Joanne E. Davis

Unlocking the secrets of cytotoxic granule proteins.

Cytotoxic lymphocytes largely comprise CD8+ cytotoxic T cells and natural killer cells and form the major defense of higher organisms against virus‐infected and transformed cells. A key function of cytotoxic lymphocytes is to detect and eliminate potentially harmful cells by inducing them to undergo apoptosis. This is achieved through two principal pathways, both of which require direct but transient contact between the killer cell and its target. The first, involving ligation of TNF receptor‐like molecules such as Fas/CD95 by their cognate ligands, results in mobilization of conventional, programmed cell‐death pathways centered on activation of pro‐apoptotic caspases. This review concentrates on the second pathway, in which the toxic contents of secretory vesicles of the cytotoxic lymphocyte are secreted toward the target cell, and some toxins penetrate into the target cell cytoplasm and nucleus. In addition to invoking a powerful stimulus to caspase activation, this “granule‐exocytosis mechanism” provides a variety of additional strategies for overcoming inhibitors of the caspase cascade that may be elaborated by viruses. The key molecular players in this process are the pore‐forming protein perforin and a family of granule‐bound serine proteases or granzymes. The molecular functions of perforin and granzymes are under intense investigation in many laboratories including our own, and recent advances will be discussed. In addition, this review discusses the evidence pointing to the importance of perforin and granzyme function in pathophysiological situations as diverse as infection with intracellular pathogens, graft versus host disease, susceptibility to transplantable and spontaneous malignancies, lymphoid homeostasis, and the tendency to auto‐immune diseases.

Proapoptotic functions of cytotoxic lymphocyte granule constituents in vitro and in vivo.

Recent advances in our understanding of cytolytic effector mechanisms include the partial characterization of caspase-independent apoptotic pathways triggered by granzymes, a realization of the vital importance of perforin and granzymes in the defence against certain virus infections in vivo and the first description of hereditary immunodeficiency due to disordered perforin expression in humans.

Granzyme A and B‐deficient killer lymphocytes are defective in eliciting DNA fragmentation but retain potent in vivo anti‐tumor capacity

Recent studies have demonstrated that granzymes A and B make an important contribution to the clearance of the orthopoxvirus ectromelia, and in graft versus host disease. To test whether granzymes are generally necessary for lymphocyte‐mediated cytotoxicity in vivo, we assessed the cytotoxic capacity of granzyme A and/or B–deficient lymphocytes in several perforin‐dependent settings. Splenocytes and allogeneic CTL of granzyme A and/or B‐deficient mice were defective for induction of DNA fragmentation, but induced significant membrane damage and target cell death. These results correlated well with the behavior of granzyme A/B‐deficient CTL and NK cells in three different perforin‐dependent tumor models. In a classical assay of NK cell‐mediated rejection, granzyme A and/or B‐deficient mice inoculated with RMA‐S cells were as susceptible to tumor as wild‐type mice. Perforin‐deficient mice were also considerably more susceptible to tumor initiation by methylcholanthrene than granzyme A and/or B‐deficient mice. Furthermore, rejection of the K1735‐melanoma expressing MHC class I and II molecules was mediated by adoptively transferred H‐2b anti‐k CTL from immunized granzyme A and/or B‐deficient mice. In summary, these data suggest that granzymes A and B are not critical for most anti‐tumor effector functions of NK cells and CTL that are perforin mediated.

Anti-viral strategies of cytotoxic T lymphocytes are manifested through a variety of granule-bound pathways of apoptosis induction.

Cytotoxic T cells and natural killer cells together constitute a major defence against virus infection, through their ability to induce apoptotic death in infected cells. These cytolytic lymphocytes kill their targets through two principal mechanisms, and one of these, granule exocytosis, is essential for an effective in vivo immune response against many viruses. In recent years, the authors and other investigators have identified several distinct mechanisms that can induce death in a targeted cell. In the present article, it is postulated that the reason for this redundancy of lethal mechanisms is to deal with the array of anti‐apoptotic molecules elaborated by viruses to extend the life of infected cells. The fate of such a cell therefore reflects the balance of pro‐apoptotic (immune) and anti‐apoptotic (viral) strategies that have developed over eons of evolutionary time.

Purification of natural killer cell cytotoxic granules for assaying target cell apoptosis

We compared two methods originally devised to purify cytoplasmic granules from granulocytes for their capacity to produce cytotoxic granules from natural killer cell lines, suitable for use in target cell apoptosis assays. Both methods utilised nitrogen cavitation to efficiently lyse cells, followed by density gradient fractionation on Percoll to separate the granules from other organelles and granule debris. The first method, originally described by Millard and colleagues, employed DNase I to reduce the viscosity of the initial cell lysate, but the resulting granule fractions were found to contain residual nuclease activity that made them unsuitable for use in apoptosis assays that measure DNA fragmentation. An alternative method described by Borregaard and colleagues utilised a cell relaxation buffer without DNase I. Cytotoxic granules isolated from the NK tumor cell line YT by this protocol were localised predominantly to the densest Percoll fractions, with a density of approximately 1.13 g/ml. These granule fractions were rich in perforin and enzymatically active granzyme B, and induced potent Ca(2+)-dependent lysis and DNA fragmentation of Jurkat cells. Corresponding fractions from non-cytolytic cells, or YT granule extracts incubated with EGTA were unable to mediate significant target cell damage. Cytotoxic granule extracts purified through the Borregaard method were therefore free of nonspecific nuclease activity, and most suitable for studying the mechanism of target cell death induced through the perforin/Ca(2+)-dependent granule pathway.

