The cell stress and immunity cycle in cancer: Toward next generation of cancer immunotherapy.

The cellular stress and immunity cycle is a cornerstone of organismal homeostasis. Stress activates intracellular and intercellular communications within a tissue or organ to initiate adaptive responses aiming to resolve the origin of this stress. If such local measures are unable to ameliorate this stress, then intercellular communications expand toward immune activation with the aim of recruiting immune cells to effectively resolve the situation while executing tissue repair to ameliorate any damage and facilitate homeostasis. This cellular stress-immunity cycle is severely dysregulated in diseased contexts like cancer. On one hand, cancer cells dysregulate the normal cellular stress responses to reorient them toward upholding growth at all costs, even at the expense of organismal integrity and homeostasis. On the other hand, the tumors severely dysregulate or inhibit various components of organismal immunity, for example, by facilitating immunosuppressive tumor landscape, lowering antigenicity, and increasing T-cell dysfunction. In this review we aim to comprehensively discuss the basis behind tumoral dysregulation of cellular stress-immunity cycle. We also offer insights into current understanding of the regulators and deregulators of this cycle and how they can be targeted for conceptualizing successful cancer immunotherapy regimen.

Early memory differentiation and cell death resistance in T cells predicts melanoma response to sequential anti-CTLA4 and anti-PD1 immunotherapy.

Immune checkpoint blockers (ICBs)-based immunotherapy has revolutionised oncology. However, the benefits of ICBs are limited to only a subset of patients. Herein, the biomarkers-driven application of ICBs promises to increase their efficacy. Such biomarkers include lymphocytic IFNγ-signalling and/or cytolytic activity (granzymes and perforin-1) footprints, whose levels in pre-treatment tumours can predict favourable patient survival following ICB-treatment. However, it is not clear whether such biomarkers have the same value in predicting survival of patients receiving first-line anti-CTLA4 ICB-therapy, and subsequently anti-PD1 ICB-therapy (i.e., sequential ICB-immunotherapy regimen). To address this, we applied highly integrated systems/computational immunology approaches to existing melanoma bulk-tumour transcriptomic and single-cell (sc)RNAseq data originating from immuno-oncology clinical studies applying ICB-treatment. Interestingly, we observed that CD8+/CD4+T cell-associated IFNγ-signalling or cytolytic activity signatures fail to predict tumour response in patients treated with anti-CTLA4 ICB-therapy as a first-line and anti-PD1 ICB-therapy in the second-line setting. On the contrary, signatures associated with early memory CD8+/CD4+T cells (integrating TCF1-driven stem-like transcriptional programme), capable of resisting cell death/apoptosis, better predicted objective response rates to ICB-immunotherapy, and favourable survival in the setting of sequential ICB-immunotherapy. These observations suggest that sequencing of ICB-therapy might have a specific impact on the T cell-repertoire and may influence the predictive value of tumoural immune biomarkers.

Trial watch: anticancer vaccination with dendritic cells.

Dendritic cells (DCs) are critical players at the intersection of innate and adaptive immunity, making them ideal candidates for anticancer vaccine development. DC-based immunotherapies typically involve isolating patient-derived DCs, pulsing them with tumor-associated antigens (TAAs) or tumor-specific antigens (TSAs), and utilizing maturation cocktails to ensure their effective activation. These matured DCs are then reinfused to elicit tumor-specific T-cell responses. While this approach has demonstrated the ability to generate potent immune responses, its clinical efficacy has been limited due to the immunosuppressive tumor microenvironment. Recent efforts have focused on enhancing the immunogenicity of DC-based vaccines, particularly through combination therapies with T cell-targeting immunotherapies. This Trial Watch summarizes recent advances in DC-based cancer treatments, including the development of new preclinical and clinical strategies, and discusses the future potential of DC-based vaccines in the evolving landscape of immuno-oncology.

A spatial architecture-embedding HLA signature to predict clinical response to immunotherapy in renal cell carcinoma.

