Qualitative differences in T‐cell activation by dendritic cell‐derived extracellular vesicle subtypes
2017; Springer Nature; Volume: 36; Issue: 20 Linguagem: Inglês
10.15252/embj.201696003
ISSN1460-2075
AutoresMercedes Tkach, Joanna Kowal, Andrés E. Zucchetti, Lotte Enserink, Mabel Jouve, Danielle Lankar, Michael Saitakis, Lorena Martín‐Jaular, Clotilde Théry,
Tópico(s)Cell Adhesion Molecules Research
ResumoArticle21 September 2017free access Source DataTransparent process Qualitative differences in T-cell activation by dendritic cell-derived extracellular vesicle subtypes Mercedes Tkach orcid.org/0000-0002-8011-9444 Institut Curie, PSL Research University, INSERM U932, Paris, France Search for more papers by this author Joanna Kowal orcid.org/0000-0001-7849-6040 Institut Curie, PSL Research University, INSERM U932, Paris, France Search for more papers by this author Andres E Zucchetti Institut Curie, PSL Research University, INSERM U932, Paris, France Search for more papers by this author Lotte Enserink Institut Curie, PSL Research University, INSERM U932, Paris, France Search for more papers by this author Mabel Jouve Institut Curie, PSL Research University, INSERM U932, Paris, France Search for more papers by this author Danielle Lankar Institut Curie, PSL Research University, INSERM U932, Paris, France Search for more papers by this author Michael Saitakis Institut Curie, PSL Research University, INSERM U932, Paris, France Search for more papers by this author Lorena Martin-Jaular orcid.org/0000-0002-1511-8576 Institut Curie, PSL Research University, INSERM U932, Paris, France Search for more papers by this author Clotilde Théry Corresponding Author [email protected] orcid.org/0000-0001-8294-6884 Institut Curie, PSL Research University, INSERM U932, Paris, France Search for more papers by this author Mercedes Tkach orcid.org/0000-0002-8011-9444 Institut Curie, PSL Research University, INSERM U932, Paris, France Search for more papers by this author Joanna Kowal orcid.org/0000-0001-7849-6040 Institut Curie, PSL Research University, INSERM U932, Paris, France Search for more papers by this author Andres E Zucchetti Institut Curie, PSL Research University, INSERM U932, Paris, France Search for more papers by this author Lotte Enserink Institut Curie, PSL Research University, INSERM U932, Paris, France Search for more papers by this author Mabel Jouve Institut Curie, PSL Research University, INSERM U932, Paris, France Search for more papers by this author Danielle Lankar Institut Curie, PSL Research University, INSERM U932, Paris, France Search for more papers by this author Michael Saitakis Institut Curie, PSL Research University, INSERM U932, Paris, France Search for more papers by this author Lorena Martin-Jaular orcid.org/0000-0002-1511-8576 Institut Curie, PSL Research University, INSERM U932, Paris, France Search for more papers by this author Clotilde Théry Corresponding Author [email protected] orcid.org/0000-0001-8294-6884 Institut Curie, PSL Research University, INSERM U932, Paris, France Search for more papers by this author Author Information Mercedes Tkach1, Joanna Kowal1,†, Andres E Zucchetti1, Lotte Enserink1, Mabel Jouve1, Danielle Lankar1, Michael Saitakis1, Lorena Martin-Jaular1 and Clotilde Théry *,1 1Institut Curie, PSL Research University, INSERM U932, Paris, France †Present address: Ludwig Institute for Cancer Research, Department of Oncology, University of Lausanne, Lausanne, Switzerland *Corresponding author. Tel: +33156246716; E-mail: [email protected] EMBO J (2017)36:3012-3028https://doi.org/10.15252/embj.201696003 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Exosomes, nano-sized secreted extracellular vesicles (EVs), are actively studied for their diagnostic and therapeutic potential. In particular, exosomes secreted by dendritic cells (DCs) have been shown to carry MHC-peptide complexes allowing efficient activation of T lymphocytes, thus displaying potential as promoters of adaptive immune responses. DCs also secrete other types of EVs of different size, subcellular origin and protein composition, whose immune capacities have not been yet compared to those of exosomes. Here, we show that large EVs (lEVs) released by human DCs are as efficient as small EVs (sEVs), including exosomes, to induce CD4+ T-cell activation in vitro. When released by immature DCs, however, lEVs and sEVs differ in their capacity to orient T helper (Th) cell responses, the former favouring secretion of Th2 cytokines, whereas the latter promote Th1 cytokine secretion (IFN-γ). Upon DC maturation, however, these functional differences are abolished, and all EVs become able to induce IFN-γ. Our results highlight the need to comprehensively compare the functionalities of EV subtypes in all patho/physiological systems where exosomes are claimed to perform critical roles. Synopsis Dendritic cell (DC)-derived extracellular vesicle (EV) subtypes show functional heterogeneity depending on the DC maturation stage, thus highlighting the importance of characterizing all EV subtypes for their function. Human primary dendritic cell (DC)-derived EVs of different sizes, including large EVs and mixed exosomes and non-exosomal small EVs, induce equally well activation and proliferation of allogeneic T lymphocytes. When produced by immature DCs, large EVs induce prominent secretion of Th2-associated cytokines, whereas medium and small EVs induce secretion of Th1-associated cytokines. The different qualitative activities of large and small/medium EVs are due to different ratios of EV surface-exposed T cell-binding proteins CD40, DC-SIGN (abundant only on small/medium EVs) and CD80 (present on all EVs). When produced by IFN-γ-matured DCs, all EVs induce prominent secretion of Th1-associated cytokines, thus displaying similar functional activities. Introduction Cells secrete into their environment different types of membrane-enclosed structures, collectively called extracellular vesicles (EVs), which play an important role in cell-to-cell communication (Raposo & Stoorvogel, 2013; Colombo et al, 2014; Yanez-Mo et al, 2015). EVs have been shown to contain proteins and nucleic acids that can be transferred to recipient cells, modifying their physiological state (Kosaka et al, 2016; Tkach & Théry, 2016). Live cells release very heterogeneous EV populations that have distinct structural and biochemical properties depending on their subcellular site of origin. Exosomes are a subtype of EVs that are formed as intraluminal vesicles of multivesicular compartments of the endocytic pathway, and they therefore present a mean diameter of 50–150 nm, similar to the diameter of intraluminal vesicles. On the other hand, microvesicles display a diverse range of sizes (100–1,000 nm in diameter) and are formed and released by budding from the cells’ plasma membrane. Cells undergoing apoptosis also release EVs, generally called “apoptotic bodies”, with a wide range of sizes (from 50 nm to bigger than 1,000 nm) (Crescitelli et al, 2013; Colombo et al, 2014). EVs are commonly isolated and concentrated by differential ultracentrifugation (Thery et al, 2006). At each step of centrifugation, enrichment of different EVs is obtained. Large vesicles (lEVs) are pelleted at low speed centrifugation (2,000 × g), while vesicles smaller than 150 nm constitute the majority of EVs recovered at the last step of high-speed ultracentrifugation (100,000 × g) (Kowal et al, 2016). In the literature, the latter EVs are generally called exosomes (Gould & Raposo, 2013). However, we now know that this small EV pellet is composed not only of exosomes but also of other non-exosomal vesicles (Kowal et al, 2016). We will thus use the term “small EVs (sEVs)” instead of “exosomes” in this article. In the past years, numerous studies have described functions of sEVs, in all pathological and physiological systems, from cancer to cardiovascular function, as well as development or neurobiology (Yanez-Mo et al, 2015). Induction of T lymphocyte-dependent immune responses by sEVs secreted by professional antigen presenting cells like B lymphocytes (Raposo et al, 1996) or dendritic cells (DCs) (Zitvogel et al, 1998; Thery et al, 2002; Segura et al, 2005) provided the first evidence that secretion of EVs could play a communication role between cells (Thery et al, 2009; Robbins & Morelli, 2014). DCs play a fundamental role in the initiation of immune responses (Merad et al, 2013) by presenting exogenous antigens to CD8+ T lymphocytes as peptides loaded onto MHC class