Francesca Luciani

CD95 (APO‐1/Fas) linkage to the actin cytoskeleton through ezrin in human T lymphocytes: a novel regulatory mechanism of the CD95 apoptotic pathway

CD95 (APO‐1/Fas) is a member of the tumor necrosis factor receptor family, which can trigger apoptosis in a variety of cell types. However, little is known of the mechanisms underlying cell susceptibility to CD95‐mediated apoptosis. Here we show that human T cells that are susceptible to CD95‐mediated apoptosis, exhibit a constitutive polarized morphology, and that CD95 colocalizes with ezrin at the site of cellular polarization. In fact, CD95 co‐immunoprecipitates with ezrin exclusively in lymphoblastoid CD4+ T cells and primary long‐term activated T lymphocytes, which are prone to CD95‐mediated apoptosis, but not in short‐term activated T lymphocytes, which are refractory to the same stimuli, even expressing equal levels of CD95 on the cell membrane. Pre‐treatment with ezrin antisense oligonucleotides specifically protected from the CD95‐mediated apoptosis. Moreover, we show that the actin cytoskeleton integrity is essential for this function. These findings strongly suggest that the CD95 cell membrane polarization, through an ezrin‐mediated association with the actin cytoskeleton, is a key intracellular mechanism in rendering human T lymphocytes susceptible to the CD95‐mediated apoptosis.

Immune Surveillance Properties of Human NK Cell-Derived Exosomes

Exosomes are nanovesicles released by normal and tumor cells, which are detectable in cell culture supernatant and human biological fluids, such as plasma. Functions of exosomes released by “normal” cells are not well understood. In fact, several studies have been carried out on exosomes derived from hematopoietic cells, but very little is known about NK cell exosomes, despite the importance of these cells in innate and adaptive immunity. In this paper, we report that resting and activated NK cells, freshly isolated from blood of healthy donors, release exosomes expressing typical protein markers of NK cells and containing killer proteins (i.e., Fas ligand and perforin molecules). These nanovesicles display cytotoxic activity against several tumor cell lines and activated, but not resting, immune cells. We also show that NK-derived exosomes undergo uptake by tumor target cells but not by resting PBMC. Exosomes purified from plasma of healthy donors express NK cell markers, including CD56+ and perforin, and exert cytotoxic activity against different human tumor target cells and activated immune cells as well. The results of this study propose an important role of NK cell-derived exosomes in immune surveillance and homeostasis. Moreover, this study supports the use of exosomes as an almost perfect example of biomimetic nanovesicles possibly useful in future therapeutic approaches against various diseases, including tumors.

Effect Of Human Natural Killer and γδ T Cells on the Growth of Human Autologous Melanoma Xenografts in SCID Mice

Natural killer (NK) cells were first identified for their ability to kill tumor cells of different origin in vitro. Similarly, γδ T lymphocytes display strong cytotoxic activity against various tumor cell lines. However, the ability of both the NK and γδ cells to mediate natural immune response against human malignant tumors in vivo is still poorly defined. Severe combined immunodeficient (SCID) mice have been successfully engrafted with human tumors. In this study, the antitumor effect of local as well as of systemic treatments based on NK cells or Vδ1 or Vδ2 γ/δ T lymphocytes against autologous melanoma cells was investigated in vivo. The results show that all three of the populations were effective in preventing growth of autologous human melanomas when both tumor and lymphoid cells were s.c. inoculated at the same site. However, when lymphoid cells were infused i.v., only NK cells and Vδ1 γ/δ T lymphocytes could either prevent or inhibit the s.c. growth of autologous melanoma. Accordingly, both NK cells and Vδ1 γδ T lymphocytes could be detected at the s.c. tumor site. In contrast, Vδ2 γδ T lymphocytes were only detectable in the spleen of the SCID mice. Moreover, NK cells maintained their inhibitory effect on tumor growth even after discontinuation of the treatment. Indeed they were present at the tumor site for a longer period. These data support the possibility to exploit NK cells and Vδ1 γδ T lymphocytes in tumor immunotherapy. Moreover, our study emphasizes the usefulness of human tumor/SCID mouse models for preclinical evaluation of immunotherapy protocols against human tumors.

