T84-intestinal epithelial exosomes bear MHC class II/peptide complexes potentiating antigen presentation by dendritic cells: Function of intestinal epithelial exosomes

International audienceBackground and aims: Intestinal epithelial cells release antigen presenting vesicles (exosomes) bearing MHC class II/peptide complexes stimulating specific immune responses in vivo. To further characterize the role of human epithelial exosomes in antigen presentation, their capacity to load antigenic peptides, to bind immune target cells and to induce T cell activation was analyzed in vitro. Methods: The capacity of exosomes derived from the HLA-DR4 expressing, intestinal epithelial cell line T84, to load the HLA-DR4-specific peptide 3H-HSA 64-76 and to activate a HLA-DR4-restricted T cell hybridoma, was tested in the presence or absence of human monocyte-derived dendritic cells (DCs). Interaction of FITC-labeled exosomes with T cells and DCs was analyzed by flow cytometry and confocal microscopy. Results: T84-derived exosomes, enriched in CD9, CD81, CD82 and A33 antigen, were capable of binding specifically HSA 64-76 peptide on HLA-DR4 molecules and of interacting preferentially with DCs. HSA-loaded exosomes were unable to activate the T cell hybridoma directly, but induced a productive T cell activation through DCs. When HSA peptide was bound to exosomal HLA-DR4 molecules instead of in a soluble form, the threshold of peptide presentation by DCs was markedly decreased (x10-3). Conclusions: Exosomes released by intestinal epithelial cells bear exogenous peptides complexed to MHC class II molecules and interact preferentially with DCs, strongly potentiating peptide presentation to T cells. Epithelial exosomes constitute a powerful link between luminal antigens and local immune cells by mediating the transfer of tiny amounts of luminal antigenic information and facilitating immune surveillance at mucosal surfaces

Rôle fonctionnel des exosomes epithéliaux dans l immunité intestinale

PARIS7-Bibliothèque centrale (751132105) / SudocSudocFranceF

Essential Roles and Regulation of the <em>Legionella pneumophila</em> Collagen-Like Adhesin during Biofilm Formation

<div><p>Legionellosis is mostly caused by <em>Legionella pneumophila</em> (<em>Lp</em>) and is defined by a severe respiratory illness with a case fatality rate ranging from 5 to 80%. In a previous study, we showed that a glycosaminoglycan (GAG)-binding adhesin of <em>Lp</em>, named Lcl, is produced during legionellosis and is unique to the <em>L. pneumophila</em> species. Importantly, a mutant depleted in Lcl (<em>Δlpg2644</em>) is impaired in adhesion to GAGs and epithelial cells and in biofilm formation. Here, we examine the molecular function(s) of Lcl and the transcriptional regulation of its encoding gene during different stages of the biofilm development. We show that the collagen repeats and the <em>C</em>-terminal domains of Lcl are crucial for the production of biofilm. We present evidence that Lcl is involved in the early step of surface attachment but also in intercellular interactions. Furthermore, we address the relationship between Lcl gene regulation during biofilm formation and quorum sensing (QS). In a static biofilm assay, we show that Lcl is differentially regulated during growth phases and biofilm formation. Moreover, we show that the transcriptional regulation of <em>lpg2644,</em> mediated by a prototype of QS signaling homoserine lactone (3OC12-HSL), may play a role during the biofilm development. Thus, transcriptional down-regulation of <em>lpg2644</em> may facilitate the dispersion of Lp to reinitiate biofilm colonization on a distal surface.</p> </div

<i>Legionella</i> species, <i>E. coli</i> strains and plasmids used in this study.

<p>Sg: serogroup.</p

Lcl is expressed at the cell surface of <i>L. pneumophila</i> and shows a cluster distribution.

<p>Localization of Lcl on the cell surface. (A) GFP-Lp02 (left) and GFP-Lp02 Δ<i>lpg2644</i> (right) cells were immuno-labelled using anti- Lcl antibodies followed by alexa 555 (red) conjugated secondary antibodies. Micrographs shown are merged images of 0.2 µm confocal z-stacks. Scale bars, 0.5 µm. (B) Surface localization of Lcl (red) as indicated by its co-localization with membrane labelling anti-Lp1 antibodies (top) and FM 4–64 dye (bottom). Images are confocal xy plane following deconvolution. Scale bars, 0.5 µm. (C) The localization of Lcl (red line) was determined by comparing its fluorescence intensity along the white lines from B, with cytoplasmic GFP (green line) and the outer and inner membrane markers anti-Lp1 (blue line, left) and FM 4–64 (blue line, right), respectively.</p

Representative 3D composite images of mixed culture biofilms.

