br Materials and Methods br Results br Discussion Understand

Materials and Methods
Results
Discussion Understanding the biology of human DC surface receptors and the functional consequences of the actions of individual receptors is fundamental for the rational design of medicines for cancers, inflammatory diseases (including autoimmune diseases) and microbial infections. Of the many different receptors expressed on the surface of DCs, lectin-like receptors are considered to be one of the major pattern-recognition receptor families. Some of these receptors, Dectin-1 (Duluc et al., 2014; Joo et al., 2015; LeibundGut-Landmann et al., 2007), DCIR (Fujikado et al., 2008), DC-SIGN (Geijtenbeek and Gringhuis, 2009), LOX-1 (Joo et al., 2014), and DC-ASGPR (Li et al., 2012), are known to play important roles in shaping the quality and quantity of host immune responses. However, the ability of these receptors to capture purchase pitavastatin and deliver them to intracellular compartments makes them novel targets for DC antigen delivery to enhance antigen cross-presentation to T cells. Nonetheless, one major question still remains: “Which targeted receptor results in optimal antigen cross-presentation to T cells?” This study has demonstrated that CD40 is superior to nine other lectins and scavenger receptors at cross-presenting antigen to CD8+ T cells. This was confirmed with both a tumor-associated self antigen and different forms of viral antigens. Interestingly, however, DC lectins (e.g., LOX-1 Dectin-1 and Langerin) were superior to CD40 at presenting antigens to CD4+ T cells. To further understand such functional specialization of CD40, we examined the subcellular and intracellular localization of receptor-bound mAbs in DCs. Previous studies (Burgdorf et al., 2007, 2008) showed that early endosomes are essential for the cross-presentation of antigens. Recently, Cohn (Cohn et al., 2013) and Chatterjee (Chatterjee et al., 2012) also showed that antigen delivery to early endosomes could result in enhanced antigen cross-presentation to CD8+ T cells, although antigens in late endosomes and lysosomes can also be cross-presented. However, these late compartments are far less efficient for cross-presentation of some antigens. This was due to a higher concentration of lysosomal enzymes, which degrade antigens before they can be released into the cytosol. In line with this, inhibiting proteolysis enhances the ability of the late compartments to cross-present accumulated antigens (Chatterjee et al., 2012). In this study, we found that significant fractions of receptor-bound αLOX-1 and αDectin-1 mAbs also localized to the early endosomes, although targeting CD40 was far more efficient at eliciting CD8+ T cell responses than targeting LOX-1 or Dectin-1. Quantitative analysis of the intracellular compartments across nine different donors further revealed that αCD40 mAb localized mainly to the early endosomes, but αLOX-1 and αDectin-1 localized to both the early and late endosomes. This suggested that there could be other critical factors in addition to the roles of early endosomes that can further influence the efficiency of antigen cross-presentation by DCs via MHC class I molecules. Accordingly, we showed that a large fraction of αCD40 mAb remained at the plasma membrane even after a 1-h incubation at 37°C, whereas the majority of both αLOX-1 and αDectin-1 mAbs were internalized into endosomal vesicles. Slow internalization to early endosomes or rapid antigen recycling, as speculated previously (Chatterjee et al., 2012; Cohn et al., 2013), could result in increased antigen stability, followed by prolonged antigen presentation and enhanced CD8+ T cell responses, as we have demonstrated in Fig. 6. In addition to such distinct properties of antibody-bound CD40 versus the lectins (LOX-1 and Dectin-1) described above, one may also consider the possible contribution of αCD40-mediated activation signals in the enhanced antigen cross-presentation after targeting CD40. Previous studies in mice (Bennett et al., 1998; Ridge et al., 1998; Schoenberger et al., 1998) showed that interactions between APCs (including DCs) and helper T cells via CD40–CD40L has been suggested to activate APCs to become fully competent for CD8+ T cell priming. Recent studies have also shown that other DC activators, including toll-like receptor (TLR) ligands and type 1 IFN, can also promote antigen cross-presentation (Datta et al., 2003; Maurer et al., 2002; Schulz et al., 2005; Watts et al., 2007; Wei et al., 2010). However, recombinant fusion proteins of αCD40 and antigens used in this study were not able to induce DCs to secrete cytokines or chemokines or induce surface phenotype maturation. This was in line with the previous observation (Chatterjee et al., 2012) that the enhanced antigen cross-presentation by CD40-targeted human DCs was not due to the CD40-mediated activation signals. Nonetheless, questions regarding the possible contribution of CD40 signaling in enhanced antigen cross-presentation may need to be more carefully studied in the future. Apart from the question on mechanistic insights, we may also need to consider the possible effects of αCD40 bound to CD40 on CD40–CD40L interaction in vivo, although this may not be a critical issue if a proper DC activator is included as an adjuvant in the CD40 targeting vaccines, as most likely it would be (Fig. 7).