Nanosized UCMSC-derived extracellular vesicles but not conditioned medium exclusively inhibit the inflammatory response of stimulated T cells: implications for nanomedicine

Abstract

Undesired immune responses have drastically hampered outcomes after allogeneic organ transplantation and cell therapy, and also lead to inflammatory diseases and autoimmunity. Umbilical cord mesenchymal stem cells (UCMSCs) have powerful regenerative and immunomodulatory potential, and their secreted extracellular vesicles (EVs) are envisaged as a promising natural source of nanoparticles to increase outcomes in organ transplantation and control inflammatory diseases. However, poor EV preparations containing highly-abundant soluble proteins may mask genuine vesicular-associated functions and provide misleading data. Here, we used Size-Exclusion Chromatography (SEC) to successfully isolate EVs from UCMSCs-conditioned medium. These vesicles were defined as positive for CD9, CD63, CD73 and CD90, and their size and morphology characterized by NTA and cryo-EM. Their immunomodulatory potential was determined in polyclonal T cell proliferation assays, analysis of cytokine profiles and in the skewing of monocyte polarization. In sharp contrast to the non-EV containing fractions, to the complete conditioned medium and to ultracentrifuged pellet, SEC-purified EVs from UCMSCs inhibited T cell proliferation, resembling the effect of parental UCMSCs. Moreover, while SEC-EVs did not induce cytokine response, the non-EV fractions, conditioned medium and ultracentrifuged pellet promoted the secretion of pro-inflammatory cytokines by polyclonally stimulated T cells and supported Th17 polarization. In contrast, EVs did not induce monocyte polarization, but the non-EV fraction induced CD163 and CD206 expression and TNF-α production in monocytes. These findings increase the growing evidence confirming that EVs are an active component of MSC's paracrine immunosuppressive function and affirm their potential for therapeutics in nanomedicine. In addition, our results highlight the importance of well-purified and defined preparations of MSC-derived EVs to achieve the immunosuppressive effect.

Keywords: exosomes; immunomodulation; inflammation; nanosized extracellular vesicles; size exclusion chromatography.; umbilical cord mesenchymal stem cell.

Figure 1

A: Bright-field images of UCMSC in culture media without (left) or with IFNγ (right). Scale bars = 100 µm. B-C: UCMSCs primed with IFNγ expressed unchanged levels of CD73, lower levels of CD90 and higher levels of HLA-DR (class II MHC) on surface. B: Representative histograms of unconditioned (blue line) and IFNγ-primed UCMSCs (red line) labeled for CD73, CD90 and HLA-DR. Isotype controls are depicted as shaded areas. C: MFI values for CD73, CD90 and HLA-DR of UCMSCs after culture without (white circles) or with IFNγ (black circles). Data is shown for nine independent experiments. ***p<0.001 by Paired T test; **p<0.01 by Wilcoxon matched-pairs signed rank test. D-E: Proliferation and viability of T cells stimulated with anti-CD2/CD3/CD28 coated microbeads in the absence or presence of unconditioned or IFNγ-primed UCMSCs at 1:15, 1:60, 1:240, 1:480, 1:960 or 1:1,920 cell ratios were analyzed by CFSE loss and FSC/SSC gating, respectively. D: Bars represent means ±SD of proliferation relative to their PBS control. E: Bars represent means ± SD of the percentage of viable cells. Data accounts for seven independent experiments from different donor samples, performed in triplicates. Statistical differences are indicated for groups with *p<0.05 and **p<0.01 by One sample T test to the 100%.

Figure 2

EVs were successfully isolated by SEC. A: Scheme of the methodological procedure followed for the generation of the different study fractions obtained from UCMSC 48h-culture: supernatant was cleared of debris by centrifugation to obtain conditioned media (CM); concentrated CM (CCM) and eluted CM (ECM) were collected after ultrafiltration; CCM was loaded to the SEC column and fractions collected. B-C: UCMSC-EVs were found on fractions 5-7 while protein eluted after fraction 8 on both samples coming from unconditioned (B) and IFNγ-conditioned UCMSCs (C). SEC eluted fractions were checked for EV markers (CD9 and CD63), MSC markers (CD73 and CD90) by bead-based flow cytometry (left axis). Protein elution was monitored by absorption at 280nm (right axis). D-E: Cryo-EM images confirmed UCMSC-EVs presence in pooled EV fractions (F5-7) of unconditioned (D) and IFNγ-conditioned SEC preparations (E). Images of 20,000x and 30,000x magnifications are shown, with 200nm and 100nm scale bars, respectively. F: Box plot of the fold increase in protein content, CD9 and CD63 MFI of pooled EV fractions obtained from IFNγ-primed UCMSCs relative to unconditioned UCMSCs. Medians of ten independent experiments are depicted as horizontal bars, outliers as points. G: CD73, CD90, HLA-ABC (MHC-I) and HLA-DR (MHC-II) expression on EVs from unconditioned (white dots) and corresponding IFNγ-primed MSCs (black dots) are shown normalized to their CD9 MFI. Each dot corresponds to an independent experiment (n=10 and 6).

