Fig 2.

Pretreatment with inflammatory cytokines leads to upregulation of major histocompatibility complex class I (MHCI), MHCII and vascular adhesion molecule-1 (VCAM-1), and renders allogeneic rat mesenchymal stem cells (rMSCs) susceptible to cytotoxic lysis by alloantigen-specific T cells. (A) MSCs were treated with 100 U/ml IFN-γ, IL-1β or IFN-γ + IL-1β for 24 hr and analysed for MHCI, MHCII and VCAM-1 expression by FACS. IFN-γ (thick black line) and IFN-γ + IL-1β (grey line) induced upregulation of MHCI and MHCII, while MSCs stimulated with IL-1β alone (dotted line) did not change MHCII expression and only slightly increased MHCI expression compared to untreated MSCs (thin black line). VCAM-1 expression was induced by IL-1β and IFN-γ + IL-1β, but not by IFN-γ alone. Isotype controls are shown in filled grey (representative experiment from >3). (B) Alloantigen-specific cytotoxic T cells (CTLs) were generated in a mixed lymphocyte culture of LEW and γ-irradiated DA T cells. Syngeneic LEW or allogeneic DA rMSCs, either untreated or pretreated with 100 U/ml IFN-γ, IL-1β or IFN-γ + IL-1β for 24 hr, were stained with the fluorescent dye calcein and cocultured with alloantigen-specific CTLs in an effector to target ratio of 100:1 or 50:1 for 4 hr. MSCs that are lysed by CTLs release calcein into the cell culture supernatant and fluorescence of the supernatant is proportional to the amount of cells lysed. Percentage of specific lysis is calculated in relation to spontaneous release of calcein of MSCs in medium alone and maximum release of calcein by Triton-X treated MSCs. Shown is a representative experiment with means of five replicates ± S.D. (**P < 0.01; ***P < 0.001 compared to untreated DA MSC; ^#P < 0.05; ^##P < 0.01 compared to DA MSC pretreated with IFN-γ + IL-1β; student's t-test).