MicroRNA-Containing T-Regulatory-Cell-Derived Exosomes Suppress Pathogenic T Helper 1 Cells

1Inflammation after infection, autoimmunity, or allergy is controlled to prevent immune-mediated pathologies. T regulatory (Treg) cells prevent such inflammatory damage, but the precise mechanisms of suppression are incompletely understood. RNA interference (RNAi), pioneered in plants and worms, provides a mechanism of sequence-specific gene silencing, which functions in a cell-autonomous and non-cell-autonomous manner. Multivesicular bodies (MVBs) release intracellular vesicles formed from endosomal membrane, including exosomes, that have encapsulated cystolic contents. This exosomal pathway can sample miRNAs from donor cells and transfer miRNAs and other bioactive material between cells, providing a vehicle for intercellular communication. Indeed, miRNA-containing exosomes have been isolated from various bodily fluids and can regulate gene expression in various target cells.

2Within the immune system, exosomes are released by a variety of innate and adaptive immune cells with thymus-derived exosomes capable of influencing lymphocyte differentiation. Furthermore, T-lymphocyte-derived exosomes can transfer miRNAs to dendritic cells, modulating gene expression in recipient cells. In this study, we tested whether Treg cells release exosomes and whether miRNA-containing exosomes contribute to Treg-cell-mediated control of immune responses. In summary, we observed that Treg cells released a significant quantity of miRNA-containing exosomes and that miRNAs were transferred to Th1 cells in vitro and in vivo. Coculture and transcriptional analyses identified that Treg cells transferred Let-7d to Th1 cells, suppressing Th1 cell proliferation and IFN-γ secretion. More importantly, miRNA biogenesis and the ability of Treg cells to release exosomes were both required for Treg cells to suppress Th1 cell proliferation in vivo and prevent systemic disease.

3Exosome release was quantified from different lymphocytes, including CD4+ and CD8+ naive T cells, T helper 1 (Th1) and Th17 cells, Treg cells, and CD19+ B cells, using CD63 as an exosomal surface marker, which correlated with other exosome markers including CD9 and CD81 (Figures S1A–S1C available online). After activation of various T cells, with anti-CD3 and anti-CD28, or B cells, with CD40L and LPS, Treg cells released more CD63+ exosomes per cell than other lymphocytes, despite only a minor change in cell number (Figures 1A, S1D, and S1E). Factors that regulate Treg cells, including interleukin 2 (IL-2), Amphiregulin (Areg), or all-trans retinoic acid (AT-RA), also regulated exosome release (Figures S1F and S1G) without any appreciable change in Treg cell number or metabolic activity over the culture period (Figure S1H). Despite 60% fewer exosomes released from naive T cells compared to Treg cells, exosome release from naive CD4+ T cells was also regulated by IL-2 (Figure S1G). Exosome release is regulated by changes in intracellular calcium (Ca2+), hypoxia, and sphingolipid ceramide synthesis. Similarly, exosome release by Treg cells was regulated by the calcium ionophore, monensin, hypoxia, and ceramide biosynthesis (Figure S1F).

4miRNAs are essential for Treg-cell-autonomous functions. However, miRNAs have also been observed in exosomes, released from Jurkat T cell lines and other T cell clones, raising the possibility that miRNAs may also function in a non-T-cell-autonomous manner. Using genome-wide miRNA analysis, we found that Treg-cell-derived exosomes contained both premature and mature miRNAs (Figures 1B–1E, Table S1). Furthermore, the composition of miRNAs in Treg-cell-derived exosomes was distinct from that of parental Treg cells. Of note, the most abundant miRNAs in Treg-cell-derived exosomes (miR-466 family, miR-195, and miR-16) have either proapoptotic or antiproliferative properties. Microarray analysis of Th1- and Th2-cell-derived exosomes revealed a different suite of miRNAs to those identified in Treg-cell-derived exosomes (Figures S1I–S1K), suggesting that different T cells release different miRNAs. In addition to miRNAs, genome-wide mRNA analysis identified many mRNA transcripts enriched in Treg-cell-derived exosomes (Figure S2 and Table S2). Unbiased pathway analysis of mRNA transcripts in Treg-cell-derived exosomes identified a profile consistent with the suppression of T cells (Figure S2D), suggesting that Treg-cell-derived exosomes may be involved in T cell regulation.

