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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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Inhibition of exosome biogenesis impairs transfer of exosomal miRNAs and proteins through the IS.(a) J77 cells were cultured in exosome-depleted medium for 24 h, in the presence of inhibitors of nSMase2 (manumycin-A, Manu) or BIG2 (brefeldin, Brefel). Purified exosomes secreted by equal numbers of control or treated cells were analysed by immunoblotting for the presence of the exosome marker CD81. Densitometric analyses were performed, and the ratio between the treated and the control condition is shown. (b) CD81 immunoblot of exosomes purified from equal numbers of cells transduced with control or nSMase2 shRNA. Densitometric analyses were performed, and the ratio between the silenced and the control condition is shown. (c) FACS analysis of the CD63-GFP content of Raji recipients cells after conjugation with J-335 cells in the presence of manumycin or brefeldin. Data are the percentage±s.e.m. of Raji-GFP-positive recipient cells relative to the SEE-loaded control condition. n=5 independent experiments; P≤0.001 (one-sample t-test). (d) FACS analysis of the CD63-GFP content of Raji recipient cells after coculture with J77-CD63GFP cells expressing shnSMase2 or siHrs. Data are the percentage±s.e.m. of Raji-GFP-positive recipient cells relative to the SEE-loaded control condition. n=8 independent experiments, *P=0.0005 (one-sample t-test). (e) Quantitative reverse transcription PCR (qRT–PCR) analysis of miR-335 in Raji cells sorted after conjugation with J-335 cells in the presence of manumycin or brefeldin. n=3 independent experiments; *P=0.03 (unpaired t-test). (f) qRT–PCR analysis of miR-335 in Raji cells sorted after conjugation with J-335 transduced with shnSMase 2 or shControl. n=4 independent experiments; *P=0.012 (unpaired t-test); error bars represent s.e.m.
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f6: Inhibition of exosome biogenesis impairs transfer of exosomal miRNAs and proteins through the IS.(a) J77 cells were cultured in exosome-depleted medium for 24 h, in the presence of inhibitors of nSMase2 (manumycin-A, Manu) or BIG2 (brefeldin, Brefel). Purified exosomes secreted by equal numbers of control or treated cells were analysed by immunoblotting for the presence of the exosome marker CD81. Densitometric analyses were performed, and the ratio between the treated and the control condition is shown. (b) CD81 immunoblot of exosomes purified from equal numbers of cells transduced with control or nSMase2 shRNA. Densitometric analyses were performed, and the ratio between the silenced and the control condition is shown. (c) FACS analysis of the CD63-GFP content of Raji recipients cells after conjugation with J-335 cells in the presence of manumycin or brefeldin. Data are the percentage±s.e.m. of Raji-GFP-positive recipient cells relative to the SEE-loaded control condition. n=5 independent experiments; P≤0.001 (one-sample t-test). (d) FACS analysis of the CD63-GFP content of Raji recipient cells after coculture with J77-CD63GFP cells expressing shnSMase2 or siHrs. Data are the percentage±s.e.m. of Raji-GFP-positive recipient cells relative to the SEE-loaded control condition. n=8 independent experiments, *P=0.0005 (one-sample t-test). (e) Quantitative reverse transcription PCR (qRT–PCR) analysis of miR-335 in Raji cells sorted after conjugation with J-335 cells in the presence of manumycin or brefeldin. n=3 independent experiments; *P=0.03 (unpaired t-test). (f) qRT–PCR analysis of miR-335 in Raji cells sorted after conjugation with J-335 transduced with shnSMase 2 or shControl. n=4 independent experiments; *P=0.012 (unpaired t-test); error bars represent s.e.m.

Mentions: To confirm that miRNA transfer is mediated by exosomes, we blocked exosome production in J-335 cells. Ceramide, biosynthesis of which is regulated by neutral sphingomyelinase-2 (nSMase2), triggers the budding of exosomes into MVBs, and inhibition of nSMase2 therefore reduces the secretion of CD63-containing exosomes35 and miRNAs36. Accordingly, the secretion of exosomes by J77 cells was impaired when nSMase2 activity was reduced either by addition of the inhibitor manumycin-A (Fig. 6a) or by small hairpin RNA (shRNA) silencing (Fig. 6b and Supplementary Fig. S7a). Targeting of nSMase2 activity inhibited the IS-dependent transfer of CD63-GFP (Fig. 6c,d) and miRNA-335 (Fig. 6e,f). Transfer of CD63-GFP and miR-335 was also blocked by brefeldin (Fig. 6c,e), which inhibits the guanine nucleotide-exchange protein BIG2 and regulates the constitutive release of exosome-like vesicles37. Exosome secretion and CD63-GFP transfer by J-335 cells was also inhibited by shRNA silencing of the Rab GTPase Rab27a, which is implicated in the exosomal release pathway38 (Supplementary Fig. S7b–d). In contrast, transfer of CD63-GFP occurred normally from Hrs-interfered J77 T cells (Supplementary Fig. S7e and Fig. 6d), in agreement with previous studies that demonstrated that the ESCRT system is unnecessary for the release of exosomes and miRNA3536.


