1. Introduction
Immunogenic cell death (ICD) defines various RCD processes which, upon stimulation by endogenous antigenic components from dying or dead cells, lead to an enhanced T cell-dependent immune response
[1][2][3]. Immunogenicity derives from the synergy of these antigens with DAMPs (also termed alarmins), which act as adjuvants when secreted/excreted in the microenvironment of dying cells
[1][2][4]. ICD is also characterized by the production of high levels of reactive oxygen species (ROS) and is highly related to endoplasmic reticulum (ER) stress, often resulting in an unfolded protein response (UPR)
[5].
For years, the common conception regarding the way by which cells die has been fallaciously limited to the dipole apoptosis/necrosis
[4][6]. The apoptotic process, historically considered as solely representing RCD, is characterized by programmed morphological changes occurring in the apoptotic cell, including the initial DNA fragmentation and the subsequent formation of apoptotic bodies, i.e., membrane vesicles enwrapping intracellular material
[7][8]. These changes often promote the recognition and engulfment of the shrunken apoptotic cell or smaller membranous portions thereof by phagocytes, in a non-immunologically-mediated manner
[6][8][9]. Thus, apoptotic cell death has been considered as immunologically “silent” and, subsequently, immunologically tolerated
[3][10][11][12]. On the other hand, necrosis and, later on, necroptosis, have been strongly associated with inflammation
[3][6][8][13][14]. Nonetheless, scientific data clarified this long-held misconception and rendered some RCD types as potential inducers of adaptive immune responses
[6][15]. Galluzzi et al.
[16] and Kroemer et al.
[17] elegantly described that the immunological outcome of RCD, i.e., whether immunogenic or tolerogenic, depends on the presence of antigens (antigenicity), on potent and immunostimulatory adjuvant-like signals (adjuvanticity) and on the “shaping” of an immune-permissive microenvironment. In the therapeutic management of tumors, treatment with low-dose chemotherapy (e.g., anthracyclins) or low-dose ionizing radiation (IR; e.g., γ-rays) induces ICD. The latter is associated with tumor-antigen shedding and translocation or release of DAMPs (e.g., calreticulin (CRT), high-mobility group box 1 protein (HMGB1), adenosine triphosphate (ATP)) by dying cells, which bind to innate immune receptors on antigen-presenting cells (APCs); the concomitantly released type I interferons (IFNs) and interleukin (IL)-1β modulate the microenvironment to support APC maturation and trafficking to the draining lymph nodes, where they dictate T cell activation and proliferation; tumor-reactive T cells further traffic to the tumor, rapidly eliminate cancer cells and ultimately, culminate in tumor antigen-specific immunological memory (
Figure 1). On the contrary, apoptotic non-inflammatory RCD induced for example by high-dose chemotherapy or radiation, causes blebbing of the tumor-cell membrane, loss of tumor-antigen(s) and of DAMP secretion/excretion, impedes APC activation and secretion of pro-inflammatory cytokines, and consequently, inhibits the activities of effector T cells, finally leading to immunosuppression
[18][19].
Unlike the accidental/necrotic cell death (ACD) caused by various physical, chemical, and mechanical cell injuries, RCD is elegantly controlled by a plethora of molecular signaling pathways. Well-characterized RCD modalities reported to stimulate immunogenic properties through the release of DAMPs are shown in Figure 2.
Figure 2. An overview of ICD mechanisms and key regulatory molecules. The main ICD modalities shown are apoptosis and necroptosis (A), ferroptosis (B), parthanatos (C), and pyroptosis (D). Arrows indicate the pathway flow and the relative regulatory molecules. AIF, apoptosis-inducing factor; ASC, apoptosis-associated speck-like protein containing a CARD; BAK, Bcl-2 homologous antagonist killer; BAX, Bcl-2-like protein 4; FADD, FAS-associated death domain protein; GPX4, glutathione peroxidase 4; MLKL, mixed lineage kinase domain-like pseudokinase; NLRs, NOD-like receptors; PARP1, poly(ADP-ribose) polymerase-1; RIPK, receptor-interacting protein kinase; ROS, reactive oxygen species; TLR, Toll-like receptor; and TRADD, tumor necrosis factor receptor type 1-associated death domain protein.
