Multipotent mesenchymal stromal cells (MSCs) are widely used for cell therapy. Treatment with interferon-γ(IFNγ) increases the immunomodulating properties of MSCs. When administered intravenously, MSCs interact with lymphocytes. It is impossible to follow the fate of MSCs in the recipient organism. The aim of this study was to investigate the properties of MSCs after their interaction with lymphocytes in vitro.
Bone marrow MSCs were co-cultured for 4 days with activated and non-activated lymphocytes. A portion of the MSCs was pretreated with IFNγ.
HLA-DR expression on the MSCs increased when these cells were co-cultured with lymphocytes and after they were treated with IFNγ. The activated lymphocytes induced significantly higher HLA-DR expression levels than did IFNγ treatment. IFNγ increased the viability of MSCs when these cells were co-cultured with lymphocytes. The immunomodulating properties of MSCs were amplified after IFNγ priming and co-cultivation with lymphocytes; therefore, this amplification was not dependent on the IFNγ source. IFNγ treatment and lymphocyte interactions induced increases in the relative expression levels (RELs) of ICAM1 and factors involved in immunomodulation in the MSCs.
IFNγ stabilizes MSCs while maintaining their viability. The data suggest that MSCs obtained from the hematopoietic cells donor or autologous should be used for cell therapy.
Due to their immunomodulatory properties, MSCs are used for the treatment of acute graft versus host disease (aGVHD), autoimmune diseases, inflammatory bowel diseases, and diabetes mellitus. However, the use of MSCs is not always effective. According to some studies, the immunoregulatory potential of MSCs increases after their activation, and these cells become more effective in suppressing the immune response. It is expected that some molecules can be used for MSC activation. Currently, the possibility of activating MSCs with various substances, such as IFN-γ, TNF-α, and IL17, is being investigated. It has been shown that IFN-γ is needed for the manifestation of the immunosuppressive properties of MSCs: MSCs do not have an effect on the proliferation of IFN-γ-deficient T-cells. MSCs activated by IFN-γ (MSCs-γ) have an increased anti-inflammatory effect, and blocking the receptors for IFN-γ leads to a significant decrease in immunosuppression. In contrast, pretreatment of MSCs with IFN-γ leads to an increase in their immunomodulatory properties. However, MSCs-γ became more immunogenic due to an increase in the expression of MHC-I and the appearance of MHC-II on the cell surface. After MSCs interact with IFN-γ, they began to produce high levels of IDO1, soluble enzyme that cleaves tryptophan, resulting in the accumulation of kynurenine, which has immunosuppressive properties. The efficiency of the IFN-γ treatment depends on its concentration since IFN-γ does not change the immunomodulatory properties of MSCs at a low concentration (50 U/ml), whereas it enhances the immunosuppressive properties of MSCs at a high concentration (500 U/ml). The activated MSCs show increased expression levels of IL6 and IDO1, leading to more efficient inhibition of T-cell and NK-cell proliferation. MSCs with enhanced IDO1secretion can more effectively suppress the T-cell-mediated immune response by inhibiting proliferation and inducing apoptosis in T-cells. MSCs-γ boost the number of regulatory T-cells, which may be partly due to increased expression of PD-L1. Additionally, the activated MSCs are characterized by more pronounced suppression of CD25 expression on CD4+ effector Tcells and the suppression of NK-cells compared to non-activated MSCs. The expression levels of IFN-γ, TNF-α and IL2 decrease in MSCs-γ, but the expression level of PGE2 does not change, which also contributes to the immunomodulatory properties of these cells. MSCs do not express the co-stimulatory molecules CD80, CD83, and CD86 even under the influence of IFN-γ, and CD40 expression data are contradictory. After MSCs are primed by IFN-γ, they begin to express MHC II molecules that promote T-cell activation and cytotoxic reactions. Clinical use of MSCs-γ can lead to complications including acute immune responses. The application of MSCs-γ in animal models has been effective. Crohn’s disease has been modeled in mice that have been administered dextran coupled with trinitrobenzene sulfonate. Administration of human MSCs-γ in these mice led to better survival, and the mice gained weight as the severe symptoms of colitis subsided. Thus, the ability to increase the therapeutic efficacy of MSCs has been illustrated.
