Ana Henriques

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.

Frequency and functional activity of Th17, Tc17 and other T-cell subsets in Systemic Lupus Erythematosus

To compare frequency and functional activity of peripheral blood (PB) Th(c)17, Th(c)1 and Treg cells and the amount of type 2 cytokines mRNA we recruited SLE patients in active (n=15) and inactive disease (n=19) and healthy age- and gender-matched controls (n=15). The study of Th(c)17, Th(c)1 and Treg cells was done by flow cytometry and cytokine mRNA by real-time PCR. Compared to NC, SLE patients present an increased proportion of Th(c)17 cells, but with lower amounts of IL-17 per cell and also a decreased frequency of Treg, but with increased production of TGF-beta and FoxP3 mRNA. Iotan active compared to inactive SLE, there is a marked decreased in frequency of Th(c)1 cells, an increased production of type 2 cytokines mRNA and a distinct functional profile of Th(c)17 cells. Our findings suggest a functional disequilibrium of T-cell subsets in SLE which may contribute to the inflammatory process and disease pathogenesis.

Cytokine production by monocytes, neutrophils, and dendritic cells is hampered by long-term intensive training in elite swimmers

Elite level athletes seem to be prone to illness especially during heavy training phases. The aim of this study was to investigate the influence of long term intensive training on the functional features of innate immune cells from high competitive level swimmers, particularly the production of inflammatory mediators and the possible relationship with upper respiratory symptoms (URS) occurrence. A group of 18 swimmers and 11 healthy non athletes was studied. Peripheral blood samples were collected from athletes after 36xa0h of resting recovery from exercise at four times during the training season and at three times from non athletes. Samples were incubated in the presence or absence of LPS and IFN-γ and the frequency of cytokine-producing cells and the amount produced per cell were evaluated by flow cytometry. In addition, plasma cortisol levels were measured and URS recorded through daily logs. The athletes, but not the controls, showed a decrease in the number of monocytes, neutrophils, and dendritic cell (DC) subsets and in the amount of IL-1β, IL-6, IL-12, TNF-α, and MIP-1β produced after stimulation, over the training season. Differences were most noticeable between the first and second blood collections (initial increase in training volume). Athlete’s cortisol plasma levels partially correlated with training intensity and could help explain the reduced in vitro cell response to stimulation. Our results support the idea that long-term intensive training may affect the function of innate immune cells, reducing their capacity to respond to acute challenges, possibly contributing to an elevated risk of infection.

S4 13 - PV cell penetrating peptide and cationic liposomes act synergistically to mediate intracellular delivery of plasmid DNA

Cell penetrating peptides have been successfully used to mediate the intracellular delivery of a wide variety of molecules of pharmacological interest. The main aim of the present work was to evaluate the potential of the S413‐PV cell penetrating peptide to mediate the intracellular delivery of plasmid DNA, aiming at its use in gene therapy applications. The S413‐PV cell penetrating peptide is a chimeric peptide that results from the combination of a cell penetrating sequence derived from the Dermaseptin S4 peptide with the nuclear localization signal present in the Simian Virus 40 (SV40) large T antigen.

Functional characterization of peripheral blood dendritic cells and monocytes in systemic lupus erythematosus

With the purpose of contributing to a better knowledge of the APCs functional activity in SLE, we evaluated the distribution and functional ability to produce pro-inflammatory cytokines (TNF-α, IL-1β, IL-6 and IL-12) of peripheral blood (PB) monocytes and DC (tDC), particularly myeloid (mDC) and CD14−/lowCD16+ DC subpopulations comparing them with those obtained from healthy individuals. The study was performed in 34 SLE patients with diverse disease activity scores (SLEDAI) and 13 healthy age- and sex-matched controls (NC). Our results show an overall decrease in absolute number and relative frequency of tDC in SLE patients with active disease when compared to those with inactive disease and NC, although this decrease did not seem to have an effect on the distribution of PB DC subsets. The monocytes number in SLE patients was similar to those found in NC, whereas a higher frequency of monocytes producing cytokines as well as the amount of each cytokine per cell found without stimulation was particularly observed in those patients with active disease. After stimulation, we observed a higher frequency of IL-12-producing monocytes in active SLE patients. On the other hand, we found among DCs higher frequencies of cytokine-producing CD14−/lowCD16+ DCs and a higher amount of cytokines produced per cell, particularly in active disease. These findings support an increased production of inflammatory cytokines by APCs in active SLE, mostly associated with alterations in CD14−/lowCD16+ DC subset homeostasis that might contribute to explain the dynamic role of these cells in disease pathogenesis.

