Elena Catalán

Perforin Rapidly Induces Plasma Membrane Phospholipid Flip-Flop

The cytotoxic cell granule secretory pathway is essential for host defense. This pathway is fundamentally a form of intracellular protein delivery where granule proteases (granzymes) from cytotoxic lymphocytes are thought to diffuse through barrel stave pores generated in the plasma membrane of the target cell by the pore forming protein perforin (PFN) and mediate apoptotic as well as additional biological effects. While recent electron microscopy and structural analyses indicate that recombinant PFN oligomerizes to form pores containing 20 monomers (20 nm) when applied to liposomal membranes, these pores are not observed by propidium iodide uptake in target cells. Instead, concentrations of human PFN that encourage granzyme-mediated apoptosis are associated with pore structures that unexpectedly favor phosphatidylserine flip-flop measured by Annexin-V and Lactadherin. Efforts that reduce PFN mediated Ca influx in targets did not reduce Annexin-V reactivity. Antigen specific mouse CD8 cells initiate a similar rapid flip-flop in target cells. A lipid that augments plasma membrane curvature as well as cholesterol depletion in target cells enhance flip-flop. Annexin-V staining highly correlated with apoptosis after Granzyme B (GzmB) treatment. We propose the structures that PFN oligomers form in the membrane bilayer may include arcs previously observed by electron microscopy and that these unusual structures represent an incomplete mixture of plasma membrane lipid and PFN oligomers that may act as a flexible gateway for GzmB to translocate across the bilayer to the cytosolic leaflet of target cells.

Protein Kinase C-θ (PKC-θ) in Natural Killer Cell Function and Anti-Tumor Immunity.

The protein kinase C-θ (PKCθ), which is essential for T cell function and survival, is also required for efficient anti-tumor immune surveillance. Natural killer (NK) cells, which express PKCθ, play a prominent role in this process, mainly by elimination of tumor cells with reduced or absent major histocompatibility complex class-I (MHC-I) expression. This justifies the increased interest of the use of activated NK cells in anti-tumor immunotherapy in the clinic. The in vivo development of MHC-I-deficient tumors is much favored in PKCθ−/− mice compared with wild-type mice. Recent data offer some clues on the mechanism that could explain the important role of PKCθ in NK cell-mediated anti-tumor immune surveillance: some studies show that PKCθ is implicated in signal transduction and anti-tumoral activity of NK cells elicited by interleukin (IL)-12 or IL-15, while others show that it is implicated in NK cell functional activation mediated by certain killer-activating receptors. Alternatively, the possibility that PKCθ is involved in NK cell degranulation is discussed, since recent data indicate that it is implicated in microtubule-organizing center polarization to the immune synapse in CD4+ T cells. The implication of PKC isoforms in degranulation has been more extensively studied in cytotoxic T lymphocyte, and these studies will be also summarized.

Granzyme B of cytotoxic T cells induces extramitochondrial reactive oxygen species production via caspase-dependent NADPH oxidase activation.

Induction of reactive oxygen species (ROS) is a hallmark of granzyme B (gzmB)‐mediated pro‐apoptotic processes and target cell death. However, it is unclear to what extent the generated ROS derive from mitochondrial and/or extra‐mitochondrial sources. To clarify this point, we have produced a mutant EL4 cell line, termed EL4‐ρ0, which lacks mitochondrial DNA, associated with a decreased mitochondrial membrane potential and a defective ROS production through the electron transport chain of oxidative phosphorylation. When incubated with either recombinant gzmB plus streptolysin or ex vivo gzmB+ cytotoxic T cells, EL4‐ρ0 cells showed phosphatydylserine translocation, caspase 3 activation, Bak conformational change, cytochrome c release and apoptotic morphology comparable to EL4 cells. Moreover, EL4‐ρ0 cells produced ROS at levels similar to EL4 under these conditions. GzmB‐mediated ROS production was almost totally abolished in both cell lines by the pan‐caspase inhibitor, Z‐VAD‐fmk. However, addition of apocynin, a specific inhibitor of nicotinamide adenine dinucleotide phosphate (NADPH) oxidases, led to a significant reduction of ROS production and cell death only in EL4‐ρ0 but not EL4 cells. These data suggest that gzmB‐induced cell death is accompanied by a caspase‐dependent pathway of extra‐mitochondrial ROS production, most probably through activation of NADPH oxidase.

