Qingdong Guan1, Yun Li2, Tanner Shpiruk3, Swaroop Bhagwat4, Donna A Wall5. 1. Department of Pediatrics & Child Health, University of Manitoba, Winnipeg, Canada; Department of Immunology, University of Manitoba, Winnipeg, Canada; Cellular Therapy Laboratory, CancerCare Manitoba, Winnipeg, Canada; The Institute of Clinical Research and Translational Medicine, Gansu Provincial Hospital, Lanzhou, China. Electronic address: qdguan@gmail.com. 2. Department of Pediatrics & Child Health, University of Manitoba, Winnipeg, Canada. 3. Department of Pediatrics & Child Health, University of Manitoba, Winnipeg, Canada; Cellular Therapy Laboratory, CancerCare Manitoba, Winnipeg, Canada. 4. Department of Immunology, University of Manitoba, Winnipeg, Canada; Cellular Therapy Laboratory, CancerCare Manitoba, Winnipeg, Canada. 5. Department of Pediatrics & Child Health, University of Manitoba, Winnipeg, Canada; Department of Immunology, University of Manitoba, Winnipeg, Canada; Cellular Therapy Laboratory, CancerCare Manitoba, Winnipeg, Canada; Blood and Marrow Transplant/Cellular Therapy, Division of Haematology/Oncology, The Hospital for Sick Children and the University of Toronto, Toronto, Canada. Electronic address: donna.wall@sickkids.ca.
Abstract
AIM: Establishment of a potency assay in the manufacturing of clinical-grade mesenchymal stromal cells (MSCs) has been a challenge due to issues of relevance to function, timeline and variability of responder cells. In this study, we attempted to develop a potency assay for MSCs. METHODS: Clinical-grade bone marrow-derived MSCs were manufactured. The phenotype and immunosuppressive functions of the MSCs were evaluated based on the International Society for Cellular Therapy guidelines. Resting MSCs licensed by interferon (IFN)-γ exposure overnight were evaluated for changes in immune suppression and immune-relevant proteins. The relationship of immune-relevant protein expression with immunosuppression of MSCs was analyzed. RESULTS: MSC supressed third-party T-lymphocyte proliferation with high inter-donor and inter-test variability. The suppression of T-lymphocyte proliferation by IFN-γ-licensed MSCs correlated with that by resting MSCs. Many cellular proteins were up-regulated after IFN-γ exposure, including indoleamine 2,3-dioxygenase 1 (IDO-1), programmed death ligand 1 (PD-L1), vascular cell adhesion molecule 1 (VCAM-1), intercellular adhesion molecule 1 (ICAM-1) and bone marrow stromal antigen 2 (BST-2). The expression levels of IDO-1 and PD-L1 on licensed MSCs, not VCAM-1, ICAM-1 or BST-2 on licensed MSCs, correlated with MSC suppression of third-party T-cell proliferation. CONCLUSION: A flow cytometry-based assay of MSCs post-IFN-γ exposure measuring expression of intracellular protein IDO-1 and cell surface protein PD-L1 captures two mechanisms of suppression and offers the potential of a relevant, rapid assay for MSC-mediated immune suppression that would fit with the manufacturing process.
AIM: Establishment of a potency assay in the manufacturing of clinical-grade mesenchymal stromal cells (MSCs) has been a challenge due to issues of relevance to function, timeline and variability of responder cells. In this study, we attempted to develop a potency assay for MSCs. METHODS: Clinical-grade bone marrow-derived MSCs were manufactured. The phenotype and immunosuppressive functions of the MSCs were evaluated based on the International Society for Cellular Therapy guidelines. Resting MSCs licensed by interferon (IFN)-γ exposure overnight were evaluated for changes in immune suppression and immune-relevant proteins. The relationship of immune-relevant protein expression with immunosuppression of MSCs was analyzed. RESULTS: MSC supressed third-party T-lymphocyte proliferation with high inter-donor and inter-test variability. The suppression of T-lymphocyte proliferation by IFN-γ-licensed MSCs correlated with that by resting MSCs. Many cellular proteins were up-regulated after IFN-γ exposure, including indoleamine 2,3-dioxygenase 1 (IDO-1), programmed death ligand 1 (PD-L1), vascular cell adhesion molecule 1 (VCAM-1), intercellular adhesion molecule 1 (ICAM-1) and bone marrow stromal antigen 2 (BST-2). The expression levels of IDO-1 and PD-L1 on licensed MSCs, not VCAM-1, ICAM-1 or BST-2 on licensed MSCs, correlated with MSC suppression of third-party T-cell proliferation. CONCLUSION: A flow cytometry-based assay of MSCs post-IFN-γ exposure measuring expression of intracellular protein IDO-1 and cell surface protein PD-L1 captures two mechanisms of suppression and offers the potential of a relevant, rapid assay for MSC-mediated immune suppression that would fit with the manufacturing process.
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