The granzyme b/serpinb9 nexus controls lymphocyte viability following lysosomal membrane permeabilisation

Cytotoxic lymphocytes (CLs), which include natural killer (NK) cells and cytotoxic T lymphocytes (CTL), contain lysosome-related organelles (LROs) that perform the normal degradative functions of the lysosome, in addition to storage and release of powerful cytotoxins employed to kill virally infected or abnormal cells. Of these cytotoxins, granzyme B (GrB) is the most potent. GrB has also been implicated in activation (restimulation)-induced cell death of both NK and T cells, but the underlying mechanism and its regulation are unclear. SERPINB9 (Sb9) is an intracellular serine protease inhibitor which is most highly expressed in the cytosol and nucleus of CLs. It is a very specific and efficient inhibitor of GrB. Sb9 is thought to protect the CL from any mis-directed GrB, thus prolonging the life of the lymphocyte. Work described in this thesis shows that restimulation of previously activated human or mouse lymphocytes induces lysosomal membrane permeabilisation (LMP), followed by GrB release from LROs into the CL cytosol: if not controlled by Sb9, cytosolic GrB induces cell death. LMP induced by model lysosomal stressors indicates that GrB release is both time- and concentration-dependent. Also, the effectiveness of Sb9 decreases as LMP increases, until eventually cytosolic GrB overwhelms the serpin. In CLs from gene-targeted mice, GrB is the major effector of LMP-mediated death in T cells but additional effectors are released in NK cells, making GrB redundant. Limited LMP also occurs constitutively in proliferating lymphocytes and in NK cells engaged with targets in vitro. In Ectromelia virus-infected lymph nodes, working NK cells lacking Sb9 are more susceptible to OrB-mediated death. Taken together, these data show that a basal level of LMP occurs in proliferating and activated lymphocytes, and is increased on restimulation. LMP releases GrB from LROs into the cytoplasm and its ensuing interaction with Sb9 dictates whether or not the lymphocyte survives. Thus, the GrB-Sb9 nexus may represent a mechanism oflimiting lymphocyte lifespan.