Background/Objectives: Acute leukemia (AL) alters both hematopoiesis and the bone marrow stromal microenvironment. Attempts to develop a culture of multipotent mesenchymal stromal cells (MSCs) from AL patients’ bone marrow are not always successful, as opposed to healthy donors’ bone marrow. Methods: To unveil the reason, healthy donors’ MSCs were cultured in the presence of sera from healthy donors (control group) or AL patients at the onset of the disease, in short- and long-term remission, and before and after allogeneic hematopoietic stem cell transplantation (allo-HSCT). Results: The cell yield in the presence of patient sera was lower than in the control, regardless of the AL stage. It was assumed that the patients either lacked growth factors to sustain MSCs, or there were inhibitors of MSC growth present. The serum’s ability to support MSC growth correlated with platelet count and albumin and calcium concentrations in patients’ blood. Platelet-derived growth factors—PDGFA and PDGFB—are known to induce MSC growth. Their concentration in the serum of AL patients and healthy donors was analyzed. A decrease in PDGFA concentration was found in the sera of patients compared to healthy donors. PDGFB concentration was lower at disease onset, increased during remission and decreased again during relapse. PDGFB concentration correlated with platelet count, while PDGFA concentration did not. AL patients’ sera reflected systemic disturbances affecting MSC growth. So far, decreases in PDGFs, albumin and calcium concentration, as well as platelet count, are the parameters that might be among the causes of this observation.

Introduction: Multipotent mesenchymal stromal cells are progenitors of the bone marrow stromal microenvironment that support hematopoiesis. Mitochondria, which can be transferred between cells via nanotubes or extracellular vesicles, play a key role in the functions of mesenchymal stromal cells. In a murine model, donor hematopoietic stem and progenitor cells transfer functional mitochondria to bone marrow mesenchymal stromal cells of the recipient. The aim of this study was to find out whether such transfer occurs in humans after allogeneic hematopoietic stem cell transplantation. Methods: This study included nine patients with acute leukemia who received a reduced intensity conditioning regimen. Donor hematopoietic stem and progenitor cells mobilized into peripheral blood were the source of transplanted stem cells. Total DNA was isolated from bone marrow mesenchymal stromal cells of each patient before and after transplantation and their respective donors’ leukocytes. A fragment of mitochondrial DNA including the full-length control region was sequenced. The mitochondrial DNA sequence of each patient’s mesenchymal stromal cells was compared before and after the procedure and with the respective donor leukocytes. Results: Donor mitochondrial DNA was not detected in the mesenchymal stromal cells of any patient after transplantation even as trace amounts. Co-culturing donor leukocytes with intact and irradiated mesenchymal stromal cells in vitro did not lead to detection of donor mitochondrial DNA transfer. Conclusion: The data show that there is no mitochondrial transfer from donor hematopoietic stem and progenitor cells to recipient mesenchymal stromal cells after transplantation. Thus, the results indicate that one cannot count on improved mesenchymal stromal cell metabolism due to mitochondrial transfer. It is necessary to look for other ways to restore the stromal microenvironment.

In patients with acute leukemia (AL), malignant cells and therapy modify the properties of multipotent mesenchymal stromal cells (MSCs) and their descendants, reducing their ability to maintain normal hematopoiesis. The aim of this work was to elucidate the alterations in MSCs at the onset and after therapy in patients with AL. The study included MSCs obtained from the bone marrow of 78 AL patients (42 AML and 36 ALL) and healthy donors. MSC growth characteristics, gene expression pattern, proteome and secretome were studied using appropriate methods. The concentration of MSCs in the bone marrow, proliferative potential, the expression of several genes, proteomes and secretomes were altered in AL-MSCs. Stromal progenitors had been affected differently in ALL and AML patients. In remission, MSC functions remain impaired despite the absence of tumor cells and the maintenance of benign hematopoietic cells. AL causes crucial and, to a large extent, irreversible changes in bone marrow MSCs.

