Luis Ignacio Sánchez-Abarca

The effect of mesenchymal stem cells on the viability, proliferation and differentiation of B-lymphocytes

Mesenchymal stem cells are multipotent non-hematopoietic progenitor cells capable of differentiating into various lineages including osteoblasts, chondrocytes and adipocytes. The findings of this study indicate that mesenchymal stem cells promote survival and inhibit proliferation and maturation of B cells, and support a role of these cells in the immune response. Background Mesenchymal stem cells are multilineage non-hematopoietic progenitor cells that play a key role in supporting the lymphohematopoietic system. Their distribution in bone marrow and secondary lymphoid organs allows an intimate interaction with T- and B-lymphocytes. While their effect on T-lymphocytes has been extensively analyzed, data on the effect of mesenchymal stem cells on B cells are more limited. We analyzed the effects of mesenchymal stem cells on B-lymphocytes and the pathways involved in these effects. Design and Methods The effect of MSC on the proliferation and viability of B cells was evaluated using MTT assays, annexin/7-amino-actinomycin D and propidium iodide staining. The B-cell maturation pattern was established using flow cytometry based on the expression of different markers related to the differentiation of B cells, such as CD38, CD138, CD19 and CCR7, and to the expression of surface and intracellular immunoglobulins. Finally, western blot assays were used to identify the pathways involved in the effects of mesenchymal stem cells on B-lymphocytes. Results Mesenchymal stem cells increased viability and blocked the cell cycle of B-lymphocytes in the G0/G1 phase. In vitro exposure of B cells to plasmacytoid dendritic cells induced B-cell differentiation as shown by an increased number of CD38++/CD138++ cells, which also displayed higher levels of cytoplasmic immunoglobulin and lower levels of CD19, CCR7 and surface immunoglobulin. Interestingly, this maturation pattern was inhibited by adding mesenchymal stem cells to the culture. Finally, mesenchymal stem cells modified the phosphorylation pattern of the extracellular response kinase 1/2 and p38 pathways which are both involved in B-cell viability, proliferation and activation. Conclusions Mesenchymal stem cells increase B-cell viability while inhibiting proliferation, arresting B-lymphocytes in the G0/G1 phase of the cell cycle. The presence of mesenchymal stem cells blocked B-cell differentiation as assessed by flow cytometry. Finally, mesenchymal stem cells modified the activation pattern of the extracellular response kinase and the p38 mitogen-activated protein kinase pathways in B-lymphocytes.

Immunomodulatory effect of 5-azacytidine (5-azaC): potential role in the transplantation setting

Cytokine genes are targets of multiple epigenetic mechanisms in T lymphocytes. 5-azacytidine (5-azaC) is a nucleoside-based DNA methyltransferase inhibitor that induces demethylation and gene reactivation. In the current study, we analyzed the effect of 5-azaC in T-cell function and observed that 5-azaC inhibits T-cell proliferation and activation, blocking cell cycle in the G(0) to G(1) phase and decreasing the production of proinflammatory cytokines such as tumor necrosis factor-alpha and interferon-gamma. This effect was not attributable to a proapoptotic effect of the drug but to the down-regulation of genes involved in T-cell cycle progression and activation such as CCNG2, MTCP1, CD58, and ADK and up-regulation of genes that induce cell-growth arrest, such as DCUN1D2, U2AF2, GADD45B, or p53. A longer exposure to the drug leads to demethylation of FOXP3 promoter, overexpression of FOXP3, and expansion of regulatory T cells. Finally, the administration of 5-azaC after transplantation prevented the development of graft-versus-host disease, leading to a significant increase in survival in a fully mismatched bone marrow transplantation mouse model. In conclusion, the current study shows the effect of 5-azaC in T lymphocytes and illustrates its role in the allogeneic transplantation setting as an immunomodulatory drug, describing new pathways that must be explored to prevent graft-versus-host disease.

p22phox-dependent NADPH oxidase activity is required for megakaryocytic differentiation.