5To test whther RNA was transferred between immune cells, we developed a flow-cytometry-based system using Treg cells transfected with fluorescent oligonucleotide duplexes (FL-dsRNA) (Figure 2A), allowing us to track Treg-cell-derived dsRNA in single cells. When we cocultured Treg cells with other leukocytes, Treg cells transferred FL-dsRNA to ∼60% of cocultured naive T cells (CD3+CD4+CD25–) or other Treg cells (CD3+CD4+Foxp3rfp+CD25+) and to ∼40% of cocultured DCs (CD11c+MHCIIhi) or B cells (CD3–CD19+) (Figure 2B). Using an in vitro suppression assay with a decreasing number of FL-dsRNA-transfected Treg cells to conventional T effector (Teff) cells, we observed a dose-dependent transfer of FL-dsRNA to Teff cells (Figure 2C), with the amount of FL-dsRNA transferred correlated with the decrease in target cell proliferation. These data suggest that transfer of RNA from Treg cells to conventional T cells may be involved in the suppression of conventional T cells.

6RNA can be transferred from T cells to antigen-presenting cells (APCs) and between various APC populations. However, addition of APCs to Treg cell and conventional T cell cocultures had no impact on RNA transfer to conventional T cells (Figure 2D). HIV-1 virus can be transferred between T cells in nanotubules in a cell-contact-dependent manner. However, when Treg cells and conventional T cells were cultured in a transwell system, physically separating Treg cell and conventional T cells beyond the reach of nanotubes, RNA transfer was only slightly reduced (Figure 2E), suggesting transfer of RNA by an extracellular microvesicle-related mechanism.

7To determine whether FL-dsRNA was being transferred into the cytoplasm of recipient conventional T cells from donor Treg cells, rather than simply decorating the surface of recipient cells, we cocultured Treg cells and conventional T cells and then FACS purified each population after coculture. Using anti-CD45.1 membrane stain on the recipient cell, we verified that the FL-dsRNA was indeed delivered inside the recipient cell (Figure 2F).

8Next, we took advantage of the dependency on Dicer for mature miRNA processing to formally test whether miRNAs were transferred from Treg cells to conventional T cells in vitro. Treg cells from miRNA-sufficient CD45.2 WT mice were cocultured with miRNA-deficient conventional T cells from congenic CD45.1+Cd4CreDicerfl/flR26eyfp (Dicer–/–) mice, which were labeled with cell trace violet (Figure 3A). This system allowed us to (1) accurately discriminate and separate each population, (2) assess proliferation of Dicer–/– conventional T cells, and (3) repurify Dicer–/– conventional T cells after coculture to identify the presence of any acquired miRNAs. As expected, Treg cells suppressed (Figure 3B) and transcriptionally altered (Figure 3C) Dicer–/– T cells. Several inflammatory cytokines (Il22, Il2, il17a, Ifng) were downregulated in “suppressed” Dicer–/– T cells, with the concomitant increase in several regulatory genes (Cd73, Foxp3, Ikzf2 [encoding Helios], and Cd103) (Figure 3C, Table S3), relative to Dicer–/– T cells cultured alone. Concurrently, Dicer–/– conventional T cells acquired three mature (miR-155, Let-7b, and Let-7d) and one pre-miRNA (Hp_miR-344d-2) from Treg cells (Figures 3D and S2E). Using a Dicer-sufficient (WT) congenic system with CD45.2+ WT Treg cells and CD45.1+ WT conventional T cells, we also observed an increase in miR-155, Let-7d, and Let-7b in Dicer-sufficient WT conventional T cells, when cocultured with WT Treg cells (Figure S2F), further supporting the observation that miRNAs were transferred between cells. Finally, using CD45.2+ mir155–/– conventional T cells as recipient cells, cocultured with CD45.1+ mir155+/+ (WT) Treg cells, we confirmed the transfer of miR-155 from Treg to mir155–/– conventional T cells (Figure S2G). Taken together these data support the notion that Treg cells transfer specific miRNAs to conventional T cells, suppressing effector gene expression and proliferation.

