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Advances in single-cell level analytical techniques, especially cytometric approaches, have led to profound innovation in biomedical research, particularly in the field of clinical immunology. This has resulted in an expansion of high-dimensional data, posing great challenges for comprehensive and unbiased analysis. Conventional manual analysis is thus becoming untenable to handle these challenges. Furthermore, most newly developed computational methods lack flexibility and interoperability, hampering their accessibility and usability. Here, we adapted Seurat, an R package originally developed for single-cell RNA sequencing (scRNA-seq) analysis, for high-dimensional flow cytometric data analysis. Based on a 20-marker antibody panel and analyses of T-cell profiles in both adult blood and cord blood (CB), we showcased the robust capacity of Seurat in flow cytometric data analysis, which was further validated by Spectre, another high-dimensional cytometric data analysis package, and conventional manual analysis. Importantly, we identified a unique CD8+ T-cell population defined as CD8+CD45RA+CD27+CD161+ T cell that was predominantly present in CB. We characterised its IFN-γ-producing and potential cytotoxic properties using flow cytometry experiments and scRNA-seq analysis from a published dataset. Collectively, we identified a unique human CB CD8+CD45RA+CD27+CD161+ T-cell subset and demonstrated that Seurat, a widely used package for scRNA-seq analysis, possesses great potential to be repurposed for cytometric data analysis. This facilitates an unbiased and thorough interpretation of complicated high-dimensional data using a single analytical pipeline and opens a novel avenue for data-driven investigation in clinical immunology.
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Linfocitos T CD8-positivos , Sangre Fetal , Citometría de Flujo , Antígenos Comunes de Leucocito , Humanos , Citometría de Flujo/métodos , Sangre Fetal/inmunología , Sangre Fetal/citología , Antígenos Comunes de Leucocito/metabolismo , Antígenos Comunes de Leucocito/inmunología , Linfocitos T CD8-positivos/inmunología , Miembro 7 de la Superfamilia de Receptores de Factores de Necrosis Tumoral/metabolismo , Miembro 7 de la Superfamilia de Receptores de Factores de Necrosis Tumoral/inmunología , Subgrupos de Linfocitos T/inmunología , Subgrupos de Linfocitos T/metabolismo , Subfamilia B de Receptores Similares a Lectina de Células NK/inmunología , Subfamilia B de Receptores Similares a Lectina de Células NK/metabolismo , Programas Informáticos , Análisis de la Célula Individual/métodos , Inmunofenotipificación/métodos , AdultoRESUMEN
Mapping the dynamics of immune cell populations over time or disease-course is key to understanding immunopathogenesis and devising putative interventions. We present TrackSOM, a novel method for delineating cellular populations and tracking their development over a time- or disease-course cytometry datasets. We demonstrate TrackSOM-enabled elucidation of the immune response to West Nile Virus infection in mice, uncovering heterogeneous subpopulations of immune cells and relating their functional evolution to disease severity. TrackSOM is easy to use, encompasses few parameters, is quick to execute, and enables an integrative and dynamic overview of the immune system kinetics that underlie disease progression and/or resolution.
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Fiebre del Nilo Occidental , Virus del Nilo Occidental , Ratones , Animales , Virus del Nilo Occidental/fisiología , Fiebre del Nilo Occidental/patología , Inmunidad , Análisis por ConglomeradosRESUMEN
Systemic suppression of adaptive immune responses is a major way in which UV radiation contributes to skin cancer development. Immune suppression is also likely to explain how UV protects from some autoimmune diseases, such as multiple sclerosis. However, the mechanisms underlying UV-mediated systemic immune suppression are not well understood. Exposure of C57BL/6 mice to doses of UV known to suppress systemic autoimmunity led to the accumulation of cells within the skin-draining lymph nodes and away from non-skin-draining lymph nodes. Transfer of CD45.1+ cells from nonirradiated donors into CD45.2+ UV-irradiated recipients resulted in preferential accumulation of donor naive T cells and a decrease in activated T cells within skin-draining lymph nodes. A single dose of immune-suppressive UV was all that was required to cause a redistribution of naive and central memory T cells from peripheral blood to the skin-draining lymph nodes. Specifically, CD69-independent increases in sphingosine-1-phosphate (S1P) receptor 1-negative naive and central memory T cells occurred in these lymph nodes. Mass spectrometry analysis showed UV-mediated activation of sphingosine kinase 1 activity, resulting in an increase in S1P levels within the lymph nodes. Topical application of a sphingosine kinase inhibitor on the skin prior to UV irradiation eliminated the UV-induced increase in lymph node S1P and T cell numbers. Thus, exposure to immunosuppressive UV disrupts T cell recirculation by manipulating the S1P pathway.
