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

Complementing antibody profiles: Assessing antibody function on antigen microarrays

Antibody effector functions other than neutralization depend on interactions with soluble and cellular components of the immune system. Antigen recognition is usually oligoclonal, with the different clones of antibodies belonging to different classes, subclasses, glycoforms and having different affinities and epitope specificities. Thus, composition of immune complexes determines biological effects mainly via interactions with FcR and complement proteins. Antibodies are capable of triggering any of the three pathways of complement activation and antigen recognition of complex antigens often results in the activation of more than one pathway. These events can be tracked in a multiplex format using antigen microarrays, where complement products bind to elements of the microarray. By controlling cation concentrations and detecting various complement components (C1q, C4, C3) contribution of the different pathways can be identified. Parallel measurement of antibodies and complement proteins provides a novel way of looking at interactions between antigen and antibodies. We propose the use of immune complex signatures, composite depictions of antibody and complement content of immune complexes characterizing healthy and diseased populations. Normalized interquartile ranges of antibody binding (IgM, IgG) and complement deposition (C4, C3) are projected onto radar charts to produce patterns that can distinguish normal and altered immune responses. We propose that comprehensive interaction studies of serum antibodies and complement with arrays of antigens can generate functional antibody profiles and help better understand immunological disease mechanism

Mannóz-kötő lektin és C1q szerepe dendritikus sejtek és makrofágok funkcióiban és immunregulációs folyamatokban = The role of mannose binding lectin (MBL) and Clq on various functions of dendritic cells and macrophages and on immunoregulation

Munkánk célja annak kiderítése volt, hogy az in vivo körülmények között IC-hez kapcsolódó C1q és MBL komplement fehérjék befolyásolják-e a dendritikus sejtek (DC-k) működését. A C1q-t erősen kötötték a sejtek, viszont az MBL nem kötődött A sejteket C1q-val fedett felszínen tenyésztettük, az C1q-val borított antigén felszínt modellezve ilyen módon. Az immobilizált C1q erősen stimulálta az MDC-k érését, amit a CD83, CD86 és MHCII membránfehérjék expresszió fokozódása jelzett. Ezeknek a molekuláknak az expressziója megközelítette az LPS által indukált mértéket. Kimutattuk, hogy az immobilizált C1q-val aktivált MDC-k fokozott allogén T-sejt proliferációt váltanak ki. A fokozás mértéke hasonló volt az LPS-kezelt MDC által kiváltott hatáshoz. Igazoltuk, hogy a C1q kötődése MDC-khez az NF-kB transzkripciós faktor magi transzlokációját váltja ki, és fokozott IL-12, IL-6, TNF-alfa és IL-10 szekréciót okoz. Kimutattuk, hogy az immobilizált C1q Th1 típusú választ indukál, mivel a C1q-aktivált MDC-k kokultúrában IFN-gamma termelő allogén T-sejteket stimulálnak. Jelen munkánkban bizonyítottuk, hogy az MDC-khez kötődő C1q képes fokozni az adaptív immunválaszt, ugyanakkor T-sejt toleranciát is indukálhat, az adott szöveti környezett által meghatározott módon. | The aim of our present study was to investigate whether additional components known to be associated with immune complexes (ICs);C1q and MBL formed in vivo also influence dendritic cell (DC) maturation. Monocyte derived DCs (MDC) strongly bind C1q but they do not interact with MBL. MDCs were cultured on immobilized C1q, in order to mimick the multimeric configuration presented on ICs. Cultivating of imMDCs on immobilized C1q leads to enhanced expression of CD83, CD86 and MHCII, almost to the same extent as in the presence of LPS. Our results also demonstrate that MDCs matured on immobilized C1q activate allogeneic T-cells to a similar extent as LPS-matured cells, pointing to a novel role for C1q and emphasizing the biological relevance of these findings in vivo. C1q binding causes NF-kB translocation to the nucleus in imMDCs, and that cultivating imMDCs on immobilized C1q results in elevated production of IL-12, IL-6 TNF-alfa and IL-10. We demonstrated that immobilized C1q directs a Th1 response, since allogeneic T cells co-cultured with C1q-matured DCs are IFN-gamma producers. In the present study we demonstrate that in addition to IgG, another serum protein, C1q, is also a potent maturation factor, which can induce phenotypic and functional changes in MDCs. Our data suggest that C1q could be a major signal for DCs to induce either an enhanced adaptive immune response or T cell tolerance, depending on their tissue environment

Az adaptív immunválasz kialakulása és szabályozása fiziológiás és kóros körülmények között: dendritikus sejtek, hízósejtek és a komplementrendszer szerepének vizsgálata = Regulation of adaptive immune responses under normal and pathological conditions; role of dendritic cells, mastocytes and the complement system

