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Supplementary Data from Plexin-A4 Mediates Cytotoxic T-cell Trafficking and Exclusion in Cancer

Supplementary Data from Efficacy of CD40 Agonists Is Mediated by Distinct cDC Subsets and Subverted by Suppressive Macrophages

Agonistic αCD40 therapy has been shown to inhibit cancer progression in only a fraction of patients. Understanding the cancer cell–intrinsic and microenvironmental determinants of αCD40 therapy response is therefore crucial to identify responsive patient populations and to design efficient combinatorial treatments. Here, we show that the therapeutic efficacy of αCD40 in subcutaneous melanoma relies on preexisting, type 1 classical dendritic cell (cDC1)–primed CD8+ T cells. However, after administration of αCD40, cDC1s were dispensable for antitumor efficacy. Instead, the abundance of activated cDCs, potentially derived from cDC2 cells, increased and further activated antitumor CD8+ T cells. Hence, distinct cDC subsets contributed to the induction of αCD40 responses. In contrast, lung carcinomas, characterized by a high abundance of macrophages, were resistant to αCD40 therapy. Combining αCD40 therapy with macrophage depletion led to tumor growth inhibition only in the presence of strong neoantigens. Accordingly, treatment with immunogenic cell death–inducing chemotherapy sensitized lung tumors to αCD40 therapy in subcutaneous and orthotopic settings. These insights into the microenvironmental regulators of response to αCD40 suggest that different tumor types would benefit from different combinations of therapies to optimize the clinical application of CD40 agonists.Significance:This work highlights the temporal roles of different dendritic cell subsets in promoting CD8+ T-cell–driven responses to CD40 agonist therapy in cancer.

Supplementary Figure from Efficacy of CD40 Agonists Is Mediated by Distinct cDC Subsets and Subverted by Suppressive Macrophages

Supplementary materials and methods

Supplementary Figures 1-6. Suppl. Fig. 1. Mel-ILP induces release of certain cytokines, however does not affect infiltration of immune cells into tumor bed. Suppl. Fig. 2. Melphalan induces apoptosis of melanoma cells. Suppl. Fig. 3 Melphalan induces exposure of danger signals. Suppl. Fig. 4. Dying human melanoma cells induce danger signaling-independent in vitro stimulation of immune cells. Suppl. Fig. 5. In vitro treated melanoma cells do not strongly activate NK cells. Suppl. Fig. 6. (A) The phenotypic maturation of murine iDCs (B) Effect of antibodybased elimination of CD4+ and CD8+ cells in prophylactic mice vaccination experiment

Supplementary Tables 1-3. Supplementary Table 1. Meta-data analysis of clinical response to melphalan-based isolated limbtreatment procedures in melanoma patients. Supplementary Table 2. Meta-data analysis of clinical response to melphalan/TNF or Mel/TNF/IFNγ combination therapies for isolated limb-treatment procedures in melanoma patients. Supplementary Table 3. Follow up information about the patients whose tumour samples were used for analysis of infiltration of immune cells and expression levels of cytokines.

Systemic chemotherapy generally has been considered immunosuppressive, but it has become evident that certain chemotherapeutic drugs elicit immunogenic danger signals in dying cancer cells that can incite protective antitumor immunity. In this study, we investigated whether locoregionally applied therapies, such as melphalan, used in limb perfusion for melanoma (Mel-ILP) produce related immunogenic effects. In human melanoma biopsies, Mel-ILP treatment upregulated IL1B, IL8, and IL6 associated with their release in patients' locoregional sera. Although induction of apoptosis in melanoma cells by melphalan in vitro did not elicit threshold levels of endoplasmic reticulum and reactive oxygen species stress associated with danger signals, such as induction of cell-surface calreticulin, prophylactic immunization and T-cell depletion experiments showed that melphalan administration in vivo could stimulate a CD8+ T cell–dependent protective antitumor response. Interestingly, the vaccination effect was potentiated in combination with exogenous calreticulin, but not tumor necrosis factor, a cytokine often combined with Mel-ILP. Our results illustrate how melphalan triggers inflammatory cell death that can be leveraged by immunomodulators such as the danger signal calreticulin. Cancer Res; 75(8); 1603–14. ©2015 AACR.