Figure 3

Only the pooled EV fraction reduced T cell polyclonal proliferation. A: The three different pooled SEC fractions: EV, proximal non-EV (“non-EV prox”) and distal non-EV fractions (“non-EV dist”) were analyzed for T cell proliferation suppression capacity compared to the CCM, ECM, full CM, and ultracentrifuged pellet (UC Pellet). B, C: Proliferation of T cells stimulated with anti-CD2/CD3/CD28 beads (10:1 ratio) was analyzed by CFSE loss in the presence of pooled UCMSC-EVs, proximal and distal non-EV fractions, CCM, ECM and CM (B) or the UC Pellet (C). Quantities were adjusted to 2.5x10⁵ initial UCMSC. Bars represent proliferation relative to their PBS control. C, D: Viability of stimulated T cells assessed by FSC-A/SSC-A. Data represent means ± SD for seven (B, D) and four (C, E) independent experiments. Statistical differences are indicated for groups with p<0.05 by Kruskall-Wallis; #p<0.05 by Wilcoxon Signed Rank test (to the 100%); *p<0.05 and **p<0.01 by Mann-Whitney test.

Figure 4

Cytokines found in the supernatants of T cell proliferation assays corresponding to Figure 3 were analyzed by CBA (IL-2, IL-4, IL-6, IL-10, TNF-α, IFNγ, and IL-17A) and TGF-β1 ELISA. Bars represent means ± SD of seven (A) and four (B) independent experiments. Statistical differences are indicated for groups with *p<0.05 by Kruskall-Wallis; *p<0.05 and **p<0.01 by Mann-Whitney test.

Figure 5

Concentrated EV and non-EV fractions but not CM inhibit T cell polyclonal proliferation. A: EV, proximal non-EV fractions and CM were vacuum-concentrated and checked for T cell proliferation suppression capacity. B: Proliferation of T cells stimulated with anti-CD2/CD3/CD28 beads (10:1 ratio) was analyzed by CFSE loss in the presence of vacuum-concentrated UCMSC-EVs, proximal non-EV fractions or CM. Quantities were adjusted to 2.5x10⁵ initial UCMSC, and dosage dependency was studied diluting samples 1/2 and 1/10 in PBS. Bars represent means ± SD of proliferation relative to their PBS control, for three independent experiments. C: Viability of stimulated T cells assessed by FSC-A/SSC-A. Data represent means ± SD for five independent experiments. Statistical differences are indicated for groups with p<0.05 by Kruskall-Wallis; #p<0.01 by Wilcoxon Signed Rank test (to the 100%); *p<0.05 and **p<0.01 by Mann-Whitney test.

Figure 6

Non-EV fractions and CM induce an inflammatory response on stimulated T cells. Cytokines found in the supernatants of T cell proliferation assays corresponding to Figure 5 were analyzed by CBA (IL-2, IL-4, IL-6, IL-10, TNF-α and IFNγ) and TGF-β1 and IL-17 ELISA. Bars represent means ± SD of three independent experiments for IL-2, IL-4, IL-6, IL-10, TNF-α and IFNγ and five independent experiments for TGF-β1 and IL-17. Statistical differences are indicated for groups with *p<0.05, **p<0.01 and ***p<0.001 by One-way ANOVA with Tukey's post hoc analysis.

Figure 7

UCMSC-EVs do not affect CD80, CD163 and CD206 polarization marker expression in monocytes, while the non-EV fraction and CM induce increased expression of CD163 and CD206. Monocytes were cultured for 48h with polarizing cytokines to induce an inflammatory M1 (LPS+IFNγ) or anti-inflammatory M2a (IL-4) and M2c (IL-10) phenotypes, or with the EV, proximal non-EV fractions or full CM. A: mRNA fold change of the M1 marker CD80 and M2 markers CD163 and CD206, as analyzed by real time PCR. Values are relative to 18S and expressed as a fold change to the PBS-treated monocytes in a log₂ scale. B: Levels of TNF-α and IL-10 in the supernatants of monocytes as measured by ELISA. Data are expressed as mean + SD and account for two different UCMSC and four monocyte donors. Statistical differences are indicated for groups with *p<0.05 by Kruskall-Wallis; *p<0.05 by Mann-Whitney test.