ABSTRACT
BACKGROUND: Multiple lines of evidence support peripheral organs in the initiation or progression of Lewy body disease (LBD), a spectrum of neurodegenerative diagnoses that include Parkinson's Disease (PD) without or with dementia (PDD) and dementia with Lewy bodies (DLB). However, the potential contribution of the peripheral immune response to LBD remains unclear. This study aims to characterize peripheral immune responses unique to participants with LBD at single-cell resolution to highlight potential biomarkers and increase mechanistic understanding of LBD pathogenesis in humans. METHODS: In a case-control study, peripheral mononuclear cell (PBMC) samples from research participants were randomly sampled from multiple sites across the United States. The diagnosis groups comprise healthy controls (HC, n = 159), LBD (n = 110), Alzheimer's disease dementia (ADD, n = 97), other neurodegenerative disease controls (NDC, n = 19), and immune disease controls (IDC, n = 14). PBMCs were activated with three stimulants (LPS, IL-6, and IFNa) or remained at basal state, stained by 13 surface markers and 7 intracellular signal markers, and analyzed by flow cytometry, which generated 1,184 immune features after gating. RESULTS: The model classified LBD from HC with an AUROC of 0.87 ± 0.06 and AUPRC of 0.80 ± 0.06. Without retraining, the same model was able to distinguish LBD from ADD, NDC, and IDC. Model predictions were driven by pPLCγ2, p38, and pSTAT5 signals from specific cell populations under specific activation. The immune responses characteristic for LBD were not associated with other common medical conditions related to the risk of LBD or dementia, such as sleep disorders, hypertension, or diabetes. CONCLUSIONS AND RELEVANCE: Quantification of PBMC immune response from multisite research participants yielded a unique pattern for LBD compared to HC, multiple related neurodegenerative diseases, and autoimmune diseases thereby highlighting potential biomarkers and mechanisms of disease.
Subject(s)
Leukocytes, Mononuclear , Lewy Body Disease , Parkinson Disease , Humans , Parkinson Disease/immunology , Parkinson Disease/metabolism , Lewy Body Disease/immunology , Male , Female , Aged , Case-Control Studies , Leukocytes, Mononuclear/metabolism , Leukocytes, Mononuclear/immunology , Biomarkers/metabolism , Middle Aged , Cohort Studies , Aged, 80 and over , Lewy Bodies/pathology , Lewy Bodies/metabolism , Single-Cell Analysis/methodsABSTRACT
Introduction: Innate lymphoid cells (ILCs) are enriched at mucosal surfaces where they respond rapidly to environmental stimuli and contribute to both tissue inflammation and healing. Methods: To gain insight into the role of ILCs in the pathology and recovery from COVID-19 infection, we employed a multi-omics approach consisting of Abseq and targeted mRNA sequencing to respectively probe the surface marker expression, transcriptional profile and heterogeneity of ILCs in peripheral blood of patients with COVID-19 compared with healthy controls. Results: We found that the frequency of ILC1 and ILC2 cells was significantly increased in COVID-19 patients. Moreover, all ILC subsets displayed a significantly higher frequency of CD69-expressing cells, indicating a heightened state of activation. ILC2s from COVID-19 patients had the highest number of significantly differentially expressed (DE) genes. The most notable genes DE in COVID-19 vs healthy participants included a) genes associated with responses to virus infections and b) genes that support ILC self-proliferation, activation and homeostasis. In addition, differential gene regulatory network analysis revealed ILC-specific regulons and their interactions driving the differential gene expression in each ILC. Discussion: Overall, this study provides mechanistic insights into the characteristics of ILC subsets activated during COVID-19 infection.
