Abstract 4865

Introduction:

Mesenquimal Stromal Cells (MSCs) possess immunosuppressive properties, becoming these cells a promising subject of study for future approaches in cell-based therapy. During co-culture with activated lymphocytes and probably through T-cell derived cytokines, MSCs are activated in order to become suppressive. Once MSCs are triggered, they acquire an inhibitory profile increasing the release of immunoregulatory factors, such as IDO, IFNγ, PGE2, NO and TGF-β, responsible for T-cell inhibition. In addition, expression of adhesion molecules, such as ICAM-1, and generation of regulatory T cells subsets, such as CD69+ T cells, are important in immunosuppressive property of human MSCs. However, the exact mechanisms underlying the immunomodulatory functions of MSCs remain largely unknown. NF-kB comprises a family of inducible transcription factors that serve as important regulators of the host immune and inflammatory responses. The NF-kB signals are activated via canonical (mediated mainly by RelA-p50) and/or non-canonical (mediated by RelB-p52) pathways in response to diverse stimuli. Given the immunomodulatory properties of MSCs and the possible involvement of NF-Bk pathway in this effect, here, it was explored the role of non-canonical NF-KB signaling in the immunomodulatory capacity of MSCs cocultured with activated T cells.

Methods:

siRNAs targeting RelB were transfected into MSC with lipofectamine. siRNAs C- were used as negative control and siRNA-FITC as transfection control. After 24 hours, immunomagnetically purified CD3+ T cells were stained with CFSE, activated by anti-CD2/CD3/CD28 beads and cultured in the presence of MSCs. After 3 days, flow cytometry was performed to observe the transfection efficiency, T cell proliferation and percentage of CD69+ T regulatory cells. Additionally, RNA from MSCs was extracted and RelB and ICAM-1 mRNA expression were quantified by Real-time PCR.

Results:

The transfection efficiency was around 75% and RelB mRNA level was reduced by 80% in MSCs transfected with siRNA RelB compared to siRNAs C- transfected cells. Compared to MSCs previously transfected with siRNA C- and co-cultured with activated T cells, MSCs transfected with siRNA RelB resulted increasing of 22% in T cell proliferation and decreasing of 9,2% and 30% in the CD69+ regulatory T cells generation and ICAM-1 expression respectively.

Conclusion:

Non-canonical NF-kB pathway, mediated by RelB, may be partially involved in acquisition of the inhibitory profile by decreasing T cells proliferation, and increasing the expression of ICAM-1 and the generation of CD69+ regulatory T cells.

Disclosures:

No relevant conflicts of interest to declare.

Author notes

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Asterisk with author names denotes non-ASH members.