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New drugs that precisely target the immune mechanisms critical for cytotoxic T lymphocyte (CTL) and natural killer (NK) cell driven pathologies are desperately needed. In this perspective, we explore the cytolytic protein perforin as a target for therapeutic intervention. Perforin plays an indispensable role in CTL/NK killing and controls a range of immune pathologies, while being encoded by a single copy gene with no redundancy of function. An immunosuppressant targeting this protein would provide the first-ever therapy focused specifically on one of the principal cell death pathways contributing to allotransplant rejection and underpinning multiple autoimmune and postinfectious diseases. No drugs that selectively block perforin-dependent cell death are currently in clinical use, so this perspective will review published novel small molecule inhibitors, concluding with in vivo proof-of-concept experiments performed in mouse models of perforin-mediated immune pathologies that provide a potential pathway toward a clinically useful therapeutic agent.

The anti-tumor efficacy of adoptively transferred T cells requires their in vivo persistence and memory polarization. It is unknown if human chimeric antigen receptor (CAR)-expressing T cells can also undergo memory polarization. We examined the functional status of CAR CD8(+) T cells, re-directed to Lewis Y antigen (LeY-T), throughout a period of ex vivo expansion. Immediately before culture CD8(+) T cells comprised a mixture of phenotypes including naive (CD45RA(+)/CCR7(+)/CD27(+)/CD28(+)/perforin-), central memory (CM, CD45RA(-)/CCR7(lo)/CD27(+)/CD28(+)/perforin(lo)), effector memory (EM, CD45RA(-)/CCR7(-)/CD27(+)/CD28(+)/perforin(mod)) and effector (Eff, CD45RA(+)/CCR7(-)/CD27(-)/CD28(-)/perforin(hi)) cells. After transduction and expansion culture of peripheral blood mononuclear cells from normal donors or multiple myeloma patients, CD8(+) LeY-T cells polarized to EM- and CM-like phenotype. CD8(+) LeY-T cells differed from starting CD8(+) CM and EM T cells in that CD27, but not CD28, was downregulated. In addition, CD8(+) LeY-T cells expressed high levels of perforin, similar to starting CD8(+) Eff. CD8(+) LeY-T cells also showed hallmarks of both memory and Eff function, underwent homeostatic proliferation in response to interleukin (IL)-15, and showed interferon (IFN)-γ production and cytotoxicity in response to Le-Y antigen on OVCAR-3 (human ovarian adenocarcinoma) cells. This study confirms CD8(+) LeY-T cells have a CM- and EM-like phenotype and heterogeneous function consistent with potential to persist in vivo after adoptive transfer.

The induction of cell death by cytotoxic T-lymphocytes (CTL) or natural killer (NK) cells is one of the main ways by which higher organisms protect themselves from rogue cells, including those infected by a virus, or posing a risk of cancer. Considering the rapidity of viral replication and spread to uninfected cells, CTL and NK are extremely efficient killers. This is at least partly due to the variety of pathways that these cytolytic lymphocytes (CL) can use to ensure the death of a cell. Primarily, CL utilize two independently initiated pathways involving either ligation of death receptors or perforin mediated trafficking of granzyme B to the target cell cytosol to activate a family of death proteases (caspases) in the target cell. The caspases then orchestrate the orderly dismantling of that cell by cleavage of a set of critical substrates. If caspases are inactivated, due either to mutations in proteins that signal their activation or direct inhibition by a viral gene product, CL can utilize a caspase-independent pathway to ensure the death of the target cell. Here we will discuss the mechanisms by which these stellar killers achieve their goal.

The structurally related TCR-zeta and Fc receptor for IgE (Fc epsilon RI)-gamma are critical signaling components of the TCR and Fc epsilon RI, respectively. Although chimeric Ab receptors containing zeta and gamma signaling chains have been used to redirect CTL to tumors, a direct comparison of their relative efficacy has not previously been undertaken. Here, in naive T lymphocytes, we compare the signaling capacities of the zeta and gamma subunits within single-chain variable domain (scFv) chimeric receptors recognizing the carcinoembryonic Ag (CEA). Using a very efficient retroviral gene delivery system, high and equivalent levels of scFv-zeta and scFv-gamma receptors were expressed in T cells. Despite similar levels of expression and Ag-specific binding to colon carcinoma target cells, ligation of scFv-anti-CEA-zeta chimeric receptors on T cells resulted in greater cytokine production and direct cytotoxicity than activation via scFv-anti-CEA-gamma receptors. T cells expressing scFv-zeta chimeric receptors had a greater capacity to control the growth of human colon carcinoma in scid/scid mice or mouse colon adenocarcinoma in syngeneic C57BL/6 mice. Overall, these data are the first to directly compare and definitively demonstrate the enhanced potency of T cells activated via the zeta signaling pathway.