Introduction. During enzyme replacement therapy in patients with Gaucher disease (GD) with recombinant glucocerebrosidase (GCase), regression of bone manifestations is possible, but with prolonged therapy osteonecrosis may occur. These changes may be due to impaired differentiation of multipotent mesenchymal stromal cells (MSCs). Aim: to study changes in the MSCs of healthy donors and a patient with GD when cultured in the presence of GCase. Material and methods. MSCs were isolated from the bone marrow of 17 healthy donors and a female patient with GD by a standard method and cultured in the presence of various concentrations of GCase after the second passage from 2 to 7 weeks. Cell proliferation and the ability to differentiate were analyzed, including after induction. The assessment was carried out by differential staining, elution, and expression of differentiation marker genes by real-time PCR. Results. Low concentrations of recombinant GCase (0.25-1.5 U/ml) did not affect the proliferative activity of MSCs. Prolonged cultivation of MSCs in the presence of low doses of GCase led to a change in the differentiation potential of these cells in the direction of adipogenesis. Concentrations of GCase of 3-5 U/ml inhibited the proliferation of MSCs and caused significant changes in cell differentiation. High doses of the enzyme (7-10 U/ml) had a cytotoxic effect and led to cell death within one passage. The proliferative and differentiation potential of the MSCs of a patient with GD differed significantly from the cells of healthy donors in all the parameters studied. Conclusion. The cultivation of donor MSCs in the presence of recombinant GCase alters the proliferation and differentiation potential of these cells. These changes depend on the dose of the enzyme in the medium and the duration of cultivation.

In patients with acute leukemia, not only normal hematopoiesis, but also bone marrow stromal microenvironment is damaged. Multipotent mesenchymal stromal cells (MSC) are essential for the formation and function of the stromal microenvironment. Analysis of changes in MSC is important for the development of new approaches to leukemia therapy. The metabolism of mitochondria in MSC, relative content of mitochondrial DNA, and expression levels of genes encoding PGC-1α and Nrf2 proteins, important regulators of biogenesis, were studied using real-time PCR. Relative content of mitochondrial DNA does not change in MSC of acute leukemia patients at the onset of disease or in remission. Relative expression level of the gene encoding PGC-1α protein in MSC does not change significantly. However, relative expression level of the gene encoding Nrf2, an important antioxidant activity regulator, insignificantly decreases in patients at the onset of acute leukemia, and this decrease becomes significant upon reaching remission.

Multipotent mesenchymal stromal cells (MSCs) support hematopoiesis and regulate immune response. Mitochondria (Mt) play a key role in functions of the MSCs. Mt can be transferred between cells via nanotubes or extracellular vesicles. It has been shown on a murine model that donor hematopoietic stem and progenitor cells (HSPCs) transfer functional Mt to bone marrow MSCs of the recipient.The aim of the work was to find out whether Mt are transferred from transplanted HSPCs to the recipient's MSCs after allogeneic hematopoietic stem cells transplantation (allo-HSCT) in humans.The study included 8 patients with acute leukemia after allo-HSCT. In all cases, the patients received reduced intensity conditioning regimen (RIC) and mobilized peripheral blood was used as the stem cell source. Total DNA was isolated from bone marrow MSCs of each patient before and after allo-HSCT and their respective donors. A fragment of mtDNA 15967–605 (1208 bp) was sequenced, including full-length control region. Obtained primary structures were compared to the Cambridge sequence (NC_012920.1). The results of the patients after allo-HSCT were compared with the results of the same patient prior to allo-HSCT and their donor.Donor mtDNA was not detected in any of the patients’ samples after allo-HSCT even in trace amounts. In vitro co-culture of donor mononuclear cells with patient MSCs did not lead to detection of donor mtDNA in the MSCs.This data show that there is no Mt transfer from donor HSPCs to recipient MSCs after allo-HSCT. The discrepancy with the murine model may be due to the fact that the mice were irradiated before transplantation, and all the patients in this study received a RIC regimen. The results indicate that one cannot count on improvement of MSCs metabolism after allo-HSCT due to Mt transfer. It is necessary to look into other ways to restore the stromal microenvironment.

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.

In acute leukemia, the stromal microenvironment of the bone marrow that regulates hematopoiesis is modified under the influence of malignant cells. Chemotherapy also adversely affects stromal cells. Multipotent mesenchymal stromal cells (MSC) are involved in the formation of the stromal microenvironment and in the regulation of normal and tumor hematopoietic cells. The properties of MSC from the bone marrow of patients with acute myeloid and lymphoid leukemia were studied at the onset of the disease and after achieving remission. The immunophenotype and the level of gene expression were analyzed in MSC of 34 patients. In MSC from patients with acute leukemia, the expression of CD105 and CD274 was significantly reduced in comparison with MSC from healthy donors. At the onset of the disease, the expression of IL6, JAG1, PPARG, IGF1, and PDGFRA was enhanced, while the expression of IL1B, IL8, SOX9, ANG1, and TGFB was reduced. All these changes affect the course of the disease in patients and can be the targets of therapeutic intervention.

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.

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.

