Vloga cistatina F in cisteinskih katepsinov pri delovanju citotoksičnih limfocitov T : doktorska disertacija

Prunk, Mateja (avtor), Kos, Janko (mentor)

Citotoksični limfociti T CD8+ (CTL) in naravne celice ubijalke (celice NK) imajo ključno vlogo v imunskem odzivu proti celicam, okuženim z virusi in proti rakavim celicam, saj jih lahko neposredno ubijejo. CTL in celice NK se bistveno razlikujejo v načinih aktivacije, vendar pa so molekularni mehanizmi, ki jih uporabljajo za ubijanje tarčnih celic, pri obeh vrstah citotoksičnih limfocitov enaki. Najpomembnejše je ubijanje preko perforin/grancimske poti, pri kateri citotoksični limfociti izločijo vsebino posebnih sekretornih lizosomov, ki jih imenujemo citotoksična zrna. Glavne citotoksične molekule, prisotne v citotoksičnih zrnih, so perforin, protein, ki omogoči vstop grancimom v citosol tarčne celice, ter grancimi, družina serinskih peptidaz, ki v tarčni celici sprožijo celično smrt. Tako perforin kot grancimi se sintetizirajo v neaktivni prekurzorski obliki, za njihovo aktivacijo pa je potrebno proteolitično procesiranje, kjer imajo ključno vlogo cisteinski katepsini. Za aktivacijo grancimov je tako potrebno delovanje katepsinov C in H, medtem ko katepsin L aktivira perforin. Aktivnost cisteinskih katepsinov je uravnavana na različnih nivojih, pomembno je uravnavanje z njihovimi endogenimi proteinskim inhibitorji, cistatini. Med cistatini je v citotoksičnih limfocitih še posebej pomembno delovanje cistatina F, saj je edini cistatin, ki se nahaja v endosomih/lizosomih in lahko uravnava delovanje cisteinskih katepsinov, prisotnih v teh veziklih. Poleg tega se cistatin F iz celic izloča, zunajcelični cistatin F pa lahko privzamejo druge celice, privzeti cistatin F nato uravnava delovanje njihovih endogenih cisteinskih katepsinov. Nedavno je bilo pokazano, da se nivo cistatina F poviša po vzpostavitvi delne anergije celic NK, tj. stanja zmanjšane citotoksičnosti in povečanega izločanja citokinov. Poleg tega je zunajcelični cistatin F zmanjšal tako aktivnost grancimov A in B kot samo citotoksičnost celic NK, kar kaže na pomembno vlogo cistatina F pri uravnavanju delovanja celic NK. Vloga cistatina F pri delovanju CTL pa nasprotno še ni poznana, poleg tega ni dokazan vpliv zunajceličnega cistatina F na aktivnost cisteinskih katepsinov v človeških citotoksičnih limfocitih. V okviru doktorske disertacije smo tako najprej raziskali pomen cistatina F pri anergiji CTL. Najprej smo vzpostavili dva anergična modela na celični liniji TALL-104, z uporabo nizke koncentracije ionomicina ter z imunosupresivnim citokinom transformirajočim rastnim dejavnikom β (TGFβ). Ugotovili smo, da je pri obeh modelih citotoksična sposobnost celic TALL-104 močno zmanjšana proti različnim tarčnim celicam, to je celicam K-562, ki jih ubijajo tudi celice NK, ter proti celicam Raji, ki jih celice NK ne ubijajo. Potrdili smo, da pri naših modelih ni vpliva na živost celic ter da ubijanje tarčnih celic temelji na perforin/grancimski poti. Zmanjšana citotoksičnost je pri obeh modelih sovpadala s povišanimi nivoji aktivne monomerne oblike cistatina F ter celokupnih nivojev cistatina F, medtem ko je bil nivo neaktivne dimerne oblike po tretiranju s TGFβ zmanjšan, po tretiranju z nizkimi koncentracijami ionomicina pa povišan. Aktivacija CTL je povzročila zmanjšanje nivojev tako monomerne kot dimerne oblike cistatina F, medtem ko je nivo cistatina F po aktivaciji anergičnih celic ostal povišan. Potrdili smo tudi interakcijo cistatina F s