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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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Related in: MedlinePlus

Ag recognition induces transfer of exosomes from T cell to APC.(a) Left: J77-CD63-GFP donor cells were conjugated with SEE-primed or unprimed Raji cells (recipient, in blue). Right: Raji-CD63-GFP donor cells (±SEE) were conjugated with blue-labeled J77 recipients. After 16 h, cocultures were analysed by flow cytometry. Bar chart shows the percentage±s.e.m. of positive recipient cells (n=9, P<0.001, Student's t-test). (b) J77 cells transfected with CD69-GFP or GFP were conjugated with SEE-primed Raji cells (blue) and analysed as in a. (c) FACS analysis of the percentage of GFP-positive Raji cells after incubation with J77-CD63-GFP cells in contact with coculture as in a (control), in the presence of inhibitors of actin (latrunculin-A, LatA or cytochalasin-D, CytD) or tubulin (nocodazol, NCD), or after incubation with separation of donors and recipients by a 0.4 μm pore-size transwell membrane (Twell). T cells in transwells were activated with CD3+CD28 Abs and Raji cells were loaded with SEE. (d) J77-CD63-GFP donor cells were conjugated with a 1:1 mix of two B cell lines: Raji cells (black) and BLS-1 cells (blue) and analysed as in a. Bar chart shows percentages±s.e.m. of positive recipient cells (n=6, P<0.005, Mann–Whitney test). Maximal projections of confocal images and the DIC of a triple conjugated formed by J77-CD63-GFP (green), Raji cells (black asterisk) and BLS-1 (CMAC stained and blue asterisk), stained for CD3 (red) are shown, scale bar shown in e. (e) Confocal analysis of Raji cells that acquired CD63-GFP directly from J77-CD63-GFP after IS formation (IS-dependent transfer) or after external administration of CD63-GFP exosomes isolated from J77-CD63GFP supernatants (non-synaptic uptake). Cells were stained for MHC-II (red). Images show maximal projections of confocal images, one representative confocal section, and the DIC images. Plots show cell perimeter fluorescence intensity profiles of the green and the red signals. ARU: arbitrary relative units. Scale bar, 10 ìm. (f) Raji cells (±SEE) were treated as in e. FACS analysis of the effect of trypsin on the CD63-GFP content of Raji cells that acquired exosomes by IS-dependent transfer or non-synaptic uptake. White bars, control; grey bars, 10 min trypsin; black bars, 10 min+10 min trypsin. Bar chart shows percentages±s.e.m. of positive cells (n=3).
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f4: Ag recognition induces transfer of exosomes from T cell to APC.(a) Left: J77-CD63-GFP donor cells were conjugated with SEE-primed or unprimed Raji cells (recipient, in blue). Right: Raji-CD63-GFP donor cells (±SEE) were conjugated with blue-labeled J77 recipients. After 16 h, cocultures were analysed by flow cytometry. Bar chart shows the percentage±s.e.m. of positive recipient cells (n=9, P<0.001, Student's t-test). (b) J77 cells transfected with CD69-GFP or GFP were conjugated with SEE-primed Raji cells (blue) and analysed as in a. (c) FACS analysis of the percentage of GFP-positive Raji cells after incubation with J77-CD63-GFP cells in contact with coculture as in a (control), in the presence of inhibitors of actin (latrunculin-A, LatA or cytochalasin-D, CytD) or tubulin (nocodazol, NCD), or after incubation with separation of donors and recipients by a 0.4 μm pore-size transwell membrane (Twell). T cells in transwells were activated with CD3+CD28 Abs and Raji cells were loaded with SEE. (d) J77-CD63-GFP donor cells were conjugated with a 1:1 mix of two B cell lines: Raji cells (black) and BLS-1 cells (blue) and analysed as in a. Bar chart shows percentages±s.e.m. of positive recipient cells (n=6, P<0.005, Mann–Whitney test). Maximal projections of confocal images and the DIC of a triple conjugated formed by J77-CD63-GFP (green), Raji cells (black asterisk) and BLS-1 (CMAC stained and blue asterisk), stained for CD3 (red) are shown, scale bar shown in e. (e) Confocal analysis of Raji cells that acquired CD63-GFP directly from J77-CD63-GFP after IS formation (IS-dependent transfer) or after external administration of CD63-GFP exosomes isolated from J77-CD63GFP supernatants (non-synaptic uptake). Cells were stained for MHC-II (red). Images show maximal projections of confocal images, one representative confocal section, and the DIC images. Plots show cell perimeter fluorescence intensity profiles of the green and the red signals. ARU: arbitrary relative units. Scale bar, 10 ìm. (f) Raji cells (±SEE) were treated as in e. FACS analysis of the effect of trypsin on the CD63-GFP content of Raji cells that acquired exosomes by IS-dependent transfer or non-synaptic uptake. White bars, control; grey bars, 10 min trypsin; black bars, 10 min+10 min trypsin. Bar chart shows percentages±s.e.m. of positive cells (n=3).

