Cytotoxicity as a form of immunogenic cell death leading to efficient tumor antigen cross‐priming

Antigen cross‐priming of CD8+ T cells is a critical process necessary for the effective expansion and activation of CD8+ T cells endowed with the ability to recognize and destroy tumor cells. The cross‐presentation of tumor antigens to cross‐prime CD8+ T cells is mainly mediated, if not only, by a subset of professional antigen‐presenting cells termed type‐1 conventional dendritic cells (cDC1). The demise of malignant cells can be immunogenic if it occurs in the context of premortem stress. These ways of dying are termed immunogenic cell death (ICD) and are associated with biochemical features favoring cDC1 for the efficient cross‐priming of tumor antigens. Immunosurveillance and the success of immunotherapies heavily rely on the ability of cytotoxic immune cells, primarily CD8+ T cells and NK cells, to detect and eliminate tumor cells through mechanisms collectively known as cytotoxicity. Recent studies have revealed the significance of NK‐ and CTL‐mediated cytotoxicity as a prominent form of immunogenic cell death, resulting in mechanisms that promote and sustain antigen‐specific immune responses. This review focuses on the mechanisms underlying the cross‐presentation of antigens released during tumor cell killing by cytotoxic immune cells, with an emphasis on the role of cDC1 cells. Indeed, cDC1s are instrumental in the effectiveness of most immunotherapies, underscoring the significance of tumor antigen cross‐priming in contexts of immunogenic cell death. The notion of the potent immunogenicity of cell death resulting from NK or cytotoxic T lymphocyte (CTL)‐mediated cytotoxicity has far‐reaching implications for cancer immunotherapy.


| INTRODUC TI ON
Whether immune cytotoxicity is a terminator of ongoing immune responses or an activating event for stronger cytotoxicity is an important matter to understanding immune regulation, especially against cancer.To understand and dissect the functional consequences of the upcoming notion that immune cytotoxicity leads to stronger tumor antigen recognition and anti-tumor effector immune responses, we have to consider the function of dendritic cells mediating tumor antigen cross-presentation, the concept of immunogenic cell death, and the molecular events associated to T-and NK-mediated cytotoxicity.

| DENDRITI C CELL S AND cD C1 FUN C TIONS
Fifty years ago, in 1973, dendritic cells (DCs) were discovered by Ralph Steinman and Zanvil Cohn as a novel immune cell type, morphologically distinct from macrophages. 1 Since their discovery, dendritic cells have proven to be the key bridge between innate and adaptive immunity due to their unique ability to uptake, process, and present antigens to T lymphocytes.Hence, DCs are critical for initiating and orchestrating adaptive immune responses.Apart from antigen presentation, DCs also crucially provide co-stimulation (CD80/ CD86) and paracrine cytokine secretion (critically IL-12 and IL15) to fully activate antigen-cognate T cells 2 (Figure 1).Importantly, costimulation and cytokine secretion are not constitutive in DCs but are inducible by microbial presence, tissue damage, and/or ongoing T-helper immune responses. 3e field of deep single-cell phenotyping has significantly advanced our understanding of dendritic cells, uncovering distinct cell subsets characterized by variations in their development, phenotypic profiles, tissue distribution, ontogenic requirements, and specialized functions.Among the DC subpopulations, three types of dendritic cells can be readily identified: conventional dendritic cells (cDCs), plasmacytoid DCs (pDCs), and monocyte-derived DCs (MoDC). 4Conventional dendritic cells (cDCs) originate from common DC precursors (CDPs) in the bone marrow and can be further categorized into two subsets: conventional type-1 dendritic cells (cDC1) and conventional type-2 dendritic cells (cDC2).cDC1s are highly specialized in cross-presenting exogenous antigens on major histocompatibility complex class I (MHC-I) molecules for CD8 + T-cell priming.Ontogenically, these cells transcriptionally rely on interferon regulatory factor 8 (IRF8), 5 basic leucine zipper transcriptional factor ATF-like 3 (Batf3), inhibitor of DNA binding 2 (Id2) 6 and nuclear factor interleukin 3 regulated (Nfil3) for their development in the bone marrow.This cDC1 cell subset is distinguished by the selective expression of the X-C Motif Chemokine Receptor 1 (XCR1) and the C-type lectin domain family 9 member A (CLEC9A or DNGR-1).In mice, cDC1s encompass lymphoid-resident CD8α + DCs and migratory CD103 + DCs, while the human counterparts express CD141 (BDCA3).cDC1s are key immune cells in antiviral cytotoxic T lymphocyte responses and the cancer immunity cycle. 7,8nversely, cDC2s are specialized in presenting exogenous antigens on MHC-II for CD4 + T cell priming and depend on interferon regulatory factor 4 (IRF4) for their ontogenic development.Regarding cell surface markers, cDC2 can be best identified by the expression of CD172a (SIRPα), CD11b, and the human-specific marker CD1c (BDCA1).
