Toll-Like Receptor Agonists Induce Secretion of Immunomodulatory Soluble Factors from Human Mesenchymal Stem Cells.

Human mesenchymal stem cells (MSCs) suppress T-cell activation and proliferation by inducible expression and secretion of soluble inhibitory factors. We have previously shown that MSCs require activation by antigen-presenting cells (APCs) to secrete these factors. Toll-like receptors (TLRs) and their associated agonists have critical roles in APC activation, maturation and function. Therefore, we investigated whether TLR agonists induce cytokine and chemokine production from MSCs and if such soluble factors mediate inhibition of T-cell alloreactivity. Human MSCs (CD45-CD105+CD90+CD80-CD73+HLA−I+) were expanded from normal volunteer bone marrow aspirate specimens. MSCs were characterized morphologically by light microscopy, phenotypically by flow cytometry and functionally by ex vivo cell culture stimulation and inhibition of T-cell IFN-γ production. Cytokine and chemokine induction of third and fourth-passage MSCs co-cultured in triplicate with established TLR agonists were measured and compared to soluble factor induction from human IL-1β stimulated MSCs. TLR agonists included formalin-fixed Staphylococcus aureus Cowan A strain (SAC, TLR-2), Pam₃CysSerLys₄ (Pam₃Cys, TLR-2), polyinosine:polycytidylic acid (poly I:C, TLR-3), Salmonella enteriditis lipopolysaccharide (LPS, TLR-4), R848 (TLR7/8) and unmethylated CpG oligodeoxynucleotides 1826 and 2216 (TLR-9). 24h MSC-culture supernatants were assessed for levels of soluble factors using standard and multiplex ELISA and for inhibition of T-cell alloreactivity using an established mixed lymphocyte reaction (MLR) IFN-γ ELISPOT. MLR was also performed in the presence of TLR agonists alone and agonist-stimulated MSCs. In general, TLR-MSC supernatants contained 2 to 5-fold lower levels of inducible IL-6, IL-8, IL-10 and RANTES than IL-1β-MSC supernatants. LPS- and poly I:C-MSC supernatants had comparable levels of inducible factors as IL-1β-MSC supernatant. Neither stimulation with IL-1β nor TLR agonists induced phenotypic changes in MSCs, as measured by surface expression of MSC markers as well as activation markers (HLA-DR, CD40, CD40L, CD80, HLA-I and HLA-II). However, supernatant from TLR-MSC cultures (CpG 1826 and poly I:C) and from IL-1β-MSC cultures did inhibit T-cell IFN-γ production. For example, percent IFN-γ inhibition using supernatant from MSC-CpG 1826 culture versus supernatant from IL-1β-MSC culture was 46.5% ± 22.8 and 66.8% ± 13.5, respectively (mean ± SEM, n=3 separate experiments involving different donor MSCs). Likewise, TLR- and IL-1β-stimulated MSCs themselves inhibited T-cell IFN-γ. Together, these results demonstrate that ex vivo culture with TLR agonists activates human MSCs to inhibit ex vivo T-cell alloreactivity likely via inducible soluble factors. Optimization of cell culture conditions is needed to define the soluble factors mediating this inhibitory effect. Our results suggest the presence of a potentially novel immunomodulatory interface at which TLR agonists are uniquely positioned to influence immune effector cell and mesenchymal stem cell responses.