Abstract
Background aims
Extracellular vesicles (EVs), including exosomes and microvesicles, are released by
almost all cells and found in all body fluids. Unknown proportions of EVs transmit
specific information from their cells of origin to specific target cells and are key
mediators in intercellular communication processes. Depending on their origin, EVs
can modulate immune responses, either acting as pro- or anti-inflammatory. With the
aim to analyze the immunomodulating activities of EV preparations, especially those
from mesenchymal stromal cells (MSCs) in vitro, a multi-donor mixed lymphocyte reaction (mdMLR) assay was established and stressed
for its reproducibility.
Methods
To this end, human peripheral blood-derived mononuclear cells (PBMCs) of 12 different
healthy donors were pooled warranting mutual allogeneic cross-reactivity, even following
an optimized freezing and thawing procedure. After thawing, mixed PBMCs were cultured
for 5 days in the absence or presence of EVs to be tested. Reflecting allogeneic reactions,
in the absence of EVs, pooled PBMCs form characteristic satellite colonies whose appearance
can be modulated by EVs. More quantifiable, the strength of the allogenic reaction
is reflected by the content of activated CD4 and CD8 T cells being recognized by means
of their CD25 and CD54 expression.
Results
Of note, connected to the use of primary cells, independent multi-donor PBMC pools
differed in their capability to activate their cultured T cells. Thus, throughout
the study, only pooled PBMC batches were used whose activated T-cell contents exceeded
25% of the total T-cell population at culture day 5 and whose contents were reproducibly
reduced in the presence of immunomodulatory active MSC-EVs. T-cell activation–suppressing
effects of the MSC-EV preparations tested were in all cases accompanied by the impact
on monocytes. In the presence of immunomodulatory active MSC-EVs, more monocytes were
harvested from mdMLR cultures than in their absence. Furthermore, in the absence of
immunomodulatory EVs, most monocytes appeared as non-classical (CD14+CD16+) monocytes, whereas immunomodulatory active MSC-EVs promoted the appearance of classical
(CD14++CD16–) and intermediate (CD14++CD16+) monocyte subpopulations.
Conclusions
Overall, the obtained results qualify the mdMLR assay as a robust experimental tool
for the evaluation of immunomodulatory potentials of given MSC-EV samples. However,
further assay development is required to develop and qualify an authority-acceptable
potency assay for clinically applicable MSC-EV products.
Keywords
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References
- Biological properties of extracellular vesicles and their physiological functions.J Extracell Vesicles. 2015; 4: 27066
- Mesenchymal stem/stromal cell-derived extracellular vesicles and their potential as novel immunomodulatory therapeutic agents.Int J Mol Sci. 2017; 18
- Indirect activation of naive CD4+ T cells by dendritic cell-derived exosomes.Nat Immunol. 2002; 3: 1156-1162
- Exosomes and tumor-mediated immune suppression.J Clin Invest. 2016; 126: 1216-1223
- Dendritic cell-derived exosomes as maintenance immunotherapy after first line chemotherapy in NSCLC.Oncoimmunology. 2016; 5e1071008
- Vaccination of metastatic melanoma patients with autologous dendritic cell (DC) derived-exosomes: results of the first phase I clinical trial.J Transl Med. 2005; 3: 10
- A phase I study of dexosome immunotherapy in patients with advanced non-small cell lung cancer.J Transl Med. 2005; 3: 9
- Eradication of established murine tumors using a novel cell-free vaccine: dendritic cell-derived exosomes.Nat Med. 1998; 4: 594-600
- Clinical potential of mesenchymal stem/stromal cell-derived extracellular vesicles.Stem Cell Investig. 2017; 4: 84
- Mesenchymal stem cell-derived microvesicles protect against acute tubular injury.J Am Soc Nephrol. 2009; 20: 1053-1067
- Exosome secreted by MSC reduces myocardial ischemia/reperfusion injury.Stem Cell Res. 2010; 4: 214-222
- MSC-derived exosomes: a novel tool to treat therapy-refractory graft-versus-host disease.Leukemia. 2014; 28: 970-973
