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The intestine is a site of diverse functions including digestion, nutrient absorption, immune surveillance, and microbial symbiosis. Intestinal microRNAs (miRNAs) are detectable in faeces and regulate barrier integrity, host-microbe interactions and the immune response, potentially offering valuable non-invasive tools to study intestinal health. However, current experimental methods are suboptimal and heterogeneity in study design limits the utility of faecal miRNA data. Here, we develop an optimised protocol for faecal miRNA detection and report a reproducible murine faecal miRNA profile in healthy mice. We use this pipeline to study faecal miRNAs during infection with the gastrointestinal helminth, Trichuris muris , revealing roles for miRNAs in fibrosis and wound healing. Intestinal fibrosis was confirmed in vivo using Hyperion® imaging mass cytometry, demonstrating the efficacy of this approach. Further applications of this optimised pipeline to study host-microbe interactions and intestinal disease will enable the generation of hypotheses and therapeutic strategies in diverse contexts. Here, Layton et al describe a non-invasive method to study intestinal disease by profiling faecal microRNAs, revealing intestinal fibrosis in chronic helminth infection.

Cell-based gene therapies, including chimeric antigen receptor-T, T-cell receptor-T, and tumor-infiltrating lymphocyte therapies, have transformed the treatment landscape for certain cancers, yet their efficacy in solid tumors remains limited. Next-generation therapies aim to overcome biological barriers, enhance potency and safety, and streamline development timelines through innovative approaches. Recent advances in genome editing technologies have identified hundreds of gene edits that improve T-cell functionality in preclinical models. However, the limited direct translatability of these findings and the impracticality of testing each of the individual edits in a traditional clinical trial highlight the need for more efficient strategies.This article provides an overview of genome-wide screens that identify gene knockouts and knock-ins to enhance T-cell function and the limitations with translating these results to human trials. Next, we propose a novel clinical trial design for testing multiple gene modifications simultaneously within a single T-cell infusion product. This approach would enable head-to-head evaluation of edits in an internally controlled setting, accelerating the identification of promising candidate edits. Key considerations for Chemistry, Manufacturing, and Controls, non-clinical evaluation, and clinical protocols are discussed, with an emphasis on patient safety and ethical transparency.This framework is informed by insights shared at the “Unlocking Complex Cell-based Gene Therapies” workshop, held on May 6, 2024. Co-hosted by Friends of Cancer Research and the Parker Institute for Cancer Immunotherapy, the event brought together participants from academia, the US Food and Drug Administration, and patient advocacy groups. By fostering collaboration among these stakeholders, this innovative approach aims to accelerate the development of effective cell-based therapies for complex diseases.

BackgroundImmune checkpoint inhibitors (ICIs) have led to enduring responses in subsets of patients with cancer. However, these responses carry the risk of immune-related adverse events (irAEs), which can diminish the overall benefit of ICI treatment. While associations between irAE development and overall survival have been increasingly documented, there is a need for further understanding of these connections in large prospective real-world cohorts.MethodsThe Resistance Drivers for Immuno-Oncology Patients Interrogated by Harmonized Molecular Datasets (RADIOHEAD) study, a pan-tumor, prospective cohort of 1,070 individuals undergoing standard of care first-line ICI treatment, aims to identify factors driving irAEs and clinical response. Clinical data and longitudinal blood samples were collected prospectively at multiple time points from 49 community-based oncology clinics across the USA. Structured, harmonized clinical data underwent unbiased statistical analysis to uncover predictors of real-world overall survival (rwOS) and risk factors for irAEs.ResultsAcross 1,070 participants’ treatment courses, RADIOHEAD accumulated over 4,500 clinical data points. Patients experiencing any irAE (25.4%, n=272) exhibited significantly improved rwOS in the pan-tumor cohort (n=1,028, HR=0.41, 95% CI=(0.31, 0.55)). This association persisted when adjusting for age and metastatic disease in multivariate time-dependent Cox proportional hazard analysis, and was consistent across major tumor subtypes, including lung cancer and melanoma. Skin and endocrine irAEs of any grade were strongly associated with improved rwOS (Cox proportional hazard analysis, skin, p=2.03e−05; endocrine, p=0.0006). In this real-world cohort, the irAE rate appeared lower than those reported in clinical trials. Patients receiving corticosteroids prior to initiation of ICI treatment had significantly worse survival outcomes than non-users (HR 1.37, p=0.0054), with a stronger association with systemic steroid use (HR 1.75, p=0.0022). The risk of irAE was increased by exposure to combination immunotherapy relative to monotherapy (OR 4.17, p=2.8e−7), zoster vaccine (OR 2.4, p=5.2e−05), and decreased by prior chemotherapy (OR 1.69, p=0.0005).ConclusionThe RADIOHEAD cohort is a well-powered, real-world cohort that clearly demonstrates the association between irAE development with improved response and baseline steroid use with worse response to ICI treatment after adjustment for survival bias.

