Background The inhibitory costimulatory molecule Cytotoxic T-Lymphocyte Antigen-4 (CTLA-4) plays a crucial role in conveying immune tolerance in both inflammatory disorders and regenerative processes such as bone healing. During regenerative processes, mesenchymal stromal cells (MSC) provide the building bricks for reestablishing structural integrity but do also control inflammation by their immunomodulatory activities under restrictive microenvironmental conditions such as hypoxia. Here, we hypothesize these cells to support the control of inflammation via CTLA-4 in order to facilitate tissue regeneration such as bone fracture healing.
Objectives Therefore, we analyzed expression of CTLA-4 by human MSC and their ability to convey immune suppression.
Methods MSC were isolated from bone marrow of patients undergoing total hip replacement and characterized (i) by surface marker staining using flow cytometry and (ii) via assessing their osteogenic and adipogenic differentiation potential. MSC were cultured under normoxic (∼18% O2) and hypoxic (<1.5% O2) conditions, respectively. CTLA-4 expression in MSC was analyzed on protein and on transcriptional levels by flow cytometry, immunoblotting, ELISA and by quantitative PCR, respectively. To assess the functionality of CTLA-4 expression, MSC were co-cultured with peripheral blood mononuclear cells (PBMC) isolated by density gradient centrifugation from venous blood of healthy donors. The cells were stimulated for 48h with PHA (5μg/ml) or left untreated and challenged using either CTLA-4-Ig or anti-CTLA-4-antibody, incubated under normoxic (∼18% O2) or hypoxic (<1.5% O2) conditions and analyzed for TNFα secretion by suspension assay.
Results The MSC phenotype of bone-marrow derived cells could be verified according to their surface marker expression and their osteogenic and adipogenic differentiation capacity. On transcriptional level, MSC express both the full-length and - to a higher extent - the soluble CTLA-4 isoforms with a higher mRNA abundance under normoxic as compared to hypoxic conditions. Extra- and intracellular analysis of CTLA-4 expression on protein level, demonstrated a significant shift of the whole MSC population (p<0,01). CTLA-4 expression and secretion by MSC was confirmed using immunoblot and ELISA, respectively. Co-culture of MSC with PHA-activated PBMC significantly reduced the amount of secreted TNFα (p<0,05) which could be reversed by anti-CTLA-4-antibody (p<0,05), under both normoxic and compared to hypoxic conditions, respectively.
Conclusions We clearly demonstrate the existence of CTLA-4 on hMSC and its functionality with regard to the inhibition of PHA-induced TNFα secretion by PBMC. We also demonstrate that the expression of CTLA-4 (i) contributes to the immunomodulatory capacity of hMSC and (ii) supports the 'immune privileged' status of these cells.
Disclosure of Interest None declared