WITHDRAWN: Knockout of β2 Microglobulin Potentiates The Effects of IFN-γ Priming On The Survival and Immunomodulatory Capacities of Mesenchymal Stem Cells
Abstract Background MSCs have long been thought to be immune-privileged with low levels of major histocompatibility complex (MHC) class I and rare expression of MHC class II. However, growing evidence indicates that these cells may not actually be hypoimmunogenic, particularly when exposed to cytokines such as IFN-γ. IFN-γ primed increase of MHC class I expression can promote the rejection of allogenic MSCs in the host recipient. A strategy to overcome this drawback is urgently required. Methods We knocked out β2-microglobulin (B2M) in MSCs, which is a component of MHC class I, using the ribonucleoprotein (RNP)-mediated clustered regularly interspaced short palindromic repeats (CRISPR)- CRISPR-associated protein 9 (Cas9) system. The expression of MSC surface markers, MHC class I, and B2M was assayed by flow cytometry and western blotting. Upon co-culture of MSCs with CD4+ and CD8+ T cells, the survival and proliferation of both cell types were examined by cell counting kit (CCK-8) and carboxy fluorescein succinimidyl ester (CFSE), respectively. The levels of immunomodulatory molecules in MSCs were evaluated by both enzyme-linked immunosorbent assay (ELISA) and western blotting. Results B2M-knockout MSCs expressed low levels of MHC class I even upon IFN-γ priming, but maintained their native properties as evidenced by the expression of specific surface markers. CD8+ T cell proliferation was also far less stimulated by B2M-knockout MSCs than by control cells. Under these conditions, B2M-knockout MSCs had a significantly longer survival duration (> 2.4 fold) than did control cells. B2M-knockout MSCs showed significantly elevated levels of immune-modulatory molecules including indoleamine 2, 3-dioxygenase 1 (IDO-1), prostaglandin E2 (PGE2), C-C motif chemokine ligand 2 (CCL-2), and interleukin-6 (IL-6); conversely, B2M-knockout MSCs produced significantly lower levels of proinflammatory molecules (e.g., IL-1b, CXCL10) compared with control cells. Conclusion The loss of B2M in MSCs potentiated the immunomodulatory effects of IFN-γ priming while mitigating its potential inflammatory effects. B2M-knockout MSCs are a potentially promising treatment for immune-related inflammatory diseases..
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Preprint| Erscheinungsjahr: |
2024 |
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| Erschienen: |
2024 |
| Enthalten in: |
ResearchSquare.com - (2024) vom: 15. März Zur Gesamtaufnahme - year:2024 |
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| Sprache: |
Englisch |
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| Beteiligte Personen: |
HAN, A.Reum [VerfasserIn] |
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| Links: |
Volltext [kostenfrei] |
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| doi: |
10.21203/rs.3.rs-864687/v1 |
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XRA033465819 |
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| 520 | |a Abstract Background MSCs have long been thought to be immune-privileged with low levels of major histocompatibility complex (MHC) class I and rare expression of MHC class II. However, growing evidence indicates that these cells may not actually be hypoimmunogenic, particularly when exposed to cytokines such as IFN-γ. IFN-γ primed increase of MHC class I expression can promote the rejection of allogenic MSCs in the host recipient. A strategy to overcome this drawback is urgently required. Methods We knocked out β2-microglobulin (B2M) in MSCs, which is a component of MHC class I, using the ribonucleoprotein (RNP)-mediated clustered regularly interspaced short palindromic repeats (CRISPR)- CRISPR-associated protein 9 (Cas9) system. The expression of MSC surface markers, MHC class I, and B2M was assayed by flow cytometry and western blotting. Upon co-culture of MSCs with CD4+ and CD8+ T cells, the survival and proliferation of both cell types were examined by cell counting kit (CCK-8) and carboxy fluorescein succinimidyl ester (CFSE), respectively. The levels of immunomodulatory molecules in MSCs were evaluated by both enzyme-linked immunosorbent assay (ELISA) and western blotting. Results B2M-knockout MSCs expressed low levels of MHC class I even upon IFN-γ priming, but maintained their native properties as evidenced by the expression of specific surface markers. CD8+ T cell proliferation was also far less stimulated by B2M-knockout MSCs than by control cells. Under these conditions, B2M-knockout MSCs had a significantly longer survival duration (> 2.4 fold) than did control cells. B2M-knockout MSCs showed significantly elevated levels of immune-modulatory molecules including indoleamine 2, 3-dioxygenase 1 (IDO-1), prostaglandin E2 (PGE2), C-C motif chemokine ligand 2 (CCL-2), and interleukin-6 (IL-6); conversely, B2M-knockout MSCs produced significantly lower levels of proinflammatory molecules (e.g., IL-1b, CXCL10) compared with control cells. Conclusion The loss of B2M in MSCs potentiated the immunomodulatory effects of IFN-γ priming while mitigating its potential inflammatory effects. B2M-knockout MSCs are a potentially promising treatment for immune-related inflammatory diseases. | ||
| 650 | 4 | |a Biology |7 (dpeaa)DE-84 | |
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| 700 | 1 | |a Shin, Ha Rim |4 aut | |
| 700 | 1 | |a Kweon, Jiyeon |4 aut | |
| 700 | 1 | |a Lee, Sang Eun |4 aut | |
| 700 | 1 | |a Go, Jinyoung |4 aut | |
| 700 | 1 | |a Lee, Soo Bin |4 aut | |
| 700 | 1 | |a Chang, Eun-Ju |4 aut | |
| 700 | 1 | |a Kim, Yongsub |4 aut | |
| 700 | 1 | |a Kim, Seong Who |4 aut | |
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