2. Apoptosis
Apoptosis is the most extensively studied type of RCD. An apoptotic cell undergoes a variety of rigorously programmed processes that affect its morphology, including the condensation and fragmentation of chromatin, the rupture of the nucleus, and a decrease of cellular volume and blebbing, which, finally, result in the formation of apoptotic bodies. Apoptotic bodies contain intracellular material and organelles and are eventually cleared by non-professional (e.g., macrophages) and professional (e.g., dendritic cells (DCs)) phagocytes, via a process known as efferocytosis
[2][8]. Based on the stimulus that initiates the apoptotic cascade, apoptosis may be mediated by two distinct pathways, the extrinsic and the intrinsic
[2][20][21].
The extrinsic or receptor-mediated pathway is initiated by the stimulation of death receptors, belonging to the tumor necrosis factor (TNF) family, such as CD95 (APO-1/Fas) or TNF-related apoptosis-inducing ligand (TRAIL), that activate caspase-8, the primary initiator of the caspase cascade. Caspase-8 is responsible for the direct cleavage of downstream effector caspases, such as caspase-3
[2][7][21][22].
The intrinsic or mitochondrial pathway is initiated by stress-induced signals, followed by the release and accumulation of apoptogenic, mitochondria-derived factors in the cytoplasm, such as cytochrome c, apoptosis-inducing factor (AIF), the second mitochondria-derived activator of caspase/direct inhibitor of apoptosis-binding protein with low pI (Smac/DIABLO), the serine protease high-temperature requirement A2 (HtrA2/Omi), and endonuclease G. Accumulation of cytochrome c in the cytoplasm triggers the formation of the cytochrome c/apoptotic protease-activating factor 1 (Apaf-1) apoptosome complex, which recruits and activates pro-caspase-9, subsequently resulting in the activation of caspase-3. Smac/DIABLO and HtrA2/Omi interact with and antagonize the inhibitor-of-apoptosis proteins (IAPs), whereas AIF and endonuclease G translocate to the nucleus, thus promoting DNA condensation
[2][7][20][21][23].
Numerous studies have shown that apoptosis mediated by either the extrinsic or the intrinsic pathway can be immunogenic
[24]. Albert et al. first observed that human DCs can efficiently present antigens derived from apoptotic monocytes previously infected with influenza A virus and stimulate major histocompatibility complex (MHC) class I-restricted CD8+ cytotoxic T lymphocytes (CTLs)
[25]. It was further shown that protein cleavage, generated by activated caspases during apoptosis, facilitates antigen processing and cross-presentation by DCs
[26][27]. At the same time, the release of DAMPs by apoptotic cells triggers immune responses. Specifically, tumor cells treated with doxorubicin (DX) and other anthracyclines, oxaliplatin (OXP) or IR, elicit anticancer immune responses in vivo and thus, grant protection to mice against tumor growth
[15][27][28]. This procedure involves the release of various DAMPs by apoptotic cells, including CRT, members of the heat shock protein (HSP) family, such as HSP70 and HSP90, HMGB1 and ATP. These molecules are essential prerequisites for characterizing an RCD case as immunogenic
[27]. The aforementioned data suggest that apoptotic cells may be immunogenic, whereas necrotic cells induce excessive inflammation, due to the massive release of DAMPs, but are incapable of eliciting potent CD8+ T cell responses
[24][29].