Subject(s)
COVID-19 , Immunity, Innate , Lymphocytes , Adult , Aged , Female , Humans , Male , Middle Aged , COVID-19/immunology , COVID-19/genetics , Gene Expression Profiling , Gene Regulatory Networks , Lymphocytes/immunology , Lymphocytes/metabolism , Multiomics , Single-Cell Analysis , TranscriptomeABSTRACT
BACKGROUND: Fludarabine in combination with cyclophosphamide (FC) is the standard lymphodepletion regimen for CAR T-cell therapy (CAR T). A national fludarabine shortage in 2022 necessitated the exploration of alternative regimens with many centers employing single-agent bendamustine as lymphodepletion despite a lack of clinical safety and efficacy data. To fill this gap in the literature, we evaluated the safety, efficacy, and expansion kinetics of bendamustine as lymphodepletion prior to axicabtagene ciloleucel (axi-cel) therapy. METHODS: 84 consecutive patients with relapsed or refractory large B-cell lymphoma treated with axi-cel and managed with a uniform toxicity management plan at Stanford University were studied. 27 patients received alternative lymphodepletion with bendamustine while 57 received FC. RESULTS: Best complete response rates were similar (73.7% for FC and 74% for bendamustine, p=0.28) and there was no significant difference in 12-month progression-free survival or overall survival estimates (p=0.17 and p=0.62, respectively). The frequency of high-grade cytokine release syndrome and immune effector cell-associated neurotoxicity syndrome was similar in both the cohorts. Bendamustine cohort experienced lower proportions of hematological toxicities and antibiotic use for neutropenic fever. Immune reconstitution, as measured by quantitative assessment of cellular immunity, was better in bendamustine cohort as compared with FC cohort. CAR T expansion as measured by peak expansion and area under the curve for expansion was comparable between cohorts. CONCLUSIONS: Bendamustine is a safe and effective alternative lymphodepletion conditioning for axi-cel with lower early hematological toxicity and favorable immune reconstitution.
Subject(s)
Bendamustine Hydrochloride , Biological Products , Lymphoma, Large B-Cell, Diffuse , Humans , Bendamustine Hydrochloride/therapeutic use , Bendamustine Hydrochloride/administration & dosage , Male , Female , Middle Aged , Aged , Lymphoma, Large B-Cell, Diffuse/drug therapy , Biological Products/therapeutic use , Biological Products/adverse effects , Adult , Immunotherapy, Adoptive/methods , Immunotherapy, Adoptive/adverse effects , Antigens, CD19/immunology , Antigens, CD19/therapeutic useABSTRACT
Native top-down mass spectrometry (nTDMS) allows characterization of protein structure and noncovalent interactions with simultaneous sequence mapping and proteoform characterization. The majority of nTDMS studies utilize purified recombinant proteins, with significant challenges hindering application to endogenous systems. To perform native top-down proteomics (nTDP), where endogenous proteins from complex biological systems are analyzed by nTDMS, it is essential to separate proteins under nondenaturing conditions. However, it remains difficult to achieve high resolution with MS-compatible online chromatography while preserving protein tertiary structure and noncovalent interactions. Herein, we report the use of online mixed-bed ion exchange chromatography (IEC) to enable separation of endogenous proteins from complex mixtures under nondenaturing conditions, preserving noncovalent interactions for nTDP analysis. We have successfully detected large proteins (>146 kDa) and identified endogenous metal-binding and oligomeric protein complexes in human heart tissue lysate. The use of a mixed-bed stationary phase allowed retention and elution of proteins over a wide range of isoelectric points without altering the sample or mobile phase pH. Overall, our method provides a simple online IEC-MS platform that can effectively separate proteins from complex mixtures under nondenaturing conditions and preserve higher-order structure for nTDP applications.
