Hélder Trindade

Mesenchymal stem cells from umbilical cord matrix, adipose tissue and bone marrow exhibit different capability to suppress peripheral blood B, natural killer and T cells

IntroductionThe ability to self-renew, be easily expanded in vitro and differentiate into different mesenchymal tissues, render mesenchymal stem cells (MSCs) an attractive therapeutic method for degenerative diseases. The subsequent discovery of their immunosuppressive ability encouraged clinical trials in graft-versus-host disease and auto-immune diseases. Despite sharing several immunophenotypic characteristics and functional capabilities, the differences between MSCs arising from different tissues are still unclear and the published data are conflicting.MethodsHere, we evaluate the influence of human MSCs derived from umbilical cord matrix (UCM), bone marrow (BM) and adipose tissue (AT), co-cultured with phytohemagglutinin (PHA)-stimulated peripheral blood mononuclear cells (MNC), on T, B and natural killer (NK) cell activation; T and B cells’ ability to acquire lymphoblast characteristics; mRNA expression of interleukin-2 (IL-2), forkhead box P3 (FoxP3), T-bet and GATA binding protein 3 (GATA3), on purified T cells, and tumor necrosis factor-alpha (TNF-α), perforin and granzyme B on purified NK cells.ResultsMSCs derived from all three tissues were able to prevent CD4+ and CD8+ T cell activation and acquisition of lymphoblast characteristics and CD56dim NK cell activation, wherein AT-MSCs showed a stronger inhibitory effect. Moreover, AT-MSCs blocked the T cell activation process in an earlier phase than BM- or UCM-MSCs, yielding a greater proportion of T cells in the non-activated state. Concerning B cells and CD56bright NK cells, UCM-MSCs did not influence either their activation kinetics or PHA-induced lymphoblast characteristics, conversely to BM- and AT-MSCs which displayed an inhibitory effect. Besides, when co-cultured with PHA-stimulated MNC, MSCs seem to promote Treg and Th1 polarization, estimated by the increased expression of FoxP3 and T-bet mRNA within purified activated T cells, and to reduce TNF-α and perforin production by activated NK cells.ConclusionsOverall, UCM-, BM- and AT-derived MSCs hamper T cell, B cell and NK cell-mediated immune response by preventing their acquisition of lymphoblast characteristics, activation and changing the expression profile of proteins with an important role in immune function, except UCM-MSCs showed no inhibitory effect on B cells under these experimental conditions. Despite the similarities between the three types of MSCs evaluated, we detect important differences that should be taken into account when choosing the MSC source for research or therapeutic purposes.

Effect of human bone marrow mesenchymal stromal cells on cytokine production by peripheral blood naive, memory, and effector T cells

IntroductionThe different distribution of T cells among activation/differentiation stages in immune disorders may condition the outcome of mesenchymal stromal cell (MSC)-based therapies. Indeed, the effect of MSCs in the different functional compartments of T cells is not completely elucidated.MethodsWe investigated the effect of human bone marrow MSCs on naturally occurring peripheral blood functional compartments of CD4+ and CD8+ T cells: naive, central memory, effector memory, and effector compartments. For that, mononuclear cells (MNCs) stimulated with phorbol myristate acetate (PMA) plus ionomycin were cultured in the absence/presence of MSCs. The percentage of cells expressing tumor necrosis factor-alpha (TNF-α), interferon gamma (IFNγ), and interleukin-2 (IL-2), IL-17, IL-9, and IL-6 and the amount of cytokine produced were assessed by flow cytometry. mRNA levels of IL-4, IL-10, transforming growth factor-beta (TGF-β), and cytotoxic T-lymphocyte-associated protein 4 (CTLA4) in purified CD4+ and CD8+ T cells, and phenotypic and mRNA expression changes induced by PMA + ionomycin stimulation in MSCs, were also evaluated.ResultsMSCs induced the reduction of the percentage of CD4+ and CD8+ T cells producing TNF-α, IFNγ, and IL-2 in all functional compartments, except for naive IFNγ+CD4+ T cells. This inhibitory effect differentially affected CD4+ and CD8+ T cells as well as the T-cell functional compartments; remarkably, different cytokines showed distinct patterns of inhibition regarding both the percentage of producing cells and the amount of cytokine produced. Likewise, the percentages of IL-17+, IL-17+TNF-α+, and IL-9+ within CD4+ and CD8+ T cells and of IL-6+CD4+ T cells were decreased in MNC-MSC co-cultures. MSCs decreased IL-10 and increased IL-4 mRNA expression in stimulated CD4+ and CD8+ T cells, whereas TGF-β was reduced in CD8+ and augmented in CD4+ T cells, with no changes for CTLA4. Finally, PMA + ionomycin stimulation did not induce significant alterations on MSCs phenotype but did increase indoleamine-2,3-dioxygenase (IDO), inducible costimulatory ligand (ICOSL), IL-1β, IL-8, and TNF-α mRNA expression.ConclusionsOverall, our study showed that MSCs differentially regulate the functional compartments of CD4+ and CD8+ T cells, which may differentially impact their therapeutic effect in immune disorders. Furthermore, the influence of MSCs on IL-9 expression can open new possibilities for MSC-based therapy in allergic diseases.

