Noelia Casares

Calreticulin exposure dictates the immunogenicity of cancer cell death.

Anthracyclin-treated tumor cells are particularly effective in eliciting an anticancer immune response, whereas other DNA-damaging agents such as etoposide and mitomycin C do not induce immunogenic cell death. Here we show that anthracyclins induce the rapid, preapoptotic translocation of calreticulin (CRT) to the cell surface. Blockade or knockdown of CRT suppressed the phagocytosis of anthracyclin-treated tumor cells by dendritic cells and abolished their immunogenicity in mice. The anthracyclin-induced CRT translocation was mimicked by inhibition of the protein phosphatase 1/GADD34 complex. Administration of recombinant CRT or inhibitors of protein phosphatase 1/GADD34 restored the immunogenicity of cell death elicited by etoposide and mitomycin C, and enhanced their antitumor effects in vivo. These data identify CRT as a key feature determining anticancer immune responses and delineate a possible strategy for immunogenic chemotherapy.

Inhibition of Macroautophagy Triggers Apoptosis

ABSTRACT Mammalian cells were observed to die under conditions in which nutrients were depleted and, simultaneously, macroautophagy was inhibited either genetically (by a small interfering RNA targeting Atg5, Atg6/Beclin 1-1, Atg10, or Atg12) or pharmacologically (by 3-methyladenine, hydroxychloroquine, bafilomycin A1, or monensin). Cell death occurred through apoptosis (type 1 cell death), since it was reduced by stabilization of mitochondrial membranes (with Bcl-2 or vMIA, a cytomegalovirus-derived gene) or by caspase inhibition. Under conditions in which the fusion between lysosomes and autophagosomes was inhibited, the formation of autophagic vacuoles was enhanced at a preapoptotic stage, as indicated by accumulation of LC3-II protein, ultrastructural studies, and an increase in the acidic vacuolar compartment. Cells exhibiting a morphology reminiscent of (autophagic) type 2 cell death, however, recovered, and only cells with a disrupted mitochondrial transmembrane potential were beyond the point of no return and inexorably died even under optimal culture conditions. All together, these data indicate that autophagy may be cytoprotective, at least under conditions of nutrient depletion, and point to an important cross talk between type 1 and type 2 cell death pathways.

Caspase-dependent immunogenicity of doxorubicin-induced tumor cell death

Systemic anticancer chemotherapy is immunosuppressive and mostly induces nonimmunogenic tumor cell death. Here, we show that even in the absence of any adjuvant, tumor cells dying in response to anthracyclins can elicit an effective antitumor immune response that suppresses the growth of inoculated tumors or leads to the regression of established neoplasia. Although both antracyclins and mitomycin C induced apoptosis with caspase activation, only anthracyclin-induced immunogenic cell death was immunogenic. Caspase inhibition by Z-VAD-fmk or transfection with the baculovirus inhibitor p35 did not inhibit doxorubicin (DX)-induced cell death, yet suppressed the immunogenicity of dying tumor cells in several rodent models of neoplasia. Depletion of dendritic cells (DCs) or CD8+T cells abolished the immune response against DX-treated apoptotic tumor cells in vivo. Caspase inhibition suppressed the capacity of DX-killed cells to be phagocytosed by DCs, yet had no effect on their capacity to elicit DC maturation. Freshly excised tumors became immunogenic upon DX treatment in vitro, and intratumoral inoculation of DX could trigger the regression of established tumors in immunocompetent mice. These results delineate a procedure for the generation of cancer vaccines and the stimulation of anti-neoplastic immune responses in vivo.

AIF deficiency compromises oxidative phosphorylation

Apoptosis‐inducing factor (AIF) is a mitochondrial flavoprotein that, after apoptosis induction, translocates to the nucleus where it participates in apoptotic chromatinolysis. Here, we show that human or mouse cells lacking AIF as a result of homologous recombination or small interfering RNA exhibit high lactate production and enhanced dependency on glycolytic ATP generation, due to severe reduction of respiratory chain complex I activity. Although AIF itself is not a part of complex I, AIF‐deficient cells exhibit a reduced content of complex I and of its components, pointing to a role of AIF in the biogenesis and/or maintenance of this polyprotein complex. Harlequin mice with reduced AIF expression due to a retroviral insertion into the AIF gene also manifest a reduced oxidative phosphorylation (OXPHOS) in the retina and in the brain, correlating with reduced expression of complex I subunits, retinal degeneration, and neuronal defects. Altogether, these data point to a role of AIF in OXPHOS and emphasize the dual role of AIF in life and death.

A novel dendritic cell subset involved in tumor immunosurveillance

The interferon (IFN)-γ–induced TRAIL effector mechanism is a vital component of cancer immunosurveillance by natural killer (NK) cells in mice. Here we show that the main source of IFN-γ is not the conventional NK cell but a subset of B220+Ly6C− dendritic cells, which are atypical insofar as they express NK cell-surface molecules. Upon contact with a variety of tumor cells that are poorly recognized by NK cells, B220+NK1.1+ dendritic cells secrete high levels of IFN-γ and mediate TRAIL-dependent lysis of tumor cells. Adoptive transfer of these IFN-producing killer dendritic cells (IKDCs) into tumor-bearing Rag2−/−Il2rg−/− mice prevented tumor outgrowth, whereas transfer of conventional NK cells did not. In conclusion, we identified IKDCs as pivotal sensors and effectors of the innate antitumor immune response.

Contribution of IL-17–producing γδ T cells to the efficacy of anticancer chemotherapy

IL-17 production by γδ T cells is required for tumor cell infiltration by IFN-γ–producing CD8+ T cells and inhibition of tumor growth in response to anthracyclines.

