Abstract:
Unbalanced T-cell responses, such as occurring during graft-versus-host disease (GvHD) in patients after allogenic transplantation, can be fatal. Human mesenchymal stromal cells (MSCs) are already used for the treatment of GvHD, however, their isolation requires a bone marrow puncture and their expansion needs in vitro care for several weeks. This study focused on the investigation of different immunosuppressive cell types and on the systematic comparison of their functional capacity to suppress T-cell function, in order to find an alternative for MSCs. Polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs) and regulatory T cells (Tregs) were isolated by magnetic separation from peripheral blood. In order to investigate the T-cell suppressive capacity of each cell type, cells were co-cultured with stimulated responder cells and after 4-5 days of incubation, T-cell proliferation and secretion of Interferon-γ (IFNγ) were analyzed by flow cytometry and ELISA, respectively. Furthermore, the requirement of cell-to-cell contact was investigated by transwell experiments and the available cell numbers after isolation were assessed, with respect to clinical application.
MSCs, Tregs, and freshly isolated PMN-MDSCs inhibited T-cell proliferation and secretion of IFNγ in a concentration-dependent manner. Thereby, PMN-MDSCs showed the strongest inhibition of T-cell proliferation compared to MSCs and Tregs, but cell number of MDSCs was limited in peripheral blood of healthy donors. Thus, PMN-MDSCs were generated by cytokine induction from peripheral blood mononuclear cells (PBMCs) and from bone marrow mononuclear cells (BMMCs) in vitro. BMMC-derived PMN-MDSCs effectively suppressed T-cell proliferation and dampened secretion of IFNγ, while PMN-MDSCs generated from PBMCs showed weaker inhibition. The effects of in vitro-generated PMN-MDSCs were partially dependent on cell-to-cell contact, similar to freshly isolated PMN-MDSCs. In order to increase the T-cell suppressive effect at lower cell concentrations, MSCs and freshly isolated PMN-MDSCs, as well as MSCs and BMMC-derived PMN MDSCs, were combined for the analysis of T-cell suppression and compared with the effect of each cell type alone. MSCs combined with freshly isolated PMN-MDSCs demonstrated no additional effect in this study, whereas MSCs combined with BMMC-derived PMN-MDSCs showed an increased T-cell suppressive effect than each cell type alone. However, the available cell number of BMMC-derived PMN-MDSCs was too low for a clinical application. In another set of experiments, isolated CD34+ hematopoietic stem cells (HSCs) were cultured and stimulated with a mix of cytokines and growth factors to generate MDSCs. The HSC-derived MDSCs strongly suppressed T-cell proliferation and the secretion of IFNγ in a concentration-dependent manner. The inhibition of IFNγ release by HSC-derived MDSCs was greater than the reduction by freshly isolated PMN-MDSCs or BMMC-derived PMN-MDSCs. In addition, a great expansion of CD33+ MDSCs was detected during HSC-derived generation.
Overall, this study demonstrates a systematical comparison of the T-cell suppressive effect of various immunomodulatory cell types (MSCs, freshly isolated PMN-MDSCs, and six differently in vitro generated MDSCs). Freshly isolated as well as generated MDSCs strongly suppressed T cell proliferation and the secretion of the pro-inflammatory cytokine IFNγ in vitro. The HSC-derived MDSCs showed strong suppression of effector T cells and IFNγ release as well as great expansion rates, therefore these cells might represent a novel cellular therapeutic to dampen excessive T-cell responses by adoptive transfer of MDSCs for the management of GvHD.
mesenchymal stromal cell