Title ULOGA RECEPTORA NCR1 U PRIROĐENOM I STEČENOM IMUNOLOŠKOM ODGOVORU NA CITOMEGALOVIRUS Title (english) THE ROLE OF NCR1 RECEPTOR IN NAIVE AND ADAPTIVE IMMUNE RESPONSE TO CYTOMEGALOVIRUS Author Antonija Miletić Mentor Stipan Jonjić (mentor)Committee member Astrid Krmpotić (predsjednik povjerenstva)Committee member Danka Grčević (član povjerenstva)Committee member Ivica Pavić (član povjerenstva)Committee member Stipan Jonjić (član povjerenstva)Granter University of Rijeka Granter University of Rijeka Defense date and country 2019-12-12, Croatia Scientific / art field, BIOMEDICINE AND HEALTHCARE Universal decimal classificationUDC ) 61 - Medical sciences ThesaurusMESH - Medical Subject Headings )
Muromegalovirus
immunology
Herpesviridae Infections
immunology
Natural Cytotoxicity Triggering Receptor 1
Receptors, Natural Killer Cell
T-Lymphocytes
Mice, Inbred BALB C
Abstract Cilj istraživanja: Prirodnoubilačke stanice (engl. Natural killer cells, stanice NK) pripadaju stanicama prirođene imunosti i od iznimnog su značaja u kontroli velikog broja bakterijskih i virusnih infekcija. Novija istraživanja ukazuju na njihovu imunoregulacijsku ulogu prilikom formiranja specifičnog imunološkog odgovora. NCR1 je aktivacijski receptor izražen na stanicama NK. Cilj ovog istraživanja bio je ispitati ulogu receptora NCR1 u imunološkom nadzoru virusne infekcije mišjim citomegalovirusom (MCMV). Također, jedan od glavnih ciljeva bio je i istražiti mehanizme virusne regulacije staničnih liganada za navedeni receptor i karakterizirati virusne gene (proteine) odgovorne za taj učinak.
Materijali i metode: U ovom istraživanju uz kontrolne, BALB/c miševe, koristila sam i NCR1-deficijentne miševe (NCR1gfp/gfp) kako bi ispitala ulogu receptora NCR1 u nadzoru infekcije MCMV-om. Test virusnih čistina koristila sam kako bi odredila virusni titar u organima. Fenotipskom analizom pojedinih imunoloških stanica protočnom citometrijom pratila sam njihovo sazrijevanje i aktivacijski status tijekom infekcije. Kvalitetu formiranog MCMV-specifičnog humoralnog odgovora utvrdila sam na temelju rezultata dobivenih različitim testovima kao što su ELISA, ELISPOT, test aviditeta, neutralizacijski test kao i in vivo test protektivnog potencijala protutijela. Kako bi ispitala značaj virusne regulacije liganada za NCR1 u kontroli virusne infekcije, virusne gene uključene u regulaciju identificirala sam analizom izražaja NCR1-liganada na površini stanica B12 inficiranih različitim virusnim mutantama i divljim tipom virusa (engl. wild type, WT) koji je služio kao kontrola.
Rezultati: Fenotipska analiza stanica NK iz NCR1gfp/gfp miševa inficiranih MCMV-om ukazuje na promijenjeno sazrijevanje i aktivaciju analiziranih stanica. Također, izražaj receptora CXCR3 na stanicama NK smanjen je kod NCR1gfp/gfp miševa što za posljedicu ima lošiju migraciju stanica NK u medijastinalni limfni čvor (MLČ). Broj CD103+ migracijskih konvencionalnih dendritičkih stanica kao i broj efektorskih memorijskih CD4+ limfocita T u plućima inficiranih NCR1gfp/gfp miševa je također bio smanjen. Broj folikularnih pomagačkih CD4+ limfocita T (stanice Tfh) i limfocita B germinativnog centra analiziranih u MLČ inficiranih NCR1gfp/gfp miševa bio je značajno manji u usporedbi sa analiziranim stanicama u kontrolnim miševima što je rezultiralo smanjenim brojem limfocita B koji produciraju protutijela visokoafinitetna za MCMV.
Zaključak: U ovom istraživanju pokazala sam važnu ulogu receptora NCR1 u modulaciji humoralnog imunološkog odgovora posredovanoj stanicama NK. Narušena migracija ‘íscrpljenih’ stanica NK u regionalne limfne čvorove, negativno se odrazila na formiranje specifičnog imunološkog odgovora, rezultirajući lošijim sazrijevanjem stanica Tfh i konačno lošijom stimulacijom imunološkog odgovora posredovanog limfocitima B.
Abstract (english) Objectives: Natural killer cells are cytotoxic lymphocytes classified as a part of the innate immunity well known for their high importance in the early control of wide number of bacterial and virus infections. Recent studies suggest their indispensable immunoregulatory role during the formation of adaptive immune response. NCR1 is activating receptor expressed on the surface of NK cells Aim of this study is to investigate the role of NCR1 receptor in the immune surveillance of viral, MCMV infection. Also, one of the main aims of the study was to investigate the mechanisms which virus use to regulate expression of NCR1 ligands on infected cells and to characterize virus genes involved in this process.
Materials and Methods: In this study I used control, BALB/c mice and NCR1-deficient mice (NCR1gfp/gfp) to investigate the role of NCR1 receptor in the control of virus infection. Standard plaque assay was used to analyze virus titers in organs. I used phenotypic analysis of different immune cells by flow cytometry to monitor their maturation and activation status during infection. The quality of humoral response was assessed by different tests including ELISA, ELISPOT, avidity assay, neutralization assay and in vivo protective capacity assay. To determine the significance of virus regulation of the NCR1 ligand(s) expression for the in vivo control of virus infection, I identified the virus genes included in the downregulation of NCR1 ligands by infecting the NCR1-ligand expressing, B12 cell line with different virus mutants and wild type (WT) virus used as control.
Results: NK cells from MCMV-infected NCR1gfp/gfp mice showed altered maturation and activation profile in comparison to NK cells in control mice in all investigated organs. In addition, the expression of CXCR3 receptor was diminished in these mice leading to poor migration of NK cells to the mediastinal lymph nodes. The number of CD103+ migratory dendritic cells as well as the number of effector memory CD4 T cells in lungs of NCR1gfp/gfp infected mice was also lower. Finally, the number of Tfh cells and GC B cells found in MLN of infected NCR1gfp/gfp mice was significantly lower than in control resulting in reduced number of MCMV-specific antibody-secreting B cells and affinity-matured antibodies.
Conclusion: In this study I provide evidence for the important role of NCR1 in the NK cell-mediated modulation of the humoral immune response. Impaired migration of ‘exhausted’ NK cells to the regional lymph nodes, affected the development of the adaptive immune response, resulting in lower Tfh CD4 T cell maturation and ultimately inferior induction of B cell response.
Keywords
receptor NCR1
stanice NK
MCMV
virusna infekcija
imunoregulacija
CD4+ limfociti T
limfociti B
virus-specifična protutijela
ligandi za NCR1
Keywords (english)
NCR1 receptor
NK cells
MCMV
virus infection
immunoregulation
CD4 T cells
B cells
virus-specific antibodies
NCR1 ligands
Language croatian URN:NBN urn:nbn:hr:184:677781 Study programme Title: Biomedicine Postgraduate (doctoral) study programme Type of resource Text File origin Born digital Access conditions Access restricted to students and staff of home institution Terms of use Created on 2020-02-18 13:41:56