I molecules (a process called cross-presentation), or to CD4+ T lymphocytes as peptides bound to MHC class II molecules. Consistently, human DC-derived sEVs have been shown to bear functional MHC class I and class II molecules that can be loaded with specific peptides to activate cognate T cells (Hsu et al, 2003; Admyre et al, 2006), demonstrating the capacity of DCs to spread immune responses through sEVs. These findings motivated the use of DC-derived sEVs to boost anti-tumour immune responses in cancer clinical trials (Escudier et al, 2005; Morse et al, 2005; Besse et al, 2016), although with limited clinical effects. We have recently demonstrated that human DCs secrete simultaneously abundant and heterogeneous EVs of different sizes and from different intracellular origins, which all bear MHC class I and class II molecules (Kowal et al, 2016). These other types of EVs may thus represent alternative immunotherapy tools. However, side-by-side comparison of the immune effects of sEVs and other EVs has never been comprehensively performed. Therefore, it is unclear whether exosomes, despite the numerous studies describing their functions, are the best or only EVs with interesting therapeutic potential. Here, we have isolated different subtypes of EVs simultaneously released by live human primary DCs and characterized their effects on primary CD4+ T lymphocytes in vitro. Our results show that both lEVs and sEVs secreted by immature DCs efficiently induce activation of T cells, but these activated T cells release different cytokines. Particularly, lEVs promote the secretion of Th2 cytokines like IL-4, IL-5 and IL-13 while sEVs induce the secretion of the Th1 cytokine IFN-γ. sEVs bearing the IFN-γ-inducing activity were recovered in intermediate floating fractions of a density gradient, with the most efficient sEVs in a subfraction not enriched in bona fide exosomes (Kowal et al, 2016). In addition, the most efficient IL-13-inducing EVs were recovered in floating fractions of the gradient displaying a higher density than sEVs. We could assign part of the activity of EVs to some transmembrane receptors involved in T-cell activation: CD40 and DC-SIGN for sEVs, and CD80 for lEVs. Upon DC maturation; however, all types of EVs became equally efficient to induce IFN-γ secretion by Th lymphocytes, that is the kind of immune response expected to be efficient in anti-tumour immunotherapy. Results Characterization of different types of EVs secreted by human DCs We have shown that human primary DCs differentiated in vitro from monocytes secrete a heterogeneous range of EVs, which can be in part separated by their pelleting properties (Thery et al, 2006; Kowal et al, 2016). In this previous work, we extensively characterized by transmission electron microscopy (TEM) and Western blot (WB) the lEVs recovered by low speed centrifugation (2,000 × g = 2K), intermediate speed (10,000 × g = 10K) centrifugation and sEVs pelleted by high-speed (100,000 × g = 100K) ultracentrifugation. Our previous measuring of EV sizes on TEM pictures had shown that 95% of EVs were smaller than 200 nm in the 100K pellet, 85% in the 10K pellet and less than 50% in the 2K pellet (Kowal et al, 2016). Nanoparticle Tracking Analysis (NTA, Fig 1A) showed similar distributions, with 74.4% particles smaller than 200 nm in the 100K pellet (median size of 155.6 nm), less than 45.1% in the 2K pellet, which contained vesicles as big as 500 nm (median size of 199.7 nm), and a combination of both in the intermediate 10K pellet (median size 168.2 nm). The 2K pellet contained generally lower numbers of particles than the 100K, as quantified by NTA (Fig 1B), but more total proteins (Kowal et al, 2016), suggesting its enrichment in large, protein-containing EVs. This was confirmed by scanning electron microscopy (SEM) imaging of the pellets loaded on slides coated with poly-l-lysine, which ensures efficient capture of all vesicles (Fig 1C). We observed numerous spherically shaped vesicles larger than 500 nm in diameter in the 2K pellet: these lEVs are clearly distinct from irregular-shape debris (arrow, Fig 1C, 2K panel). The 2K pellet also contains EVs of about 200–500 nm in diameter, and a few even smaller EVs. While analysing the morphology of the whole