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Unidirectional transfer of microRNA-loaded exosomes from T cells to antigen-presenting cells.

Mittelbrunn M, Gutiérrez-Vázquez C, Villarroya-Beltri C, González S, Sánchez-Cabo F, González MÁ, Bernad A, Sánchez-Madrid F - Nat Commun (2011)

Bottom Line: We investigate whether miRNAs are exchanged during cognate immune interactions, and demonstrate the existence of antigen-driven unidirectional transfer of miRNAs from the T cell to the APC, mediated by the delivery of CD63+ exosomes on immune synapse formation.Moreover, miRNAs transferred during immune synapsis are able to modulate gene expression in recipient cells.Thus, our results support a mechanism of cellular communication involving antigen-dependent, unidirectional intercellular transfer of miRNAs by exosomes during immune synapsis.

View Article: PubMed Central - PubMed

Affiliation: Centro Nacional de Investigaciones Cardiovasculares, Melchor Fernández Almagro, 3. 28029, Madrid, Spain.

ABSTRACT
The immune synapse is an exquisitely evolved means of communication between T cells and antigen-presenting cells (APCs) during antigen recognition. Recent evidence points to the transfer of RNA via exosomes as a novel mode of intercellular communication. Here we show that exosomes of T, B and dendritic immune cells contain microRNA (miRNA) repertoires that differ from those of their parent cells. We investigate whether miRNAs are exchanged during cognate immune interactions, and demonstrate the existence of antigen-driven unidirectional transfer of miRNAs from the T cell to the APC, mediated by the delivery of CD63+ exosomes on immune synapse formation. Inhibition of exosome production by targeting neutral sphingomyelinase-2 impairs transfer of miRNAs to APCs. Moreover, miRNAs transferred during immune synapsis are able to modulate gene expression in recipient cells. Thus, our results support a mechanism of cellular communication involving antigen-dependent, unidirectional intercellular transfer of miRNAs by exosomes during immune synapsis.

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Uptake of CD63-GFP exosomes by immune cells.(a) Untransfected J77 T cells and J77 T cells stably expressing CD63-GFP (J77-CD63-GFP cells) were cultured in exosome-depleted medium for 24 h and exosomes were purified from supernatants by ultracentrifugation. Exosomes were labelled with anti-CD63-phycoerythrin and analysed by flow cytometry. (b) Uptake of CD63-GFP exosomes by T cells and B cells (recipient cells). Untransfected cells were incubated with CD63-GFP exosomes for 16 h and analysed by flow cytometry. Data represent the percentage of GFP-positive cells (±s.e.m.) of three independent experiments. Open bars, no exosomes; striped bars, Raji exosomes; filled bars, J77 exosomes. (c) Confocal microscopy detection of CD63-GFP (green) on the surface of recipient cells (Raji) after incubation with J77-CD63-GFP exosomes. Cell membranes were stained for the cell-surface molecule CD45 (red) and nuclei were stained with HOESCHT (blue). Images show maximal projections of confocal images (projection), one representative confocal section (section) and the DIC images. Scale bar, 10 ìm.
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f2: Uptake of CD63-GFP exosomes by immune cells.(a) Untransfected J77 T cells and J77 T cells stably expressing CD63-GFP (J77-CD63-GFP cells) were cultured in exosome-depleted medium for 24 h and exosomes were purified from supernatants by ultracentrifugation. Exosomes were labelled with anti-CD63-phycoerythrin and analysed by flow cytometry. (b) Uptake of CD63-GFP exosomes by T cells and B cells (recipient cells). Untransfected cells were incubated with CD63-GFP exosomes for 16 h and analysed by flow cytometry. Data represent the percentage of GFP-positive cells (±s.e.m.) of three independent experiments. Open bars, no exosomes; striped bars, Raji exosomes; filled bars, J77 exosomes. (c) Confocal microscopy detection of CD63-GFP (green) on the surface of recipient cells (Raji) after incubation with J77-CD63-GFP exosomes. Cell membranes were stained for the cell-surface molecule CD45 (red) and nuclei were stained with HOESCHT (blue). Images show maximal projections of confocal images (projection), one representative confocal section (section) and the DIC images. Scale bar, 10 ìm.

Mentions: To analyse the capacity of cells to take up immune exosomes, we generated Raji B and J77 T cells stably expressing the exosomal marker CD63 fused to green fluorescent protein (GFP). The tetraspanin CD63 is very abundant in exosomes, and inside cells localizes mainly to MVBs and lysosomes, with only a small pool present at the plasma membrane30. Cytometry and western blot analyses confirmed the presence of CD63-GFP in exosomes released by these cells (Fig. 2a and Supplementary Fig. S2). The purified CD63-GFP exosomes were then incubated with non-transfected J77 cells or Raji cells (recipient cells) for 16 h. Flow cytometry analysis revealed that both J77 T and Raji B cells have the capacity to take up immune exosomes (Fig. 2b). It is important to highlight that Raji B cells take up T cell-derived exosomes to a greater extent than their own exosomes and vice versa. Moreover, CD63-GFP was detected at the surface of recipient cells by confocal microscopy (Fig. 2c), suggesting that exosomes are not internalized but remain attached to the recipient plasma membrane.