- Umbilical cord mesenchymal stem cells derived extracellular vesicles can safely ameliorate the progression of chronic kidney diseases.Biomater Res. 2016; 20: 21
- Exosomes derived from bone marrow mesenchymal stem cells as treatment for severe COVID-19.Stem Cells Dev. 2020; 29: 747-754
- First-in-human intracochlear application of human stromal cell-derived extracellular vesicles.J Extracell Vesicles. 2021; 10: e12094
- Critical considerations for the development of potency tests for therapeutic applications of mesenchymal stromal cell-derived small extracellular vesicles.Cytotherapy. 2021; 23: 373-380
- Treatment of severe acute graft-versus-host disease with third party haploidentical mesenchymal stem cells.Lancet. 2004; 363: 1439-1441
- Mesenchymal stromal cells: a new tool against graft-versus-host disease?.Biol Blood Marrow Transplant. 2012; 18: 822-840
- The mesenchymal stromal cells dilemmaࣧdoes a negative phase III trial of random donor mesenchymal stromal cells in steroid-resistant graft-versus-host disease represent a death knell or a bump in the road?.Cytotherapy. 2013; 15: 2-8
- A phase 3 randomized study of remestemcel-L versus placebo added to second-line therapy in patients with steroid-refractory acute graft-versus-host disease.Biol Blood Marrow Transplant. 2020; 26: 835-844
- A phase 3, single-arm, prospective study of remestemcel-L, ex vivo culture-expanded adult human mesenchymal stromal cells for the treatment of pediatric patients who failed to respond to steroid treatment for acute graft-versus-host disease.Biol Blood Marrow Transplant. 2020; 26: 845-854
- Study 275: updated expanded access program for remestemcel-L in steroid-refractory acute graft-versus-host disease in children.Biol Blood Marrow Transplant. 2020; 26: 855-864
- Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement.Cytotherapy. 2006; 8: 315-317
- Functional heterogeneity of mesenchymal stem cells: implications for cell therapy.J Cell Biochem. 2012; 113: 2806-2812
- Donor variation in the growth properties and osteogenic potential of human marrow stromal cells.J Cell Biochem. 1999; 75: 424-436
- Heterogeneity among human bone marrow-derived mesenchymal stem cells and neural progenitor cells.Haematologica. 2003; 88: 126-133
- Strategies to address mesenchymal stem/stromal cell heterogeneity in immunomodulatory profiles to improve cell-based therapies.Acta Biomater. 2021;
- Mesenchymal stromal cell variables influencing clinical potency: the impact of viability, fitness, route of administration and host predisposition.Cytotherapy. 2021; 23: 368-372
- Human mesenchymal and murine stromal cells support human lympho-myeloid progenitor expansion but not maintenance of multipotent haematopoietic stem and progenitor cells.Cell Cycle. 2016; 15: 540-545
- Extracellular vesicles improve post-stroke neuroregeneration and prevent postischemic immunosuppression.Stem Cells Transl Med. 2015; 4: 1131-1143
- Mesenchymal stem cell-derived extracellular vesicles ameliorate inflammation-induced preterm brain injury.Brain Behav Immun. 2017; 60: 220-232
- Mesenchymal stromal cell-derived extracellular vesicles protect the fetal brain after hypoxia-ischemia.Stem Cells Transl Med. 2016; 5: 754-763
- Small extracellular vesicles obtained from hypoxic mesenchymal stromal cells have unique characteristics that promote cerebral angiogenesis, brain remodeling and neurological recovery after focal cerebral ischemia in mice.Basic Res Cardiol. 2021; 116: 40
- Mesenchymal stromal cell-derived extracellular vesicles reduce neuroinflammation, promote neural cell proliferation and improve oligodendrocyte maturation in neonatal hypoxic-ischemic brain injury.Front Cell Neurosci. 2020; 14601176
- Independent human mesenchymal stromal cell-derived extracellular vesicle preparations differentially attenuate symptoms in an advanced murine graft-versus-host-disease model.Cytotherapy. 2023; https://doi.org/10.1016/j.jcyt.2023.03.008
- Anti-inflammatory mesenchymal stromal cell-derived extracellular vesicles improve pathology in Niemann-Pick type C disease.Biomedicines. 2021; 9: 1864
- Mesenchymal stromal cell-derived small extracellular vesicles induce ischemic neuroprotection by modulating leukocytes and specifically neutrophils.Stroke. 2020; 51: 1825-1834
