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.
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