3. Necroptosis
Necroptosis is a type of RCD, morphologically resembling necrosis. It is triggered by the stimulation of TNF receptors, such as TNF receptor 1 (TNFR1) and Fas
[2][30], or pattern recognition receptors (PRRs), such as DNA-dependent activator of interferon-regulatory factors (DAI) and Toll-like receptors (TLRs) 3 and 4
[2][31]. Via signal transduction, the receptor-interacting protein kinase (RIPK) 1 is activated and RIPK3 is consequently recruited. RIPK3 further activates the mixed lineage kinase domain-like pseudokinase (MLKL), which promotes cell membrane breaching and cell death, with simultaneous spilling of intracellular content that contains pro-inflammatory cytokines and DAMPs
[2]. The aforementioned DAMPs, together with the released cytokines and chemokines, render necroptotic cells immunogenic and thus, able to elicit CD8+ T cell-mediated responses, including potent anticancer responses
[4][3], since necroptosis bypasses tumor cell resistance to apoptosis.
4. Pyroptosis
Pyroptosis is an RCD modality initiated by intracellular and extracellular homeostatic perturbations, associated with the innate arm of immunity
[7]. Specifically, it is triggered in response to pathogenic infections, e.g., with
Salmonella spp.
[32]. Similarly to necroptosis, pyroptotic cells present a necrotic morphology, characterized by plasma membrane rupture that results in the release of their cellular con
tent [7][32]. In general, it occurs in phagocytes, such as macrophages, DCs and neutrophils, although it has been observed in other cell types as well. The mechanism of pyroptosis is strongly linked to the enzymatic activity of caspases, especially of caspase-1, and its activation associates with inflammasomes—cytosolic structures assembled by activated specific PRRs
[33]. This activation results in the release of IL-1β, a pyrogenic cytokine that induces fever and recruits immune cells to the infected tissue, and IL-18, which conditionally promotes either T helper (Th) 1 or Th2 immune responses. Furthermore, as the pyroptotic process involves membrane breaching, it consequently leads to the release, among other intracellular components, of DAMPs, such as HMGB1, several S100 proteins, and IL-18α
[32][33]. Strong evidence suggests that neutrophil pyroptosis may play a pivotal role in sepsis
[34].
5. Ferroptosis
Ferroptosis also shares common morphological characteristics with necrosis and is triggered by cellular homeostatic disturbances, associated with impaired regulation of intracellular iron levels, leading to a lethal iron-dependent accumulation of lipid hydroperoxides
[35][36]. This severe lipid peroxidation is associated with the release of immunostimulatory DAMPs, such as HMGB1, and cytokines, such as IL-1β and IL-18, by the ferroptotic cell, thus rendering the ferroptotic process immunogenic
[37][38].
6. Parthanatos
Parthanatos is another form of RCD that features necrotic-like morphology. It is the result of severe/prolonged alkylating DNA damage and is driven by hyperactivation of a specific component of the DNA damage response. It is also involved in the pathogenesis of several conditions, such as ischemia-reperfusion injury, hypoxia, inflammation, myocardial infarction, glutamate excitotoxicity and Parkinson’s disease
[7][39]. The key molecule implicated in the mechanism of parthanatos is poly(ADP-ribose) polymerase-1 (PARP-1), a nuclear protein that plays an important role in DNA repair, genomic stability, and transcription
[39][40]. In cells facing excessive DNA damage, overactivation of PARP-1 eventually drives cells to RCD, as a result of the depletion of cellular energy, the mitochondrial release of AIF, and the production of excess poly(ADP-ribose) (PAR) polymers
[39]. Activation of PARP-1 induces the release of immunogenic alarmins, primarily of HMGB1.
Overall, based on this evidence, various RCD processes can likely be deemed as forms of ICD. Some additional death types include anoikis (an apoptotic RCD modality)
[1][7][41][42], mitochondrial permeability transition (MPT)-driven necrosis
[7], entotic cell death (entosis)
[7][43], the neutrophil extracellular trap (NET) cell death or NETosis
[7][43][44], lysosome-dependent cell death (LDCD)
[7][43], autophagy-dependent cell death (ADCD)
[7][43], autosis
[7][43][45], alkaliptosis
[43], and oxeiptosis
[43], but their detailed analysis is beyond the scope of this re
vise
warch. Major types of ICD along with their morphological characteristics and immunologic profiles are briefly presented in
Table 1, and regulatory molecules required for ICD induction and coordination of the process are comparatively shown in
Figure 2.
