Abstract
Abstract 4049
Aim of the European Consortium CASCADE is to standardize GMP-grade production and clinical use of Mesenchymal Stromal Cells (MSC) to treat skin and corneal wounds. MSC possess immunogenicity and immunomodulatory properties that must be carefully addressed before clinical use. CASCADE Immunological Unit is aimed to set up and validate a wide panel of functional assays to fully characterize in a standardized and reproducible manner the immunomodulatory properties of MSC obtained inside CASCADE Units from bone marrow, adipose tissue, cord blood, and amniotic membrane (BM, AT, CB, AM) through different GMP-grade expansion protocols including platelet lysate- and fetal calf serum-based culture conditions.
Immune cells were isolated using indirect immunomagnetic depletion; samples with less than 96% of purity were discarded. For the experiments, MSC were expanded in the same medium used for production and harvested at 70% confluence. Primed MSC were obtained by 48h-treatment with 10 ng/ml of rh-INFγ and 15 ng/ml of rh-TNFα.
Cocultures were set up by plating primed or unprimed MSC in 96 or 48 flat bottomed – well plates; CFSE-stained T, B, NK cells were seeded at different effector cell:MSC ratios. Cells were harvested after 4 or 6 days of coculture for proliferation evaluation by FACS analysis. T cells were stimulated with mitogenic αCD3 plus αCD28 antibodies at 0.5 μg/ml each; B cells were activated with CD40L at 50 ng/ml, its enhancer at 5 g/ml, IL-2 20 UI/ml, CpG 2006 2.5 μg/mL, and F(ab')2 anti-IgM/IgA/IgG 2 μg/mL; NK cells were activated with 100 U/ml rh-IL2. To identify the molecular mechanisms involved in immunomodulatory properties of MSC, coculture of immune effector cells and MSC were performed in the presence of specific inhibitors, after identifying their non-toxic and effective concentrations: 1 mM for L-1MT (IDO inhibitor), 2 μM for snPP (HO-1 inhibitor), 5 μM for NS-398 (COX2 inhibitor), 1 mM for L-NMMA (iNOS inhibitor) and 10 μg/ml for anti-IFNγ neutralizing antibody.
We also studied the capacity of resting and primed MSC to sustain the survival of unstimulated T, B, and NK cells through the evaluation of the percentage of caspase-3negCD45pos viable immune cells after 4 to 6 days in culture with or without MSC.
For MSC immunogenicity assay, the proliferation of allogeneic T was evaluated at day 5 of culture by incorporation of 3H-Thymidine; in addition, NK cells were activated for 2 days with 100 U/ml of rh-IL2 whereas resting or primed MSC were loaded with non radioactive fluorophore (BaTDA) or with Cr51 and used as target cells.
Inflammatory milieu significantly upregulated MHC class I and II, CD54, CD106, CD40, CD274, CD112, CD155 expression, and downregulated NKG2D ligands (ULBP 1–3, MICA/B) 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. On the contrary, only primed MSC were capable of suppressing B cell proliferation. In addition, MSC inhibited apoptosis of resting T, B, and NK cells, while inflammatory priming increased their pro-survival activity. T cell/MSC coculture showed that activation of IDO and HO-1 was the main mechanism involved in MSC immune modulation, as the addition of specific inhibitors (L-1-MT and snPP) significantly reverted the phenomenon. MSC never promoted allogeneic T cell proliferation; by contrast, IL-2-activated NK cells could efficiently recognize and kill allogenic unprimed MSC. However, MSC became insensitive to NK cells once primed with inflammatory cytokines. 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.
Disclosures:
No relevant conflicts of interest to declare.
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