The anti-cancer drug, phenoxodiol, kills primary myeloid and lymphoid leukemic blasts and rapidly proliferating T cells

The plasma electron transport system is a relatively newly-discovered potential target for anti-leukemia drugs. In this paper Herst and coworkers describe the effects of phenoxodiol, an inhibitor of this pathway on leukemia cell lines and primary as well as on resting and activated T cells. The ability of phenoxodiol to kill rapidly proliferating lymphocytes might make this drug a promising candidate for the treatment of pathologically-activated lymphocytes. Background The redox-active isoflavene anti-cancer drug, phenoxodiol, has previously been shown to inhibit plasma membrane electron transport and cell proliferation and promote apoptosis in a range of cancer cell lines and in anti-CD3/anti-CD28-activated murine splenocytes but not in non-transformed WI-38 cells and human umbilical vein endothelial cells. Design and Methods We determined the effects of phenoxodiol on plasma membrane electron transport, MTT responses and viability of activated and resting human T cells. In addition, we evaluated the effect of phenoxodiol on the viability of leukemic cell lines and primary myeloid and lymphoid leukemic blasts. Results We demonstrated that phenoxodiol inhibited plasma membrane electron transport and cell proliferation (IC50 46 μM and 5.4 μM, respectively) and promoted apoptosis of rapidly proliferating human T cells but did not affect resting T cells. Phenoxodiol also induced apoptosis in T cells stimulated in HLA-mismatched allogeneic mixed lymphocyte reactions. Conversely, non-proliferating T cells in the mixed lymphocyte reaction remained viable and could be restimulated in a third party mixed lymphocyte reaction, in the absence of phenoxodiol. In addition, we demonstrated that leukemic blasts from patients with primary acute myeloid leukemia (n=22) and acute lymphocytic leukemia (n=8) were sensitive to phenoxodiol. The lymphocytic leukemic blasts were more sensitive than the myeloid leukemic blasts to 10 μM phenoxodiol exposure for 24h (viability of 23±4% and 64±5%, respectively, p=0.0002). Conclusions The ability of phenoxodiol to kill rapidly proliferating lymphocytes makes this drug a promising candidate for the treatment of pathologically-activated lymphocytes such as those in acute lymphoid leukemia, or diseases driven by T-cell proliferation such as auto-immune diseases and graft-versus-host disease.

Induction of potent NK cell-dependent anti-myeloma cytotoxic T cells in response to combined mapatumumab and bortezomib

There is increasing evidence that some cancer therapies can promote tumor immunogenicity to boost the endogenous antitumor immune response. In this study, we used the novel combination of agonistic anti-TRAIL-R1 antibody (mapatumumab, Mapa) with low dose bortezomib (LDB) for this purpose. The combination induced profound myeloma cell apoptosis, greatly enhanced the uptake of myeloma cell apoptotic bodies by dendritic cell (DC) and induced anti-myeloma cytotoxicity by both CD8+ T cells and NK cells. Cytotoxic lymphocyte expansion was detected within 24 h of commencing therapy and was maximized when myeloma-pulsed DC were co-treated with low dose bortezomib and mapatumumab (LDB+Mapa) in the presence of NK cells. This study shows that Mapa has two distinct but connected modes of action against multiple myeloma (MM). First, when combined with LDB, Mapa produced powerful myeloma cell apoptosis; secondly, it promoted DC priming and an NK cell-mediated expansion of anti-myeloma cytotoxic lymphocyte (CTL). Overall, this study indicates that Mapa can be used to drive potent anti-MM immune responses.

B cells in GVHD: friend or foe?

To the editor: We were pleased to read the recent review by Shimabukuro-Vornhagen et al, “The role of B cells in the pathogenesis of graft-versus-host disease,” which highlights the importance of B cells after bone marrow transplantation, as B cells have tended to be overlooked as a contributor

Comparison of gene expression and flow cytometry for immune profiling in chronic lymphocytic leukaemia

Understanding how cancer and cancer therapies affect the immune system is integral to the rational application of immunotherapies. Flow cytometry is the gold standard method of peripheral blood immune cell profiling. However, the requirement for viable cells can limit its applicability, especially in studies of retrospective clinical cohorts. We aimed to determine if gene expression, analysed using the NanoString platform, could be used to quantify the immune populations present in cryopreserved peripheral blood mononuclear cell (PBMC) samples from patients with chronic lymphocytic leukaemia. Cell abundance scores derived from gene expression analysis were significantly correlated with the population frequency quantified by flow cytometry for all subsets analysed, including T cells, NK cells and Monocytes. This study demonstrates that gene expression analysis can be applied to cryopreserved PBMC and provides a concordant and complementary understanding of the immune profile to flow cytometry.

Strategies to enhance the graft versus tumour effect after allogeneic haematopoietic stem cell transplantation

Relapse of haematological malignancies after allogeneic haematopoietic stem cell transplant is a major cause of mortality. The immunological mechanisms that may lead to disease relapse may include immunological immaturity prior to reconstitution of the allogeneic immune system, tumour antigen downregulation or promotion of T-cell exhaustion by interactions with the tumour microenvironment. Current therapeutic strategies for post-transplant relapse are limited in their efficacy and alternative approaches are required. In this review, we discuss the mechanisms of T and NK-cell immune evasion that facilitate relapse of haematological malignancies after allogeneic stem cell transplantation, and explore emerging strategies to augment the allogeneic immune system in order to construct a more potent graft versus tumour response.