gms | German Medical Science

45. Kongress der Deutschen Gesellschaft für Rheumatologie, 31. Jahrestagung der Deutschen Gesellschaft für Orthopädische Rheumatologie, 27. Jahrestagung der Gesellschaft für Kinder- und Jugendrheumatologie

06.09. - 09.09.2017, Stuttgart

Human Mesenchymal Stromal Cells reduce TNFα secretion of activated PBMC via CTLA-4

Meeting Abstract

  • Timo Gaber - Charité-Universitätsmedizin Berlin, Medizinische Klinik mit Schwerpunkt Rheumatologie und klinische Immunologie, Berlin
  • Kerstin Schönbeck - Deutsches Rheuma-Forschungszentrum (DRFZ), AG Buttgereit, Berlin
  • Cam Loan Tran - Charité - Universitätsmedizin Berlin, Medizinische Klinik mit Schwerpunkt Rheumatologie und klinische Immunologie, Berlin
  • Cindy Strehl - Charité - Universitätsmedizin Berlin, Medizinische Klinik mit Schwerpunkt Rheumatologie und klinische Immunologie, Berlin
  • Eric Röhner - Friedrich-Schiller-Universität, Orthopädie, Jena
  • Georg Matziolis - Friedrich-Schiller-University, Department of Orthopaedics, Jena
  • Gerd-Rüdiger Burmester - Charité - Universitätsmedizin Berlin, Medizinische Klinik mit Schwerpunkt Rheumatologie und klinische Immunologie, Berlin
  • Frank Buttgereit - Charité-Universitätsmedizin Berlin, Medizinische Klinik mit Schwerpunkt Rheumatologie und klinische Immunologie, Berlin
  • Paula Hoff - Charité-Universitätsmedizin Berlin, Medizinische Klinik mit Schwerpunkt Rheumatologie und klinische Immunologie, Berlin

Deutsche Gesellschaft für Rheumatologie. Deutsche Gesellschaft für Orthopädische Rheumatologie. Gesellschaft für Kinder- und Jugendrheumatologie. 45. Kongress der Deutschen Gesellschaft für Rheumatologie (DGRh), 31. Jahrestagung der Deutschen Gesellschaft für Orthopädische Rheumatologie (DGORh), 27. Jahrestagung der Gesellschaft für Kinder- und Jugendrheumatologie (GKJR). Stuttgart, 06.-09.09.2017. Düsseldorf: German Medical Science GMS Publishing House; 2017. DocER.09

doi: 10.3205/17dgrh084, urn:nbn:de:0183-17dgrh0841

Published: September 4, 2017

© 2017 Gaber et al.
This is an Open Access article distributed under the terms of the Creative Commons Attribution 4.0 License. See license information at http://creativecommons.org/licenses/by/4.0/.


Outline

Text

Background: The inhibitory costimulatory molecule Cytotoxic T-Lymphocyte Antigen-4 (CTLA-4) plays a crucial role in conveying immune tolerance in inflammatory disorders and in regenerative processes such as bone healing. During regenerative processes, mesenchymal stromal cells (MSC) provide the building bricks for reestablishing structural integrity but 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. 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 level 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: 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 could be confirmed using immunoblot and ELISA, respectively. Co-culture of MSC with PHA-activated PBMC significantly reduced the amount of secreted TNFa (p<0,05) which could be reversed by anti-CTLA-4-antibody (p<0,05), under both normoxic and compared to hypoxic conditions, respectively.

Conclusion: We could clearly demonstrate the existence of CTLA-4 on hMSC and its functionality with regard to the inhibition of PHA-induced TNF secretion by PBMC. We could 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.