It has recently been appreciated that NK cells exhibit many features reminiscent of adaptive immune cells. Considerable heterogeneity exists with respect to the ligand specificity of individual NK cells and as such, a subset of NK cells can respond, expand, and differentiate into memory-like cells in a ligand-specific manner. MHC I-binding inhibitory receptors, including those belonging to the Ly49 and KIR families, are expressed in a variegated manner, which creates ligand-specific diversity within the NK cell pool. However, how NK cells determine which inhibitory receptors to express on their cell surface during a narrow window of development is largely unknown. In this manuscript, we demonstrate that signals from activating receptors are critical for induction of Ly49 and KIR receptors during NK cell development; activating receptor-derived signals increased the probability of the Ly49 bidirectional Pro1 promoter to transcribe in the forward versus the reverse direction, leading to stable expression of Ly49 receptors in mature NK cells. Our data support a model where the balance of activating and inhibitory receptor signaling in NK cells selects for the induction of appropriate inhibitory receptors during development, which NK cells use to create a diverse pool of ligand-specific NK cells.

[This corrects the article DOI: 10.1371/journal.pbio.1002526.].

BackgroundProteomics holds great promise for cancer immunotherapy, with intensive efforts being exerted for early disease identification, patients selection, and adverse event prediction. Despite this potential, the high cost and low throughput of existing tools to profile circulating proteins render such studies prohibitively slow and costly, limiting their wide-spread application. As a result, proteomics studies in the field have been constrained to sample sizes in the 10s and 100s , restricting the power to discover key biomarkers. Here, we leverage a novel proteomics tool, the nELISA, to quantify ~600 circulating proteins across ~3000 samples from the RADIOHEAD (Resistance Drivers for Immuno-Oncology Patients Interrogated by Harmonized Molecular Datasets) cohort, a prospective study of 1070 immunotherapy naive pan-tumor patients on standard of care immune checkpoint inhibitor (ICI) therapy regimens from community oncology clinics.MethodsThe Nomic platform is a highly multiplexed immunoassay technology that enables the profiling of hundreds of proteins across 1536 samples per instrument daily, at significantly reduced costs. The method miniaturizes sandwich immunoassays by placing antibody pairs on the surface of color-coded microparticles, which can then be analyzed via high-throughput flow cytometry.ResultsUsing this technology, our preliminary data identified several markers of response to treatment; for example, PD-1 inhibitors result in increased circulating levels of soluble PD-1, and ICIs increase levels of several chemokines including CXCL9 and CXCL10, as seen in several other small-scale studies. We also identified several markers potentially predicting response to treatment and irAEs, which will require much larger datasets for validation. The scalable nature of the nELISA platform now allows us to validate these findings in a large longitudinal cohort, providing the power needed for such a broad biomarker discovery effort. In this presentation, we share the results of applying nELISA to the RADIOHEAD blood serum samples from pretreatment, early on-treatment, 6-month, and 12-month timepoints. For participants who experienced immune-related adverse events, additional samples were collected upon presentation and in follow-up visits - these samples were also analyzed in this study.ConclusionsPairing nELISA protein profiling of these longitudinal samples with associated demographic metadata and clinical outcomes provides an opportunity to identify clinically actionable mechanisms for ICI resistance and adverse events, discover targets for combination therapies and post-ICI treatment, and inform system biology approaches to elucidate disease pathways. Here, we highlight biomarkers and protein signatures related to patient outcomes, to reveal additional insights and further accelerate research in the field of cancer immunotherapy.