Table 1. The classification and characteristics of various cell death modalities. Important regulators mediating each death type are listed in the last column.
Cell Death Modality |
Classification |
Morphological Characteristics |
Immunologic Profile |
Regulators |
Necrosis |
ACD |
cell swelling; DNA fragmentation; membrane rupture; loss of cell organelles |
Tolerogenic/immunogenic |
None |
Apoptosis |
RCD |
cell shrinkage/rounding; nuclear condensation/fragmentation; nuclear membrane rupture; membrane blebbing; apoptotic body formation |
Tolerogenic/immunogenic |
Death receptors, BAX, BAK, AIF, caspases 2, 3, 6, 7, 8, and 9 |
Necroptosis |
RCD |
cell/mitochondrial swelling; membrane rupture; chromatin condensation; loss of cell organelles |
Immunogenic |
TLRs, TCR, RIPK1, RIPK3, MLKL |
Pyroptosis |
RCD |
cell swelling; membrane permeabilization/rupture; DNA condensation/ fragmentation |
Immunogenic |
CASP1, CASP11, GSDMD, NLRs, ALRs |
Ferroptosis |
RCD |
mitochondrial shrinkage; reduced mitochondrial cristae; mitochondrial membrane rupture |
Immunogenic |
System XC−, GPX4, TFRC, ACSL4, LPCAT3, ALOX15, GLS2, DPP4, NCOA4, BAP1, BECN1, PEBP1, CARS, VDAC2/3, RAB7A, HSP90, ALK4/5 |
Parthanatos |
RCD |
chromatin condensation; DNA fragmentation; membrane rupture; inconsistent mitochondrial membrane; no apoptotic body formation |
Immunogenic |
PARP-1, AIFM1, MIF, OGG1 |
Anoikis |
RCD |
cell shrinkage/rounding; nuclear condensation/fragmentation; nuclear membrane rupture; membrane blebbing; apoptotic body formation; detachment from substrate/other cells |
Tolerogenic/immunogenic |
Death receptors, BAX, BAK, AIF, caspases 2, 3, 6, 7, 8, and 9 |
MPT-driven necrosis |
RCD |
similar to necrosis; loss of mitochondrial inner membrane impermeability; mitochondrial membrane dissipation/breakdown |
Immunogenic |
CYPD (PPIF) |
Entotic cell death
(Entosis) |
RCD |
cell-in-cell formation |
Tolerogenic/immunogenic |
RhoA, ROCKI/II, E-cadherin, α-catenin, actomyosin, LC3, ATGs |
Neutrophil extracellular trap cell death (NETosis) |
RCD |
membrane rupture; nuclear membrane dissolvement; chromatin decondensation/release |
Tolerogenic/immunogenic |
NOX4, PAD4, ELANE, MMP, MPO, ELANE, MMP, MPO |
Lysosome-dependent cell death
(LDCD) |
RCD |
lysosome/plasma membrane rupture |
Immunogenic |
BECN1, Na+/K+-ATPase, AMPK, Ras-like protein A |
Autophagy-dependent cell death (ADCD) |
RCD |
vacuolization (large intracellular vesicles); enlargement of cell organelles; depletion of cell organelles |
Immunogenic |
UKL1, PI3KIII, ATGs, LC3 |
Autosis |
RCD |
enhanced cell-substrate adherence; ER fragmentation/breakdown; cell swelling; chromatin condensation |
Immunogenic |
Na+/K+-ATPase |
Alkaliptosis |
RCD |
similar to necrosis |
Immunogenic |
IKBKB, NF-κB |
Oxeiptosis |
RCD |
similar to apoptosis |
Tolerogenic |
KEAP1, PGAM5, AIFM1 |