HLA Mismatched MSC Suppress T Lymphocyte Allo responses in Vitro and Do Not Induce Immunological Memory in Recipients of MSC Infusion

Multipotent mesenchymal stromal cells (MSC) are increasingly used to treat refractory graft-versus-host-disease and other complications in HLA-matched and mismatched hematopoietic stem cell transplantation (HSCT) patients. We evaluated immunogenicity of human allogeneic MSC infused post-transplant to HLA-mismatched, i.e. patients undergoing HSCT. We compared recipient lympho cyte response to MSC and peripheral blood lymphocytes (PBL) from the MSC or third party donors before and after infusion, and lymphocyte responses to MSC and to PBL from the MSC donor in primary and secondary challenge using 3H-tymidine. MSC recipients displayed in median responses less than 500 counts per minute (CPM) to infused third party MSC 1 week to 6 months following infusion. However, the recipients responded normally to MSC donor lymphocytes, >1000 CPM (p<0.005), and third party lymphocytes, >5000 CPM (p<0.005). MSC failed to prime responder lymphocytes to rechallenge with PBL, as the response was <10 000 versus >30 000 CPM for the corresponding control (p<0.05). On MSC rechallenge of lymphocytes primed with PBL from the MSC donor, only responses <500 CPM occurred. MSC upregulated lymphocyte gene expression of CD25, IFN-γ, FoxP3, CTLA-4 and IL-10 upon MSC/PBL co-culture and MSC presence in mixed lymphocyte cultures. Unprimed and primed responder lymphocytes expanded and proliferated poorly to MSC as stimuli, evaluated by flow cytometry. The MSC failed to induce CD25+ (activated) or CD57+ (effector) CD4+ or CD8+ subsets and only inconsistently induced FoxP3+ regulatory T lymphocytes. These results confirm in vivo and in vitro that infused MSC are weakly immunogenic and do not induce significant immunological memory in HLA-disparate recipients.