Toll-Like Receptor-Stimulated CD14⁺ Monocytes Induce Soluble Factors That Activate Human Mesenchymal Stem Cells To Inhibit T-Cell Alloreactivity.

Human mesenchymal stem cells (MSCs) produce soluble factors that inhibit T-cell proliferation and alloreactivity. We have previously shown that MSCs require activation by CD14⁺ monocytes in cell culture. Toll-like receptors (TLRs) critically modulate antigen-presenting cell (APC) activation, maturation and function. Therefore, we aimed to determine whether TLR agonists could enhance CD14-mediated MSC activation and subsequent MSC-mediated T-cell inhibition. TLR agonists and human IL-1β were used to stimulate CD14⁺ cells isolated from peripheral blood of normal donors. TLR agonists included formalin-fixed Staphylococcus aureus Cowan A strain (SAC, TLR-2), Pam₃CysSerLys₄ (Pam₃Cys, TLR-2), Salmonella enteriditis lipopolysaccharide (LPS, TLR-4), and R848 (Resiquimod, TLR7/8) in complete RPMI media (heat-inactivated FBS, glutamine, and antibiotics). TLR-stimulated CD14⁺ cells and supernatants from TLR-stimulated CD14⁺ cells were then used to stimulate third- and fourth-passage human MSCs (CD45⁻CD105⁺CD90⁺CD80⁻CD73⁺HLA-I⁺) expanded from normal volunteer bone marrow aspirate specimens. After a 24-hour culture with stimulant cells or supernatants, stimuli were removed, MSCs were washed twice with sterile PBS, and then were cultured for an additional 24 h in FBS-free RPMI media. Supernatants from TLR-stimulated CD14⁺ cell cultures and from washed MSC cultures were used to measure cytokine and chemokine production and to inhibit T-cell alloreactivity using an established mixed lymphocyte reaction (MLR) IFN-γ ELISPOT. MLR was also performed in the presence of TLR agonists and media alone. TLR stimulation resulted in high-level soluble factor induction (IL-1β, IL-6, TNF-α, and RANTES) from CD14⁺ cells. For example, levels of inducible IL-6 measured in CD14⁺ cell culture supernatants following LPS, SAC and R848 stimulation were 5.2 (70.3 ± 26.2 ng/ml), 4.7 (64.1 ± 16.3 ng/ml), and 4.6 (63.4 ± 13.1 ng/ml)-fold higher than after IL-1β stimulation (13.7 ± 4.0 ng/ml) (Mean ± SEM, 4 independent experiments). Supernatants of TLR-stimulated CD14⁺ cells induced higher levels of soluble factors from MSCs than stimulation with CD14⁺ cells themselves, suggesting that soluble factors from CD14⁺ cells activate MSCs. Not all supernatants of TLR-stimulated CD14⁺ cells were similar in their capacity to activate MSC-mediated inhibition of T-cell alloreactivity. For example, supernatants of Pam₃Cys-, R848- and SAC-stimulated CD14⁺ cells all induced high levels of TNF-α in washed MSC cultures; but only supernatant from Pam₃Cys-stimulated CD14⁺ cells resulted in MSC-mediated inhibition of T-cell alloreactivity (63.7 ± 12.9 % inhibition, Mean ± SEM, 4 independent ELISPOTs). Ongoing studies are being performed to define these immunomodulatory factors. Together, these results suggest that ex vivo TLR stimulation enhances soluble factor production from CD14⁺ cells, which, in turn, increases MSC production of immunomodulatory factors that mediate inhibition of T-cell alloreactivity.