Antigén-bemutató sejtek és polarizált TH-limfociták kapcsolata; kostimulációs kölcsönhatások és az immunológiai szinapszis = The antigen presenting cell - T cell contact; costimulation and the immunological synapse

Öt (1 TH1, 2 TH2 és 2 TH0) hosszútávon stabilnak bizonyult egér TH hibridómát állítottunk elő és jellemeztünk részletesen. Vizsgáltuk az IL-4, GATA-3, IFNgamma és Tbet génexpressziókat, a GM-CSF, IL-2, IL-4 és IFNgamma szekréciós képességet, a TH fenotípus stabilitását, az aktiválhatóság, a homing tulajdonság és a membrán raftok szempontjából jellemző sejtfelszíni markerek megjelenését, a kalcium-válasz kiválthatóságát és karakterisztikáját, a nyugvó és aktivációt követő fehérje foszforilációs mintázatot, valamint a membrán koleszterin tartalmának és a raftok mennyiségének összefüggéseit, továbbá a TCR raft asszociáltságát. Ezek alapján jellegzetes különbségeket mutattunk ki az eltérő fenotípusú TH sejtek között. Az antigén bemutató sejtek tulajdonságainak feltérképezése kapcsán különböző B-limfocita és makrofág eredetű sejtvonalak tulajdonságait vizsgáltuk a sejttípusra jellemző illetve az antigén bemutató kapacitás szempontjából kiemelt sejtfelszíni molekulák jelenléte és a sejt funkcionális képességei szempontjából. Megállapítottuk a membrán gangliozid (GM1, GM3) (raft)-mennyiség és az antigén bemutató valamint fagocita képesség összefüggését. A membránban a raft komponens koleszterin tartalmának vizsgálatához anti-koleszterin egér monoklonális ellenanyagokat fejlesztettünk ki, melyek nemcsak a sejtmentes és sejtes közegben történő koleszterin kimutatásra bizonyultak alkalmasnak hanem segítségükkel bizonyos koleszterin-függő sejtfunkciók modulálhatók. | Five (1 TH1, 2 TH2 és 2 TH0) mouse TH hybridomas with long-term stability were established and characterized in detail. IL-4, GATA-3, IFNgamma and Tbet gene expressions, GM-CSF, IL-2, IL-4 and IFNgamma secreting capacity, phenotypical stability, expression of cell surface markers involved in activation and homing processes or markers of the membrane rafts in correlation with the membrane cholesterol content, induction and characteristics of the calcium response, the protein tyrosine phosphorylation patterns before and after activation, and TCR recruitment into membrane rafts were investigated. Characteristic differences were found in most parameters among the TH cells of different effector phenotype. APC cell lines of B-cell and monocyte/macrophage origin were also characterized for their cell surface antigen expression in correlation with their functional features. A significant correlation was found between the raft marker membrane ganglioside (GM1, GM3) expression and the antigen presenting capacity or the phagocitic activity in macrophage cells. Cholesterol specific mouse monoclonal antibodies were generated to detect raft-associated and free cholesterol in the membrane. On the basis of our preliminary results these antibodies may also act directly as modulators of certain immune functions

Membrane nanotubes are ancient machinery for cell-to-cell communication and transport. Their interference with the immune system

Nanotubular connections between mammalian cell types came into the focus only two decades ago, when “live cell super-resolution imaging” was introduced. Observations of these long-time overlooked structures led to understanding mechanisms of their growth/withdrawal and exploring some key genetic and signaling factors behind their formation. Unbelievable level of multiple supportive collaboration between tumor cells undergoing cytotoxic chemotherapy, cross-feeding” between independent bacterial strains or “cross-dressing” collaboration of immune cells promoting cellular immune response, all via nanotubes, have been explored recently. Key factors and "calling signals" determining the spatial directionality of their growth and their overall in vivo significance, however, still remained debated. Interestingly, prokaryotes, including even ancient archaebacteria, also seem to use such NT connections for intercellular communication. Herein, we will give a brief overview of current knowledge of membrane nanotubes and depict a simple model about their possible “historical role”

A T-sejt aktiváció/celluláris immunválasz szabályozása: membrán mikrodomén-függő immunmoduláció és az ösztrogén közvetített nem-genomiális hormonális hatások vizsgálata = Regulation of the T-cell mediated immune response: investigation of membrane microdomain-dependent immunomodulation and estrogen-mediated nongenomial hormonal effects

