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OBJECTIVE: The aim of this study was to determine whether single-cell and plasma proteomic elements of the host's immune response to surgery accurately identify patients who develop a surgical site complication (SSC) after major abdominal surgery. SUMMARY BACKGROUND DATA: SSCs may occur in up to 25% of patients undergoing bowel resection, resulting in significant morbidity and economic burden. However, the accurate prediction of SSCs remains clinically challenging. Leveraging high-content proteomic technologies to comprehensively profile patients' immune response to surgery is a promising approach to identify predictive biological factors of SSCs. METHODS: Forty-one patients undergoing non-cancer bowel resection were prospectively enrolled. Blood samples collected before surgery and on postoperative day one (POD1) were analyzed using a combination of single-cell mass cytometry and plasma proteomics. The primary outcome was the occurrence of an SSC, including surgical site infection, anastomotic leak, or wound dehiscence within 30âdays of surgery. RESULTS: A multiomic model integrating the single-cell and plasma proteomic data collected on POD1 accurately differentiated patients with (n = 11) and without (n = 30) an SSC [area under the curve (AUC) = 0.86]. Model features included coregulated proinflammatory (eg, IL-6- and MyD88- signaling responses in myeloid cells) and immunosuppressive (eg, JAK/STAT signaling responses in M-MDSCs and Tregs) events preceding an SSC. Importantly, analysis of the immunological data obtained before surgery also yielded a model accurately predicting SSCs (AUC = 0.82). CONCLUSIONS: The multiomic analysis of patients' immune response after surgery and immune state before surgery revealed systemic immune signatures preceding the development of SSCs. Our results suggest that integrating immunological data in perioperative risk assessment paradigms is a plausible strategy to guide individualized clinical care.
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Fístula Anastomótica/epidemiologia , Proteínas Sanguíneas/análise , Proteínas Alimentares/sangue , Deiscência da Ferida Operatória/epidemiologia , Infecção da Ferida Cirúrgica/epidemiologia , Adulto , Estudos de Coortes , Procedimentos Cirúrgicos do Sistema Digestório , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Modelos Teóricos , Prognóstico , Estudos Prospectivos , Proteoma , Análise de Célula ÚnicaRESUMO
OBJECTIVE: Systemic sclerosis (SSc) is a connective tissue disease with poorly understood pathogenesis and limited treatment options. Patient mortality is rooted predominantly in the development of pulmonary and cardiac complications. The overactivated immune system is assumed to sustain the inflammatory signature of this autoimmune disease. Here, we investigate the potential of immunoregulatory invariant natural killer T (iNKT) cells to inhibit proinflammatory B cell responses in an in vitro model of inflammation. METHODS: B cells from healthy volunteers (n = 17) and patients with SSc (n = 15) were used for functional testing upon lipopolysaccharide (LPS) stimulation in a co-culture system with third-party iNKT cells. Cytokine production was measured with antibody-based immunoassays (ELISA) and intracellular cytokine staining. RESULTS: iNKT cells strongly inhibited the production of proinflammatory interleukin-6 by B cells upon stimulation with LPS in both healthy volunteers and patients with SSc. In a Transwell assay, cell contact between B cells and iNKT cells proved necessary for this inhibitory effect. Similarly, blocking of CD1d on the surface of B cells abolished the immunoregulatory effect of iNKT cells on B cells. B cell subsets with higher expression of CD1d, namely unswitched memory B cells, were more susceptible to iNKT cell inhibition. CONCLUSION: Our in vitro data underline the potential of iNKT cells in the control of SSc and provide a rationale for the use of novel iNKT cell-based therapeutic strategies in the context of autoimmune diseases.