Immune-checkpoint blockers (ICB) provide limited benefit against T cell-depleted tumours, calling for therapeutic innovation. Here, we aimed at designing a new type of dendritic cell (DC) vaccine by unbiased computational integration of multi-omics data from cancer patients. In a first attempt, a DC vaccine designed to present tumor antigens from cancer cells succumbing to immunogenic cancer cell death (ICD) and to elicit high type I interferon (IFN) responses failed to induce the regression of mouse tumors lacking T cell infiltrates. In lymph nodes (LNs), instead of activating CD4+and CD8+T cells, DCs stimulated immunosuppressive PD-L1+LN-associated macrophages (LAMs) via a type I IFN response. Moreover, DC vaccines of this type stimulated pre-existing, T cell-suppressive, PD-L1+tumour-associated macrophages (TAMs). This created a T cell-suppressive circuit of PD-L1+macrophages, spanning across LNs and tumours. Accordingly, DC vaccines synergised with PD-L1 blockade to deplete PD-L1+macrophages, suppress myeloid inflammation affecting the tumor bed and draining lymph nodes, and de-inhibit effector/stem-like memory T cells, eventually causing tumour regression. The synergistic interaction between the DC vaccine and PD-L1 blockade was lost when DCs were manipulated to loseIfnar1orCcr7or when macrophages were depleted. Interestingly, clinical DC vaccines also potentiated lymphocyte-suppressive PD-L1+TAMs in patients bearing T cell-depleted tumours. Altogether, our results reveal the existence of a novel PD-L1+LAM/TAM-driven immunosuppressive pathway that can be elicited by DC vaccines, yet can be subverted for improving the outcome of immunotherapy.

Immunogenic cell death (ICD) has emerged as a key component of therapy-induced anti-tumor immunity. Over the past few years, ICD was found to play a pivotal role in a wide variety of novel and existing treatment modalities. The clinical application of these techniques in cancer treatment is still in its infancy. Glioblastoma (GBM) is the most lethal primary brain tumor with a dismal prognosis despite maximal therapy. The development of new therapies in this aggressive type of tumors remains highly challenging partially due to the cold tumor immune environment. GBM could therefore benefit from ICD-based therapies stimulating the anti-tumor immune response. In what follows, we will describe the mechanisms behind ICD and the ICD-based (pre)clinical advances in anticancer therapies focusing on GBM. ispartof: GENES AND IMMUNITY vol:23 issue:1 pages:1-11 ispartof: location:England status: published

FOXP3-expressing regulatory T cells (T

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.

Immune-checkpoint blockade (ICB) combined with neoadjuvant chemotherapy improves pathological complete response in breast cancer. To understand why only a subset of tumors respond to ICB, patients with hormone receptor-positive or triple-negative breast cancer were treated with anti-PD1 before surgery. Paired pre- versus on-treatment biopsies from treatment-naive patients receiving anti-PD1 (n = 29) or patients receiving neoadjuvant chemotherapy before anti-PD1 (n = 11) were subjected to single-cell transcriptome, T cell receptor and proteome profiling. One-third of tumors contained PD1-expressing T cells, which clonally expanded upon anti-PD1 treatment, irrespective of tumor subtype. Expansion mainly involved CD8+ T cells with pronounced expression of cytotoxic-activity (PRF1, GZMB), immune-cell homing (CXCL13) and exhaustion markers (HAVCR2, LAG3), and CD4+ T cells characterized by expression of T-helper-1 (IFNG) and follicular-helper (BCL6, CXCR5) markers. In pre-treatment biopsies, the relative frequency of immunoregulatory dendritic cells (PD-L1+), specific macrophage phenotypes (CCR2+ or MMP9+) and cancer cells exhibiting major histocompatibility complex class I/II expression correlated positively with T cell expansion. Conversely, undifferentiated pre-effector/memory T cells (TCF7+, GZMK+) or inhibitory macrophages (CX3CR1+, C3+) were inversely correlated with T cell expansion. Collectively, our data identify various immunophenotypes and associated gene sets that are positively or negatively correlated with T cell expansion following anti-PD1 treatment. We shed light on the heterogeneity in treatment response to anti-PD1 in breast cancer. Transcriptomic and proteomic profiling of breast cancer biopsies identifies baseline features of the tumor immune microenvironment associated with T cell clonal expansion following neoadjuvant anti-PD-1 treatment.

