2224 Abstracts / Molecular Immunology 47 (2010) 2198–2294 164 Complement receptors and hormonal contraceptives: Expres- sion and role of CR1/CR3 in mediating the oxidative burst of neutrophils Renata I. Bertozi, Maria Regina Torqueti, Ana Elisa C.S. Azzolini, Luciana S. Pereira-Crott, Yara M. Lucisano-Valim, Cleni M. Marzocchi-Machado Faculdade de Ciências Farmacêuticas de Ribeirão Preto-USP, Ribeirão Preto-São Paulo, Brazil Complement receptors (CR) in neutrophils can mediate oxida- tive burst (OB) and the cell functions and inflammatory processes may be impaired by inherited/acquired dysfunctions involving these receptors. Of recent interest are the known antioxidant and pro oxidant properties of estrogens and their effects on neutrophil functions. We evaluated the effect of three different hormonal contraceptives (HC) upon the OB mediated by CR and their expres- sion on neutrophils. Volunteer women (n = 40) were assigned to one of four groups: control, HC non-users (n = 17); HC-1 (30 g ethinylestradiol (E)/3 mg drosperinone (D); n = 11); HC-2 (20 g E/3 mg D; n = 5) and HC-3 (20 g E/0.075 mg gestodene; n = 7). Normal human sera (NHS) or HC sera were used as complement sources to opsonise zymosan (opZy). Neutrophils (1 × 10 6 /mL) were stimulated with opZy (1 mg/mL). The OB was measured by luminol-chemiluminescence. CR1 and CR3 expressions were eval- uated by flow cytometry using specific antibodies (E11 and ICRF 44 clones, respectively). A significant reduced expression of CR1 and CR3 was observed in neutrophils from women of the HC-2 (p = 0.0268) and HC-3 (p = 0.0216) groups compared to control. The reduced expression of CR1 and CR3 did not affect OB when neu- trophils were stimulated with opZy/NHS. However, the decrease of OB was significant in neutrophils from women of the HC-3 group (p = 0.0231) when stimulated with opZy/HC-3 sera compared to opZy/NHS. Data show that different HC influence neutrophil CR and the opsonising activity of serum complement. These results suggest that efficient coating of Zy particles with C3b/iC3b is more impor- tant to the role of CR in promoting the activation of NADPH oxidase, the enzyme responsible for the OB, than their numbers on neu- trophil membranes. The modulation of the complement system by HC may have significant consequences to inflammatory and home- ostatic processes involving neutrophils, complement and hormonal regulation. Financial support: FAPESP and CNPq. doi:10.1016/j.molimm.2010.05.084 165 Ectosomes of stored platelets (PLT-Ect) Salima Sadallah, Ceylan Eken, Perrine Martin, Jürg Schifferli Immunonephrology Laboratory, Biomedicine Department, University Hospital Basel, Basel, Switzerland Patients with low or dysfunctional platelets are treated with platelet concentrates to prevent and treat bleeding. Platelets are prepared in a plasma rich solution and conserved up to 4 days before transfusion. During that time they release small vesicles by budding from the cell surface (ectocytosis) so that the transfused patient receives not only functional platelets but also a shower of these vesicles (PLT-Ect). We decided to analyze the structural and functional properties of PLT-Ect. We purified PLT-Ect from platelet concentrates having been stored for 4 days by differential centrifugation. By EM PLT-Ect size ranged from 200 nm to 1 m. By FACS they were CD61+ without contamination with leukocyte- derived particles (no CD45 signals). Annexin V binding attested for the presence of phophatidylserine (PS). PLT-Ect expressed CD59, CD55, CD36, CD47, and tissue factor. Since platelets are stored in medium containing plasma, we analyzed whether PLT-Ect would acquire and activate complement. C1q as well as C3d fragments were bound to PLT-Ect. Factor H known to binds to platelet inte- grin alphaIIbbeta3 was bound as well. In a hirudin whole blood assay, 15% of PLT-Ect bound to erythrocytes with only 2% binding when EDTA was added indicating further complement activation by PLT-Ect under in vivo conditions. We then analyzed the capacity of PLT-Ect to modulate macrophage (M) and dendritic cell (DC) acti- vation. Both type of cells activated with Zymosan had a reduced release of pro- inflammatory cytokines when co-incubated with PLT-Ect. In addition PLT-Ect interfered with the in vitro differenti- ation of monocytes towards fully functional iDCs. However when PLT-Ect were added to monocytes during their differentiation to M in the presence of NHS, they induced an opposite effect, i.e. the cells initially exposed to PLT-Ect, released significantly more TNFa and IL6 when activated with Zymosan. Thus, despite many anti- inflammatory properties PLT-Ect induce monocyte to differentiate into an aggressive M phenotype when serum is present. Whether this is due to the presence of complement remains to be studied. doi:10.1016/j.molimm.2010.05.085 166 The complement regulator CD46 regulates unconventional gammadelta T cell responses Gaelle Le Friec a,1 , Pierre Vantourout b,c,1 , John Cardone a , Andrew Roberts b,c , Adrian Hayday b,c,d , Claudia Kemper a a Division of Immunology, Infection and Inflammatory Diseases, MRC Centre for Transplantation, King’s College London, Guy’s Hospital, Lon- don SE1 9RT, UK b London Research Institute, Cancer Research UK, London WC2A 3PX, UK c DIIID, Peter Gorer Department of Immunobiology, King’s College Lon- don, Guy’s Hospital, London SE1 9RT, UK d Biomedical Research Centre, King’s Health Partners, Guy’s Hospital, London SE1 9RT, UK CD46, an ubiquitous transmembrane glycoprotein, is commonly expressed in four isoforms with two possible cytoplasmic domains, CYT-1 and CYT-2. CD46 prevents complement deposition on host tissue by binding and inactivating the opsonins C3b/C4b. Impor- tantly, CD46 is also a costimulatory molecule on human CD4+ T cells and prevents immune activation-induced tissue pathology by regulating the timely switch from IFN-gamma to IL-10 in Th1 cells. CD46-mediated induction of IL-10 stringently requires the intracellular domain CYT-1 of CD46 and a defect in CD46-mediated IFN-gamma/IL-10 switching is connected with autoimmunity (i.e. multiple sclerosis and rheumatoid arthritis). Although conven- tional CD4+ T cell activation defines the delayed adaptive response, it is now known that unconventional gammadelta T cells play an important role in the early phases of immune responses. To assess if CD46-mediated T cell regulation extends to gammadelta T cells, we measured cytokine production by this T cell sub- set upon stimulation with HMBPP (Vg9/Vd2 agonist) and CD46 crosslinking. Surprisingly, CD46-costimulation of gammadelta T cells provoked negligible IL-10 production but induced substantial suppression of effector function (IFN-gamma and TNF-alpha secre- tion) and decrease in IL-2R expression. Intriguingly, CYT-1-bearing, 1 These authors contributed equally to the study.