There is an urgent need for human immunodeficiency virus (HIV) vaccines that induce robust mucosal immunity. Influenza A viruses (both H1N1 and H3N2) were engineered to express simian immunodeficiency virus (SIV) CD8 T-cell epitopes and evaluated following administration to the respiratory tracts of 11 pigtail macaques. Influenza virus was readily detected from respiratory tract secretions, although the infections were asymptomatic. Animals seroconverted to influenza virus and generated CD8 and CD4 T-cell responses to influenza virus proteins. SIV-specific CD8 T-cell responses bearing the mucosal homing marker ␤7 integrin were induced by vaccination of naïve animals. Further, SIV-specific CD8 T-cell responses could be boosted by recombinant influenza virus-SIV vaccination of animals with already-established SIV infection. Sequential vaccination with influenza virus-SIV recombinants of different subtypes (H1N1 followed by H3N2 or vice versa) produced only a limited boost in immunity, probably reflecting T-cell immunity to conserved internal proteins of influenza A virus. SIV challenge of macaques vaccinated with an influenza virus expressing a single SIV CD8 T cell resulted in a large anamnestic recall CD8 T-cell response, but immune escape rapidly ensued and there was no impact on chronic SIV viremia. Although our results suggest that influenza virus-HIV vaccines hold promise for the induction of mucosal immunity to HIV, broader antigen cover will be needed to limit cytotoxic T-lymphocyte escape.

CTL mediate anti-viral immunity via targeted exocytosis of cytolytic granules containing perforin and members of the granzyme (grz) serine protease family. Here, we provide the first analysis of grzA protein expression by murine anti-viral CTL. During the progression of influenza A virus infection, CTL expressed two divergent cytolytic phenotypes: grzA À B 1 and grzA 1 B 1 . CTL lacked grzA expression during the initial rounds of antigen-driven division. High levels of grzA were expressed by influenza-specific CTL early post infection (day 6), particularly in tissues associated with the infected respiratory tract (bronchoalveolar lavage, lung). Following resolution of influenza infection, a small population of memory CTL expressed grzA. Interestingly, individual influenza A virus-derived epitopespecific CTL expressed different levels of grzA. The grzA expression hierarchy was determined to be K b PB1 703 5 D b F2 62 5 K b NS2 114 4D b NP 366 5 D b PA 224 and inversely correlated with CTL magnitude. Therefore following influenza infection, a CTL cytolytic hierarchy was established relating to the different profiles of antigen expression and relative immunodominance. Analysis of CTL grzA expression during influenza virus immunity has enabled a more detailed insight into the cytolytic mechanisms of virus elimination.

Marginal zone (MZ) B cells produce broad-spectrum antibodies that protect against infection early in life. In some instances, antibody production requires MZ B cells to display pathogen antigens bound to major histocompatibility complex class II (MHC II) molecules to T cells. We describe the trogocytic acquisition of these molecules from conventional dendritic cells (cDCs). Complement component 3 (C3) binds to murine and human MHC II on cDCs. MZ B cells recognize C3 with complement receptor 2 (CR2) and trogocytose the MHC II–C3 complexes, which become exposed on their cell surface. The ubiquitin ligase MARCH1 limits the number of MHC II–C3 complexes displayed on cDCs to prevent their elimination through excessive trogocytosis. Capture of C3 by MHC II thus enables the transfer of cDC-like properties to MZ B cells.