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Ganglios Linfáticos/inmunología , Esclerosis Múltiple/radioterapia , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Piel/patología , Animales , Circulación Sanguínea , Células Cultivadas , Humanos , Memoria Inmunológica , Terapia de Inmunosupresión , Activación de Linfocitos , Lisofosfolípidos/metabolismo , Ratones , Ratones Endogámicos C57BL , Transducción de Señal , Piel/efectos de la radiación , Esfingosina/análogos & derivados , Esfingosina/metabolismo , Receptores de Esfingosina-1-Fosfato/metabolismo , Rayos Ultravioleta , Terapia UltravioletaRESUMEN
Multiple sclerosis (MS) is a disease in which the immune system damages components of the central nervous system (CNS), leading to the destruction of myelin and the formation of demyelinating plaques. This often occurs in episodic "attacks" precipitated by the transmigration of leukocytes across the blood-brain barrier (BBB), and repeated episodes of demyelination lead to substantial losses of axons within and removed from plaques, ultimately leading to progressive neurological dysfunction. Within leukocyte populations, macrophages and T and B lymphocytes are the predominant effectors. Among current immunotherapies, oral cladribine's impact on lymphocytes is well characterised, but little is known about its impact on other leukocytes such as monocytes and dendritic cells (DCs). The aim of this study was to determine the transmigratory ability of monocyte and DC subsets in healthy subjects and untreated and cladribine-treated relapse-remitting MS (RRMS) patients using a well-characterised model of the BBB. Peripheral blood mononuclear cells from subjects were added to an in vitro transmigration assay to assess cell migration. Our findings show that while prior treatment with oral cladribine inhibits the migration of intermediate monocytes, it has no impact on the transmigration of DC subsets. Overall, our data indicate a previously unrecognised role of cladribine on intermediate monocytes, known to accumulate in the brain active MS lesions.
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Monocitos , Esclerosis Múltiple , Humanos , Cladribina/farmacología , Cladribina/uso terapéutico , Esclerosis Múltiple/tratamiento farmacológico , Barrera Hematoencefálica , Leucocitos MononuclearesRESUMEN
B cells play a major role in multiple sclerosis (MS), with many successful therapeutics capable of removing them from circulation. One such therapy, alemtuzumab, is thought to reset the immune system without the need for ongoing therapy in a proportion of patients. The exact cells contributing to disease pathogenesis and quiescence remain to be identified. We utilized mass cytometry to analyze B cells from the blood of patients with relapse-remitting MS (RRMS) before and after alemtuzumab treatment, and during relapse. A complementary RRMS cohort was analyzed by single-cell RNA sequencing. The R package "Spectre" was used to analyze these data, incorporating FlowSOM clustering, sparse partial least squares-discriminant analysis and permutational multivariate analysis of variance. Immunoglobulin (Ig)A+ and IgG1 + B-cell numbers were altered, including higher IgG1 + B cells during relapse. B-cell linker protein (BLNK), CD40 and CD210 expression by B cells was lower in patients with RRMS compared with non-MS controls, with similar results at the transcriptomic level. Finally, alemtuzumab restored BLNK, CD40 and CD210 expression by IgA+ and IgG1 + B cells, which was altered again during relapse. These data suggest that impairment of IgA+ and IgG1 + B cells may contribute to MS pathogenesis, which can be restored by alemtuzumab.