Granulysin induces apoptotic cell death and cleavage of the autophagy regulator Atg5 in human hematological tumors.

Granulysin is a protein present in the granules of human CTL and NK cells, with cytolytic activity against microbes and tumors. Previous work demonstrated that granulysin caused cell death through mitochondrial damage with release of AIF and cytochrome c. However, the molecular mechanism and, especially, the type of cell death were still not well defined. In the present work we show that granulysin-induced cell death is apoptotic, with phosphatidylserine exposure preceding membrane breakdown and with caspase 3 activation. Granulysin-induced apoptosis is prevented in Jurkat cells over-expressing Bcl-xL or Bcl2, or lacking Bak and Bax or Bim expression, suggesting a central role of the mitochondrial apoptotic pathway. This apoptotic process is initiated by intracellular Ca(2+) increase and mitochondrial ROS generation. We have tested granulysin against other hematological tumor cells such as multiple myeloma cell lines, and cells from B cell chronic lymphocytic leukemia (B-CLL) patients, finding different degrees of sensitivity. We also show that granulysin induces the cleavage of Atg5 in the complex formed with Atg12, without affecting autophagy. In conclusion, granulysin induces apoptosis on hematological tumor cells and on cells from B-CLL patients, opening the door to research on its use as a new anti-tumoral treatment.

Essential complicity of perforin-granzyme and FAS-L mechanisms to achieve tumor rejection following treatment with anti-CD137 mAb

BackgroundTreatment with agonist anti-CD137 (4-1BB) immunostimulatory monoclonal antibodies elicits complete tumor regressions in a number of transplanted hematological and solid malignancies in mice. Rejection is mainly dependent on cytotoxic T lymphocytes (CTL) and IFNγ, although a role for NK cells and dendritic cells has been observed in some tumor models. Rejection of EG7-derived thymomas has been shown to be CTL-dependent but not NK-dependent.FindingsIn this therapeutic setting, we show that both the perforin-granzyme and FasL effector systems are readily expressed by CD8+ T lymphocytes infiltrating the EG7 lymphomas which are undergoing rejection. Using knock-out mice, we demonstrate that both effector cytolytic systems are involved in the execution of complete immune rejections against EG7 established tumors. In accordance, EG7 tumor cells were susceptible in vitro to both killing mechanisms acting in a synergistic fashion.ConclusionsCD137-elicited rejection of EG7-derived tumors involves the interplay of at least two final effector cytolytic mechanisms that act in cooperation.

MHC-I modulation due to changes in tumor cell metabolism regulates tumor sensitivity to CTL and NK cells

Tumor cells have a tendency to use glucose fermentation to obtain energy instead of mitochondrial oxidative phosphorylation (OXPHOS). We demonstrated that this phenotype correlated with loss of ERK5 expression and with reduced MHC class I expression. Consequently, tumor cells could evade cytotoxic T lymphocyte (CTL)-mediated immune surveillance, but also increase their sensitivity to natural killer (NK) cells. These outcomes were evaluated using two cellular models: leukemic EL4 cells and L929 transformed fibroblasts and their derived ρ° cell lines, which lack mitochondrial DNA. We have also used a L929 cell sub-line that spontaneously lost matrix attachment (L929dt), reminiscent of metastasis generation, that also downregulated MHC-I and ERK5 expression. MHC-I expression is lower in ρ° cells than in the parental cell lines, but they were equally sensitive to CTL. On the contrary, ρ° cells were more sensitive to activated NK cells than parental cells. On the other hand, L929dt cells were resistant to CTL and NK cells, showed reduced viability when forced to perform OXPHOS, and surviving cells increased MHC-I expression and became sensitive to CTL. The present results suggest that when the reduction in MHC-I levels in tumor cells due to glycolytic metabolism is partial, the increase in sensitivity to NK cells seems to predominate. However, when tumor cells completely lose MHC-I expression, the combination of treatments that increase OXPHOS with CTL-mediated immunotherapy could be a promising therapeutic approach.

