Constitutive Expression of the Granzyme B Inhibitor PI-9 Protects Leukemic Cells from Granzyme B Induced Apoptosis.

Constitutive expression of the granzyme B inhibitor PI-9 protects leukemic cells from granzyme B induced apoptosis Introduction Apoptosis induced in leukemic blasts by alloreactive Cytotoxic T-Lymphocytes (CTL) is the molecular mechanism of cell death involved in the Graft versus leukemia (GvL) reaction following allogeneic bone marrow (BMT) and peripheral stem cell transplantation (PBSCT). Apoptosis in alloreactivity is mediated mainly by the granule exocytosis pathway. Granules rich in serine proteases and perforin are released into the target cell upon recognition by CTL. Of these proteases granzyme B (GrB) has been shown to be the most important effector for apoptosis induction by activating Caspase 3 and subsequently the apoptotic effector stage. Thus, resistance to GvL might be mediated at least in part by resistance to granzyme B induced apoptosis. The human serine proteinase inhibitor 9 (PI-9) is the only known endogenous proteinase inhibitor capable to inhibit proteolytic activity of granzyme B. Therefore, PI-9 may constitute a mechanism of GvL resistance. We investigated a number of human leukemic cell of myeloid and lympoid recognition for their pattern of PI-9 expression and the functional relevance on apoptosis by granzyme B. Methods and Results Following cell lines were investigated: the lymphoblastic cell lines Daudi and Jurkat, the myeloblastic cell lines K562 and U937 and two EBV transformed primary B-Cells (LCL) from volunteer donors. Furthermore we established PI-9 high expressing clones from U937 (U937/PI-9+) that is normally low expressing. PI-9 expression in cell lines was measured by western blot. Daudi, U937/PI-9+ and LCL1 showed a high expression level of PI-9. K562, LCL2 and U937 showed a low expression level of PI-9. Jurkat showed no expression of PI-9.

Cytotoxic granules rich in granzyme B were purified using the human NK cell line YT. Cells were incubated with granzyme B granules and intracellular granzyme B activity was measured using a colorimetric assay based on the substrate IEPD. Granzyme B activity inversely correlated to the levels of PI-9 expression in all cell types. Furthermore, Caspase 3 activity was assessed by intracytoplasmatic staining for active Caspase 3 using a fluorochrome coupled antibody against human active Caspase 3 fragment.

Caspase 3 activity also was directly correlated to granzyme B activity, i.e. negatively correlated to the level of PI-9 expression, respectively.

Discussion Our data show a clear correlation of PI-9 expression levels and inhibition of granzyme B activity in a panel of myeloblastic and lymphoblastic leukemia derived cell lines as well as EBV-transformed B-cells. Furthermore, granzyme B activity was correlated to downstream Caspase 3 activation.

Thus, leukemic cells constitutively expressing PI-9 exhibit intrinsic resistance towards granzyme B dependent Caspase 3 activation and apoptosis induction, respectively.

This mechanism may be involved in resistance to GvL following allogeneic transplantation. Further studies involving primary leukemic cells might reveal if leukemias with high PI-9 expression are at higher risk of relapse post hematopoetic SCT.