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Esclerosis Múltiple Recurrente-Remitente , Esclerosis Múltiple , Alemtuzumab/uso terapéutico , Enfermedad Crónica , Humanos , Inmunoglobulina A , Inmunoglobulina G , Esclerosis Múltiple/tratamiento farmacológico , Esclerosis Múltiple Recurrente-Remitente/tratamiento farmacológico , RecurrenciaRESUMEN
As the size and complexity of high-dimensional (HD) cytometry data continue to expand, comprehensive, scalable, and methodical computational analysis approaches are essential. Yet, contemporary clustering and dimensionality reduction tools alone are insufficient to analyze or reproduce analyses across large numbers of samples, batches, or experiments. Moreover, approaches that allow for the integration of data across batches or experiments are not well incorporated into computational toolkits to allow for streamlined workflows. Here we present Spectre, an R package that enables comprehensive end-to-end integration and analysis of HD cytometry data from different batches or experiments. Spectre streamlines the analytical stages of raw data pre-processing, batch alignment, data integration, clustering, dimensionality reduction, visualization, and population labelling, as well as quantitative and statistical analysis. Critically, the fundamental data structures used within Spectre, along with the implementation of machine learning classifiers, allow for the scalable analysis of very large HD datasets, generated by flow cytometry, mass cytometry, or spectral cytometry. Using open and flexible data structures, Spectre can also be used to analyze data generated by single-cell RNA sequencing or HD imaging technologies, such as Imaging Mass Cytometry. The simple, clear, and modular design of analysis workflows allow these tools to be used by bioinformaticians and laboratory scientists alike. Spectre is available as an R package or Docker container. R code is available on Github (https://github.com/immunedynamics/spectre).
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Algoritmos , Análisis de la Célula Individual , Análisis por Conglomerados , Citometría de Flujo/métodos , Programas InformáticosRESUMEN
High-dimensional cytometry represents an exciting new era of immunology research, enabling the discovery of new cells and prediction of patient responses to therapy. A plethora of analysis and visualization tools and programs are now available for both new and experienced users; however, the transition from low- to high-dimensional cytometry requires a change in the way users think about experimental design and data analysis. Data from high-dimensional cytometry experiments are often underutilized, because of both the size of the data and the number of possible combinations of markers, as well as to a lack of understanding of the processes required to generate meaningful data. In this article, we explain the concepts behind designing high-dimensional cytometry experiments and provide considerations for new and experienced users to design and carry out high-dimensional experiments to maximize quality data collection.
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Citometría de Flujo , HumanosRESUMEN
The ultraviolet (UV) radiation contained in sunlight is a powerful immune suppressant. While exposure to UV is associated with protection from the development of autoimmune diseases, particularly multiple sclerosis, the precise mechanism by which UV achieves this protection is not currently well understood. Regulatory B cells play an important role in preventing autoimmunity and activation of B cells is a major way in which UV suppresses adaptive immune responses. Whether UV-protection from autoimmunity is mediated by the activation of regulatory B cells has never been considered before. When C57BL/6 mice were exposed to low, physiologically relevant doses of UV, a unique population of B cells was activated in the skin draining lymph nodes. As determined by flow cytometry, CD1d(low)CD5(-)MHC-II(hi)B220(hi) UV-activated B cells expressed significantly higher levels of CD19, CD21/35, CD25, CD210 and CD268 as well as the co-stimulatory molecules CD80, CD86, CD274 and CD275. Experimental autoimmune encephalomyelitis (EAE) in mice immunized with MOG/CFA was reduced by exposure to UV. UV significantly inhibited demyelination and infiltration of inflammatory cells into the spinal cord. Consequently, UV-exposed groups showed elevated IL-10 levels in secondary lymphoid organs, delayed EAE onset, reduced peak EAE score and significantly suppressed overall disease incidence and burden. Importantly, protection from EAE could be adoptively transferred using B cells isolated from UV-exposed, but not unirradiated hosts. Indeed, UV-protection from EAE was dependent on UV activation of lymph node B cells because UV could not protect mice from EAE who were pharmacologically depleted of B cells using antibodies. Thus, UV maintenance of a pool of unique regulatory B cells in peripheral lymph nodes appears to be essential to prevent an autoimmune attack on the central nervous system.