Plasmacytoid DCs (pDCs) are well-known for being a significant source of type-I interferons (IFNs) in response to viral infections, while monocyte-derived DCs (MoDC) primarily emerge from monocyte precursors in response to inflammation. 3,9The distinct functional specialization of dendritic cells enables them to initiate specific immune responses in diverse immunological scenarios and contexts.

| CROSS -PRIMING AND CROS S -PR E S E NTATI O N
The process of cross-presentation, mainly performed by cDC1, involves the uptake, processing, and loading of extracellular antigens onto MHC-I molecules by professional antigen-presenting cells (APCs).The subsequent recognition of these cross-presented antigens by naive or memory antigen-specific CD8 + T cells and their activation into cytotoxic CD8 + T cells is referred to as cross-priming (Figure 1).This essential phenomenon, crucial for immunity against viruses and tumor cells, was first discovered by Michael Bevan et al. 10,11 in 1976, using models of cell-associated antigens in mice (minor histocompatibility antigens and ovalbumin).Surprisingly, it was found that the class I MHC alleles of the immunized host, rather than those expressed by the injected cells, actually presented the antigens and therefore restricted the cytotoxic immune response. 10,11is discovery indicated the need for certain host cells to take up antigens from third-party cells and present them to CD8 + T lymphocytes, which then could become cytotoxic T lymphocytes. 12,13e exact cellular and molecular mechanisms underlying antigen cross-presentation are not fully understood, but two main pathways have been experimentally proposed.In the cytosolic pathway, phagocytosed antigens are released or translocated from endosomes to the cytosol, trimmed by the proteasome, and translocated to the lumen of the endoplasmic reticulum for loading onto MHC-I molecules.The vacuolar pathway proposes the vesicular trafficking from endosomes to MHC-I-containing compartments in a proteasomeindependent manner.In either case, the slow acidification of cDC1 endosomes to prolong the persistence of undigested proteins and special features regarding intracellular vesicle trafficking appear to be key for this ability to redirect engulfed antigenic material to the MHC class I presentation pathway functions. 14Both routes of crosspresentation may actually coexist.
More recently, a novel phenomenon called cross-dressing has been identified, involving the direct transfer of peptide-bound class I MHC-I molecules from tumor cells to dendritic cells as a result of trogocytosis, also leading to CD8 + T cell priming. 15This mechanism can be performed by cDC2 cells under tissue environments rich in type I Interferons. 16

| cD C1 CELL S IN ANTIC AN CER IMMUNIT Y
Despite their paucity in numbers, cDC1s have a key role in generating effective immune responses against tumors.Mice lacking cDC1 due to Batf3 deficiency or genetic disruption of an enhancer located 32 kilobases (kb) downstream of the Irf8 transcriptional start site show impaired antitumor immunity and lack of CD8 + T-cell crosspriming against tumor antigens. 7,17,18Likewise, mice carrying a genetic deletion of the transcription factor Nfil3, also called E4BP4, led to compromised development of CD8α + dendritic cells and impaired in vivo cross-priming of CD8 + T cells against cell-associated antigens. 19Similarly, specific depletion in mouse models expressing the diphtheria toxin receptor under the specific cDC1 promoter XCR1 results in enhanced tumor growth. 20I G U R E 1 Mechanisms of cross-priming, immunogenic Cell death (ICD), and cellular cytotoxicity in antitumor immunity.These phenomena result in a virtuous cycle in antitumor immunity that can be exploited for cancer immunotherapy.