HDAC6 regulates the dynamics of lytic granules in cytotoxic T lymphocytes

ABSTRACT HDAC6 is a tubulin deacetylase involved in many cellular functions related to cytoskeleton dynamics, including cell migration and autophagy. In addition, HDAC6 affects antigen-dependent CD4+ T cell activation. In this study, we show that HDAC6 contributes to the cytotoxic function of CD8+ T cells. Immunization studies revealed defective cytotoxic activity in vivo in the absence of HDAC6. Adoptive transfer of wild-type or Hdac6−/− CD8+ T cells to Rag1−/− mice demonstrated specific impairment in CD8+ T cell responses against vaccinia infection. Mechanistically, HDAC6-deficient cytotoxic T lymphocytes (CTLs) showed defective in vitro cytolytic activity related to altered dynamics of lytic granules, inhibited kinesin-1–dynactin-mediated terminal transport of lytic granules to the immune synapse and deficient exocytosis, but not to target cell recognition, T cell receptor (TCR) activation or interferon (IFN)γ production. Our results establish HDAC6 as an effector of the immune cytotoxic response that acts by affecting the dynamics, transport and secretion of lytic granules by CTLs. Summary: HDAC6 is required for the cytolytic function of CD8 T cells in vivo and in vitro by controlling lytic granule dynamics.

Mouse cytotoxic T cell-derived granzyme B activates the mitochondrial cell death pathway in a Bim-dependent fashion.

Background: Cytotoxic T cells employ perforin and granzyme B to kill tumor cells. Results: Bim-deficient 3T9-transformed MEF cells are resistant to gzmB-induced apoptosis. Conclusion: The mitochondrial apoptotic pathway activated via Bim is critically involved in apoptosis induced by mouse granzyme B. Significance: Learning how granzyme B induces apoptosis in different tumor cell types will help to predict and increase the efficacy of cancer immunotherapy. Cytotoxic T cells (Tc) use perforin and granzyme B (gzmB) to kill virus-infected cells and cancer cells. Recent evidence suggests that human gzmB primarily induces apoptosis via the intrinsic mitochondrial pathway by either cleaving Bid or activating Bim leading to the activation of Bak/Bax and subsequent generation of active caspase-3. In contrast, mouse gzmB is thought to predominantly induce apoptosis by directly processing pro-caspase-3. However, in certain mouse cell types gzmB-mediated apoptosis mainly occurs via the mitochondrial pathway. To investigate whether Bim is involved under the latter conditions, we have now employed ex vivo virus-immune mouse Tc that selectively kill by using perforin and gzmB (gzmB+Tc) as effector cells and wild type as well as Bim- or Bak/Bax-deficient spontaneously (3T9) or virus-(SV40) transformed mouse embryonic fibroblast cells as targets. We show that gzmB+Tc-mediated apoptosis (phosphatidylserine translocation, mitochondrial depolarization, cytochrome c release, and caspase-3 activation) was severely reduced in 3T9 cells lacking either Bim or both Bak and Bax. This outcome was related to the ability of Tc cells to induce the degradation of Mcl-1 and Bcl-XL, the anti-apoptotic counterparts of Bim. In contrast, gzmB+Tc-mediated apoptosis was not affected in SV40-transformed mouse embryonic fibroblast cells lacking Bak/Bax. The data provide evidence that Bim participates in mouse gzmB+Tc-mediated apoptosis of certain targets by activating the mitochondrial pathway and suggest that the mode of cell death depends on the target cell. Our results suggest that the various molecular events leading to transformation and/or immortalization of cells have an impact on their relative resistance to the multiple gzmB+Tc-induced death pathways.