9The adoptive transfer of Treg-cell-depleted CD4+CD45RBhi T cells into T-cell-deficient mice leads to systemic inflammation, which can be prevented by the cotransfer of Treg cells (Figures S3A–S3E). Despite the loss of miRNAs, Dicer–/– CD45RBhi T cells also caused a similar wasting disease with systemic inflammation (Figures 4A and 4B) and IFN-γ production (Figures 4C, 4D, and S3F–S3H), which could be prevented by cotransfer of WT, but not Dicer–/–, Treg cells. Because Dicer–/– CD45RBhi cells retained pathogenicity and sensitivity to Treg-cell-mediated control, we were able to test whether miRNAs were transferred to Dicer–/– CD45RBhi cells in vivo. After 5 weeks, “pathogenic” CD4+Dicer–/– YFP+ (Dicer–/– CD45RBhi cells transferred alone) or “regulated” CD4+Dicer–/– YFP+ (Dicer–/– CD45RBhi cells cotransferred with WT Treg cells) were recovered ex vivo to determine whether Dicer–/– cells acquired miRNAs in vivo (Figure S4A). Consistent with a suppressed state, regulated CD4+Dicer–/– YFP+ cells had reduced Ifng and Tnf expression (Figure 4D), compared to pathogenic CD4+Dicer–/– YFP+ cells. miRNA analysis of CD4+Dicer–/– CD45RBhi cells pretransfer and pathogenic and regulated CD4+Dicer–/– YFP+ cells isolated ex vivo confirmed our in vitro observations (Figure 3) and identified the presence of miR-155, Let-7b, and Let-7d in regulated CD4+Dicer–/– YFP+ cells, when WT Treg cells had been cotransferred (Figure 4E). In contrast, miR-155, Let-7b, and Let-7d weres not observed in pathogenic CD4+Dicer–/– YFP+ cells, when no Treg cells were transferred, suggesting that WT Treg cells either supported or directly transferred miRNAs to Dicer–/– cells. Relative to a housekeeping small RNA, RNU6B, regulated CD4+Dicer–/– YFP+ cells had almost as much miR-155, Let-7b, and Let-7d as WT Treg cells pretransfer, suggesting that a large amount of RNA was being transferred. Of note, WT Treg cells recovered ex vivo had elevated expression of miR-155, Let-7b, and Let-7d compared to WT Treg cells pretransfer (Figures 4E and S4B), suggesting that activated Treg cells also increase transcription of these miRNAs.

10Within miRNA-sufficient Rag2–/– hosts, it was conceivable that the regulated CD4+Dicer–/– YFP+ cells acquired miRNAs from non-Treg cells. We therefore used an additional control of Dicer–/– Treg cells, cotransferred with Dicer–/– CD45RBhi cells. Dicer–/– Treg cells failed to suppress disease. Furthermore, Dicer–/–CD4+YFP+ cells from mice that were cotransferred with Dicer–/– Treg cells did not have measurable miR-155, Let-7b, or Let-7d (Figure 4E). These data demonstrate that Treg-cell-mediated suppression is accompanied by the transfer of these three, and possibly other, miRNAs from Treg cells.

11Exosome release requires Rab27a and Rab27b for docking multivesicular endosomes (MVE) to Rab27 effectors on the plasma membrane. To test the role of Rab27 and exosome release, we purified Treg cells from Rab27ashen/ashenRab27b–/– double knockout mice (Rab27-DKO) and stimulated these cells, as above. Compared to WT and Dicer–/– Treg cells, Rab27-DKO Treg cells failed to release exosomes with no change in cell viability (Figures 5A and 5B), confirming the requirement of Rab27 for exosome release from Treg cells. Under hypoxic conditions, exosome release was significantly curtailed in WT and Dicer–/– Treg cells (Figure 5B). Rab27-DKO Treg cells also failed to transfer FL-dsRNA from Treg cells to conventional Teff cells (Figure 5C), indicating that a Rab27-regulated exosomal pathway was responsible for transferring RNA between T cells. Furthermore, when cocultured with Th1 cells, Rab27-DKO Treg cells failed to suppress Th1 cells, similar to Dicer–/– Treg cells (Figure 5D; ). These data demonstrate that Rab27 is essential for (1) exosome release from Treg cells, (2) RNA transfer from Treg cells to other T cells, and (3) proficient Treg-cell-mediated suppression in vitro.