Mentions: To investigate whether the IS promotes the transfer of exosomes from T cells to APCs, CD63-GFP T cells were cultured with Raji cells (stained blue with chloromethyl derivative of aminocoumarin (CMAC)) for 16 h, by which most stage conjugates will have separated. The coculture was analysed by flow cytometry for the transfer of CD63-GFP. SEE-pulsed Raji cells acquired CD63-GFP from the T cells, whereas transfer in the absence of SEE was negligible (Fig. 4a). No transfer of GFP signal was detected when the assay was performed in the opposite direction (from Raji-CD63-GFP to J77 cells; Fig. 4a) or when using J77 cells overexpressing non-exosomal membrane or cytoplasmic proteins (CD69-GFP and GFP; Fig. 4b). In addition, we also detected transfer of other molecules related to exosomes and vesicles, such as CD38 (ref. 31) and LAT (Supplementary Fig. S4).


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)

Ag recognition induces transfer of exosomes from T cell to APC.(a) Left: J77-CD63-GFP donor cells were conjugated with SEE-primed or unprimed Raji cells (recipient, in blue). Right: Raji-CD63-GFP donor cells (±SEE) were conjugated with blue-labeled J77 recipients. After 16 h, cocultures were analysed by flow cytometry. Bar chart shows the percentage±s.e.m. of positive recipient cells (n=9, P<0.001, Student's t-test). (b) J77 cells transfected with CD69-GFP or GFP were conjugated with SEE-primed Raji cells (blue) and analysed as in a. (c) FACS analysis of the percentage of GFP-positive Raji cells after incubation with J77-CD63-GFP cells in contact with coculture as in a (control), in the presence of inhibitors of actin (latrunculin-A, LatA or cytochalasin-D, CytD) or tubulin (nocodazol, NCD), or after incubation with separation of donors and recipients by a 0.4 μm pore-size transwell membrane (Twell). T cells in transwells were activated with CD3+CD28 Abs and Raji cells were loaded with SEE. (d) J77-CD63-GFP donor cells were conjugated with a 1:1 mix of two B cell lines: Raji cells (black) and BLS-1 cells (blue) and analysed as in a. Bar chart shows percentages±s.e.m. of positive recipient cells (n=6, P<0.005, Mann–Whitney test). Maximal projections of confocal images and the DIC of a triple conjugated formed by J77-CD63-GFP (green), Raji cells (black asterisk) and BLS-1 (CMAC stained and blue asterisk), stained for CD3 (red) are shown, scale bar shown in e. (e) Confocal analysis of Raji cells that acquired CD63-GFP directly from J77-CD63-GFP after IS formation (IS-dependent transfer) or after external administration of CD63-GFP exosomes isolated from J77-CD63GFP supernatants (non-synaptic uptake). Cells were stained for MHC-II (red). Images show maximal projections of confocal images, one representative confocal section, and the DIC images. Plots show cell perimeter fluorescence intensity profiles of the green and the red signals. ARU: arbitrary relative units. Scale bar, 10 ìm. (f) Raji cells (±SEE) were treated as in e. FACS analysis of the effect of trypsin on the CD63-GFP content of Raji cells that acquired exosomes by IS-dependent transfer or non-synaptic uptake. White bars, control; grey bars, 10 min trypsin; black bars, 10 min+10 min trypsin. Bar chart shows percentages±s.e.m. of positive cells (n=3).
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Related In: Results  -  Collection

