Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2020 Apr 6;10(11):5011-5028.
doi: 10.7150/thno.42742. eCollection 2020.

Extracellular vesicles from human umbilical cord blood plasma modulate interleukin-2 signaling of T cells to ameliorate experimental autoimmune encephalomyelitis

Sueon Kim  1   2 , Ji-Young Maeng  1   2 , Seung-Joo Hyun  1   2 , Hyun-Jung Sohn  3 , Su-Yeon Kim  3 , Cheol-Hwa Hong  1   2 , Tai-Gyu Kim  1   2   3
Affiliations

Extracellular vesicles from human umbilical cord blood plasma modulate interleukin-2 signaling of T cells to ameliorate experimental autoimmune encephalomyelitis

Sueon Kim et al. Theranostics. .

Abstract

Human umbilical cord blood (UCB) cell-derived extracellular vesicles (EV) reportedly play immunosuppressive roles; however, UCB plasma-derived extracellular vesicles (CBP EVs) remain poorly studied. We examined the immunosuppressive potential of CBP EVs compared to that of adult blood plasma-derived extracellular vesicles (ABP EVs) in vitro and constructed an experimental autoimmune encephalomyelitis (EAE) model. Methods: CBP EVs were isolated by ultracentrifugation and their proteomic profiling was performed using the high-resolution liquid chromatography with tandem mass spectrometry. Human T lymphocytes or mouse splenocytes labeled with carboxyfluorescein succinimidyl ester were incubated with CBP EV to measure the immunosuppressive function of CBP EV. The effect on T-cell polarization was analyzed by flow cytometry and enzyme-linked immunospot assay. The matrix metalloproteinase (MMP) function in CBP EV was specifically inhibited using a chemical inhibitor. The efficacy of CBP EVs in the EAE mouse model was determined by scoring the symptoms and analyzing cell phenotype and cytokines using mouse splenocytes. We generated genetically engineered artificial EVs using HLA/MIC-null HEK293T (H1ME-5) cell line to characterize the immunosuppressive effect of CBP EV. Results: CBP EVs primarily inhibited the proliferation of T cells by reducing the production of IL-2. Specifically, CBP EV-derived matrix metallopeptidase cleaved the IL-2 receptor α (CD25) on the surface of activated T cells, consequently downregulating IL-2 signaling in response to IL-2R engagement. Although the inhibition of MMP activity in CBP EVs abrogated CD25 cleavage and restored IL-2 production in activated T cells, the immunosuppressive response was not fully recovered. Thus, we further analyzed changes in immunosuppressive cells such as regulatory T cells and bone marrow-derived suppressor cells by CBP EV. Further, GAL-3, GAL-7, S100-A7, MMP-9, MMP-8, HSP-72, and PIP were highly enriched in CBP EV-mimics in which they served as pivotal mediators of CBP EV-induced immunosuppressive effects. Therefore, we generated genetically engineered GAL-3, GAL-7, S100-A7, MMP-9, MMP-8, HSP-72, and PIP-EVs using HLA/MIC-null HEK293T cells to characterize the immunosuppressive effect of these molecules. Among these, MMP-9 and HSP-72-enriched EVs showed the most significant T cell immunosuppression. Conclusion: CBP EVs inhibited T cell proliferation and EAE development by modulating IL-2 signaling and immunosuppressive cell fate. CBP EVs contain critical components for immunosuppression and that CBP EV mimics, specifically those expressing MMP-9 and HSP-72, may offer a novel promising strategy for the treatment of various autoimmune diseases.

Keywords: experimental autoimmune encephalomyelitis (EAE); interleukin-2 (IL-2) signaling; matrix metalloproteinase-9 (MMP-9); regulatory T cell (Treg); umbilical cord blood plasma-derived extracellular vesicles (CBP EVs).

PubMed Disclaimer

Conflict of interest statement

Competing Interests: The authors have declared that no competing interest exists.