12Beyond a failure to release exosomes, purified splenic Rab27-DKO Treg cells were very similar to WT Treg cells, with a comparable frequency (Figure S5A), transcriptional profile (Figures S5B, qRT-PCR, and S5C, microarray), and intracellular and surface Treg cell marker and tetraspanin expression profile (Figure S5D), indicating that Rab27 was not required for Treg cell development or many other features of Treg cells. Furthermore, the deletion of Rab27 did not impact the ability of Rab27-DKO Th cells to secrete cytokines (Figure S5E), migrate, and mediate disease when transferred into Rag2–/– hosts in vivo (Figures S5F–S5H), indicating that Rab27a and Rab27b, unlike SNAREs and Rho GTPases, are primarily required for the exosomal pathway.

13To test whether Rab27-dependent exosome release contributed to Treg cell function in vivo, we transferred WT or Rab27-DKO Treg cells with WT CD45RBhi T cells into Rag2–/– hosts. Similar to miRNA-ablated Dicer–/– Treg cells, Rab27-DKO Treg cells failed to prevent disease with significantly elevated Ifng expression (Figure 5E), colon shortening (Figure 5F), and weight loss (Figure 5G) with IFN-γ+ T cell recruitment (Figure 5H), despite a similar frequency of Treg cells observed (Figure S6A). Failure of Rab27-DKO Treg cells to control effector T cells also led to significant colonic and systemic inflammation and Ifng expression (Figures 5I, S6B, and S6C), demonstrating that Rab27-dependent exosome release was essential for Treg cell function in vitro and in vivo.

14Using Dicer–/– CD4+CD45RBhi effector cells cotransferred with WT, Dicer–/–, or Rab27-DKO Treg cells, we identified increased amounts of miR-155, Let-7b, and Let-7d in ex vivo regulated CD4+Dicer–/– YFP+ cells that were cotransferred with WT, but not Dicer–/– or Rab27-DKO, Treg cells (Figure 5J). These data support and extend our in vitro and in vivo observations, indicating that both Dicer and Rab27 sufficiency in Treg cells is required for the transfer of miRNAs, suppression of effector T cells, and prevention of disease. Of note, Rab27-DKO mice suffered from mild pulmonary inflammation with increased granulocyte influx and abnormal airway epithelium. Upon further examination, aged Rab27-DKO mice had inflammatory foci in the lung, liver, and colon, which may be due to compromised Treg-cell-mediated immune regulation (Figure S6D).

15To determine whether miRNAs in Treg-cell-derived exosomes were suppressive, we purified exosomes released from WT and Dicer–/– Treg cells. WT, but not Dicer–/–, Treg cells suppressed Th1 cell proliferation, as previously reported. Similarly, 107 exosomes from WT, but not Dicer–/–, Treg cells suppressed Th1 cell proliferation (Figures 6A, 6B, S6E, and S6F) and IFN-γ secretion (Figure 6C), indicating that exosomes were suppressive in a Dicer-dependent manner. Although Th1 cells could transfer RNA to conventional T cells in vitro (Figure S6G), Th1-cell-derived exosomes had no impact on Th1 cell differentiation (Figure S6H). Th1 cells had reduced amounts of Let-7d, compared to naive T cells (Figure S7A), and Th1-cell-derived exosomes were devoid of Let-7d (Figure S1I), providing one explanation of why Th1-cell-derived exosomes could not suppress Th1 cells.

16Following the observation that miR-155, Let-7b, and Let-7d were transferred from Treg cells to conventional T cells, we tested whether miR-155, Let-7b, or Let-7d, alone or in combination, were responsible for suppression of Th1 cells. Transfection of Th1-cell-polarized WT or Dicer–/– T cells with miRNA mimics of miR-155, Let-7b, Let-7d, or all three miRNAs increased the expression of each respective miRNA (Figure 6D). Consistent with previous reports, miR-155 enhanced IFN-γ and TNF-α secretion, without any appreciable change in proliferation (Figures 6E and 6F), indicating that miR-155 promoted, rather than inhibited, Th1 cell responses. Transfection of Th1 cells with Let-7b had little impact on IFN-γ production or proliferation (Figures 6E and 6F). In contrast, transfection with Let-7d, alone or in combination with either Let-7b or miR-155, significantly reduced Tnf and Ifng mRNA (Figure 6E), Th1 cell proliferation, and IFN-γ secretion (Figure 6F), suggesting that the transfer of Let-7d from Treg cells to Th1 cells may be an important intermediary of Treg-cell-mediated suppression of Th1 cells.