To specifically address the underlying mechanisms that impair the cross-priming of CD8 + T cells, mouse models in which crosspresentation is hindered have been developed.In this regard, diminished cross-presentation of tumor antigens by Sec22-deficient dendritic cells (DCs) also resulted in decreased antitumor immune responses and accelerated tumor growth. 21Moreover, the small GTPase RAB43, highly expressed on CD8α + DCs and localized to the Golgi and cytoplasmic vesicles, was identified as crucial for cross-presentation of both cell-associated and soluble antigens. 22e BEACH (Beige and Chediak-Higashi) domain-containing protein WDFY4 (WD repeat-and FYVE domain-containing protein 4) was also found to play a crucial role in the cross-presentation of cellassociated antigens by cDC1s in mice, and such knock-out mice also suffer from an altered immune-mediated tumor control. 23The targeted deletion of CD40 and MHCII in cDC1s revealed these moieties as crucial components of the process of DC licensing by activated CD4 + T cells since their loss abrogates effective CD8 + T cell crosspriming, thereby hampering the spontaneous rejection of a highly immunogenic fibrosarcoma. 18re recently, perforin-2 was shown to facilitate the endosomal delivery of antigens to the cytosol in cross-presenting dendritic cells, leading to the impairment of cross-priming and CD8 + T cell responses in vivo. 24All of these mouse models have provided crucial insights into the mechanisms underlying antigen crosspresentation and T cell cross-priming, emphasizing not only the pivotal role of cDC1s in initiating and maintaining potent antitumor immune responses but also highlighting that cross-priming is one of the most important functions exerted by cDC1 cells in cancer.
In humans, gene signatures denoting the presence and abundance of cDC1 in the tumor microenvironment (TME) are associated with the level of T-cell infiltration in tumors. 25This association has been found to be predictive of patient survival and responsiveness to immunotherapy with checkpoint inhibitors 26,27 across different tumor types, including luminal breast cancer (LBC) 28 and melanoma. 29All these pieces of work collectively demonstrated a notable correlation between an augmented expression of the functional cDC1 signature and enhanced patient survival and responsiveness to immunotherapy.These findings highlight the importance of considering not only the quantity but also the functional state of cDC1 to predict patient outcomes. 29,30C1 cells are required for the therapeutic effects of most immunotherapies and several other cancer treatments.Our group and several others have shown that cDC1 cells are not only an absolute requirement for the effect of anti-PD-1/anti-PD-L1-based immune checkpoint blockade [31][32][33][34][35] even in the neoadjuvant setting, 36 but also for the beneficial effect of other immunostimulatory antibodies such as anti-CD137 agonistic mAbs. 31,353][44][45] The abscopal effect promoted by the combination of immunotherapy and local radiotherapy is also contingent on cDC1mediated cross-priming. 46

| IMMUNOG ENI C CELL DE ATH AND CROSS -PRIMING
In the often-referred cancer immunity cycle model proposed by Mellman and Chen, the killing of tumor cells by cytotoxic lymphocytes would induce antigen uptake by local dendritic cells, allowing migration to the tumor-draining lymph nodes (TdLN), and the priming/activation of CD8 + T cells to allow more rounds of tumor cell killing 47 (Figure 1).However, many aspects of this paradigm were not formally proven at the time it was originally proposed.For example, to allow proper cross-priming of tumor antigens released by tumor cells killed by cytotoxicity, several of the stimuli provided by the dying tumor cells need to promote antigen capture of the apoptotic cell remains by cDC1.This requires adequate activation of cDC1 and migration to the TdLN, where antigen cross-presentation should occur.In favor of the existence of this step in the tumor-immunity cycle is the phenomenon called "antigen spreading" characterized by the detection of T-cell reactivity to new tumor antigens undetectable before treatment.This has been shown to occur as a result of an ample array of efficacious immunotherapies, spanning from vaccines to adoptive T-cell therapy with TCR-transduced or CAR-T cells. 42,48itope spreading suggested that immune cell cytotoxicity might be followed by tumor antigen cross-priming.The treatment of cancer patients with immune checkpoint blockade, which ultimately works through CD8 + T cell-mediated killing of cancer cells, often causes the appearance of new TCR clonotypes, indirectly suggesting that cross-priming of new antigens provided by immune cell killing is effectively happening in cancer patients. 49e uptake of tumor antigen from cells undergoing cell death by dendritic cells, particularly cDC1, and its cross-presentation have been extensively described. 50,51Mechanistically, several receptors have been identified to mediate the uptake of apoptotic cell material, mainly in experiments performed with the CD8α + cDC1 population residing in the spleen.These include Tim-3 and AXL/RBP-1. 5211c, an integrin chain highly expressed in dendritic cells and often used as a cell surface marker for these immune cell subsets, can also mediate antigen uptake, at least in cDC2, but its potential role in cDC1 cells and cancer has not yet been studied. 