A T49696 OTKA projekt (3 év) támogatásával elért főbb eredmények: Kimutattuk, hogy a sejtmembrán "tutajok" szfingolipid és koleszterin alkotóelemei fontos szabályozói a T sejtek aktiváció/sejthalál egyensúlyának, valamint a polarizált helper T sejtek válaszképességének. Megmutattuk ezen folyamatokban a ceramidok, a Kv és Cav ioncsatornák alapvető szerepét is. Az általunk leírt szignálintegrációs modell újabb immunmodulációs lehetőségeket kínál. Kimutattuk, hogy az ösztrogén steroid hormonok gyors, nem-genomiális jeleket indukálnak T és B limfocitákon (foszforiláció, szelektív kalcium szignál, stb.), egy még nem azonosított membránreceptoron keresztül, és fokozzák a T sejt-függő, antigén-indukált ellenanyagtermelést. Eredményeink elősegíthetik az ösztrogénszint és egyes autoimmunbetegségek közötti összefüggések hátterének mélyebb megértését. Új, koleszterin-specifikus IgG monoklonális ellenanyagokat (AC1, AC8) állítottunk elő, melyeknek megmutattuk celluláris-koleszterin diagnosztikai célokra történő alkalmazhatóságát. Ezen ellenanyagok képesek a HIV-infekció/ termelés gátlására monocita-makrofág és T sejteken in vitro, elsősorban a célsejtek plazmamembránjának (lipid tutajok, HIV receptorok) molekuláris átrendezése révén. A projekt eredményeit felhasználva 2 PhD tudományos fokozat született; 6 referált tudományos közlemény nemzetközi folyóiratban; 4 új kongresszusi absztrakt, melyekből folyamatos a publikálás (3 publikáció-kész közlemény) | Main results of the project T49696 supported by OTKA (3 years): We have shown that two lipid constituents of rafts (sphingolipids or cholesterol) are critical in regulating the balance of activation/cell death signaling in T cells or the functional responses of polarized Th1 or Th2 cells. Specific, important contribution of ceramides, Kv and Cav ion channels was also shown in these processes, offering new possibilites of immunomodulation. Rapid, non-genomial signals (selective phosphorylation and Ca2+ signals or NFκB nuclear translocation) of estrogen steroid hormones have been shown in both T and B lymphocytes, that may be partly responsible for augmentation of the T-dependent humoral immune response observed in in vivo mice studies. These effects, mediated by a yet unidentified E2 membrane receptor, may help us to reveal and understand the relationship between estrogen levels and several autoimmune diseases. We generated novel cholesterol-specific IgG antibodies (AC1 and AC8), that may serve as diagnostic markers of clustered cholesterol (cell-free or cellular). As one of their most intriguing biological activity - inhibition of HIV entry/production in vitro - has been elucidated and shown that the major mechanism of action is remodeling of the target cells' plasma membrane (rafts and HIV receptors). 2 PhD degrees were received based on these results; 6 reviewed research articles; 4 new Congress Abstracts (publication is continuous: 3 publication-ready manuscripts

Jelátviteli kompartmentek és proteolózis az immunválasz, valamint az immun- és idegrendszer közötti kommunikáció szabályozásában = Signaling compartments and proteolysis in regulation of immune responses and the communication between the immune- and nervous systems