Transient reactive oxygen species (ROS) production is currently proving to be an important mechanism in the regulation of intracellular signalling, but reports showing the involvement of ROS in important biological processes, such as cell differentiation, are scarce. In this study, we show for the first time that ROS production is required for megakaryocytic differentiation in K562 and HEL cell lines and also in human CD34+ cells. ROS production is transiently activated during megakaryocytic differentiation, and such production is abolished by the addition of different antioxidants (such as N-acetyl cysteine, trolox, quercetin) or the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase inhibitor diphenylene iodonium. The inhibition of ROS formation hinders differentiation. RNA interference experiments have shown that a p22phox-dependent NADPH oxidase activity is responsible for ROS production. In addition, the activation of ERK, AKT and JAK2 is required for differentiation, but the activation of phosphatidylinositol 3-kinase and c-Jun N-terminal kinase seems to be less important. When ROS production is prevented, the activation of these signalling pathways is partly inhibited. Taken together, these results show that NADPH oxidase ROS production is essential for complete activation of the main signalling pathways involved in megakaryocytopoiesis to occur. We suggest that this might also be important for in vivo megakaryocytopoiesis.

Treatment with bortezomib of human CD4+ T cells preserves natural regulatory T cells and allows the emergence of a distinct suppressor T-cell population

In vitro depletion of alloreactive T cells using the proteasome inhibitor bortezomib is a promising approach to prevent graft-versus-host disease (GVHD) after allogeneic stem cell transplantation. The findings of this study strengthen the idea of using bortezomib in the prevention of GVHD, not only because of its selective cytotoxic effect on activated T cells, but also due to its ability to preserve and/or generate regulatory T cells. Background In vitro depletion of alloreactive T cells using the proteasome inhibitor bortezomib is a promising approach to prevent graft-versus-host disease after allogeneic stem cell transplantation. We have previously described the ability of bortezomib to selectively eliminate alloreactive T cells in a mixed leukocyte culture, preserving non-activated T cells. Due to the role of regulatory T cells in the control of graft versus host disease, in the current manuscript we have analyzed the effect of bortezomib in regulatory T cells. Design and Methods Conventional or regulatory CD4+ T cells were isolated with immunomagnetic microbeads based on the expression of CD4 and CD25. The effect of bortezomib on T-cell viability was analyzed by flow cytometry using 7-amino-actinomycin D staining. To investigate the possibility of obtaining an enriched regulatory T-cell population in vitro with the use of bortezomib, CD4+ T cells were cultured during four weeks in the presence of anti-CD3 and anti-CD28 antibodies, IL-2 and bortezomib. The phenotype of these long-term cultured cells was studied, analyzing the expression of CD25, CD127 and FOXP3 by flow cytometry, and mRNA levels were determined by RT-PCR. Their suppressive capacity was assessed in co-culture experiments, analyzing proliferation and IFN-γ and CD40L expression of stimulated responder T cells by flow cytometry. Results We observed that naturally occurring CD4+CD25+ regulatory T cells are resistant to the pro-apoptotic effect of bortezomib. Furthermore, we found that long-term culture of CD4+ T cells in the presence of bortezomib promotes the emergence of a regulatory T-cell population that significantly inhibits proliferation, IFN-γ production and CD40L expression among stimulated effector T cells. Conclusions These results reinforce the proposal of using bortezomib in the prevention of graft versus host disease and, moreover, in the generation of regulatory T-cell populations, that could be used in the treatment of multiple T-cell mediated diseases.