17Target prediction analyses identified many putative Let-7d targets involved in Th1 cells (Il12r, Tbx21, Stat1 and Stat4, and Ifng). We therefore analyzed the miRNA and mRNA profile of FACS-purified IFN-γyfp+ Th1 cells, compared to naive T cells, and identified reduced premature and mature Let-7d with an increase in many Let-7d target genes (Figure S7A). Specifically, Let-7d was predicted to target Ptgs2 (Cox-2), which was elevated in Th1 cells (Figures S6 and S7). Testing the requirement of Cox-2 using a selective Cox-2 inhibitor (celecoxib), we noted that Cox-2 inhibition ablated IFN-γ secretion (Figure S6J), indicating that Cox-2 is required for Th1 cell responses. Thus, Let-7d may target Cox-2 in Th1 cells to regulate IFN-γ secretion.

18To test whether Let-7d from Treg-cell-derived exosomes contributed to the suppression of Th1 cell responses in vitro, we transfected Th1 cells with Let-7d hairpin inhibitors, to inhibit endogenous or exogenous Let-7d, and cocultured these cells with Treg cells or exosomes purified from Treg cells. Let-7d inhibitor treatment alone did not impact Th1 cell proliferation or IFN-γ secretion in the absence of Treg cells (Figures 7A, 7B, and S7E). The addition of Treg cells or Treg-cell-derived exosomes suppressed Th1 cell proliferation (Figures 7A and 7B) and IFN-γ secretion (Figure 7C). However, Treg cells and Treg-cell-derived exosomes failed to suppress Let-7d inhibitor-treated Th1 cells, indicating that sequestering endogenous or exogenous Let-7d in Th1 cells prevented Treg cell and Treg cell exosome-mediated suppression (Figures 7A–7C). To further test whether Let-7d from Treg cells contributed to Treg cell- and Treg-cell-derived exosome-mediated suppression, we transfected Treg cells with Let-7d hairpin inhibitors or control inhibitors (Figures 7D and S7). Let-7d inhibitor treatment reduced Let-7d by ∼50% in Treg cells and by ∼75% in Treg-cell-derived exosomes without any appreciable impact on cell viability (Figure 7D). Let-7d inhibitor-treated Treg cells, but not control inhibitor-treated Treg cells (Figure S7), were compromised in their ability to suppress Th1 cells, and Let-7d-depleted exosomes had completely lost their ability to suppress Th1 cells (Figures 7D and 7E). Although it remains possible that Let-7d inhibitors were transferred with Let-7d to Th1 cells in this experimental setup, taken together with the reduced Let-7d in Th1 cells (Figure S7) and the fact that Let-7d inhibitor-treated Th1 cells when cultured alone proliferated and secreted IFN-γ similar to control Th1 cells (Figures 7A and 7B, black bars), these data indicate that Let-7d contributes to Treg-cell-mediated suppression and more specifically, that Let-7d was responsible for Treg-cell-derived exosome-mediated suppression.

19Finally, we tested whether Let-7d was required for Treg-cell-mediated suppression in vivo by transferring mock or Let-7d inhibitor-treated WT Treg cells into Rag2–/– hosts with WT CD45RBhi cells. Transfected Treg cells survive only 7–10 days in vivo, so we transferred Treg cells weekly for 3 weeks (weeks 2, 3, and 4) before analysis at week 5. Mock-transfected Treg cells prevented weight loss, Ifng responses, and intestinal inflammation (Figures 7F–7H). In contrast, Let-7d inhibitor-treated Treg cells, which released Let-7d-depleted exosomes (Figure 7C), failed to prevent disease with significant weight loss, Ifng expression, and colonic inflammation (Figures 7F–7H). Collectively, these data support a mechanism of Treg-cell-mediated suppression through the release of miRNA-containing exosomes. Furthermore, we identified an important contribution of Let-7d in mediating this suppression in vitro and in vivo.

20The control of immune responses is critical for survival, with compromised Treg-cell-mediated control resulting in multiorgan lymphocytic infiltration, organ failure, and death. In this study, we identified a mechanism of Treg-cell-mediated suppression via non-cell-autonomous gene regulation, mediated by miRNA-containing exosomes. Exosome-deficient, miRNA-deficient, or Let-7d-ablated Treg cells all failed to transfer miRNAs and failed to prevent lethal systemic inflammation. These data support a growing paradigm of non-cell-autonomous gene regulation previously reported in plants and animals and now demonstrated here, functioning as an important feature of immune regulation.