Subject(s)
Proteomics , Chromatography, Ion Exchange/methods , Humans , Proteomics/methods , Myocardium/chemistry , Mass Spectrometry/methods , Complex Mixtures/chemistry , Proteins/chemistry , Proteins/analysis , Proteins/isolation & purificationABSTRACT
BACKGROUNDPatients hospitalized for COVID-19 exhibit diverse clinical outcomes, with outcomes for some individuals diverging over time even though their initial disease severity appears similar to that of other patients. A systematic evaluation of molecular and cellular profiles over the full disease course can link immune programs and their coordination with progression heterogeneity.METHODSWe performed deep immunophenotyping and conducted longitudinal multiomics modeling, integrating 10 assays for 1,152 Immunophenotyping Assessment in a COVID-19 Cohort (IMPACC) study participants and identifying several immune cascades that were significant drivers of differential clinical outcomes.RESULTSIncreasing disease severity was driven by a temporal pattern that began with the early upregulation of immunosuppressive metabolites and then elevated levels of inflammatory cytokines, signatures of coagulation, formation of neutrophil extracellular traps, and T cell functional dysregulation. A second immune cascade, predictive of 28-day mortality among critically ill patients, was characterized by reduced total plasma Igs and B cells and dysregulated IFN responsiveness. We demonstrated that the balance disruption between IFN-stimulated genes and IFN inhibitors is a crucial biomarker of COVID-19 mortality, potentially contributing to failure of viral clearance in patients with fatal illness.CONCLUSIONOur longitudinal multiomics profiling study revealed temporal coordination across diverse omics that potentially explain the disease progression, providing insights that can inform the targeted development of therapies for patients hospitalized with COVID-19, especially those who are critically ill.TRIAL REGISTRATIONClinicalTrials.gov NCT04378777.FUNDINGNIH (5R01AI135803-03, 5U19AI118608-04, 5U19AI128910-04, 4U19AI090023-11, 4U19AI118610-06, R01AI145835-01A1S1, 5U19AI062629-17, 5U19AI057229-17, 5U19AI125357-05, 5U19AI128913-03, 3U19AI077439-13, 5U54AI142766-03, 5R01AI104870-07, 3U19AI089992-09, 3U19AI128913-03, and 5T32DA018926-18); NIAID, NIH (3U19AI1289130, U19AI128913-04S1, and R01AI122220); and National Science Foundation (DMS2310836).
Subject(s)
COVID-19 , Severity of Illness Index , Adult , Aged , Female , Humans , Male , Middle Aged , COVID-19/immunology , COVID-19/mortality , COVID-19/blood , Cytokines/blood , Cytokines/immunology , Longitudinal Studies , MultiomicsABSTRACT
Multiparameter flow cytometry is a common tool for assessing responses of T, B, and other cells to pathogens or vaccines. Such responses are likely to be important for predicting the efficacy of an HIV vaccine, despite the elusive findings in HIV vaccine trials to date. Fortunately, flow cytometry has evolved to be capable of readily measuring 30-40 parameters, providing the ability to dissect detailed phenotypes and functions that may be correlated with disease protection. Nevertheless, technical hurdles remain, and standardization of assays is still largely lacking. Here an optimized protocol for antigen-specific T cell monitoring is presented, with specific variations for particular markers. It covers the analysis of multiple cytokines, cell surface proteins, and other functional markers such as CD107, CD154, CD137, etc. References are given to published panels of 8-28 colors.
Subject(s)
Flow Cytometry , T-Lymphocytes , Flow Cytometry/methods , Humans , T-Lymphocytes/immunology , Cytokines/metabolism , Immunophenotyping/methods , BiomarkersABSTRACT
Introduction: Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS) presents substantial challenges in patient care due to its intricate multisystem nature, comorbidities, and global prevalence. The heterogeneity among patient populations, coupled with the absence of FDA-approved diagnostics and therapeutics, further complicates research into disease etiology and patient managment. Integrating longitudinal multi-omics data with clinical, health,textual, pharmaceutical, and nutraceutical data offers a promising avenue to address these complexities, aiding in the identification of underlying causes and providing insights into effective therapeutics and diagnostic strategies. Methods: This study focused on an exceptionally severe ME/CFS patient with hypermobility spectrum disorder (HSD) during a period of marginal symptom improvements. Longitudinal cytokine profiling was conducted alongside the collection of extensive multi-modal health data to explore the dynamic nature of symptoms, severity, triggers, and modifying factors. Additionally, an updated severity assessment platform and two applications, ME-CFSTrackerApp and LexiTime, were introduced to facilitate real-time symptom tracking and enhance patient-physician/researcher communication, and evaluate response to medical intervention. Results: Longitudinal cytokine profiling revealed the significance of Th2-type cytokines and highlighted synergistic activities between mast cells and eosinophils, skewing Th1 toward Th2 immune responses in ME/CFS pathogenesis, particularly in cognitive impairment and sensorial intolerance. This suggests a potentially shared underlying mechanism with major ME/CFS comorbidities such as HSD, Mast cell activation syndrome, postural orthostatic tachycardia syndrome (POTS), and small fiber neuropathy. Additionally, the data identified potential roles of BCL6 and TP53 pathways in ME/CFS etiology and emphasized the importance of investigating adverse reactions to medication and supplements and drug interactions in ME/CFS severity and progression. Discussion: Our study advocates for the integration of longitudinal multi-omics with multi-modal health data and artificial intelligence (AI) techniques to better understand ME/CFS and its major comorbidities. These findings highlight the significance of dysregulated Th2-type cytokines in patient stratification and precision medicine strategies. Additionally, our results suggest exploring the use of low-dose drugs with partial agonist activity as a potential avenue for ME/CFS treatment. This comprehensive approach emphasizes the importance of adopting a patient-centered care approach to improve ME/CFS healthcare management, disease severity assessment, and personalized medicine. Overall, these findings contribute to our understanding of ME/CFS and offer avenues for future research and clinical practice.