DC by SEM, we observed various protrusions on the plasma membrane, including spherical structures of 100–250 nm, and larger tubules and ruffles that we speculate can lead to the formation of EVs (Fig EV1A). The 100K pellet contains a vast majority of the smallest EVs (< 100 nm, Fig 1C, 100K panel, enlargement), but also some rare intermediate size EVs. The 10K pellet contains a mixture of all these EVs. SEM therefore represents a complementary imaging technique providing a comprehensive view of the size and shape of EVs, and demonstrating the actual vesicular nature of the lEVs. Figure 1. Characterization of DC-derived EVs recovered in successive differential ultracentrifugation pellets A, B. The different pellets of EVs isolated from DCs (2K = 2,000 × g; 10K = 10,000 × g; 100K = 100,000 × g) were analysed by Nanoparticle Tracking Analysis (NTA). Each line corresponds to the mean ± SEM of four acquired donor samples for each pellet. A wide range of particle size is observed in the 2K and 10K pellet, whereas the size range of particles in the 100K pellets is more restricted (A). The number of purified particles secreted per million cells tracked by NTA for each pellet (2K, 10K and 100K) was calculated using the NTA3.2 software (B) (n = 4, one symbol per donor). Red line indicates median. C. The 2K, 10K and 100K pellets were loaded on poly-lysine-coated slides, fixed and analysed by scanning EM (SEM). Inset in the 100K pellet image shows another field with higher magnification. Arrow in 2K panel indicates a cell debris. Scale bars = 1 μm. D. The whole number of MHC II molecules on the EVs was quantified coupling the different pellets to beads and incubating them with mouse antibodies against HLA-DR/DQ/DP. The absolute number of MHC II molecules per pellet was determined using calibration beads (see Materials and Methods for assay description) (n = 4, one symbol per donor). Red line indicates median. E. The successive pellets were analysed by flow cytometry, to measure the overall level of surface expression of various markers. EVs were detected in a FSC/SSC gate, which did not contain any events when dilutions of antibodies in filtered PBS in the absence of EV pellets were analysed (upper panel). EVs were stained for the CD9 tetraspanin and immune molecules (HLA-ABC, HLA-DR and CD86) (red histogram). Isotype antibodies were used as control (black line). The specific mean fluorescence intensity (MFI with antibody–MFI with isotype control) was calculated as value of global molecule exposure on the bulk EV pellets. Representative histograms are shown in the lower left panel, and the quantification of the specific MFI are shown in the lower right panel (n = 8–12, one symbol per donor). Red line indicates median. *P < 0.05; **P < 0.01; (Friedman test). Download figure Download PowerPoint Click here to expand this figure. Figure EV1. SEM of immature DCs and whole-mount TEM analysis of each pellet DCs were loaded on poly-lysine-coated slides, fixed and analysed by SEM. Two DCs are shown as representative images. Scale bars = 1 μm for the whole cell and 100 nm for the inset. Whole-mount EVs were stained for MHC II (PAG10) and analysed by TEM. Images are shown for the different pellets. Download figure Download PowerPoint By WB and quantitative proteomic analyses, we had observed that the tetraspanin CD9 was present in all pellets but enriched in the sEVs, whereas equivalent levels of the immunologically relevant MHC class I and class II molecules were recovered in all EV pellets (Kowal et al, 2016). Thus, we next analysed whether the relevant immune molecules (i.e. MHC molecules) were equally displayed at the surface of all EVs, information that could not be retrieved from the analysis by WB of intracellular epitopes in lysates of EVs. We thereby used a FACS-based calibration assay previously developed (Viaud et al, 2011) to quantify the absolute number of MHC class II (HLA-DR, DQ and DP) molecules and observed not significantly different levels in all pellets (mean ± SEM: 2K = 7.6 × 1008 ± 1.9 × 1008, 10K = 6.7 × 1008 ± 9.5 × 1007 and 100K = 8.8 × 1008 ± 3.2 × 1008 molecules secreted on EVs per million cells) (Fig 1D). We have also analysed the presence of CD9, MHC class I (HLA-A/B/C), MHC class II (HLA-DR) and the co-stimulatory molecule CD86 by direct flow cytometry with antibodies recognizing extracellular domains from each protein (Fig 1E). Forward scatter (FSC) and side scatter (SSC) parameters of the flow cytometer were set to log scale and low threshold, to allow detection of antibody-dependent fluorescent signal in EV preparations above the background signal given by antibodies in the absence of EVs (Fig 1E, upper right panel), and by isotype control antibodies on EVs. Note that the flow cytometer used here cannot differentiate FSC/SSC properties of EVs or polystyrene beads of 100 nm, 400 nm or 1 μm in diameter (data not shown). Thus, it provides information about EVs as a bulk population and not single EVs. By using this assay, we could corroborate the presence of all the molecules in all EV pellets, although a clear enrichment of CD9 and CD86 was detected in sEVs. Besides, all different vesicles showed variable contents of MHC class I and II, with higher levels of HLA-DR on the 100K compared to the 2K pellet. Finally, by TEM, we observed MHC class II staining on the surface of several (but not all) EVs in the 10K and 100K pellet, and a particular pattern of MHC class II distribution on lEVs (> 500 nm), which often displayed high content of MHC class II on membrane patches (Fig EV1B). CD4+ T cells are activated by all DC-derived EV subtypes Since the 2K, 10K and 100K pellets all contain MHC class II, we decided to analyse whether the EVs could exert any immunomodulatory effect on CD4+ T cells. Given the biochemical complexity of EVs (proteins, lipids, nucleic acids), it is impossible to quantify their concentration in a rigorous manner in terms of molarity, as is done for single molecules. Furthermore, we observed that while the 2K pellet contained, in general, more proteins and fewer particles than the other two pellets, the 100K pellet contained, conversely, more particles and less proteins (Fig 1B and Kowal et al, 2016). Therefore, choosing either protein amount or particle number to equalize the amount of EVs used in functional assays would have biased the results in favour of either the 100K or the 2K pellet, respectively, leading to, potentially, opposite conclusions. We thus chose to expose T cells to EV pellets recovered from a given number of secreting DCs, that is a given volume of conditioned medium. Table 1 indicates the average particle number and protein content of each pellet used in such experiments. We first evaluated how the different EVs interacted with T cells, as compared to DC-derived sEVs, which are known to be recruited by these cells (Nolte-’t Hoen et al, 2009). We incubated freshly isolated total CD4+ T cells for 18 h with the different EVs labelled with DiO, and analysed them by confocal microscopy (Fig 2A) and flow cytometry (Figs 2B and EV2). Some CD4+ T cells became DiO+ upon treatment with each of the different EV pellets, with similar percentages of DiO+ cells observed in all conditions (Fig 2B), suggesting that CD4+ T cells bind all of the EV subtypes similarly. We did not observe any DiO-labelled CD4+ T cell when we used as control the DiO-labelled pellets of non-conditioned medium (Fig EV2). We then assessed T-cell activation by measuring the up-regulation of the early T-cell activation marker CD69. The three pellets induced surface expression of CD69 on allogeneic CD4+ T cells when compared to untreated cells (control), and the 100K was slightly more efficient than the 2K and the 10K (Fig 2C). Upon prolonged culture, CD4+ T cells proliferated in the presence of all EV pellets in a dose-dependent manner and at similar rates (Fig 2D). At the highest dose (EVs from 8 × 106 DCs), 2K, 10K and 100K pellets induced similar proliferations of CD4+ T cells, 19.8% (±2.9%), 16.6% (±2.8%) and 17.5% (±3.0%), respectively. T-cell proliferation was due to specific recognition of allogeneic MHC class II molecules in all EV subtypes, as it was decreased in the presence of antibodies against HLA-DP/DR/DQ (Fig 2E), and not observed when T cells were exposed to autologous EVs secreted by DCs of the same donor (Fig 2F). Note that to perform the latter experiment, we had to keep both autologous and allogeneic CD4+ T cells frozen during the 6 days of DC culture required for