katepsinoma C in H z imunoprecipitacijo v TALL-104, ter ko-lokalizacijo s katepsini C, H in L v TALL-104 in primarnih človeških limfocitih T CD8+ (pCTL). Nato smo pokazali, da je višji nivo aktivnega monomernega cistatina F povezan ne le z manjšo citotoksičnostjo, temveč tudi z zmanjšano specifično aktivnostjo katepsinov C, H in L ter končne efektorske peptidaze perforin/grancimske poti, grancimom B. Dobljeni rezultati opredeljujejo pomen cistatina F pri anergiji CTL. V naslednjem delu raziskav smo najprej potrdili lokalizacijo cistatina F v citotoksičnih zrncih, saj je cistatin F ko-lokaliziral s perforinom v celicah TALL-104, poleg tega smo dokazali ko-lokalizacijo cistatina F in grancima B z ligacijskim testom bližine v celicah TALL-104 in pCTL. Nato smo ocenili vpliv zunajceličnega cistatina F na katepsine C, H in L. Dodajanje rekombinantnega človeškega neskrajšanega ali N-končno skrajšanega cistatina F je zmanjšalo aktivnosti katepsinov C, H in L v celicah TALL-104, pri čemer je bil vpliv N-končno skrajšane različice na aktivnosti katepsinov C in H nekoliko večji, vendar ni bil statistično značilen. Preverili smo, ali inhibicija katepsina L vpliva na aktivacijo katepsina C. Pokazali smo, da v celicah HeLa transfekcija s cistatinom F zmanjša aktivacijo katepsina C, vendar pa dodajanje cistatina F celicam TALL-104 na procesiranje katepsina C ni vplivalo. Poleg tega inhibicija katepsina L ni imela vpliva na procesiranje perforina, najverjetneje so zmanjšano aktivnost katepsina L nadomestile druge peptidaze. Po drugi strani pa je dodajanje neskrajšane in N-končno skrajšane različice cistatina F izrazito zmanjšalo aktivnost efektorskih peptidaz citotoksičnosti, grancimov A in B, vpliv N-končno skrajšane različice je bil statistično značilno večji od vpliva neskrajšane različice. V zadnjem sklopu raziskav smo dokazali, da dodajanje obeh različic cistatina F, neskrajšane in N-končno skrajšane, tudi neposredno zmanjša citotoksičnost celic TALL-104. S tem smo dokazali, da zunajcelični cistatin F lahko regulira citotoksično delovanje CTL preko vpliva na aktivnost cisteinskih katepsinov C in H ter s tem na aktivacijo grancimov A in B. Poleg tega pa smo pokazali, da lahko citotoksičnost CTL zmanjša tudi vsebnost cistatina F v tarčnih celicah, saj so celice TALL-104 manj učinkovito ubijale celice K-562, ki smo jih inkubirali s cistatinom F in so cistatin F privzele, kot celice K-562, ki jih nismo inkubirali s cistatinom F. Rezultati kažejo, da cistatin F v tarčnih celicah lahko deluje zaščitno za tarčne celice in prepreči njihovo ubijanje s strani citotoksičnih celic. V okviru doktorske disertacije smo tako pokazali vpletenost cistatina F v vzpostavitev in vzdrževanje anergičnega stanja CTL ter vpliv privzemanja zunajceličnega cistatina F, ki neposredno uravnava citotoksičnost CTL preko delovanja na pro-grancimski konvertazi, katepsina C in H. Hkrati smo pokazali, da lahko na citotoksičnost CTL vpliva tudi cistatin F, ki se nahaja v tarčnih celicah, kar je predvsem zanimivo v luči protitumorskega imunskega odziva, saj predstavlja enega od mehanizmov, kako bi se rakave celice izognile imunskemu nadzoru. Z uravnavanjem aktivacije cistatina F preko uravnavanja aktivnosti peptidaz, ki aktivirajo cistatin F, oziroma z uravnavanjem privzemanja cistatina F preko vpliva na njegov glikozilacijski profil, bi lahko uravnavali citotoksičnost CTL, kar bi bilo pomembno pri imunoterapiji raka ter boleznih, pri katerih je moteno citotoksično delovanje citotoksičnih limfocitov.