Több új immunreceptor-kölcsönhatást (FcR/CR/BCR; sejthalál-R/TCR), és több szabályozó mechanizmust azonosítottunk a lipid raftok és más membrán mikro-kompartmentek, valamint limfocita adaptor fehérjék részéről, melyek fontosak a limfociták effektor funkcióiban ill. a sejthalál folyamatában. Leírtuk az utóbbi folyamatokban kritikus miozin motorfehérje izotípusok néhány molekuláris kapcsolatának szerkezeti hátterét és szabályozási lehetőségét. Kimutattuk egyes komplement (C) faktorok és receptorok sokrétű szerepét az immunválasz szabályozásában és, hogy a belőlük származtatott peptidekkel az allergiás hízósejtválasz gátolható. Leírtuk a C-rendszer szabályozó szerepét a Sclerosis Multiplex (SM) állatmodelljében is. Kimutattuk a tripszin-szerű proteáz aktivitás szabályozó szerepét a B-sejt válaszban, és valószínűsítettük szerepüket a mielin bázikus fehérje specifikus hasításában, mely fontos eleme lehet az SM kialakulásának. Eredményeink alapját képezik szelektív immunmodulánsok kifejlesztésének autoimmun ill. allergiás kórképekben. Kimutattunk egyes immunrendszeri effektor- és idegrendszeri funkciók között fellépő, a citokin hálózat ill. steroid hormonok (ösztrogének, glukortikoidok) útján megvalósuló kommunikációs útvonalakat. Új módszereket is kifejlesztettünk (pl. antigén-targetingre alkalmas egyláncú ellenanyag konstrukciók) és a projekt támogatásával beszereztünk egy konfokális mikroszkópot, melyen több új metodikát optimalizáltunk a celluláris kommunikáció vizsgálatára. | We identified several novel immunoreceptor cross-talk elements (FcR/CR/BCR; cell death-R/TCR) and regulatory mechanisms by lipid rafts, other membrane-compartments, as well as adaptor proteins, which are essential in the lymphocytes' effector functions and cell death. Structural and regulatory aspects of some important molecular interactions of myosin motor protein isotypes, essential players in the above processes, were also described. Multiple regulatory functions of the complement system in the immune response and the inhibitory potential of C3a-derived peptides in the allergic mast cell response were identified. The regulatory potential of the complement system was also shown in an animal model of Sclerosis Multiplex (SM). We have shown that trypsin-like protease activities are involved the B cell response through formation of soluble receptors and that trypsin-4, specifically cleaving MBP, may be a critical element in development of SM. Our results form a basis for development of selective immunomodulators for autoimmune- and allergic diseases. We explored several novel communication pathways between effector mechanisms of the immune system and certain nervous system functions, through the inflammatory cytokine network and steroid hormones. New methods and molecular constructs (e.g. antigen-targeting by engineered single-chain antibodies) and several modern confocal imaging techniques for studying cellular communication were also developed with the support of the grant

Stromal Cells Serve Drug Resistance for Multiple Myeloma via Mitochondrial Transfer: A Study on Primary Myeloma and Stromal Cells

SIMPLE SUMMARY: Mitochondrial transfer plays a crucial role in the acquisition of drug resistance in multiple myeloma, but its exact mechanism is not yet clear; moreover, overcoming the drug resistance that it causes is also a major challenge. Our research on primary myeloma cell cultures reveals that mitochondrial transfer is bi-directional between bone marrow stromal cells and myeloma cells, occurring via tunneling nanotubes and partial cell fusion with extreme increases under the influence of chemotherapeutic drugs, whereupon survival and adenosine triphosphate levels increase, while mitochondrial superoxide levels decrease in myeloma cells. These changes and the elevation of superoxide levels in stromal cells are proportional to the amount of incorporated mitochondria derived from the other cell type and to the concentration of the used drug. Although the inhibition of mitochondrial transfer is limited between stromal and myeloma cells, the supportive effect of stromal cells can be effectively averted by influencing the tumor metabolism with an inhibitor of oxidative phosphorylation in addition to chemotherapeutics. ABSTRACT: Recently, it has become evident that mitochondrial transfer (MT) plays a crucial role in the acquisition of cancer drug resistance in many hematologic malignancies; however, for multiple myeloma, there is a need to generate novel data to better understand this mechanism. Here, we show that primary myeloma cells (MMs) respond to an increasing concentration of chemotherapeutic drugs with an increase in the acquisition of mitochondria from autologous bone marrow stromal cells (BM-MSCs), whereupon survival and adenosine triphosphate levels of MMs increase, while the mitochondrial superoxide levels decrease in MMs. These changes are proportional to the amount of incorporated BM-MSC-derived mitochondria and to the concentration of the used drug, but seem independent from the type and mechanism of action of chemotherapeutics. In parallel, BM-MSCs also incorporate an increasing amount of MM cell-derived mitochondria accompanied by an elevation of superoxide levels. Using the therapeutic antibodies Daratumumab, Isatuximab, or Elotuzumab, no similar effect was observed regarding the MT. Our research shows that MT occurs via tunneling nanotubes and partial cell fusion with extreme increases under the influence of chemotherapeutic drugs, but its inhibition is limited. However, the supportive effect of stromal cells can be effectively avoided by influencing the metabolism of myeloma cells with the concomitant use of chemotherapeutic agents and an inhibitor of oxidative phosphorylation

Some new faces of membrane microdomains: A complex confocal fluorescence, differential polarization, and FCS imaging study on live immune cells

