TNF‐a/IL‐1B Cytokine Licensing Enhances Immunomodulatory Potential of Allogeneic Mesenchymal Stromal Cells

Aims/Purpose: Mesenchymal stromal cells (MSCs) have shown great potential for therapeutic treatment of inflammatory diseases; however, their immunomodulatory potential can be further enhanced by cytokine licensing. The aim of this work was to assess changes in MSC surface characterization profile and immunomodulatory function following licensing with TNF‐α/IL‐1β pro‐inflammatory cytokines.Methods: MSCs were isolated from the bone marrow of FVB mice (H2kq) and licensed with a combination of 50 ng/mL TNF‐α and 50 ng/mL IL‐1β or cultured in growth medium alone. MSCs were harvested following 72‐h cytokine stimulation and added to immune cell potency assays. Characterization of MSC surface marker expression was assessed by flow cytometry. The immunology potential of FVB MSCs as an allogeneic cell therapy was assessed in vitro through co‐culture with Balb/c (H2kd) αCD3/CD28 activated T lymphocytes or Balb/c IFN‐γ/LPS‐stimulated macrophages.Results: TNF‐α/IL‐1β licensing was found to induce surface marker phenotype alterations compared with unlicenced MSCs, with increased expression of CD73, CD44, Sca‐1, CD29 and PD‐L1 observed. TNF‐α/IL‐1β licensed MSCs also secreted significantly higher quantities of nitric oxide compared with their untreated counterparts at two separate timepoints, 24‐h and 42‐h post‐stimulation. TNF‐α/IL‐1β MSCs significantly suppressed both CD4+ and CD8+ T cell proliferation at three different rations (1:5, 1:10, 1:50 MSCs: T cells) and significantly increased expansion of CD4+CD25++FoxP3+ regulatory T cells following co‐culture. TNF‐α/IL‐1β MSCs were found to significantly decrease levels of IFN‐γ from proliferating T cells. TNF‐α/IL‐1β MSCs were positive for CD80 expression had significantly reduced MHC I expression and could increase expansion of CD206 on M1‐like macrophages. Both T cell and macrophage co‐cultures with TNF‐α/IL‐1β MSCs showed a significant increase in IL‐6.Conclusions: Our results show the potential to enhance the therapeutic effects of MSC through cytokine licensing. Currently, TNF‐α/IL‐1β MSCs are being tested in ocular surface injury models for therapeutic efficacy.Keywords: Mesenchymal Stromal Cells, Cytokine Licensing, Immunomodulation, TNF‐α/IL‐1β.