• Jezik: Slovenski jezik
• Ključne besede: biomedicina, klinična biokemija, imunski sistem, molekularna imunologija, citotoksični limfociti T, naravne celice ubijalke, citotoksičnost, perforin, grancim, cisteinski katepsini, katepsin C, katepsin H, katepsin L, cistatini, cistatin F, doktorske disertacije
• Vrsta gradiva: Doktorska disertacija
• Tipologija: 2.08 - Doktorska disertacija
• Organizacija: FFA - Fakulteta za farmacijo
• Kraj izida: Ljubljana
• Založnik: [M. Prunk]
• Leto izida: 2019
• Št. strani: XVIII, 205 str.
• PID: 20.500.12556/RUL-137320
• UDK: 577.27(043.3)
• COBISS.SI-ID: 299697920
• Datum objave v RUL: 10.06.2022

Sekundarni jezik

• Jezik: Angleški jezik
• Naslov: Role of cystatin F and cysteine cathepsins in the function of cytotoxic T lymphocytes

Izvleček

Cytotoxic T cells CD8+ (CTLs) and natural killer cells (NK cell) are crucial cell effectors of the immune response against virus infected cells and tumour cells because of their ability to directly kill target cells. Activation of CTLs and NK cells is triggered by different mechanisms, however, both cell types use the same molecular mechanisms for target cell killing. The most important is the perforin/granzyme pathway, where cytotoxic lymphocytes secrete content of secretory lysosomes, termed cytotoxic granules. The main cytotoxic molecules of cytotoxic granules are perforin, a pore forming protein that enables entry of granules' content into the cytosol of the target cell, and granzymes, a family of serine peptidases that, once in the target cell, trigger cell death. Perforin as well as granzymes are synthesised in an inactive pro-form and need to be proteolitically activated by a process where cysteine cathepsins are crucial. Activation of granzymes depends on cathepsins C and H, while cathepsin L is involved in perforin activation. Activity of cysteine cathepsins is regulated by different means, importantly, their activity is regulated by their endogenous protein inhibitors, the cystatins. Among cystatins the role of cystatin F is especially important in cytotoxic lymphocytes, since it is the only cystatin localised in the endosomes/lysosomes and thus capable of regulation of endogenous peptidases present in these vesicles. In addition, cystatin F can also be secreted from cells and internalised by by-stander cells, where it can regulate activity of endogenous cysteine cathepsins. It was recently demonstrated, that cystatin F levels are increased in split anergic NK cells, that is the cells that lose their cytotoxicity, but secrete increased levels of cytokines. Additionally, it was shown that extracellular cystatin F can attenuate activity of granzymes A and B as well as cytotoxicity of NK cells, implying an important role for cystatin F in cytotoxic function of NK cells. The role of cystatin F in CTLs, on the other hand, is not known, and the impact of extracellular cystatin F on activity of cysteine cathepsins in human cytotoxic lymphocytes was not demonstrated yet. In the doctoral thesis we first studied the involvement of cystatin F in anergy of CTLs. We established two anergic models on cell line TALL-104, using either low concentrations of ionomycin or immunosuppressive cytokine, transforming growth factor β (TGFβ). Cytotoxicity of TALL-104 cells was significantly decreased in both models, shown against different target cells, i.e. NK-sensitive target K-562 cells and NK-resistant target Raji cells. We confirmed that in our models there is no effect on viability and that target cell killing depends on the perforin/granzyme pathway. Furthermore, decreased cytotoxicity in both models correlated with increased levels of the active monomeric form of cystatin F and total cystatin F, while the inactive dimeric form of cystatin F was decreased after TGFβ treatment and increased when anergy was induced