BackgroundPredicting the risk of immune-related adverse events (IrAEs) in patients receiving immune checkpoint blockade (ICB) therapy is crucial for optimizing safety and treatment outcomes. While single biomarkers have been implicated in risk stratification, multi-modal data integration can greatly enhance prediction accuracy.1–3 Here, we developed a blood-based, multi-modal predictor for estimating severe irAE risk prior to ICB initiation.MethodsClinical annotations, HLA genotyping, and bulk RNA-sequencing data from peripheral blood were analyzed across three cohorts: cancer patients prior to ICB therapy (Cohorts 1 and 2) and individuals with inflammatory bowel disease (IBD) or healthy controls (Cohort 3) (table 1). Events with grades ≥3 were defined as severe IrAEs. RNA-seq data were preprocessed to evaluate features such as immune signatures,4 cell composition,5 immunotypes,6 and TCR diversity. For overall IrAE risk estimation, the CatBoost classifier was used to train predictors on clinical data,7 while a logistic regression model was trained on integrated clinical and transcriptomic data. A genetic-based model based on HLA alleles was also developed to predict specific IrAE types.ResultsA CatBoost classifier trained to assess IrAE risk based on clinical data from Train Set 1 predicted severe irAEs in Test Sets 1 (AUC=0.72) and 2 (AUC=0.59). Age, therapy type, and diagnosis were the major risk determinants. We improved the model’s predictive capacity by combining its output with transcriptomic data (12 preprocessed features) and training a logistic regression classifier on Train Set 1, subsequently achieving AUC=0.79 in Test Set 1 and AUC=0.63 in Test Set 2. Features related to immune regulation (such as CD4+ T-cell activation, myeloid-cell-mediated suppression, and Treg activity) were strongly associated with severe IrAEs. Finally, using train and test sets from Cohort 3, we developed a CatBoost classifier based on 46 HLA alleles associated with risk of and protection against autoimmune colitis. This genetic-based model predicted the likelihood of colitis with AUC=0.68 in patients with severe IrAEs from Cohort 1, revealing a threefold HLA-driven increase in colitis risk (OR=2.8).ConclusionsOur multi-modal, machine-learning approach provides a robust framework for assessing severe IrAE risk before ICB initiation. Early identification and monitoring of high-risk patients will enable physicians to mitigate IrAEs in a timely manner, thus improving patient outcomes.ReferencesAli O, Berner F, Bomze D, Fässler M, Diem S, Cozzio A, Jörger M, Früh M, Driessen C, Lenz TL, Flatz L. Human leukocyte antigen variation is associated with adverse events of checkpoint inhibitors. Eur J Cancer. 2019;107:8–14.Ye W, Olsson-Brown A, Watson R, Cheung V, Morgan R, Nassiri I, Cooper R, Taylor C, Akbani U, Brain O, Matin R, Coupe N, Middleton M, Coles M, Sacco J, Payne M, Fairfax B. Checkpoint-blocker-induced autoimmunity is associated with favourable outcome in metastatic melanoma and distinct T-cell expression profiles. Br J Cancer. 2021;124(11):1661–1669.Kim KH, Hur JY, Cho J, Ku BM, Koh J, Koh JY, Sun JM, Lee SH, Ahn JS, Park K, Ahn MJ, Shin EC. Immune-related adverse events are clustered into distinct subtypes by T-cell profiling before and early after anti-PD-1 treatment. Oncoimmunology. 2020;9(1):1722023.1–12.Bolshakov E, Vasileva T, Kust S, Frank A, Savchenko M, Wang I, Conroy T, Merriam NR, Markova K, Brunovlenskaia-Bogoiavlenskaia A, Ushakova E, Ambarian S, Mulyukina A, Shilov E, Arutyunyan N, Shchetsova A, Shulga P, Spirin D, Terenteva A, Goldberg MF, Lawless A, Boland GM, Sullivan RJ, Zaytcev A. Identifying a composite signature for predicting immune-related adverse events in advanced melanoma patients treated with immune checkpoint blockade. J Immunother Cancer. 2024;12:e144.Zaitsev A, Chelushkin M, Dyikanov D, Cheremushkin I, Shpak B, Nomie K, Zyrin V, Nuzhdina E, Lozinsky Y, Zotova A, Degryse S, Kotlov N, Baisangurov A, Shatsky V, Afenteva D, Kuznetsov A, Paul SR, Davies DL, Reeves PM, Lanuti M, Goldberg MF, Tazearslan C, Chasse M, Wang I, Abdou M, Aslanian SM, Andrewes S, Hsieh JJ, Ramachandran A, Lyu Y, Galkin I, Svekolkin V, Cerchietti L, Poznansky MC, Ataullakhanov RI, Fowler N, Bagaev A. Precise reconstruction of the TME using bulk RNA-seq and a machine learning algorithm trained on artificial transcriptomes. Cancer Cell. 2022;40(8):879–894.e16.Dyikanov D, Zaitsev A, Vasileva T, Wang I, Sokolov A, Bolshakov E, Frank A, Turova P, Golubeva O, Gantseva A, Kamysheva A, Shpudeiko P, Krauz I, Abdou M, Chasse M, Conroy T, Merriam NR, Alesse JE, English N, Shpak B, Shchetsova A, Tikhonov E, Filatov I, Radko A, Bolshakova A, Kachalova A, Lugovykh N, Bulahov A, Kilina A, Asanbekov S, Zheleznyak I, Skoptsov P, Alekseeva E, Johnson JM, Curry JM, Linnenbach AJ, South AP, Yang EJ, Morozov K, Terenteva A, Nigmatullina L, Fastovetz D, Bobe A, Balabanian L, Nomie K, Yong ST, Davitt CJH, Ryabykh A, Kudryashova O, Tazearslan C, Bagaev A, Fowler N, Luginbuhl AJ, Ataullakhanov R, Goldberg MF. Comprehensive peripheral blood immunoprofiling reveals five immunotypes with immunotherapy response characteristics in patients with cancer. Cancer Cell. 2024;42(5):759–779.e12.Quandt Z, Lucas A, Liang SI, Yang E, Stone S, Fadlullah MZH, Bayless NL, Marr SS, Thompson MA, Padron LJ, Bucktrout S, Butterfield LH, Tan AC, Herold KC, Bluestone JA, Anderson MS, Spencer CN, Young A, Connolly JE. Associations between immune checkpoint inhibitor response, immune-related adverse events, and steroid use in RADIOHEAD: a prospective pan-tumor cohort study. J Immunother Cancer. 2025 May 12;13(5):e011545.Ethics ApprovalThis study involves human participants and was approved by WCG IRB Protocol #20182579. Participants gave informed consent to participate in the study before taking part.Abstract 1094 Table 1Cohort description