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Autoinmunidad/efectos de la radiación , Linfocitos B Reguladores/efectos de la radiación , Sistema Nervioso Central/inmunología , Encefalomielitis Autoinmune Experimental/prevención & control , Activación de Linfocitos/efectos de la radiación , Luz Solar , Animales , Anticuerpos Monoclonales/administración & dosificación , Anticuerpos Monoclonales/inmunología , Antígenos CD/metabolismo , Linfocitos B Reguladores/inmunología , Linfocitos B Reguladores/metabolismo , Sistema Nervioso Central/patología , Encefalomielitis Autoinmune Experimental/inducido químicamente , Encefalomielitis Autoinmune Experimental/inmunología , Ensayo de Inmunoadsorción Enzimática , Femenino , Citometría de Flujo , Antígenos de Histocompatibilidad Clase II/metabolismo , Inyecciones Intraperitoneales , Interleucina-10/metabolismo , Interleucina-10/efectos de la radiación , Ganglios Linfáticos/citología , Ganglios Linfáticos/inmunología , Ratones , Ratones Endogámicos C57BL , Glicoproteína Mielina-Oligodendrócito/toxicidadRESUMEN
Ultraviolet (UV) wavelengths in sunlight are the prime cause of skin cancer in humans with both the UVA and UVB wavebands making a contribution to photocarcinogenesis. UV has many different biological effects on the skin that contribute to carcinogenesis, including suppression of adaptive immunity, sunburn and altering the migration of mast cells into and away from irradiated skin. Many molecular mechanisms have been identified as contributing to skin responses to UV. Recently, using gene set enrichment analysis of microarray data, we identified the alternative complement pathway with a central role for factor B (fB) in UVA-induced immunosuppression. In the current study we used mice genetically deficient in fB (fB-/- mice) to study the functional role of the alternative complement pathway in skin responses to UV. We found that fB is required for not only UVA but also UVB-induced immunosuppression and solar-simulated UV induction of the oedemal component of sunburn. Factor B-/- mice had a larger number of resident skin mast cells than control mice, but unlike the controls did not respond to UV by increasing mast cell infiltration into the skin. This study provides evidence for a function role for fB in skin responses to UV radiation. Factor B regulates UVA and UVB induced immunosuppression, UV induced oedema and mast cell infiltration into the skin. The alternative complement pathway is therefore an important regulator of skin responses to UV.
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Factor B del Complemento/metabolismo , Edema/fisiopatología , Hipersensibilidad Tardía/fisiopatología , Mastocitos/efectos de la radiación , Piel/efectos de la radiación , Rayos Ultravioleta/efectos adversos , Animales , Movimiento Celular/fisiología , Movimiento Celular/efectos de la radiación , Factor B del Complemento/genética , Modelos Animales de Enfermedad , Edema/etiología , Femenino , Hipersensibilidad Tardía/etiología , Masculino , Mastocitos/fisiología , Ratones Endogámicos C57BL , Ratones Noqueados , Piel/fisiopatología , Quemadura Solar/etiología , Quemadura Solar/fisiopatologíaRESUMEN
Advancements in cytometry technologies have enabled quantification of up to 50 proteins across millions of cells at single cell resolution. Analysis of cytometry data routinely involves tasks such as data integration, clustering, and dimensionality reduction. While numerous tools exist, many require extensive run times when processing large cytometry data containing millions of cells. Existing solutions, such as random subsampling, are inadequate as they risk excluding rare cell subsets. To address this, we propose SuperCellCyto, an R package that builds on the SuperCell tool which groups highly similar cells into supercells. SuperCellCyto is available on GitHub ( https://github.com/phipsonlab/SuperCellCyto ) and Zenodo ( https://doi.org/10.5281/zenodo.10521294 ).
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Investigación , Análisis de la Célula Individual , Análisis por Conglomerados , Programas InformáticosRESUMEN
Vessels that encapsulate tumour clusters (VETC) is a distinct histologic vascular pattern associated with a novel mechanism of metastasis. First described in human cancers in 2004, its prevalence and prognostic significance in hepatocellular carcinoma (HCC) has only been appreciated in the past decade with a rapidly increasing body of literature. A robust biomarker of aggressive disease, the VETC pattern is easy to recognise but relies on histologic examination of tumour tissue for its diagnosis. Radiological recognition of the VETC pattern is an area of active research and is becoming increasingly accurate. As a prognostic marker, VETC has consistently proven to be an independent predictor of disease recurrence and overall survival in patients with HCC undergoing resection and liver transplantation. It can also guide treatment by predicting response to other therapies such as transarterial chemoembolisation and sorafenib. Without prospective randomised-controlled trials or routine evaluation of VETC in clinical practice, there are currently no firm treatment recommendations for VETC-positive tumours, although some perspectives are provided in this review based on the latest knowledge of their pathogenesis - a complex interplay between tumour angiogenesis and the immune microenvironment. Nevertheless, VETC has great potential as a future biomarker that could take us one step closer to precision medicine for HCC.
