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  Human basophils as antigen presenting cells?: development of a co-culture system and functional studies
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 Ophav:
Poulsen, Britta Cathrina1, Forfatter
Skov, Søren1, Vejleder
Poulsen, Lars K.1, Vejleder
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1Det Biovidenskabelige Fakultet, København, Danmark, diskurs:7002              
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 Abstract: AbstractMore than 10% of the European population is now considered to suffer from at least one type of allergic disease. Even though, the immunological events causing allergic disease have been studied for decades the immunological response behind the development of allergy has still not been unraveled. So far the dendritic cell has been regarded as the antigen presenting cell responsible for initiating the allergic response. However, three articles published in nature in July 2009, showed that the mouse basophil could function as an antigen presenting cell and induce an allergic Th2 response in mice.The aim of this thesis was to increase the understanding of the early development of an allergic reaction, by studying the capacity of the human basophil to function as antigen presenting cells, including comparison of its ability to induce proliferation in CD4+ T cells to that induced by a DC precursor the monocyte. To accomplish this, suitable methods for purifying CD4+ T cells, basophils, and monocytes neededto be studied, so that co-cultures could be established.Furthermore, for the human basophil to function as an antigen presenting cell it needs to posses certain qualities: it must be able to take up antigen, process it, and present it on MHC class II molecules to CD4+ T cells. Hence, the other part of this thesis was to investigate whether human basophils possess some of the abilities essential to an antigen presenting cells.Experiments were performed in vitro using buffy coat derived human peripheral blood mononuclear cells from which basophils, CD4+ T cells, and monocytes were isolated. The function of human basophils as antigen presenting cells was assessed by their ability to induce proliferation of CFSE stained CD4+ T cells in the presence of antigen, and the proliferation was measured by flow cytometry. With the focus on developing a co-culture system that could be used to study the function of human basophils as antigen presenting cells, suitable methods for purifying basophils, CD4+ T cells, and monocytes, were established. Human basophils failed to induce proliferation of CD4+ T cells, when co-cultured. However, it was found that freshly isolated human basophils expressed MHC class II molecules, and that they can take up antigen in presence of IL-3, both traits essential for antigen presenting cells. Since the uptake of antigen was dependent on IL-3 this might explain, why the basophils in the co-culture were not able to induce proliferation of CD4+ T cells under natural conditions. During this thesis, a co-culture system has been developed that can be used as a frame for studying the function of basophils as antigen presenting cells, and in addition it was shown that freshly isolated human basophils can express HLA-DR, and can take up antigen in presence of IL-3.
 Abstract: Over 10 % af Europas befolkning lider nu af en eller flere allergiske sygdomme så som høfeber og fødevareallergi. Selv om der i årtier er blevet forsket i det immunologiske respons, som fører til udviklingen af allergiske sygdomme, vides det stadig ikke, hvordan folk udvikler allergi.Hidtil er dendritiske celler blevet betragtet som den gruppe af antigen præsenterende celler, der igangsætter det allergiske respons. Imidlertid har tre artikler, publiceret i Nature i juli 2009, vist at basofile celler i mus kan fungere som antigen præsenterende celler og fremkalde et allergisk Th2 respons hos mus.Formålet med dette speciale var at øge forståelsen for, hvordan et allergisk respons udvikles, ved at undersøge humane basofile cellers evne til at fungere som antigen præsenterende celler, og at sammenligne deres egenskaber til at inducere et proliferativt respons i CD4+ T celler med et respons induceret af en forløber for den dendritiske celle, monocytten. For at kunne studere dette blev forskellige metoder til at oprense basofile celler, CD4+ T celler og monocytter undersøgt, med henblik på etablering af co-kulturer. For at kunne fungere som antigen præsenterende celler skal humane basofile celler besidde bestemte egenskaber. De skal være i stand til at optage og forarbejde antigen, samt kunne præsentere antigen på MHC klasse II molekyler til CD4+ T celler. Derfor omhandlede en del af specialet en undersøgelse af, hvorvidt de human basofile celler besidder de evner, som er nødvendige for at kunne fungere som antigen præsenterende celler.Eksperimenter blev udført in vitro med buffy coats, hvorfra humane perifere blod mononukleære celler blev oprenset for videre at kunne oprense basofile, CD4+ T celler samt monocytter fra disse. Hvorvidt de humane basofile celler fungerede som antigen præsenterende, blev vurderet ud fra deres evne til at inducere et proliferativt respons i CFSE farvede CD4+ T celler ved tilstedeværelse af antigen. Det proliferative respons blev målt vha. flow cytometri. Med fokus på at udvikle et co-kultur system, der kunne bruges til at undersøge funktionen af de humane basofile som antigen præsentere celler, blev hensigtsmæssige metoder til oprensning af basofile celler, CD4+ T-celler, og monocytter etableret.Det mislykkedes at få de human basofile til at inducere proliferation i CD4+ T celler. Til gengæld blev det fundet at humane basofile celler udtrykker MHC klasse II molekyler, og at de kan optage antigen under tilstedeværelse af IL-3. Da optag af antigen viste sig at være afhængigt af IL-3 kan dette måske forklare, hvorfor de humane basofile celler ikke inducerede proliferation i de CD4+ T celler under neutrale forhold.Under dette speciale er en co-kultur til at studere de humane basofile cellers egenskaber som antigen præsenterende celler blevet udviklet. Derudover er det blevet vist at nyoprensede humane basofile celler udtrykker MHC klasse II molekyler og at de kan optage antigen, hvis IL-3 er tilstede.
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Bogmærk denne post: https://diskurs.kb.dk/item/diskurs:40005:1
 Type: Speciale
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Detaljer

Sprog: English - eng
 Datoer: 2010
 Sider: 84 pages
 Publiceringsinfo: København, Danmark : Institut for Veterinær Sygdomsbiologi
 Indholdsfortegnelse: -
 Note: Biology - Biotechnology, Biologi - Bioteknologi, Final semester, Afsluttende semester
 Type: Speciale
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