prikaz prve stranice dokumenta Odgovor CD8 limfocita T u miševa cijepljenih rekombinantnim citomegalovirusnim vektorom koji izražava NKG2D ligand RAE-1γ
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doctoral thesis
Odgovor CD8 limfocita T u miševa cijepljenih rekombinantnim citomegalovirusnim vektorom koji izražava NKG2D ligand RAE-1γ
2022. urn:nbn:hr:184:647874
Šustić, Marko
University of Rijeka
Faculty of Medicine
Department of Histology and Embryology

Cite this document

Šustić, M. (2022). Odgovor CD8 limfocita T u miševa cijepljenih rekombinantnim citomegalovirusnim vektorom koji izražava NKG2D ligand RAE-1γ (Doctoral thesis). Rijeka: University of Rijeka, Faculty of Medicine. Retrieved from https://urn.nsk.hr/urn:nbn:hr:184:647874

Šustić, Marko. "Odgovor CD8 limfocita T u miševa cijepljenih rekombinantnim citomegalovirusnim vektorom koji izražava NKG2D ligand RAE-1γ." Doctoral thesis, University of Rijeka, Faculty of Medicine, 2022. https://urn.nsk.hr/urn:nbn:hr:184:647874

Šustić, Marko. "Odgovor CD8 limfocita T u miševa cijepljenih rekombinantnim citomegalovirusnim vektorom koji izražava NKG2D ligand RAE-1γ." Doctoral thesis, University of Rijeka, Faculty of Medicine, 2022. https://urn.nsk.hr/urn:nbn:hr:184:647874

Šustić, M. (2022). 'Odgovor CD8 limfocita T u miševa cijepljenih rekombinantnim citomegalovirusnim vektorom koji izražava NKG2D ligand RAE-1γ', Doctoral thesis, University of Rijeka, Faculty of Medicine, accessed 19 November 2024, https://urn.nsk.hr/urn:nbn:hr:184:647874

Šustić M. Odgovor CD8 limfocita T u miševa cijepljenih rekombinantnim citomegalovirusnim vektorom koji izražava NKG2D ligand RAE-1γ [Doctoral thesis]. Rijeka: University of Rijeka, Faculty of Medicine; 2022 [cited 2024 November 19] Available at: https://urn.nsk.hr/urn:nbn:hr:184:647874

M. Šustić, "Odgovor CD8 limfocita T u miševa cijepljenih rekombinantnim citomegalovirusnim vektorom koji izražava NKG2D ligand RAE-1γ", Doctoral thesis, University of Rijeka, Faculty of Medicine, Rijeka, 2022. Available at: https://urn.nsk.hr/urn:nbn:hr:184:647874