Multipotent mesenchymal stromal cells (MSCs) are an object of intense investigation due to their therapeutic potential. MSCs have been well studied in vitro, while their fate after implantation in vivo has been poorly analyzed. We studied the properties of MSCs from the bone marrow (BM-MSC) before and after implantation under the renal capsule using a mini pig model. Autologous BM-MSCs were implanted under the kidney capsule. After 2.5 months, ectopic foci containing bones, foci of ectopic hematopoiesis, bone marrow stromal cells and muscle cells formed. Small pieces of the implant were cultivated as a whole. The cells that migrated out from these implants were cultured, cloned, analyzed and were proven to meet the most of criteria for MSCs, therefore, they are designated as MSCs from the implant—IM-MSCs. The IM-MSC population demonstrated high proliferative potential, similar to BM-MSCs. IM-MSC clones did not respond to adipogenic differentiation inductors: 33% of clones did not differentiate, and 67% differentiated toward an osteogenic lineage. The BM-MSCs revealed functional heterogeneity after implantation under the renal capsule. The BM-MSC population consists of mesenchymal precursor cells of various degrees of differentiation, including stem cells. These newly discovered properties of mini pig BM-MSCs reveal new possibilities in terms of their manipulation.

Topic: 3. Acute myeloid leukemia - Biology & Translational Research Background: Multipotent mesenchymal stromal cells (MSCs), their descendants and the proteins secreted by them, are essential hematopoiesis regulators. In patients with acute myeloid leukemia (AML) malignant cells change MSCs’ properties and reduce their ability to maintain normal hematopoiesis. A detailed study of MSCs secretome at the onset of the disease and in remission may elucidate the role of MSCs in supporting cancer cells and restoration of normal hematopoiesis. Aims: The aim of this work was to study general changes in secretome of MSCs in patients with AML at the onset of the disease and in remission. Methods: The study included MSCs obtained from the bone marrow of 13 AML patients: (3 male, 10 female, median age 38) and 21 donors (10 male, 11 female, median age 35) that were used as control. All donors and patients signed informed consent. MSCs were cultured by standard method. The analysis of proteins contained in the MSCs-conditioned medium was performed on an Orbitrap Q Exactive HF-X mass spectrometer equipped with a nano-electrospray source and a high-pressure nanoflow chromatograph (UPLC Ultimate 3000) with a C-18 (100um) reverse-phase column x 300mm. The results were analyzed in Scaffold 5 (version 5.1.0) for validation and meta-analysis. For functional enrichment analysis we selected the proteins that had ǀlogFCǀ>2 and the differences in secretion of which were significant (p<0.05). Results: A total of 2833 proteins were identified, of which 582 were differentially secreted by patients’ and donors’ MSCs (Figure). Some proteins, including those involved in proteolysis, cellular protein metabolic process and regulation of hydrolase activity, were secreted by MSCs of patients at AML onset, but not in donors’. MSCs from primary patients secreted higher levels of proteins related to immune regulation, cell adhesion, and extracellular matrix organization. MSCs of patients at the onset of AML secreted lower levels of proteins responsible for platelet activity, aging, differentiation, and angiogenesis. Protein secretion by patients’ MSCs differs little between onset of AML and remission. Proteins belonging to the extracellular matrix organization were reduced in remission compared to the onset of the disease. Upon reaching remission secretion of proteins related to lysosomes and exosomes increased. The most pronounced differences between the MSCs’ secretome composition at the onset and that in remission were revealed in comparison with donors. The most differences of donors’ and AML MSCs’ secretome were the same for onset and remission. The unique differences between AML onset and donors were decreased levels of proteins related to ossification, transport and immune response. In remission, but not at the onset, proteins responsible for cell adhesion, immune response, complement and coagulation cascades were reduced compared to donors. Extracellular matrix organization and vesicular transport were disorganized: every comparison revealed alterations in different proteins related to these processes. Summary/Conclusion: AML causes crucial changes in the MSCs’ secretome. Some secretome modifications caused by the tumor are reversed upon achieving remission, but not all of them. Chemotherapy leads to new alterations. Thus, in remission, in the absence of tumor cells and with the formation of normal hematopoietic cells, the functions of MSCs remain impaired. The study was supported by a grant from the Russian Science Foundation project № 22-15-00018, https://rscf.ru/project/22-15-00018/Figure. Diferentially secreted proteins Keywords: Secretion, Acute myeloid leukemia, Mesenchymal stem cell

Multipotent mesenchymal stromal cells (MSC) were administered to patients after allogeneic hematopoietic stem cell transplantation to prevent the development of acute graft—versus— host disease (GVHD). The injection of MSC did not always prevent the development of GVHD. The aim of the work was to compare the secretome of MSC effective and ineffective in the prevention of GVHD. MSC were obtained from the bone marrow of hematopoietic stem cells donors. The secretome was studied using a TripleTOF 5600+ mass spectrometer with a NanoSpray III ion source coupled to a NanoLC Ultra 2D Plus nano-HPLC System. A total of 1,965 proteins were analyzed. Analysis of the secretome of effective and ineffective MSC samples revealed significant differences in the secretion of 1,119 proteins associated with ribosomes, exosomes, focal contacts, and others. Analysis of proteins secreted by MSC can be used to identify prognostically effective samples.