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Células T Matadoras Naturais , Escleroderma Sistêmico , Citocinas/metabolismo , Humanos , Interleucina-6/metabolismo , Lipopolissacarídeos , Ativação Linfocitária , Escleroderma Sistêmico/metabolismo , Escleroderma Sistêmico/terapiaRESUMO
PURPOSE OF REVIEW: Postoperative complications including infections, cognitive impairment, and protracted recovery occur in one-third of the 300 million surgeries performed annually worldwide. Complications cause personal suffering along with a significant economic burden on our healthcare system. However, the accurate prediction of postoperative complications and patient-targeted interventions for their prevention remain as major clinical challenges. RECENT FINDINGS: Although multifactorial in origin, the dysregulation of immunological mechanisms that occur in response to surgical trauma is a key determinant of postoperative complications. Prior research, primarily focusing on inflammatory plasma markers, has provided important clues regarding their pathogenesis. However, the recent advent of high-content, single-cell transcriptomic, and proteomic technologies has considerably improved our ability to characterize the immune response to surgery, thereby providing new means to understand the immunological basis of postoperative complications and to identify prognostic biological signatures. SUMMARY: The comprehensive and single-cell characterization of the human immune response to surgery has significantly advanced our ability to predict the risk of postoperative complications. Multiomic modeling of patients' immune states holds promise for the discovery of preoperative predictive biomarkers, ultimately providing patients and surgeons with actionable information to improve surgical outcomes. Although recent studies have generated a wealth of knowledge, laying the foundation for a single-cell atlas of the human immune response to surgery, larger-scale multiomic studies are required to derive robust, scalable, and sufficiently powerful models to accurately predict the risk of postoperative complications in individual patients.
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Complicações Pós-Operatórias , Proteômica , Biomarcadores , Humanos , Imunidade , PrognósticoRESUMO
Stroke is a leading cause of cognitive impairment and dementia, but the mechanisms that underlie post-stroke cognitive decline are not well understood. Stroke produces profound local and systemic immune responses that engage all major innate and adaptive immune compartments. However, whether the systemic immune response to stroke contributes to long-term disability remains ill-defined. We used a single-cell mass cytometry approach to comprehensively and functionally characterize the systemic immune response to stroke in longitudinal blood samples from 24 patients over the course of 1 year and correlated the immune response with changes in cognitive functioning between 90 and 365 days post-stroke. Using elastic net regularized regression modelling, we identified key elements of a robust and prolonged systemic immune response to ischaemic stroke that occurs in three phases: an acute phase (Day 2) characterized by increased signal transducer and activator of transcription 3 (STAT3) signalling responses in innate immune cell types, an intermediate phase (Day 5) characterized by increased cAMP response element-binding protein (CREB) signalling responses in adaptive immune cell types, and a late phase (Day 90) by persistent elevation of neutrophils, and immunoglobulin M+ (IgM+) B cells. By Day 365 there was no detectable difference between these samples and those from an age- and gender-matched patient cohort without stroke. When regressed against the change in the Montreal Cognitive Assessment scores between Days 90 and 365 after stroke, the acute inflammatory phase Elastic Net model correlated with post-stroke cognitive trajectories (r = -0.692, Bonferroni-corrected P = 0.039). The results demonstrate the utility of a deep immune profiling approach with mass cytometry for the identification of clinically relevant immune correlates of long-term cognitive trajectories.
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Cognição/fisiologia , Acidente Vascular Cerebral/imunologia , Acidente Vascular Cerebral/fisiopatologia , Idoso , Idoso de 80 Anos ou mais , Isquemia Encefálica/complicações , Proteína de Ligação a CREB/metabolismo , Transtornos Cognitivos/etiologia , Transtornos Cognitivos/imunologia , Disfunção Cognitiva/complicações , Disfunção Cognitiva/imunologia , Estudos de Coortes , Feminino , Humanos , Imunoglobulina M , Estudos Longitudinais , Masculino , Pessoa de Meia-Idade , Neutrófilos , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais , Acidente Vascular Cerebral/complicações , SobreviventesRESUMO
Postoperative cognitive decline (POCD) is the predominant complication affecting elderly patients following major surgery, yet its prediction and prevention remain challenging. Understanding biological processes underlying the pathogenesis of POCD is essential for identifying mechanistic biomarkers to advance diagnostics and therapeutics. This longitudinal study involving 26 elderly patients undergoing orthopedic surgery aimed to characterize the impact of peripheral immune cell responses to surgical trauma on POCD. Trajectory analyses of single-cell mass cytometry data highlighted early JAK/STAT signaling exacerbation and diminished MyD88 signaling post-surgery in patients who developed POCD. Further analyses integrating single-cell and plasma proteomic data collected before surgery with clinical variables yielded a sparse predictive model that accurately identified patients who would develop POCD (AUC = 0.80). The resulting POCD immune signature included one plasma protein and ten immune cell features, offering a concise list of biomarker candidates for developing point-of-care prognostic tests to personalize perioperative management of at-risk patients. The code and the data are documented and available at https://github.com/gregbellan/POCD . Teaser: Modeling immune cell responses and plasma proteomic data predicts postoperative cognitive decline.