Human Bone Marrow-Derived Mesenchymal Stromal Cells Differentially Inhibit Cytokine Production by Peripheral Blood Monocytes Subpopulations and Myeloid Dendritic Cells

The immunosuppressive properties of mesenchymal stromal/stem cells (MSC) rendered them an attractive therapeutic approach for immune disorders and an increasing body of evidence demonstrated their clinical value. However, the influence of MSC on the function of specific immune cell populations, namely, monocyte subpopulations, is not well elucidated. Here, we investigated the influence of human bone marrow MSC on the cytokine and chemokine expression by peripheral blood classical, intermediate and nonclassical monocytes, and myeloid dendritic cells (mDC), stimulated with lipopolysaccharide plus interferon (IFN)γ. We found that MSC effectively inhibit tumor necrosis factor- (TNF-) α and macrophage inflammatory protein- (MIP-) 1β protein expression in monocytes and mDC, without suppressing CCR7 and CD83 protein expression. Interestingly, mDC exhibited the highest degree of inhibition, for both TNF-α and MIP-1β, whereas the reduction of TNF-α expression was less marked for nonclassical monocytes. Similarly, MSC decreased mRNA levels of interleukin- (IL-) 1β and IL-6 in classical monocytes, CCL3, CCL5, CXCL9, and CXCL10 in classical and nonclassical monocytes, and IL-1β and CXCL10 in mDC. MSC do not impair the expression of maturation markers in monocytes and mDC under our experimental conditions; nevertheless, they hamper the proinflammatory function of monocytes and mDC, which may impede the development of inflammatory immune responses.

Changes in naïve and memory T-cells in elite swimmers during a winter training season.

High intensity training regimens appear to put athletes at a higher risk of illness. As these have been linked to alterations in the proportions of differentiated T cells, how training load affects these populations could have important implications for athlete susceptibility to disease. This study examined the effect of a winter training season on the proportions of circulating naïve and memory T cells subsets of high competitive level swimmers. Blood samples were taken at rest at 4 time-points during the season: before the start of the season (t0-September), after 7weeks of an initial period of gradually increasing training load (t1-November), after 6weeks of an intense training cycle (t2-February) and 48h after the main competition (t3-April) and from eleven non-athlete controls at 2 similar time-points (t2 and t3). CD4, CD8 and gamma-delta (γδ) T cells expressing the naïve (CCR7(+)CD45RA(+)), central-memory (CM-CCR7(+)CD45RA(-)), effector-memory (EM-CCR7(-)CD45RA(-)) and terminal effector (TEMRA-CCR7(-)CD45RA(+)) were quantified by flow cytometry. Statistical analyses were performed using multilevel modeling regression. Both T CD4(+) naïve and CM presented a linear increase in response to the first moment of training exposure, and had an exponential decrease until the end of the training exposure. As for TCD4(+) EM, changes were observed from t2 until the end of the training season with an exponential trend, while TCD4(+) TEMRA increased linearly throughout the season. TCD8(+) naïve increased at t1 and decreased exponentially thereafter. TCD8(+) TEMRA values decreased at t1 and increased exponentially until t3. γδT-EM had an increase at t1 and an exponential decrease afterwards. In contrast, γδT-TEMRA decreased at t1 and exponentially increased during the remaining 20weeks of training. An increase in TEMRA and EM T cells alongside a decrease in naïve T cells could leave athletes more susceptible to illness in response to variation in training stimulus during the season.