Mesenchymal stem cells are functionally abnormal in patients with immune thrombocytopenic purpura

BACKGROUND AIMS Immune thrombocytopenic purpura (ITP) is a bleeding disorder characterized by an accelerated destruction of platelets as a result of the presence of autoreactive antibodies. Patients with ITP also display activated platelet-autoreactive T cells. Mesenchymal stem cells (MSC) inhibit both T- and B-cell activation and may have functional impairments in autoimmune disorders. METHODS We analyzed the potential role of MSC in the pathogenesis of ITP. RESULTS MSC from ITP showed an impaired proliferative capacity and a lower capability of inhibiting activated T-cell proliferation compared with healthy donors. While MSC from controls showed a decreased expression of p27 after stimulation with platelet-derived growth factor, this effect was not observed in MSC from patients. Furthermore, MSC from healthy donors down-regulated p16 upon exposure to platelet-released supernatant, while this effect was not observed for ITP. Interestingly, caspase 9 expression was higher in MSC from ITP. CONCLUSIONS These abnormalities suggest a role of MSC malfunction in the physiopathology of the disease and may have therapeutic implications.

Microvesicles from Mesenchymal Stromal Cells Are Involved in HPC-Microenvironment Crosstalk in Myelodysplastic Patients.

Exosomes/microvesicles (MVs) provide a mechanism of intercellular communication. Our hypothesis was that mesenchymal stromal cells (MSC) from myelodysplastic syndrome (MDS) patients could modify CD34+ cells properties by MVs. They were isolated from MSC from MDS patients and healthy donors (HD). MVs from 30 low-risk MDS patients and 27 HD were purified by ExoQuick-TC™ or ultracentrifugation and identified by transmission electron microscopy, flow cytometry (FC) and western blot for CD63. Incorporation of MVs into CD34+ cells was analyzed by FC, and confocal and fluorescence microscopy. Changes in hematopoietic progenitor cell (HPC) properties were assessed from modifications in microRNAs and gene expression in CD34+ cells as well as viability and clonogenic assays of CD34+ cells after MVs incorporation. Some microRNAs were overexpressed in MVs from patients MSC and two of them, miR-10a and miR-15a, were confirmed by RT-PCR. These microRNAs were transferred to CD34+ cells, modifying the expression of MDM2 and P53 genes, which was evaluated by RT-PCR and western blot. Finally, examining CD34+ cells properties after incorporation, higher cell viability (p = 0.025) and clonogenic capacity (p = 0.037) were observed when MVs from MDS patients were incorporated. In summary, we show that BM-MSC release MVs with a different cargo in MDS patients compared with HD. These structures are incorporated into HPC and modify their properties.

Depletion of alloreactive T-cells in vitro using the proteasome inhibitor bortezomib preserves the immune response against pathogens

Current graft-versus-host disease (GVHD) inhibition approaches lead to abrogation of pathogen-specific T-cell responses. We propose an approach to inhibit GVHD without hampering immunity against pathogens: in vitro depletion of alloreactive T cells with the preoteasome inhibitor bortezomib. We show that PBMCs stimulated with allogeneic cells and treated with bortezomib greatly reduce their ability to produce IFN-γ when re-stimulated with the same allogeneic cells, but mainly preserve their ability to respond to citomegalovirus stimulation. Unlike in vivo administration of immunosuppressive drugs or other strategies of allodepletion, in vitro allodepletion with bortezomib maintains pathogen-specific T cells, representing a promising alternative for GVHD prophylaxis.

The novel combination of sirolimus and bortezomib prevents graft-versus-host disease but maintains the graft-versus-leukemia effect after allogeneic transplantation.