Figures

Figure 1
Figure 1
Characterization of exosomal membrane vesicles purified from human umbilical CBP. (A) CBP EVs bound to latex beads coated with individual EV-specific marker antibodies were analyzed by flow cytometry. Latex beads coated with complete exosomal preparations were included for comparison. The plots represent intensities derived from EV-specific antibodies (CD9, CD63, CD81 and HSP72) with the corresponding bead-only controls. (B) Dot-blot analysis of CBP EV lysates. The corresponding dots were evaluated using an EV antibody array kit. EV-specific antibody spots provided signals of varying intensities. The values shown are the means of values obtained in three independent experiments. Error bars = standard error of the mean (SEM). “Blank” indicates the negative control, also indicated by Φ, and GM130 represents cell debris. The mean intensity was analyzed using ImageJ software. (C) Particles with a lipid bilayer structure and the expected size (80-100 nm) were observed by TEM. Scale bars, 100 nm. (D) The CBP EVs are 81 ± 1.4 nm in size and 1.64×1012 ± 2.37×1010 particles/mL in number.
Figure 2
Figure 2
Confirmation of CBP EV-mediated immunosuppression in stimulated human CD4+ and CD8+ T cells. (A-B) Representative experimental data demonstrating that CBP EVs inhibited human CD4+ and CD8+ T cell stimulation. (C) The immunosuppressants, rapamycin and cyclosporine, were used as the standard positive controls. The intensity of CFSE-labeled T cells was acquired by flow cytometry and further analyzed using ModFit LT 4.0 software. (D-E) Human CD4+ and CD8+ T cells were activated using CD3/CD28 DYNABEAD in the presence of CBP EVs or ABP EVs dose-dependently. The intensity of CFSE-labeled T cells was acquired by flow cytometry and was further analyzed using ModFit LT 4.0 software. All experiments were repeated a minimum of five times using different batches of CBP EVs (one batch of plasma EVs = the sum of 10 units of cord blood). Statistically significant differences (ANOVA test): *p < 0.1, **p < 0.05, ***p < 0.01. Activated PBMCs indicate the positive control = Φ.
Figure 3
Figure 3
Mechanism underlying CBP EV-mediated immunosuppression in CD3/CD28 Dynabead-stimulated human T cells. (A) CBP EVs induced apoptosis of Human T cells activated by CD3/CD28 DYNABEADs. CD4+ T cells were harvested after 156 h and stained with Annexin V-FITC, 7AAD, anti-CD4-APC antibodies. The cells gated on CD3+ are shown. The assay data were analyzed using FlowJo v10. (B) This experiment was repeated five times independently. Statistically significant differences (ANOVA test): *p < 0.1, **p < 0.05, ***p < 0.01. (C) CBP EVs-induced G0/G1 cell cycle arrest of human CD3+ T cells was activated by CD3/CD28 DYNABEAD. Human CD3+ T cells were stimulated with DYNABEAD and CBP EVs were added at the same time. After 156 h of incubation, the cells were stained with propidium iodide for 20 min and assessed by flow cytometry to reveal the effect of CBP EVs on cell cycle progress. These data were analyzed using ModFit LT 4.0 software. (D) This experiment was repeated three times independently. One-way ANOVA was used to calculate the significance between groups: *p < 0.1 **p < 0.05 ***p < 0.01.
Figure 4
Figure 4
