Quality Controls Of Immune Regulatory Properties Of Ex-Vivo, GMP-Grade Expanded Mesenchymal Stromal Cells For Clinical Use

Aim of CASCADE is to standardize GMP-grade production and clinical use of Mesenchymal Stromal Cells (MSC). Immunological Unit is aimed at setting up and validating a standardized panel of functional assays to fully characterize the immunomodulatory properties of MSC obtained from bone marrow and adipose tissue through different GMP-grade expansion protocols (platelet lysate- vs. fetal calf serum).

Immune cells were isolated using indirect immunomagnetic depletion (purity >96%). MSC were expanded in the same medium used for production and harvested at 70% confluence. Primed MSC (pMSC) were obtained after 48h-treatment with rh-IFNγ and rh-TNFα. MSC or pMSC were cocultured with T, B, NK cells for 4 or 6 days, and proliferation was evaluated by CFDA-SE dilution. T cells were stimulated with αCD3 + αCD28 antibodies; B cells were activated with CD40L, its enhancer, IL-2, CpG 2006, and anti-IgM/IgA/IgG; NK cells were activated with 100 U/ml rhIL-2. Cocultures were performed also with specific molecule inhibitors: L-1MT (IDO), snPP (HO-1), NS-398 (COX2), L-NMMA (iNOS) and anti-IFNγ antibody. For MSC immunogenicity assay, allogeneic T cell proliferation was evaluated at day 5 of culture; in addition, NK cells were activated for 2 days with rh-IL2, and MSC and pMSC were used as target cells.

Inflammatory milieu significantly upregulated MHC class I and II, CD54, CD106, CD40, CD274, CD112, CD155 expression, and downregulated NKG2D ligands and mesenchymal markers (CD73, CD90, CD105). AT-derived MSC expressed less MHC class II, CD200 and CD106 molecules than BM-MSC. MSC coculture inhibited T and NK cell proliferation without inducing apoptosis, and this effect was greater in presence of primed MSC. Only primed MSC were capable of suppressing B cell proliferation. MSC inhibited apoptosis of resting T, B, and NK cells, while inflammatory priming increased their pro-survival activity. Activation of IDO and HO-1 was the main mechanism involved in MSC immune modulation. MSC never promoted allogeneic T cell proliferation; by contrast, IL-2-activated NK cells could efficiently recognize and kill allogenic unprimed MSC, while primed MSC became insensitive to NK cells. Some differences were observed depending on the origin and culture conditions of clinical-grade MSC.

All the experimental protocols to assess MSC inhibitory effects on immune effector cells have been standardized and will be applied for the release of GMP-grade MSC produced inside the CASCADE Consortium.