Destruction of tumor cells is a key function of lymphocytes, but the molecular processes driving it are unclear. Analysis of signal molecules indicated that mitogen-activated protein kinase (MAPK)/extracellular regulated kinase 2 critically controlled lytic function in human NK cells. We now have evidence to indicate that target ligation triggers a Ras-independent MAPK pathway that is required for lysis of the ligated tumor cell. Target engagement caused NK cells to rapidly activate MAPK within 5 min, and PD098059 effectively blocked both MAPK activation and tumoricidal function in NK cells. Target engagement also rapidly activated Ras, detected as active Ras-GTP bound to GST-Raf-RBD, a GST fusion protein linked to the Raf protein fragment containing the Ras-GTP binding domain. However, Ras inactivation by pharmacological disruption with the farnesyl transferase inhibitor, FTI-277, had no adverse effect on the ability of NK cells to lyse tumor cells or to express MAPK activation upon target conjugation. Notably, MAPK inactivation with PD098059, but not Ras inactivation with FTI-277, could interfere with perforin and granzyme B polarization within NK cells toward the contacted target cell. Using vaccinia delivery of N17 Ras into NK cells, we demonstrated that IL-2 activated a Ras-dependent MAPK pathway, while target ligation used a Ras-independent MAPK pathway to trigger lysis in NK cells.

Immune surveillance by cytotoxic lymphocytes against cancer has been postulated for decades, but direct evidence for the role of cytotoxic lymphocytes in protecting against spontaneous malignancy has been lacking. As the rejection of many experimental cancers by cytotoxic T lymphocytes and natural killer cells is dependent on the pore-forming protein perforin (pfp), we examined pfp-deficient mice for increased cancer susceptibility. Here we show that pfp-deficient mice have a high incidence of malignancy in distinct lymphoid cell lineages (T, B, NKT), indicating a specific requirement for pfp in protection against lymphomagenesis. The susceptibility to lymphoma was accentuated by simultaneous lack of expression of the p53 gene, mutations in which also commonly predispose to human malignancies, including lymphoma. In contrast, the incidence and age of onset of sarcoma was unaffected in p53-deficient mice. Pfp-deficient mice were at least 1,000-fold more susceptible to these lymphomas when transplanted, compared with immunocompetent mice in which tumor rejection was controlled by CD8(+) T lymphocytes. This study is the first that implicates direct cytotoxicity by lymphocytes in regulating lymphomagenesis.

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.

The redirection of autologous lymphocytes to predefined tumor target Ags has considerable potential for the immunotherapeutic treatment of cancer; however, robust experimental systems for comparing various approaches have not been developed. Herein, we have generated a single chain variable domain anti-carcinoembryonic Ag (CEA) Fcepsilon receptor I gamma-chain fusion (scFv anti-CEA) receptor and demonstrated high-level expression of this chimeric receptor in naive mouse T lymphocytes by retroviral gene transduction. These gene-modified CTL were able to lyse CEA+ targets and secrete high levels of IFN-gamma following Ag stimulation. Depletion studies demonstrated that specific tumor cell cytotoxicity was mediated by gene-modified CD8+ T cells. Importantly, in increasingly stringent tests of efficacy in vivo, transduced CTL were sequentially shown to reject CEA+ colon carcinoma cells in a Winn assay and then reject established s.c. colon carcinoma in scid or syngeneic mice. Furthermore, using gene-targeted and scFv anti-CEA receptor-transduced donor CTL, perforin and IFN-gamma were demonstrated to be absolutely critical for the eradication of colon carcinoma in mice. In summary, we have developed a highly efficient gene transfer system for evaluating chimeric receptor expression in cytotoxic lymphocytes. This series of experiments has revealed the utility of scFv anti-CEA chimeras in providing mouse T cells the capacity to reject colon carcinoma in an Ag- and perforin-specific manner.