The phenotypical and functional characteristics of cord blood monocytes and CD14−/low/CD16+ dendritic cells can be relevant to the development of cellular immune responses after transplantation

Umbilical cord blood (UCB) has been used as an alternative source of haematopoietic progenitors for transplantation presenting advantages over bone marrow (BM) that are related with known shortages of newborns immune system at adaptive and innate levels. Using flow cytometry, we studied the expression of Toll-like receptors (TLRs) and chemokine receptors (CKRs) and the production of pro-inflammatory cytokines by monocytes and CD14(-/low)/CD16(+)DCs from peripheral blood (PB; n=10), and umbilical cord blood (UCB; n=10). CKRs and cytokines were studied before and after stimulation of cells with LPS plus IFN-gamma. We also identified the two populations in normal bone marrow samples (BM; n=5). BM presented lower frequencies of both studied populations when compared to UCB and PB. CD14(-/low)/CD16(+)DCs presented a pattern of TLR expression different from mature monocytes reflecting distinct functions for these two populations. UCB cells presented reduced expression of TLR-4 and lower capability to produce cytokines prior stimulation. The populations studied presented different patterns of CKR expression reflecting distinct migratory pathways. Moreover, UCB cells presented higher expressions of CXCR4 and CCR7 that may be involved in immune system maturation and stem cell homing. Monocytes and CD14(-/low)/CD16(+)DCs present functional and phenotypical characteristics that may contribute to the lower incidence and severity of GVHD.

Changes in natural killer cell subpopulations over a winter training season in elite swimmers.

Immune changes and increased susceptibility to infection are often reported in elite athletes. Infectious episodes can often impair training and performance with consequences for health and sporting success. This study monitored the occurrence of episodes of upper respiratory symptoms (URS) and the variation in circulating NK cells, CD56bright and CD56dim NK cells subpopulations, over a winter swimming season. Nineteen national elite swimmers and 11 non-athlete controls participated in this study. URS episodes were monitored using daily log books. Blood samples were taken at rest at four time points during the season: before the start of the season (t1—middle September), after 7xa0weeks of an initial period of gradually increasing training load (t2—early November), after 6xa0weeks of an intense training cycle (t3—late February) and 48xa0h after the main competition (t4—early April) and from the controls at three similar time points (t1—early November; t2—late February; t3—early April). In the swimmers, the occurrence of URS clustered around the periods of elevated training load (67xa0%). No URS were reported at equivalent time points in the non-athletes. Athletes showed a decrease in the percentage (t2xa0=xa021xa0%; t3xa0=xa027xa0%; t4xa0=xa017xa0%) and absolute counts of circulating NK cells (t2xa0=xa035xa0%; t3xa0=xa022xa0%; t4xa0=xa022xa0%), coinciding with the periods of increased training load, never recovering to the initial values observed at the start of the season. The reduction in the CD56dim and an increase in the CD56bright NK cell subpopulations were significant at t2 and t3 (pxa0<xa00.05). Concomitant with the fall in values of NK cells, in athletes that shown more than three URS episodes, a moderate correlation (rxa0=xa00.493; pxa0=xa00.036) was found between CD56bright/CD56dim ratio and the number of URS episodes after the more demanding training phase (t3). At t3, a lower value of CD56 cell counts was found in the group who reported three or more URS episodes (txa0=xa02.239; pxa0=xa00.032). A progressive significant decrease in the expression of CD119, the receptor for IFN-γ, on the CD56dim cells was found over the season and an elevation in Granzyme B expression was coincident with the more demanding training phases. Periods of highly demanding training seem to have a negative impact on innate immunity mediated by NK cell subsets, which could partially explain the higher frequency of URS observed during these training phases.

