Cystatins, cysteine peptidase inhibitors, as regulators of immune cell cytotoxicity

Cystatins comprise a superfamily of evolutionarily related proteins, present in all living organisms, from protozoa to mammals. They act as inhibitors of cysteine peptidases although they can also function independently of their inhibitory function. Cysteine cathepsins are implicated in various physiological and pathological processes. In the immune response they are involved in antigen processing and presentation, the cytotoxicity of natural killer (NK) cells and cytotoxic T lymphocytes (CTL), migration and adhesion of immune cells, cytokine and growth factor regulation and toll-like receptor signalling. Cystatins are probably involved in the regulation of all these processes; importantly, cystatin F has a crucial role in the regulation of immune cell cytotoxicity. NK cells and CTLs exploit the granzyme/perforin pathway for target cell killing, with perforin and granzymes as crucial effector molecules. Granzymes are synthesized as inactive pro-granzymes and need to be proteolytically activated by cathepsins C and H. Cystatin F is the main regulator of the activity of cathepsins C and H in cytotoxic cells and, consequently, regulates their cytotoxicity. The role of cystatins and cysteine cathepsins in the immune response is presented, with emphasis on their role in the regulation of cytotoxicity of NK cells and CTLs.</p

Expression of the Immune Checkpoint Protein VISTA Is Differentially Regulated by the TGF-β1 - Smad3 Signaling Pathway in Rapidly Proliferating Human Cells and T Lymphocytes.

Immune checkpoint proteins play crucial roles in human embryonic development but are also used by cancer cells to escape immune surveillance. These proteins and biochemical pathways associated with them form a complex machinery capable of blocking the ability of cytotoxic immune lymphoid cells to attack cancer cells and, ultimately, to fully suppress anti-tumor immunity. One of the more recently discovered immune checkpoint proteins is V-domain Ig-containing suppressor of T cell activation (VISTA), which plays a crucial role in anti-cancer immune evasion pathways. The biochemical mechanisms underlying regulation of VISTA expression remain unknown. Here, we report for the first time that VISTA expression is controlled by the transforming growth factor beta type 1 (TGF-β)-Smad3 signaling pathway. However, in T lymphocytes, we found that VISTA expression was differentially regulated by TGF-β depending on their immune profile. Taken together, our results demonstrate the differential biochemical control of VISTA expression in human T cells and various types of rapidly proliferating cells, including cancer cells, fetal cells and keratinocytes

Role of cystatin F and cysteine cathepsins in the function of cytotoxic T lymphocytes

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.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\u27 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

Cathepsin X activity does not affect NK-target cell synapse but is rather distributed to cytotoxic granules

Cathepsin X is a lysosomal peptidase that is involved in tumour progression and represents a potential target for therapeutic interventions. In addition, it regulates important functions of immune cells and is implicated in the modulation of tumour cell–immune cell crosstalk. Selective cathepsin X inhibitors have been proposed as prospective antitumour agents to prevent cancer progressionhowever, their impact on the antitumour immune response has been overlooked. Previous studies indicate that the migration and adhesion of T cells and dendritic cells are affected by diminished cathepsin X activity. Meanwhile, the influence of cathepsin X inhibition on natural killer (NK) cell function has not yet been explored. Here, we examined the localization patterns of cathepsin X and the role of its inhibitors on the cytotoxicity of cell line NK-92, which is used for adoptive cellular immunotherapy in cancer patients. NK-92 cells depend on lymphocyte function-associated antigen 1 (LFA-1) to form stable immunoconjugates with target cells, providing, in this way, optimal cytotoxicity. Since LFA-1 is a substrate for cathepsin X activity in other types of cells, we hypothesized that cathepsin X could disturb the formation of NK-92 immunoconjugates. Thus, we employed cathepsin X reversible and irreversible inhibitors and evaluated their effects on the NK-92 cell interactions with target cells and on the NK-92 cell cytotoxicity. We show that cathepsin X inhibition does not impair stable conjugate formation or the lytic activity of NK-92 cells. Similarly, the conjugate formation between Jurkat T cells and target cells was not affected by cathepsin X activity. Unlike in previous migration and adhesion studies on T cells, in NK-92 cells cathepsin X was not co-localized with LFA-1 at the plasma membrane but was, rather, redistributed to the cytotoxic granules and secreted during degranulation

