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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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Exosomal miRNA-335 is transferred from T cell to APC in an Ag-specific manner.(a) Levels of miR-335 were assessed by quantitative reverse transcription PCR (qRT–PCR) in primary dendritic cells and T lymphoblasts, and in Raji and J77 cells. J77-CD63-GFP cells were stably transduced with miR-335 (J-335 cells) or miR-101 (J-101 cells), and miR-335 levels were determined by qRT–PCR in cells and derived exosomes. Data are representative of three experiments (mean and s.e.m.) (b) miR-335 levels in SEE-primed Raji cells sorted 24 h after conjugation with J-335 cells. J-101 were used as control donor cells. Left panel, Data are representative of seven independent experiments (mean and s.e.m), P=0.014 (one-sample t-test). Right panel, n=5 independent experiments; P=0.04 (one-sample t-test); error bars represent s.e.m. (c) miR-335 levels in HA-primed HOM-2 cells sorted 24 h after conjugation with CH7C17 cells overexpressing miR-335 (C-335). CH7C17 cells overexpressing miR-101 (C-101) were used as control donor cells. Data are representative of five experiments (mean and s.e.m.). (d) miR-335 and miR-92a levels in SEE-primed Raji cells sorted 24 h after conjugation with primary T lymphoblasts-expressing miR-335 and miR-92a endogenously. Data are representative of three experiments (mean and s.e.m.), *P=0.026 (one-sample t-test) AU, arbitrary units.
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f5: Exosomal miRNA-335 is transferred from T cell to APC in an Ag-specific manner.(a) Levels of miR-335 were assessed by quantitative reverse transcription PCR (qRT–PCR) in primary dendritic cells and T lymphoblasts, and in Raji and J77 cells. J77-CD63-GFP cells were stably transduced with miR-335 (J-335 cells) or miR-101 (J-101 cells), and miR-335 levels were determined by qRT–PCR in cells and derived exosomes. Data are representative of three experiments (mean and s.e.m.) (b) miR-335 levels in SEE-primed Raji cells sorted 24 h after conjugation with J-335 cells. J-101 were used as control donor cells. Left panel, Data are representative of seven independent experiments (mean and s.e.m), P=0.014 (one-sample t-test). Right panel, n=5 independent experiments; P=0.04 (one-sample t-test); error bars represent s.e.m. (c) miR-335 levels in HA-primed HOM-2 cells sorted 24 h after conjugation with CH7C17 cells overexpressing miR-335 (C-335). CH7C17 cells overexpressing miR-101 (C-101) were used as control donor cells. Data are representative of five experiments (mean and s.e.m.). (d) miR-335 and miR-92a levels in SEE-primed Raji cells sorted 24 h after conjugation with primary T lymphoblasts-expressing miR-335 and miR-92a endogenously. Data are representative of three experiments (mean and s.e.m.), *P=0.026 (one-sample t-test) AU, arbitrary units.

Mentions: To demonstrate delivery of exosomal miRNA from T cell to APC after IS formation, we stably overexpressed miR-335 in J77-CD63-GFP T cells (Fig. 5a). This miRNA is not normally expressed in the donor (J77) or recipient (Raji) cells, but is sorted to the exosomes of primary immune cells (DCs and T lymphoblasts; Figs 1c and 5a, and Supplementary Data 1). J77-CD63-GFP cells expressing miR-335 (J-335) were cocultured with unprimed or SEE-primed Raji B cells (stained blue with CMAC), and after 24 h the Raji cells were sorted by flow cytometry (Supplementary Fig. S6) and their miR-335 content analysed by reverse transcription PCR. miR-335 was transferred to Raji cells only in the presence of SEE (Fig. 5b). Raji cells that acquired high amounts of CD63-GFP contained correspondingly high amounts of miR-335, demonstrating correlation between the transfer of miR-335 and exosomal proteins (Fig. 5b). To demonstrate that miR-335 is not expressed de novo in Raji cells after IS formation, we repeated the experiment with J77-CD63-GFP cells stably overexpressing miR-101 (J-101); conjugation with these cells did not induce expression of miR-335 in Raji cells (Fig. 5b). The ability of a peptide antigen-specific IS to transfer miRNA was further demonstrated in HA-loaded CH7C17 cells overexpressing miR-335 (C-335) and conjugated to Hom-2 cells (Fig. 5c), demonstrating antigen-specific directional transfer of exosomal miRNA from T cell to APC. We also investigated the transfer of endogenous miRNAs by primary SEE-specific T lymphocytes. These cells transferred endogenous miR-335 and miR-92 to Raji APCs in a SEE-specific manner (Fig. 5d).