NK cells are part of the innate immune system, and play an important role in viral and tumor defense. Improving natural killer (NK) cell function could be beneficial for enhancing anti-tumor or anti-viral responses. However, efforts to improve NK cell function by disrupting negative regulators that target proximal signaling pathways paradoxically results in less responsive NK cells. This is often attributed to their ability to tune their responsiveness. In this thesis, I found that NK cells are extremely sensitive to loss of inhibitory ligand or mediators of inhibitory signaling. Using adoptive transfer and mixed chimera models, I found that MHC class I expression is necessary both in cis and trans for NK cells to possess full functionality. Furthermore, using an acute model of genetic targeting, I found that temporal ablation of SHP-1 was sufficient to drive hyporesponsiveness, and the loss of even a single allele of SHP-1 had profound effects on NK cell responses. However, the data also showed that tuned NK cells could still be stimulated to respond via analogs of secondary messengers of signaling, suggesting that NK cell tuning targets proximal signaling pathways. To improve NK cell function but avoid NK cell tuning, I targeted a distal negative regulator of signaling. I found that genetic deletion of diacylglycerol kinase zeta (DGKζ), a negative regulator of diacylglycerol-mediated signaling, enhances NK cell function due to its distal position in the signaling cascade. Upon activating receptor stimulation, NK cells from mice lacking DGKζ display increased cytokine production and cytotoxicity in an ERK-dependent manner. This enhancement of NK cell function is NK cell-intrinsic and developmentally independent. Importantly, DGKζ deficiency does not affect inhibitory NK cell receptor expression or function. Thus, DGKζ KO mice display enhanced clearance of a TAP-deficient tumor. I therefore propose that enzymes that negatively regulate distal signaling pathways such as DGKζ represent novel targets for augmenting the therapeutic potential of NK cells.