IFNα signaling through PKC-θ is essential for antitumor NK cell function

We have previously shown that the development of a major histocompatibility complex class I (MHC-I)-deficient tumor was favored in protein kinase C-θ knockout (PKC-θ−/−) mice compared to that occurring in wild-type mice. This phenomenon was associated with scarce recruitment of natural killer (NK) cells to the tumor site, as well as impaired NK cell activation and reduced cytotoxicity ex vivo. Poly-inosinic:cytidylic acid (poly I:C) treatment activated PKC-θ in NK cells depending on the presence of a soluble factor produced by a different splenocyte subset. In the present work, we sought to analyze whether interleukin-15 (IL-15) and/or interferon-α (IFNα) mediate PKC-θ-dependent antitumor NK cell function. We found that IL-15 improves NK cell viability, granzyme B expression, degranulation capacity and interferon-γ (IFNγ) secretion independently of PKC-θ. In contrast, we found that IFNα improves the degranulation capability of NK cells against target cancer cells in a PKC-θ-dependent fashion both ex vivo and in vivo. Furthermore, IFNα induces PKC-θ auto-phosphorylation in NK cells, in a signal transduction pathway involving both phosphatidylinositol-3-kinase (PI3K) and phospholipase-C (PLC) activation. PKC-θ dependence was further implicated in IFNα-induced transcriptional upregulation of chemokine (C-X-C motif) ligand 10 (CXCL10), a signal transducer and activator of transcription-1 (STAT-1)-dependent target of IFNα. The absence of PKC-θ did not affect IFNα-induced STAT-1 Tyr701 phosphorylation but affected the increase in STAT-1 phosphorylation on Ser727, attenuating CXCL10 secretion. This connection between IFNα and PKC-θ in NK cells may be exploited in NK cell-based tumor immunotherapy.

Antigen-specific primed cytotoxic T cells eliminate tumour cells in vivo and prevent tumour development, regardless of the presence of anti-apoptotic mutations conferring drug resistance

Cytotoxic CD8+ T (Tc) cells are the main executors of transformed and cancer cells during cancer immunotherapy. The latest clinical results evidence a high efficacy of novel immunotherapy agents that modulate Tc cell activity against bad prognosis cancers. However, it has not been determined yet whether the efficacy of these treatments can be affected by selection of tumoural cells with mutations in the cell death machinery, known to promote drug resistance and cancer recurrence. Here, using a model of prophylactic tumour vaccination based on the LCMV-gp33 antigen and the mouse EL4 T lymphoma, we analysed the molecular mechanism employed by Tc cells to eliminate cancer cells in vivo and the impact of mutations in the apoptotic machinery on tumour development. First of all, we found that Tc cells, and perf and gzmB are required to efficiently eliminate EL4.gp33 cells after LCMV immunisation during short-term assays (1–4 h), and to prevent tumour development in the long term. Furthermore, we show that antigen-pulsed chemoresistant EL4 cells overexpressing Bcl-XL or a dominant negative form of caspase-3 are specifically eliminated from the peritoneum of infected animals, as fast as parental EL4 cells. Notably, antigen-specific Tc cells control the tumour growth of the mutated cells, as efficiently as in the case of parental cells. Altogether, expression of the anti-apoptotic mutations does not confer any advantage for tumour cells neither in the short-term survival nor in long-term tumour formation. Although the mechanism involved in the elimination of the apoptosis-resistant tumour cells is not completely elucidated, neither necroptosis nor pyroptosis seem to be involved. Our results provide the first experimental proof that chemoresistant cancer cells with mutations in the main cell death pathways are efficiently eliminated by Ag-specific Tc cells in vivo during immunotherapy and, thus, provide the molecular basis to treat chemoresistant cancer cells with CD8 Tc-based immunotherapy.