Human Umbilical Cord Blood-derived Stromal Cells Suppress Xenogeneic Immune Cell Response In Vitro

Aim

To explore immunological properties of human umbilical cord blood-derived stromal cells (hUCBDSC) and their effect on xenogeneic immune cells in vitro.

Methods

Immunological phenotype of freshly isolated and cryopreserved hUCBDSCs was evaluated by flow cytometry. Xenogeneic splenic T-cells were stimulated by phytohemaglutinin A (PHA) or dendritic cells in the absence or presence of hUCBDSCs. T-cell proliferation was measured by cell counting kit-8 after 7-day incubation. The proportion of apoptotic cells and CD4⁺CD25⁺Foxp3⁺ regulatory T-cells (Tregs) was determined in T-cells activated by PHA in the absence or presence of hUCBDSCs by flow cytometry. Phenotype of dendritic cells, cultured alone or with hUCBDSCs, was analyzed by flow cytometry.

Results

Levels of immune molecule expression on freshly isolated hUCBDSCs were as follows: human leukocyte antigen-I (HLA-I) (84.1 ± 2.9%), HLA-II (1.6 ± 0.3%), CD80 (0.8 ± 0.1%), CD86 (0.8 ± 0.1%), CD40 (0.6 ± 0.1%), and CD40L (0.5 ± 0.1%), which was not influenced by cryopreservation. T-cell proliferation in the presence of hUCBDSCs was significantly lower than that of positive control. The coculture led to a 10-fold increase (from 1.2 ± 0.3% to 12.1 ± 1.4%, P < 0.001) in the proportion of CD4⁺CD25⁺Foxp3⁺ regulatory T-cells (Tregs) and a reversion of mature dendritic cells, as indicated by the down-regulation of major histocompatibility complex (MHC)-II molecule (49.3% vs 25.9%, P = 0.001), CD80 (47.2% vs 23.3%, P = 0.001), and CD86 (40.6% vs 25.1%, P = 0.002). When subjected to annexin V binding and propidium iodide uptake assay, the hUCBDSCs did not show the ability to induce apoptosis of xenogeneic T-cells.

Conclusion

These results demonstrate low immunogenicity and immunomodulation effect of the hUCBDSCs. Reversion of mature dendritic cells and increase in Treg proportion, but not cell apoptosis, can possibly contribute to the suppression of xenogeneic T-cell proliferation by the hUCBDSCs.Adherent spindle-shaped, fibroblastoid cells were isolated from the bone marrow by Friedenstein (1) and others (2-4), and are also referred to as multipotent mesenchymal stromal cells (MSC) (5) because of their self-renewal and multilineage differentiation capacity at the population level. One of the main biological functions of bone marrow MSCs is to regulate proliferation, differentiation, and maturation of hematopoietic cells through cell-to-cell interactions and secretion of cytokines and growth factors. Besides, MSCs also probably contribute to regulation of maturation, proliferation, and function of lymphocytes and have been shown to participate in the positive selection of T lymphocytes in the thymus in murine models (6-10).In fact, a variety of studies with human, baboon, and murine MSCs have demonstrated the inability of MSCs to elicit a proliferative response of allogeneic lymphocytes, but instead they showed the capability of MSCs to suppress T lymphocyte activation and proliferation in vitro when stimulated by alloantigens, mitogens, and CD3 and CD28 antibodies (11-17). Different mechanisms are proposed to explain the suppressive effect of MSCs depending on the stimuli. Some animal models, related to alloreactive immunity, autoimmunity, or tumor immunity, have been established to examine the immunomodulatory function of MSCs in vivo. Graft vs host disease (GVHD), induced by donor T-cells, constitutes the most frequent complication after allogeneic hematopoietic cell transplantation (HCT) (18,19). Early and repeated systemic administration of ex-vivo expanded MSCs reduced the incidence and severity of GVHD in mice (20). It was shown clinically that the infusion of MSCs was safe and effective in the treatment of GVHD (21,22). MSCs have been an attractive candidate for cell therapy for GVHD.Although MSCs have also been isolated from other tissues such as adipose tissue (23), placenta (24), amniotic fluid (25), and umbilical cord blood (26,27), most studies on immunomodulatory function focused on bone marrow MSCs. However, the bone marrow collection is an invasive procedure and the number and expansion capacity of bone marrow MSCs decline with age (28,29). Due to easy collection and younger age of donor cells, umbilical cord blood is one of the most attractive alternative sources of MSCs. Our laboratory has previously isolated a novel population of adherent fibroblast-like cells from human umbilical cord blood (hUCB) CD34⁺cells, called hUCB-derived stromal cells (hUCBDSCs) and confirmed their supportive effect on hematopoiesis in vitro (30).Previous studies have usually investigated human MSCs-allogeneic immune cell reaction in vitro (11,14,31-33). To test the feasibility of replacing human immune cells with xenogeneic counterparts in vitro is of significance in establishing an animal model for further in vivo study of immunological properties of human MSCs, such as their inhibitory effect on GVHD. In the present study, we focused on immunological properties of hUCBDSCs and their effect on xenogeneic immune cells and demonstrated that hUCBDSCs suppressed xenogeneic T-cell activation induced by mitogen PHA and dendritic cells, in addition to its low immunogenicity per se, such as the lack of human leukocyte antigen-II (HLA-II) and some costimulator expression.