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Adoption of high-content omic technologies in clinical studies, coupled with computational methods, has yielded an abundance of candidate biomarkers. However, translating such findings into bona fide clinical biomarkers remains challenging. To facilitate this process, we introduce Stabl, a general machine learning method that identifies a sparse, reliable set of biomarkers by integrating noise injection and a data-driven signal-to-noise threshold into multivariable predictive modeling. Evaluation of Stabl on synthetic datasets and five independent clinical studies demonstrates improved biomarker sparsity and reliability compared to commonly used sparsity-promoting regularization methods while maintaining predictive performance; it distills datasets containing 1,400-35,000 features down to 4-34 candidate biomarkers. Stabl extends to multi-omic integration tasks, enabling biological interpretation of complex predictive models, as it hones in on a shortlist of proteomic, metabolomic and cytometric events predicting labor onset, microbial biomarkers of pre-term birth and a pre-operative immune signature of post-surgical infections. Stabl is available at https://github.com/gregbellan/Stabl .
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High-multiplex tissue imaging (HMTI) approaches comprise several novel immunohistological methods that enable in-depth, spatial single-cell analysis. Over recent years, studies in tumor biology, infectious diseases, and autoimmune conditions have demonstrated the information gain accessible when mapping complex tissues with HMTI. Tumor biology has been a focus of innovative multiparametric approaches, as the tumor microenvironment (TME) contains great informative value for accurate diagnosis and targeted therapeutic approaches: unraveling the cellular composition and structural organization of the TME using sophisticated computational tools for spatial analysis has produced histopathologic biomarkers for outcomes in breast cancer, predictors of positive immunotherapy response in melanoma, and histological subgroups of colorectal carcinoma. Integration of HMTI technologies into existing clinical workflows such as molecular tumor boards will contribute to improve patient outcomes through personalized treatments tailored to the specific heterogeneous pathological fingerprint of cancer, autoimmunity, or infection. Here, we review the advantages and limitations of existing HMTI technologies and outline how spatial single-cell data can improve our understanding of pathological disease mechanisms and determinants of treatment success. We provide an overview of the analytic processing and interpretation and discuss how HMTI can improve future routine clinical diagnostic and therapeutic processes.
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Neoplasias da Mama , Neoplasias Colorretais , Melanoma , Humanos , Feminino , Microambiente TumoralRESUMO
Oral mucosal pathologies comprise an array of diseases with worldwide prevalence and medical relevance. Affecting a confined space with crucial physiological and social functions, oral pathologies can be mutilating and drastically reduce quality of life. Despite their relevance, treatment for these diseases is often far from curative and remains vastly understudied. While multiple factors are involved in the pathogenesis of oral mucosal pathologies, the host's immune system plays a major role in the development, maintenance, and resolution of these diseases. Consequently, a precise understanding of immunological mechanisms implicated in oral mucosal pathologies is critical (1) to identify accurate, mechanistic biomarkers of clinical outcomes; (2) to develop targeted immunotherapeutic strategies; and (3) to individualize prevention and treatment approaches. Here, we review key elements of the immune system's role in oral mucosal pathologies that hold promise to overcome limitations in current diagnostic and therapeutic approaches. We emphasize recent and ongoing multiomic and single-cell approaches that enable an integrative view of these pathophysiological processes and thereby provide unifying and clinically relevant biological signatures.
