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A clathrin/dynamin- and mannose-6-phosphate receptor-independent pathway for granzyme B-induced cell death
Joseph A Trapani1, Vivien R Sutton, Kevin Y T Thia
1Cancer Immunology Laboratory, Peter MacCallum Cancer Institute, Melbourne 8006, Australia. j.trapani@pmci.unimelb.edu.au
Abstract:
The 280-kD cation-independent mannose-6-phosphate receptor (MPR) has been shown to play a role in endocytic uptake of granzyme B, since target cells overexpressing MPR have an increased sensitivity to granzyme B-mediated apoptosis. On this basis, it has been proposed that cells lacking MPR are poor targets for cytotoxic lymphocytes that mediate allograft rejection or tumor immune surveillance. In the present study, we report that the uptake of granzyme B into target cells is independent of MPR. We used HeLa cells overexpressing a dominant-negative mutated (K44A) form of dynamin and mouse fibroblasts overexpressing or lacking MPR to show that the MPR/clathrin/dynamin pathway is not required for granzyme B uptake. Consistent with this observation, cells lacking the MPR/clathrin pathway remained sensitive to granzyme B. Exposure of K44A-dynamin-overexpressing and wild-type HeLa cells to granzyme B with sublytic perforin resulted in similar apoptosis in the two cell populations, both in short and long term assays. Granzyme B uptake into MPR-overexpressing L cells was more rapid than into MPR-null L cells, but the receptor-deficient cells took up granzyme B through fluid phase micropinocytosis and remained sensitive to it. Contrary to previous findings, we also demonstrated that mouse tumor allografts that lack MPR expression were rejected as rapidly as tumors that overexpress MPR. Entry of granzyme B into target cells and its intracellular trafficking to induce target cell death in the presence of perforin are therefore not critically dependent on MPR or clathrin/dynamin-dependent endocytosis.
Insights
Mannose-6-phosphate receptor (MPR) is not essential for granzyme B uptake. Cells lacking MPR remain sensitive to granzyme B-induced apoptosis, challenging previous assumptions about immune surveillance and allograft rejection.
Area of Science:
- Immunology
- Cell Biology
- Molecular Biology
Background:
- The cation-independent mannose-6-phosphate receptor (MPR) was previously thought to mediate granzyme B uptake, influencing target cell sensitivity to apoptosis.
- This led to the hypothesis that MPR-deficient cells are less susceptible to cytotoxic lymphocytes, impacting allograft rejection and tumor immune surveillance.
Purpose of the Study:
- To investigate whether MPR is essential for granzyme B uptake into target cells.
- To determine if MPR-deficient cells are indeed less sensitive to granzyme B-mediated apoptosis.
- To clarify the role of MPR in cellular immune responses.
Main Methods:
- Utilized HeLa cells overexpressing dominant-negative dynamin (K44A) to disrupt clathrin/dynamin-dependent endocytosis.
- Employed mouse fibroblasts with varying MPR expression levels (overexpressing or null).
- Assessed granzyme B uptake and apoptosis induction in these cell models using granzyme B and perforin.
Main Results:
- Granzyme B uptake and subsequent apoptosis were observed in cells regardless of MPR expression or clathrin/dynamin pathway involvement.
- MPR-deficient cells showed slower but significant granzyme B uptake via fluid phase micropinocytosis, maintaining sensitivity to apoptosis.
- Mouse tumor allografts lacking MPR were rejected at rates comparable to MPR-overexpressing tumors.
Conclusions:
- Granzyme B entry into target cells and its role in inducing apoptosis are not critically dependent on the mannose-6-phosphate receptor (MPR).
- Clathrin/dynamin-dependent endocytosis is not the primary pathway for granzyme B uptake.
- Cellular sensitivity to granzyme B and immune surveillance mechanisms like allograft rejection are MPR-independent.