Natural tumor surveillance capabilities of the host were investigated in six different mouse tumor models where endogenous interleukin (IL)-12 does or does not dictate the efficiency of the innate immune response. Gene-targeted and lymphocyte subset-depleted mice were used to establish the relative importance of natural killer (NK) and NK1.1(+) T (NKT) cells in protection from tumor initiation and metastasis. In the models examined, CD3(-) NK cells were responsible for tumor rejection and protection from metastasis in models where control of major histocompatibility complex class I-deficient tumors was independent of IL-12. A protective role for NKT cells was only observed when tumor rejection required endogenous IL-12 activity. In particular, T cell receptor Jalpha281 gene-targeted mice confirmed a critical function for NKT cells in protection from spontaneous tumors initiated by the chemical carcinogen, methylcholanthrene. This is the first description of an antitumor function for NKT cells in the absence of exogenously administered potent stimulators such as IL-12 or alpha-galactosylceramide.

In this study we directly compared the in vitro responses of T-cells from normal donors and melanoma patients to Melan-A27-35 and Melan-A26-35. These peptides have been previously used in peptide-based vaccination studies. Following three stimulations with peptide-pulsed antigen-presenting cells in vitro, Melan-A-specific cytolytic T-lymphocytes (CTLs) were generated from seven of 20 subjects; two of the seven subjects responded reproducibly to both Melan-A27-35 and Melan-A26-35, three to only Melan-A27-35 and two to only Melan-A26-35. However, CTLs generated with either Melan-A27-35 or Melan-A26-35 showed cross recognition, and both types of CTL could recognize naturally processed antigen displayed on HLA-A2+ tumour cells. Furthermore, Melan-A-specific CTLs could also be generated by stimulating peripheral blood mononuclear cells with autologous melanoma cells. Our results suggest that some subjects may have a bias in their CTL repertoire which favours the generation of Melan-A27-35 specific CTLs, while others may favour Melan-A26-35 specific CTLs. It is also likely that CTL precursors capable of detecting both peptides may have different affinities to the two Melan-A peptides. Since it is difficult to predict the CTL responses to Melan-A peptide in a given individual, we suggest vaccinating with both Melan-A27-35 and Melan-A26-35 peptides in clinical trials.

A chimeric receptor, consisting of the single-chain variable (scFv) domains of an anti-erbB-2 mAb linked via a CD8 membrane-proximal hinge to the Fc receptor gamma chain, was expressed in the mouse cytotoxic T lymphocyte (CTL) hybridoma cell line, MD45. This cell line was grafted with the additional specificity to recognise and bind erbB-2-expressing breast carcinoma target cells T47D, MCF-7 and BT-20 in a non-MHC-restricted manner. Tumour cell lysis was antigen-specific since erbB-2-negative tumours were insensitive to lysis by MD45-scFv-anti-erbB-2-gamma clones, and lysis of erbB-2+ tumour targets was inhibited in the presence of an anti-erbB-2 mAb. Furthermore, target cell death correlated with the level of chimeric receptor expression on the effector MD45 subclones. Redirected MD45 CTL utilised Fas ligand to induce target cell death since soluble Fas-Fc fusion protein completely inhibited cytolysis. The sensitivity of tumour target cells to Fas ligand was further enhanced by treating them with interferon-gamma, a regulator of Fas and downstream signalling components of the Fas pathway. Overall, this study has demonstrated the requirement for successful activation of Fas ligand function in conjunction with cytokine treatment for effective lysis of breast carcinoma target cells mediated by redirected CTL.

In the MD45 mouse cytotoxic T lymphocyte (CTL) hybridoma cell line, we have expressed a chimeric receptor, consisting of the single-chain variable domains (scFv) of anti-carcinoma embryonic antigen (CEA) mAb linked to Fcgamma receptor (FcgammaR) chain via a CD8 hinge. Transfected MD45 subclones lysed CEA-positive human colon carcinoma cell lines in an antigen-specific and FasL-dependent manner. The degree of lysis correlated with the level of chimeric receptor expressed on transduced MD45 subclones. The requirement for an intact Y65TGL motif in the signaling gamma chain suggested that interaction of the chimeric receptor with target cell CEA induced the cytotoxicity of MD45-scFv subclones. However, MD45 expressing a Y65F mutant chimera still displayed minor levels of lysis following PMA stimulation, suggesting that PMA could bypass gamma chain induction of functional FasL. Pretreatment of Fas-resistant CEA-positive colon carcinoma target cells with IFN-gamma increased their sensitivity of MD45-scFv subclones and FasL-mediated lysis. This study has demonstrated the successful activation of FasL function via a chimeric receptor introduced into lymphocytes and the susceptibility of human colon carcinoma to combined cytokine and CTL treatment.

