Polyclonal anti-human T-lymphocyte immunoglobulin(ATG) have been recently shown, in two randomized studies, to significantly reduce the incidence of graft versus host disease (GVHD) post allogeneic ...stem cell transplantation (HSCT) from both sibling and unrelated donors. Induction of regulatory T cells is suggested as one of the possible mechanisms involved. The aim of our current study was to further characterize the T cell populations induced by ATG treatment and to delineate the mechanisms involved in ATG-induced tolerance in patients receiving intravenous ATG (ATG-Fresenius ® S, Neovii Biotech) as part of their pre HSCT conditioning.
Phenotypic characterization of regulatory cells markers revealedthattwo days culture of peripheral blood mononuclear cells (PBMCs) with ATG-F (30-120 µg/ml) resulted in significant increase in CD25 expression on CD4+ T cells. The percentages of cells expressing CTLA4, GITR, CD95 and FoxP3 was also significantly elevated on CD4+ cells compared to rabbit IgG-treated PBMCs. In addition, expression of CD127 and VLA-4 molecules was significantly decreased on CD4+CD25+ cells upon ATG-F treatment (p<0.01).
Next, tolerance ability of ATG-F-induced cells was examined. Addition of ATG-F-treated cells to autologous PBMCs stimulated with antiCD3/antiCD28 antibodies resulted in significant (50-75%) inhibition of cell proliferation (p<0.001), measured by CFSE and Ki67 staining. Moreover, CD69 cell expression and interferon-γ (IFNγ) proinflammatory cytokine secretion were reduced by 50-60% and 65-90%, respectively, in the presence of ATG-F-activated Treg cells (p<0.01).
Importantly, addition of cyclosporine A to the induction culture with ATG-F interfered with the ATG-induced regulatory phenotype acquisition, suggesting the involvement of interleukin-2 in ATG-mediated activity.
In order to purify the tolerizing population, sorting of CD4+CD25+CD127-low cells (considered as viable regulatory T cells) from ATG-F-treated culture was performed. Sorted cells demonstrated greater suppressive potency than bulked pre-sorted cell population when added to autologous stimulated PBMCs. Of note, Treg-depleted fraction was still able to suppress the proliferation, albeit less efficiently then sorted Treg cells, suggesting that ATG-F is capable to induce multiple immune suppressive cell populations that should be further defined.
To explore the possible involvement of soluble factor(s)-mediated mechanisms, in addition to the involvement of cell to cell contact mechanism, conditioned medium (CM) produced by ATG-F-primed cells was applied on stimulated autologous PBMCs. Addition of CM produced by ATG-F-treated cells, but not IgG-treated cells, resulted in significant suppression (30-65%, p<0.01) of T cell proliferation and activation, indicating the presence of soluble factors secreted by ATG-F-primed suppressive cells. Indeed, significant dose- and time-dependent induction of TGFβ secretion was observed in ATG-treated cells. To this end, addition of TGFβ receptor kinase inhibitor SB-431542 interfered with suppressive activity of ATG-F-primed cells, enabling partial rescue of proliferation and IFNγ secretion in response to antiCD3/antiCD28 activation. Similar results were obtained with anti-TGFβ neutralizing antibodies.
Finally, characterization of phenotype and frequencies of regulatory immune populations in peripheral blood of 26 patients undergoing allogeneic transplantation with conditioning regimen including ATG-F (15mg/kg) was performed. Consistent with our ex vivo results, transient increase in percent of circulating CD4+CD25+CD127-low cells was detected in the ATG-F treated patients on days 14, 21 and 28 after HSCT. Furthermore, elevated levels of TGFβ were detected in the patients' plasma at day 28 and remaining high at day 60 post HSCT.
Our results demonstratethat in vitro treatment with ATG-F is capableto induce functional Treg cells. Suppressive ability of ATG-F-induced cells was both contact and soluble-factors dependent and was partially promoted by TGFβ. Patients undergoing allogeneic HSCT with ATG-F-including conditioning demonstrated increased frequencies of circulating Treg cells and elevated plasma levels of TGFβ. Altogether, our data further support the use of ATG-F, a potent inducer of Treg cells, for prevention of GVHD post HSCT and potentially other therapeutic applications.
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No relevant conflicts of interest to declare.
Introduction: Multiple myeloma (MM) is an incurable hematological malignancy characterized by proliferation of malignant plasma cells in the bone marrow (BM). Interactions between MM cells and BM ...milieu facilitate disease progression and therapy resistance. Chemokine receptor CXCR4 and its cognate ligand CXCL12 are implicated in these processes and are associated with poor prognosis. Sphingosine-1-phosphate (S1P) pathway is involved in cancer progression, including oncogenesis, cell survival and cell migration, therefore representing an attractive target for anti-cancer therapy. FTY720 (fingolimod) is a modulator of S1P signaling system that exhibit immunosuppressive and anti-cancer properties. The role of S1P system and FTY720 modulator in MM is less defined. The aim of this study was to explore the functional consequences of possible cross-talk between the CXCR4/CXCL12 and the S1P axes in MM cells and to evaluate the effect of S1P targeting with FTY720 as potential anti-MM therapeutic strategy.