No abstract

No abstract

Bone marrow stromal cell-2 (BST-2) has major roles in viral tethering and modulation of interferon production. Here we investigate BST-2 as a receptor for the delivery of antigen to dendritic cells (DCs). We show that BST-2 is expressed by a panel of mouse and human DC subsets, particularly under inflammatory conditions. The outcome of delivering antigen to BST-2 expressed by steady state and activated plasmacytoid DC (pDC) or conventional CD8 + and CD8 − DCs was determined. T-cell responses were measured for both MHC class I (MHCI) and MHC class II (MHCII) antigen presentation pathways in vitro.Delivering antigen via BST-2 was compared with that via receptors DEC205 or Siglec-H. We show that despite a higher antigen load and faster receptor internalisation, when antigen is delivered to steady state or activated pDC via BST-2, BST-2-targeted activated conventional DCs present antigen more efficiently. Relative to DEC205, BST-2 was inferior in its capacity to deliver antigen to the MHCI cross-presentation pathway. In contrast, BST-2 was superior to Siglec-H at initiating either MHCI or MHCII antigen presentation. In summary, BST-2 is a useful receptor to target with antigen, given its broad expression pattern and ability to access both MHCI and MHCII presentation pathways with relative efficiency.Keywords: Antigen-presenting cells r Antigen presentation/processing r Dendritic cells r VaccinationAdditional supporting information may be found in the online version of this article at the publisher's web-site Moffat et al. Eur. J. Immunol. 2013. 43: 595-605 Introduction Bone marrow stromal cell antigen-2 (BST-2; also CD317, PDCA-1, tetherin and HM1.24) is a widely utilised plasmacytoid dendritic cell (pDC) marker due to its high expression by pDCs in steady state mouse model systems [1]. BST-2 is an interesting molecule with unusual topology in that it contains a large extracellular domain, a cytoplasmic N-terminus and a C-terminus that is GPIanchored in the plasma membrane [2]. Major functions for BST-2 include modulation of pDC interferon (IFN)-α production [1,3] and the tethering of newly formed enveloped viral particles to the infected cell surface [4]. BST-2 virus tethering capacity was first described for HIV [4] and is now considered to occur for many, if not all, enveloped viruses [5]. Here we speculate that BST-2 tethering facilitates pDC antigen presentation. A major role for pDCs in efficient antigen presentation, particularly relative to conventional DCs (cDCs) is controversial [6], however, it is generally accepted that pDCs, particularly following activation, have the capacity to present antigen via MHC class I (MHCI) and MHC class II (MHCII) [7]. BST-2 may act as a receptor that confers anti-viral antigen presentation properties to pDCs, given the high expression of BST-2 by this cell type. In support of this hypothesis is the observation that BST-2 can be utilised as a target molecule for antigen delivery in vivo [8][9][10] and can modulate CD8 + T-cell priming to viral antigen [11]. Incorporatin...

i RNA-specific adenosine deaminase (ADAR)-mediated adenosine-to-inosine (A-to-I) editing is a critical arm of the antiviral response. However, mechanistic insights into how A-to-I RNA editing affects viral infection are lacking. We posited that inosine incorporation into RNA facilitates sensing of nonself RNA by innate immune sensors and accordingly investigated the impact of inosine-modified RNA on Toll-like receptor 7 and 8 (TLR7/8) sensing. Inosine incorporation into synthetic single-stranded RNA (ssRNA) potentiated tumor necrosis factor alpha (TNF-␣) or alpha interferon (IFN-␣) production in human peripheral blood mononuclear cells (PBMCs) in a sequence-dependent manner, indicative of TLR7/8 recruitment. The effect of inosine incorporation on TLR7/8 sensing was restricted to immunostimulatory ssRNAs and was not seen with inosine-containing short doublestranded RNAs or with a deoxy-inosine-modified ssRNA. Inosine-mediated increase of self-secondary structure of an ssRNA resulted in potentiated IFN-␣ production in human PBMCs through TLR7 recruitment, as established through the use of a TLR7 antagonist and Tlr7-deficient cells. There was a correlation between hyperediting of influenza A viral ssRNA and its ability to stimulate TNF-␣, independent of 5=-triphosphate residues, and involving Adar-1. Furthermore, A-to-I editing of viral ssRNA directly enhanced mouse Tlr7 sensing, when present in proportions reproducing biologically relevant levels of RNA editing. Thus, we demonstrate for the first time that inosine incorporation into immunostimulatory ssRNA can potentiate TLR7/8 activation. Our results suggest a novel function of A-to-I RNA editing, which is to facilitate TLR7/8 sensing of phagocytosed viral RNA.

Cytokine polyfunctionality has recently emerged as a correlate of effective CTL immunity to viruses and tumors. Although the determinants of polyfunctionality remain unclear, there are published instances of a link between the production of multiple effector molecules and the peptide plus MHC class I molecule avidity of T cell populations. Influenza A virus infection of C57BL/6J mice induces CTL populations specific for multiple viral epitopes, each with varying proportions of monofunctional (IFN-γ+ only) or polyfunctional (IFN-γ+TNF-α+IL-2+) CTLs. In this study, we probe the link between TCR avidity and polyfunctionality for two dominant influenza epitopes (DbNP366 and DbPA224) by sequencing the TCR CDR3β regions of influenza-specific IFN-γ+ versus IFN-γ+IL-2+ cells, or total tetramer+ versus high-avidity CTLs (as defined by the peptide plus MHC class I molecule-TCR dissociation rate). Preferential selection for particular clonotypes was evident for the high-avidity DbPA224-specific set but not for any of the other subsets examined. These data suggest that factors other than TCRβ sequence influence cytokine profiles and demonstrate no link between differential avidity and polyfunctionality.