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)

Inhibition of exosome biogenesis impairs transfer of exosomal miRNAs and proteins through the IS.(a) J77 cells were cultured in exosome-depleted medium for 24 h, in the presence of inhibitors of nSMase2 (manumycin-A, Manu) or BIG2 (brefeldin, Brefel). Purified exosomes secreted by equal numbers of control or treated cells were analysed by immunoblotting for the presence of the exosome marker CD81. Densitometric analyses were performed, and the ratio between the treated and the control condition is shown. (b) CD81 immunoblot of exosomes purified from equal numbers of cells transduced with control or nSMase2 shRNA. Densitometric analyses were performed, and the ratio between the silenced and the control condition is shown. (c) FACS analysis of the CD63-GFP content of Raji recipients cells after conjugation with J-335 cells in the presence of manumycin or brefeldin. Data are the percentage±s.e.m. of Raji-GFP-positive recipient cells relative to the SEE-loaded control condition. n=5 independent experiments; P≤0.001 (one-sample t-test). (d) FACS analysis of the CD63-GFP content of Raji recipient cells after coculture with J77-CD63GFP cells expressing shnSMase2 or siHrs. Data are the percentage±s.e.m. of Raji-GFP-positive recipient cells relative to the SEE-loaded control condition. n=8 independent experiments, *P=0.0005 (one-sample t-test). (e) Quantitative reverse transcription PCR (qRT–PCR) analysis of miR-335 in Raji cells sorted after conjugation with J-335 cells in the presence of manumycin or brefeldin. n=3 independent experiments; *P=0.03 (unpaired t-test). (f) qRT–PCR analysis of miR-335 in Raji cells sorted after conjugation with J-335 transduced with shnSMase 2 or shControl. n=4 independent experiments; *P=0.012 (unpaired t-test); error bars represent s.e.m.
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f6: Inhibition of exosome biogenesis impairs transfer of exosomal miRNAs and proteins through the IS.(a) J77 cells were cultured in exosome-depleted medium for 24 h, in the presence of inhibitors of nSMase2 (manumycin-A, Manu) or BIG2 (brefeldin, Brefel). Purified exosomes secreted by equal numbers of control or treated cells were analysed by immunoblotting for the presence of the exosome marker CD81. Densitometric analyses were performed, and the ratio between the treated and the control condition is shown. (b) CD81 immunoblot of exosomes purified from equal numbers of cells transduced with control or nSMase2 shRNA. Densitometric analyses were performed, and the ratio between the silenced and the control condition is shown. (c) FACS analysis of the CD63-GFP content of Raji recipients cells after conjugation with J-335 cells in the presence of manumycin or brefeldin. Data are the percentage±s.e.m. of Raji-GFP-positive recipient cells relative to the SEE-loaded control condition. n=5 independent experiments; P≤0.001 (one-sample t-test). (d) FACS analysis of the CD63-GFP content of Raji recipient cells after coculture with J77-CD63GFP cells expressing shnSMase2 or siHrs. Data are the percentage±s.e.m. of Raji-GFP-positive recipient cells relative to the SEE-loaded control condition. n=8 independent experiments, *P=0.0005 (one-sample t-test). (e) Quantitative reverse transcription PCR (qRT–PCR) analysis of miR-335 in Raji cells sorted after conjugation with J-335 cells in the presence of manumycin or brefeldin. n=3 independent experiments; *P=0.03 (unpaired t-test). (f) qRT–PCR analysis of miR-335 in Raji cells sorted after conjugation with J-335 transduced with shnSMase 2 or shControl. n=4 independent experiments; *P=0.012 (unpaired t-test); error bars represent s.e.m.
Mentions: To confirm that miRNA transfer is mediated by exosomes, we blocked exosome production in J-335 cells. Ceramide, biosynthesis of which is regulated by neutral sphingomyelinase-2 (nSMase2), triggers the budding of exosomes into MVBs, and inhibition of nSMase2 therefore reduces the secretion of CD63-containing exosomes35 and miRNAs36. Accordingly, the secretion of exosomes by J77 cells was impaired when nSMase2 activity was reduced either by addition of the inhibitor manumycin-A (Fig. 6a) or by small hairpin RNA (shRNA) silencing (Fig. 6b and Supplementary Fig. S7a). Targeting of nSMase2 activity inhibited the IS-dependent transfer of CD63-GFP (Fig. 6c,d) and miRNA-335 (Fig. 6e,f). Transfer of CD63-GFP and miR-335 was also blocked by brefeldin (Fig. 6c,e), which inhibits the guanine nucleotide-exchange protein BIG2 and regulates the constitutive release of exosome-like vesicles37. Exosome secretion and CD63-GFP transfer by J-335 cells was also inhibited by shRNA silencing of the Rab GTPase Rab27a, which is implicated in the exosomal release pathway38 (Supplementary Fig. S7b–d). In contrast, transfer of CD63-GFP occurred normally from Hrs-interfered J77 T cells (Supplementary Fig. S7e and Fig. 6d), in agreement with previous studies that demonstrated that the ESCRT system is unnecessary for the release of exosomes and miRNA3536.

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