An important challenge in the real-world management of patients with advanced clear-cell renal cell carcinoma (aRCC) is determining who might benefit from immune checkpoint blockade (ICB). Here we performed a comprehensive multiomics mapping of aRCC in the context of ICB treatment, involving discovery analyses in a real-world data cohort followed by validation in independent cohorts. We cross-connected bulk-tumor transcriptomes across >1,000 patients with validations at single-cell and spatial resolutions, revealing a patient-specific crosstalk between proinflammatory tumor-associated macrophages and (pre-)exhausted CD8+ T cells that was distinguished by a human leukocyte antigen repertoire with higher preference for tumoral neoantigens. A cross-omics machine learning pipeline helped derive a new tumor transcriptomic footprint of neoantigen-favoring human leukocyte antigen alleles. This machine learning signature correlated with positive outcome following ICB treatment in both real-world data and independent clinical cohorts. In experiments using the RENCA-tumor mouse model, CD40 agonism combined with PD1 blockade potentiated both proinflammatory tumor-associated macrophages and CD8+ T cells, thereby achieving maximal antitumor efficacy relative to other tested regimens. Thus, we present a new multiomics and spatial map of the immune-community architecture that drives ICB response in patients with aRCC.

Targeting conserved TIM3+VISTA+ tumor-associated macrophages overcomes resistance to cancer immunotherapy.

Despite the success of immunotherapy, overcoming immunoresistance in cancer remains challenging. We identified a unique niche of tumor-associated macrophages (TAMs), coexpressing T cell immunoglobulin and mucin domain-containing 3 (TIM3) and V-domain immunoglobulin suppressor of T cell activation (VISTA), that dominated human and mouse tumors resistant to most of the currently used immunotherapies. TIM3+VISTA+ TAMs were sustained by IL-4-enriching tumors with low (neo)antigenic and T cell-depleted features. TIM3+VISTA+ TAMs showed an anti-inflammatory and protumorigenic phenotype coupled with inability to sense type I interferon (IFN). This was established with cancer cells succumbing to immunogenic cell death (ICD). Dying cancer cells not only triggered autocrine type I IFNs but also exposed HMGB1/VISTA that engaged TIM3/VISTA on TAMs to suppress paracrine IFN-responses. Accordingly, TIM3/VISTA blockade synergized with paclitaxel, an ICD-inducing chemotherapy, to repolarize TIM3+VISTA+ TAMs to proinflammatory TAMs that killed cancer cells via tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) signaling. We propose targeting TIM3+VISTA+ TAMs to overcome immunoresistant tumors.

Lymph node and tumor-associated PD-L1+ macrophages antagonize dendritic cell vaccines by suppressing CD8+ T cells.

Current immunotherapies provide limited benefits against T cell-depleted tumors, calling for therapeutic innovation. Using multi-omics integration of cancer patient data, we predict a type I interferon (IFN) responseHIGH state of dendritic cell (DC) vaccines, with efficacious clinical impact. However, preclinical DC vaccines recapitulating this state by combining immunogenic cancer cell death with induction of type I IFN responses fail to regress mouse tumors lacking T cell infiltrates. Here, in lymph nodes (LNs), instead of activating CD4+/CD8+ T cells, DCs stimulate immunosuppressive programmed death-ligand 1-positive (PD-L1+) LN-associated macrophages (LAMs). Moreover, DC vaccines also stimulate PD-L1+ tumor-associated macrophages (TAMs). This creates two anatomically distinct niches of PD-L1+ macrophages that suppress CD8+ T cells. Accordingly, a combination of PD-L1 blockade with DC vaccines achieves significant tumor regression by depleting PD-L1+ macrophages, suppressing myeloid inflammation, and de-inhibiting effector/stem-like memory T cells. Importantly, clinical DC vaccines also potentiate T cell-suppressive PD-L1+ TAMs in glioblastoma patients. We propose that a multimodal immunotherapy and vaccination regimen is mandatory to overcome T cell-depleted tumors.

Single cell dynamics of tumor specificity vs bystander activity in CD8+ T cells define the diverse immune landscapes in colorectal cancer.

