus, Vetsuisse Faculty, University of Bern, Switzerland Received 22 November 2007; received in revised form 19 February 2008; accepted 4 March 2008 Abstract Synthetic agonists of TLR9 containing novel DNA structures and R 0 pG (wherein R = 1-(2 0 -deoxy-b-D-ribofuranosyl)-2-oxo-7- deaza-8-methyl-purine) motifs, referred to as immune modulatory oligonucleotides (IMOs), have been shown to stimulate T H -1- type-immune responses and potently reverse allergen-induced T H -2 responses to T H -1 responses in vitro and in vivo in mice. In order to investigate the immunomodulatory potential of IMOs in dogs, canine peripheral blood mononuclear cells (PBMC) from healthy dogs were stimulated with three different IMOs and a control IMO, alone or in combination with concanavalin A (ConA). Lipopolysaccharide (LPS) was used as a positive control for B lymphocyte activation. Carboxyfluorescein diacetate succinimidyl ester and phenotype staining was used to tag proliferating T and B lymphocytes (CD5 + and CD21 + ) by flow cytometry. Real-time PCR and ELISA were processed to assay cytokine production of IFN-g, IL-10, TGF-b, IL-6 and IL-10. Like LPS, IMOs alone induced neither proliferation of CD5 + T cells nor CD21 + B cells, but both LPS and IMO had the capacity to co-stimulate ConA and induced proliferation of B cells. In combination with ConA, one of the IMOs (IMO1) also induced proliferation of T cells. IMO1 also significantly enhanced the expression of IFN-g on the mRNA and protein level in canine PBMC, whereas expression of IL-10, TGF-b and IL-4 mRNAs was not induced by any of the IMOs. These results indicate that in canine PBMC from healthy dogs, IMO1 was able to induce a T H -1 immune response including T- and B-cell proliferation. # 2008 Elsevier B.V. All rights reserved. Keywords: TLR9 agonists; Immunomodulatory oligonucleotides; Flow cytometry; Canine lymphocyte proliferation; Cytokines; Toll-like receptor 9 1. Introduction The immune system of vertebrates recognizes unmethylated cytidine–phosphate–guanosine (CpG) dinucleotides, which are present in bacterial DNA, as a danger signal (Krieg, 2001). CpG dinucleotides trigger a protective immune response that improves the ability of the host to eliminate the pathogen (Wagner, 1999). This www.elsevier.com/locate/vetimm Available online at www.sciencedirect.com Veterinary Immunology and Immunopathology 124 (2008) 120–131 Abbreviations: CFSE, carboxyfluorescein diacetate succinimidyl ester; CpG, cytidine-phosphate-guanosine; IMO, immunomodulatory oligonucleotide; ODN, oligodeoxynucleotide; PE, phycoerythrin; RT, room temperature; TLR9, toll-like receptor 9; Treg, T regulatory cells. * Corresponding author. Tel.: +41 31 631 23 15; fax: +41 31 631 22 75. E-mail address: michelle.imhof@kkh.unibe.ch (M. Im Hof). 1 These authors contributed equally. 0165-2427/$ – see front matter # 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.vetimm.2008.03.002