Molecular Changes in Immunological Characteristics of Bone Marrow Multipotent Mesenchymal Stromal Cells in Lymphoid Neoplasia.

Immune system and bone marrow stromal cells play an important role in maintaining normal hematopoiesis. Lymphoid neoplasia disturbs not only development of immune cells, but other immune response mechanisms as well. Multipotent mesenchymal stromal cells (MSCs) of the bone marrow are involved in immune response regulation through both intercellular interactions and secretion of various cytokines. In hematological malignancies, the bone marrow stromal microenvironment, including MSCs, is altered. Aim of this study was to describe the differences of MSCs' immunological function in the patients with acute lymphoblastic leukemia (ALL) and diffuse large B-cell lymphoma (DLBCL). In ALL, malignant cells arise from the early precursor cells localized in bone marrow, while in DLBCL they arise from more differentiated B-cells. In this study, only the DLBCL patients without bone marrow involvement were included. Growth parameters, surface marker expression, genes of interest expression, and secretion pattern of bone marrow MSCs from the patients with ALL and DLBCL at the onset of the disease and in remission were studied. MSCs from the healthy donors of corresponding ages were used as controls. It has been shown that concentration of MSCs in the bone marrow of the patients with ALL is reduced at the onset of the disease and is restored upon reaching remission; in the patients with DLBCL this parameter does not change. Proliferative capacity of MSCs did not change in the patients with ALL; however, the cells of the DLBCL patients both at the onset and in remission proliferated significantly faster than those from the donors. Expression of the membrane surface markers and expression of the genes important for differentiation, immunological status maintenance, and cytokine secretion differed significantly in the MSCs of the patients from those of the healthy donors and depended on nosology of the disease. Secretomes of the MSCs varied greatly; a number of proteins associated with immune response regulation, differentiation, and maintenance of hematopoietic stem cells were depleted in the secretomes of the cells from the patients. Lymphoid neoplasia leads to dramatic changes in the functional immunological status of MSCs.

Acute Myeloid Leukemia Causes Serious and Partially Irreversible Changes in Secretomes of Bone Marrow Multipotent Mesenchymal Stromal Cells.

In patients with acute myeloid leukemia (AML), malignant cells modify the properties of multipotent mesenchymal stromal cells (MSCs), reducing their ability to maintain normal hematopoiesis. The aim of this work was to elucidate the role of MSCs in supporting leukemia cells and the restoration of normal hematopoiesis by analyzing ex vivo MSC secretomes at the onset of AML and in remission. The study included MSCs obtained from the bone marrow of 13 AML patients and 21 healthy donors. The analysis of proteins contained in the MSCs-conditioned medium demonstrated that secretomes of patient MSCs differed little between the onset of AML and remission; pronounced differences were observed between MSC secretomes of AML patients and healthy donors. The onset of AML was accompanied by a decrease in the secretion of proteins related to ossification, transport, and immune response. In remission, but not at the onset, secretion of proteins responsible for cell adhesion, immune response, and complement was reduced compared to donors. We conclude that AML causes crucial and, to a large extent, irreversible changes in the secretome of bone marrow MSCs ex vivo. In remission, functions of MSCs remain impaired despite the absence of tumor cells and the formation of benign hematopoietic cells.

T Cell and Cytokine Dynamics in the Blood of Patients after Hematopoietic Stem Cell Transplantation and Multipotent Mesenchymal Stromal Cell Administration.

