A galektin-1, mint a mesenchymalis őssejtek által termelt immunszuppresszív faktor = Determining the role of Galectin-1 as an immunosuppressive factor produced by bone marrow derived mesenchymal stem cells

A pályázatban a mesenchymalis őssejteknek (MSC) az immunválasz kimenetelét befolyásoló faktorait, ezen belül a galektin (Gal)-1 szerepét vizsgáltuk a T sejtek aktivációja során. Meghatároztuk a humán, illetve egér csontvelői MSC sejtvonalak Gal-1 termelését, valamint kimutattuk a Gal-1 hiányos egér MSC által termelt egyéb faktorokat (TGF-béta, COX2, NOS2, IDO és PD-L1). Új eredményeink: -A Gal-1 hozzájárul az MSC antiproliferatív hatásához, azonban hiányában más immunszuppresszív faktorok is gátolják a T sejtek osztódását. Eredményeink alapján sem a Gal-1, sem a PGE2, NO vagy IDO szerepe nem kizárólagos, együttes működésük vezet a T sejtek szuppressziójához. -Az MSC-termelte Gal-1 közvetlen sejt-sejt kapcsolatban váltja ki az aktivált T sejtek pusztulását, ennek lépései megegyeznek az alacsony dózisú rekombináns Gal-1 apoptotikus mechanizmusával. -Diabéteszes egérmodellben az MSC pozitív hatása nem függ a Gal-1 termeléstől. Más in vivo kísérletben az MSC kezelése gátolja az egerek anafilaxiás reakcióját, és ez a hatás a Gal-1 hiányában részben csökken. In vivo nem csak a T sejtek, de egyéb immunsejtek működését is befolyásolja az MSC, mint például a B sejtek ellenanyag-válaszát. Eredményeink az mutatják, hogy az MSC által termelt Gal-1 immunszuppresszív hatása nyomon követhető in vitro tesztekben, de nem minden esetben nyilvánul meg az in vivo eseményekben. Ez a mesenchymalis őssejtek körültekintő jellemzésének elvégzésére figyelmeztet a terápiás alkalmazásuk előtt. | We examined the immunosuppressive function of MSCs on T cells, focusing on their galectin (Gal)-1 expression. We determined the Gal-1 production of human and mouse bone marrow-derived MSCs, and detected the further immunosuppressive factors (TGF-beta, COX2, NOS2, IDO and PD-L1) expressed by MSCs isolated from Gal-1 knockout mice. Our new results achieved by testing these MSC lines are: -Gal-1 contributes to the anti-proliferative effect of Gal-1, however in the absence of Gal-1 other immunosuppressive factors inhibit the proliferation of T cells. -MSC-derived Gal-1 induces T cell apoptosis in direct cell-to-cell interaction with the same mechanism as recombinant Gal-1 does applied at low concentration. -The positive impact of MSCs on the outcome of diabetes in streptozotocin-treated mice does not depend on their Gal-1 production. In other in vivo experiment, MSCs inhibit the anaphylactic reaction to ovalbumin, and this effect is reduced using Gal-1 knockout MSCs. The in vivo action of MSCs is not restricted on T cells as the antibody production of B cells is also influenced by MSC-derived Gal-1. Our results show that the MSC-derived Gal-1 has an immunosuppressive effect in vitro, however its manifestation is variable in different in vivo experiments. This caution on the thorough characterization of MSCs before their therapeutic use

Immunmoduláló humán galektin-1 által kiváltott T-sejt apoptózis molekuláris mechanizmusa = Molecular mechanism of T-cell apoptosis induced by human galectin-1

