To successfully feed, ticks inject pharmacoactive molecules into the vertebrate host including cystatin cysteine protease inhibitors. However, the molecular and cellular events modulated by tick saliva remain largely unknown. Here, we describe and characterize a novel immunomodulatory cystatin, Iristatin, which is upregulated in the salivary glands of feeding Ixodes ricinus ticks. We present the crystal structure of Iristatin at 1.76 Å resolution. Purified recombinant Iristatin inhibited the proteolytic activity of cathepsins L and C and diminished IL-2, IL-4, IL-9, and IFN-γ production by different T-cell populations, IL-6 and IL-9 production by mast cells, and nitric oxide production by macrophages. Furthermore, Iristatin inhibited OVA antigen-induced CD4+ T-cell proliferation and leukocyte recruitment in vivo and in vitro. Our results indicate that Iristatin affects wide range of anti-tick immune responses in the vertebrate host and may be exploitable as an immunotherapeutic.

Department of Medical Biology Faculty of Science University of South Bohemia in České Budějovice Branišovská 1760c 37005 České Budějovice Czech Republic

Institute for Immunology University Medical Center of the Johannes Gutenberg University Mainz Langenbeckstrasse 1 Mainz 55131 Germany

Institute of Organic Chemistry and Biochemistry Academy of Sciences of the Czech Republic Flemingovo nám 2 16610 Prague Czech Republic

Laboratory of Genomics and Proteomics of Disease Vectors Biology Centre CAS Institute of Parasitology Branišovská 1160 31 37005 České Budějovice Czech Republic

Laboratory of Malaria and Vector Research National Institute of Allergy and Infectious Diseases National Institutes of Health 12735 Twinbrook Parkway Rockville MD 20852 USA

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