Consensus guidelines for the detection of immunogenic cell death

Abstract

Apoptotic cells have long been considered as intrinsically tolerogenic or unable to elicit immune responses specific for dead cell-associated antigens. However, multiple stimuli can trigger a functionally peculiar type of apoptotic demise that does not go unnoticed by the adaptive arm of the immune system, which we named "immunogenic cell death" (ICD). ICD is preceded or accompanied by the emission of a series of immunostimulatory damage-associated molecular patterns (DAMPs) in a precise spatiotemporal configuration. Several anticancer agents that have been successfully employed in the clinic for decades, including various chemotherapeutics and radiotherapy, can elicit ICD. Moreover, defects in the components that underlie the capacity of the immune system to perceive cell death as immunogenic negatively influence disease outcome among cancer patients treated with ICD inducers. Thus, ICD has profound clinical and therapeutic implications. Unfortunately, the gold-standard approach to detect ICD relies on vaccination experiments involving immunocompetent murine models and syngeneic cancer cells, an approach that is incompatible with large screening campaigns. Here, we outline strategies conceived to detect surrogate markers of ICD in vitro and to screen large chemical libraries for putative ICD inducers, based on a high-content, high-throughput platform that we recently developed. Such a platform allows for the detection of multiple DAMPs, like cell surface-exposed calreticulin, extracellular ATP and high mobility group box 1 (HMGB1), and/or the processes that underlie their emission, such as endoplasmic reticulum stress, autophagy and necrotic plasma membrane permeabilization. We surmise that this technology will facilitate the development of next-generation anticancer regimens, which kill malignant cells and simultaneously convert them into a cancer-specific therapeutic vaccine.

Keywords: APC, antigen-presenting cell; ATF6, activating transcription factor 6; ATP release; BAK1, BCL2-antagonist/killer 1; BAX, BCL2-associated X protein; BCL2, B-cell CLL/lymphoma 2 protein; CALR, calreticulin; CTL, cytotoxic T lymphocyte; DAMP, damage-associated molecular pattern; DAPI, 4′,6-diamidino-2-phenylindole; DiOC6(3), 3,3′-dihexyloxacarbocyanine iodide; EIF2A, eukaryotic translation initiation factor 2A; ER, endoplasmic reticulum; FLT3LG, fms-related tyrosine kinase 3 ligand; G3BP1, GTPase activating protein (SH3 domain) binding protein 1; GFP, green fluorescent protein; H2B, histone 2B; HMGB1; HMGB1, high mobility group box 1; HSP, heat shock protein; HSV-1, herpes simplex virus type I; ICD, immunogenic cell death; IFN, interferon; IL, interleukin; MOMP, mitochondrial outer membrane permeabilization; PDIA3, protein disulfide isomerase family A; PI, propidium iodide; RFP, red fluorescent protein; TLR, Toll-like receptor; XBP1, X-box binding protein 1; autophagy; calreticulin; endoplasmic reticulum stress; immunotherapy; member 3; Δψm, mitochondrial transmembrane potential.

Figure 1.

Molecular and cellular mechanisms of immunogenic cell death. Cancer cells succumb to specific stimuli (e.g., anthracyclines, oxaliplatin, some forms of radiation therapy, photodynamic therapy) while emitting a spatiotemporally ordered combination of damage-associated molecular patterns (DAMPs). These signals include (but are not limited to) the pre-apoptotic exposure of the endoplasmic reticulum chaperone calreticulin (CALR) on the surface of dying cells, the secretion of ATP during the blebbing phase of apoptosis, and the release of the nuclear protein high mobility group box 1 (HMGB1) upon plasma membrane permeabilization. Upon binding to specific receptors, immunogenic cell death (ICD)-associated DAMPs promote the recruitment of antigen-presenting cells (APCs) and stimulate their ability to take up particulate material and cross-present dead cell-associated antigens to CD8⁺ cytotoxic T lymphocytes (CTLs) while secreting interleukin (IL)-1β. The consequent adaptive immune response also involves γδ T lymphocytes that produce IL-17. Both γδ T cells and αβ CTLs mediate direct antineoplastic effects by secreting interferon γ (IFNγ) and via the granzyme-perforin pathway. In addition, some CTLs acquire a memory phenotype, underlying the establishment of long-term immunological protection.

Figure 2.

Assays for the evaluation of immunogenic cell death in vivo. (A) Vaccination assays. Murine cancer cells of choice are exposed in vitro to a putative inducer of immunogenic cell death (ICD), 1 μM mitoxantrone (positive control) or 50 μM cisplatin (negative control) for a predetermined time (normally 6–24 hours), then washed, resuspended in PBS, and eventually injected s.c. into one flank (vaccination site) of immunocompetent syngeneic mice (ideally 5–10 per group). One week later, mice are challenged with living cancer cells of the same type, which are inoculated s.c. into the contralateral flank (challenge site). Tumor incidence and growth are routinely monitored at both injection sites over a 1-2 months period. The development of neoplastic lesions at the vaccination site indicates that the stimulus under investigation is unable to cause cell death (under the circumstances under investigation) to a degree that is compatible with the elicitation of adaptive immunity. Conversely, in the absence of tumors at the vaccination site, the ability of the experimental maneuver under evaluation to promote bona fide ICD inversely correlates with the number of neoplastic lesions developed at the challenge site. As an indication, neoplastic cells exposed in vitro to 1 μM mitoxantrone for 6 hours and maintained in culture for additional 18 hours vaccinate approximately 80% of mice against a challenge with living cells of the same type. (B) Therapeutic assays. Immunocompetent and immunodeficient syngeneic mice bearing grafted, genetically-driven or chemically-induced subcutaneous or orthotopic tumors are treated with a putative ICD inducer, mitoxantrone (positive control) or cisplatin (negative control) at therapeutic doses, followed by the monitoring of tumor size over a 1–3 weeks period. In this setting, bona fide ICD inducers mediate optimal antineoplastic effects in immunocompetent, but not in immunodeficient, mice. Since this is also the case of therapeutic interventions that exert off-target immunostimulatory effects, this assay cannot be employed alone to discriminate between ICD and non-immunogenic cell death (nICD). Please note that all curves represented in this figure do not depict primary data but have been created for the sake of exemplification.