No abstract

The efficiency of antigen (Ag) processing by dendritic cells (DCs) is vital for the strength of the ensuing T-cell responses. Previously, we and others have shown that in comparison to protein vaccines, vaccination with synthetic long peptides (SLPs) has shown more promising (pre-)clinical results. Here, we studied the unknown mechanisms underlying the observed vaccine efficacy of SLPs. We report an in vitro processing analysis of SLPs for MHC class I and class II presentation by murine DCs and human monocyte-derived DCs. Compared to protein, SLPs were rapidly and much more efficiently processed by DCs, resulting in an increased presentation to CD4 + and CD8 + T cells. The mechanism of access to MHC class I loading appeared to differ between the two forms of Ag. Whereas whole soluble protein Ag ended up largely in endolysosomes, SLPs were detected very rapidly outside the endolysosomes after internalization by DCs, followed by proteasomeand transporter associated with Ag processing-dependent MHC class I presentation. Compared to the slower processing route taken by whole protein Ags, our results indicate that the efficient internalization of SLPs, accomplished by DCs but not by B or T cells and characterized by a different and faster intracellular routing, leads to enhanced CD8 + T-cell activation.Keywords: Antigen presentation/processing r Cellular immunology r CD8 + T cells r Dendritic cells Additional supporting information may be found in the online version of this article at the publisher's web-site lower efficiency compared to SLP-loaded DCs (Fig. 1B). Prestimulation of DCs with the TLR4 ligand LPS had no effect on the MHC class I presentation of OVA-protein but improved Ag presentation of SSP-OVA 8aa (data not shown) and long peptide Ag (Fig. 1C). HLA-B7-restricted presentation by human monocyte-derived DCs (MoDCs) of HIV-derived protein and SLPs was also studied. We were unable to detect cytokine production by CD8 + T cells cocultured with GAG-protein-loaded DCs. In contrast, SLP-GAG 22aa induced significant CD8 + T-cell activation (see Fig. 2 and below).Together, these data show that cross-presentation of SLPs is superior to that of proteins as examined with both mouse and human DCs. Rapid Ag presentation of SLPs by murine and human DCsThe efficiency of SLP-processing was assessed by studying the time required for DCs to present Ag on MHC class I (H2-K b )molecules. Murine DCs were incubated with a single concentration of SLPs, synthetic short peptides (SSPs) or protein for the indicated time periods. The minimal peptide, SSPs, was rapidly presented to CD8 + T cells resulting in strong activation already after 1 h. DCs loaded with SLP also activated CD8 + T cells 1 h after Ag loading but with lower potency. We excluded that SLPs were cleaved extracellularly, processed, and loaded on MHC class I and II molecules by incubating paraformaldehyde (PFA) fixed cells with the peptide Ag and observed no cross-presentation (data not shown). DCs loaded with 10 μM OVA-protein failed to induce significant CD8 + T-ce...

The staphylococcal bi-component leukocidins Panton-Valentine leukocidin (PVL) and γ-haemolysin CB (HlgCB) target human phagocytes. Binding of the toxins' S-components to human complement C5a receptor 1 (C5aR1) contributes to cellular tropism and human specificity of PVL and HlgCB. To investigate the role of both leukocidins during infection, we developed a human C5aR1 knock-in (hC5aR1) mouse model. HlgCB, but unexpectedly not PVL, contributed to increased bacterial loads in tissues of hC5aR1 mice. Compared to humans, murine hC5aR1 neutrophils showed a reduced sensitivity to PVL, which was mediated by the toxin's F-component LukF-PV. By performing a genome-wide CRISPR-Cas9 screen, we identified CD45 as a receptor for LukF-PV. The human-specific interaction between LukF-PV and CD45 provides a molecular explanation for resistance of hC5aR1 mouse neutrophils to PVL and probably contributes to the lack of a PVL-mediated phenotype during infection in these mice. This study demonstrates an unsuspected role of the F-component in driving the sensitivity of human phagocytes to PVL.

Neutrophils contain high levels of chymotrypsin-like serine proteases (NSPs) within their azurophilic granules that have a multitude of functions within the immune system. In response, the pathogen Staphylococcus aureus has evolved three potent inhibitors (Eap, EapH1, and EapH2) that protect the bacterium as well as several of its secreted virulence factors from the degradative action of NSPs. We previously showed that these so-called EAP domain proteins represent a novel class of NSP inhibitors characterized by a non-covalent inhibitory mechanism and a distinct target specificity profile. Based upon high levels of structural homology amongst the EAP proteins and the NSPs, as well as supporting biochemical data, we predicted that the inhibited complex would be similar for all EAP/NSP pairs. However, we present here evidence that EapH1 and EapH2 bind the canonical NSP, Neutrophil Elastase (NE), in distinct orientations. We discovered that alteration of EapH1 residues at the EapH1/NE interface caused a dramatic loss of affinity and inhibition of NE, while mutation of equivalent positions in EapH2 had no effect on NE binding or inhibition. Surprisingly, mutation of residues in an altogether different region of EapH2 severely impacted both the NE binding and inhibitory properties of EapH2. Even though EapH1 and EapH2 bind and inhibit NE and a second NSP, Cathepsin G, equally well, neither of these proteins interacts with the structurally related, but non-proteolytic granule protein, azurocidin. These studies expand our understanding of EAP/NSP interactions and suggest that members of this immune evasion protein family are capable of diverse target recognition modes.

Staphylococcus aureus is a well-known colonizer of the human skin and nose, but also a human pathogen that causes a wide spectrum of diseases. It is well established that S. aureus secretes an arsenal of virulence factors that have evolved to circumvent the human immune system. A major group of S. aureus virulence factors is the bi-component β-barrel pore-forming toxins, also known as leukocidins. These poreforming toxins target specific cells of the innate and adaptive immune system by interacting with specific receptors expressed on the cell membrane. Even though still heavily debated, clinical and epidemiological studies suggest the involvement of one of the bi-component toxin, Panton-Valentine Leukocidin (PVL), as an important factor contributing to the epidemic spread and increased virulence of CA-MRSA strains. However, the host-and cell-specificity of PVL and other leukocidins, and the lack of adequate in vivo models, fuels the controversy and impairs the appropriate assessment of their role in S. aureus pathophysiology. Currently, the mechanisms of pore-formation and the contribution of PVL and other leukocidins to S. aureus pathophysiology are incompletely understood. This review summarizes our current understanding of leukocidin pore-formation, knowledge gaps, and highlights recent findings identifying novel host-factors involved in the toxin-host interface. As a result, this review furthers emphasizes the complexity behind S. aureus leukocidin cytotoxicity and the challenges associated in the quest to study and understand these major virulence factors.