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Multiômica , Qualidade de Vida , Humanos , BiomarcadoresRESUMO
Ischemic stroke (IS) is the leading cause of acquired disability and the second leading cause of dementia and mortality. Current treatments for IS are primarily focused on revascularization of the occluded artery. However, only 10% of patients are eligible for revascularization and 50% of revascularized patients remain disabled at 3 months. Accumulating evidence highlight the prognostic significance of the neuro- and thrombo-inflammatory response after IS. However, several randomized trials of promising immunosuppressive or immunomodulatory drugs failed to show positive results. Insufficient understanding of inter-patient variability in the cellular, functional, and spatial organization of the inflammatory response to IS likely contributed to the failure to translate preclinical findings into successful clinical trials. The inflammatory response to IS involves complex interactions between neuronal, glial, and immune cell subsets across multiple immunological compartments, including the blood-brain barrier, the meningeal lymphatic vessels, the choroid plexus, and the skull bone marrow. Here, we review the neuro- and thrombo-inflammatory responses to IS. We discuss how clinical imaging and single-cell omic technologies have refined our understanding of the spatial organization of pathobiological processes driving clinical outcomes in patients with an IS. We also introduce recent developments in machine learning statistical methods for the integration of multi-omic data (biological and radiological) to identify patient-specific inflammatory states predictive of IS clinical outcomes.
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Isquemia Encefálica , AVC Isquêmico , Acidente Vascular Cerebral , Humanos , Acidente Vascular Cerebral/diagnóstico por imagem , Acidente Vascular Cerebral/etiologia , Isquemia Encefálica/diagnóstico por imagem , Isquemia Encefálica/etiologia , Multiômica , Neuroimagem/métodos , Inflamação/terapiaRESUMO
High-content omic technologies coupled with sparsity-promoting regularization methods (SRM) have transformed the biomarker discovery process. However, the translation of computational results into a clinical use-case scenario remains challenging. A rate-limiting step is the rigorous selection of reliable biomarker candidates among a host of biological features included in multivariate models. We propose Stabl, a machine learning framework that unifies the biomarker discovery process with multivariate predictive modeling of clinical outcomes by selecting a sparse and reliable set of biomarkers. Evaluation of Stabl on synthetic datasets and four independent clinical studies demonstrates improved biomarker sparsity and reliability compared to commonly used SRMs at similar predictive performance. Stabl readily extends to double- and triple-omics integration tasks and identifies a sparser and more reliable set of biomarkers than those selected by state-of-the-art early- and late-fusion SRMs, thereby facilitating the biological interpretation and clinical translation of complex multi-omic predictive models. The complete package for Stabl is available online at https://github.com/gregbellan/Stabl.
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Oral squamous cell carcinoma (OSCC), a prevalent and aggressive neoplasm, poses a significant challenge due to poor prognosis and limited prognostic biomarkers. Leveraging highly multiplexed imaging mass cytometry, we investigated the tumor immune microenvironment (TIME) in OSCC biopsies, characterizing immune cell distribution and signaling activity at the tumor-invasive front. Our spatial subsetting approach standardized cellular populations by tissue zone, improving feature reproducibility and revealing TIME patterns accompanying loss-of-differentiation. Employing a machine-learning pipeline combining reliable feature selection with multivariable modeling, we achieved accurate histological grade classification (AUC = 0.88). Three model features correlated with clinical outcomes in an independent cohort: granulocyte MAPKAPK2 signaling at the tumor front, stromal CD4+ memory T cell size, and the distance of fibroblasts from the tumor border. This study establishes a robust modeling framework for distilling complex imaging data, uncovering sentinel characteristics of the OSCC TIME to facilitate prognostic biomarkers discovery for recurrence risk stratification and immunomodulatory therapy development.
