Title Karakterizacija imunoregulatorne uloge najviše izraženoga virusnog transkripta tijekom infekcije citomegalovirusom
Title (english) Characterization of immunoregulatory role of the most abundant viral transcript during cytomegalovirus infection
Author Jelena Železnjak https://orcid.org/0000-0001-6619-3675
Mentor Astrid Krmpotić (mentor)
Committee member Stipan Jonjić (predsjednik povjerenstva)
Committee member Alemka Markotić (član povjerenstva)
Committee member Đurđica Cekinović Grbeša (član povjerenstva)
Committee member Astrid Krmpotić (član povjerenstva)
Granter University of Rijeka Faculty of Medicine (Department of Histology and Embryology) Rijeka
Defense date and country 2019, Croatia
Scientific / art field, discipline and subdiscipline BIOMEDICINE AND HEALTHCARE Basic Medical Sciences
Universal decimal classification (UDC ) 61 - Medical sciences
Abstract Cilj istraživanja: Kako bi izbjegao odgovor CD8+ limfocita T, mišji citomegalovirus (MCMV) kompromitira izražaj molekula MHC I na površini inficirane stanice. Međutim, nedostatak MHC I na površini može potaknuti aktivaciju stanica NK. Stoga, MCMV kodira protein m04 koji veže mali dio MHC I i odvodi ih na površinu stanice gdje vežu inhibicijske receptore Ly49 i utišavaju stanicu NK. Ipak, iako proteina m04 unutar stanice ima mnogo, samo mali dio odlazi na površinu, a afinitet vezanja kompleksa m04 i MHC I je nizak. Naša preliminarna istraživanja ukazivala su na ulogu transkripta MAT u navedenoj interakciji. Stoga je cilj ovog rada razjasniti funkciju transkripta MAT u regulaciji imunosnog odgovora domaćina, s naglaskom na receptore Ly49 i stanice NK.
Materijali i metode: Kako bi razjasnili ulogu receptora Ly49 u prepoznavanju virusom-inficiranih stanica, koristili smo reporterske stanice koje izražavaju pojedini receptor Ly49. Za određivanje uloge proteina MATp1 u sazrijevanju i izražavanju MHC I, metabolički smo obilježili MHC I te ih imunoprecipitirali. Mehanizam interakcije između MATp1, m04 i MHC I odredili smo metodom ko-imunoprecipitacije i testom interakcija temeljem blizine vezanja. Pritom smo koristili novo razvijeno monoklonsko protutijelo α-m04. S ciljem određivanja uloge MATp1 u kontroli infekcije in vivo, primijenili smo test virusnih čistina. Također smo proveli funkcionalne testove i fenotipsku analizu stanica NK i CD8+ limfocita T pomoću protočne citometrije.
Rezultati: Protein MATp1 stupa u interakcije s molekulama MHC I (H-2Dd) i m04 i time omogućuje dolazak kompleksa m04/H-2Dd na površinu inficirane stanice. Takve MATp1/m04-promijenjene molekule MHC I vežu inhibicijske receptore Ly49A snažnije nego same molekule MHC I. To dovodi do inhibicije stanica NK i nemogućnosti kontrole MCMV-a u ranim danima infekcije. Kao odgovor na to, domaćin je razvio aktivacijske receptore Ly49D2, Ly49L i Ly49P koji specifično prepoznaju MATp1/m04-promijenjene molekule MHC I. Ipak, virus je mutirao MATp1 i m04 te izbjegao prepoznavanje od strane aktivacijskih receptora Ly49 te stvorio nove inačice MCMV-a. MATp1 također negativno utječe i na virus-specifičan odgovor CD8+ limfocita T tijekom infekcije.
Zaključak: Ovaj rad odgovara na dosad nerazjašnjeno pitanje kako MCMV izbjegava prepoznavanje od strane stanica NK u većini mišjih sojeva usprkos smanjenom površinskom izražaju molekula MHC I, otkriva novi temeljni mehanizam virusne imunosubverzije koji uključuje interakciju virusom-promijenjenih molekula MHC I i inhibicijskih receptora Ly49 te pokazuje posljedicu navedene interakcije, odnosno evoluciju virus-specifičnih aktivacijskih receptora Ly49 ovisnih o MHC I kao odgovor na prvobitnu virusnu strategiju.
Abstract (english) Objectives: To avoid recognition by CD8+ T cells, mouse cytomegalovirus (MCMV) downregulates MHC I molecules from the surface of infected cells. However, complete downregulation of MHC I makes infected cells susceptible to NK cells lysis. Therefore, MCMV encodes for m04 protein that binds some MHC I molecules in the infected cells, escorts them to the cell surface where they engage inhibitory Ly49 receptors and inhibit NK cell response strongly than uninfected cells. Although m04 protein is abundant inside the cell, only small proportion goes to the cell surface and binds to MHC I with surprisingly low affinity. Our preliminary results indicate the role of MAT transcript in the mentioned interaction. Therefore, the aim of this study is to clarify the function of MAT transcript in the regulation of the immune response, with an emphasis on Ly49 receptors and NK cells.
Materials and Methods: To clarify the role of Ly49 receptors in the recognition of viral-infected cells, we used reporter cells expressing Ly49 receptor. To determine the role of MATp1 protein in maturation and expression of MHC I molecules, we have metabolically labeled MHC I and immunoprecipitated them. The mechanism of interaction between MATp1, m04 and MHC I was determined using co-immunoprecipitations and proximity ligation assay (PLA). Newly generated monoclonal antibody α-m04 was used in these methods. We used the plaque assay in order to determine the role of MATp1 on the control of MCMV infection in vivo. We also performed functional tests and phenotype analysis of NK cells and CD8+ T lymphocytes by flow cytometry.
Results: MATp1 interacts with MHC I (H-2Dd) and m04, thereby enabling m04/H-2Dd complex to reach the cell surface. There, MATp1/m04-modified altered-self MHC I molecules bind inhibitory Ly49A receptors more strongly than MHC I molecules alone. This results in NK cell inhibition and inability of viral control in early days post infection. To counter this, host developed activating Ly49D2, Ly49L and Ly49P receptors specific for MATp1/m04-modified altered-self MHC I molecules. However, MCMV mutated MATp1 and m04 therefore escaping the recognition by activating Ly49 receptors and consequently created new variants of MCMV. MATp1 also negatively affects the virus-specific CD8+ T cell response during infection.
Conclusion: Results of this research answer a long-standing conundrum of how MCMV avoids recognition by NK cells in most mouse strains despite reduced surface levels of MHC I, reveals a fundamental new mechanism of viral immune evasion involving the interaction of the virally-altered MHC I molecules and inhibitory Ly49 receptors and demonstrate how this interaction forced the evolution of virus-specific activating MHC I-restricted Ly49 receptors.
Keywords
Imunosni nadzor
m04/gp34
Mišji citomegalovirus
Molekule MHC I
Receptori Ly49
Stanice NK
Transkript MAT
Keywords (english)
Immunoevasion
Ly49 receptors
m04/gp34
MHC I molecules
Mouse cytomegalovirus
NK cells
Transcript MAT
Language croatian
URN:NBN urn:nbn:hr:184:658660
Promotion 2019
Study programme Title: 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 resource Text
File origin Born digital
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Created on 2019-09-10 14:11:50