A magasabbrendű szervezetek immunhomeosztázisát a veleszületett és az adaptív immunrendszer együtt tartja fenn; egymással szorosan összefonódva, folyamatosan együttműködve. E két rendszer sejtes és molekuláris elemei - köztük a limfociták, makrofágok, dendritikus sejtek, hízósejtek, továbbá a komplementrendszer és a különböző citokinek - számos ponton hatnak egymásra úgy a fiziológiás, mint a kóros immunfolyamatok esetében. A pályázati munka során újabb kölcsönhatásokat írtunk le e két rendszer között, megismertük az egyes folyamatok molekuláris mechanizmusát, és a veleszületett immunrendszer egyes elemeink számos új funkcióját tártuk fel. Mindezzel jelentősen hozzájárultunk a veleszületett elemek adaptív választ befolyásoló ill. irányító hatásának további megismeréséhez. A pályázati munkába hét doktoranduszt vontunk be, akik közül öten már megszerezték a PhD fokozatot. Eredményeinket tizenkét, nemzetközi szaklapban megjelent publikációban közöltük; a cikkek - IF: 49.5. | The immunehomeostasis of higher vertebrates is maintained by the constant coordination and cooperation of the elements of the innate and adaptive immune system. Their cellular and molecular constituents - such as lymphocytes, macrophages, dendritic cells, mast cells, and the complement system and various cytokines - exert their effect on each other at several points under both physiological and pathological conditions. During the period of this research grant we could get further insight into the molecular mechanisms of these interactions; we learnt more about the possible ways how innate elements may influence or direct adaptive responses, moreover, we revealed some novel functions of certain elements. Seven doctoral students have been involved in this research - five of them already defended their thesis. 12 papers have been published in peer-reviewed journals; 'IF: 49.5

Characterization of factors influencing on-chip complement activation to optimize parallel measurement of antibody and complement proteins on antigen microarrays.

Binding of immunoglobulins and complement fragments to targets of adaptive immune responses can be monitored using collections of arrayed antigens and is used to generate profiles of antibody binding and function. The collection of reliable data on these reactions on a large scale requires the establishment of criteria from sample collection through reaction conditions to normalization strategies. We characterized the detection of IgG, complement C3 and C4 under conditions that better resemble in vivo events than most serological assays and are also relevant for in vitro diagnostic purposes. Immune complex formation was modeled using nitrocellulose-based protein arrays and the effects of factors like anticoagulant use, serum dilution, time and bivalent cation concentrations were assessed. Blood samples from healthy controls (n=24) and patients with systemic autoimmune disease (n=60) were collected and correlations between classical laboratory tests and chip-based reference proteins were evaluated to optimize normalization schemes. Best signal-to-noise ratio and acceptable masking of IgG by complement C3 fragments was achieved at modest, five to ten-fold serum dilutions. C3 binding to captured human IgG was found to correlate best with serum C3 concentrations and C3 activity and is therefore an ideal reference feature for normalization of biological and methodological variations in complement activity and detection

Anti-factor B autoantibody in dense deposit disease

Dense deposit disease (DDD), also known as membranoproliferative glomerulonephritis type II, is a rare kidney disorder that is associated with dysregulation of the alternative pathway of complement. Autoantibodies against the C3bBb convertase termed C3 nephritic factor are common in DDD patients. Here we report an autoantibody that binds to complement factor B in a DDD patient who was negative for C3 nephritic factor. This anti-factor B autoantibody recognized an epitope within the Bb fragment and was able to bind to the C3bBb convertase. Upon binding, the anti-factor B autoantibody stabilized the convertase against both intrinsic and factor H-mediated extrinsic decay and thus enhanced C3 consumption. Functional analyses demonstrated that, in contrast to C3 nephritic factor, the anti-factor B autoantibody inhibited complement-mediated lysis in vitro due to inhibition of the C5 convertase and the terminal complement pathway. Analysis of C5a plasma levels indicated that not all C5 convertases are inhibited by the autoantibodies in the patient in vivo. Antigen array experiments confirmed the presence of anti-factor B autoantibodies and also revealed complement activating anti-C1q antibodies in the patient's plasma. In summary, the present report describes a new autoantibody in DDD that binds to factor B and to the alternative pathway C3 convertase and alters the kinetics of complement activation and regulation. (C) 2010 Elsevier Ltd. All rights reserved

Serological and Genetic Evidence for Altered Complement System Functionality in Systemic Lupus Erythematosus: Findings of the GAPAID Consortium.