CD8+ T cell activation via immune checkpoint blockade (ICB) is successful in microsatellite instable (MSI) colorectal cancer (CRC) patients. By comparison, the success of immunotherapy against microsatellite stable (MSS) CRC is limited. Little is known about the most critical features of CRC CD8+ T cells that together determine the diverse immune landscapes and contrasting ICB responses. Hence, we pursued a deep single cell mapping of CRC CD8+ T cells on transcriptomic and T cell receptor (TCR) repertoire levels in a diverse patient cohort, with additional surface proteome validation. This revealed that CRC CD8+ T cell dynamics are underscored by complex interactions between interferon-γ signaling, tumor reactivity, TCR repertoire, (predicted) TCR antigen-specificities, and environmental cues like gut microbiome or colon tissue-specific 'self-like' features. MSI CRC CD8+ T cells showed tumor-specific activation reminiscent of canonical 'T cell hot' tumors, whereas the MSS CRC CD8+ T cells exhibited tumor unspecific or bystander-like features. This was accompanied by inflammation reminiscent of 'pseudo-T cell hot' tumors. Consequently, MSI and MSS CRC CD8+ T cells showed overlapping phenotypic features that differed dramatically in their TCR antigen-specificities. Given their high discriminating potential for CD8+ T cell features/specificities, we used the single cell tumor-reactive signaling modules in CD8+ T cells to build a bulk tumor transcriptome classification for CRC patients. This "Immune Subtype Classification" (ISC) successfully distinguished various tumoral immune landscapes that showed prognostic value and predicted immunotherapy responses in CRC patients. Thus, we deliver a unique map of CRC CD8+ T cells that drives a novel tumor immune landscape classification, with relevance for immunotherapy decision-making.

Trial watch: chemotherapy-induced immunogenic cell death in oncology.

Immunogenic cell death (ICD) refers to an immunologically distinct process of regulated cell death that activates, rather than suppresses, innate and adaptive immune responses. Such responses culminate into T cell-driven immunity against antigens derived from dying cancer cells. The potency of ICD is dependent on the immunogenicity of dying cells as defined by the antigenicity of these cells and their ability to expose immunostimulatory molecules like damage-associated molecular patterns (DAMPs) and cytokines like type I interferons (IFNs). Moreover, it is crucial that the host's immune system can adequately detect the antigenicity and adjuvanticity of these dying cells. Over the years, several well-known chemotherapies have been validated as potent ICD inducers, including (but not limited to) anthracyclines, paclitaxels, and oxaliplatin. Such ICD-inducing chemotherapeutic drugs can serve as important combinatorial partners for anti-cancer immunotherapies against highly immuno-resistant tumors. In this Trial Watch, we describe current trends in the preclinical and clinical integration of ICD-inducing chemotherapy in the existing immuno-oncological paradigms.

Multiomics and spatial mapping characterizes human CD8+ T cell states in cancer.

Clinically relevant immunological biomarkers that discriminate between diverse hypofunctional states of tumor-associated CD8+ T cells remain disputed. Using multiomics analysis of CD8+ T cell features across multiple patient cohorts and tumor types, we identified tumor niche-dependent exhausted and other types of hypofunctional CD8+ T cell states. CD8+ T cells in "supportive" niches, like melanoma or lung cancer, exhibited features of tumor reactivity-driven exhaustion (CD8+ TEX). These included a proficient effector memory phenotype, an expanded T cell receptor (TCR) repertoire linked to effector exhaustion signaling, and a cancer-relevant T cell-activating immunopeptidome composed of largely shared cancer antigens or neoantigens. In contrast, "nonsupportive" niches, like glioblastoma, were enriched for features of hypofunctionality distinct from canonical exhaustion. This included immature or insufficiently activated T cell states, high wound healing signatures, nonexpanded TCR repertoires linked to anti-inflammatory signaling, high T cell-recognizable self-epitopes, and an antiproliferative state linked to stress or prodeath responses. In situ spatial mapping of glioblastoma highlighted the prevalence of dysfunctional CD4+:CD8+ T cell interactions, whereas ex vivo single-cell secretome mapping of glioblastoma CD8+ T cells confirmed negligible effector functionality and a promyeloid, wound healing-like chemokine profile. Within immuno-oncology clinical trials, anti-programmed cell death protein 1 (PD-1) immunotherapy facilitated glioblastoma's tolerogenic disparities, whereas dendritic cell (DC) vaccines partly corrected them. Accordingly, recipients of a DC vaccine for glioblastoma had high effector memory CD8+ T cells and evidence of antigen-specific immunity. Collectively, we provide an atlas for assessing different CD8+ T cell hypofunctional states in immunogenic versus nonimmunogenic cancers.