The COVID-19 pandemic poses a major burden on healthcare and economic systems across the globe. Even though a majority of the population develops only minor symptoms upon SARS-CoV-2 infection, a significant number are hospitalized at intensive care units (ICU) requiring critical care. While insights into the early stages of the disease are rapidly expanding, the dynamic immunological processes occurring in critically ill patients throughout their recovery at ICU are far less understood. Here, we have analysed whole blood samples serially collected from 40 surviving COVID-19 patients throughout their recovery in ICU using high-dimensional cytometry by time-of-flight (CyTOF) and cytokine multiplexing. Based on the neutrophil-to-lymphocyte ratio (NLR), we defined four sequential immunotypes during recovery that correlated to various clinical parameters, including the level of respiratory support at concomitant sampling times. We identified classical monocytes as the first immune cell type to recover by restoration of HLA-DR-positivity and the reduction of immunosuppressive CD163 + monocytes, followed by the recovery of CD8 + and CD4 + T cell and non-classical monocyte populations. The identified immunotypes also correlated to aberrant cytokine and acute-phase reactant levels. Finally, integrative analysis of cytokines and immune cell profiles showed a shift from an initially dysregulated immune response to a more coordinated immunogenic interplay, highlighting the importance of longitudinal sampling to understand the pathophysiology underlying recovery from severe COVID-19. Supplementary Information The online version contains supplementary material available at 10.1007/s00018-021-03808-8.

Contains fulltext : 283345.pdf (Publisher’s version ) (Open Access) 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.

Agonistic αCD40 therapy has been shown to inhibit cancer progression in only a fraction of patients. Understanding the cancer cell–intrinsic and microenvironmental determinants of αCD40 therapy response is therefore crucial to identify responsive patient populations and to design efficient combinatorial treatments. Here, we show that the therapeutic efficacy of αCD40 in subcutaneous melanoma relies on preexisting, type 1 classical dendritic cell (cDC1)–primed CD8+ T cells. However, after administration of αCD40, cDC1s were dispensable for antitumor efficacy. Instead, the abundance of activated cDCs, potentially derived from cDC2 cells, increased and further activated antitumor CD8+ T cells. Hence, distinct cDC subsets contributed to the induction of αCD40 responses. In contrast, lung carcinomas, characterized by a high abundance of macrophages, were resistant to αCD40 therapy. Combining αCD40 therapy with macrophage depletion led to tumor growth inhibition only in the presence of strong neoantigens. Accordingly, treatment with immunogenic cell death–inducing chemotherapy sensitized lung tumors to αCD40 therapy in subcutaneous and orthotopic settings. These insights into the microenvironmental regulators of response to αCD40 suggest that different tumor types would benefit from different combinations of therapies to optimize the clinical application of CD40 agonists. Significance: This work highlights the temporal roles of different dendritic cell subsets in promoting CD8+ T-cell–driven responses to CD40 agonist therapy in cancer.

