21:T43a,Antigen presentation to T cells in major histocompatibility complex class II (MHC class II) requires the conversion of early endo/phagosomes into lysosomes by a process called maturation. Maturation is driven by the phosphoinositide kinase PIKfyve. Blocking PIKfyve activity by small molecule inhibitors caused a delay in the conversion of phagosomes into lysosomes and in phagosomal acidification, whereas production of reactive oxygen species (ROS) increased. Elevated ROS resulted in reduced activity of cathepsin S and B, but not X, causing a proteolytic defect of MHC class II chaperone invariant chain Ii processing. We developed a novel universal MHC class II presentation assay based on a bio-orthogonal "clickable" antigen and showed that MHC class II presentation was disrupted by the inhibition of PIKfyve, which in turn resulted in reduced activation of CD4⁺ T cells. Our results demonstrate a key role of PIKfyve in the processing and presentation of antigens, which should be taken into consideration when targeting PIKfyve in autoimmune disease and cancer.22:T475,In dendritic cells, the NADPH oxidase 2 complex (NOX2) is recruited to the phagosomal membrane during antigen uptake. NOX2 produces reactive oxygen species (ROS) in the lumen of the phagosome that kill ingested pathogens, delay antigen breakdown and alter the peptide repertoire for presentation to T cells. How the integral membrane component of NOX2, cytochrome b₅₅₈ (which comprises CYBB and CYBA), traffics to phagosomes is incompletely understood. In this study, we show in dendritic cells derived from human blood-isolated monocytes that cytochrome b₅₅₈ is initially recruited to the phagosome from the plasma membrane during phagosome formation. Cytochrome b₅₅₈ also traffics from a lysosomal pool to phagosomes and this is required to replenish oxidatively damaged NOX2. We identified syntaxin-7, SNAP23 and VAMP8 as the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins mediating this process. Our data describe a key mechanism of how dendritic cells sustain ROS production after antigen uptake that is required to initiate T cell responses.

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