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OBJECTIVE: Systemic sclerosis (SSc) is a connective tissue disease with a significant morbidity and reduced survival of patients. Effective treatment and clinical control of the disease remain challenging. In particular, the development of pulmonary and cardiac fibrosis and pulmonary hypertension are severe complications responsible for excessive mortality. Currently available treatment strategies only alleviate symptoms and slow disease progression. Here, we investigated the therapeutic potential of ibrutinib, a Bruton's tyrosine kinase (BTK) inhibitor used in B cell malignancies, to alter B cell pathology in SSc in an in vitro model of autoimmunity. METHODS: PBMCs and sorted B cells of 24 patients with SSc were used for functional testing after stimulation with hypomethylated DNA fragments (CpG) to induce an innate immune response. The effects of ibrutinib on cytokine production, autoantibody release, and activation of the transcription factor NFκB were evaluated. RESULTS: Ibrutinib was able to reduce the production of the profibrotic hallmark cytokines IL-6 and TNF-α mainly from the effector B cell population in patients with SSc. Importantly, small doses of ibrutinib (0.1 µM) preserved the production of immunoregulatory IL-10 while effectively inhibiting hyperactivated, profibrotic effector B cells. In a flow cytometry analysis of phosphorylated NFκB, an important transcription factor in the induction of innate immune responses in B cells, significantly less activation was observed with ibrutinib treatment. CONCLUSION: Our data could pave the avenue for a clinical application of ibrutinib for patients with SSc as a novel treatment option for the underlying pathogenetic immune imbalance contributing to disease onset and progression.
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Adenina/análogos & derivados , Linfócitos B/efeitos dos fármacos , Piperidinas/farmacologia , Adenina/farmacologia , Adulto , Tirosina Quinase da Agamaglobulinemia/antagonistas & inibidores , Tirosina Quinase da Agamaglobulinemia/metabolismo , Idoso , Idoso de 80 Anos ou mais , Linfócitos B/citologia , Linfócitos B/metabolismo , Células Cultivadas , Citocinas/metabolismo , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , NF-kappa B/metabolismo , Fosforilação/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Escleroderma Sistêmico/tratamento farmacológico , Escleroderma Sistêmico/metabolismo , Escleroderma Sistêmico/patologiaRESUMO
Glucocorticoids (GC) are a controversial yet commonly used intervention in the clinical management of acute inflammatory conditions, including sepsis or traumatic injury. In the context of major trauma such as surgery, concerns have been raised regarding adverse effects from GC, thereby necessitating a better understanding of how GCs modulate the immune response. Here we report the results of a randomized controlled trial (NCT02542592) in which we employ a high-dimensional mass cytometry approach to characterize innate and adaptive cell signaling dynamics after a major surgery (primary outcome) in patients treated with placebo or methylprednisolone (MP). A robust, unsupervised bootstrap clustering of immune cell subsets coupled with random forest analysis shows profound (AUC = 0.92, p-value = 3.16E-8) MP-induced alterations of immune cell signaling trajectories, particularly in the adaptive compartments. By contrast, key innate signaling responses previously associated with pain and functional recovery after surgery, including STAT3 and CREB phosphorylation, are not affected by MP. These results imply cell-specific and pathway-specific effects of GCs, and also prompt future studies to examine GCs' effects on clinical outcomes likely dependent on functional adaptive immune responses.
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Imunidade Adaptativa/efeitos dos fármacos , Artroplastia de Quadril/efeitos adversos , Glucocorticoides/farmacologia , Ferimentos e Lesões/etiologia , Ferimentos e Lesões/imunologia , Doença Aguda , Idoso , Estudos de Casos e Controles , Método Duplo-Cego , Fadiga/tratamento farmacológico , Feminino , Humanos , Masculino , Metilprednisolona/farmacologia , Metilprednisolona/uso terapêutico , Inibidor de NF-kappaB alfa/metabolismo , Dor/tratamento farmacológico , Fenótipo , Fosforilação , Fator de Transcrição STAT3/metabolismo , Resultado do TratamentoRESUMO
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
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High-throughput single-cell analysis technologies produce an abundance of data that is critical for profiling the heterogeneity of cellular systems. We introduce VoPo (https://github.com/stanleyn/VoPo), a machine learning algorithm for predictive modeling and comprehensive visualization of the heterogeneity captured in large single-cell datasets. In three mass cytometry datasets, with the largest measuring hundreds of millions of cells over hundreds of samples, VoPo defines phenotypically and functionally homogeneous cell populations. VoPo further outperforms state-of-the-art machine learning algorithms in classification tasks, and identified immune-correlates of clinically-relevant parameters.
