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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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Synaptically transferred miR-335 downregulates target gene expression in the APC.(a) Raji cells were transfected with reporter constructs consisting of the luciferase sequence placed upstream of the full-length 3′-UTR of either the miR-335-target gene SOX4 or the control gene UBE2F. Luciferase activity was assayed 24 h after coculture of SEE-loaded Raji with J77-miR-335 cells or control J-101 cells (synaptic transfer) or after addition of derived exosomes to SEE-loaded Raji cells (non-synaptic uptake). (b) Experiments as in a performed with Raji recipient cells expressing the wild-type of mutated seed sequence for miR-335 from the SOX4 3′-UTR. Luciferase activities are shown relative to control incubations with untreated Raji cells. Data are means±s.e.m.; n=5 independent experiments, *P<0.05 (Newman–Keuls multiple comparison test). White bars, control; grey bars, synaptic transfer; black bars, non-synaptic transfer.
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f7: Synaptically transferred miR-335 downregulates target gene expression in the APC.(a) Raji cells were transfected with reporter constructs consisting of the luciferase sequence placed upstream of the full-length 3′-UTR of either the miR-335-target gene SOX4 or the control gene UBE2F. Luciferase activity was assayed 24 h after coculture of SEE-loaded Raji with J77-miR-335 cells or control J-101 cells (synaptic transfer) or after addition of derived exosomes to SEE-loaded Raji cells (non-synaptic uptake). (b) Experiments as in a performed with Raji recipient cells expressing the wild-type of mutated seed sequence for miR-335 from the SOX4 3′-UTR. Luciferase activities are shown relative to control incubations with untreated Raji cells. Data are means±s.e.m.; n=5 independent experiments, *P<0.05 (Newman–Keuls multiple comparison test). White bars, control; grey bars, synaptic transfer; black bars, non-synaptic transfer.

Mentions: To determine whether synaptically transferred miRNA-335 is functional in the recipient APC, we carried out luciferase reporter assays with full-length 3′-UTR constructs of the miR-335 target SOX4 and the miR-335-insensitive UBE2F39. Raji cells transfected with luciferase constructs were cocultured with J-335 cells and luciferase activity was assessed. Expression from the SOX4 3′-UTR was significantly reduced in SEE-primed Raji cells that had been in contact with J-335 cells, whereas expression from the UBE2F 3′-UTR was unaffected (Fig. 7a). Expression from these 3′-UTR reporters was not affected by coculture of Raji cells with J-101 cells, indicating that the process is not a general effect of IS formation, but rather involves the direct transfer of the specific miRNA. Reduction in luciferase activity after conjugate formation was also detected in Raji cells expressing base pairs 449–509 of the SOX4 3′-UTR, which encompasses the miR-335 seed sequence; and inhibition of luciferase expression was abolished by mutation of this sequence (Fig. 7b). The IS thus guides the transfer of functional miRNA from the T cell to the APC. Contrasting with IS-dependent miRNA transfer, addition of exosomes isolated from J-335 cells to transfected Raji cells had no effect on luciferase activity (Fig. 7a,b), indicating that any miR-335 acquired in this way was non-functional.


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)

Synaptically transferred miR-335 downregulates target gene expression in the APC.(a) Raji cells were transfected with reporter constructs consisting of the luciferase sequence placed upstream of the full-length 3′-UTR of either the miR-335-target gene SOX4 or the control gene UBE2F. Luciferase activity was assayed 24 h after coculture of SEE-loaded Raji with J77-miR-335 cells or control J-101 cells (synaptic transfer) or after addition of derived exosomes to SEE-loaded Raji cells (non-synaptic uptake). (b) Experiments as in a performed with Raji recipient cells expressing the wild-type of mutated seed sequence for miR-335 from the SOX4 3′-UTR. Luciferase activities are shown relative to control incubations with untreated Raji cells. Data are means±s.e.m.; n=5 independent experiments, *P<0.05 (Newman–Keuls multiple comparison test). White bars, control; grey bars, synaptic transfer; black bars, non-synaptic transfer.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f7: Synaptically transferred miR-335 downregulates target gene expression in the APC.(a) Raji cells were transfected with reporter constructs consisting of the luciferase sequence placed upstream of the full-length 3′-UTR of either the miR-335-target gene SOX4 or the control gene UBE2F. Luciferase activity was assayed 24 h after coculture of SEE-loaded Raji with J77-miR-335 cells or control J-101 cells (synaptic transfer) or after addition of derived exosomes to SEE-loaded Raji cells (non-synaptic uptake). (b) Experiments as in a performed with Raji recipient cells expressing the wild-type of mutated seed sequence for miR-335 from the SOX4 3′-UTR. Luciferase activities are shown relative to control incubations with untreated Raji cells. Data are means±s.e.m.; n=5 independent experiments, *P<0.05 (Newman–Keuls multiple comparison test). White bars, control; grey bars, synaptic transfer; black bars, non-synaptic transfer.
Mentions: To determine whether synaptically transferred miRNA-335 is functional in the recipient APC, we carried out luciferase reporter assays with full-length 3′-UTR constructs of the miR-335 target SOX4 and the miR-335-insensitive UBE2F39. Raji cells transfected with luciferase constructs were cocultured with J-335 cells and luciferase activity was assessed. Expression from the SOX4 3′-UTR was significantly reduced in SEE-primed Raji cells that had been in contact with J-335 cells, whereas expression from the UBE2F 3′-UTR was unaffected (Fig. 7a). Expression from these 3′-UTR reporters was not affected by coculture of Raji cells with J-101 cells, indicating that the process is not a general effect of IS formation, but rather involves the direct transfer of the specific miRNA. Reduction in luciferase activity after conjugate formation was also detected in Raji cells expressing base pairs 449–509 of the SOX4 3′-UTR, which encompasses the miR-335 seed sequence; and inhibition of luciferase expression was abolished by mutation of this sequence (Fig. 7b). The IS thus guides the transfer of functional miRNA from the T cell to the APC. Contrasting with IS-dependent miRNA transfer, addition of exosomes isolated from J-335 cells to transfected Raji cells had no effect on luciferase activity (Fig. 7a,b), indicating that any miR-335 acquired in this way was non-functional.

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