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

Simple Summary Cytotoxic T lymphocytes kill cancer or virally infected cells by exocytosis of lytic granules. This leads to perforin-mediated granzyme entry into the target cell, consequently killing the target cell. Granzymes and perforin are activated by cysteine cathepsins whose activity is regulated by the protein inhibitor cystatin F. Since cystatin F can be secreted by a range of cancer and immune cells in tumour microenvironments, we here investigated whether extracellular cystatin F can be taken up by and affect the function of cytotoxic T lymphocytes. We demonstrated cystatin F uptake into cytotoxic T lymphocytes, down-regulation of target peptidases, and reduced target cell killing. Overall, our results indicate that cystatin F is an important mediator that can impair the killing efficiency of cytotoxic T lymphocytes and thus suggest that it is a possible target for cancer immunotherapy. 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.