Unidirectional transfer of microRNA-loaded exosomes from T cells to antigen-presenting cells.

Mittelbrunn M, Gutiérrez-Vázquez C, Villarroya-Beltri C, González S, Sánchez-Cabo F, González MÁ, Bernad A, Sánchez-Madrid F - Nat Commun (2011)

Uptake of CD63-GFP exosomes by immune cells.(a) Untransfected J77 T cells and J77 T cells stably expressing CD63-GFP (J77-CD63-GFP cells) were cultured in exosome-depleted medium for 24 h and exosomes were purified from supernatants by ultracentrifugation. Exosomes were labelled with anti-CD63-phycoerythrin and analysed by flow cytometry. (b) Uptake of CD63-GFP exosomes by T cells and B cells (recipient cells). Untransfected cells were incubated with CD63-GFP exosomes for 16 h and analysed by flow cytometry. Data represent the percentage of GFP-positive cells (±s.e.m.) of three independent experiments. Open bars, no exosomes; striped bars, Raji exosomes; filled bars, J77 exosomes. (c) Confocal microscopy detection of CD63-GFP (green) on the surface of recipient cells (Raji) after incubation with J77-CD63-GFP exosomes. Cell membranes were stained for the cell-surface molecule CD45 (red) and nuclei were stained with HOESCHT (blue). Images show maximal projections of confocal images (projection), one representative confocal section (section) and the DIC images. Scale bar, 10 ìm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC3104548&req=5

f2: Uptake of CD63-GFP exosomes by immune cells.(a) Untransfected J77 T cells and J77 T cells stably expressing CD63-GFP (J77-CD63-GFP cells) were cultured in exosome-depleted medium for 24 h and exosomes were purified from supernatants by ultracentrifugation. Exosomes were labelled with anti-CD63-phycoerythrin and analysed by flow cytometry. (b) Uptake of CD63-GFP exosomes by T cells and B cells (recipient cells). Untransfected cells were incubated with CD63-GFP exosomes for 16 h and analysed by flow cytometry. Data represent the percentage of GFP-positive cells (±s.e.m.) of three independent experiments. Open bars, no exosomes; striped bars, Raji exosomes; filled bars, J77 exosomes. (c) Confocal microscopy detection of CD63-GFP (green) on the surface of recipient cells (Raji) after incubation with J77-CD63-GFP exosomes. Cell membranes were stained for the cell-surface molecule CD45 (red) and nuclei were stained with HOESCHT (blue). Images show maximal projections of confocal images (projection), one representative confocal section (section) and the DIC images. Scale bar, 10 ìm.
Mentions: To analyse the capacity of cells to take up immune exosomes, we generated Raji B and J77 T cells stably expressing the exosomal marker CD63 fused to green fluorescent protein (GFP). The tetraspanin CD63 is very abundant in exosomes, and inside cells localizes mainly to MVBs and lysosomes, with only a small pool present at the plasma membrane30. Cytometry and western blot analyses confirmed the presence of CD63-GFP in exosomes released by these cells (Fig. 2a and Supplementary Fig. S2). The purified CD63-GFP exosomes were then incubated with non-transfected J77 cells or Raji cells (recipient cells) for 16 h. Flow cytometry analysis revealed that both J77 T and Raji B cells have the capacity to take up immune exosomes (Fig. 2b). It is important to highlight that Raji B cells take up T cell-derived exosomes to a greater extent than their own exosomes and vice versa. Moreover, CD63-GFP was detected at the surface of recipient cells by confocal microscopy (Fig. 2c), suggesting that exosomes are not internalized but remain attached to the recipient plasma membrane.

Bottom Line: We investigate whether miRNAs are exchanged during cognate immune interactions, and demonstrate the existence of antigen-driven unidirectional transfer of miRNAs from the T cell to the APC, mediated by the delivery of CD63+ exosomes on immune synapse formation.Moreover, miRNAs transferred during immune synapsis are able to modulate gene expression in recipient cells.Thus, our results support a mechanism of cellular communication involving antigen-dependent, unidirectional intercellular transfer of miRNAs by exosomes during immune synapsis.

View Article: PubMed Central - PubMed

Affiliation: Centro Nacional de Investigaciones Cardiovasculares, Melchor Fernández Almagro, 3. 28029, Madrid, Spain.

ABSTRACT
The immune synapse is an exquisitely evolved means of communication between T cells and antigen-presenting cells (APCs) during antigen recognition. Recent evidence points to the transfer of RNA via exosomes as a novel mode of intercellular communication. Here we show that exosomes of T, B and dendritic immune cells contain microRNA (miRNA) repertoires that differ from those of their parent cells. We investigate whether miRNAs are exchanged during cognate immune interactions, and demonstrate the existence of antigen-driven unidirectional transfer of miRNAs from the T cell to the APC, mediated by the delivery of CD63+ exosomes on immune synapse formation. Inhibition of exosome production by targeting neutral sphingomyelinase-2 impairs transfer of miRNAs to APCs. Moreover, miRNAs transferred during immune synapsis are able to modulate gene expression in recipient cells. Thus, our results support a mechanism of cellular communication involving antigen-dependent, unidirectional intercellular transfer of miRNAs by exosomes during immune synapsis.

Show MeSH
Related in: MedlinePlus