A galektin-1 egy új immunszuppresszív stratégiát szolgáltathat a a gyulladásos és autoimmun betegségek gyógyításában. A Gal-1 a tumorok rossz prognózisának is kiváló diagnosztikus markere. Jelen ismereteink szerint a Gal-1 immunreguláló hatása az aktivált T-sejtek apoptózisának indukcióján alapul. A Gal-1 immunválasz szabályozó hatása miatt felvetődik gyakorlati, terápiás felhasználása. Ezért alapvető, hogy, a pályázat célkitűzése szerint, a Gal-1 által kiváltott sejthalál mechanizmusát a részletes módon megismerjük. Összehasonlító vizsgálatunkkal, mely tisztázott egy régi ellentmondást, bizonyítottuk, hogy a Gal-1, koncentrációjától függően, más mechanizmus szerint váltja ki a T-sejt pusztulást. Tisztáztuk, hogy a fiziológiás sejthalált a sejtkötött és az alacsony koncentrációban alkalmazott szolubilis Gal-1 váltja ki. A Gal-1 termelő tumor sejt a vele kölcsönhatásba kerülő T-sejtben az Lck kináz és tirozin foszforilált fehérjék specifikus átrendeződését indukálja, mely a sejt-sejt kölcsönhatás felületével ellentétes oldalon figyelhető meg. A Gal-1 a GM1-gangliozid és a CD7 közvetítésével internalizálódik, ez a jelenség tehető felelőssé a Gal-1 késleltetett hatásáért. A Gal-1 monomer és dimer formában fordulhat elő, de nem egyértelmű, hogy melyik forma a fiziológiailag aktív . Ex vivo kísérletünkkel igazoltuk, hogy a monomer Gal-1, a dimerhez hasonló T-sejt apoptózist okoz, egy a szénhidrát kötő helytől független fehérje kötő motívumot feltételezve | Galectin-1 is considered as a novel immunosuppressive strategy in immune diseases. It is also a valuable marker in the prognosis of malignant tumors. The immunosuppressive effect of Gal-1 is attributed to its cytotoxic effect on activated T cells. Due to its immunoregulatory effect, its therapeutical application has been suugested. Therefore, in according to the aim of the project, we characterized the most detailed mechanism of the Gal-1 induced T-cell apoptosis. We resolved a long lasting conflict in the literature. In a comparative work it has been proved that Gal-1 acts as an apoptotic agent by different mechanism depending on its concentration. It has been clarified that the cell-derived or low concentration soluble Gal-1 represent the physiological function of this protein. It has also been determined that during cell-cell interaction, Lck and tyrosine phosphorylated proteins localize to the distal membrane region on a Gal-1 dependent fashion. Gal-1 is rapidly internalized via GM1 gangliozide and CD7 and this event may be responsible for the delayed apoptotic action of Gal-1. Gal-1 can occur in monomer and dimmer form. However it is not revealed which form represents the biologically active form. In ex vivo experiment we showed that the monomer Gal-1 is similarly effective in induction of T-cell apoptosis as the dimmer is. These results suggest that in addition to the sugar binding site Gal-1 also contain a protein binding motif

Genetics, molecular and cell biology of apoptotic cell death

Apoptotic cell death is an integral part of development and cell turnover in multicellular organisms. Since early 1970’s, when apoptosis was defined on morphological basis, plethora of genes has been identified participating in initiation, execution and regulation of cell death. This article reviews these latest advances and describes our present understanding of the sequential events of apoptotic cell death, from the early steps of death receptor initiated and mitochondrial pathways to activation of caspases, and finally, the proper corpse clearance. It also discusses dysregulation of apoptosis, leading to various pathologies, such as cancer, autoimmune disease and neurodegenerative disorders

Polyploid Adipose Stem Cells Shift the Balance of IGF1/IGFBP2 to Promote the Growth of Breast Cancer

Background: The close proximity of adipose tissue and mammary epithelium predispose involvement of adipose cells in breast cancer development. Adipose-tissue stem cells (ASCs) contribute to tumor stroma and promote growth of cancer cells. In our previous study, we have shown that murine ASCs, which undergo polyploidization during their prolonged in vitro culturing, enhanced the proliferation of 4T1 murine breast cancer cells in IGF1 dependent manner. Aims: In the present study, our aim was to clarify the regulation of ASC-derived IGF1. Methods: 4T1 murine breast carcinoma cells were co-transplanted with visceral fat-derived ASCs (vASC) or with the polyploid ASC.B6 cell line into female BALB/c mice and tumor growth and lung metastasis were monitored. The conditioned media of vASCs and ASC.B6 cells were subjected to LC-MS/MS analysis and the production of IGFBP2 was verified by Western blotting. The regulatory effect was examined by adding recombinant IGFBP2 to the co-culture of ASC.B6 and 4T1. Akt/protein kinase B (PKB) activation was detected by Western blotting. Results: Polyploid ASCs promoted the tumor growth and metastasis more potently than vASCs with normal karyotype. vASCs produced the IGF1 regulator IGFBP2, which inhibited proliferation of 4T1 cells. Downregulation of IGFBP2 by polyploidization of ASCs and enhanced secretion of IGF1 allowed survival signaling in 4T1 cells, leading to Akt phosphorylation. Conclusions: Our results implicate that ASCs in the tumor microenvironment actively regulate the growth of breast cancer cells through the IGF/IGFBP system

Novel role for galectin-1 in T-cells under physiological and pathological conditions