Results: The partners of the S1P pathway (S1P receptor 1 and sphingosine kinase 1 (SPHK1)) and CXCL12 chemokine were found to be co-expressed in MM cell lines and primary BM samples from MM patients. Increased mRNA levels of SPHK1 and CXCL12 were detected in MM BM samples (n=24) comparing to BM from healthy donors (n=7) (p<0.01). In vitro treatment of MM cell lines (n=6) with FTY720 modulator resulted in time- and dose-dependent cell death (IC50 2.8 – 5.3 µM). Further characterization of cell death mechanisms revealed that FTY720 treatment induced MM cell apoptosis with mitochondrial involvement, cytochrome C release and caspase 3 activation.
Interestingly, suppressive potential of FTY720 negatively correlated with CXCR4 expression on MM cells. Enforced expression of CXCR4 reduced the sensitivity to FTY720, whereas silencing of endogenous CXCL12 increased the sensitivity of MM cells to FTY720-mediated cell death. These results suggested the CXCR4 axis to be directly regulated by S1P pathway. In support, we have found that FTY720 treatment significantly reduced CXCR4-dependent MM cell adhesion to fibronectin and abrogated MM migration toward CXCL12. Activation of signaling pathways, such as MAPK and Akt, in response to CXCL12 stimulation was also fully blocked by FTY720 pre-treatment. In addition to functional suppression, FTY720 directly and profoundly reduced CXCR4 cell-surface levels in a dose-dependent manner. Importantly, none of the suppressive effects of FTY720 (neither apoptosis, nor migration or adhesion inhibition) were dependent on protein phosphatase 2A (PP2A) activation, suggesting alternative mechanism of action.
To further investigate down-stream molecular machinery involved in FTY720-mediated CXCR4 targeting in MM cells, the intra-cellular levels of different signaling mediators were evaluated. We identified the mTOR pathway to be regulated by CXCR4 and targeted by FTY720. FTY720 treatment suppressed mTOR signaling in MM cells, as demonstrated by de-phosphorylation of p70S6K and S6. Forced expression of CXCR4 and interaction with BM stromal cells antagonized with FTY720-mediated apoptosis and prevented FTY720-induced S6 de-phosphorylation. While, combination of FTY720 with mTOR inhibitor RAD001 resulted in significantly increased cell death, effectively abrogating CXCR4- and stroma-dependent resistance to FTY720 and suppressing mTOR signaling in MM cells.
Finally, in a recently developed novel xenograft model of CXCR4-dependent systemic MM with BM involvement, in vivo FTY720 effectively reduced tumor burden in two third of the treated mice, decreasing both the levels of M protein in blood and the number of MM cells in BM.
Conclusions: Taken together, our findings demonstrate cross talk between S1P and CXCR4/CXCL12 signaling pathways that may be of importance for MM cell survival and localization of the MM cells in CXCL12-expressing protective niches in the BM. Moreover, this is, to our knowledge, the first evidence that CXCR4 can be directly targeted with FTY720 modulator, thus restricting the tumor-promoting activities of S1P and CXCR4/CXCL12 axes. In addition, mTOR pathway was recognized as down-stream molecular partner being involved in FTY720-mediated anti-myeloma activities. Combining FTY720 with mTOR inhibitors may thus serve as promising novel therapeutic strategy in MM.
Peled:BioLineRx: Research Funding.
Here we describe a Paleocene-aged methane seep locality and an associated layer of sunken wood, from Fossildalen on Spitsbergen, Svalbard, hosted in offshore to prodelta siltstones of the Basilika ...Formation, Van Mijenfjorden Group. The fossiliferous seep carbonates were first identified in museum collections from expeditions in the 1920s and 1970s, and subsequently sampled as ex-situ blocks in the field in 2015. The isotopically light composition (δ13C values approaching −50‰ V-PDB), and characteristic petrographic textures of the carbonates combined with the isotopically light archaeal lipids are consistent with their formation at fossil hydrocarbon seep environment. The invertebrate fauna within the carbonates is of moderate diversity (17 species) and has a shallow water affinity. Wood specimens within the carbonates contain the borings and shells of wood-boring bivalves. The seep fauna is dominated by a species of the thyasirid genus Conchocele, common to other seeps of similar age. The data shed new light onto the history of methane seepage on Svalbard, and the evolution and ecology of seep and wood-fall faunas during the latest Cretaceous–earliest Paleogene time interval.
•We describe Paleocene shallow water seep and wood-fall environments from Spitsbergen.•Both seep and wood-fall faunas are heavily dominated by thyasirid bivalve Conchocele.•The associated species are mostly shallow water background molluscs.•Paleocene seep and wood-fall environments from Spitsbergen spatially overlapped.•They are most comparable to Recent forest litter accumulations in New Zealand fiords.
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