Background We have previously shown that bortezomib induces a depletion of alloreactive T cells and allows the expansion of T cells with suppressive properties. In the current study, we analyzed the potential synergistic effect of bortezomib in conjunction with sirolimus in order to reduce-graft-versus-host disease without hampering graft-versus-leukemia effect in the allogeneic transplant setting. Design and Methods We evaluated the effect of sirolimus, bortezomib or the combination of both in the proliferation and activation of in vitro stimulated T lymphocytes. Pathways involved in this synergy were also analyzed using Western blot assays. Finally, BALB/c mice receiving C57BL/6 allogeneic donor bone marrow with splenocytes were used to measure in vivo the effect of this novel combination on the risk of graft-versus-host disease. Results The combination of both drugs synergistically inhibited both activation and proliferation of stimulated T cells. Also, the production of Th1 cytokines (IFN γ, IL-2 and TNF) was significantly inhibited. This effect was due, at least in part, to the inhibition of Erk and Akt phosphorylation. In vivo, the combination reduced the risk of graft-versus-host disease without hampering graft-versus-leukemia effect, as shown in mice receiving graft-versus-host disease prophylaxis with sirolimus plus bortezomib being infused with tumor WEHI cells plus C57BL/6 donor BM and splenocytes. Conclusions The current study reveals a synergistic effect of the combination sirolimus and bortezomib to prevent graft-versus-host disease while maintaining the graft-versus-leukemia effect.

Caspase-8 inhibition represses initial human monocyte activation in septic shock model.

In septic patients, the onset of septic shock occurs due to the over-activation of monocytes. We tested the therapeutic potential of directly targeting innate immune cell activation to limit the cytokine storm and downstream phases. We initially investigated whether caspase-8 could be an appropriate target given it has recently been shown to be involved in microglial activation. We found that LPS caused a mild increase in caspase-8 activity and that the caspase-8 inhibitor IETD-fmk partially decreased monocyte activation. Furthermore, caspase-8 inhibition induced necroptotic cell death of activated monocytes. Despite inducing necroptosis, caspase-8 inhibition reduced LPS-induced expression and release of IL-1β and IL-10. Thus, blocking monocyte activation has positive effects on both the pro and anti-inflammatory phases of septic shock. We also found that in primary mouse monocytes, caspase-8 inhibition did not reduce LPS-induced activation or induce necroptosis. On the other hand, broad caspase inhibitors, which have already been shown to improve survival in mouse models of sepsis, achieved both. Thus, given that monocyte activation can be regulated in humans via the inhibition of a single caspase, we propose that the therapeutic use of caspase-8 inhibitors could represent a more selective alternative that blocks both phases of septic shock at the source.

Immunomodulatory Effect of Vitamin D after Allogeneic Stem Cell Transplantation: Results of a Prospective Multicenter Clinical Trial

Purpose: We describe the results of a prospective multicenter phase I/II trial evaluating the impact of the use of vitamin D (VitD) from day −5 to +100 on the outcome of patients undergoing allogeneic transplantation (EudraCT: 2010-023279-25; ClinicalTrials.gov: NCT02600988). Experimental Design: A total of 150 patients were included in three consecutive cohorts of 50 patients each group: control group (CG, not receive VitD); low-dose group (LdD, received 1,000 IU VitD daily); and high-dose group (HdD, 5,000 IU VitD daily). We measured levels of VitD, cytokines, and immune subpopulations after transplantation. Results: No significant differences were observed in terms of cumulative incidence of overall and grades 2–4 acute GVHD in terms of relapse, nonrelapse mortality, and overall survival. However, a significantly lower cumulative incidence of both overall and moderate plus severe chronic GVHD (cGVHD) at 1 year was observed in LdD (37.5% and 19.5%, respectively) and HdD (42.4% and 27%, respectively) as compared with CG (67.5% and 44.7%, respectively; P < 0.05). In multivariable analysis, treatment with VitD significantly decreased the risk of both overall (for LdD: HR = 0.31, P = 0.002; for HdD: HR = 0.36, P = 0.006) and moderate plus severe cGVHD (for LdD: HR = 0.22, P = 0.001; for HdD: HR = 0.33, P = 0.01). VitD modified the immune response, decreasing the number of B cells and naïve CD8 T cells, with a lower expression of CD40L. Conclusions: This is the first prospective trial that analyzes the effect of VitD postransplant. We observed a significantly lower incidence of cGVHD among patients receiving VitD. Interestingly, VitD modified the immune response after allo-SCT. Clin Cancer Res; 22(23); 5673–81. ©2016 AACR.