Some exogenous antigens, such as heat shock proteins or apoptotic bodies, gain access to the MHC class I processing pathway and initiate CTL responses, a process called cross-priming. To be efficient in vivo, this process requires endocytosis of the antigen by dendritic cells via receptors which remain unidentified. Here, we report that scavenger receptors are the main HSP binding structures on human dendritic cells and identify LOX-1 as one of these molecules. A neutralizing anti-LOX-1 mAb inhibits Hsp70 binding to dendritic cells and Hsp70-induced antigen cross-presentation. In vivo, to target LOX-1 with a tumor antigen using an anti-LOX-1 mAb induces antitumor immunity. Thus, the scavenger receptor LOX-1 is certainly a promising target for cancer immunotherapy.

Outer membrane protein A (OmpA) is a conserved major component of the outer membrane of Enterobacteriaceae. Here, we report that OmpA from Klebsiella pneumoniae (KpOmpA) activates macrophages and dendritic cells (DCs) in a TLR2-dependent way. However, TLR2 does not account for binding of KpOmpA to innate immune cells. KpOmpA binds the scavenger receptors (SRs) LOX-1 and SREC-I, but not other members of the same family. LOX-1 colocalizes and cooperates with TLR2 in triggering cellular responses. The TLR2-activated functional program includes production of the long pentraxin PTX3, a soluble pattern recognition receptor involved in resistance against diverse pathogens. PTX3, in turn, binds KpOmpA but does not affect recognition of this microbial moiety by cellular receptors. KpOmpA-elicited in vivo inflammation is abrogated in TLR2(-/-) mice and significantly reduced in PTX3(-/-) mice. Thus, SR-mediated KpOmpA recognition and TLR2-dependent cellular activation set in motion a nonredundant PTX3-mediated humoral amplification loop of innate immunity.

Arenaviruses such as Lassa virus (LASV) can cause severe hemorrhagic fever in humans. As a major impediment to vaccine development, delayed and weak neutralizing antibody (nAb) responses represent a unifying characteristic of both natural infection and all vaccine candidates tested to date. To investigate the mechanisms underlying arenavirus nAb evasion we engineered several arenavirus envelope-chimeric viruses and glycan-deficient variants thereof. We performed neutralization tests with sera from experimentally infected mice and from LASV-convalescent human patients. NAb response kinetics in mice correlated inversely with the N-linked glycan density in the arenavirus envelope protein’s globular head. Additionally and most intriguingly, infection with fully glycosylated viruses elicited antibodies, which neutralized predominantly their glycan-deficient variants, both in mice and humans. Binding studies with monoclonal antibodies indicated that envelope glycans reduced nAb on-rate, occupancy and thereby counteracted virus neutralization. In infected mice, the envelope glycan shield promoted protracted viral infection by preventing its timely elimination by the ensuing antibody response. Thus, arenavirus envelope glycosylation impairs the protective efficacy rather than the induction of nAbs, and thereby prevents efficient antibody-mediated virus control. This immune evasion mechanism imposes limitations on antibody-based vaccination and convalescent serum therapy.

Increasing evidence suggests that neutrophils may participate in the regulation of adaptive immune responses, and can reach draining lymph nodes and crossprime naive T cells. The aim of this study was to identify the mechanism(s) involved in the migration of neutrophils to the draining lymph nodes. We demonstrate that a subpopulation of human and mouse neutrophils express CCR7. CCR7 is rapidly expressed at the membrane upon stimulation. In vitro, stimulated human neutrophils migrate in response to the CCR7 ligands CCL19 and CCL21. In vivo, injection of complete Freund adjuvant induces a rapid recruitment of neutrophils to the lymph nodes in wild-type mice but not in Ccr7 ؊/؊ mice. Moreover, intradermally injected interleukin-17-and granulocyte-macrophage colonystimulating factor-stimulated neutrophils from wild-type mice, but not from Ccr7 ؊/؊ mice, migrate to the draining lymph nodes. These results identify CCR7 as a chemokine receptor involved in the migration of neutrophils to the lymph nodes. IntroductionPolymorphonuclear neutrophils are the most abundant immune cells in human blood (60% of leukocytes). Every day, 10 11 neutrophils transit through the adult human circulation. 1 They are the first type of leukocytes recruited at the site of infection, where they represent the major infiltrating cells. Their migration and subsequent activation are controlled by chemokines and cytokines such as chemokine (C-X-C motif) ligand 8 (CXCL8) and tumor necrosis factor-␣ (TNF-␣). At the inflammatory site, microorganisms are phagocytosed by neutrophils, destroyed via oxygendependent or oxygen-independent mechanisms, or sequestered in extracellular traps. 1,2 Neutrophils also contain an important arsenal of microbicidal mediators. 3 Neutrophils are short-lived, terminally differentiated cells that have long been considered to be the prototypic innate immune cells and to have a restricted number of properties. 4,5 Nevertheless, recent studies have shown that the biologic properties of neutrophils are more complicated than was previously thought. Neutrophils exhibit characteristic features classically dedicated to professional antigen-presenting cells. 6,7 Under certain circumstances, neutrophils display a dendritic cell (DC)-like phenotype, as evidenced by the expression of the DC markers CD83, CD80, CD86, and major histocompatibility complex II (MHC-II) molecules. Neutrophils may present antigens in an MHC-II-dependent manner and induce the proliferation of antigen-specific T cells. [6][7][8] More recently, we reported that neutrophils cross-prime naive CD8 ϩ T lymphocytes. 9 Finally, neutrophils may also influence the polarization of antigen-specific T-cell responses. [10][11][12] Interestingly, previous studies have reported that, after the capture of antigens in the periphery, neutrophils may migrate to the lymph nodes. [13][14][15] In a murine model of Toxoplasma gondii infection, parasites are transported by neutrophils from the infection site to draining lymph nodes. 15 Similarly, after an injection of ovalbumin (O...

In recent years, unprecedented DNA sequencing capacity provided by next generation sequencing (NGS) has revolutionized genomic research. Combining the Illumina sequencing platform and a scFv library designed to confine diversity to both CDR3, >1.9 × 107 sequences have been generated. This approach allowed for in depth analysis of the library’s diversity, provided sequence information on virtually all scFv during selection for binding to two targets and a global view of these enrichment processes. Using the most frequent heavy chain CDR3 sequences, primers were designed to rescue scFv from the third selection round. Identification, based on sequence frequency, retrieved the most potent scFv and valuable candidates that were missed using classical in vitro screening. Thus, by combining NGS with display technologies, laborious and time consuming upfront screening can be by-passed or complemented and valuable insights into the selection process can be obtained to improve library design and understanding of antibody repertoires.