21The mechanism of exosome release from Treg cells appears similar to other cells, with the exception of monensin treatment, which reduced exosome release from Treg cells, in contrast to exosome release from the K562 cell line. This might be due to the particularly sensitive nature of T cells to calcium changes or might indicate that Golgi-mediated protein secretions support exosome release. Several overlapping pathways appear to regulate Foxp3 stability and exosome release, including inactivation of HIF-1α, ceramide biosynthesis, IL-2 signaling, and Amphiregulin signaling, suggesting that Foxp3 might regulate exosome release or that exosome-associated pathways might support Foxp3+ cells. In support of the latter, we identified that miR-155, which is involved in Treg cell development, was transferred from Treg cell to conventional T cell with the concomitant upregulation of several Treg-cell-associated genes in recipient cells (Figure 3).

22Whether exosome contents are preferentially packaged within the parent cell or whether they are randomly assorted is not well understood. We previously identified significant miRNA heterogeneity between distinct Treg cell populations and found that such heterogeneity was influenced by the cytokine microenvironment. We report here that the miRNA content of Treg-cell-derived exosomes is distinct from the miRNA content of Th1- or Th2-cell-derived exosomes, indicating that cell/context-specific miRNAs may be packaged into exosomes. It is tempting to speculate that the local cytokine environment that shapes the miRNA profile of Treg cells also influences the exosome content of divergent T cells and that specific miRNA-containing exosomes are targeted to different effector cells. Here, we identified that Let-7d was preferentially packaged and transferred to Th1 cells, suppressing Th1 cell proliferation and IFN-γ secretion; whether different miRNAs are delivered to different Th cells is unclear.

23Similarly, the rate of packaging and transfer of miRNAs between cells has not been elucidated. Recent reports indicate that at the immunological synapse between T cells and APCs, a microvesicle-rich pocket is formed, providing a focused and protected region between cells, potentially allowing mass and rapid transfer of material. Whether miRNAs are transferred in these regions between T cell and APC was unclear from this study. However, for T cell-to-T cell transfer, the precise mechanism of exosome delivery is yet to be clarified. Furthermore, the precise mechanism of exosome uptake by recipient cell remains largely unknown.

24Regulatory T cells have a growing repertoire of means to inhibit and prevent immune cell activation. With respect to suppression of conventional T cells, current mechanisms include short-range, cell-contact-dependent (cytolysis and inhibitory receptor engagement) and potentially longer-range, cell-contact-independent (IL-2 consumption and suppressive cytokine secretions, such as IL-10, TGF-β, and IL-35) mechanisms. However, a combination of cell contact and secreted mechanisms is most likely optimal for Treg-cell-mediated suppression. From this study we can add non-cell-autonomous gene silencing as a mode of cell-contact-independent Treg-cell-mediated suppression, via transfer of miRNAs. We observed that isolated Treg-cell-derived exosomes can suppress conventional T cells; however, this was not as efficient as Treg cells, indicating that exosome-mediated transfer and additional mechanisms are indeed required for optimal suppression. Although cell contact was not required for the transfer of RNA between cells, whether cell contact allows more rapid and focused transfer, as mentioned above, is currently under investigation. Together these observations suggest that exosome-mediated transfer of miRNAs may collaborate with other Treg cell mechanisms for optimal suppression. For example, Treg cells anchored to other T cells via galectins or to APCs via CTLA-4 might be required for most efficient delivery of miRNA-containing exosomes to target cells. From data presented here, the ability of Treg cells to release microvesicles in a Rab27-dependent manner, and specifically transfer Let-7d to target cells, was required for optimal Treg-cell-mediated suppression in vitro and in vivo. Whether this mechanism works in concert with other previously described Treg-cell-associated mechanisms or not is currently unclear.