Potent phagocytic activity discriminates metastatic and primary human malignant melanomas: a key role of ezrin.

Features of phagocytosis have been observed in human tumors, but the phagocytic apparatus of tumor cells and the mechanism(s) underlying this phenomenon have yet to be defined. To address the phenomenon of phagocytosis, its underlying mechanism(s), and its possible role in tumor biology, we used human melanoma cells as a prototypic model. Our results showed that a process of phagocytosis of apoptotic cells occurs in vivo in human melanoma. This finding was consistent with evidence that human melanoma cells in vitro express all of the known lysosomal and phagocytic markers on their cytoplasmic vesicles and that a process of phagocytosis occurs in these vesicles. However, exclusively human melanoma cells deriving from metastatic lesions possess an efficient phagocytic machinery responsible for a macrophage-like activity against latex beads, yeast, and apoptotic cells of different origins, which was comparable to that of human primary macrophages. Moreover, the actin-binding protein ezrin was expressed on phagocytic vacuoles of melanoma cells and of cells deriving from a human adenocarcinoma; both treatment with cytochalasin B and specific inhibition of ezrin synthesis strongly affected the phagocytic activity of melanoma cells. This suggests that the association with the actin cytoskeleton is a crucial requirement for the development of this phenomenon. Hence our data provide evidence for a potent phagocytic activity exerted by metastatic melanoma cells possibly involved in determining the level of aggressiveness of human melanoma. This suggests that the assessment of phagocytic activity may be exploited as a new tool to evaluate the malignancy of human melanoma. Moreover, our data suggest that gene therapy or drug treatments aimed at inhibiting actin assembly to the phagosomal membranes may be proposed as a new strategy for the control of tumor aggressiveness.

Identification and Relevance of the CD95-binding Domain in the N-terminal Region of Ezrin

The CD95 (Fas/APO-1) linkage to the actin cytoskeleton through ezrin is an essential requirement for susceptibility to the CD95-mediated apoptosis in CD4+ T cells. We have previously shown that moesin was not involved in the binding to CD95. Here we further support the specificity of the ezrin/CD95 binding, showing that radixin did not bind CD95. The ezrin region specifically and directly involved in the binding to CD95 was located in the middle lobe of the ezrin FERM domain, between amino acids 149 and 168. In this region, ezrin, radixin, and moesin show 60–65% identity, as compared with the 86% identity in the whole FERM domain. Transfection of two different human cell lines with a green fluorescent protein-tagged ezrin mutated in the CD95-binding epitope, induced a marked inhibition of CD95-mediated apoptosis. In these cells, the mutated ezrin did not co-localize or co-immunoprecipitate with CD95. Further analysis showed that the mutated ezrin, while unable to bind CD95, was fully able to bind actin, thus preventing the actin linkage to CD95. Altogether, our results support the specificity of ezrin in the association to CD95 and the importance of the ezrin-to-CD95 linkage in CD95-mediated apoptosis. Moreover, this study suggests that a major role of ezrin is to connect CD95 to actin, thus allowing the CD95 polarization on the cells and the occurrence of the following multiple cascades of the CD95 pathway.

Adoptive transfer of an anti-MART-127–35-specific CD8+ T cell clone leads to immunoselection of human melanoma antigen-loss variants in SCID mice

The identification of appropriate mouse models could be useful in carefully evaluating the actual role of the in vivo development of antigen‐loss variants during antigen‐specific vaccine therapy of human tumors. In this study we investigated the level of efficacy of a MART‐1/Melan‐A‐specific CD8+ T cell clone against its autologous melanoma in a severe combined immunodeficiency (SCID) mouse model, in which the tumor cells expressed in vivo heterogeneous and suboptimal levels of MART‐1. The subcutaneous co‐injection of the MART‐1/Melan‐A‐reactive T cell clone A42 with MART‐1/Melan‐A+ autologous human melanoma cells into SCID mice caused a total inhibition of tumor growth. However, the systemic treatment with A42 clone lymphocytes resulted inonly 50–60% inhibition of tumor growth, although the T cell clone targeted the tumors and the MART‐1+ cells virtually disappeared from the tumors. This study suggests that an immunotherapybased on the expansion of an antigen‐specific T cell clone generated in vitro is highly efficient in abolishing tumor growth when the target antigen is fully expressed, but leads to in vivoimmunoselection of antigen‐loss variants in the presence of suboptimal levels of antigen expression. Furthermore, this work shows that human tumors/SCID mouse models may be useful in evaluating thein vivo efficacy of adoptive immunotherapies.