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Objectives: SARS-CoV-2 infection causes a spectrum of clinical disease presentation, ranging from asymptomatic to fatal. While neutralising antibody (NAb) responses correlate with protection against symptomatic and severe infection, the contribution of the T-cell response to disease resolution or progression is still unclear. As newly emerging variants of concern have the capacity to partially escape NAb responses, defining the contribution of individual T-cell subsets to disease outcome is imperative to inform the development of next-generation COVID-19 vaccines. Methods: Immunophenotyping of T-cell responses in unvaccinated individuals was performed, representing the full spectrum of COVID-19 clinical presentation. Computational and manual analyses were used to identify T-cell populations associated with distinct disease states. Results: Critical SARS-CoV-2 infection was characterised by an increase in activated and cytotoxic CD4+ lymphocytes (CTL). These CD4+ CTLs were largely absent in asymptomatic to severe disease states. In contrast, non-critical COVID-19 was associated with high frequencies of naïve T cells and lack of activation marker expression. Conclusion: Highly activated and cytotoxic CD4+ T-cell responses may contribute to cell-mediated host tissue damage and progression of COVID-19. Induction of these potentially detrimental T-cell responses should be considered when developing and implementing effective COVID-19 control strategies.
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The breakdown of the blood-brain barrier (BBB) and the trans-endothelial migration of lymphocytes are central events in the development of multiple sclerosis (MS). Autoreactive T cells are major players in MS pathogenesis, which are rapidly depleted following alemtuzumab treatment. This modulation, in turn, inhibits CNS inflammation, but alemtuzumab's effect on T cell migration into the CNS has been less studied. Human brain endothelial cells were stimulated with pro-inflammatory cytokines to mimic an inflamed BBB in vitro. Peripheral blood mononuclear cells from healthy controls, untreated or alemtuzumab-treated patients with relapsing-remitting MS (RRMS) were added to the BBB model to assess their transmigratory capacity. Here, the migration of CD4+ effector memory T (TEM) and CD8+ central memory T (TCM) cells across the BBB was impaired in alemtuzumab-treated patients. Naïve T (Tnaïve) cells were unable to migrate across all groups. CD38 was lowly expressed on CD8+ TCM cells, particularly for RRMS patients, compared to CD8+ Tnaïve cells. CD62L expression was lower on CD4+ TEM cells than CD4+ Tnaïve cells and decreased further in alemtuzumab-treated patients. These data suggest that repopulated memory T cells are phenotypically different from naïve T cells, which may affect their transmigration across the BBB in vitro.
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Multiple sclerosis (MS) is a chronic, demyelinating disease of the central nervous system (CNS) induced by immune dysregulation. Cladribine has been championed for its clinical efficacy with relatively minor side effects in treating MS. Although it is proposed that cladribine exerts an anti-migratory effect on lymphocytes at the blood-brain barrier (BBB) in addition to its lymphocyte-depleting and modulating effects, this has not been properly studied. Here, we aimed to determine if cladribine treatment influences trans-endothelial migration of T cell subsets across an inflamed BBB. Human brain endothelial cells stimulated with pro-inflammatory cytokines were used to mimic the BBB. Peripheral blood mononuclear cells were obtained from healthy controls, untreated and cladribine-treated MS patients. The trans-endothelial migration of CD4+ effector memory T (TEM) and CD8+ central memory T (TCM) cells was reduced in cladribine-treated MS patients. CD28 expression was decreased on both CD4+ TEM and CD8+ TCM cells, suggesting lowered peripheral activation of these cells thereby maintaining the integrity of the BBB. In addition, these cells have likely reconstituted following cladribine treatment, revealing a long-term anti-migratory effect. These results highlight new mechanisms by which cladribine acts to control MS pathogenesis.
