Innate signaling regulates cross‐priming at the level of DC licensing and not antigen presentation

Abstract

Innate stimuli, such as TLR ligands, are known to greatly facilitate cross-priming. Currently it is unclear whether innate stimuli enhance cross-priming at the level of crosspresentation or at the level of T-cell priming. In this study, we addressed this question by measuring cross-presentation as well as cross-priming by virus-like particles (VLP) displaying peptide p33 derived of lymphocytic choriomeningitis virus. Innate stimuli were varied by either packaging different TLR ligands into virus-like particles or using mice deficient in two key molecules of TLR-signaling, namely the adaptor molecule MyD88 as well as IFN-a/b receptor. While efficient cross-presentation occurred despite strongly reduced activation of DC in the absence of TLR ligand-mediated signals, T-cell priming was abolished. Thus, innate stimuli regulate cross-priming at the level of DC licensing for T-cell activation and not antigen presentation.Key words: Antigen presentation/processing . Cross-presentation/priming . DC Introduction CD8 1 T cells play an important role in defense against infectious agents. Activated CD8 1 T cells differentiate into CTL that kill infected cells. CTL recognize endogenously synthesized antigenic peptides complexed with MHC class I molecules on the surface of APC. Since not all pathogens directly infect APC, it is necessary that APC are able to internalize exogenous antigens and present them on MHC class I molecules for CD8 1 T-cell recognition, a process known as cross-presentation [1][2][3]. Such cross-presented antigens may make an important contribution to the induction of CTL responses, in particular for non-replicating antigens, such as virus-like particles (VLP) [4]. The main cell types involved in cross-presentation are professional APC, such as DC [5,6] as well as macrophages [7,8] but not B cells [9].The primary function of immature DC is to sample foreign antigens for T-cell and perhaps B-cell priming. To this end, DC either sit at strategic positions within lymphoid organs or act as sentinels in peripheral tissues. In any event, for optimal T-cell priming, DC need pathogen-derived signals in order to mature [10]. In a first step, DC are activated by various pathogen-derived molecules (known as PAMP), including ligands for TLR, lectins, intracellular nucleotide-binding oligomerization domain receptors or retinoic acid induced gene [11][12][13][14]. So far 11 TLR have been identified in mice [15]. TLR recognize invariant viral or bacterial structures. A number of receptors are specialized in recognizing nucleic acids, such as dsRNA (TLR3), ssRNA (TLR7 and TLR8), and DNA rich in non-methlylated CG motifs (CpG, TLR9). The interaction of microbial ligands with innate immune system receptors initiates the maturation of DC. Stimulation of DC with such innate stimuli results in licensed DC. Such DC express intermediate levels of cell surface costimulatory molecules. Endogenous mediators, such as TNF family members, chemokines, or other cytokines facilitate the full differentiation of DC, expressing high leve...

Innate Immunity Mediates Follicular Transport of Particulate but Not Soluble Protein Antigen

“…This resulted in VLPs devoid of RNA (Qb-VLPPG) (29). MyD88-dependent cytotoxic T cell priming with Qb-VLPs has previously been shown to be abrogated with Qb-VLPPG indicating that innate stimuli were removed and only the protein component of Qb was present (29). In addition, Qb-VLPPG was deposited on FDCs as efficiently as Qb-VLP in naive mice (data not shown).…”

“…To produce Qb-VLPs without packaged RNA, which serves as a stimulator of innate immunity, VLPs were disassembled and reassembled in the presence of the polyanionic molecule polyglutamic acid. This resulted in VLPs devoid of RNA (Qb-VLPPG) (29). MyD88-dependent cytotoxic T cell priming with Qb-VLPs has previously been shown to be abrogated with Qb-VLPPG indicating that innate stimuli were removed and only the protein component of Qb was present (29).…”

“…To produce Qb devoid of packaged innate stimuli, VLPs were reassembled in the presence of the polyanionic molecule polyglutamic acid. The resulting VLPs were devoid of RNA (Qb-VLPPG) but exhibited the shape and size of the original Qb-VLPs as demonstrated by electron microscopy (29). Qb-VLPs and Qb-dimer were conjugated to Alexa 488 or Alexa 647 dyes using Alexa Fluor Protein Labeling Kits (Invitrogen) according to the manufacturer's instructions.…”

Cyclophosphamide Synergizes with Type I Interferons through Systemic Dendritic Cell Reactivation and Induction of Immunogenic Tumor Apoptosis

“…Apoptotic cell uptake by DC and CD8 cross-priming are strongly enhanced by IFN-I CD8a þ DCs are specialized for cross-presentation of dead cell-derived Ag; however, appropriate activation signals are needed to license DC for cross-priming (30,31). We asked whether IFN-I could act as such signal-stimulating DC for CD8 T-cell cross-priming against MAFO-EG7-derived Ag.…”

Universal vaccine against influenza virus: Linking TLR signaling to anti‐viral protection

“…Successful RNA digestion was verified by the disappearance of the fluorescent EtBr signal on the gel, which colocalizes with the coomassie brilliant blue band in RNA-containing VLPs. The procedure has previously been shown by electron miscroscopy to not impact on the structure of the VLPs [38]. …”