The dendritic cell (DC) immunoreceptors (DCIR) and DC-immunoactivating receptors (DCAR) represent a subfamily of cell surface C-type lectin receptors (CLR), whose multifunctional capacities range from classical Ag uptake and immunoregulatory mechanisms to the involvement in DC ontogeny. On the basis of the generation of specific mAbs, we functionally characterized mouse DCAR1 (mDCAR1) as a member of the DCIR/DCAR family. Expression of mDCAR1 was strongly tissue dependent. mDCAR1 expression on DCs was restricted to the CD8+ DC subset in spleen and thymus and on subpopulations of CD11b+ myeloid cells in bone marrow and spleen, whereas the molecule was not detectable on both cell types in lymph nodes and peripheral blood. With respect to the function of CLRs as pattern recognition receptors, Ag delivered via mDCAR1 was internalized, was trafficked to early and late endosomes/lysosomes and, as a consequence, induced cellular and humoral responses in vivo even in the absence of CD40 stimulation. Intriguingly, upon triggering mDCAR1, CD8+ DCs increased the secretion of bioactive IL-12, whereas IL-10 release is markedly reduced, thereby indicating that Ag recognized by mDCAR1 induces enhanced proinflammatory responses. These data indicate that mDCAR1 is a functional receptor on cells of the immune system and provides further insights into the regulation of immune responses by CLRs.

A transcellular shuttle system was generated for the delivery of non-covalently linked payloads across blood-brain barrier (BBB) endothelial cells. Transcytosis-enabling shuttles are composed of bispecific antibodies (bsAbs) that simultaneously bind transferrin receptor (TfR) and haptens such as digoxigenin or biocytinamide. Haptenylated payloads are attached to these vehicles via non-covalent hapten-antibody complexation. This enables targeting to and internalization into human BBB-derived microvascular endothelial hCMEC/D3 cells. In contrast to other shuttles, this system does not require special affinities or formats of their TfR-binding moieties for transcytosis and subsequent release. Non-covalent payload complexation to bsAb is flexible and robust, works for a multitude of payloads and enables separation of payloads from shuttles during transcytosis. Released payloads can enter the brain without connected bsAb entities, minimizing potential interference with distribution or functionality. Intracellular separation of shuttle and payload and recycling to cell surfaces may also enable recharging of the cell-bound BBB shuttle with payload for subsequent (merry-go-round) transport cycles.

C‐type lectins (CLEC) comprise a superfamily of cell surface proteins, whose multifunctional capacity has been shown to address both, innate and adaptive immune response. Here, we describe mDCAR1 a novel CLEC belonging to the recently defined murine dendritic cell (DC) immuno‐receptor (mDCIR)/DC immuno‐activating receptor (mDCAR) family.mDCAR1 expression was found to be restricted to mononuclear phagocytes in bone marrow. Extensive phenotypical characterization defined them as CCR2−CD62L−CX3CR1+Ly6C− cells, thereby belonging to the recently described ‘resident’ monocytes, which correspond to CD14+ CD16+ human monocytes. In contrast, the CCR2+CD62L+CX3CR1lowLy6C+ ‘inflammatory’ monocytes were mDCAR1−. Noteworthy, in thymus, mDCAR1 expression was found on CD11chighCD8alpha+CD205+CD11b−B220−BP‐1+ lymphoid‐related DC, which play an important role in central tolerance induction.With respect to the classical function of CLEC as antigen uptake receptors for pathogens and self‐proteins, mimicking the ligation of mDCAR1 by labeling with specific mAb shows the internalization of the mDCAR1/ligand complex. Crosslinking of mDCAR1 further initiates signal transduction resulting in tyrosine‐phosphorylation of proteins.The expression pattern of mDCAR1 and the evidence of a classical antigen‐uptake mechanism are first steps to uncover the role of this representative of the CLEC receptor family.