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

Abstract

BackgroundCystatin 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.Materials and methodsLevels of cystatin F were assessed by western blot and interactions of cystatin F with cathepsins C, H and L were analysed by immunoprecipitation and confocal microscopy. In TALL-104 cells specific activities of the cathepsins and granzyme B were determined using peptide substrates.ResultsTwo models of reduced T cell cytotoxicity of TALL-104 cell line were established, either by treatment by ionomycin or by immunosuppressive transforming growth factor beta. Reduced cytotoxicity correlated with increased levels of cystatin F and with attenuated activities of cathepsins C, H and L and of granzyme B. Co-localisation of cystatin F and cathepsins C, H and L and interactions between cystatin F and cathepsins C and H were demonstrated.ConclusionsCystatin F is designated as a possible regulator of T cell cytotoxicity, similar to its role in natural killer cells.

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

“…All of these studies demonstrated uptake by cells that do not express endogenous cystatin F. In NK cells that express endogenous cystatin F, extracellular cystatin F uptake was demonstrated to potentiate the suppressive function of endogenous cystatin F regarding granzyme function and cell cytotoxicity [13]. As the internalised cystatin F in cystatin F-expressing cells must compete with endogenous intracellular cystatin F, the target peptidases might differ between internalised and endogenous cystatin F. However, here we demonstrate that the target of internalised extracellular cystatin F in TALL-104 cells is also cathepsin C, similar to endogenous cystatin F that also forms immune complexes with cathepsin C [11,16].…”

“…We first assessed recombinant cystatin F uptake into TALL-104 cells, a model cell line of cytotoxic T lymphocytes [16]. Western blot analysis demonstrated that his-tag-labelled recombinant cystatin F is indeed internalised into TALL-104 cells (Figure 1a,b).…”

“…In cytotoxic cells, cystatin F is localised in cytotoxic granules in which it co-localises with its target cathepsins C, H, and L [11,15,16] indicating that cystatin F regulates their activities and thus the activation of the effector molecules of the perforin/granzyme pathway [8,17]. Indeed, it was demonstrated that extracellular cystatin F decreases the activities of granzymes A and B as well as the cytotoxicity of NK-92 and primary human NK cells [13].…”

“…In addition, both dimeric and monomeric cystatin F levels were increased in split anergic primary NK cells [15], a state characterised by decreased cytotoxicity and increased cytokine secretion [18,19]. Similarly, cystatin F levels were increased while the activities of cathepsins C, H, and L and granzyme B were decreased in the cytotoxic T CD8+ cell line (TALL-104) when a hyporesponsive anergic state was induced by ionomycin or transforming growth factor beta [16]. Likewise, the overexpression of cystatin F in mouse CTLs was shown to attenuate cathepsin C activity [11], and internalisation of cystatin F secreted from mouse CTLs into cystatin F-null mouse CTLs was also demonstrated [14].…”

Cysteine Cathepsins in Tumor-Associated Immune Cells

“…Endogenous cysteine cathepsin inhibitor, cystatin F, is a major regulator of cathepsin C, H, and L activities in cytotoxic immune cells (84, 85, 236). Notably, when a state of functional anergy was induced, in either NK cells or CTL, cystatin F levels increased, while levels of cathepsins C, H, L, and granzyme B levels and activities declined (84, 236). Even though cystatin F is, to a greater extent, targeted to endosomes, part of it is secreted as an inactive dimer.…”