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001-es BibID:	BIBFORM004836
Első szerző:	Gombos Imre
Cím:	Rafting MHC-II domains in the APC (presynaptic) plasma membrane and the thresholds for T-cell activation and immunological synapse formation / Gombos, I., Detre, C., Vamosi, G., Matko, J.
Dátum:	2004
Megjegyzések:	Glycosphingolipid- and cholesterol-rich membrane microdomains (rafts) in T-cells are important in triggering and regulation of T(H)-cell activation in immunological synapses (IS), which in turn may control the T-cell repertoire in lymph nodes and at the periphery. It is less known, however, how the "presynaptic side" controls formation and function of IS. We investigated here activation signals and synapse formation frequency of murine IP12-7 T(H) hybridoma cell specific to influenza virus HA-peptide upon stimulation with two B-lymphoma cells, A20 and 2PK3, pulsed with peptide antigen. Confocal microscopic colocalization and FRET data consonantly revealed clustered distribution and constitutive raft-association of a major fraction of MHC-II molecules in both APCs. Costimulatory molecules (CD80 and CD86), not associated constitutively with rafts, were expressed at much lower level in A20 cells. T-cells responded to 2PK3 APC with much higher signal strength than to A20 cells, in good correlation with the frequency of IS formation, as assessed by microscopic conjugation assay. Disruption of rafts by cholesterol depletion in 2PK3 cells largely decreased the magnitude of T(H) cell activation signals, especially at low peptide antigen doses, similarly to masking CD4 with mAb on T-cells. The frequency of IS formation was reduced by blocking LFA-1 on T-cells and CD80 on APCs, by lowering the temperature below the phase transition of the membrane or by disrupting actin cytoskeleton. These data together suggest that the surface density and affinity/stability of peptide-MHC-II complexes and the costimulatory level are primary determinants for an efficient TCR recognition and the strength of the subsequent T-cell signals, as well as of the IS formation, which additionally requires a cytoskeleton-dependent remodeling of APC surface after the initial TCR signal. The threshold of T-cell activation can be further set by rafting MHC-II domains via concentrating high affinity ligands and promoting thereby T-cells for sensing low density antigen. Our data also demonstrate that B-cells, similarly to dendritic cells, could also provide T-cells with antigen-independent weak survival signals, likely associated with integrin engagement.
Tárgyszavak:	Orvostudományok Elméleti orvostudományok idegen nyelvű folyóiratközlemény külföldi lapban Animals Antigen-Presenting Cells Antigens Cell Membrane Cells Cholesterol Cytoskeleton Dendritic Cells Histocompatibility Antigens Histocompatibility Antigens Class II Hungary immunology Ligands Lymph Nodes Lymphocyte Activation Lymphoma,B-Cell Membrane Microdomains Mice Peptide Fragments Research Support Synapses T-Lymphocytes Temperature Time Factors Tumor Cells,Cultured ultrastructure
Megjelenés:	Immunology Letters. - 92 : 1-2 (2004), p. 117-124. -
További szerzők:	Detre, Cynthia Vámosi György (1967-) (biofizikus) Matkó János (1952-) (biológus)
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001-es BibID:	BIBFORM005182
Első szerző:	Gombos Imre
Cím:	Some new faces of membrane microdomains : a complex confocal fluorescence, differential polarization, and FCS imaging study on live immune cells / Gombos, I., Steinbach, G., Pomozi, I., Balogh, A., Vamosi, G., Gansen, A., Laszlo, G., Garab, G., Matko, J.
Dátum:	2008
ISSN:	552-4930 (Electronic)
Megjegyzések:	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 diI 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 diI 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.
Tárgyszavak:	Orvostudományok Elméleti orvostudományok idegen nyelvű folyóiratközlemény külföldi lapban analysis Animals Antibodies article Cell Line,Tumor Cells Cells,Cultured chemistry Cholesterol Comparative Study Confocal Fluorescence Fluorescence Polarization Humans Hungary immunology Lipids Membrane Microdomains methods Mice Mice, Inbred BALB C Mice, Inbred C3H Microscopy Microscopy, Confocal Microscopy, Fluorescence Microscopy, Polarization Proteins Rats Rats, Sprague-Dawley Research Research Support Signal Transduction Spectrometry, Fluorescence Support
Megjelenés:	Cytometry. Part A. - 73 : 3 (2008), p. 220-229. -
További szerzők:	Steinbach Gábor Pomozi István Balogh Andrea Vámosi György (1967-) (biofizikus) Gansen, Alexander László Glória Garab Győző Matkó János (1952-) (biológus)
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