Mounting evidence indicates that immunogenic therapies engaging the unfolded protein response (UPR) following endoplasmic reticulum (ER) stress favor proficient cancer cell-immune interactions, by stimulating the release of immunomodulatory/proinflammatory factors by stressed or dying cancer cells. UPR-driven transcription of proinflammatory cytokines/chemokines exert beneficial or detrimental effects on tumor growth and antitumor immunity, but the cell-autonomous machinery governing the cancer cell inflammatory output in response to immunogenic therapies remains poorly defined. Here, we profiled the transcriptome of cancer cells responding to immunogenic or weakly immunogenic treatments. Bioinformatics-driven pathway analysis indicated that immunogenic treatments instigated a NF-κB/AP-1-inflammatory stress response, which dissociated from both cell death and UPR. This stress-induced inflammation was specifically abolished by the IRE1α-kinase inhibitor KIRA6. Supernatants from immunogenic chemotherapy and KIRA6 co-treated cancer cells were deprived of proinflammatory/chemoattractant factors and failed to mobilize neutrophils and induce dendritic cell maturation. Furthermore, KIRA6 significantly reduced the in vivo vaccination potential of dying cancer cells responding to immunogenic chemotherapy. Mechanistically, we found that the anti-inflammatory effect of KIRA6 was still effective in IRE1α-deficient cells, indicating a hitherto unknown off-target effector of this IRE1α-kinase inhibitor. Generation of a KIRA6-clickable photoaffinity probe, mass spectrometry, and co-immunoprecipitation analysis identified cytosolic HSP60 as a KIRA6 off-target in the IKK-driven NF-κB pathway. In sum, our study unravels that HSP60 is a KIRA6-inhibitable upstream regulator of the NF-κB/AP-1-inflammatory stress responses evoked by immunogenic treatments. It also urges caution when interpreting the anti-inflammatory action of IRE1α chemical inhibitors. ispartof: CELL DEATH AND DIFFERENTIATION vol:29 issue:1 pages:230-245 ispartof: location:England status: published

Simple Summary Over the last 5 years, immune checkpoint inhibitors (ICIs) are increasingly used in the treatment of non-small cell lung cancer (NSCLC) as either monotherapy or in combination with chemo- and/or radiotherapy. However, despite these advances, outcome still remains poor for most patients and there is still a lot of room to improve prognosis in these patients. To date, we have no tools that allow us to identify the patients that will benefit from chemo- and/or radiotherapy combined with immunotherapy, what treatment-induced immune changes can be expected, and what are the most optimal treatment combinations. Therefore, prognostic and predictive immunological biomarkers are urgently needed. This prospective exploratory study aimed to identify potential prognostic and predictive immune-related proteins that are associated with progression-free survival in patients with stage I/III NSCLC. The results of this trial provide a good starting point to implement blood-based immune profiling analyses in future clinical trials. Abstract 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.

Cytotoxic T cell (CTL) infiltration of the tumor carries the potential to limit cancer progression, but their exclusion by the immunosuppressive tumor microenvironment hampers the efficiency of immunotherapy. Here, we show that expression of the axon guidance molecule Plexin-A4 (Plxna4) in CTLs, especially in effector/memory CD8+ T cells, is induced upon T-cell activation, sustained in the circulation, but reduced when entering the tumor bed. Therefore, we deleted Plxna4 and observed that Plxna4-deficient CTLs acquired improved homing capacity to the lymph nodes and to the tumor, as well as increased proliferation, both achieved through enhanced Rac1 activation. Mice with stromal or hematopoietic Plxna4 deletion exhibited enhanced CTL infiltration and impaired tumor growth. In a melanoma model, adoptive transfer of CTLs lacking Plxna4 prolonged survival and improved therapeutic outcome, which was even stronger when combined with anti–programmed cell death protein 1 (PD-1) treatment. PLXNA4 abundance in circulating CTLs was augmented in melanoma patients versus healthy volunteers but decreased after the first cycle of anti–PD-1, alone or in combination with anti–cytotoxic T-Lymphocyte Associated Protein 4 (CTLA-4), in those patients showing complete or partial response to the treatment. Altogether, our data suggest that Plxna4 acts as a “checkpoint,” negatively regulating CTL migration and proliferation through cell-autonomous mechanisms independent of the interaction with host-derived Plxna4 ligands, semaphorins. These findings pave the way toward Plxna4-centric immunotherapies and propose Plxna4 detection in circulating CTLs as a potential way to monitor the response to immune checkpoint blockade in patients with metastatic melanoma.