Secreted, extracellular galectin-1 (exGal-1) but not intracellular Gal-1 (inGal-1) has been described as a strong immunosuppressive protein due to its major activity of inducing apoptosis of activated T-cells. It has previously been reported that T-cells express Gal-1 upon activation, however its participation in T-cell functions has remained largely elusive. To determine function of Gal-1 expressed by activated T-cells we have carried out a series of experiments. We have shown that Gal-1, expressed in Gal-1-transgenic Jurkat cells or in activated T-cells, remained intracellularly indicating that Gal-1-induced T-cell death was not a result of an autocrine effect of the de novo expressed Gal-1. Rather, a particular consequence of the inGal-1 expression was that T-cells became more sensitive to exGal-1 added either as a soluble protein or bound to the surface of a Gal-1-secreting effector cell. This was also verified when the susceptibility of activated T-cells from wild type or Gal-1 knockout mice to Gal-1-induced apoptosis were compared. Murine T-cells expressing Gal-1 were more sensitive to the cytotoxicity of the exGal-1 than their Gal-1 knockout counterparts. We also conducted a study with activated T-cells from patients with systemic lupus erythematosus (SLE), a disease in which dysregulated T-cell apoptosis has been well described. SLE T-cells expressed lower amounts of Gal-1 than healthy T-cells and were less sensitive to exGal-1. These results suggested a novel role of inGal-1 in T-cells as a regulator of T-cell response to exGal-1, and its likely contribution to the mechanism in T-cell apoptosis deficiency in lupus

Licensing by Inflammatory Cytokines Abolishes Heterogeneity of Immunosuppressive Function of Mesenchymal Stem Cell Population

When mesenchymal stem cells (MSCs) are used for therapy of immunological pathologies, they get into an inflammatory environment, altering the effectiveness of the treatment. To establish the impact of environmental inflammatory factors on MSCs' immunofunction in the mirror of intrinsic heterogeneity of mouse MSC population, individual MSC clones were generated and characterized. Adipogenic but not osteogenic differentiation and pro-angiogenic activity of five independent MSC cell lines were similar. Regarding osteogenic differentiation, clones MSC3 and MSC6 exhibited poorer capacity than MSC2, MSC4, and MSC5. To study the immunosuppressive heterogeneity, in vitro and in vivo experiments have been carried out using T-cell proliferation assay and delayed-type hypersensitivity (DTH) response, respectively. A remarkable difference was found between the clones in their ability to inhibit T-cell proliferation in the following order: MSC2MSC5>MSC4>MSC3>>MSC6. Nevertheless, the differences between the immunosuppressive activities of the individual clones disappeared on pretreatment of the cells with pro-inflammatory cytokines, a procedure called licensing. Stimulation of all clones with IFN- and TNF- resulted in elevation of their inhibitory capability to a similar level. Nitric oxide (NO) and prostaglandin E2 (PGE2) were identified as major mediators of immunofunction of the MSC clones. The earlier findings were also supported by in vivo results. Without licensing, MSC2 inhibited DTH response, while MSC6 did not affect DTH response. In contrast, prestimulation of MSC6 with inflammatory cytokines resulted in strong suppression by this clone as well. Here, we have showed that MSC population is functionally heterogeneous in terms of immunosuppressive function; however, this variability is largely reduced under pro-inflammatory conditions

Identification of Galectin-1 as a Critical Factor in Function of Mouse Mesenchymal Stromal Cell-Mediated Tumor Promotion

Bone marrow derived mesenchymal stromal cells (MSCs) have recently been implicated as one source of the tumor-associated stroma, which plays essential role in regulating tumor progression. In spite of the intensive research, the individual factors in MSCs controlling tumor progression have not been adequately defined. In the present study we have examined the role of galectin-1 (Gal-1), a protein highly expressed in tumors with poor prognosis, in MSCs in the course of tumor development. Co-transplantation of wild type MSCs with 4T1 mouse breast carcinoma cells enhances the incidence of palpable tumors, growth, vascularization and metastasis. It also reduces survival compared to animals treated with tumor cells alone or in combination with Gal-1 knockout MSCs. In vitro studies show that the absence of Gal-1 in MSCs does not affect the number of migrating MSCs toward the tumor cells, which is supported by the in vivo migration of intravenously injected MSCs into the tumor. Moreover, differentiation of endothelial cells into blood vessel-like structures strongly depends on the expression of Gal-1 in MSCs. Vital role of Gal-1 in MSCs has been further verified in Gal-1 knockout mice. By administering B16F10 melanoma cells into Gal-1 deficient animals, tumor growth is highly reduced compared to wild type animals. Nevertheless, co-injection of wild type but not Gal-1 deficient MSCs results in dramatic tumor growth and development. These results confirm that galectin-1 is one of the critical factors in MSCs regulating tumor progression