53CLEC9-A is a C-type lectin that is almost exclusively expressed on cDC1 and was originally discovered as a receptor for polymerized F-actin that can mediate antigen uptake and allow subsequent antigen crosspresentation. 54Yet, CLEC9A-deficient mice show no impairment in tumor growth or anti-tumor immune responses. 54,55The group of Caetano Reis Sousa recently discovered that tumors produced high levels of a protein that inhibits CLEC9A functions, gelsolin, showing that in conditions in which gelsolin cannot be expressed, CLEC9A becomes important in anti-tumor immunity. 56Other receptors have also been described allowing the uptake of material from apoptotic tumor cells, as in the case of Tim-4, which is expressed on the surface of cDC1 cells in the lung and mediates the uptake of tumor antigens.Tim-4 is progressively lost from the surface of such cDC1 cells upon tumor progression. 57e main mechanisms by which dendritic cells are attracted to dying tumor cells and whether they mainly uptake tumor antigen material from the dying tumor cell bodies or by the released vesicles during the apoptotic process have not yet been elucidated, but antigen uptake from exosomes 58 and larger vesicular bodies similar to the ones produced in apoptotic cells 59 has been described.Importantly, membrane-associated antigens are more efficiently introduced into the cross-priming machinery than soluble or cytoplasmic ones. 60,61key notion is that antigen uptake by itself does not necessarily promote cDC1 activation and antigen cross-presentation.To allow effective antigen cross-priming, cDC1s have to be subjected to adequate stimulation in the TME, particularly by stressed dying cancer cells or the presence of microbially denoting molecular patterns. 50,62,63cording to the Nomenclature Committee on Cell Death (NCCD), immunogenic cell death (ICD) refers to a regulated form of cell death that activates an adaptive immune response specific to antigens expressed by dying cells in immunocompetent hosts. 64ring ICD, cancer cells release damage-associated molecular patterns (DAMPs) that are recognized by pattern recognition receptors (PRRs) expressed by immune cells, especially dendritic cells, which initiate an immune response against tumor neoantigens through cross-presentation to T cells.
One of the key events in ICD is the translocation of CALRETIC-ULIN (CRT), together with its cofactor ERp57, from the endoplasmic reticulum to the outer side of the plasma membrane of dying cancer cells. 65This exposed CRT acts as an "eat me" signal for dendritic cells, which is recognized by the low-density lipoprotein receptor-related protein 1 (LRP1 or CD91), promoting DC-mediated phagocytosis and antigen cross-presentation. 66[69][70] Adenosine triphosphate (ATP) release during ICD has a dual effect, depending on the receptor it binds to.Interaction with the purinergic receptor P2Y (P2RY2) acts as a "find-me" signal, 71 attracting DCs and macrophages while binding to the purinergic receptor P2X7 stimulates NLRP3 (NACHT, LRR, and PYD domains-containing protein 3) inflammasome activation, resulting in an immunostimulatory effect. 72Aberrant RNA molecules or DNA fragments detected by RNA or DNA sensors, such as TLR3 or cGAS-STING trigger the production of type I IFNs.IFNα/β in turn stimulates the expression of CXCL10, a CXCR3 + T cell chemoattractant, in an autocrine manner. 73,74Type I IFN activates T and NK cells and promotes the activation and cross-presentation of antigens by cDC1s. 75Annexin A1 (ANXA1) released upon plasma membrane permeabilization facilitates DC migration to tumor cells through the formyl peptide receptor 1 (FPR1). 76Tumor cell-derived HMGB1 (high mobility group protein B1) release, via stimulation of TLR4 on DCs, induces antigen cross-presentation by preventing the fusion of phagosomes and lysosomes while upregulating costimulatory surface receptors and cytokines. 77though ICD is characterized by these molecular hallmarks, tumors have developed various strategies to evade immune cell recognition, including the loss of CRT, LRP1, and HMGB1 expression, single nucleotide polymorphisms on several receptors, upregulation of CRT antagonist CD47, and favoring the expression of ATPdegrading ectoenzymes like CD39 and CD73 on malignant cells.Furthermore, tumors may upregulate T cell immunoglobulin mucin receptor 3 (TIM-3) on DCs, sequester HMGB1, or exhibit deficiencies in signaling transducers to subvert immunosurveillance. 78,79

| C Y TOTOXI CIT Y IS A FORM OF IMMUNOG ENI C CELL DE ATH
Cellular cytotoxicity, carried out by the attack of CD8 + T cells and NK cells on tumor cells, has recently emerged as a form of ICD.Following the killing by CD8 + T cells and NK cells, cancer cells expose and release CRT and HMGB1, acting as alarmins for DC activation (Figure 1).This process results in the release of cell debris from the killed cancer cells and prevents tumor engraftment when this cell debris is inoculated to mice as a vaccine.Such protection is completely dependent on Batf3-dependent cDC1s. 80The significance of cellular cytotoxicity as a form of immunogenic cell death is further supported by the study conducted by Jaime-Sanchez et al., 81 who demonstrated the expression of ICD signals upon CD8-mediated killing.The use of different transplantable tumor models in these pieces of work reinforces the notion of antigen spreading, with cDC1 emerging as the primary cellular subset responsible for generating a broad immune response against tumor antigens.
