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Serbian Association for Cancer Research SDIRSACR

L10

Immune Checkpoint Inhibitors Modulate Cystatin F Expression in Cytotoxic T Cells from Melanoma
Patients

Emanuela Senjor1, Simona Miceska2, Biljana Mileva Boshkoska1, Tanja Mesti2, Janja Ocvirk2,
Janko Kos3, Milica Perišić Nanut1

¹Jožef Stefan Institute, Ljubljana
2Institute of Oncology Ljubljana, 1000 Ljubljana, Slovenia
3Faculty of Pharmacy, University of Ljubljana

Keywords: melanoma, cystatins, immune checkpoint inhibitors, cytotoxic T lymphocytes

Background: Despite their superior clinical efficacy over conventional chemotherapy and radiotherapy, immune
checkpoint inhibitors (ICIs) remain ineffective in over 60% of cancer patients. Moreover, cytotoxic immune cells
targeting tumors can also damage healthy tissue, leading to potentially life-threatening adverse events. These
limitations underscore the urgent need for early, reliable predictive biomarkers to improve patient management and
reduce treatment costs. Even among initial responders, only a minority experience durable benefit, largely due to
diverse resistance mechanisms—particularly in solid tumors, where the immunosuppressive tumor microenvironment
(TME) plays a central role. Additionally, dysfunctional CTL effector activity following release from checkpoint blockade
further limits tumor cell killing and therapeutic efficacy. A deeper understanding of resistance pathways and CTL
dysfunction is critical for the development of effective combination strategies aimed at enhancing ICI responses.
Therapies that target complementary immune escape mechanisms may yield synergistic effects and improve clinical
outcomes. Insights into CTL impairment may also guide optimization of other immunotherapies, including CAR T
cell therapy. Emerging biomarkers could help identify the most effective T cell subsets for adoptive cell transfer in
combination with ICIs. Cytotoxic T lymphocytes (CTLs) eliminate cancer cells by releasing cytotoxic molecules—such
as the pore-forming protein perforin and apoptosis-inducing granzymes—into the immunological synapse between
the effector and target cell. These effector molecules are synthesized as inactive precursors and require activation
via proteolytic processing by cysteine cathepsins. Cystatin F, an endogenous inhibitor of these cathepsins, has been
identified as a potent negative regulator of CTL cytotoxic function. In natural killer (NK) cells, which share the same
granule-mediated cytotoxic pathway, reduced killing capacity—termed “split anergy”—is linked to increased cystatin
F and decreased levels of active granzyme convertases (cathepsins C and H). Similarly, cystatin F expression is elevated
in CTLs rendered anergic by ionomycin or TGF-β, correlating with diminished cytotoxicity. Ex vivo long-term stimulation
of CD4⁺ T cells also induces expression of granzymes A and B, cathepsins C and H, perforin, and cystatin F. We further
established a model using PD-1–expressing cytotoxic TALL-104 cells, in which preliminary data show that anti–PD-1
antibody treatment leads to upregulation of cystatin F, suggesting a link between ICI therapy and modulation of CTL
effector function. Cystatin F is initially synthesized as a disulfide-linked dimer, which is inactive as a cathepsin inhibitor.
Following synthesis, it is directed to the endo/lysosomal compartment via the mannose-6-phosphate (M6P) pathway,
facilitated by multiple N-linked glycans. Within endo/lysosomes, cystatin F undergoes proteolytic cleavage by the
cysteine peptidase cathepsin V, converting it into its active form. In this monomeric state, cystatin F potently inhibits
lysosomal peptidases, specifically cathepsins C and H. Its ability to translocate to endo/lysosomes—essential for
activation—is influenced by the type of N-glycosylation it undergoes. Our recent unpublished findings reveal that the
glycosylation profile of cystatin F shifts from high-mannose to complex-type N-glycosylation in NK cells with differing
cytotoxic capacities, including NK-92 cells, primary NK cells, and IL-2–activated “super-charged” NK cells. These results
suggest that modulation of cystatin F glycosylation or its interaction with M6PR can affect its trafficking and subsequent
activation. Since only the monomeric form of cystatin F inhibits cathepsins C, H, and L within cytotoxic granules,
targeting the activation process represents a promising strategy to restore cytotoxic function.
Our recently published work supports this concept: treatment with a small-molecule inhibitor of cathepsin V (compound
7) reduced cystatin F monomerization and activation, leading to enhanced cytotoxicity in NK-92 cells. Furthermore,
cathepsin V inhibition increased granule-mediated killing by primary NK cells against stem-like glioma cells (NCH421k),
underscoring the therapeutic potential of this approach. Cystatin F expression has been shown to increase in the tissue
of certain tumors as the disease progresses. Survival analysis of RNA sequencing data from the TCGA dataset, including
patients with low-grade gliomas and grade IV glioblastoma, indicates that higher cystatin F expression correlates with
shorter overall survival. This finding is particularly significant given that both the monomeric and dimeric active forms
of cystatin F can be internalized by immune effector cells through the mannose-6-phosphate receptor (M6PR) pathway,
targeting the endo/lysosomal compartment. Uptake of extracellular cystatin F has been demonstrated to reduce the
susceptibility of glioblastoma cells to NK cell–mediated cytotoxicity. Additionally, internalization of both forms of

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