Systemic lupus erythematosus is a chronic autoimmune disease with multifactorial ethiopathogenesis. The complement system is involved in both the early and late stages of disease development and organ damage. To better understand autoantibody mediated complement consumption we examined ex vivo immune complex formation on autoantigen arrays. We recruited patients with SLE (n = 211), with other systemic autoimmune diseases (n = 65) and non-autoimmune control subjects (n = 149). Standard clinical and laboratory data were collected and serum complement levels were determined. The genotype of SNP rs1143679 in the ITGAM gene was also determined. Ex vivo formation of immune complexes, with respect to IgM, IgG, complement C4 and C3 binding, was examined using a functional immunoassay on autoantigen microarray comprising nucleic acids, proteins and lipids. Complement consumption of nucleic acids increased upon binding of IgM and IgG even when serum complement levels were decreased due to consumption in SLE patients. A negative correlation between serum complement levels and ex vivo complement deposition on nucleic acid autoantigens is demonstrated. On the contrary, complement deposition on tested protein and lipid autoantigens showed positive correlation with C4 levels. Genetic analysis revealed that the non-synonymous variant rs1143679 in complement receptor type 3 is associated with an increased production of anti-dsDNA IgG antibodies. Notwithstanding, homozygous carriers of the previously reported susceptible allele (AA) had lower levels of dsDNA specific IgM among SLE patients. Both the non-synonymous variant rs1143679 and the high ratio of nucleic acid specific IgG/IgM were associated with multiple organ involvement. In summary, secondary complement deficiency in SLE does not impair opsonization of nucleic-acid-containing autoantigens but does affect other antigens and potentially other complement dependent processes. Dysfunction of the receptor recognizing complement opsonized immune complexes promotes the development of class-switched autoantibodies targeting nucleic acids

Exacerbation of collagen induced arthritis by Fcγ receptor targeted collagen peptide due to enhanced inflammatory chemokine and cytokine production

Antibodies specific for bovine type II collagen (CII) and Fcγ receptors play a major role in collagen-induced arthritis (CIA), a mouse model of rheumatoid arthritis (RA). Our aim was to clarify the mechanism of immune complex-mediated inflammation and modulation of the disease. CII pre-immunized DBA/1 mice were intravenously boosted with extravidin coupled biotinylated monomeric CII-peptide epitope (ARGLTGRPGDA) and its complexes with biotinylated FcγRII/III specific single chain Fv (scFv) fragment. Disease scores were monitored, antibody titers and cytokines were determined by ELISA, and binding of complexes was detected by flow cytometry and immune histochemistry. Cytokine and chemokine secretion was monitored by protein profiler microarray. When intravenously administered into collagen-primed DBA/1 mice, both CII-peptide and its complex with 2.4G2 scFv significantly accelerated CIA and increased the severity of the disease, whereas the monomeric peptide and monomeric 2.4G2 scFv had no effect. FcγRII/III targeted CII-peptide complexes bound to marginal zone macrophages and dendritic cells, and significantly elevated the synthesis of peptide-specific IgG2a. Furthermore, CII-peptide containing complexes augmented the in vivo secretion of cytokines, including IL-10, IL-12, IL-17, IL-23, and chemokines (CXCL13, MIP-1, MIP-2). These data indicate that complexes formed by the CII-peptide epitope aggravate CIA by inducing the secretion of chemokines and the IL-12/23 family of pro-inflammatory cytokines. Taken together, these results suggest that the in vivo emerging immune complexes formed with autoantigen(s) may trigger the IL-12/23 dependent pathways, escalating the inflammation in RA. Thus blockade of these cytokines may be beneficial to downregulate immune complex-induced inflammation in autoimmune arthritis

Label-free Microfluidic Sensing by Detection of Interaction-triggered Change in Blood Flow Characteristics

Abstract Microfluidic devices exploit combined physical, chemical and biological phenomena that could be unique in the sub-millimeter dimensions. The blood is a non-Newtonian fluid, containing particulate and soluble elements, which penetrates the whole body carrying a wealth of biomedical information. The design of microfluidic devices capable of extracting immediately this information is the current goal of development Point-of-Care (POC) medical devices. We examined the characteristics of blood flow in specially designed microfluidic devices having different geometric structure and material composition with the aim of defining suitable conditions for sensitive detection of changes in the interactions between particulate elements of the blood and the adequately modified surfaces of the microfluidic system. As a model experiment we demonstrated the fast analysis of the AB0 blood group system, applying respective antibody reagents and capillary blood samples with different blood groups. We showed that by tuning the hydrophilicity of the surface and capillary dimensions of the microfluidic system it is possible to detect precisely the red blood cell binding to the capillary walls by monitoring the flow rate characteristics in an autonomous microfluidic system. Our proof-of-concept results point to a novel direction in blood analysis in autonomous microfluidic systems and also provide the basis for the construction of a simple quantitative device for blood group determination