Multipotent mesenchymal stromal cells (MSCs) are currently under intensive investigation for the treatment and prevention of graft-versus-host disease (GVHD) after allogeneic hematopoietic stem cell transplantation (allo-HSCT), owing to their substantial immunomodulatory properties. The responses of recipients to MSC infusion following allo-HSCT are not yet well understood. T cells are central to the adaptive immune system, protecting the organism from infection and malignant cells. Memory T cells with different phenotypes, gene expression profiles, and functional properties are critical for immune processes regulation. The aim of this study was to study the dynamics of memory T cell subpopulations and cytokines in the blood of allo-HSCT recipients after MSC administration. In clinical trial NCT01941394, patients after allo-HSCT were randomized into 2 groups, one receiving standard GVHD prophylaxis and the other also receiving MSC infusion on the day of leukocyte recovery to 1000 cells/µL (engraftment, day E0). Blood samples of patients from both groups were analyzed on days E0, E+3, and E+30. T cell subpopulations were studied by flow cytometry, and cytokine concentrations were evaluated by the Bio-Plex Pro Human Cytokine Panel. Administration of MSCs to patients on day E0 did not affect the overall dynamics of restoration of absolute numbers and proportions of T and B lymphocytes after 3 and 30 days. At 3 days after MSC injection, only the numbers of CD8+ effector cells (CD8+TE, CD8+TM, and CD8+EM) were found to increase significantly. A significant increase in the number of CD4+ cells after 30 days compared to day E0 was observed only in patients who received MSCs, indicating faster recovery of the CD4+ cell population following MSC injection. An increase in CD8+ cell number by day E+30 was significant regardless of MSC administration. To characterize the immune status of patients following allo-HSCT in more detail, changes in the cytokine concentration in the peripheral blood of patients on days E0, E+3, and E+30 after MSC administration were investigated. On day E+30, significant increases in the numbers of CD4+CM and activated CD4+CD25+ cells were observed. The concentrations of proinflammatory and anti-inflammatory cytokines IL-6, IL-8, IL-17, TNF-α, and IFN-γ were increased significantly in patients injected with MSCs. Analysis of growth factor levels showed that in the group of patients who received MSCs, the concentrations of G-CSF, GM-CSF, PDGFbb, FGFb, and IL-5 increased by day E+30. Among the cytokines involved in regulation of the immune response, concentrations of IL-9, eotaxin, IP-10, MCP-1, and MIP-1a were increased after 30 days irrespective of MSC administration. The administration of MSCs exerts a positive effect on the restoration of T cell subpopulations and immune system recovery in patients after allo-HSCT.

Humoral Effect of a B-Cell Tumor on the Bone Marrow Multipotent Mesenchymal Stromal Cells.

The properties of bone marrow (BM)-derived multipotent mesenchymal stromal cells (MSCs) are altered in the patients with the diffuse large B cell lymphoma (DLBCL) without BM involvement. It was suggested that plasma from the patients contains soluble factors that affect MSCs. Plasma and BM-derived MSCs from the DLBCL patients at the onset of the disease and one month after the end of treatment were studied. Concentration of the plasma cytokines and gene expression in the MSCs were evaluated by the Bio-Plex Pro Human Cytokine Panel kit to measure 27 analytes and real-time PCR. Plasma and MSCs from the healthy donors were used as controls. Analysis of cytokines in the plasma from healthy donors and patients before and one month after the end of treatment revealed significant differences in the concentration of 14 out of 27 cytokines. Correlations between the levels of secreted cytokines were altered in the plasma from patients indicating that the immune response regulation was disturbed. Cultivation of the MSCs from the healthy donors in the medium supplemented with the plasma from patients led to the changes in the MSC properties, similar to those observed in the MSCs from patients. The BM-derived MSCs were shown to participate in the humoral changes occurring in the DLBCL patients. For the first time, it was shown that the precursors of the stromal microenvironment - multipotent mesenchymal stromal cells - are altered in the patients with DLBCL without bone marrow involvement due to the humoral effect of the tumor and the response of organism to it. Comprehensive analysis of the results shows that, when remission is achieved in the patients with DLBCL, composition of the plasma cytokines normalizes, but does not reach the level observed in the healthy donors. The discovery of a new aspect of the effect of the tumor B-cells on the organism could help to reveal general regularities of the humoral effect of various tumors on the bone marrow stromal cells.