their differentiation and subsequent EV isolation, which resulted in a weaker responsiveness of T cells to the 10K and 100K pellets (compare Fig 2F and E). Finally, activation by allogeneic EVs was only observed on total CD4+ T cells: none of the EV pellets were able to induce proliferation of isolated naïve T cells (Fig 2G), as it was previously shown for mouse DC-derived sEVs (Thery et al, 2002). These observations indicate that all types of EVs, not only sEVs, induce direct activation of allogeneic CD4+ T cells in an MHC-dependent manner, and that presence of activated/memory cells is necessary for an efficient response. Table 1. Calculation of the average number of particles and total proteins present in the EV pellets from 1, 2, 4 and 8 million DCs Million of secreting cells Total number of particles Total protein (μg) 2K 10K 100K 2K 10K 100K 8 4.11 × 1008 ± 2.27 × 1007 5.18 × 1008 ± 3.83 × 1007 6.82 × 1008 ± 4.97 × 1007 23.2 ± 2.4 13.6 ± 2.4 8.8 ± 1.6 4 2.06 × 1008 ± 1.14 × 1007 2.59 × 1008 ± 1.92 × 1007 3.41 × 1008 ± 2.48 × 1007 11.6 ± 1.2 6.8 ± 1.2 4.4 ± 0.8 2 1.03 × 1008 ± 5.69 × 1006 1.29 × 1008 ± 9.58 × 1006 1.70 × 1008 ± 1.24 × 1007 5.8 ± 0.6 3.4 ± 0.6 2.2 ± 0.4 1 5.14 × 1007 ± 2.84 × 1006 6.47 × 1007 ± 4.79 × 1006 8.52 × 1007 ± 6.21 × 1006 2.9 ± 0.3 1.7 ± 0.3 1.1 ± 0.2 Figure 2. All EV subtypes from DCs interact with and activate T cells A. DiO-labelled DC-derived EVs were cultured for 18 h with allogeneic primary total CD4+ T cells and analysed by confocal microscopy. Nuclei are stained with DAPI (DiO = green; DAPI = blue) (scale bars = 5 μm). B. The percentage of DiO+ T cells was analysed by flow cytometry. Total CD4+ T cells incubated for 18 h with DiO-labelled EVs coming from 4 × 106 DCs or cultured alone as control (n = 11, one symbol per donor). Red line indicates median. C. CD69 expression on CD4+ T cells cultured for 18 h with the subtypes of EVs released by DCs. The percentage of CD69+ CD4+ T cells for each pellet treatment is shown (n = 11 donors, one symbol per donor). Red line indicates median. *P < 0.05; ***P < 0.001 (Friedman test). D. Total CD4+ T-cell proliferation was evaluated after 6 days of culture with different amounts of DC-derived EVs. Proliferation was calculated by dilution of Cell Trace Violet dye (n = 13 donors, mean ± SEM is shown). E. Proliferation of total CD4+ T cells after culture for 6 days with EVs from 8 × 106 DCs in the presence of blocking antibodies against HLA-DR/DQ/DP or control isotype antibodies (n = 8 donors). F, G. EVs from 8 × 106 DCs were cultured with autologous or allogeneic total CD4+ T cells (F) or with total versus naïve CD4+ T (G) for 6 days to evaluate T-cell proliferation (n = 4 donors (F) and n = 10 donors (G)). Data information: (E–G) P-values were calculated using a Wilcoxon signed-rank test: *P < 0.05; **P < 0.01; ***P < 0.001. Download figure Download PowerPoint Click here to expand this figure. Figure EV2. Flow Cytometry analysis of fluorescently labelled-EV transfer to CD4+ T cellsEV pellets (2K, 10K and 100K) coming from conditioned medium of DCs cultures or the pellets from non-conditioned medium (depleted of serum EVs) were labelled with DiO lipophilic dye, washed in PBS and re-ultracentrifuged three times and cultured for 18 h with allogeneic primary total CD4+ T cells. Transfer of the dye was analysed by flow cytometry. Dot-plot graphs show the percentage of CD4+ DiO+ cells for both EV-treated and control-treated CD4+ T cells. Download figure Download PowerPoint Distinct DC-derived EVs promote the release of a specific cytokine signature by CD4+ T cells Since activation of CD4+ T cells can result in the release of different cytokines, which reflect the polarization state of the T cells, we examined CD4+ T-cell cytokine profile after 6 days of culture with allogeneic EVs. IFN-γ is the main cytokine secreted by Th1 effector cells involved in cell-mediated immunity, and IL-13, as well as IL-5 and IL-4, is preferentially produced by differentiated Th2 cells that regulate antibody-mediated humoral immune responses (Chaudhry & Rudensky, 2013). T cells activated by the 2K pellet from 1 to 8 × 106 DCs secreted higher levels of IL-13 as compared to untreated cells, but little or no IFN-γ nor IL-17 (Th17-secreted cytokine) (Fig 3A