Mesenchymal stem cells preconditioned with proinflammatory cytokines enhance T cell inhibition by the downregulation of CD25 on activated T cells, expression of immunosuppressive factors and increase of regulatory T cells.

Kisha N Sivanathan^1,2,3,4, Christopher M Hope^1,2,3,4, Robert Carroll^1,2,3,4, Stan Gronthos^1,2,3,4, P. Toby Coates^1,2,3,4

¹Clinical and Experimental Transplantation Group, Royal Adelaide Hospital, Adelaide, Australia; ²Medicine , University of Adelaide, Adelaide, Australia; ³Mesenchymal Stem Cell Group, Department of Hematology, SA Pathology, Adelaide, Australia; ⁴Central Northern Adelaide Renal Transplantation Service, , Royal Adelaide Hospital, Adelaide, Australia

Mesenchymal stem cells (MSC) require sufficient threshold of proinflammatory cytokines to activate their immunosuppressive function in vivo. Hence, we aimed to modify human bone marrow derived MSC with proinflammatory cytokines as a strategy to enhance MSC immunosuppression on T cells. To investigate the effect of exogenous proinflammatory cytokines on MSC immunosuppression, MSC were co-cultured with T cells activated by the mitogen phytohemagglutinin (PHA). The exogenous addition of interferon-gamma (IFN-γ), tumor necrosis factor-alpha (TNF-α), interleukin-1β (IL-1β), IL-2, IL-12p70 and IL-17A to these co-cultures antagonised MSC-mediated immunosuppression of T cells. However, when MSC were preconditioned with the following cytokines for 5 days, they displayed greater immunosuppressive properties on T cell proliferation by 14.8%, 22.2%, 17.1%, 23.2%, 17.2% and 27.7%, respectively, compared to unmodified-MSC (UT:MSC). Only the IFN-γ modified-MSC (MSC-γ) showed upregulation of the T cell negative co-stimulatory molecule programme-death ligand-1 (PD-L1). The neutralisation of PD-L1 however failed to restore T cell proliferative responses suggesting a partial but non-exclusive role of PD-L1 in MSC-γ immunosuppression. IL-6 gene expression significantly increased in the TNF-α and IL-1β-modified MSC. MSC-γ and TNF-α-modified MSC upregulated prostaglandin-2 gene expression relative to UT:MSC and significantly induced the indoleamine 2,3-dioxygenase gene expression in MSC. The gene expression of transforming growth factor beta-1 (TGF-β1) was unaffected following cytokine modification of MSC. Both the UT:MSC and the cytokine modified MSC showed no detectable levels of IL-10 following 5 days of culture. Furthermore, MSC-γ showed greatest reduction in the T cell activation marker CD25 on CD3⁺ T cells while the IL-17 preconditioned MSC (MSC-17) reduced CD25 levels comparable to UT:MSC. These MSC show a greater reduction of CD25 on CD8⁺ T cells than in the CD4⁺T cells. In addition, a similar increase in proportion of CD3⁺CD4⁺Foxp3⁺CD127^lowCD25^hi regulatory T cells (Tregs) was observed in T cells co-cultured with UT:MSC and MSC-17 compared to the PHA activated T cell control. A 2-fold increase in the proportion of Tregs was evident in the MSC-γ-T cell co-culture compared to UT:MSC and MSC-17. In conclusion, preconditioning MSC with proinflammatory cytokines enhances the therapeutic efficacy of MSC as immunosuppressive agents via the downregulation of CD25 on activated T cells, expression of inhibitory factors and increase of Tregs. The mechanisms of enhanced MSC-mediated immunosuppressive effect on T cells were dependent on the proinflammatory cytokines used for MSC modification.