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Hepatocellular carcinoma (HCC) is a common and deadly cancer worldwide. Many factors contribute to mortality and place an individual at high risk of developing HCC, including viral infection, alcohol intake, metabolic-associated disease, autoimmunity and genetic liver disorders. Although there are many therapeutics available, much about this disease remains to be understood. This is most evident when investigating the tumour microenvironment (TME). Both innate and adaptive immune cells have been associated with carcinogenesis within the TME of HCC patients. The ability to interrogate the TME more thoroughly with spatial technologies continues to improve, both at the experimental and analytical stages. This review provides insight into technologies available to investigate the TME, and how such technologies are beneficial for improving our understanding of HCC carcinogenesis.
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Objectives: The role of innate lymphoid cells (ILC), particularly helper ILC, in the pathogenesis of multiple sclerosis (MS) is not well understood. Here, we present a comprehensive analysis of peripheral ILC subsets in MS patients prior and after alemtuzumab administration using mass cytometry. Methods: Circulating ILC were analysed by mass cytometry in MS patients before and after alemtuzumab. These were compared with non-MS controls. MS-related shifts among ILC immunophenotypes were further elucidated by fast interpolation-based t-SNE (Flt-SNE) dimensionality reduction. Results: Neither natural killer (NK) cells nor helper ILC (ILC1, ILC2 and ILC3) levels were altered following alemtuzumab treatment. However, CD56bright NK cell expansions were observed in relapsing patients. MS patients prior to alemtuzumab further displayed proportional shifts from ILC1 to ILC2, with MS-associated decreases in CCR6+ helper ILC proportions. Conclusion: CD56bright NK cells during relapse indicate an immediate response to disease reactivation, while CCR6-related shifts among helper ILC suggest altered ILC migration to the CNS during MS.
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Immune-related adverse events represent a major hurdle to the success of immunotherapy. The immunological mechanisms underlying their development and relation to antitumor responses are poorly understood. By examining both systemic and tissue-specific immune changes induced by combination anti-CTLA-4 and anti-PD-1 immunotherapy, we found distinct repertoire changes in patients who developed moderate-severe colitis, irrespective of their antitumor response to therapy. The proportion of circulating monocytes were significantly increased at baseline in patients who subsequently developed colitis compared with patients who did not develop colitis, and biopsies from patients with colitis showed monocytic infiltration of both endoscopically and histopathologically normal and inflamed regions of colon. The magnitude of systemic expansion of T cells following commencement of immunotherapy was also greater in patients who developed colitis. Importantly, we show expansion of specific T cell subsets within inflamed regions of the colon, including tissue-resident memory CD8+ T cells and Th1 CD4+ T cells in patients who developed colitis. Our data also suggest that CD8+ T cell expansion was locally induced, while Th1 cell expansion was systemic. Together, our data show that exaggerated innate and T cell responses to combination immunotherapy synergize to propel colitis in susceptible patients.
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Colitis , Receptor de Muerte Celular Programada 1 , Humanos , Linfocitos T CD8-positivos , Inmunoterapia/efectos adversos , Colitis/inducido químicamente , Colitis/terapia , Factores Inmunológicos , Inmunidad InnataRESUMEN
SARS-CoV-2 causes a spectrum of COVID-19 disease, the immunological basis of which remains ill defined. We analyzed 85 SARS-CoV-2-infected individuals at acute and/or convalescent time points, up to 102 days after symptom onset, quantifying 184 immunological parameters. Acute COVID-19 presented with high levels of IL-6, IL-18, and IL-10 and broad activation marked by the upregulation of CD38 on innate and adaptive lymphocytes and myeloid cells. Importantly, activated CXCR3+cTFH1 cells in acute COVID-19 significantly correlate with and predict antibody levels and their avidity at convalescence as well as acute neutralization activity. Strikingly, intensive care unit (ICU) patients with severe COVID-19 display higher levels of soluble IL-6, IL-6R, and IL-18, and hyperactivation of innate, adaptive, and myeloid compartments than patients with moderate disease. Our analyses provide a comprehensive map of longitudinal immunological responses in COVID-19 patients and integrate key cellular pathways of complex immune networks underpinning severe COVID-19, providing important insights into potential biomarkers and immunotherapies.
