Title ULOGA RECEPTORA NKG2D U RAZVOJU, HOMEOSTAZI I EFEKTORSKIM FUNKCIJAMA LIMFOCITA B Title (english) THE ROLE OF NKG2D IN DEVELOPMENT, HOMEOSTASIS AND EFFECTOR FUNCTIONS OF B LYMPHOCYTES Author Maja Lenartić Mentor Bojan Polić (mentor)Committee member Stipan Jonjić (predsjednik povjerenstva)Committee member Mirela Baus Lončar (član povjerenstva)Committee member Igor Jurak (član povjerenstva)Committee member Bojan Polić (član povjerenstva)Granter University of Rijeka Defense date and country 2017-05-05, Croatia Scientific / art field, BIOMEDICINE AND HEALTHCARE Universal decimal classificationUDC ) 61 - Medical sciences Abstract Cilj istraživanja: Naša prethodna istraživanja su pokazala da nedostatak receptora NKG2D utječe na razvoj stanica NK, ali i na promjene u brojnosti limfocita B. Glavni cilj ovoga istraživanja bio je razjasniti na koji način i u kojoj fazi nedostatak receptora NKG2D utječe na razvoj i homeostazu limfocita B te ima li to za posljedicu promjene u njihovoj funkciji
Materijali i metode: Protočnom citometrijom sam utvrdila koje su B-limfocitne podpopulacije najviše pogođene NKG2D-deficijencijom. Kako bih utvrdila na koji način NKG2D-deficijencija utječe na brojnost limfocita B koristila sam se adoptivnim transferima te modelima kimeričnih miševa miješane koštane srži ili fetalne jetre. U radu sam koristila genetski modificirane miševe kako bih ispitala u kojoj fazi razvoja nedostatak NKG2D-a najviše utječe na razvoj limfocita B i koje su komponente NKG2D-signalizirajućeg puta odgovorne za isto. Različitim funkcionalnim testovima, poput ELISA i ELISPOT, ispitala sam proizvodnju protutijela od strane limfocita B1a i B2. Također, kako bih ispitala kliničku važnost učinka NKG2D-deficijencije na humoralnu imunost miševe sam inficirala F. novicidom ili ih podvrgla sepsi uzrokovanoj crijevnim bakterijama.
Rezultati: Nedostatak izražaja receptora NKG2D rezultira smanjenjem brojnosti limfocita B1a u slezeni i seroznim šupljinama te redistribucijom limfocita B2 između slezene i drugih limfnih tkiva. NKG2D-deficijencija ne utječe na preživljenje, homeostatsku proliferaciju ili udomljavanje limfocita B1a. Ipak, uočeno udomljavanje Klrk1-/- stanica B2 u većoj mjeri u ekstrasplenična tkiva mogao bi biti uzrok smanjenog broja limfocita B2 u slezeni i porasta njihova broja u peritonealnoj šupljini te timusu. Smanjena brojnost limfocita B1a je posljedica nedostatka receptora NKG2D u ranim fazama razvoja limfocita B, najvjerojatnije prije stadija zajedničkog limfocitnog prekursora (CLP). Klrk1-/- hematopoetske stanice imaju smanjenu sposobnost stvaranja limfocita B1a u usporedbi s kontrolnim stanicama. Redistribucija limfocita B2 ne utječe na jačinu humoralnog odgovora ovisnog o limfocitima T. U peritoneumu NKG2D-deficitnih miševa višestruko je smanjen broj stanica koje izlučuju prirodna protutijela. Smanjena brojnost limfocita B1a kao i njihova poremećena funkcionalnost utječe na jačinu humoralnog odgovora tipa 2 neovisnog o limfocitima T (TI-2). To ima za posljedicu visoku osjetjivost NKG2D-deficitnih miševa na bakterijske infekcije i visoku podložnost sepsi.
Zaključak: Rezultati ovog istraživanja su pokazali da jedan važan NK-stanični aktivacijski receptor specifično utječe na razvoj limfocita B, točnije na urođene limfocite B1a. Osim već poznate uloge ovoga receptora u razvoju stanica NK, istraživanje ukazuje na njegovu dodatnu ulogu u razvoju drugih populacija limfocita urođene imunosti. Također, ovaj rad po prvi put pokazuje direktnu vezu između NKG2D-deficijencije i promijenjene funkcionalnosti limfocita B1a.
Abstract (english) Objectives: Our group has previously shown that NKG2D plays an important role in the development of NK cells, but also influences numbers of B cells in the spleen. The main goal of this research was to investigate how NKG2D affects B cell numbers in the bone marrow and periphery. In addition, I aimed to investigate when in the course of B cell development NKG2D plays a major role. Finally, I investigated whether NKG2D has any impact on homing and B cell functions.
Materials and methods: Flowth cytometry was used to investigate which B cell subpopulations are mostly affected by NKG2D-deficiency. Adoptive transfers and mixed bone marrow or fetal liver chimeras were done to investigate the mechanism by which NKG2D-deficiency affects B cell numbers. To investigate in which stage of development is NKG2D involved in skewing of B1a cells I used many different genetically modified mouse strains. Different functional tests like ELISA and ELISPOT were used to investigate the functionality of Klrk1-/- B cells. To investigate clinical impact of NK2GD-deficiency, Klrk1-/- and control mice were infected with F. novicida or were subjected to cecum ligation and puncture procedure to induce sepsis.
Results: The lack of NKG2D results in low B1a cell numbers in spleen and serous cavities, and redistribution of B2 cells between the spleen and other lymphoid organs. NKG2D-deficiency does not affect survival, homeostatic proliferation or homing of B1a cells. It does, however, affect the homing of B2 cells which results in lower number of B2 cells in the spleen and increase of B2 cells numbers in extrasplenic tissues such as peritoneal cavity or thymus. Reduced numbers of B1a lymphocytes are result of NKG2D-deficiency in the earliest stages of B cell development, probably at the level or preceding CLP stage. The B1a cell formation capacity from Klrk1-/- precursors is significantly lower than from Klrk1+/+. On functional level, I noticed that redistribution of B2 cells does not affect the range of TD humoral response. However, reduced numbers of B1a cells as well as their impaired functionality has significant impact on the strenght of TI-2 humoral responses rendering NKG2D-deficient mice very sensitive to F. novicida infection and highly susceptible to experimentally-induced sepsis.
Conclusion: This research shows for the first time that an NK cell activating receptor influences the development of B cells, more precisely, innate-like B1a cells. This research presents an additional proof to support the regulatory role of NKG2D. The regulatory role of NKG2D can therefore be extended to the development of innate-like B cells. In addition, my doctoral thesis shows that the lack of NKG2D results with the impaired funcionality of B1a lymphocytes.
Keywords
B1a
limfociti
F. novicida
NKG2D
receptor
protutijela
prirodna
razvoj
sepsa
bakterijska.
Keywords (english)
antibodies
natural
development
F. novicida
lymphocytes
B1a
receptor
NKG2D
sepsis
Language croatian URN:NBN urn:nbn:hr:184:568017 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 Repository Repository of the University of Rijeka, Faculty of Medicine Created on 2022-03-17 12:27:16
