Melanoma cell lysosome secretory burst neutralizes the CTL-mediated cytotoxicity at the lytic synapse

Abstract

Human melanoma cells express various tumour antigens that are recognized by CD8+ cytotoxic T lymphocytes (CTLs) and elicit tumour-specific responses in vivo. However, natural and therapeutically enhanced CTL responses in melanoma patients are of limited efficacy. The mechanisms underlying CTL effector phase failure when facing melanomas are still largely elusive. Here we show that, on conjugation with CTL, human melanoma cells undergo an active late endosome/lysosome trafficking, which is intensified at the lytic synapse and is paralleled by cathepsin-mediated perforin degradation and deficient granzyme B penetration. Abortion of SNAP-23-dependent lysosomal trafficking, pH perturbation or impairment of lysosomal proteolytic activity restores susceptibility to CTL attack. Inside the arsenal of melanoma cell strategies to escape immune surveillance, we identify a self-defence mechanism based on exacerbated lysosome secretion and perforin degradation at the lytic synapse. Interfering with this synaptic self-defence mechanism might be useful in potentiating CTL-mediated therapies in melanoma patients.

Distinct patterns of cytolytic T‐cell activation by different tumour cells revealed by Ca2+ signalling and granule mobilization

“…The status of ICAM‐1 expression by the tumour cells was not discussed in that study and fine differences in the pattern of CTL activation (as observed in our study) may have been bypassed due to use of high concentrations of exogenous antigenic peptide . More recently, this group identified a defence mechanism of human melanoma cells based on their exacerbated lysosome secretion and cathepsin B‐mediated perforin degradation at the lytic synapse . Over‐expression of serpin‐9 (gene PI‐9), an inhibitor of GzmB activity, has also been described in metastatic melanoma.…”

Mechanisms of natural killer cell-mediated cellular cytotoxicity

“…107 Interestingly, tumor cells may use the same mechanism of secreting cathepsin B at the synapse to escape killing by cytotoxic T cells. 108 However, in cathepsin B-deficient mice, cytotoxic lymphocytes did not show enhanced apoptosis during the killing of target cells, 109 demonstrating that cathepsin B is not necessary for the self-protection of cytotoxic cells. However, it is likely that the protection of cytotoxic cells from their own lytic machinery is such an important aspect that there are several redundant mechanisms at play to guarantee the survival of the cell even if one of these mechanisms should fail.…”

“…As a final mechanism, it was proposed that cathepsin B, which also reaches the plasma membrane of cytotoxic lymphocytes during degranulation, can cleave and inactivate perforin . Interestingly, tumor cells may use the same mechanism of secreting cathepsin B at the synapse to escape killing by cytotoxic T cells . However, in cathepsin B‐deficient mice, cytotoxic lymphocytes did not show enhanced apoptosis during the killing of target cells, demonstrating that cathepsin B is not necessary for the self‐protection of cytotoxic cells.…”

Dynamic visualization of the whole process of cytotoxic T lymphocytes killing B16 tumor cells in vitro

“…5 The mechanisms of CTLs killing tumor cells unfold in several ways: through the secretion of perforin and granzyme B, through the interaction of FasL and Fas on the cell surface, and through the NKG2D pathway. [6][7][8][9][10] Before ACT, the activity and cytotoxicity of CTLs against target tumor cells need to be evaluated in vitro. Some methods-such as flow cytometry, immunohistochemistry, real-time PCR, western blot, and a few others-have already analyzed the functions of CTLs.…”

“…Even though these methods revealed some important facts about CTLs' killing abilities, other dynamic details, especially the special interaction between CTLs and target tumor cells and the whole process of CTLs successfully killing tumor cells are still not well understood. [7][8][9][11][12][13] The confocal imaging enables tracking several cellular or molecular events dynamically at high resolution in real time via multichannel parallel detections. [14][15][16][17] In order to visualize the quick changing interactions between tumor cells and CTLs, fluorescent proteins (FPs) and fluorochromes are used to label tumor cells [18][19][20] and CTLs.…”