Lipid rafts are cholesterol- and glycosphingolipid-rich plasma membrane microdomains, which control signal transduction, cellular contacts, pathogen recognition, and internalization processes. Their stability/lifetime, heterogeneity remained still controversial, mostly due to the high diversity of raft markers and cellular models. The correspondence of the rafts of living cells to liquid ordered (Lo) domains of model membranes and the effect of modulating rafts on the structural dynamics of their bulk membrane environment are also yet unresolved questions. Spatial overlap of various lipid and protein raft markers on live cells was studied by confocal laser scanning microscopy, while fluorescence polarization of DiIC18(3) and Bodipy-phosphatidylcholine was imaged with differential polarization CLSM (DP-CLSM). Mobility of the dil probe under different conditions was assessed by fluorescence correlation spectroscopic (FCS). GM1 gangliosides highly colocalized with GPI-linked protein markers of rafts and a new anti -cholesterol antibody (AC8) in various immune cells. On the same cells., albeit not fully excluded from rafts, diI colocalized much less with raft markers of both lipid and protein nature, suggesting the Lo membrane regions are not equivalents to lipid rafts. The DP-CLSM technique was capable of imaging probe orientation and heterogeneity of polarization in the plasma membrane of live cells, reflecting differences in lipid order/packing. This property-in accordance with dil mobility assessed by FCS-was sensitive to modulation of rafts either through their lipids or proteins. Our complex imaging analysis demonstrated that two lipid probes-G(M1) and a new anti-cholesterol antibody-equivocally label the membrane rafts on a variety of cell types, while some raft-associated proteins (MHC-II, CD48, CD59, or CD90) do not colocalize with each other. This indicates the compositional heterogeneity of rafts. Usefulness of the DP-CLSM technique in imaging immune cell surface, in terms of lipid order/packing heterogeneities, was also shown together with its sensitivity to monitor biological modulation of lipid rafts. (c) 2007 International Society for Analytical Cytology

Sex hormone-binding globulin provides a novel entry pathway for estradiol and influences subsequent signaling in lymphocytes via membrane receptor

The complex effects of estradiol on non-reproductive tissues/cells, including lymphoid tissues and immunocytes, have increasingly been explored. However, the role of sex hormone binding globulin (SHBG) in the regulation of these genomic and non-genomic actions of estradiol is controversial. Moreover, the expression of SHBG and its internalization by potential receptors, as well as the influence of SHBG on estradiol uptake and signaling in lymphocytes has remained unexplored. Here, we found that human and mouse T cells expressed SHBG intrinsically. In addition, B lymphoid cell lines as well as both primary B and T lymphocytes bound and internalized external SHBG, and the amount of plasma membrane-bound SHBG decreased in B cells of pregnant compared to non-pregnant women. As potential mediators of this process, SHBG receptor candidates expressed by lymphocytes were identified in silico, including estrogen receptor (ER) alpha. Furthermore, cell surface-bound SHBG was detected in close proximity to membrane ERs while highly colocalizing with lipid rafts. The SHBG-membrane ER interaction was found functional since SHBG promoted estradiol uptake by lymphocytes and subsequently influenced Erk1/2 phosphorylation. In conclusion, the SHBG-SHBG receptor-membrane ER complex participates in the rapid estradiol signaling in lymphocytes, and this pathway may be altered in B cells in pregnant women

Nanotubes connecting B lymphocytes: High impact of differentiation-dependent lipid composition on their growth and mechanics

Nanotubes (NTs) are thin, long membranous structures forming novel, yet poorly known communication pathways between various cell types. Key mechanisms controlling their growth still remained poorly understood. Since NT-forming capacity of immature and mature B cells was found largely different, we investigated how lipid composition and molecular order of the membrane affect NT-formation. Screening B cell lines with various differentiation stages revealed that NT-growth linearly correlates with membrane ganglioside levels, while it shows maximum as a function of cholesterol level. NT-growth of B lymphocytes is promoted by raftophilic phosphatidylcholine and sphingomyelin species, various glycosphingolipids, and docosahexaenoic acid-containing inner leaflet lipids, through supporting membrane curvature, as demonstrated by comparative lipidomic analysis of mature versus immature B cell membranes. Targeted modification of membrane cholesterol and sphingolipid levels altered NT-forming capacity confirming these findings, and also highlighted that the actual lipid raft number may control NT-growth via defining the number of membrane-F-actin coupling sites. Atomic force microscopic mechano-manipulation experiments further proved that mechanical properties (elasticity or bending stiffness) of B cell NTs also depend on the actual membrane lipid composition. Data presented here highlight importance of the lipid side in controlling intercellular, nanotubular, regulatory communications in the immune system