Increased cystatin F levels correlate with decreased cytotoxicity of cytotoxic T cells

Cystatin F is a protein inhibitor of cysteine peptidases, expressed predominantly in immune cells and localised in endosomal/lysosomal compartments. In cytotoxic immune cells cystatin F inhibits both the major pro-granzyme convertases, cathepsins C and H that activate granzymes, and cathepsin L, that acts as perforin activator. Since perforin and granzymes are crucial molecules for target cell killing by cytotoxic lymphocytes, defects in the activation of either granzymes or perforin can affect their cytotoxic potential

Extracellular Cystatin F Is Internalised by Cytotoxic T Lymphocytes and Decreases Their Cytotoxicity

Cystatin F is a protein inhibitor of cysteine cathepsins, peptidases involved in the activation of the effector molecules of the perforin/granzyme pathway. Cystatin F was previously shown to regulate natural killer cell cytotoxicity. Here, we show that extracellular cystatin F has a role in regulating the killing efficiency of cytotoxic T lymphocytes (CTLs). Extracellular cystatin F was internalised into TALL-104 cells, a cytotoxic T cell line, and decreased their cathepsin C and H activity. Correspondingly, granzyme A and B activity was also decreased and, most importantly, the killing efficiency of TALL-104 cells as well as primary human CTLs was reduced. The N-terminally truncated form of cystatin F, which can directly inhibit cathepsin C (unlike the full-length form), was more effective than the full-length inhibitor. Furthermore, cystatin F decreased cathepsin L activity, which, however, did not affect perforin processing. Cystatin F derived from K-562 target cells could also decrease the cytotoxicity of TALL-104 cells. These results clearly show that, by inhibiting cysteine cathepsin proteolytic activity, extracellular cystatin F can decrease the cytotoxicity of CTLs and thus compromise their function

Extracellular cystatin F is internalised by cytotoxic T lymphocytes and decreases their cytotoxicity

Cystatin F is a protein inhibitor of cysteine cathepsins, peptidases involved in the activation of the effector molecules of the perforin/granzyme pathway. Cystatin F was previously shown to regulate natural killer cell cytotoxicity. Here, we show that extracellular cystatin F has a role in regulating the killing efficiency of cytotoxic T lymphocytes (CTLs). Extracellular cystatin F was internalised into TALL-104 cells, a cytotoxic T cell line, and decreased their cathepsin C and H activity. Correspondingly, granzyme A and B activity was also decreased and, most importantly, the killing efficiency of TALL-104 cells as well as primary human CTLs was reduced. The N-terminally truncated form of cystatin F, which can directly inhibit cathepsin C (unlike the full-length form), was more effective than the full-length inhibitor. Furthermore, cystatin F decreased cathepsin L activity, which, however, did not affect perforin processing. Cystatin F derived from K-562 target cells could also decrease the cytotoxicity of TALL-104 cells. These results clearly show that, by inhibiting cysteine cathepsin proteolytic activity, extracellular cystatin F can decrease the cytotoxicity of CTLs and thus compromise their function

New inhibitors of cathepsin V impair tumor cell proliferation and elastin degradation and increase immune cell cytotoxicity

Cathepsin V is a human lysosomal cysteine peptidase with specific functions during pathological processes and is as such a promising therapeutic target. Peptidase inhibitors represent powerful pharmacological tools for regulating excessive proteolytic activity in various diseases. Cathepsin V is highly related to cathepsin L but differs in tissue distribution, binding site morphology, substrate specificity, and function. To validate its therapeutic potential and extend the number of potent and selective cathepsin V inhibitors, we used virtual high-throughput screening of commercially available compound libraries followed by an evaluation of kinetic properties to identify novel potent and selective cathepsin V inhibitors. We identified the ureido methylpiperidine carboxylate derivative, compound 7, as a reversible, selective, and potent inhibitor of cathepsin V. It also exhibited the most preferable characteristics for further evaluation with in vitro functional assays that simulate the processes in which cathepsin V is known to play an important role. Compound 7 exerted significant effects on cell proliferation, elastin degradation, and immune cell cytotoxicity. The latter was increased because compound 7 impaired conversion of immunosuppressive factor cystatin F to its active monomeric form. Taken together, our results present novel potent inhibitors of cathepsin V and provide new hit compounds for detailed development and optimization. Further, we demonstrate that cathepsin V is a potential target for new approaches to cancer therapy