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Formación de Anticuerpos , COVID-19/inmunología , Inmunidad Adaptativa , Adulto , Anciano , Anticuerpos Antivirales/sangre , Linfocitos B/citología , Linfocitos B/metabolismo , COVID-19/patología , COVID-19/virología , Femenino , Humanos , Inmunidad Innata , Interleucina-18/metabolismo , Interleucina-6/metabolismo , Masculino , Persona de Mediana Edad , Receptores CXCR3/metabolismo , Receptores de Interleucina-6/metabolismo , SARS-CoV-2/inmunología , SARS-CoV-2/aislamiento & purificación , Índice de Severidad de la Enfermedad , Células TH1/citología , Células TH1/metabolismo , Adulto JovenRESUMEN
OBJECTIVES: Disease-modifying therapies (DMTs) targeting B cells are amongst the most effective for preventing multiple sclerosis (MS) progression. IgG3 antibodies and their uncharacterised B-cell clones are predicted to play a pathogenic role in MS. Identifying subsets of IgG3 + B cells involved in MS progression could improve diagnosis, could inform timely disease intervention and may lead to new DMTs that target B cells more specifically. METHODS: We designed a 31-parameter B-cell-focused mass cytometry panel to interrogate the role of peripheral blood IgG3 + B cells in MS progression of two different patient cohorts: one to investigate the B-cell subsets involved in conversion from clinically isolated syndrome (CIS) to MS; and another to compare MS patients with inactive or active stages of disease. Each independent cohort included a group of non-MS controls. RESULTS: Nine distinct CD20+IgD-IgG3 + B-cell subsets were identified. Significant changes in the proportion of CD21+CD24+CD27-CD38- and CD27+CD38hiCD71hi memory B-cell subsets correlated with changes in serum IgG3 levels and time to conversion from CIS to MS. The same CD38- double-negative B-cell subset was significantly elevated in MS patients with active forms of the disease. A third CD21+CD24+CD27+CD38- subset was elevated in patients with active MS, whilst narrowband UVB significantly reduced the proportion of this switched-memory B-cell subset. CONCLUSION: We have identified previously uncharacterised subsets of IgG3 + B cells and shown them to correlate with autoimmune attacks on the central nervous system (CNS). These results highlight the potential for therapies that specifically target IgG3 + B cells to impact MS progression.
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CD8+CD57+ terminal effector T (TTE) cells are a component of marrow-infiltrating lymphocytes and may contribute to the altered immune responses in multiple myeloma (MM) patients. We analyzed TTE cells in the bone marrow (BM) and peripheral blood (PB) of age-matched controls and patients with monoclonal gammopathy of undetermined significance (MGUS), smoldering MM (SMM), and newly diagnosed (ND) MM using flow cytometry, mass cytometry, and FlowSOM clustering. TTE cells are heterogeneous in all subjects, with BM containing both CD69- and CD69+ subsets, while only CD69- cells are found in PB. Within the BM-TTE compartment, CD69- and CD69+ cells are found in comparable proportions in controls, while CD69- cells are dominant in MGUS and SMM and predominantly either CD69- or CD69+ cells in NDMM. A positive relationship between CD69+TTE and CD69-TTE cells is observed in the BM of controls, lost in MGUS, and converted to an inverse relationship in NDMM. CD69-TTE cells include multiple oligoclonal expansions of T-cell receptor/Vß families shared between BM and PB of NDMM. Oligoclonal expanded CD69-TTE cells from the PB include myeloma-reactive cells capable of killing autologous CD38hi plasma cells in vitro, involving degranulation and high expression of perforin and granzyme. In contrast to CD69-TTE cells, oligoclonal expansions are not evident within CD69+TTE cells, which possess low perforin and granzyme expression and high inhibitory checkpoint expression and resemble T resident memory cells. Both CD69-TTE and CD69+TTE cells from the BM of NDMM produce large amounts of the inflammatory cytokines interferon-γ and tumor necrosis factor α. The balance between CD69- and CD69+ cells within the BM-TTE compartment may regulate immune responses in NDMM and contribute to the clinical heterogeneity of the disease.