(H-303) Efferocytosis of Viable, but not heat-inactivated allogeneic human MSCs converts human monocytes to an immunosuppressive phenotype

Introduction:: Mesenchymal stromal cells (MSCs) have been widely utilized as an immunotherapy to combat pathological inflammation that occurs in diseases like graft versus host disease and Crohn’s disease. Despite their use in 100’s of clinical trials, the precise mechanisms by which MSCs suppress inflammation are still the subject of intense research and debate. While MSCs actively produce numerous immunosuppressive factors, intravenously infused MSCs are rapidly cleared from the body limiting the time these factors have to act. Despite this limited persistence, a potent immunotherapeutic response is still observed upon infusion of MSCs. Recently it has been hypothesized that MSCs achieve their durable effects despite their limited persistence by polarizing monocytes toward immunosuppressive phenotypes via efferocytosis. We sought to answer the question, if efferocytosis of MSCs by monocytes is responsible, are viable MSCs even required to elicit an immunosuppressive effect?


Materials and Methods:: To facilitate efferocytosis, human peripheral blood mononuclear cells (PBMCs) and umbilical-cord MSCs were co-cultured for 24hr in non-adherent polypropylene V-bottom microplates. To assess monocyte efferocytosis of MSCs, MSCs were stained with CellBrite Orange (CBO) prior to co-culture. MSCs were either viable or heat-inactivated prior to the 24hr co-culture. Following the co-culture, efferocytosis was then assessed by flow cytometry by the percent of monocytes (CD14+) cells positive for cell bright orange.

To assess immunosuppressive potential of efferocytosis-licensed monocytes, PBMCs were stained with CFSE proliferation dye prior to co-culture with MSCs in the non-adherent V-bottom plates. Following 24 hours of efferocytosis, PBMCs were collected and either re-plated to a flat-bottom microplate or underwent depletion of non-efferocytosed MSCs before being re-plated to allow us to determine the contribution of residual non-efferocytosed MSCs on T-cell suppression. T cells were then stimulated using anti-CD3/CD28 Dynabeads. After 4-days, samples were collected and assessed for T cell proliferation by flow cytometry using CFSE dilution of CD3+ gated cells as a marker of T-cell inflammation. All data was analyzed using FlowJo and GraphPad Prism 9.


Results, Conclusions, and Discussions:: Results

Viable and HI-MSCs experienced equivalent levels of efferocytosis by monocytes showing heat inactivation does not impair monocyte efferocytosis of MSCs. While the amount of efferocytosis was the same between viable and HI-MSC, only monocytes that efferocytosed viable MSC displayed an immunosuppressive phenotype toward T-cells. Efferocytosis of viable, but not heat inactivated MSC led monocytes to display a dose-dependent increase in the level of suppression of T-cell proliferation. When non-efferocytosed MSCs were removed prior to T-cell activation, the same level of T-cell suppression was observed, demonstrating that efferocytosis of viable MSCs leads to conversion of monocytes to a phenotype that is directly suppressive of T-cells.

Figure. (A) Schematic of the culture procedure used to allow human monocytes to efferocytose labeled MSCs followed by stimulation of T-cells with CD3-CD28 dynabeads. (B) Images from a z-stack of immunohistochemically stained monocytes after efferocytosis of MSCs CBO+ MSC debris (red) within CD14+ monocytes (green). (C) Percent efferocytosis of CBO+ viable or HI-MSCs within CD14+ monocytes showing dose-dependent MSC efferocytosis by monocytes. (D)Represen€ive CFSE flow cytometry plots reveal efferocytosis of viable but not HI-MSC results in T-cell suppression. (E) Percent suppression of T cells following efferocytic-licensing of monocytes with viable or HI-MSCs compared to control. (F) Viable MSC efferocytic-licensing with and without removal of non-phagocytosed MSCs results in suppression of T cell proliferation.

Discussion & Conclusion

This work provides clear evidence that efferocytosis of viable MSCs by monocytes is a mechanism of action by which MSCs elicit an immunosuppressive response toward T-cell mediated inflammation. Furthermore, it highlights the importance of good manufacturing, cryopreservation, and quality control practices in the production of MSC-based immunotherapies. Even though efferocytosis of dead MSCs by monocytes is a mechanism of action, it still depends on the quality and initial health of the MSC product. Future work is needed to identify the viable MSC attributes that dictate whether monocytes convert to an immunosuppressive phenotype after efferocytosing MSCs.




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