and B). By contrast, T cells activated by the 10K and 100K pellet secreted IFN-γ and IL-17, but only low amounts of IL-13 (Fig 3A and B), even at the highest dose of EV used. The highest dose of EVs (i.e. secreted by 8 × 106 DCs) was used to analyse secretion of other cytokines by T cells exposed to it: the other Th2 cytokines (IL-4 and IL-5) were also significantly secreted by T cells exposed to the 2K pellet, whereas the 10K and 100K pellets induced secretion of IL-6 (Fig 3B) and of TNF-α (100K pellet, Fig EV3A). The 10K and 100K pellets were also able to promote production of IL-5 and IL-13 in some cases, but not in a significantly different manner from spontaneous secretion by untreated CD4+ T cells. We also measured other cytokines in the supernatants, such as IL-9 (Th9-secreted cytokine) and IL-10 (secreted by Treg) (Chaudhry & Rudensky, 2013), but we did not observe any significant differences compared to their basal levels secreted by non-activated T cells (Fig EV3A). IFN-γ and IL-13 were the two cytokines produced in the greatest amounts in our cultures upon EV treatment (ranges of 0.2–2 ng/ml). We thus used the ratio of secreted IFN-γ versus IL-13 as an indication of the relative proportions of Th1 versus Th2 lymphocytes. The median IFN-γ/IL-13 ratio was below 1 (0.45) for the 2K-stimulated CD4+ T cells, whereas it was above 1 (3.1 and 3.8) for the 10K- and 100K-stimulated T cells, respectively (Fig 3C). This suggests that lEVs can tip the balance towards Th2, whereas medium and sEVs tip it towards Th1 lymphocytes. To study cytokine production by individual CD4+ T cells that proliferated upon EV stimulation (6 days culture), we performed intracellular staining for IFN-γ, IL-4 and IL-17 after 4 h stimulation with PMA and ionomycin. The percentage of different cytokine-producing subpopulations (Fig EV3B and C) and their relative proportions (Fig EV3D) were calculated for the proliferating CD4+ T cells. We observed that in all activation conditions, cytokine-expressing cells were in majority single IFN-γ producers (Fig EV3C). Cells activated by the 2K pellet, however, were more frequently IL-4 single-positive (10.2%) than cells activated by the 10K and 100K pellets (2–4%, Fig EV3C and D), whereas the latter were more frequently multifunctional, especially by producing simultaneously IFN-γ and IL-17A (10.7%), than cells activated by the 2K pellet (3.2%) (Fig EV3C and D). In addition, among the proliferating CD4+ T cells, the 10K and 100K pellets were more efficient at inducing cytokine-producing cells (58–61%) than the 2K pellets (51%) (Fig EV3D). Therefore, even if all types of EVs efficiently induce CD4+ T-cell activation, the resulting functionality of Th cells is different. Figure 3. CD4+ T cells release different cytokines upon stimulation by DC-derived EV subtypes The concentration of IL-13, IFN-γ and IL-17 was measured by cytometric bead array (CBA) in supernatants after 6 days of culture of total CD4+ T cells with EVs released by 1 to 8 × 106 DCs (n = 9, mean ± SEM is shown). The concentration of IL-4, IL-5, IL-6, IL-13, IFN-γ and IL-17 was measured by CBA in supernatants after 6 days of culture of total CD4+ T cells with the highest dose of EVs (the released by 8 × 106 DCs). Each individual DC-EV donor was used on T cells from two different donors, providing two biological replicates. Results are expressed as fold induction compared to untreated CD4+ T cells (n = 11, one symbol per individual DC-EV:T-cell combination). Red line indicates median. The range of cytokines secreted by untreated CD4+ T cells was 8.3–15 pg/ml of IL-4; 18–109.1 pg/ml of IL-5; 12.4–961.1 pg/ml of IL-13; 18–115.2 pg/ml of IL-6; 23–894.4 pg/ml of IFN-γ; 20–152.5 pg/ml of IL17. Th1 to Th2 ratio was calculated by dividing the absolute amount of IFN-γ (pg/ml) to the absolute amount of IL-13 (pg/ml) secreted by a given T-cell donor exposed to the different pellets obtained from an individual DC donor (n = 20 individual DC-EV:T-cell combination). The ratio is represented as a box plot (25th to 75th percentiles, the line represents the median; whiskers, min to max, points represent outliers as calculated by Tukey's test). Data information: (B and C) P-values were
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