Analysis of the differential molecular functions and biological processes of ABP EV and CBP EV, confirming the immunosuppressive response by MMP-expressing CBP EV. (A) Proteomic analysis of various biological functions of human CBP EVs and ABP EVs using the ExoCarta database and SBC analysis system. (B) Proteomic analysis of human CBP EVs and ABP EVs, as well as various molecular functions of immunosuppression screened using the ExoCarta database and SBC Analysis system. The red box indicates a higher relevance of CBP EVs due to functional classification. (C) MMP and TIMP antibody arrays using 300 μg of CBP EV or ABP EV lysate; the corresponding dots were evaluated using an EV antibody array kit. The MMP and TIMP antibody spots that produced signals of varying intensities were calculated using ImageJ software. This experiment was repeated three times independently. (D) CBP EV-mediated suppression via MMP inhibition results in the partial restoration of T cell proliferation. T cells were stimulated with DYNABEAD in the presence of CBP EVs and then treated with or without GM6001 before comparison via CFSE analysis at 156 h. The CFSE-labeled cells were acquired by FACSCanto, and the cells were gated on CD4+ events. (E) The percentage of cells in each generation was calculated using ModFit LT 4.0 software. This experiment was repeated six times independently. Statistically significant differences (ANOVA test): *p < 0.1, **p < 0.05, ***p < 0.01.
Figure 5
Figure 5
Mouse cross-reactivity of T cell suppression and IL-2 downregulation by human CBP EVs. (A) Immunosuppressive effects of CBP EVs were examined by DYNABEAD-stimulated mouse CD4+ T cell proliferation. This experiment was repeated five times independently. Statistically significant differences (ANOVA test): *p < 0.1, **p < 0.05, ***p < 0.01. (B) Immunosuppressive effects of CBP EVs were examined by DYNABEAD-stimulated mouse CD8+ T cell proliferation. This experiment was repeated five times independently. Statistically significant differences (ANOVA test): *p < 0.1, **p < 0.05, ***p < 0.01. (C) CBP EVs significantly decreased human T cell proliferation and downregulated IL-2, IFN-γ, and IL-6 secretion, which are associated with the differentiation of Th17 and Th1 cells. The cytokine level at 156 h in the harvested culture supernatant was analyzed using the human cytometric bead array. This experiment was repeated three times independently. (D) CBP EVs not only downregulated IL-2 secretion in human T cells but also reduced IL-2 in mice. The changes in cytokine levels were observed using the mouse cytometric bead array. This experiment was repeated three times independently.
Figure 6
Figure 6
Changing patterns of IL-2 levels in experimental autoimmune encephalomyelitis (EAE) mice treated with EVs. (A) Development of EAE was reduced in CBP EV-treated EAE mice. EAE was induced in 15 C57BL/6 mice following co-treatment with MOG/CFA and PTx immunization. CBP EVs and ABP EVs were injected intravenously twice on days 0 and 7 into five EAE mice at a 100-μg dose (black arrow). The mice were assigned to different groups to analyze the clinical score after EAE induction: EAE mice (n = 5) versus CBP EV- or ABP EV- injected EAE mice (n = 5). EAE indicates positive control = Φ. (B) The maximum and minimum scores for each EAE group are shown. (C) Representative micrographs of the brain and spleen 24 h after injection of PKH67-stained EVs into the tail vein. EAE mice without EVs were used as the negative control. Blue - DAPI and green - PKH67-stained EVs. All scale bars are 20µm. (D) Hematoxylin and eosin (H&E) staining on the brain section of EAE induced animals which treated by EVs. Digital images were collected under a color bright-field setting using a ×20 objective. Scale bars represent 100 µm. (E-F) Quantitative comparison of the levels of cellular infiltration number and inflammation area within the brain sections from EAE mice treated with EVs. Images were processed using Gen5 software (n = 3). (G) Detection of MOG35-55-induced cytokine recall response in whole splenocytes during acute EAE. C57BL/6 mice were immunized with MOG33-55 in CFA and tested 22 days after immunization. All tested mice exhibited clinical symptoms of EAE. The frequency of MOG peptide-specific IFN-γ-, IL-2-, and IL-17-secreting cells was determined for comparison with that of the EAE control, CBP EV-treated EAE, and ABP EV-treated EAE groups using the ELISPOT assay. MOG peptide-specific T cells were analyzed during re-stimulation with MOG peptides (25 μg/mL) (n = 3).