MSCs have been well characterized in vitro. However, what happens to MSCs after intravenous injection remains unclear. It is known that 2 weeks after infusion, MSCs could not be detected in the recipient organism. Typically, MSCs are administered intravenously, and their interaction with lymphocytes occurs in the blood and tissues. MSCs express multiple adhesion molecules on their surface, which allows them to interact with lymphocytes. Lymphocytes secrete cytokines that act on the MSCs. As a result, properties of the MSCs, such as their ability to modulate the immune response and their trophic function, can change. The immune system of the recipient thus gains the ability to recognize these foreign MSCs, and the MSCs lose their immune privilege.
The aim of this study was to investigate the changes in the main properties of MSCs after these cells interact with allogeneic lymphocytes. The MSCs were co-cultured with lymphocytes for 4 days, and their basic properties were analyzed over time. The interaction of MSCs with lymphocytes led to the appearance of HLA-DR expression on the surface of MSCs. CD90 expression was reduced over time, and IDO1, CFH, PTGES, IL6, and CSF1 expression was elevated. These changes may affect the duration of exogenous MSC survival in the recipient organism.
Multipotent mesenchymal stromal cells (MSCs)
MSCs were isolated from the bone marrow of 13 donors (7 males and 6 females), ranging in age from 22 to 62 years (median: 27 years). After informed consent was obtained, samples were collected during the aspiration of hematopoietic stem cells for allogeneic transplantation at the Department of Bone Marrow Transplantation. The protocol was approved by the local medical ethics committee.
The MSCs were derived from 5-10 ml of donor bone marrow. For the separation of mononuclear cells, the bone marrow was mixed with an equal volume of alpha-МЕМ (ICN) containing 0.2% methylcellulose (1500 cP, Sigma-Aldrich). After 40 min, most of the erythrocytes and granulocytes had precipitated, while the mononuclear cells remained in suspension. The suspended (upper) fraction was aspirated and centrifuged for 10 min at 450×g.
The cells from the sediment were resuspended in a standard culture medium that was composed of alpha-MEM supplemented with 10% fetal bovine serum (Hy-Clone), 2 mМ L-glutamine (ICN), 100 U/ml penicillin (Ferein) and 50 micro gm/ml streptomycin (Ferein). The cells (3×106) were cultured in T25 culture flasks (Corning-Costar). When a confluent monolayer of cells had formed, the cells were washed with 0.02% EDTA (ICN) in a physiological solution (Sigma-Aldrich) and then trypsinized (ICN). The cells were seeded at 4×103cells per cm2 of flask growth area. The cultures were maintained at 37°C in 5% CO2. The number of harvested cells was counted directly; cell viability was checked by trypan blue dye exclusion staining.
Total RNA was extracted from the MSCs at passage 1 by the standard method, and cDNA was synthesized using a mixture of random hexamers and oligo (dT) primers. Gene expression levels were quantified by real-time quantitative PCR using hydrolysis probes (TaqMan) and an ABI Prism 7500 (Life Technologies). Gene-specific primers were designed by the authors and synthesized by Syntol R&D. All primers and probe sequences are provided in Supplement 1. The relative expression levels (RELs)of the genes were determined by normalizing the expression of each target gene to the levels of βACTIN and GAPDH and calculated using the ΔΔCt method for each MSC sample.
HLA-DR expression of on the surface of MSCs after these cells interact with lymphocytes
In the MSCs co-cultured with both autologous and allogeneic lymphocytes, the level of HLA-DR surface expression gradually increased by 1.4 times on day 1 and then by 1.7, 1.7 and 2 times (p=0.03) on days 2, 3 and 4, respectively (Figure 2A, B). It is known that an increase in HLA-DR expression on MSCs can occur after their treatment with immunomodulatory agents without T-cell activation.