After 2 consecutive stimulations, the proliferated cells were sorted and restimulated for 24 hours with fresh LCs and HIV gag p24 protein to evaluate IFN- secretion by Luminex. DC-SIGN, another lectin receptor. DCIR targeting also induced main CD8+ T-cell responses against self (MART-1) and viral (HIV gag) antigens. Addition of Toll-like receptor (TLR) 7/8 agonist enhanced DCIR-mediated cross-presentation as well as cross-priming, particularly when combined with a CD40 transmission. TLR7/8 activation was associated with increased expansion of the primed CD8+ T cells, high production of interferon- and tumor necrosis factor-, and reduced levels of type 2Cassociated cytokines. Thus, antigen targeting via the human DCIR receptor allows activation of specific CD8+ T-cell immunity. Introduction Dendritic cells (DCs) play a key role in initiating and controlling the magnitude and the quality of adaptive immune responses.1,2 DCs decode and integrate signals received from their environment and ferry this information to cells of the adaptive immune system. The presence of subsets, which possess specialized as well as shared phenotype and functions, brings out another level of complexity to the DC system of antigen-presenting cells (APCs).3C5 Microbes can directly activate DCs through a variety of pattern recognition receptors, such as Toll-like receptors (TLRs),6 cell surface C-type lectin receptors (CLRs),7 and intracytoplasmic NOD-like receptors.8,9 In humans, certain CLRs distinguish DC subsets, with plasmacytoid DCs (pDCs) expressing BDCA2,10 Langerhans cells (LCs) expressing Fosravuconazole Langerin,11 and interstitial DCs expressing DC-SIGN.12 Other C-type lectins are expressed on other cell types, including endothelial cells and neutrophils. CLRs, such as DCCspecific intracellular adhesion molecule-3-grabbing non-integrin (SIGN),7 can act as anchors for a large TM4SF18 number of microbes and allow their internalization. Furthermore, CLRs also act as adhesion molecules between DCs and other cell types, including endothelial cells, T cells, and neutrophils.12,13 DEC-205/CD205, a lectin of unknown function, has been extensively studied in the mouse Fosravuconazole for its ability to endocytose ligands. Targeting antigens to mouse DCs through DEC-205 in the absence of DC activation results in tolerance induction.14,15 In contrast, targeting antigens in the presence of DC activation (CD40 and TLR3 agonists) results in the generation of immunity against a variety of antigens.14,16 Most studies demonstrating induction of CD4+ T-cell responses or primary CD8+ T-cell response against antigens delivered via DEC-205 have been limited to the transgenic mouse OT-I/II system. Antigens have been targeted to mouse DCs through other surface Fosravuconazole molecules, including LOX-1 (a type II C-type lectin receptor that binds to HSP7017), mannose receptor,18 Dectin-1,19 Dectin-2,20 CD40,21 Langerin,22 Gb3 (a receptor for Shiga toxin23), DEC-205,24 and CLEC9A, which was recently reported to primary naive CD8+ T cells in mice.25C27 The targeting of antigens through receptors expressed on different murine DC subsets results in different functional outcomes.28,29 Targeting antigens to human DCs using conjugates of antiCDC-SIGN with keyhole limpet hemocyanin (KLH),30 antiCDEC-205 with HIV gag,31 and anti-mannose receptor with human chorionic gonadotropin hormone32 has been shown to be offered/cross-presented to blood CD4+ and CD8+ T cells, respectively, or to T-cell clones. We have turned our attention to the lectin DCIR,33 which intriguingly is usually widely expressed on different types DCs, including DCs from blood. Indeed, DCIR was initially described as expressed on blood monocytes, B cells, neutrophils, granulocytes, and dermal DCs, but not LCs, and was also recently found to be expressed on pDCs.34 Functionally, it can serve as a receptor for HIV.35 The human genome encodes only a single gene, whereas the mouse genome presents 4 DCIR-like genes: Web site; see the Supplemental Materials link at the top of the online article). This led us to construct fusion proteins based on recombinant anti-DCIR (or control IgG4 antibodies) and FluMP, but these failed to be efficiently secreted from transfected HEK293F cells. We therefore designed a strategy Fosravuconazole based on the high-affinity conversation ( 30pM) between cohesin and dockerin, 2 proteins of the cellulosome from (A.-L.F., S.Z., L. Ni, E.K., J. Quinn, S. Oh, J.B., G.Z., manuscript in preparation). The mAb.Dockerin fusion protein mAb.doc (Physique 2AII; supplemental Physique 2A) was readily secreted by transfected mammalian cells and.