Abstract—The transcription factors of NF-kB family match the external signal from pro-inflammatory cytokines and transcription of their target genes. It was previously shown that in the peripheral blood of irradiated young and nonirradiated old mice, an increased level of interleukin-1β is maintained, which is simultaneously the activator and the target gene of NF-kB signaling pathway. We suggested that during both induced by radiation and natural aging, similar processes can take place in various tissues of the body, accompanied by the activation of NF-kB. To test this hypothesis and establish the characteristics of induced and natural aging, we studied the expression of NF-kB family genes, its IKK regulatory complex and NF-kB target genes at the level of transcription in bone marrow, spleen, thymus, liver and muscles of irradiated mice three months after exposure and in the same tissues of old mice. RNA was extracted from the tissues, reverse transcription was performed followed by real-time PCR. It was shown that, in general, the pattern of changes in the expression of NF-kB differs in irradiated and old animals and, moreover, depends on the type of tissue. Biochemical processes occurring during natural and accelerated radiation aging affect NF-kB signaling pathway, but the details of its activation differ from each other under these conditions. However, general trends were identified. In the bone marrow, common for irradiation and aging was a reduction in the expression of Nemo encoding the subunit of the regulatory IKK complex NF-kB. In the thymus, common feature for these conditions was a decrease in Ikkb expression, which encodes another subunit of the same IKK complex. This indicates an important role of these genes in maintaining normal functioning of hematopoietic tissue. In older mice, expression of the gene encoding IL-1β was increased in the thymus, spleen, and liver. The production of IL-1β by these tissues may contribute to an increase in its concentration in peripheral blood.

Introduction . Current knowledge of tumour biology attests a dual genetic and epigenetic nature of cancer cell abnormalities. Tumour epigenetics research provided insights into the key pathways mediating oncogenesis and facilitated novel epigenetic therapies. Aim — an overview of intricate involvement of epigenetic change in haematological morbidity and current therapeutic approaches to target the related mechanisms. Main findings . We review the best known epigenetic marks in tumour cells, e.g. DNA cytosine methylation, methylation and acetylation of histone proteins, the underlying enzymatic machinery and its role in oncogenesis. The epigenetic profile-changing drugs are described, including DNA hypomethylating agents, histone deacetylase and methylase inhibitors. A particular focus is made on substances currently approved in haematological therapy or undergoing clinical trial phases for future clinical availability.

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.

In the bone marrow of vertebrates, two types of stem cells coexist—hematopoietic stem cells (HSCs) and mesenchymal stem cells (MSCs). Hematopoiesis only occurs when these two stem cell types and their descendants interact. The descendants of HSCs supply the body with all the mature blood cells, while MSCs give rise to stromal cells that form a niche for HSCs and regulate the process of hematopoiesis. The studies of hematopoiesis were initially based on morphological observations, later extended by the use of physiological methods, and were subsequently augmented by massive application of sophisticated molecular techniques. The combination of these methods produced a wealth of new data on the organization and functional features of hematopoiesis in the ontogenesis of mammals and humans. This review summarizes the current views on hematopoiesis in mice and humans, discusses the development of blood elements and hematopoiesis in the embryo, and describes how the hematopoietic system works in the adult organism and how it changes during aging.

Introduction. The mechanism of action of extracorporeal photopheresis (ECP) is associated with the induction of apoptosis of lymphocytes, which cause the activation of antigen-presenting cells (APC). As a result of the use of ECF, the number of T-regulatory lymphocytes increases, which induces an immunosuppressive effect. The actual problem lies in the use of cryopreserved and photo-irradiated mononuclear cells.Aim — to analyze the functional features of cryopreserved mononuclear cells after ECP.Materials and methods. The study analyzed the indicators of early and late stages of apoptosis in the concentrate of mononuclear cells of different groups, depending on the time of collection and the type of exposure on these concentrates of mononuclear cells: immediately after apheresis (groups 1.1 and 1.2), and after ECP (groups 1.3, 2.1 and 2.2), groups 1.2 and 2.2 were after cryopreservation, and 1.3 — after cryopreservation, and then after ECP. 113 samples of mononuclear cell concentrates obtained from 12 patients with chronic graft-versus-host reaction (GVHD) were analyzed. All groups of samples were cultured to determine the dynamics of changes in indicators of early and late stages of apoptosis over time.Results. The proportion of lymphocytes in the late stage of apoptosis after 2 days cultivation, after the standard ECP procedure, was comparable to the proportion of lymphocytes in the late stage of apoptosis during ECP followed by cryopreservation of the leukoconcentrate.Conclusion. It is advisable to collect mononuclear cells, perform their extracorporeal photo-irradiation, and then divide the photo-irradiated mononuclear cells into several parts both for cryopreservation and for the return of non-cryopreserved photo-irradiated mononuclear cells to the patient.

