Limits...
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.

Show MeSH

Related in: MedlinePlus

microRNA profiles of exosomes and their parental cells.(a) Microarray analysis of exosomal miRNAs versus the miRNAs of their respective donor cells. Exosomes were isolated by serial centrifugation and filtration steps from supernatants of donor cells cultured in RPMI-1640 supplemented with exosome-depleted FBS (10%). Total RNA, including microRNA, was isolated from exosomes and their donor cells and miRNA profiles were assessed by microarray technology. The panel shows the hierarchical clustering of the VSN-normalized array data in the log2 scale averaged per biological replicate for each origin (exosomes per cells) and cell type. DC, dendritic cells; J77, Jurkat-derived J77 T cell line; Raji, Raji B cell line). (b) Principal component analysis (PCA) of all normalized array data. Each point represents a hybridized sample: that is, different biological samples per origin and cell type. x axis, first principal component (PC1); y axis, second principal component (PC2). Cell samples, red; exosomal samples, blue. (c) Heatmap of the VSN-normalized data for selected miRNAs. For visualization purposes the expression profiles were centred on the median of the profile. The scale bar across the bottom depicts standard deviation change from the mean. (d) Scatter plots of the exosome versus cell averaged array data in each cell type and of Raji exosomes versus J77 exosomes. The correlation is shown.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3104548&req=5

f1: microRNA profiles of exosomes and their parental cells.(a) Microarray analysis of exosomal miRNAs versus the miRNAs of their respective donor cells. Exosomes were isolated by serial centrifugation and filtration steps from supernatants of donor cells cultured in RPMI-1640 supplemented with exosome-depleted FBS (10%). Total RNA, including microRNA, was isolated from exosomes and their donor cells and miRNA profiles were assessed by microarray technology. The panel shows the hierarchical clustering of the VSN-normalized array data in the log2 scale averaged per biological replicate for each origin (exosomes per cells) and cell type. DC, dendritic cells; J77, Jurkat-derived J77 T cell line; Raji, Raji B cell line). (b) Principal component analysis (PCA) of all normalized array data. Each point represents a hybridized sample: that is, different biological samples per origin and cell type. x axis, first principal component (PC1); y axis, second principal component (PC2). Cell samples, red; exosomal samples, blue. (c) Heatmap of the VSN-normalized data for selected miRNAs. For visualization purposes the expression profiles were centred on the median of the profile. The scale bar across the bottom depicts standard deviation change from the mean. (d) Scatter plots of the exosome versus cell averaged array data in each cell type and of Raji exosomes versus J77 exosomes. The correlation is shown.

Mentions: To determine the miRNA repertoires of exosomes secreted by immune cells, we isolated exosomes from cell supernatants of the Raji B-cell line, the Jurkat-derived J77 T cell line, and primary dendritic cells (DCs) derived from human monocytes. Exosomes were isolated by a series of microfiltration and ultracentrifugation steps29, and exosome identity was assessed by extensive protein analysis with liquid chromatography with tandem mass spectrometry (LC MS/MS) technology. About 60% of proteins found in the analysed exosome samples have been previously found in other types of exosomes; these include the tetraspanins CD63, CD81 and CD9, proteins involved in membrane transport and fusion (Rab GTPases, annexins and fotillin), and other exosomal markers such as Tsg101 (data not shown). Moreover, exosomes derived from T lymphocytes and from APCs both contained RNA. Profiling of RNA isolated from exosomes and their donor cells indicates that exosomes are highly enriched in small RNA species (Supplementary Fig. S1). Agilent microRNA microarray analysis (Agilent) showed that certain miRNAs are expressed at higher levels in exosomes than in their donor cells and vice versa (Fig. 1 and Supplementary Data 1). The hierarchical clustering and the principal component analysis of the array data grouped the samples according to their cellular or exosomal origin (Fig. 1a,b). Several miRNAs (for example, miR-760, miR-632, miR-654-5p and miR-671-5p) were significantly more abundant in exosomal samples from all cell types; others, for example miR-335, were found only in exosomes derived from the primary DCs; in contrast, others (for example, miR-101, miR-32 and miR-21*) were more highly represented in cells than in exosomes (Fig. 1c). These data indicate that specific miRNA populations are selectively sorted into exosomes. Consistently, there was lower overall similarity between the miRNA repertoire in exosomes and their corresponding cells than between the exosomes of different cellular origin (Fig. 1d).