<p>Total bacteria were labelled with SYTO62 and one strain of each 1∶1 mixture was labelled with GFP. Confocal scanning laser microscopy (CSLM) micrographs show the distribution of <i>L. pneumophila</i> within static biofilms using (A) GFP- Lp02 <i>Δlpg2644/</i>Lp02, (B) GFP-Lp02/Lp02, (C) GFP- Lp02 <i>Δlpg2644/</i>Lp02 <i>Δlpg2644</i>, (D) GFP- Lp02/Lp02 <i>Δlpg2644.</i> (Magnification 100 X).</p

Oligonucleotides used for PCR, qRT-PCR, sequencing and cloning.

<p>Oligonucleotides used for PCR, qRT-PCR, sequencing and cloning.</p

Role of Lcl during the initial attachment step of biofilm formation.

<p>The attachment of Lp02, Lp02 pBH6119, Lp02 <i>Δlpg2644</i>, and Lp02 <i>Δlpg2644</i> p<i>lpg2644</i> to the wells of a microtiter plate was measured by (A) qPCR and (B and C) fluorescence microscopy. (A) The binding of Lp02 <i>Δlpg2644</i> was significantly reduced compared to the other strains. Values are means+/−s.d. from three individual experiments. * Two-tailed Student’s t-test P-value <0.001 Lp02 <i>Δlpg2644</i> vs. all other strains. (B) Comparative analysis of the attachment of SYTO 62 stained <i>Legionella</i> strains attached to glass cover slips. (C) Among 10 representative microscopy fields, the average attachment of Lp02, Lp02 pBH6119 and complemented mutant were similar whereas Lp02 <i>Δlpg2644</i> showed a marked decrease attachment to surface. * Two-tailed Student’s t-test P-value <0.001.</p

Adherence and persistence of <i>L. pneumophila</i> in a continuous-flow chamber system.

<p>Fluorescence micrographs of GFP-expressing <i>L.pneumophila</i> strains Lp02, Lp02 <i>Δlpg2644</i> and chromosomal complemented mutant (Lp02 <i>Δlpg2644/clpg2644</i>) in a continuous-flow chamber system operated at 5.5 µl/mn with BYE medium (magnification 100 X). During the initial attachment (0 h), Lp02 and Lp02 <i>Δlpg2644/clpg2644</i> strains formed a monolayer on abiotic surface whereas Lp02 <i>Δlpg2644</i> was unable to efficiently attach to the flow chambers and detached in a flow dependent manner (2 h to 48 h). Data are representative of three independent replicates. Scale bar, 5 µm.</p

Transcriptional regulation of <i>lpg2644</i> and synthesis of Lcl in broth cultures, biofilm and in presence of 3OC₁₂-HSL homoserine lactone.

<p>Amounts of <i>lpg2644</i> transcripts and synthesis of Lcl were respectively measured by (A) qRT-PCR and (B) anti-Lcl immunoblot analysis of cell pellet (c) supernatant (s) and sessile cells/matrix (sm) fractions in Lp02 harvested from exponential (E), post-exponential (PE), mid-stationary (MS), late stationary (LS) broth cultures and biofilms (2 and 6 days old). Quantitative RT-PCR values are means+/−s.d. from three individual experiments. # two-tailed Student’s t-test P-value ≤0.01 versus MS phase, * two-tailed Student’s t-test P-value ≤0.001 versus MS phase. (C) Effect of 3OC12-HSL homoserine lactone on <i>lpg2644</i> transcription and (D) Lcl synthesis. Lp02 was grown in absence or in presence of 3OC12-HSL (10 µM) for 24 h. Transcriptional level of <i>lpg2644</i> was determined by qRT-PCR analysis. Data are representative of 3 independent experiments.* Student’s t-test P-value ≤0,001 versus untreated Lp02. (D) Synthesis of Lcl was estimated by anti-Lcl immunoblot of cell pellets (c) and supernatant fractions (s) of Lp02 and Lp02 <i>Δlpg2644</i> p<i>lpg2644</i>. The positions of the molecular size markers are indicated on the left of the blot in kDa.</p