Tumoural-CD8+T cells exhibit exhausted or dysfunctional states. Contrary to immunotherapy-responsive exhausted-CD8+T cells, the clinical features of dysfunctional-CD8+T cells are disputed. Hence, we conducted large-scale multi-omics and multi-dimensional mapping of CD8+T cell-states across multiple cancer patient-cohorts. This identified tumour-specific continuum of CD8+T cell-states across 6 human cancers, partly imprinted by organ-specific immuno-modulatory niches. Herein, melanoma and glioblastoma enriched prototypical exhausted (CD8+TEXT) and severely-dysfunctional (CD8+TSDF) states, respectively. Contrary to CD8+TEXT, CD8+TSDF displayed transcriptomic and epigenetic effector/cytolytic dysfunctions, and dysregulated effector/memory single-cell trajectories, culminating into maladaptive prodeath stress and cell-cycle defects. Suboptimal antigen-priming underscored CD8+TSDF, which was distinct from immune-checkpoints “rich” CD8+TEXT, reflecting chronic antigen-stimulation. Continuum variation also existed on tumour spatial-level, with convergent (CD8+TEXT-supportive vascular regions) and divergent features (dysfunctional CD4+T::CD8+TSDFcell-to-cell interactions) between melanoma and glioblastoma. Globally, IFNγ-IL2 disparities, paucity of intra-tumoural CD4+/CD8+T cells, and myeloid TGFβ/wound healing responses, distinguished CD8+TSDF-landscape. Within immuno-oncology clinical-trials, anti-PD1 immunotherapy failed to “reinvigorate” CD8+TSDF-landscape, and instead facilitated effector-dysfunction and TGFβ/wound healing. However, cellular immunotherapies (dendritic cell-vaccines, adoptive T-cell therapy) ameliorated assorted CD8+TSDF-landscape disparities, highlighting a roadmap for anti-glioblastoma multimodal-immunotherapy. Collectively, our study comprehensively expands clinical-knowledge on CD8+T cell-exhaustion and suggests that tumour-specific, pre-existing CD8+TEXT/TSDF-states, determine immunotherapy-responses.

Over the last two decades, a large volume of studies has established that dying and dead cancer cells exert a potent immunomodulatory effect on their immediate microenvironment, which has a major influence on the anticancer immunity [...]. ispartof: CELLS vol:10 issue:5 ispartof: location:Switzerland status: published

BNIP3 is a mitophagy receptor with context‐dependent roles in cancer, but whether and how it modulates melanoma growth in vivo remains unknown. Here, we found that elevated BNIP3 levels correlated with poorer melanoma patient’s survival and depletion of BNIP3 in B16‐F10 melanoma cells compromised tumor growth in vivo. BNIP3 depletion halted mitophagy and enforced a PHD2‐mediated downregulation of HIF‐1α and its glycolytic program both in vitro and in vivo. Mechanistically, we found that BNIP3‐deprived melanoma cells displayed increased intracellular iron levels caused by heightened NCOA4‐mediated ferritinophagy, which fostered PHD2‐mediated HIF‐1α destabilization. These effects were not phenocopied by ATG5 or NIX silencing. Restoring HIF‐1α levels in BNIP3‐depleted melanoma cells rescued their metabolic phenotype and tumor growth in vivo, but did not affect NCOA4 turnover, underscoring that these BNIP3 effects are not secondary to HIF‐1α. These results unravel an unexpected role of BNIP3 as upstream regulator of the pro‐tumorigenic HIF‐1α glycolytic program in melanoma cells., Mitophagy receptor BNIP3 facilitates skin cancer by supporting HIF1‐1α protein stability.