with low concentrations of ionomycin. Activation of CTLs led to decreased levels of monomeric and dimeric forms of cystatin F, but in anergic cells cystatin F levels remained increased after activation. We confirmed interaction of cystatin F with cathepsins C and H in TALL-104 cells by immunoprecipitation and co-localisation of cystatin F with cathepsins C, H and L in TALL-104 cells and primary human cytotoxic T CD8+ lymphocytes (pCTLs). We also found that increased levels of monomeric cystatin F levels and lower cytotoxicity correlated with decreased specific activities of cathepsins C, H and L and the final cytotoxic effector molecule, granzyme B. Our results thus demonstrate a role for cystatin F in anergy of CTLs. In the next part of our studies we first confirmed localisation of cystatin F in cytotoxic granules, since cystatin F co-localised with perforin in TALL-104 cells and also demonstrated cystatin F co-localisation with granzyme B in TALL-104 cells and pCTLs by proximity ligation assay. Next, we evaluated the effect of extracellular cystatin F on cathepsins C, H and L activities. Treatment of TALL-104 cells with either full length or terminally truncated cystatin F led to decreased activities of cathepsins C, H and L. The effect was more pronounced for N-terminally truncated cystatin F form, but was not statistically significant from full-length form. We also tested if inhibition of cathepsin L affects activation of cathepsin C. Indeed, transfection of HeLa cells with cystatin F led to decreased processing of cathepsin C, however in TALL-104 cells addition of cystatin F did not affect cathepsin C activation. Similarly, cathepsin L inhibition also did not affect perforin processing, probably other peptidases involved in perforin processing substituted for reduced cathepsin L activity. On the other hand, treatment with full-length and N-terminally truncated cystatin F substantially decreased activities of granzymes A and B, the effect of N-terminally truncated cystatin F was significantly higher compared to full-length cystatin F. Moreover, both forms of cystatin F, full-length and N-terminally truncated, significantly decreased cytotoxicity of TALL-104 cells. Thus, we demonstrated that cystatin F regulates cytotoxicity of CTLs by inhibition of cathepsins C and H, consequently decreasing activation of granzymes A and B. Lastly, we also demonstrated, that cystatin F levels in target cells affect cytotoxicity of TALL-104 cells, since target K- 562 cells incubated with cystatin F were killed less efficiently than control K-562 target cells. To conclude, we demonstrated that cystatin F is involved in establishment and maintenance of anergy of CTLs. In addition, we provide evidence of direct regulation of cytotoxicity of CTLs by extracellular cystatin F, that inhibits pro-granzyme convertases, cathepsins C and H, and consequently granzymes A and B. Furthermore, we show that cystatin F present in target cells also attenuates cytotoxicity of CTLs, a finding important for antitumor immune response, since it represents one of the mechanisms cancer cells could exploit to evade the immune surveillance. Thus, regulating cystatin F activation by regulating its activating peptidases or regulating cystatin F internalization by regulating its glycosylation profile could be useful in tailoring the cytotoxicity of CTL in the cancer immunotherapy and other pathologies with dysfunctional cytotoxic lymphocytes.

Projekti

• Financer: ARRS - Agencija za raziskovalno dejavnost Republike Slovenije
• Številka projekta: P4-0127
• Naslov: Farmacevtska biotehnologija: znanost za zdravje

• Financer: ARRS - Agencija za raziskovalno dejavnost Republike Slovenije
• Številka projekta: J4-6811
• Naslov: Vloga inhibitorjev cisteinskih proteaz v citotoksičnem delovanju naravnih celic ubijalk na tumorske celice