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)

Exosomal miRNA-335 is transferred from T cell to APC in an Ag-specific manner.(a) Levels of miR-335 were assessed by quantitative reverse transcription PCR (qRT–PCR) in primary dendritic cells and T lymphoblasts, and in Raji and J77 cells. J77-CD63-GFP cells were stably transduced with miR-335 (J-335 cells) or miR-101 (J-101 cells), and miR-335 levels were determined by qRT–PCR in cells and derived exosomes. Data are representative of three experiments (mean and s.e.m.) (b) miR-335 levels in SEE-primed Raji cells sorted 24 h after conjugation with J-335 cells. J-101 were used as control donor cells. Left panel, Data are representative of seven independent experiments (mean and s.e.m), P=0.014 (one-sample t-test). Right panel, n=5 independent experiments; P=0.04 (one-sample t-test); error bars represent s.e.m. (c) miR-335 levels in HA-primed HOM-2 cells sorted 24 h after conjugation with CH7C17 cells overexpressing miR-335 (C-335). CH7C17 cells overexpressing miR-101 (C-101) were used as control donor cells. Data are representative of five experiments (mean and s.e.m.). (d) miR-335 and miR-92a levels in SEE-primed Raji cells sorted 24 h after conjugation with primary T lymphoblasts-expressing miR-335 and miR-92a endogenously. Data are representative of three experiments (mean and s.e.m.), *P=0.026 (one-sample t-test) AU, arbitrary units.
© Copyright Policy - open-access
Related In: Results  -  Collection

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f5: Exosomal miRNA-335 is transferred from T cell to APC in an Ag-specific manner.(a) Levels of miR-335 were assessed by quantitative reverse transcription PCR (qRT–PCR) in primary dendritic cells and T lymphoblasts, and in Raji and J77 cells. J77-CD63-GFP cells were stably transduced with miR-335 (J-335 cells) or miR-101 (J-101 cells), and miR-335 levels were determined by qRT–PCR in cells and derived exosomes. Data are representative of three experiments (mean and s.e.m.) (b) miR-335 levels in SEE-primed Raji cells sorted 24 h after conjugation with J-335 cells. J-101 were used as control donor cells. Left panel, Data are representative of seven independent experiments (mean and s.e.m), P=0.014 (one-sample t-test). Right panel, n=5 independent experiments; P=0.04 (one-sample t-test); error bars represent s.e.m. (c) miR-335 levels in HA-primed HOM-2 cells sorted 24 h after conjugation with CH7C17 cells overexpressing miR-335 (C-335). CH7C17 cells overexpressing miR-101 (C-101) were used as control donor cells. Data are representative of five experiments (mean and s.e.m.). (d) miR-335 and miR-92a levels in SEE-primed Raji cells sorted 24 h after conjugation with primary T lymphoblasts-expressing miR-335 and miR-92a endogenously. Data are representative of three experiments (mean and s.e.m.), *P=0.026 (one-sample t-test) AU, arbitrary units.
Mentions: To demonstrate delivery of exosomal miRNA from T cell to APC after IS formation, we stably overexpressed miR-335 in J77-CD63-GFP T cells (Fig. 5a). This miRNA is not normally expressed in the donor (J77) or recipient (Raji) cells, but is sorted to the exosomes of primary immune cells (DCs and T lymphoblasts; Figs 1c and 5a, and Supplementary Data 1). J77-CD63-GFP cells expressing miR-335 (J-335) were cocultured with unprimed or SEE-primed Raji B cells (stained blue with CMAC), and after 24 h the Raji cells were sorted by flow cytometry (Supplementary Fig. S6) and their miR-335 content analysed by reverse transcription PCR. miR-335 was transferred to Raji cells only in the presence of SEE (Fig. 5b). Raji cells that acquired high amounts of CD63-GFP contained correspondingly high amounts of miR-335, demonstrating correlation between the transfer of miR-335 and exosomal proteins (Fig. 5b). To demonstrate that miR-335 is not expressed de novo in Raji cells after IS formation, we repeated the experiment with J77-CD63-GFP cells stably overexpressing miR-101 (J-101); conjugation with these cells did not induce expression of miR-335 in Raji cells (Fig. 5b). The ability of a peptide antigen-specific IS to transfer miRNA was further demonstrated in HA-loaded CH7C17 cells overexpressing miR-335 (C-335) and conjugated to Hom-2 cells (Fig. 5c), demonstrating antigen-specific directional transfer of exosomal miRNA from T cell to APC. We also investigated the transfer of endogenous miRNAs by primary SEE-specific T lymphocytes. These cells transferred endogenous miR-335 and miR-92 to Raji APCs in a SEE-specific manner (Fig. 5d).

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