BackgroundTumors 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.MethodsWe 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.ResultsIn 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.ConclusionsWe 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.

Epidemiological and clinical reports indicate that SARS-CoV-2 virulence hinges upon the triggering of an aberrant host immune response, more so than on direct virus-induced cellular damage. To elucidate the immunopathology underlying COVID-19 severity, we perform cytokine and multiplex immune profiling in COVID-19 patients. We show that hypercytokinemia in COVID-19 differs from the interferon-gamma-driven cytokine storm in macrophage activation syndrome, and is more pronounced in critical versus mild-moderate COVID-19. Systems modelling of cytokine levels paired with deep-immune profiling shows that classical monocytes drive this hyper-inflammatory phenotype and that a reduction in T-lymphocytes correlates with disease severity, with CD8+ cells being disproportionately affected. Antigen presenting machinery expression is also reduced in critical disease. Furthermore, we report that neutrophils contribute to disease severity and local tissue damage by amplification of hypercytokinemia and the formation of neutrophil extracellular traps. Together our findings suggest a myeloid-driven immunopathology, in which hyperactivated neutrophils and an ineffective adaptive immune system act as mediators of COVID-19 disease severity., The host immune response plays a critical role in the immunopathology of SARS-CoV2. Here the authors combine a systems biology approach to implicate monocytes as key drivers of cytokine storm and disturbed neutrophil activation in COVID-19 disease severity.

Cellular stress evoked by immunogenic anticancer treatments engaging the unfolded protein response (UPR) can elicit inflammation with conflicting therapeutic outcomes. To define cell-autonomous mechanisms coupling the UPR to molecular mediators of inflammation, we profiled the transcriptome of cancer cells responding to immunogenic or weakly immunogenic-treatments. Bioinformatics-driven pathway analysis indicated that immunogenic treatments instigated NF-κB/AP-1-inflammatory pathways, which were abolished by the IRE1α-kinase inhibitor KIRA6. Cell-free fractions of chemotherapy and KIRA6 co-treated cancer cells were deprived of pro-inflammatory/chemoattractant factors and failed to mobilize innate immune cells. Strikingly, these potent KIRA6 anti-inflammatory effects were found to be independent of IRE1α. Generation of a KIRA6-clickable photoaffinity probe, mass spectrometry and co-immunoprecipitation analysis identified cytosolic HSP60 as a KIRA6 off-target in the NF-κB pathway. In sum, our study unravels that inflammation evoked by immunogenic treatments is curtailed by KIRA6 independently of IRE1α and further suggests great caution in interpreting the anti-inflammatory action of IRE1α chemical inhibitors.

The COVID-19 pandemic poses a major burden on health-care and economic systems across the globe. Even though a majority of the population only develops minor symptoms upon SARS-CoV2 infection, a significant proportion are hospitalized at intensive care units (ICU) requiring critical care. While insights into the early stages of the disease are gradually expanding, the dynamic immunological processes occurring in critically ill patients throughout their recovery at ICU are far less understood. Here, we have analysed longitudinally collected, whole blood samples of 40 surviving COVID-19 patients during their recovery at ICU using high-dimensional cytometry by time-of-flight (CyTOF) and cytokine multiplexing. Based on the neutrophil to lymphocyte ratio (NLR), we defined 4 sequential immunotypes during recovery that correlated to various clinical parameters, including the level of respiratory support at concomitant sampling times. We also identified classical monocytes as the first immune cell type to recover by restoring HLA-DR-positivity and by reducing the immunosuppressive CD163+ monocyte population, followed by the recovery of CD8+ and CD4+ T cell, and mDC populations. The identified immunotypes also correlated to aberrant cytokine and acute-phase reactant levels. Finally, integrative analysis of cytokines and immune cell profiles showed a shift from an initially dysregulated immune response to a more coordinated immunogenic interplay, highlighting the importance of longitudinal sampling to understand the pathophysiology underlying recovery from severe COVID-19.