Cytotoxic T cells (CTL) represent the major defense mechanism against the spread of virus infection. It is believed that the pore-forming protein, perforin, facilitates the entry of a series of serine proteases (particularly granzyme B) into the target cell which ultimately leads to DNA fragmentation and apoptosis. We demonstrate here that during CTL-mediated cytolysis the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs), an enzyme implicated in the repair of double strand breaks in DNA, is specifically cleaved by an interleukin (IL)-1 beta-converting enzyme (ICE)-like protease. A serine protease inhibitor, 3,4-dichloroisocoumarin (DCl), which is known to block granzyme B activity, inhibited CTL-induced apoptosis and prevented the degradation of DNA-PKcs in cells but failed to prevent the degradation of purified DNA-PKcs by CTL extracts. However, Tyr-Val-Ala-Asp-CH2Cl (YVAD-CMK) and other cysteine protease inhibitors prevented the degradation of purified DNA-PKcs by CTL extracts. Furthermore, incubation of DNA-PKcs with granzyme B did not produce the same cleavage pattern observed in cells undergoing apoptosis and when this substrate was incubated with either CTL extracts or the ICE-like protease, CPP32. Sequence analysis revealed that the cleavage site in DNA-PKcs during CTL killing was the same as that when this substrate was exposed to CPP32. This study demonstrates for the first time that the cleavage of DNA-PKcs in this intact cell system is exclusively due to an ICE-like protease.

One mechanism used by cytotoxic T cells and natural killer cells to kill target cells involves synergy between the pore-forming protein, perforin, and a serine protease termed granzyme B, both constituents of the cytoplasmic granules of cytolytic lymphocytes. Exposing susceptible cells to perforin and granzyme B results in apoptosis, the morphological consequences of which are most clearly seen in the nucleus. It is conventionally accepted that perforin acts by perforating the target cell membrane; however, the site and mode of action of granzyme B are unknown. We have addressed this issue using Western blotting, proteolytic assays, and confocal laser scanning microscopy to demonstrate that purified human granzyme B can be taken up in large amounts and bound within nuclei. By contrast, perforin and nongranzyme serine proteases did not undergo nuclear uptake. Both unglycosylated human granzyme B (26 kDa) and that bearing high mannose glycosylation (32 kDa) were internalized and bound within nuclei, but forms greater than 32 kDa with complex carbohydrate addition were excluded. The uptake of granzymes was not dependent on net charge, as nuclei absorbed similar quantities of granzyme B at neutral pH and through a range of basic pHs but did not take up other very basic serine proteases such as the mouse mast cell protease 5. Confocal laser scanning microscopy indicated nuclear and nucleolar accumulation of fluoresceinated granzyme B by isolated nuclei. Measurement of the kinetics of nuclear import using an in vitro nuclear transport assay indicated maximal levels of nuclear accumulation of granzyme about 2.5-fold above those in the cytoplasm and nucleolar accumulation a further 3-4-fold higher. Nuclear and nucleolar accumulation were exceedingly rapid, reaching half-maximal levels within 3.3 and 7.5 min, respectively, implying that nuclear accumulation probably occurs prior to transport to the nucleolus. Our observations may provide a mechanism explaining how aspartate-specific cell death proteases access the nuclear substrate poly(ADP-ribose) polymerase, the cleavage of which is an early event in apoptosis.

The vectorial secretion of the contents of highly specialized cytoplasmic granules is of pivotal importance to the killing by cytotoxic T cells and natural killer cells. The purification and biochemical characterization of some of the granule constituents, in particular the pore-forming protein perforin, had engendered the notion that the killing of cellular targets was largely an osmotic phenomenon analogous to the insult delivered by complement attack. However, the apparent absence of membrane perforation in various examples of lymphocyte-mediated killing, and the observation that perforin alone could not account for apoptosis associated with programmed cell death, suggested that perforin activity represented, at best, only a part of the whole mechanism. More recently, the characterization of a large family of granule serine proteases (granzymes) has provided evidence that these molecules may collaborate in the killing process by inducing a 'suicide' pathway in target cells, resulting in DNA fragmentation. However, the serine proteases are inactive alone, their natural substrates have not been defined and they require access into the target cell cytoplasm via perforin-induced pores to exert their deleterious effects. Thus, we propose that the cytotoxic granule-mediated mechanism comprises at least two interdependent arms, perforin and serine proteases, that together are capable of inflicting cell death by osmotic shock and/or nuclear collapse.