Figure 7
Figure 7
Changing patterns of Treg and MDSC levels in experimental autoimmune encephalomyelitis (EAE) mice treated with EVs. (A) The Treg population in EVs-treated EAE mice splenocytes. The frequency of CD4+ gated MOG peptide-specific FOXP3+ cells was determined and compared with that of the EAE control, CBP EV-treated EAE, and ABP EV-treated EAE groups by intracellular cytokine staining (n = 3). (B) Expression profiles of CD4 versus CD8 and Foxp3 versus CD25 (gated on CD4+ T cells) for thymocyte populations in EAE, CBP EV injected EAE, ABP EV injected EAE (n=3). (C) MDSC populations in EAE mice treated with EVs. The frequencies of CD11b+Gr1+ cells were determined or compared with those of the MDSC population in the healthy control, EAE control, CBP EV-injected EAE, and ABP EV-injected EAE groups (n = 3).
Figure 8
Figure 8
CBP EV mimics enriched in MMP-9 and HSP-72 molecule exhibit immunosuppressive effects. (A) The establishment of the human leukocyte antigen (HLA) class I/MIC null HEK 293T (H1ME-5) cell line using the multiplex CRISPR/Cas9 system. H1ME-5 cells show no expression of MICA/B and HLA class I. (B) The particle number of H1ME-5 EVs was 1.96×1012 ± 6.37×1010 particles/mL. Representative NTA data indicating the size of H1ME-5 EVs (75 ± 1.4 nm). (C) Expression of the CBP EV-contained molecules GAL-3, GAL-7, S100-A7, MMP-9, MMP-8, HSP-72, and PIP transduced into H1ME-5 cells. Six days following transduction, cells positive for each molecule were sorted using a MoFlo XDP Cell Sorter. (D) CBP EVs or CBP EV mimics bound to latex beads coated with anti- GAL-3, GAL-7, S100-A7, MMP-9, MMP-8, HSP-72, and PIP antibodies were analyzed by flow cytometry. Intensities derived from GAL-3, GAL-7, S100-A7, MMP-9, MMP-8, HSP-72, and PIP with corresponding bead-only controls. (E) Representative experimental data demonstrating that CBP EV, MMP-9-, and HSP72- EV inhibited T cell stimulation. The intensity of CFSE-labeled CD3+ T cells was acquired by flow cytometry and further analyzed using the ModFit LT 4.0 software. (F) H1ME-5 EVs showed no immunosuppressive effect; only HSP72, MMP-9 expressing EV showed significant immunosuppression. The combination of EV expressing MMP-9 and HSP72 reached an immunosuppressive effect similar to that of CBP EV. The combination of EVs expressing MMP-9 and HSP72 showed a more significant effect than when using HSP72 and PIP alone. The statistical values ​​were analyzed using the results obtained from the CFSE proliferation assay. (ANOVA test): *p < 0.1, **p < 0.05, ***p < 0.01, ****p < 0.001.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Ballen KK, Gluckman E, Broxmeyer HE. Umbilical cord blood transplantation: the first 25 years and beyond. Blood. 2013;122:491–8. - PMC - PubMed
    1. Damien P, Allan DS. Regenerative Therapy and Immune Modulation Using Umbilical Cord Blood-Derived Cells. Biol Blood Marrow Tr. 2015;21:1545–54. - PubMed
    1. PrabhuDas M, Bonney E, Caron K, Dey S, Erlebacher A, Fazleabas A. et al. Immune mechanisms at the maternal-fetal interface: perspectives and challenges. Nat Immunol. 2015;16:328–34. - PMC - PubMed
    1. Shkap V, Reske A, Pipano E, Fish L, Baszler T. Immunological relationship between Neospora caninum and Besnoitia besnoiti. Vet Parasitol. 2002;106:35–43. - PubMed
    1. Chen SJ, Liu YL, Sytwu HK. Immunologic regulation in pregnancy: from mechanism to therapeutic strategy for immunomodulation. Clin Dev Immunol. 2012;2012:258391. - PMC - PubMed

Publication types

MeSH terms

开云球赛中国有限公司 | 万搏官方网站 | c7官网登录入口 | 九州平台-九州(中国) | 万搏官方网站 | 千亿球友会·(中国)官方网站 | 米兰体育网址 | 开云手机官方网站 | 江南手机网页版登录入口 |