TLR ligands differentially affect uptake and presentation of cellular antigens

Abstract

IntroductionDendritic cells (DCs) are recognized as the most efficient antigenpresenting cells that initiate antigen-specific immune responses. They reside in an immature state in peripheral tissues, where they sense their environment and take up antigens. Upon activation through inflammatory mediators or pathogen-derived products, they change their expression pattern of various cell-surface molecules and secreted mediators like cytokines and chemokines. These alterations enable DCs to migrate to lymphoid tissues, where they present antigens and induce differentiation of both naive CD4 ϩ and CD8 ϩ T lymphocytes. These functional changes are accompanied with a lower capacity to take up soluble and cellular antigens by phagocytosis or pinocytosis by mature DCs and an increased ability to stimulate T-cell responses. 1,2 Usually, antigens from exogenous sources are taken up by DCs, become processed, and are presented to CD4 ϩ T lymphocytes on MHC class II molecules while intracellular-derived peptides are loaded onto MHC class I molecules where they stimulate antigenspecific CD8 ϩ T lymphocytes. 3 However, DCs are able to present antigens taken up from their environment on MHC class I molecules in a process called cross-presentation. This mechanism enables these cells to raise immune responses against tumor cells or pathogens like viruses that do not infect themselves. [4][5][6][7][8] DCs employ various molecules to sense their environment for pathogen-associated molecular patterns (PAMPs), microbial elementary components that are not or only minimal subjected to host adaptation. The most prominent of these receptors resembles the Toll-like receptor (TLR) family. TLR family members recognize microbial components such as lipopolysaccharide (LPS), flagellin, lipopeptides, or nucleic acids, and therefore initiate specific signaling pathways that lead to distinct immune responses. 9 In our study, we analyzed the effects of TLR-mediated DC activation on uptake of cellular antigens and their crosspresentation on MHC class I molecules. We show that TLR3 or TLR4 but not TLR2 or TLR7/8 matured DCs are impaired in the phagocytosis of cellular material and subsequent cross-presentation of antigens on MHC class I molecules. Materials and methodsApproval was obtained from the ethics committee of the University of Tübingen for these studies. In our study we used buffy coats provided by the local blood bank. Informed consent was provided according to the Declaration of Helsinki. CellsHEK-293 cells (embryonal kidney; DSMZ, Braunschweig, Germany) were cultured in RP10 medium (RPMI 1640 with glutamax I, supplemented with 10% inactivated fetal calf serum [FCS] and antibiotics; Invitrogen, Karlsruhe, Germany). Generation of DCsDCs were generated from peripheral blood monocytes by magnetic cell sorting as described previously. 10 In brief, peripheral blood mononuclear cells were isolated by Ficoll/Paque (Biochrom, Berlin, Germany) density gradient centrifugation of blood obtained from buffy coats of healthy volunteers from the blood ba...

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