A first-in-class, non-invasive, immunodynamic biomarker approach for precision immuno-oncology.

Non-invasive, immuno-dynamic, biomarkers positioned in cancer patient's blood milieu with immuno-oncological applications are rare. We recently established a "first-in-class" serum functional immunodynamics status (sFIS) assay, wherein in vitro assessment of serum-induced myeloid NFkB and/or interferon (IFN) response-signaling can be performed to "mimic" in situ patient's serum immune-biology. This modality has clear implications for anticipating patient prognosis and immunotherapy-relevant stratification.

The Interface of Tumour-Associated Macrophages with Dying Cancer Cells in Immuno-Oncology.

Tumour-associated macrophages (TAMs) are essential players in the tumour microenvironment (TME) and modulate various pro-tumorigenic functions such as immunosuppression, angiogenesis, cancer cell proliferation, invasion and metastasis, along with resistance to anti-cancer therapies. TAMs also mediate important anti-tumour functions and can clear dying cancer cells via efferocytosis. Thus, not surprisingly, TAMs exhibit heterogeneous activities and functional plasticity depending on the type and context of cancer cell death that they are faced with. This ultimately governs both the pro-tumorigenic and anti-tumorigenic activity of TAMs, making the interface between TAMs and dying cancer cells very important for modulating cancer growth and the efficacy of chemo-radiotherapy or immunotherapy. In this review, we discuss the interface of TAMs with cancer cell death from the perspectives of cell death pathways, TME-driven variations, TAM heterogeneity and cell-death-inducing anti-cancer therapies. We believe that a better understanding of how dying cancer cells influence TAMs can lead to improved combinatorial anti-cancer therapies, especially in combination with TAM-targeting immunotherapies.

Trial watch: Dendritic cell (DC)-based immunotherapy for cancer.

Dendritic cell (DC)-based vaccination for cancer treatment has seen considerable development over recent decades. However, this field is currently in a state of flux toward niche-applications, owing to recent paradigm-shifts in immuno-oncology mobilized by T cell-targeting immunotherapies. DC vaccines are typically generated using autologous (patient-derived) DCs exposed to tumor-associated or -specific antigens (TAAs or TSAs), in the presence of immunostimulatory molecules to induce DC maturation, followed by reinfusion into patients. Accordingly, DC vaccines can induce TAA/TSA-specific CD8+/CD4+ T cell responses. Yet, DC vaccination still shows suboptimal anti-tumor efficacy in the clinic. Extensive efforts are ongoing to improve the immunogenicity and efficacy of DC vaccines, often by employing combinatorial chemo-immunotherapy regimens. In this Trial Watch, we summarize the recent preclinical and clinical developments in this field and discuss the ongoing trends and future perspectives of DC-based immunotherapy for oncological indications.

Identification of Potential Prognostic and Predictive Immunological Biomarkers in Patients with Stage I and Stage III Non-Small Cell Lung Cancer (NSCLC): A Prospective Exploratory Study.

Radiotherapy (RT) and chemotherapy can induce immune responses, but not much is known regarding treatment-induced immune changes in patients. This exploratory study aimed to identify potential prognostic and predictive immune-related proteins associated with progression-free survival (PFS) in patients with non-small cell lung cancer (NSCLC). In this prospective study, patients with stage I NSCLC treated with stereotactic body radiation therapy (n = 26) and patients with stage III NSCLC treated with concurrent chemoradiotherapy (n = 18) were included. Blood samples were collected before (v1), during (v2), and after RT (v3). In patients with stage I NSCLC, CD244 (HR: 10.2, 95% CI: 1.8-57.4) was identified as a negative prognostic biomarker. In patients with stage III NSCLC, CR2 and IFNGR2 were identified as positive prognostic biomarkers (CR2, HR: 0.00, 95% CI: 0.00-0.12; IFNGR2, HR: 0.04, 95% CI: 0.00-0.46). In addition, analysis of the treatment-induced changes of circulating protein levels over time (Δv2/v3-v1) also identified CXCL10 and IL-10 as negative predictive biomarkers (CXCL10, HR: 3.86, 95% CI: 1.0-14.7; IL-10, HR: 16.92 (2.74-104.36)), although serum-induced interferon (IFN) response was a positive prognostic. In conclusion, we identified several circulating immunogenic proteins that are correlated with PFS in patients with stage I and stage III NSCLC before and during treatment.

