Granzyme B and tumor cells: An enzymatic death

The ability for cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells to target and eliminate harmful cells in our body is paramount for preventing various diseases1. Central to this is the serine proteinase Granzyme B (GrB), which when released in cytotoxic secretary vesicles alongside the pore-forming protein perforin (PEN) initiate cell death in targeted cells including virally infected and tumor cells. Expanding on this role, GrB has also been shown to be expressed in a plethora of non-cytotoxic cell types including basophils2, mast cells3 and neutrophils4 suggesting a more complex role than once thought. Furthermore, efforts to develop targeted therapies utilizing GrB, while facing roadblocks, has increased interested in the therapeutic harnessing of enzymatic death.

Apart of a family of closely related serine proteases called granzymes GrB is a key component of cytotoxic granules of CTLs and NK cells6,7. In humans there are 5 different granzymes (A. B, H, K and M) and while our understanding of their functions is limited GrB is the most extensively studied family member8. Prior to release Grb exists as an inactive proenzyme that has an additional peptide sequence on its N-terminus. Only after the removal of 2 amino acids by cathepsin C or cathepsin H does Grb become activated and capable of activating death pathways9.

While still debated exactly how, classically Grb is released from CTL’s and NK cells alongside with PEN which creates a pore in the target cells membrane and nucleus allowing GrB to enter10. Once inside GrB can induce cell death via several signaling pathways. For example, GrB can cleave and activate executioner caspases 3 and 7 and initiator caspases 8 and 1010. In this scenario caspase 7 is the most sensitive to GrB, whereas, caspases 3, 8. 10 are only cleaved partially and require additional enzymes11. GrB is also capable of cleaving BID leading to the oligomerization of BAX/BAX and the release of cytochrome c from the mitochondria triggering apoptosis10.

Growing evidence suggests that GrB is also expressed non-cytotoxic cells including B cells1 among others2,3. This has led to possible new roles for GrB including in the early cell-mediated immune response to viral infection and new mechanisms for inducing cell death. For example, B cells can express and secrete GrB after stimulation by the B-cell receptor and interleukin-21 (IL-21)11. Because B cells target in an MHC-independent manner versus CTLs and NK cells this makes them likely to be the first responders in the cases of viral infection which produce IL-21 very early on12. Another key point is that GrB is capable of inducing target cell death independent of PEN since B cells do not express PEN.

Because GrB is effective in killing tumor cells that are resistant to other cytotoxic drugs there is interest in utilizing GrB as part of targeted therapy14. An example of this is with the packaging GrB as part of an immunotoxin complex, but this and other treatment designs have been limited due to several factors5. The main roadblock is the GrB inhibitor serpin B9 which can be overexpressed by tumor cells allowing tumor cells to evade and minimize the impact of GrB15. Another key limitation is that GrB has a highly positive surface charge making it capable of binding non-specifically to negatively-charged cell surfaces8.

Taken together, GrB plays an incredibly important role in the ability for cytotoxic cells like CTLs and NK cells to kill targeted cells. More recent evidence has expanded this to even include traditionally non-cytotoxic cells such as B cells. All of this and more have made GrB an interesting therapeutic option in cancer and other diseases.

Detection of human Granzyme B in FFPE metastatic lymph node by IHC.

Detection of human Granzyme B in FFPE metastatic lymph node by IHC. Antibody: Rabbit anti-Granzyme B recombinant monoclonal [BLR022E] (A700-022). Secondary: HRP-conjugated goat anti-rabbit IgG (A120-101P). Substrate: DAB.

Detection of human Granzyme B by WB of myc-tagged Granzyme A, H, and B overexpressing cell lysate.

Detection of human Granzyme B by WB of myc-tagged Granzyme A, H, and B overexpressing cell lysate. Antibodies: Rabbit anti-Granzyme B recombinant monoclonal [BLR022E] (A700-022). Lower Panel: anti-myc. Secondary: HRP-conjugated goat anti-rabbit IgG (A120-101P).


1. Hagn M, Jahrsdorfer B. 2012. Why do human B cells secrete granzyme B? Insights into a novel B-cell differentiation pathway. 1(8):1368-1375.

2. Tschopp CM, SPiegl N, Didichenko S, Lutmann W, Julius P, Virchow JC, Hack CE, Dahinden CA. 2006. Granzyme B, a novel mediator of allergic inflammation: its induction and release in blood basophils and human asthma. 108(7):2290-2299.

3. Strik MC, de Koning PJ, Kleijmeer MJ, Bladergroen BA, Wolbink AM, Griffith JM, Wouters D, Fukuoka Y, Schwartz LB, Hack CE, van Ham SM, Kummer JA. 2007. Human mast cells produce and release the cytotoxic lymphocyte associated protease granzyme B upon activation. Mol Immunol. 44(14):3462-3472.

4. Wagner C, Iking-Konert C, Denefleh B, Stegmaier S, Hug F, Hansch GM. 2004. Granzyme B and perforin: constitutive expression in human polymorphonuclear neutrophils. 103(3):1099-1104.

5. Hehmann-Titt G, Schiffer S, Berges N, Melmer G, Barth S. 2013. Improving the Therapeutic Potential of Human Granzyme B for Targeted Cancer Therapy. 2:19-49.

6. Hoves S, Trapani JA, Voskoboinik I. 2010. The battlefield of perforin/granzme cell death pathways. J Leukoc Biol. 87(2):237-43.

7. Ewen CL, Kane KP, Bleackley RC. 2012. A quarter century of granzymes. Cell Death Differ. 19(1):28-35.

8. Grossman WJ, Revell PA, Lu ZH, Johnson H, Bredemeyer AJ, Ley TJ. 2003. The orphan granzymes of humans and mice. Opin. Immunol. 15:544-552.

9. Smyth MJ, McGuire MH, Thia KY. 1995. Expression of recombinant human granzyme B. A processing and activation role for dipeptidyl peptidase I. J Immunol. 154(12):6299-6305.

10. Afonina IS, Cullen SP, Martin SJ. 2010. Cytotoxic and non-cytotoxic roles of the CTL/NK protease granzyme B. Immunol Rev. 235(1):105-116.

11. Waterhouse NJ, Sedelies KA, Trapani JA. 2006. Role of Bid-induced mitochondrial outer membrane permeabilization in granzyme B-induced apoptosis. Immunol Cell Biol. 84(1):72-78.

12. Hagn M, Schwesigner E, Ebel V, Sontheimer K, Maier J, Beyer T, Syroets T, Laumonnier Y, Fabricius D, Simmet T, Jahrsdorfer B. 2009. Human B cells secrete granzyme B when recognizing viral antigens in the context of the acute phase cytokine IL-21. J Immunol. 183(3):1838-1845.

13. Holm C, Nyvold CG, Paludan SR, Thomsen AR, Hokland M. 2006. Interleukin-21 mRNA expression during virus infections. 33(1):41-45.

14. Kurschus FC and Jenne DE. 2010. Delivery and therapeutic potential of human granzyme B. Immunol Rev. 235(1):159-171.

15. Tiacci E, Doring C, Brune V, van Noesel CJ, Klapper W, Mechtersheimer G, Falini B, Kuppers R, Hansmann ML. 2012. Analyzing primary Hodgkin and Reed-Sternberg cells to capture the molecular and cellular pathogenesis of classical Hodgkin lymphoma. 120:4609-4620.