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Algoritmos , Modelos Biológicos , Análise de Célula Única , Análise por Conglomerados , Bases de Dados como Assunto , Citometria de Fluxo , HumanosRESUMO
Allogeneic hematopoietic cell transplantation (allo-HCT) is a curative treatment option for hematologic malignancies but relapse remains the most common cause of death. Infusion of donor lymphocytes (DLIs) can induce remission and prolong survival by exerting graft-vs.-leukemia (GVL) effects. However, sufficient tumor control cannot be established in all patients and occurrence of graft-vs.-host disease (GVHD) prevents further dose escalation. Previous data indicate that invariant natural killer T (iNKT) cells promote anti-tumor immunity without exacerbating GVHD. In the present study we investigated lysis of leukemic blasts through iNKT cells from donor-derived lymphocytes for leukemia control and found that iNKT cells constitute about 0.12% of cryopreserved donor T cells. Therefore, we established a 2-week cell culture protocol allowing for a robust expansion of iNKT cells from cryopreserved DLIs (DLI-iNKTs) that can be used for further preclinical and clinical applications. Such DLI-iNKTs efficiently lysed leukemia cell lines and primary patient AML blasts ex vivo in a dose- and CD1d-dependent manner. Furthermore, expression of CD1d on target cells was required to release proinflammatory cytokines and proapoptotic effector molecules. Our results suggest that iNKT cells from donor-derived lymphocytes are involved in anti-tumor immunity after allo-HCT and therefore may reduce the risk of relapse and improve progression-free and overall survival.
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Antígenos CD1d/imunologia , Leucemia/imunologia , Linfócitos/imunologia , Células T Matadoras Naturais/imunologia , Transplante de Medula Óssea/métodos , Linhagem Celular Tumoral , Células Cultivadas , Doença Enxerto-Hospedeiro/imunologia , Efeito Enxerto vs Leucemia/imunologia , Neoplasias Hematológicas/imunologia , Transplante de Células-Tronco Hematopoéticas/métodos , Humanos , Imunoterapia Adotiva/métodos , Células Jurkat , Células K562 , Transfusão de Linfócitos/métodos , Intervalo Livre de Progressão , Doadores de Tecidos , Transplante Homólogo/métodosRESUMO
Preeclampsia is one of the most severe pregnancy complications and a leading cause of maternal death. However, early diagnosis of preeclampsia remains a clinical challenge. Alterations in the normal immune adaptations necessary for the maintenance of a healthy pregnancy are central features of preeclampsia. However, prior analyses primarily focused on the static assessment of select immune cell subsets have provided limited information for the prediction of preeclampsia. Here, we used a high-dimensional mass cytometry immunoassay to characterize the dynamic changes of over 370 immune cell features (including cell distribution and functional responses) in maternal blood during healthy and preeclamptic pregnancies. We found a set of eight cell-specific immune features that accurately identified patients well before the clinical diagnosis of preeclampsia (median area under the curve (AUC) 0.91, interquartile range [0.82-0.92]). Several features recapitulated previously known immune dysfunctions in preeclampsia, such as elevated pro-inflammatory innate immune responses early in pregnancy and impaired regulatory T (Treg) cell signaling. The analysis revealed additional novel immune responses that were strongly associated with, and preceded the onset of preeclampsia, notably abnormal STAT5ab signaling dynamics in CD4+T cell subsets (AUC 0.92, p = 8.0E-5). These results provide a global readout of the dynamics of the maternal immune system early in pregnancy and lay the groundwork for identifying clinically-relevant immune dysfunctions for the prediction and prevention of preeclampsia.