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Show All Figures
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f4: Ag recognition induces transfer of exosomes from T cell to APC.(a) Left: J77-CD63-GFP donor cells were conjugated with SEE-primed or unprimed Raji cells (recipient, in blue). Right: Raji-CD63-GFP donor cells (±SEE) were conjugated with blue-labeled J77 recipients. After 16 h, cocultures were analysed by flow cytometry. Bar chart shows the percentage±s.e.m. of positive recipient cells (n=9, P<0.001, Student's t-test). (b) J77 cells transfected with CD69-GFP or GFP were conjugated with SEE-primed Raji cells (blue) and analysed as in a. (c) FACS analysis of the percentage of GFP-positive Raji cells after incubation with J77-CD63-GFP cells in contact with coculture as in a (control), in the presence of inhibitors of actin (latrunculin-A, LatA or cytochalasin-D, CytD) or tubulin (nocodazol, NCD), or after incubation with separation of donors and recipients by a 0.4 μm pore-size transwell membrane (Twell). T cells in transwells were activated with CD3+CD28 Abs and Raji cells were loaded with SEE. (d) J77-CD63-GFP donor cells were conjugated with a 1:1 mix of two B cell lines: Raji cells (black) and BLS-1 cells (blue) and analysed as in a. Bar chart shows percentages±s.e.m. of positive recipient cells (n=6, P<0.005, Mann–Whitney test). Maximal projections of confocal images and the DIC of a triple conjugated formed by J77-CD63-GFP (green), Raji cells (black asterisk) and BLS-1 (CMAC stained and blue asterisk), stained for CD3 (red) are shown, scale bar shown in e. (e) Confocal analysis of Raji cells that acquired CD63-GFP directly from J77-CD63-GFP after IS formation (IS-dependent transfer) or after external administration of CD63-GFP exosomes isolated from J77-CD63GFP supernatants (non-synaptic uptake). Cells were stained for MHC-II (red). Images show maximal projections of confocal images, one representative confocal section, and the DIC images. Plots show cell perimeter fluorescence intensity profiles of the green and the red signals. ARU: arbitrary relative units. Scale bar, 10 ìm. (f) Raji cells (±SEE) were treated as in e. FACS analysis of the effect of trypsin on the CD63-GFP content of Raji cells that acquired exosomes by IS-dependent transfer or non-synaptic uptake. White bars, control; grey bars, 10 min trypsin; black bars, 10 min+10 min trypsin. Bar chart shows percentages±s.e.m. of positive cells (n=3).
Mentions: To investigate whether the IS promotes the transfer of exosomes from T cells to APCs, CD63-GFP T cells were cultured with Raji cells (stained blue with chloromethyl derivative of aminocoumarin (CMAC)) for 16 h, by which most stage conjugates will have separated. The coculture was analysed by flow cytometry for the transfer of CD63-GFP. SEE-pulsed Raji cells acquired CD63-GFP from the T cells, whereas transfer in the absence of SEE was negligible (Fig. 4a). No transfer of GFP signal was detected when the assay was performed in the opposite direction (from Raji-CD63-GFP to J77 cells; Fig. 4a) or when using J77 cells overexpressing non-exosomal membrane or cytoplasmic proteins (CD69-GFP and GFP; Fig. 4b). In addition, we also detected transfer of other molecules related to exosomes and vesicles, such as CD38 (ref. 31) and LAT (Supplementary Fig. S4).

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