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)

microRNA profiles of exosomes and their parental cells.(a) Microarray analysis of exosomal miRNAs versus the miRNAs of their respective donor cells. Exosomes were isolated by serial centrifugation and filtration steps from supernatants of donor cells cultured in RPMI-1640 supplemented with exosome-depleted FBS (10%). Total RNA, including microRNA, was isolated from exosomes and their donor cells and miRNA profiles were assessed by microarray technology. The panel shows the hierarchical clustering of the VSN-normalized array data in the log2 scale averaged per biological replicate for each origin (exosomes per cells) and cell type. DC, dendritic cells; J77, Jurkat-derived J77 T cell line; Raji, Raji B cell line). (b) Principal component analysis (PCA) of all normalized array data. Each point represents a hybridized sample: that is, different biological samples per origin and cell type. x axis, first principal component (PC1); y axis, second principal component (PC2). Cell samples, red; exosomal samples, blue. (c) Heatmap of the VSN-normalized data for selected miRNAs. For visualization purposes the expression profiles were centred on the median of the profile. The scale bar across the bottom depicts standard deviation change from the mean. (d) Scatter plots of the exosome versus cell averaged array data in each cell type and of Raji exosomes versus J77 exosomes. The correlation is shown.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f1: microRNA profiles of exosomes and their parental cells.(a) Microarray analysis of exosomal miRNAs versus the miRNAs of their respective donor cells. Exosomes were isolated by serial centrifugation and filtration steps from supernatants of donor cells cultured in RPMI-1640 supplemented with exosome-depleted FBS (10%). Total RNA, including microRNA, was isolated from exosomes and their donor cells and miRNA profiles were assessed by microarray technology. The panel shows the hierarchical clustering of the VSN-normalized array data in the log2 scale averaged per biological replicate for each origin (exosomes per cells) and cell type. DC, dendritic cells; J77, Jurkat-derived J77 T cell line; Raji, Raji B cell line). (b) Principal component analysis (PCA) of all normalized array data. Each point represents a hybridized sample: that is, different biological samples per origin and cell type. x axis, first principal component (PC1); y axis, second principal component (PC2). Cell samples, red; exosomal samples, blue. (c) Heatmap of the VSN-normalized data for selected miRNAs. For visualization purposes the expression profiles were centred on the median of the profile. The scale bar across the bottom depicts standard deviation change from the mean. (d) Scatter plots of the exosome versus cell averaged array data in each cell type and of Raji exosomes versus J77 exosomes. The correlation is shown.
Mentions: To determine the miRNA repertoires of exosomes secreted by immune cells, we isolated exosomes from cell supernatants of the Raji B-cell line, the Jurkat-derived J77 T cell line, and primary dendritic cells (DCs) derived from human monocytes. Exosomes were isolated by a series of microfiltration and ultracentrifugation steps29, and exosome identity was assessed by extensive protein analysis with liquid chromatography with tandem mass spectrometry (LC MS/MS) technology. About 60% of proteins found in the analysed exosome samples have been previously found in other types of exosomes; these include the tetraspanins CD63, CD81 and CD9, proteins involved in membrane transport and fusion (Rab GTPases, annexins and fotillin), and other exosomal markers such as Tsg101 (data not shown). Moreover, exosomes derived from T lymphocytes and from APCs both contained RNA. Profiling of RNA isolated from exosomes and their donor cells indicates that exosomes are highly enriched in small RNA species (Supplementary Fig. S1). Agilent microRNA microarray analysis (Agilent) showed that certain miRNAs are expressed at higher levels in exosomes than in their donor cells and vice versa (Fig. 1 and Supplementary Data 1). The hierarchical clustering and the principal component analysis of the array data grouped the samples according to their cellular or exosomal origin (Fig. 1a,b). Several miRNAs (for example, miR-760, miR-632, miR-654-5p and miR-671-5p) were significantly more abundant in exosomal samples from all cell types; others, for example miR-335, were found only in exosomes derived from the primary DCs; in contrast, others (for example, miR-101, miR-32 and miR-21*) were more highly represented in cells than in exosomes (Fig. 1c). These data indicate that specific miRNA populations are selectively sorted into exosomes. Consistently, there was lower overall similarity between the miRNA repertoire in exosomes and their corresponding cells than between the exosomes of different cellular origin (Fig. 1d).

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