CD4+/CD25+ Regulatory Cells Inhibit Activation of Tumor-Primed CD4+ T Cells with IFN-γ-Dependent Antiangiogenic Activity, as well as Long-Lasting Tumor Immunity Elicited by Peptide Vaccination

CD25+ regulatory T (T reg) cells suppress the activation/proliferation of other CD4+ or CD8+ T cells in vitro. Also, down-regulation of CD25+ T reg cells enhance antitumor immune responses. In this study, we show that depletion of CD25+ T reg cells allows the host to induce both CD4+ and CD8+ antitumoral responses following tumor challenge. Simultaneous depletion of CD25+ and CD8+ cells, as well as adoptive transfer experiments, revealed that tumor-specific CD4+ T cells, which emerged in the absence of CD25+ T reg cells, were able to reject CT26 colon cancer cells, a MHC class II-negative tumor. The antitumoral effect mediated by CD4+ T cells was dependent on IFN-γ production, which exerted a potent antiangiogenic activity. The capacity of the host to mount this antitumor response is lost once the number of CD25+ T reg cells is restored over time. However, CD25+ T reg cell depletion before immunization with AH1 (a cytotoxic T cell determinant from CT26 tumor cells) permits the induction of a long-lasting antitumoral immune response, not observed if immunization is conducted in the presence of regulatory cells. A study of the effect of different levels of depletion of CD25+ T reg cells before immunization with the peptide AH1 alone, or in combination with a Th determinant, unraveled that Th cells play an important role in overcoming the suppressive effect of CD25+ T reg on the induction of long-lasting cellular immune responses.

Abnormal Priming of CD4+ T Cells by Dendritic Cells Expressing Hepatitis C Virus Core and E1 Proteins

ABSTRACT Patients infected with hepatitis C virus (HCV) have an impaired response against HCV antigens while keeping immune competence for other antigens. We hypothesized that expression of HCV proteins in infected dendritic cells (DC) might impair their antigen-presenting function, leading to a defective anti-HCV T-cell immunity. To test this hypothesis, DC from normal donors were transduced with an adenovirus coding for HCV core and E1 proteins and these cells (DC-CE1) were used to stimulate T lymphocytes. DC-CE1 were poor stimulators of allogeneic reactions and of autologous primary and secondary proliferative responses. Autologous T cells stimulated with DC-CE1 exhibited a pattern of incomplete activation characterized by enhanced CD25 expression but reduced interleukin 2 production. The same pattern of incomplete lymphocyte activation was observed in CD4+ T cells responding to HCV core in patients with chronic HCV infection. However, CD4+ response to HCV core was normal in patients who cleared HCV after alpha interferon therapy. Moreover, a normal CD4+ response to tetanus toxoid was found in both chronic HCV carriers and patients who had eliminated the infection. Our results suggest that expression of HCV structural antigens in infected DC disturbs their antigen-presenting function, leading to incomplete activation of anti-HCV-specific T cells and chronicity of infection. However, presentation of unrelated antigens by noninfected DC would allow normal T-cell immunity to other pathogens.

Apoptosis regulation in tetraploid cancer cells

Tetraploidy can result in cancer‐associated aneuploidy. As shown here, freshly generated tetraploid cells arising due to mitotic slippage or failed cytokinesis are prone to undergo Bax‐dependent mitochondrial membrane permeabilization and subsequent apoptosis. Knockout of Bax or overexpression of Bcl‐2 facilitated the survival of tetraploid cells at least as efficiently as the p53 or p21 knockout. When tetraploid cells were derived from diploid p53 and Bax‐proficient precursors, such cells exhibited an enhanced transcription of p53 target genes. Tetraploid cells exhibited an enhanced rate of spontaneous apoptosis that could be suppressed by inhibition of p53 or by knockdown of proapoptotic p53 target genes such as BBC3/Puma, GADD45A and ferredoxin reductase. Unexpectedly, tetraploid cells were more resistant to DNA damaging agents (cisplatin, oxaliplatin and camptothecin) than their diploid counterparts, and this difference disappeared upon inhibition of p53 or knockdown of p53‐inducible ribonucleotide reductase. Tetraploid cells were also more resistant against UVC and γ‐irradiation. These data indicate the existence of p53‐dependent alterations in apoptosis regulation in tetraploid cells.

Immune Response Against Dying Tumor Cells

Publisher Summary This chapter provides clear evidence in favor of tumor escape mechanisms. Melanomas from patients experiencing partial responses after T-cell–based immunotherapies reportedly lose β 2 -microglobulin expression or down modulate the target antigen. The central dilemma of cancer immunotherapy resides in the striking contrast between the lack of spontaneous antitumor immune responses and the apparent possibility to induce active antitumor immune responses experimentally. Irradiation and chemotherapy mostly induce a type of cell death, apoptosis that is widely thought to be immunologically silent or even tolerogenic. Thus, paradoxically, the standard treatments that are used in the clinical management of both solid and diffuse tumors would suppress any possibility that the patients immune system eradicates those residual tumor cells that will ultimately cause relapse. This chapter examines this hypothetical scenario and the way the modality of tumor cell death and/or the immune system can be manipulated so dying tumor cells become immunogenic. Antitumor responses can be induced by vaccination by providing a formulation of concentrated tumor antigens whose optimal presentation is ensured in vitro or in vivo by the application of suitable immunostimulatory agents. Most of the studies performed concentrate on the dichotomy of apoptosis and necrosis incurring in important methodological problems that are discussed in this chapter.