Peripherally-driven myeloid NFkB and IFN/ISG responses predict malignancy risk, survival, and immunotherapy regime in ovarian cancer.

BACKGROUND: Tumors can influence peripheral immune macroenvironment, thereby creating opportunities for non-invasive serum/plasma immunobiomarkers for immunostratification and immunotherapy designing. However, current approaches for immunobiomarkers' detection are largely quantitative, which is unreliable for assessing functional peripheral immunodynamics of patients with cancer. Hence, we aimed to design a functional biomarker modality for capturing peripheral immune signaling in patients with cancer for reliable immunostratification. METHODS: We used a data-driven in silico framework, integrating existing tumor/blood bulk-RNAseq or single-cell (sc)RNAseq datasets of patients with cancer, to inform the design of an innovative serum-screening modality, that is, serum-functional immunodynamic status (sFIS) assay. Next, we pursued proof-of-concept analyses via multiparametric serum profiling of patients with ovarian cancer (OV) with sFIS assay combined with Luminex (cytokines/soluble immune checkpoints), CA125-antigen detection, and whole-blood immune cell counts. Here, sFIS assay's ability to determine survival benefit or malignancy risk was validated in a discovery (n=32) and/or validation (n=699) patient cohorts. Lastly, we used an orthotopic murine metastatic OV model, with anti-OV therapy selection via in silico drug-target screening and murine serum screening via sFIS assay, to assess suitable in vivo immunotherapy options. RESULTS: In silico data-driven framework predicted that peripheral immunodynamics of patients with cancer might be best captured via analyzing myeloid nuclear factor kappa-light-chain enhancer of activated B cells (NFκB) signaling and interferon-stimulated genes' (ISG) responses. This helped in conceptualization of an 'in sitro' (in vitro+in situ) sFIS assay, where human myeloid cells were exposed to patients' serum in vitro, to assess serum-induced (si)-NFκB or interferon (IFN)/ISG responses (as active signaling reporter activity) within them, thereby 'mimicking' patients' in situ immunodynamic status. Multiparametric serum profiling of patients with OV established that sFIS assay can: decode peripheral immunology (by indicating higher enrichment of si-NFκB over si-IFN/ISG responses), estimate survival trends (si-NFκB or si-IFN/ISG responses associating with negative or positive prognosis, respectively), and coestimate malignancy risk (relative to benign/borderline ovarian lesions). Biologically, we documented dominance of pro-tumorigenic, myeloid si-NFκB responseHIGHsi-IFN/ISG responseLOW inflammation in periphery of patients with OV. Finally, in an orthotopic murine metastatic OV model, sFIS assay predicted the higher capacity of chemo-immunotherapy (paclitaxel-carboplatin plus anti-TNF antibody combination) in achieving a pro-immunogenic peripheral milieu (si-IFN/ISG responseHIGHsi-NFκB responseLOW), which aligned with high antitumor efficacy. CONCLUSIONS: We established sFIS assay as a novel biomarker resource for serum screening in patients with OV to evaluate peripheral immunodynamics, patient survival trends and malignancy risk, and to design preclinical chemo-immunotherapy strategies.

Type I interferons and endoplasmic reticulum stress in health and disease.