25Of the many potential Let-7d targets in recipient Th1 cells, we identified Ptgs2 (Cox-2) as a credible candidate (Figures S6 and S7). Cox-2 is involved in Th1-cell-mediated responses and is responsible for lethal T-cell-mediated inflammation. Whether Treg-cell-derived Let-7d-containing exosomes directly target Cox-2 in Th cells, analogous to a “Cox-2 inhibitor,” is currently unclear. Supporting this pathway we observed reduced expression of Cox-2 and other Th1-cell-associated genes in suppressed T cells in vitro, concomitant with the acquisition of Let-7d from WT Treg cells. Furthermore, treatment of T cells with a COX-2 inhibitor reduced IFN-γ production by Th1 cells (Figure S6J). Given that Cox-2 inhibitors can prevent Th1-cell-mediated experimental autoimmunity and colitis, targeting of Cox-2 by Treg-cell-derived Let-7d may be a specific Treg-cell-mediated function to prevent lethal Th1-cell-mediated inflammation. In summary, this mechanism of Treg-cell-mediated suppression opens up the possibility that Treg cells package and deliver different proteins and RNA species, including miRNAs as we report here, to different cells at different times in a context-dependent manner.

26Female C57BL/6, Rag2–/–, CD45.1 Cd4CreDicerfl/fl R26eyfp (Dicer–/–), Rab27aashen/ashenb–/– (Rab27-DKO), miR155–/–, Il4gfpIfngyfpIl17aCre, R26eFP635, Foxp3rfp, and Foxp3gfp 6- to 8-week-old (or as indicated) animals were bred and kept in the specific-pathogen-free facility at the Medical Research Council, NIMR. All animal experiments were performed according to institutional guidelines and the UK Home Office regulations. A minimum of five mice per group was used in each experiment, unless indicated.

27Rag2−/− mice were injected i.v. with 5 × 105 FACS-purified CD4+CD45RBhi T cells with or without 105 CD4+CD25+ Treg cells 2 weeks later, as previously described.

28Primary cells were isolated from naive or diseased spleen, lymph nodes, or tissue, as indicated, by mechanical disruption. Full details of antibodies used can be found in Supplemental Experimental Procedures. For suppression assays, 104 Teff cells were labeled with cell trace violet (Invitrogen) as per manufacturer’s guidelines and stimulated with plate-bound anti-CD3 (1 μg/ml) and soluble CD28 (5 μg/ml) for 3 days in the presence or absence of Treg cells, at the indicated ratios.

29FACS-purified cells or isolated exosomes were stored in RLT lysis buffer at –80°C until RNA was extracted using RNeasy mini spin columns (QIAGEN). For RT-PCR, miScript RT or Quantitect RT was performed, according to manufacturer’s recommendations (QIAGEN). Real-time RT-PCR was performed on an ABI Prism 7900HT Sequence Detection System (Applied Biosystems) with relative quantities of mRNA and miRNA determined using SYBR Green PCR Master Mix (Applied Biosystems) and by the comparative threshold cycle method as described by Applied Biosystems for the ABI Prism 7700/7900HT Sequence Detection Systems. mRNA levels were normalized to HPRT and miRNA levels were normalized to RNU6B and then expressed as a relative increase or decrease compared with levels in controls, or as indicated.

30IFN-γ was measured by ELISA using cytokine capture and biotinylated detection antibodies (R&D Systems). The concentration of IFN-γ was determined from a serial-fold diluted standard curve with OD read at 450 nm in an ELISA reader.

31Cells were transfected with 100 nM of FL-dsRNA (SiGlo, Thermo Scientific Dharmacon) or BloCKiT (Invitrogen), miR-155 mimics, Let-7d mimics or hairpin inhibitors, Let-7b mimics, Cel-miR-67 hairpin inhibitors, or Cel-miR-239b hairpin inhibitors (Thermo Scientific Dharmacon) or MOCK transfected using Nucelofection reagents according to manufacturer’s recommendations (Amaxa).

32Throughout these studies, complete media with exosome-depleted FCS (following 100,000 × g centrifugation) was used. Exosomes were purified by a combination of ultracentrifugation and using Exoquick solution (SBI System Bioscience), as previously reported and tested.

33Full details of exosome isolation and other methods can be found in the Supplemental Experimental Procedures.

34M.S.W. initiated the project, carried out most of the cell biological experiments, and contributed to the experimental design and data interpretation. I.S.O., V.S.P, S.M.C., S.C., and V.P. carried out experiments and helped with transfections. T.T. and M.C.S. provided animals, helped with interpretation of experiments, and provided input for the manuscript. M.S.W supervised the project and wrote the manuscript with input from the other authors and readers.