Subset-specific alterations in frequencies and functional signatures of γδ T cells in systemic sclerosis patients

Objective and designHere, we evaluated the distribution and functional profile of circulating CD27+ and CD27− γδ T-cell subsets in systemic sclerosis (SSc) patients to assess their potential role in this disorder.Materials and methodsPeripheral blood from 39 SSc patients and 20 healthy individuals was used in this study. The TCR-γδ repertoire, cytokine production and cytotoxic signatures of circulating γδ T-cell subsets were assessed by flow cytometry. Gene expression of EOMES, NKG2D and GZMA was evaluated by quantitative RT-PCR in both purified γδ T-cell subsets.ResultsAbsolute numbers of γδ T-cell subsets were significantly decreased in SSc groups, likely reflecting their mobilization to the inflamed skin. Both γδ T-cell subsets preserved their relative proportions and Th1-type cytokine responses. However, cytotoxic properties showed significant disease-associated and subset-specific changes. SSc patients exhibited increased percentages of CD27+ γδ T cells expressing granzyme B or perforin and upregulated GZMA expression in diffuse cutaneous SSc. Conversely, EOMES and NKG2D were downregulated in both SSc γδ T-cell subsets vs. normal controls. Interestingly, patients with pulmonary fibrosis showed a biased TCR repertoire, with a selected expansion of effector Vγ9+ γδ T cells associated with increased frequency of cells expressing granzyme B, but decreased IFN-γ production.ConclusionsSignificant alterations on circulating γδ T-cell subsets suggest a deregulated (increased) cytotoxic activity and thus enhanced pathogenic potential of CD27+ γδ T cells in SSc.

Expression of CD44 and CD35 during normal and myelodysplastic erythropoiesis.

Erythroid dysplasia is a common feature of myelodysplastic syndromes (MDS). Currently available information about the immunophenotypic features of normal and dysplastic erythropoiesis is scarce and restricted to relatively few markers. Here we studied the expression of CD117, CD35 and CD44 throughout the normal (n=16) and dysplastic (n=48) bone marrow erythroid maturation. CD35 emerged as an early marker of CD34(+) erythroid-committed precursors, which is expressed before CD105 and remains positive thereafter. MDS patients (with and without morphologic dyserythropoiesis) displayed overall increased expression of CD44, associated with slight alterations on CD35 expression, suggesting that phenotypic alterations in MDS may precede morphologic dysplasia. In turn, MDS patients with anemia showed increased expression of CD117.

CD38, CD81 and BAFFR combined expression by transitional B cells distinguishes active from inactive systemic lupus erythematosus.

Abstract In view of its heterogeneous presentation and unpredictable course, clinical management of systemic lupus erythematosus (SLE) is difficult. There is a need for biomarkers and diagnostic aids to monitor SLE disease activity and severity prior to, during and after treatment. We undertook this study to search for unique phenotypic patterns in each peripheral blood (PB) B cell subset, capable of distinguishing SLE patients with inactive disease versus SLE patients with active disease versus controls by using an automated population separator (APS) visualization strategy. PB was collected from 41 SLE patients and 28 age- and gender-matched controls. We analyzed the cell surface markers (in a tube CD20/CD27/CD19/CD45/CD38/CD81/BAFFR combination) expression on PB B cell subsets using principal component analysis, implemented in the APS software tool. Overall, our analysis indicates that active SLE can be distinguished from inactive SLE on the basis of a single tube analysis, focused on the decreased expression of CD38, CD81 and BAFFR in transitional B cells. The cluster analysis of immunophenotypic profiles of B cell subsets highlighted disease-specific abnormalities on transitional B cells that emerge as promising surrogate markers for disease activity. Further validation is needed with larger samples and prospective follow-up of patients.