Differential expression and distribution of ezrin, radixin and moesin in human natural killer cells

Cytoskeleton plays a crucial role in natural killer cell function. In this study the expression and subcellular distribution of ezrin, radixin and moesin, a family of proteins that connect actin filaments to many membrane structures, were evaluated in human NK cells. The results showed that NK cells expressed all these proteins, while NK cell‐deprived peripheral blood leukocytes and purified T lymphocytes did not express radixin. Only ezrin changed its distribution following IL‐2 activation and all three ezrin, moesin and radixin were polarized on uropods of adherent natural killercells. Ezrin and radixin co‐localized with the perforin granules at the intimate sites of contact between NK and the target cells, while moesin remains uniformly distributed on the membrane of NK cells. Ezrin, radixin and perforin co‐localization was undetected in non‐lytic conjugates and inhibited by treatment with actin depolymerizing agents. These results suggest that ezrin and radixin mayexert a role in NK activity, particularly in the trafficking of perforin granules to the NK/target cells contact site. Moreover, our data suggest that radixin may represent an additional biological marker of human NK cells and that this protein may hold a specific role in NK cell function.

Murine granulocytes control human tumor growth in SCID mice

Severe combined immunodeficient (SCID) mice generally do not reject allogeneic or xenogeneic organ grafts and represent a unique model for investigating in vivo the behaviour of both normal and neoplastic human cells. However, cells from human primary tumors often do not grow in SCID mice. We have previously shown that the major reaction of SCID mice to the engraftment of human peripheral blood leukocytes is a massive granulocyte recruitment into the site of transplantation. In this study, we have investigated the role of murine granulocytes in the control of human tumor cell growth in SCID mice. We report here that murine granulocytes infiltrate and delimit the human tumor mass and that treatment of SCID mice with anti‐murine granulocyte antibody markedly improves the growth of human tumor cell lines of different origin through suppression of the host granulocyte reaction. This finding provides a new tool for improving the human tumor take in SCID mice, thus opening new perspectives for a practical in vivo preclinical test of anti‐tumor strategies. Moreover, this study, even with the limits of the known natural reaction against xenotransplants, further supports the importance of granulocytes in the control of tumor take and growth. Int. J. Cancer 87:569–573, 2000.

Implementing quality by design for biotech products: Are regulators on track?

Quality by design (QbD) is an innovative approach to drug development that has started to be implemented into the regulatory framework, but currently mainly for chemical drugs. The recent marketing authorization of the first monoclonal antibody developed using extensive QbD concepts in the European Union paves the way for future further regulatory approvals of complex products employing this cutting-edge technological concept. In this paper, we report and comment on insights and lessons learnt from the non-public discussions in the European Medicines Agencys Biologicals Working Party and Committee for Medicinal Products for Human Use on the key issues during evaluation related to the implementation of an extensive QbD approach for biotechnology-derived medicinal products. Sharing these insights could prove useful for future developments in QbD for biotech products in general and monoclonal antibodies in particular.

How to Overcome Cisplatin Resistance Through Proton Pump Inhibitors

Resistance to antitumor agents is a major cause of treatment failure in patients with cancer. Some mechanisms of tumor resistance to cytotoxic drugs may involve increased acidification of extracellular compartments. We investigated whether proton pump inhibitors (PPI), currently used in the anti-acid treatment of peptic disease, could inhibit the acidification of the tumor microenvironment and increase the sensitivity of tumor cells to cytotoxic agents.