Gliomas are among the most lethal cancers, with limited treatment options. To uncover hallmarks of therapeutic escape and tumor microenvironment (TME) evolution, we applied spatial proteomics, transcriptomics, and glycomics to 670 lesions from 310 adult and pediatric patients. Single-cell analysis shows high B7H3+ tumor cell prevalence in glioblastoma (GBM) and pleomorphic xanthoastrocytoma (PXA), while most gliomas, including pediatric cases, express targetable tumor antigens in less than 50% of tumor cells, potentially explaining trial failures. Longitudinal samples of isocitrate dehydrogenase (IDH)-mutant gliomas reveal recurrence driven by tumor-immune spatial reorganization, shifting from T-cell and vasculature-associated myeloid cell-enriched niches to microglia and CD206+ macrophage-dominated tumors. Multi-omic integration identified N-glycosylation as the best classifier of grade, while the immune transcriptome best predicted GBM survival. Provided as a community resource, this study opens new avenues for glioma targeting, classification, outcome prediction, and a baseline of TME composition across all stages.

Heart disease is the leading cause of death worldwide. A key pathogenic factor in the development of lethal heart failure is loss of terminally differentiated cardiomyocytes. However, mechanisms of cardiomyocyte death remain unclear. Here, we discovered and demonstrated that ferroptosis, a programmed iron-dependent cell death, as a mechanism in murine models of doxorubicin (DOX)- and ischemia/reperfusion (I/R)-induced cardiomyopathy. In canonical apoptosis and/or necroptosis-defective Ripk3−/−, Mlkl−/−, or Fadd−/−Mlkl−/− mice, DOX-treated cardiomyocytes showed features of typical ferroptotic cell death. Consistently, compared with dexrazoxane, the only FDA-approved drug for treating DOX-induced cardiotoxicity, inhibition of ferroptosis by ferrostatin-1 significantly reduced DOX cardiomyopathy. RNA-sequencing results revealed that heme oxygenase-1 (Hmox1) was significantly up-regulated in DOX-treated murine hearts. Administering DOX to mice induced cardiomyopathy with a rapid, systemic accumulation of nonheme iron via heme degradation by Nrf2-mediated upregulation of Hmox1, which effect was abolished in Nrf2-deficent mice. Conversely, zinc protoporphyrin IX, an Hmox1 antagonist, protected the DOX-treated mice, suggesting free iron released on heme degradation is necessary and sufficient to induce cardiac injury. Given that ferroptosis is driven by damage to lipid membranes, we further investigated and found that excess free iron accumulated inmitochondria and caused lipid peroxidation on its membrane. Mitochondria-targeted antioxidant MitoTEMPO significantly rescued DOX cardiomyopathy, supporting oxidative damage of mitochondria as a major mechanism in ferroptosis-induced heart damage. Importantly, ferrostatin-1 and iron chelation also ameliorated heart failure induced by both acute and chronic I/R in mice. These findings highlight that targeting ferroptosis serves as a cardioprotective strategy for cardiomyopathy prevention.