Sera from patients with active systemic lupus erythematosus patients enhance the toll-like receptor 4 response in monocyte subsets

BackgroundSystemic Lupus Erythematosus (SLE) is an auto-immune disease whose complex pathogenesis remains unraveled. Here we aim to explore the inflammatory ability of SLE patients’ sera upon peripheral blood (PB) monocyte subsets and myeloid dendritic cells (mDCs) obtained from healthy donors.MethodsIn this study we included 11 SLE patients with active disease (ASLE), 11 with inactive disease (ISLE) and 10 healthy controls (HC). PB from healthy donors was stimulated with patients’ sera, toll-like receptor (TLR) 4 ligand – lipopolysaccharide or both. The intracellular production of TNF-α was evaluated in classical, non-classical monocytes and mDCs, using flow cytometry. TNF-α mRNA expression was assessed in all these purified cells, after sera treatment.ResultsWe found that sera of SLE patients did not change spontaneous TNF-α production by monocytes or dendritic cells. However, upon stimulation of TLR4, the presence of sera from ASLE patients, but not ISLE, significantly increased the intracellular expression of TNF-α in classical and non-classical monocytes. This ability was related to titers anti-double stranded DNA antibodies in the serum. High levels of anti-TNF-α in the patients’ sera were associated with increased TNF-α expression by co-cultured mDCs. No relationship was found with the levels of a wide variety of other pro-inflammatory cytokines. A slight increase of TNF-α mRNA expression was observed in these purified cells when they were cultured only in the presence of SLE serum.ConclusionsOur data suggest that SLE sera induce an abnormal in vitro TLR4 response in classical and non-classical monocytes, reflected by a higher TNF-α intracellular expression. These effects may be operative in the pathogenesis of SLE.

Increased frequencies of circulating CXCL10-, CXCL8- and CCL4-producing monocytes and Siglec-3-expressing myeloid dendritic cells in systemic sclerosis patients

ObjectiveTo investigate the ex vivo pro-inflammatory properties of classical and non-classical monocytes as well as myeloid dendritic cells (mDCs) in systemic sclerosis (SSc) patients.MethodsSpontaneous production of CXCL10, CCL4, CXCL8 and IL-6 was intracellularly evaluated in classical, non-classical monocytes and Siglec-3-expressing mDCs from peripheral blood of SSc patients and healthy controls (HC) through flow cytometry. In addition, production of these cytokines was determined upon toll-like receptor (TLR) 4 plus Interferon-γ (IFN-γ) stimulation.ResultsThe frequency of non-classical monocytes spontaneously producing CXCL10 was increased in both limited (lcSSc) and diffuse cutaneous (dcSSC) subsets of SSc patients and CCL4 was augmented in dcSSc patients. The proportion of CCL4-producing mDCs was also elevated in dcSSc patients and the percentage of mDCS producing CXCL10 only in lcSSc patients. Upon stimulation, the frequency of non-classical monocytes expressing CXCL8 was increased in both patient groups and mDCs expressing CXCL8 only in lcSSc. Moreover, these parameters in unsupervised clustering analysis identify a subset of patients which are characterized by lung fibrosis and reduced pulmonary function.ConclusionsThese data point towards a role of activated non-classical monocytes and mDCs producing enhanced levels of proinflammatory cytokines in SSc, potentially contributing to lung fibrosis.

Amniotic membrane extract differentially regulates human peripheral blood T cell subsets, monocyte subpopulations and myeloid dendritic cells

The discovery of the immunoregulatory potential of human amniotic membrane (hAM) propelled several studies focusing on its application for the treatment of immunological disorders. However, there is little information regarding the effects of hAM on distinct activation and differentiation stages of immune cells. Here, we aim to investigate the effect of human amniotic membrane extract (hAME) on the pattern of cytokine production by T cells, monocytes and myeloid dendritic cells (mDCs). For this purpose, peripheral blood mononuclear cells (PBMCs) from eight healthy individuals were stimulated in vitro in the presence or absence of hAME. Mitogen-induced proliferation of PBMCs and cytokine production among the distinct T cell functional compartments, monocyte subpopulations and mDCs were evaluated. hAME displayed an anti-proliferative effect and decreased the frequency of T cells producing tumor necrosis factor (TNF)α, interferon (IFN)γ and interleukin (IL)-2, for all T cell functional compartments. The frequency of IL-17 and IL-9-producing T cells was also reduced. The inhibition of mRNA expression of granzyme B, perforin and NKG2D by CD8+ T cells and γδ T cells and the augment of FoxP3 and IL-10 in CD4+ T cells and IL-10 in regulatory T cells were also observed. Furthermore, hAME inhibited IFNγ-induced protein (IP)-10 expression by classical and non-classical monocytes, without hampering the production of TNFα and IL-6 by monocytes and mDCs. These results suggest that hAME exerts an anti-inflammatory effect on T cells, still at a different extent for distinct T cell functional compartments.