Cystatin F belongs to type II cystatins, inhibitors of cysteine peptidases. It is primarily expressed in immune cells such as monocytes, dendritic cells, natural killer cells and cytotoxic T lymphocytes. It is synthesized as a disulfide linked dimer and in this form it is secreted or targeted to endosomes/lysosomes. In endosomes/lysosomes, cystatin F is activated by monomerization facilitated by proteolytic cleavage of the 15 amino acids at its N-terminus. Proteolytic cleavage alters the inhibitory properties of cystatin F. Only cleaved cystatin F becomes a potent cathepsin C inhibitor. Inactive dimeric cystatin F, secreted from the cells, can be taken up by surrounding cells translocated to endosomes/lysosomes and activated. Increased content of cystatin F was detected in cytotoxic lymphocytes such as natural kiler cells and cytotoxic T lymphocytes. Its higher expression is associated with decreased cytotoxic function. The aim of this study was to examine the expression of cystatin F in cytotoxic T lymphocytes, treated with immune checkpoint inhibitors. To analyse cystatin F content in TALL-104, U937, and Jurkat cell lines, we used flow cytometric analysis and western blot. TALL-104 cells were activated using CD3/CD28 protein conjugated with Dynabeads or ionomycin/phorbol 12-myristate 13-acetate and upon activation, treated with immune checkpoint inhibitors. Our findings revealed that cystatin F is indeed present in TALL-104, and its expression increases upon cellular activation. The expression of cathepsins C and L as well as the expression and the activity of granzymes and perforin decreased in activated cells. Treatment of TALL-104 cells with immune checkpoint inhibitors decreased the level of cystatin F in activated cells. Our results show that cystatin F is a crucial regulator of the immune response.
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