Expression of HLA-DR by mesenchymal stromal cells in the platelet lysate era: an obsolete release criterion for MSCs?

BACKGROUND: According to the definition of the International Society for Cell and Gene Therapy (ISCT), mesenchymal stromal cells (MSCs) do not express HLA-DR. This phenotypic marker as a release criterion for clinical use was established at a time when MSCs were expanded in fetal bovine serum (FBS)-containing media. Replacement of FBS with platelet lysate (PLs) as a medium supplement induced a significantly higher fraction of MSCs to express MHC class II antigens. METHODS: As this raised concerns that such MSCs may play the role of antigen-presenting cells for T cells, in the current study, we studied major factors that may induce HLA-DR on MSCs by means of flow cytometry and real-time polymerase chain reaction. The immunomodulatory potential of MSCs was assessed by a mixed lymphocyte reaction. RESULTS: Our results demonstrated that a very low percentage of generated and expanded MSCs in FBS express HLA-DR (median: 1.1%, range: 0.3-22%) compared to MSCs generated and expanded in PLs (median: 28.4%, range: 3.3-73.7%). Analysis of the cytokine composition of ten PLs showed a significant positive correlation between the levels of IL-1ß, IL-4, IL-10, IL-17, bFGF and expression of HLA-DR, in contrast to no correlation with the age of MSC donors and HLA-DR (r = 0.21). Both MSCs expressing low and high levels of HLA-DR expressed class II transactivator (CIITA), a master gene coding for these molecules. Our results demonstrate for the first time that MSCs with constitutively high levels of HLA-DR also express moderate levels of indoleamine 2,3-dioxygenase (IDO). Treatment of MSCs with multiple doses of TGF-ß1 at passage 0 (P0) and passage 1 (P1) completely abrogated HLA-DR and IDO expression. In contrast, treatment of MSCs with a single dose of TGF-ß1 after P0 only partially reduced the expression of HLA-DR and CIITA. Remarkably, increased expression of HLA-DR on MSCs that constitutively express high levels of this antigen after overnight incubation with IFN-γ was rather unaffected by incubation with TGF-ß1. However, treatment of MSCs with TGF-ß1 for 24 h completely abrogated constitutive expression of IDO. CONCLUSIONS: Irrespective of HLA-DR expression at the population level, all MSC preparations significantly inhibited the proliferation of stimulated peripheral blood mononuclear cells, indicating that HLA-DR represents an obsolete release marker for the clinical use of MSCs.

Efficient lysis of rhabdomyosarcoma cells by cytokine-induced killer cells: implications for adoptive immunotherapy after allogeneic stem cell transplantation.

BACKGROUND: Rhabdomyosarcoma is the most common soft tissue sarcoma in childhood and has a poor prognosis. Here we assessed the capability of ex vivo expanded cytokine-induced killer cells to lyse both alveolar and embryonic rhabdomyosarcoma cell lines and investigated the mechanisms involved. DESIGN AND METHODS: Peripheral blood mononuclear cells from six healthy donors were used to generate and expand cytokine-induced killer cells. The phenotype and composition of these cells were determined by multiparameter flow cytometry, while their cytotoxic effect against rhabdomyosarcoma cells was evaluated by a europium release assay. RESULTS: Cytokine-induced killer cells efficiently lysed cells from both rhabdomyosarcoma cell lines. Antibody-mediated masking of either NKG2D molecule on cytokine-induced killer cells or its ligands on rhabdomyosarcoma cells (major histocompatibility antigen related chain A and B and UL16 binding protein 2) diminished this effect by 50%, suggesting a major role for the NKG2D molecule in rhabdomyosarcoma cell killing. No effect was observed after blocking CD11a, CD3 or TCRalphabeta molecules on cytokine-induced killer cells or CD1d on rhabdomyosar-coma cells. Remarkably, cytokine-induced killer cells used tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) to activate caspase-3, as the main caspase responsible for the execution of apoptosis. Accordingly, blocking TRAIL receptors on embryonic rhabdomyosarcoma cell lines significantly reduced the anti-tumor effect of cytokine-induced killer cells. About 50% of T cells within the cytokine-induced killer population had an effector memory phenotype, 20% had a naïve phenotype and approximately 30% of the cells had a central memory phenotype. In addition, cytokine-induced killer cells expressed low levels of activation-induced markers CD69 and CD137 and demonstrated a low alloreactive potential. CONCLUSIONS: Our data suggest that cytokine-induced killer cells may be used as a novel adoptive immunotherapy for the treatment of patients with rhabdomyosarcoma after allogeneic stem cell transplantation.