Molecular and cytogenetic characterization of expanded B-cell clones from multiclonal versus monoclonal B-cell chronic lymphoproliferative disorders

Chronic antigen-stimulation has been recurrently involved in the earlier stages of monoclonal B-cell lymphocytosis, chronic lymphocytic leukemia and other B-cell chronic lymphoproliferative disorders. The expansion of two or more B-cell clones has frequently been reported in individuals with these conditions; potentially, such coexisting clones have a greater probability of interaction with common immunological determinants. Here, we analyzed the B-cell receptor repertoire and molecular profile, as well as the phenotypic, cytogenetic and hematologic features, of 228 chronic lymphocytic leukemia-like and non-chronic lymphocytic leukemia-like clones comparing multiclonal (n=85 clones from 41 cases) versus monoclonal (n=143 clones) monoclonal B-cell lymphocytosis, chronic lymphocytic leukemia and other B-cell chronic lymphoproliferative disorders. The B-cell receptor of B-cell clones from multiclonal cases showed a slightly higher degree of HCDR3 homology than B-cell clones from mono clonal cases, in association with unique hematologic (e.g. lower B-lymphocyte counts) and cytogenetic (e.g. lower frequency of cytogenetically altered clones) features usually related to earlier stages of the disease. Moreover, a subgroup of coexisting B-cell clones from individual multiclonal cases which were found to be phylogenetically related showed unique molecular and cytogenetic features: they more frequently shared IGHV3 gene usage, shorter HCDR3 sequences with a greater proportion of IGHV mutations and del(13q14.3), than other unrelated B-cell clones. These results would support the antigen-driven nature of such multiclonal B-cell expansions, with potential involvement of multiple antigens/epitopes.

Th17 cells in systemic lupus erythematosus share functional features with Th17 cells from normal bone marrow and peripheral tissues

This study was designed to investigate the functional heterogeneity of human Th17 and how their plasticity shapes the nature of immune cell responses to inflammation and autoimmune diseases, such as systemic lupus erythematosus (SLE). We evaluated functional Th17 cell subsets based on the profile of cytokine production in peripheral blood (PB), bone marrow aspirates (BM) and lymph node biopsies (LN) from healthy individuals (nu2009=u200935) and PB from SLE patients (nu2009=u200934). Data were analysed by an automated method for merging and calculation of flow cytometric data, allowing us to identify eight Th17 subpopulations. Normal BM presented lower frequencies of Th17 (pu2009=u20090.006 and pu2009=u20090.05) and lower amount of IL-17 per cell (pu2009=u20090.03 and pu2009=u20090.02), compared to normal PB and LN biopsies. In the latter tissues were found increased proportions of Th17 producing TNF-α or TNF-α/IL-2 or IFN-γ/TNF-α/IL-2, while in BM, Th17 producing other cytokines than IL-17 was clearly decreased. In SLE patients, the frequency of Th17 was higher than in control, but the levels of IL-17 per cell were significantly reduced (pu2009<u20090.05). Among the eight generated subpopulations, despite the great functional heterogeneity of Th17 in SLE, a significant low proportion of Th17 producing TNF-α was found in inactive SLE, while active SLE showed a high proportion producing only IL-17. Our findings support the idea that the functional heterogeneity of Th17 cells could depend on the cytokine microenvironment, which is distinct in normal BM as well as in active SLE, probably due to a Th1/Th2 imbalance previously reported by our group.

Subjects with chronic lymphocytic leukaemia‐like B‐cell clones with stereotyped B‐cell receptors frequently show MDS‐associated phenotypes on myeloid cells

An increasing body of evidence suggests the potential occurrence of antigen encounter by the cell of origin in chronic lymphocytic leukaemia (CLL) and CLL‐like monoclonal B‐cell lymphocytosis (MBL). However, the scenario in which this event might occur remains unknown. In order to gain insight into this scenario we investigated the molecular, cytogenetic and haematological features of 223 CLL‐like (n = 84) and CLL (n = 139) clones with stereotyped (n = 32) versus non‐stereotyped (n = 191) immunoglobulin heavy chain variable region (IGHV) amino acid sequences. Overall, stereotyped CLL‐like MBL and CLL clones showed a unique IGHV profile, associated with higher IGHV1 and lower IGHV3 gene family usage (P = 0·03), longer IGHV complementary determining region 3 (HCDR3) sequences (P = 0·007) and unmutated IGHV (P < 0·001) versus non‐stereotyped clones. Whilst the overall size of the stereotyped B‐cell clones in peripheral blood did not appear to be associated with the CLL‐related cytogenetic profile of B‐cells (P > 0·05), it did show a significant association with the presence of myelodysplastic syndrome (MDS)‐associated immunophenotypes on peripheral blood neutrophils and/or monocytes (P = 0·01). Altogether our results point to the potential involvement of different selection forces in the expansion of stereotyped vs. non‐stereotyped CLL and CLL‐like MBL clones, the former being potentially favoured by an underlying altered haematopoiesis.