Epitope length variants balance protective immune responses and viral escape in HIV-1 infection

Cytotoxic T lymphocyte (CTL) and natural killer (NK) cell responses to a single optimal 10-mer epitope (KK10) in the human immunodeficiency virus type-1 (HIV-1) protein p24Gag are associated with enhanced immune control in patients expressing human leukocyte antigen (HLA)-B∗27:05. We find that proteasomal activity generates multiple length variants of KK10 (4–14 amino acids), which bind TAP and HLA-B∗27:05. However, only epitope forms ≥8 amino acids evoke peptide length-specific and cross-reactive CTL responses. Structural analyses reveal that all epitope forms bind HLA-B∗27:05 via a conserved N-terminal motif, and competition experiments show that the truncated epitope forms outcompete immunogenic epitope forms for binding to HLA-B∗27:05. Common viral escape mutations abolish (L136M) or impair (R132K) production of KK10 and longer epitope forms. Peptide length influences how well the inhibitory NK cell receptor KIR3DL1 binds HLA-B∗27:05 peptide complexes and how intraepitope mutations affect this interaction. These results identify a viral escape mechanism from CTL and NK responses based on differential antigen processing and peptide competition

P16-23. Antigen processing influences HIV-specific cytotoxic T lymphocyte immunodominance

International audienc

Le rôle d' "insulin-responsive aminopeptidase" dans la représentation croisée d'antigène

PARIS5-BU Méd.Cochin (751142101) / SudocSudocFranceF

IRAP Endosomes Control Phagosomal Maturation in Dendritic Cells

International audienceDendritic cells (DCs) contribute to the immune surveillance by sampling their environment through phagocytosis and endocytosis. We have previously reported that, rapidly following uptake of extracellular antigen into phagosomes or endosomes in DCs, a specialized population of storage endosomes marked by Rab14 and insulin-regulated aminopeptidase (IRAP) is recruited to the nascent antigen-containing compartment, thereby regulating its maturation and ultimately antigen cross-presentation to CD8 + T lymphocytes. Here, using IRAP –/– DCs, we explored how IRAP modulates phagosome maturation dynamics and cross-presentation. We find that in the absence of IRAP, phagosomes acquire more rapidly late endosomal markers, are more degradative, and show increased microbicidal activity. We also report evidence for a role of vesicle trafficking from the endoplasmic reticulum (ER)–Golgi intermediate compartment to endosomes for the formation or stability of the IRAP compartment. Moreover, we dissect the dual role of IRAP as a trimming peptidase and a critical constituent of endosome stability. Experiments using a protease-dead IRAP mutant and pharmacological IRAP inhibition suggest that IRAP expression but not proteolytic activity is required for the formation of storage endosomes and for DC-typical phagosome maturation, whereas proteolysis is required for fully efficient cross-presentation. These findings identify IRAP as a key factor in cross-presentation, trimming peptides to fit the major histocompatibility complex class-I binding site while preventing their destruction through premature phagosome maturation

Loss of Gadkin Affects Dendritic Cell Migration 'In Vitro'

Migration is crucial for the function of dendritic cells (DCs), which act as outposts of the immune system. Upon detection of pathogens, skin- and mucosa-resident DCs migrate to secondary lymphoid organs where they activate T cells. DC motility relies critically on the actin cytoskeleton, which is regulated by the actin-related protein 2/3 (ARP2/3) complex, a nucleator of branched actin networks. Consequently, loss of ARP2/3 stimulators and upstream Rho family GTPases dramatically impairs DC migration. However, nothing is known yet about the relevance of ARP2/3 inhibitors for DC migration. We previously demonstrated that the AP-1-associated adaptor protein Gadkin inhibits ARP2/3 by sequestering it on intracellular vesicles. Consistent with a role of Gadkin in DC physiology, we here report Gadkin expression in bone marrow-derived DCs and show that its protein level and posttranslational modification are regulated upon LPS-induced DC maturation. DCs derived from Gadkin-deficient mice were normal with regards to differentiation and maturation, but displayed increased actin polymerization. While the actin-dependent processes of macropinocytosis and cell spreading were not affected, loss of Gadkin significantly impaired DC migration in vitro, however, in vivo DC migration was unperturbed suggesting the presence of compensatory mechanisms

Mast cell-mediated inflammation relies on insulin-regulated aminopeptidase controlling cytokine export from the Golgi

International audienceBackground: Upon activation, mast cells rapidly release preformed inflammatory mediators from large cytoplasmic granules via regulated exocytosis. This acute degranulation is followed by a late activation phase involving synthesis and secretion of cytokines, growth factors and other inflammatory molecules via the constitutive pathway that remains ill-defined.Objective: Here we investigate the role for an insulin-responsive vesicle (IRV)-like endosomal compartment, marked by insulin-regulated aminopeptidase (IRAP), in the secretion of TNF-α- and IL-6 in mast cells and macrophages.Methods: Murine ko mouse models (IRAPko and kit-Wsh/sh) were used to study inflammatory disease models and to measure and mechanistically investigate cytokine secretion and degranulation in bone-marrow-derived mast cells in vitro.Results: We show that IRAPko mice are protected from TNF-α-dependent kidney injury and inflammatory arthritis. In the absence of IRAP, TNF-α and IL-6 but not IL-10 fail to be efficiently secreted. Moreover, chemical targeting of IRAP+ endosomes reduced pro-inflammatory cytokine secretion. Mechanistically, impaired TNF-α export from the Golgi and reduced co-localization of VAMP3+ TNF-α transport vesicles with Stx4 was observed in IRAPko mast cells, while VAMP8-dependent exocytosis of secretory granules was facilitated.Conclusion: Our study describes a novel role for IRAP in mast cell-mediated inflammation through the regulation of exocytic trafficking of cytokines

Gadkin is dispensable for spreading of mature BMDCs.

<p>A. Gadkin^-/- BMDCs display no alterations in cell spreading. WT and Gadkin^-/- BMDCs were matured for 24 h with LPS. Subsequently, cells were allowed to spread on fibronectin-coated cover slips for 15 to 240 min. Cells were processed for immunofluorescence and incubated with phalloidin to stain F-actin and thus visualize cell shape. Cell size was quantified at the indicated times post-plating (N = 2 independent experiments for 15 min time point and N = 4 for 30 and 240 min time points).</p

Unaltered <i>in vivo</i> DC migration in absence of Gadkin.

<p>A,B. Skin irritant-induced DC migration to lymph nodes is not altered in the absence of Gadkin. FITC in acetone/dibutylphtalate was painted onto the shaved skin of WT and Gadkin^-/- mice. The percentage of FITC-positive DCs migrating to the draining inguinal lymph nodes was assessed by flow cytometry. A. Representative examples of gating to select FITC-positive DCs for analysis (top). Lower panels show control lymph nodes devoid of FITC-labeled DCs. B. Quantification of the percentage of FITC-positive cells in draining lymph nodes (N = 5 mice per genotype from 2 independent experiments). C,D. Labeled LPS-matured WT and Gadkin^-/- BMDCs reach the draining popliteal lymph nodes with comparable efficiency after injection into foot pads. C. Representative example of gating to select transferred TAMRA- resp. DDAO-positive BMDCs for analysis. D. Quantification of the percentage of TAMRA- and DDAO-positive WT and Gadkin^-/- BMDCs in popliteal lymph nodes (LN) (N = 3 mice per genotype from 2 independent experiments).</p