Type I interferons (IFNs) comprise of pro-inflammatory cytokines created, as well as sensed, by all nucleated cells with the main objective of blocking pathogens-driven infections. Owing to this broad range of influence, type I IFNs also exhibit critical functions in many sterile inflammatory diseases and immunopathologies, especially those associated with endoplasmic reticulum (ER) stress-driven signaling pathways. Indeed, over the years accumulating evidence has indicated that the presence of ER stress can influence the production, or sensing of, type I IFNs induced by perturbations like pattern recognition receptor (PRR) agonists, infections (bacterial, viral or parasitic) or autoimmunity. In this article we discuss the link between type I IFNs and ER stress in various diseased contexts. We describe how ER stress regulates type I IFNs production or sensing, or how type I IFNs may induce ER stress, in various circumstances like microbial infections, autoimmunity, diabetes, cancer and other ER stress-related contexts.

Necroptosis in Immuno-Oncology and Cancer Immunotherapy.

Immune-checkpoint blockers (ICBs) have revolutionized oncology and firmly established the subfield of immuno-oncology. Despite this renaissance, a subset of cancer patients remain unresponsive to ICBs due to widespread immuno-resistance. To "break" cancer cell-driven immuno-resistance, researchers have long floated the idea of therapeutically facilitating the immunogenicity of cancer cells by disrupting tumor-associated immuno-tolerance via conventional anticancer therapies. It is well appreciated that anticancer therapies causing immunogenic or inflammatory cell death are best positioned to productively activate anticancer immunity. A large proportion of studies have emphasized the importance of immunogenic apoptosis (i.e., immunogenic cell death or ICD); yet, it has also emerged that necroptosis, a programmed necrotic cell death pathway, can also be immunogenic. Emergence of a proficient immune profile for necroptosis has important implications for cancer because resistance to apoptosis is one of the major hallmarks of tumors. Putative immunogenic or inflammatory characteristics driven by necroptosis can be of great impact in immuno-oncology. However, as is typical for a highly complex and multi-factorial disease like cancer, a clear cause versus consensus relationship on the immunobiology of necroptosis in cancer cells has been tough to establish. In this review, we discuss the various aspects of necroptosis immunobiology with specific focus on immuno-oncology and cancer immunotherapy.

Trial watch: chemotherapy-induced immunogenic cell death in immuno-oncology.

The term 'immunogenic cell death' (ICD) denotes an immunologically unique type of regulated cell death that enables, rather than suppresses, T cell-driven immune responses that are specific for antigens derived from the dying cells. The ability of ICD to elicit adaptive immunity heavily relies on the immunogenicity of dying cells, implying that such cells must encode and present antigens not covered by central tolerance (antigenicity), and deliver immunostimulatory molecules such as damage-associated molecular patterns and cytokines (adjuvanticity). Moreover, the host immune system must be equipped to detect the antigenicity and adjuvanticity of dying cells. As cancer (but not normal) cells express several antigens not covered by central tolerance, they can be driven into ICD by some therapeutic agents, including (but not limited to) chemotherapeutics of the anthracycline family, oxaliplatin and bortezomib, as well as radiation therapy. In this Trial Watch, we describe current trends in the preclinical and clinical development of ICD-eliciting chemotherapy as partner for immunotherapy, with a focus on trials assessing efficacy in the context of immunomonitoring.

Type I interferons and dendritic cells in cancer immunotherapy.

Type I interferons (IFNs) facilitate cancer immunosurveillance, antitumor immunity and antitumor efficacy of conventional cell death-inducing therapies (chemotherapy/radiotherapy) as well as immunotherapy. Moreover, it is clear that dendritic cells (DCs) play a significant role in aiding type I IFN-driven immunity. Owing to these antitumor properties several immunotherapies involving, or inducing, type I IFNs have received considerable clinical attention, e.g., recombinant IFNα2 or agonists targeting pattern recognition receptor (PRR) pathways like Toll-like receptors (TLRs), cGAS-STING or RIG-I/MDA5/MAVS. A series of preclinical and clinical evidence concurs that the success of anticancer therapy hinges on responsiveness of both cancer cells and DCs to type I IFNs. In this article, we discuss this link between type I IFNs and DCs in the context of cancer biology, with particular attention to mechanisms behind type I IFN production, their impact on DC driven anticancer immunity, and the implications of this for cancer immunotherapy, including DC-based vaccines.