Immune dysfunctionality of replicative senescent mesenchymal stromal cells is corrected by IFNγ priming

Abstract

Industrial-scale expansion of mesenchymal stromal cells (MSCs) is often used in clinical trials, and the effect of replicative senescence on MSC functionality is of mechanistic interest. Senescent MSCs exhibit cell-cycle arrest, cellular hypertrophy, and express the senescent marker β-galactosidase. Although both fit and senescent MSCs display intact lung-homing properties in vivo, senescent MSCs acquire a significant defect in inhibiting T-cell proliferation and cytokine secretion in vitro. IFNγ does not upregulate HLA-DR on senescent MSCs, whereas its silencing did not reverse fit MSCs’ immunosuppressive properties. Secretome analysis of MSC and activated peripheral blood mononuclear cell coculture demonstrate that senescent MSCs are significantly defective in up (vascular endothelial growth factor [VEGF], granulocyte colony-stimulating factor [GCSF], CXCL10, CCL2) or down (IL-1ra, IFNγ, IL-2r, CCL4, tumor necrosis factor-α, IL-5) regulating cytokines/chemokines. Unlike indoleamine 2,3 dioxygenase (IDO), silencing of CXCL9, CXCL10, CXCL11, GCSF, CCL2, and exogenous addition of VEGF, fibroblast growth factor-basic do not modulate MSCs’ immunosuppressive properties. Kynurenine levels were downregulated in senescent MSC cocultures compared with fit MSC counterparts, and exogenous addition of kynurenine inhibits T-cell proliferation in the presence of senescent MSCs. IFNγ prelicensing activated several immunomodulatory genes including IDO in fit and senescent MSCs at comparable levels and significantly enhanced senescent MSCs’ immunosuppressive effect on T-cell proliferation. Our results define immune functional defects acquired by senescent MSCs, which are reversible by IFNγ prelicensing.

Priming approaches to improve the efficacy of mesenchymal stromal cell-based therapies

“… [ 24 ] IFN-γ Bone marrow – In vitro Reestablished immunosuppressive effect on T-cell proliferation and did not upregulate HLA-DR of senescent MSC. [ 25 ] IFN-γ Bone marrow DSS-induced colitis model In vitro / in vivo (mice) Attenuated development of colitis, reduced pro-inflammatory cytokine levels in colon and increased migration potential. [ 26 ] IFN-γ Umbilical cord Healthy donor/tissue In vitro Increased suppression of NK cells and reduced NK-mediated cytotoxicity.…”

Identification and Characterization of a Large Source of Primary Mesenchymal Stem Cells Tightly Adhered to Bone Surfaces of Human Vertebral Body Marrow Cavities

“…A slight but non-significant increase in suppression at all ratios was observed by pre-treating vBA-MSC with IFN-γ for 18-24 hours prior to performing the suppression studies. Treatment with IFN-γ has been shown to stimulate immunosuppressive functions of MSC, with enhanced effects on senescent cells [12]. The insensitivity to priming with concentrations of IFN-γ shown previously to stimulate T cell suppression may indicate that vBA-MSC retain full immunomodulatory capacity during culture expansion.…”

“…The challenges inherent to manufacturing cellular therapies scale with the size of a manufacturing run. Effective doses of MSC for some indications are as high as 1x10 9 cells per dose, which would require manufacturing 10 trillion (10x10 12 ) cells per year to affordably meet potential demand [1][2][3][4]. Even at this level of production, with presumed economies of scale, the cost of goods sold (COGS) per dose of MSC could exceed $100,000 [3].…”

“…Of the three overarching parameters (i.e., safety, potency and identity) required to assess suitability of a manufactured cell therapy product, potency as it relates to individual donor characteristics, is most problematic due to the changing profile that occurs with expansion, as described above. This is particularly the case as MSC populations near the limits of expansion and enter into senescence which severely limits their potency [12].…”

Mesenchymal Stem Cell-Derived Extracellular Vesicles Reduce Disease Severity and Immune Responses in Inflammatory Arthritis

“…Most studies utilise pro-in ammatory IFN-γ (70,71) and/or TNF-α (72,73) to facilitate priming, although IL-1β (74) and IL-17a(75) have also been applied. Regardless of cytokine selection, the broad outcomes of exposing cells in culture to a pro-in ammatory environment is to initiate strategies that would re-establish homeostatic control in vivo, such as anti-in ammatory effects prompted through alteration of the EV cargo (76) and signalling to reduce recruitment of in ammatory mediators, expression of proin ammatory cytokines, T cell polarisation and proliferation (71,72,77), and restoration of homeostatic ratios of leukocytes and T cells (73,78). A primary mechanism involved here is IFN-γ-mediated upregulation of indoleamine 2,3-dioxygenase (IDO) (70,72,73).…”