- Individual immune-modulatory capabilities of MSC-derived extracellular vesicle (EV) preparations and recipient-dependent responsiveness.Int J Mol Sci. 2019; 20: 1642
- Perinatal derivatives: How to best validate their immunomodulatory functions.Front Bioeng Biotechnol. 2022; 10981061
- A robust potency assay highlights significant donor variation of human mesenchymal stem/progenitor cell immune modulatory capacity and extended radio-resistance.Stem Cell Res Ther. 2015; 6: 236
- An in vitro potency assay for monitoring the immunomodulatory potential of stromal cell-derived extracellular vesicles.Int J Mol Sci. 2017; 18
- CD73 activity of mesenchymal stromal cell-derived extracellular vesicle preparations is detergent-resistant and does not correlate with immunomodulatory capabilities.Cytotherapy. 2023; 25: 138-147
- Asymmetric cell division within the human hematopoietic stem and progenitor cell compartment: identification of asymmetrically segregating proteins.Blood. 2007; 109: 5494-5501
- Segregation of lipid raft markers including CD133 in polarized human hematopoietic stem and progenitor cells.Blood. 2004; 104: 2332-2338
- Scaled isolation of mesenchymal stem/stromal cell-derived extracellular vesicles.Curr Protoc Stem Cell Biol. 2020; 55: e128
- Precipitation with polyethylene glycol followed by washing and pelleting by ultracentrifugation enriches extracellular vesicles from tissue culture supernatants in small and large scales.J Extracell Vesicles. 2018; 71528109
- Minimal information for studies of extracellular vesicles 2018 (MISEV2018): a position statement of the International Society for Extracellular Vesicles and update of the MISEV2014 guidelines.J Extracell Vesicles. 2018; 71535750
- Optimisation of imaging flow cytometry for the analysis of single extracellular vesicles by using fluorescence-tagged vesicles as biological reference material.J Extracell Vesicles. 2019; 81587567
- Characterisation of exosomes derived from human cells by nanoparticle tracking analysis and scanning electron microscopy.Colloids Surf B Biointerfaces. 2011; 87: 146-150
- Contribution of naive and memory T-cell populations to the human alloimmune response.Am J Transplant. 2009; 9: 2057-2066
- Tracking donor-reactive T cells: Evidence for clonal deletion in tolerant kidney transplant patients.Sci Transl Med. 2015; 7: 272ra210
- Alloreactive T cell clonotype recruitment in a mixed lymphocyte reaction: implications for graft engineering.Exp Hematol. 2006; 34: 788-795
- Quantifying the frequency of alloreactive T cells in vivo: new answers to an old question.J Immunol. 2001; 166: 973-981
- Optimization of immunosuppressive therapy based on a multiparametric mixed lymphocyte reaction assay reduces infectious complications and mortality in living donor liver transplant recipients.Transplant Proc. 2012; 44: 555-559
- Sensitive method for measuring apoptosis and cell surface phenotype in human thymocytes by flow cytometry.Cytometry. 1994; 15: 12-20
- Comparative analysis of lymphocyte activation marker expression and cytokine secretion profile in stimulated human peripheral blood mononuclear cell cultures: an in vitro model to monitor cellular immune function.J Immunol Methods. 2004; 293: 127-142
- Scaled preparation of extracellular vesicles from conditioned media.Adv Drug Deliv Rev. 2021; 177113940
- Human T cell activation with phytohemagglutinin. The function of IL-6 as an accessory signal.J Immunol. 1988; 141: 3868-3874
- Establishment of the reference intervals of lymphocyte function in healthy adults based on IFN-γ secretion assay upon phorbol-12-myristate-13-acetate/ionomycin stimulation.Front Immunol. 2018; 9: 172
- TRAIL-receptor costimulation inhibits proximal TCR signaling and suppresses human T cell activation and proliferation.J Immunol. 2014; 193: 4021-4031
Article info
Publication history
Published online: April 22, 2023
Accepted:
March 19,
2023
Received:
November 18,
2022
Publication stage
In Press Corrected ProofIdentification
Copyright
© 2023 International Society for Cell & Gene Therapy. Published by Elsevier Inc. All rights reserved.
