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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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MVBs in T cells translocate to the IS.(a) J77 T cells were conjugated with SEE-pulsed or non-pulsed Raji B cells loaded with CMAC (blue). After 30 min, cells were fixed and stained for the MVB marker Hrs (red), CD3 and actin (both green). Plates show maximal projections of confocal images and the DIC images. (b) CH7C17 T cells were conjugated with HA-loaded or non-loaded HOM2 B cells (blue). After 30 min, cells were fixed and stained for CD63 (green), CD3 and actin (both red). Plates show maximal projections of confocal images and the DIC images, scale bars applies to a and b. (c) Percentage of T and APC cells in which CD63, Hrs and CD3 relocalized to the T cell–APC contact area in the presence of HA peptide (filled bars) or its absence (open bars). Data are the arithmetic means±s.e.m. of four experiments. *P<0.03 compared with the absence of antigen (Mann–Whitney test). (d) Live cell imaging of J77-CD63-GFP cells seeded on fibronectin-coated coverslips and conjugated with SEE-primed Raji cells (blue). Cells were monitored by time-lapse confocal microscopy at 30 s intervals. Plates show maximal projections of confocal images. Scale bars, 10 ìm.
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f3: MVBs in T cells translocate to the IS.(a) J77 T cells were conjugated with SEE-pulsed or non-pulsed Raji B cells loaded with CMAC (blue). After 30 min, cells were fixed and stained for the MVB marker Hrs (red), CD3 and actin (both green). Plates show maximal projections of confocal images and the DIC images. (b) CH7C17 T cells were conjugated with HA-loaded or non-loaded HOM2 B cells (blue). After 30 min, cells were fixed and stained for CD63 (green), CD3 and actin (both red). Plates show maximal projections of confocal images and the DIC images, scale bars applies to a and b. (c) Percentage of T and APC cells in which CD63, Hrs and CD3 relocalized to the T cell–APC contact area in the presence of HA peptide (filled bars) or its absence (open bars). Data are the arithmetic means±s.e.m. of four experiments. *P<0.03 compared with the absence of antigen (Mann–Whitney test). (d) Live cell imaging of J77-CD63-GFP cells seeded on fibronectin-coated coverslips and conjugated with SEE-primed Raji cells (blue). Cells were monitored by time-lapse confocal microscopy at 30 s intervals. Plates show maximal projections of confocal images. Scale bars, 10 ìm.

Mentions: To address whether exosomes mediate the transfer of miRNA during cognate immune interactions, we first studied the intracellular distribution of MVBs—the compartments from which exosomes arise—during the formation of an IS. In these experiments, Raji B cells were pulsed with Staphylococcus enterotoxin superantigen-E (SEE) and then incubated with TCR-Vβ8+ J77 T cells. MVB localization was assessed by immunofluorescence analysis of CD63 and two components of the ESCRT (endosomal sorting complex required for transport): Hrs, a component of the ESCRT-0 complex; and the late acting ESCRT machinery component VPS4. In the presence of SEE, which promotes formation of a fully functional IS, the MVBs of the J77 T lymphocyte lost their random cytoplasmic distribution and congregated near the IS (identified by CD3 and actin staining); in contrast, the localization of MVBs in the Raji B cell remained unchanged (Fig. 3a, and Supplementary Fig. S3). Similar results were obtained in experiments in which CH7C17 T cells, bearing an influenza hemagglutinin (HA) peptide-specific TCR, were conjugated to HA peptide-pulsed Hom2 B cells, thus confirming that antigen-induced formation of an IS polarizes T-cell MVBs to the contact site (Fig. 3b,c). Live cell imaging of CD63-GFP-expressing J77 T cells encountering SEE-pulsed Raji B cells revealed that the MVBs of the T cells move to the IS during the first 10 min (Fig. 3d, and Supplementary Movie 1).


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)

MVBs in T cells translocate to the IS.(a) J77 T cells were conjugated with SEE-pulsed or non-pulsed Raji B cells loaded with CMAC (blue). After 30 min, cells were fixed and stained for the MVB marker Hrs (red), CD3 and actin (both green). Plates show maximal projections of confocal images and the DIC images. (b) CH7C17 T cells were conjugated with HA-loaded or non-loaded HOM2 B cells (blue). After 30 min, cells were fixed and stained for CD63 (green), CD3 and actin (both red). Plates show maximal projections of confocal images and the DIC images, scale bars applies to a and b. (c) Percentage of T and APC cells in which CD63, Hrs and CD3 relocalized to the T cell–APC contact area in the presence of HA peptide (filled bars) or its absence (open bars). Data are the arithmetic means±s.e.m. of four experiments. *P<0.03 compared with the absence of antigen (Mann–Whitney test). (d) Live cell imaging of J77-CD63-GFP cells seeded on fibronectin-coated coverslips and conjugated with SEE-primed Raji cells (blue). Cells were monitored by time-lapse confocal microscopy at 30 s intervals. Plates show maximal projections of confocal images. Scale bars, 10 ìm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3104548&req=5

f3: MVBs in T cells translocate to the IS.(a) J77 T cells were conjugated with SEE-pulsed or non-pulsed Raji B cells loaded with CMAC (blue). After 30 min, cells were fixed and stained for the MVB marker Hrs (red), CD3 and actin (both green). Plates show maximal projections of confocal images and the DIC images. (b) CH7C17 T cells were conjugated with HA-loaded or non-loaded HOM2 B cells (blue). After 30 min, cells were fixed and stained for CD63 (green), CD3 and actin (both red). Plates show maximal projections of confocal images and the DIC images, scale bars applies to a and b. (c) Percentage of T and APC cells in which CD63, Hrs and CD3 relocalized to the T cell–APC contact area in the presence of HA peptide (filled bars) or its absence (open bars). Data are the arithmetic means±s.e.m. of four experiments. *P<0.03 compared with the absence of antigen (Mann–Whitney test). (d) Live cell imaging of J77-CD63-GFP cells seeded on fibronectin-coated coverslips and conjugated with SEE-primed Raji cells (blue). Cells were monitored by time-lapse confocal microscopy at 30 s intervals. Plates show maximal projections of confocal images. Scale bars, 10 ìm.
Mentions: To address whether exosomes mediate the transfer of miRNA during cognate immune interactions, we first studied the intracellular distribution of MVBs—the compartments from which exosomes arise—during the formation of an IS. In these experiments, Raji B cells were pulsed with Staphylococcus enterotoxin superantigen-E (SEE) and then incubated with TCR-Vβ8+ J77 T cells. MVB localization was assessed by immunofluorescence analysis of CD63 and two components of the ESCRT (endosomal sorting complex required for transport): Hrs, a component of the ESCRT-0 complex; and the late acting ESCRT machinery component VPS4. In the presence of SEE, which promotes formation of a fully functional IS, the MVBs of the J77 T lymphocyte lost their random cytoplasmic distribution and congregated near the IS (identified by CD3 and actin staining); in contrast, the localization of MVBs in the Raji B cell remained unchanged (Fig. 3a, and Supplementary Fig. S3). Similar results were obtained in experiments in which CH7C17 T cells, bearing an influenza hemagglutinin (HA) peptide-specific TCR, were conjugated to HA peptide-pulsed Hom2 B cells, thus confirming that antigen-induced formation of an IS polarizes T-cell MVBs to the contact site (Fig. 3b,c). Live cell imaging of CD63-GFP-expressing J77 T cells encountering SEE-pulsed Raji B cells revealed that the MVBs of the T cells move to the IS during the first 10 min (Fig. 3d, and Supplementary Movie 1).

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