Senescence-associated defective HLA-DR upregulation does not modulate immunosuppressive properties of MSCs. (A) Fit and senescent MSCs were subjected to staining of the receptor for IFNγ (CD119) and acquired through flow cytometry. (B) Fit and senescent MSCs stimulated with indicated concentrations of IFNγ for 15 minutes. P-STAT1 (Y701) Phosflow was performed subsequently and acquired through flow cytometry. Similar results were obtained in a repeat experiment with an additional 1 or 2 fit and senescent MSC donor pairs. (C) Fit and senescent MSCs were stimulated with IFNγ for 48 hours and subsequently stained with the antibodies to the surface markers for HLA-ABC, HLA-DR, B7-1, B7-2, B7H1, and B7DC for flow cytometry. (D) Cumulative HLA-DR MFI fold change derived from 2 independent fit and senescent MSC donor pairs were shown. (E) Fit and senescent MSCs were stimulated with IFNγ for 48 hours. Expression level of HLA-DR mRNA relative to GAPDH was evaluated by the quantitative SYBR green real-time PCR. δ-δ CT method was applied to calculate the fold induction of HLA-DR over the unstimulated control. Control or HLA-DR siRNA-transfected MSCs were stimulated with IFNγ for 48 hours. Cells were trypsinized, stained for HLA-DR or appropriate isotype control antibodies, and analyzed by flow cytometry. (F) Representative histogram. (G) Cumulative mean fluorescent intensity of HLA-DR and isotype control antibody stains were shown on 2 independent fit and senescent MSC donor pairs. HLA-DR or control siRNA-transfected MSCs were cultured with αCD3αCD28 Dynabeads-stimulated PBMCs. Four days post culture, T-cell proliferation was measured by Ki67 intracellular staining. (H) Representative FACS plot and effect of MSCs on (I) CD3⁺CD4⁺ and (J) CD3⁺CD8⁺ T-cell proliferation are shown. Similar results were obtained in a repeat experiment with another MSC donor.