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Metadata
TitleOdgovor CD8 limfocita T u miševa cijepljenih rekombinantnim citomegalovirusnim vektorom koji izražava NKG2D ligand RAE-1γ
Title (english)CD8 T cell response in mice vaccinated with recombinant MCMV vector expressing NKG2D ligand RAE-1γ
AuthorMarko Šustić
MentorAstrid Krmpotić (mentor)
Committee memberStipan Jonjić (predsjednik povjerenstva)
Committee memberVjekoslav Tomaić (član povjerenstva)
Committee memberHana Mahmutefendić Lučin (član povjerenstva)
GranterUniversity of Rijeka
Faculty of Medicine
(Department of Histology and Embryology)
Rijeka
Defense date and country2022-01-26, Croatia
Scientific / art field,
discipline and subdiscipline		BIOMEDICINE AND HEALTHCARE
Basic Medical Sciences
Universal decimal classification
(UDC)		61 - Medical sciences
Abstract
Cilj istraživanja: Jedna od strategija generiranja CD8 T-staničnog odgovora na antigene mikroorganizama i tumora je konstrukcija cjepnih vektora. Tijekom naših prethodnih istraživanja proizveli smo rekombinantni vektor na osnovi mišjeg citomegalovirusa koji ispoljava NKG2D ligand RAE-1γ (RAE-1γMCMV) umjesto njegovog virusnog inhibitora, m152. Pokazali smo da je RAE-1γMCMV atenuiran u odnosu na divlji tip virusa, ali neovisno o tome inducira snažniji CD8 T-stanični odgovor na ispoljeni strani epitop. Cilj ovog istraživanja je utvrditi razlike između CD8 limfocita T generiranih vektorom RAE-1γMCMV i divljim tipom vektora. Također, cilj je dobiti dublji uvid u uzročno-posljedične veze koje dovode do superiornije indukcije CD8 limfocita T u miševa cijepljenih RAE-1γMCMV-om. Materijali i metode: Kako bi se usporedio odgovor CD8 limfocita T u miševa cijepljenih vektorom koji ispoljava RAE-1γ i divljim tipom vektora, koristili su se vektori koji ispoljavaju SIINFEKL epitop te OT-1 stanice (transgenični CD8 limfociti T koji nose T-stanični receptor specifičan za epitop SIINFEKL). Memorijske OT-1 stanice inducirane s navedenim vektorima okarakterizirane su sekvencioniranjem transkriptoma i protočnom citometrijom. Protu-tumorski potencijal stanica induciranih s MCMV vektorima ispitan je u pokusima s potkožnom inokulacijom mišjeg limfoma i melanoma. Uzroci superiornije indukcije CD8 T-staničnog odgovora vektorom RAE-1γMCMV-SIINFEKL ispitani su i u in vitro pokusima ko-inkubacijom OT-1 stanica s dendritičkim stanicama inficiranim vektorima MCMV-SIINFEKL, Δm152MCMV-SIINFEKL i RAE-1γMCMV-SIINFEKL. Rezultati: RAE-1γMCMV-SIINFEKL uzrokuje superiorniji CD8 T-stanični odgovor od divljeg tipa vektora te frekvencija OT-1 stanica ostaje dugotrajno povišena u miševa inficiranih vektorom RAE-1γMCMV-SIINFEKL. Analizom transkriptoma i protočnom citometrijom utvrdili smo da stanica inducirane vektorom RAE-1γMCMV-SIINFEKL posjeduju karakteristike stanica s efektorskim fenotipom i terminalnom diferencijacijom te da posjeduju specifične funkcionalne karakteristike. Nadalje, cijepljenje miševa vektorom RAE-1γMCMV-SIINFEKL dovelo je do superiornije zaštite od potkožnog rasta tumora od cijepljenja s divljim tipom vektora. Konačno, pokazali smo da stanice koje prezentiraju antigen inficirane vektorima kojima nedostaje gen m152 (Δm152MCMV-SIINFEKL i RAE-1γMCMV-SIINFEKL) ispoljavaju znatno više kompleksa MHC-I-SIINFEKL od stanica inficiranih divljim tipom vektora i da OT-1 stanice inducirane s vektorima kojima nedostaje gen m152 pokazuju intenzivniju signalizaciju putem T-staničnog receptora tijekom prezentacije antigena što dovodi do njihove snažnije proliferacije. Zaključak: Ovaj doktorski rad pokazuje kako malene genetske promjene virusnih vektorskih cjepiva mogu imati snažne utjecaj na imunološki odgovor CD8 limfocita T.
Abstract (english)
Objectives: One of the approaches for the generation of CD8 T cell-based vaccines that would target microbial, or tumor antigens is the construction of vaccine vectors. We have constructed mouse cytomegalovirus vector expressing NKG2D ligand RAE-1γ (RAE-1γMCMV) in place of its viral inhibitor m152, which proved to be highly attenuated in vivo as compared to wild type virus, while simultaneously inducing strong CD8 T-cell response to co-expressed foreign epitope. The objective of this research was to investigate the differences between CD8 T cells generated with MCMV vector expressing RAE-1γ and control vector lacking this NKG2D ligand. Furthermore, we also wished to gain a deeper insight into the mechanisms that are driving the superior priming of CD8 T cells in mice vaccinated with RAE-1γMCMV. Materials and methods: To compare CD8 T cell response in mice immunized with RAE-1γ expressing vector and wild-type vector, we utilized vectors expressing SIINFEKL epitope and OT-1 cells (transgenic CD8 T cells carrying T-cell receptor specifically recognizing SIINFEKL epitope). Memory OT-1 cells primed with stated vectors were analysed using RNA sequencing and flow cytometry. Anti-tumor potential of these cells was tested in experiments with subcutaneous inoculation of mouse melanoma or lymphoma. The mechanisms leading to superior priming of CD8 T cells with RAE-1γMCMV-SIIFEKL vector were determined in in vitro experiments, where dendritic cells infected with MCMV-SIINFEKL, Δm152MCMV-SIINFEKL or RAE-1γMCMV-SIINFEKL were co-incubated with naive OT-1 cells. Results: RAE-1γMCMV-SIINFEKL induces superior CD8 T cell response than wild-type vector and the frequency of OT-1 cells is elevated over extended periods of time, even showing the signs of memory inflation. Transcriptomic analysis and flow cytometry showed higher expression of markers associated with effector phenotype and terminal differentiation in RAE-1γMCMV-SIINFEKL primed OT-1 cells and functional assays showed that these cells possess distinct functional features. Furthermore, immunization of mice with RAE-1 expressing vector conferred superior protection against subcutaneous tumor inoculation compared to the control vector. Finally, we demonstrated that antigen presenting cells infected with vectors lacking m152 gen (Δm152MCMV-SIINFEKL and RAE-1γMCMV-SIINFEKL) express higher levels of MHC-I-SIINFEKL complex on their surface than wild-type infected cells and that OT-1 cells primed with vectors lacking m152 exhibited enhanced T-cell receptor signaling during their priming which led to their superior proliferation. Conclusion: This doctoral thesis demonstrates that small genetical to viral vectors can have major impact on the immune response towards vectored antigens.
Keywords
Keywords (english)
Languagecroatian
URN:NBNurn:nbn:hr:184:647874
Promotion2022
ProjectNumber: 1R01DC015980-01A1
Title: Inflammation and Hearing Loss Following Congenital CMV Infection
Jurisdiction: United States
Funder: NIH
Funding stream: NATIONAL INSTITUTE ON DEAFNESS AND OTHER COMMUNICATION DISORDERS
Study programmeTitle: Biomedicine Postgraduate (doctoral) study programme
Study programme type: university
Study level: postgraduate
Academic / professional title: doktor/doktorica znanosti, područje biomedicine i zdravstvo (doktor/doktorica znanosti, područje biomedicine i zdravstvo)
Type of resourceText
File originBorn digital
Access conditionsAccess restricted to students and staff of home institution
Terms of useLicence In copyright icon
Created on2022-08-02 10:17:06