Also, MSCs alone did not activate T cells, which is not surprising as under these conditions MSCs express little to no MHC class II or co-stimulatory molecules (Fig. plated 2 days prior with 500 000 MSCs/plate and cultured in RPMI-1640 containing 10% FBS, 1 penicillin/streptomycin, and 2 mM L-glutamine supplemented with 200 U/ml IL-2 and 50 ng/ml IL-10 (R&D Systems) at 37C, 5% CO2. After 4 days, the number of OVA-specific IgG antibody forming cells (AFCs) in the non-adherent cells was assessed by enzyme-linked immunospot assay (ELISPOT). Quantitative ELISA and ELISPOT Serial twofold dilutions of supernatants or mouse IgG standard (50 ng/ml) were added to ELISA plates coated with unlabelled goat anti-mouse IgG (-specific) antibody (5 g/ml in 100 mM sodium bicarbonate buffer, pH 92). Bound IgG was detected with biotin-labelled goat anti-mouse IgG and streptavidin-HRP. The absorbance at 450 and 540 nm was measured on a dual-wavelength plate reader (Molecular Devices). Statistical analysis was performed using Student’s was tested. The MSCs arrested T cell proliferation in a dose-dependent fashion (Fig. 1e). Also, MSCs alone did not activate T cells, which is not surprising as under these conditions MSCs express little to no MHC class II or co-stimulatory molecules (Fig. 1e, grey bar). Open in a separate window Fig. 1 Mesenchymal stem cells (MSCs) differentiate into cells of the mesenchymal lineage and inhibit T cell proliferation. (aCd) MSCs were subjected to differentiation culture conditions for 21 days as described in Materials Rabbit Polyclonal to CRHR2 and methods and stained with Oil Red O, alkaline phosphatase and silver nitrate, or toluidine blue for adipose (b), bone (c) or cartilage (d) differentiation, respectively. MSCs before being subjected to differentiation conditions are shown in (a). Magnification: 20. (e) Balb/c spleen cells were cultured alone (white bar) or with anti-CD3 antibody (aCD3) in the absence (hatched bar) or presence of the indicated doses of Balb/c MSCs (black bars) for 4 days. Grey bar represents spleens cells cultured with MSCs but without aCD3. Proliferation was measured by tritiated thymidine incorporation. MSC treatment enhances autoantibody production and immune complex deposition Allogeneic MSCs have been reported to suppress MHC-unrelated T cell responses [4C8] and are considered largely non-immunogenic, given their lack of co-stimulatory molecule and MHC class II expression. Furthermore, repeated allogeneic MSC administration has been used successfully in animal models [5,26,27] and clinically to treat graft-prophylactic MSC treatment: * 005; Ispronicline (TC-1734, AZD-3480) ** 0001. (b) Anti-dsDNA IgG titres for individual animals in PBS, MSCs delivered prophylactically (MSC-P) or therapeutically (MSC-T) and cyclophosphamide (Cyclo.)-treated mice at 35 weeks of age. The line represents the mean value for each group. (c) Kidney sections stained for mouse IgG (20). Results are representative of four mice/group. (d) Average fluorescence intensity of IgG staining in glomeruli from PBS, MSCs delivered prophylactically (MSC-P) or therapeutically (MSC-T) and cyclophosphamide (Cyclo.)-treated mice. Each point represents the average intensity of immune complex deposits in all the glomeruli per field in a kidney section for each animal. The line represents the average background autofluorescence on sections stained with an isotype control antibody. Ispronicline (TC-1734, AZD-3480) PBS MSC treatment ** 0001. The increase in anti-dsDNA titres correlated with the amount of immune complex deposits in the kidney, as detected by immunofluorescent staining (Fig. 2c). The average fluorescence intensity in glomeruli from MSC-treated mice was significantly higher than in PBS-treated Ispronicline (TC-1734, AZD-3480) animals (PBS: 320 107; MSC-P: 5535 1565; MSC-T: 5376 1236; 0001). Kidneys from cyclophosphamide-treated mice exhibited little or no fluorescence above background, indicating minimal immune complex deposition in these animals (Fig. 2d). MSC treatment enhances kidney pathology and proteinuria Immune complex deposition leads to inflammation and kidney damage. To assess the pathological impact of MSC treatment on the associated enhancement.