Abstract Introduction Despite the large number of clinical studies on the use of multipotent mesenchymal stromal cells (MSCs) for the treatment and prevention of graft-versus-host disease (GVHD), the mechanism of their action in the organism is not well understood. The known data refer either to clinical effects or obtained in vitro. Due to the immunomodulatory effect of MSCs in the body, subpopulations of T cells and the concentration of cytokines involved in the immune response can change. The role of T cells and certain cytokines (TNF alpha, IL6, IL8, etc.) in the pathophysiology of GVHD is described. The aim of this investigation was to study the composition of T cells subpopulations and the concentration of cytokines in the peripheral blood of patients who received MSCs for GVHD prophylaxis. Methods The study included 21 patients who received hematopoietic stem cells donor's derived MSCs for the prevention of GVHD as part of the ClinicalTrials.gov Identifier NCT01941394 trial. The control group included 16 patients who did not receive MSCs. After signing informed consent, blood samples were taken from all patients during routine examinations on the day of restoration of the number of leukocytes to 1000 in μl (day 0), after 3 and after 30 days. MSCs were injected on day 0. None of the patients developed GVHD during this time. To analyze plasma cytokines and chemokines, the Bio-Plex Pro Human Cytokine Panel kit, 27-Plex (BioRad) was used, to determine the concentrations of IL-1β, IL-1ra, IL-2, IL-4, IL-5, IL- 6, IL-7, IL-8, IL-9, IL-10, IL-12 (p70), IL-13, IL-15, IL-17, bFGF, Eotaxin, G-CSF, GM-CSF, IFN-γ, IP-10, MCP-1 (MCAF), MIP-1α, MIP-1β, PDGF-bb, RANTES, TNF-α, VEGF according to manufacturer's recommendations. Forward and side scattering parameters determined the peripheral blood lymphocytes population and then CD4+ or CD8+ lymphocytes were gated. For each of this population the composition of memory cells subset were determined by flow cytometry. Results Significant differences were found between the 2 groups only on day 30 in the concentration of IL8 (17.0±3.2 pg/ml in the control group versus 32.8±4.1 pg/ml in the MSC group, p<0.0001). It has been shown that 30 days after MSCs, the number of CD4+ T cells in the peripheral blood of patients significantly increases compared to the group without MSCs (CD4 CM 38.9±9.0 vs 22.4±7.8, CD4 TM 97.0±32.5 vs 91.0±43.2, CD4 TE 5.0±1.9 vs 1.4±1.0, CD4 EM 34.6±20.1 vs 19.9±7.9, CD4CD25+ 27.6±6.6 vs 12.9±5.0). These cells produce IL8, which play an important role in immune cell homeostasis by activating antimicrobial neutrophils. Without the introduction of MSCs, the concentration of this protective against GVHD cytokine practically did not change within a month, whereas after the introduction of MSCs, it gradually increased almost 2 times. However, the dynamics of changes in the levels of the studied cytokines differed greatly between the 2 groups (Table). The concentration of IP10, which is involved in the development of GVHD, increased significantly faster and stronger in the group without MSCs. An increase in the concentration of other investigated cytokines associated with the activation of macrophages (MCP-1, MIP-1a, MIP-1b) did not depend on the MSCs administration. At the same time, in the MSC group, the concentration of the growth factor PDGF-bb necessary for the HSC proliferation increased significantly more actively. The IL9 concentration on day 0 was comparable to the level in healthy donors, and then gradually increased in both groups and after 30 days it was significantly higher than on day 0 in the MSC administration group. The concentration of G-CSF changed in a similar way. Conclusion Changes in the dynamics of T cells subpopulations and the concentration of cytokines in the blood after MSCs administration contribute to a faster recovery of patients after allogeneic bone marrow transplantation. The work were supported by the Russian Foundation for Basic Research, Project No. 19-29-04023. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.