Following proper activation of cDC1 cells, the antigen must be cross-presented to CD8 + T cells.cDC1 can be effectively found performing long-lasting interactions with CD8 + T cells within the tumor microenvironment, most specifically in perivascular niches. 27These interactions are suggestive of in situ antigen cross-priming and CD8 + T cell activation within the tumor tissue. 26,27In these perivascular niches, cDC1s have been found to stay in close contact with CD8 + TCF1 + , a subset of CD8 + T cells with strong proliferative potential that has been described to be the main subset responsive to anti-PD-1 blockade.cDC1 has been shown to be of great importance in the maintenance of this CD8 + TCF1 + cell population, although it is unclear if actual antigen cross-presentation is important in such function. 30,35,82However, it is incompletely defined if actual crosspriming of naïve T cells can take place within these clusters.cDC1 cells in the TME can also, upon maturation, migrate to the tumordraining lymph nodes and ferry antigen cargo, which can be presented there by migratory cDC1s themselves 83 or transferred to resident cDC1 CD8α + cells, which also efficiently prime antitumor CD8 + T cells. 84More recently, the group of Miriam Merad using single-cell transcriptomics has postulated the existence in cancer tissue of a cross-priming cDC population termed DCreg that may arise from cDC1 and cDC2 and is enabled by mediate cross-priming associated with benefits from ICI therapy in non-small cell lung cancer (NSCLC) and hepatocellular carcinoma (HCC). 85,86nce, the reciprocal interactions among CD8 + T cells, tumor cells, and cDC1 establish a beneficial cycle that amplifies anti-tumor immune responses in a positive feedback loop fashion.Understanding the intricate interplay between these processes, which form a virtuous cycle, is crucial for the design of effective and personalized cancer treatments.It is worth considering cytotoxic T and NK cells also kill cDC1 cells, and such a mechanism will act to limit the intensity of immune responses and terminate them once antigens have been cleared.This aspect warrants ongoing research efforts.

| CON CLUS I ON S AND FUTURE PER S PEC TIVE S
While significant progress has been made in understanding the functional connection between cytotoxicity and cDC1-mediated crosspriming, further research is necessary to decipher the precise molecular mechanisms governing ICD following T-and NK-cell cytotoxicity.
Tumors are postulated to co-opt mechanisms that disrupt this virtuous cycle of immunogenic cytotoxicity-cross-priming (Figure 1) that otherwise would terminate malignancies.Counteracting such immunosuppressive mechanisms that may interfere with cDC1 cross-priming in cancer-bearing hosts might be of importance.
8][89] Moreover, perhaps by mimicking or enforcing cytotoxicity in part of the tumor lesions, we could generate endogenous vaccines.Furthermore, even if cytotoxicity is immunogenic for CTL priming, it might be suboptimal, hence calling for stronger cDC1 maturation stimuli such as TLR agonists or CD40 agonists 90,91 to render more efficacious results (Figure 2).All considered, the bidirectional connection and interplay of cytotoxic lymphocytes and cDC1 cells mediating antigen cross-priming will be a fertile field of research to harvest novel immunotherapies and biomarkers for preexisting immunotherapies.Immune cytotoxicity as a form of immunogenic cell death may also have implications for organ-specific autoimmunity and viral infections.
ACK N O WLE D G E M ENTSContinuous support and scientific discussions with Dr.David Sancho (CNIC) and Dr. Ignacio Heras (CNIC) are acknowledged.FU N D I N G I N FO R M ATI O NThis work was supported by the Spanish Ministry of Economy and Competitiveness and the Spanish Ministry of Research [MINECO F I G U R E 2 Opportunities for cDC1 functional modulation to enhance crosspriming and therapeutically exploit the fact that immune cytotoxicity is a form of immunogenic cell death.