Subject(s)
Cytokines , Severity of Illness Index , Adult , Humans , Male , Cytokines/metabolismABSTRACT
Age is a major risk factor for severe coronavirus disease 2019 (COVID-19), yet the mechanisms behind this relationship have remained incompletely understood. To address this, we evaluated the impact of aging on host immune response in the blood and the upper airway, as well as the nasal microbiome in a prospective, multicenter cohort of 1031 vaccine-naïve patients hospitalized for COVID-19 between 18 and 96 years old. We performed mass cytometry, serum protein profiling, anti-severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antibody assays, and blood and nasal transcriptomics. We found that older age correlated with increased SARS-CoV-2 viral abundance upon hospital admission, delayed viral clearance, and increased type I interferon gene expression in both the blood and upper airway. We also observed age-dependent up-regulation of innate immune signaling pathways and down-regulation of adaptive immune signaling pathways. Older adults had lower naïve T and B cell populations and higher monocyte populations. Over time, older adults demonstrated a sustained induction of pro-inflammatory genes and serum chemokines compared with younger individuals, suggesting an age-dependent impairment in inflammation resolution. Transcriptional and protein biomarkers of disease severity differed with age, with the oldest adults exhibiting greater expression of pro-inflammatory genes and proteins in severe disease. Together, our study finds that aging is associated with impaired viral clearance, dysregulated immune signaling, and persistent and potentially pathologic activation of pro-inflammatory genes and proteins.
Subject(s)
COVID-19 , Humans , Aged , Adolescent , Young Adult , Adult , Middle Aged , Aged, 80 and over , SARS-CoV-2 , Prospective Studies , Multiomics , ChemokinesABSTRACT
Vaccine adjuvants increase the breadth of serum antibody responses, but whether this is due to the generation of antigen-specific B cell clones with distinct specificities or the maturation of memory B cell clones that produce broadly cross-reactive antibodies is unknown. Here, we longitudinally analyzed immune responses in healthy adults after two-dose vaccination with either a virus-like particle COVID-19 vaccine (CoVLP), CoVLP adjuvanted with AS03 (CoVLP+AS03), or a messenger RNA vaccination (mRNA-1273). CoVLP+AS03 enhanced the magnitude and durability of circulating antibodies and antigen-specific CD4+ T cell and memory B cell responses. Antigen-specific CD4+ T cells in the CoVLP+AS03 group at day 42 correlated with antigen-specific memory B cells at 6 months. CoVLP+AS03 induced memory B cell responses, which accumulated somatic hypermutations over 6 months, resulting in enhanced neutralization breadth of monoclonal antibodies. Furthermore, the fraction of broadly neutralizing antibodies encoded by memory B cells increased between day 42 and 6 months. These results indicate that AS03 enhances the antigenic breadth of B cell memory at the clonal level and induces progressive maturation of the B cell response.
Subject(s)
COVID-19 , Influenza Vaccines , Influenza, Human , Polysorbates , Squalene , alpha-Tocopherol , Adult , Humans , Memory B Cells , COVID-19 Vaccines , Antibodies, Viral , COVID-19/prevention & control , Drug CombinationsABSTRACT
T cells specific for a single antigen tend to be rare, even after expansion of memory cells. They are commonly detected by in vitro stimulation with peptides or protein, followed by staining for intracellular cytokines. In this protocol, CyTOF® mass cytometry is used to collect single-cell data on a large number of cytokines/chemokines, as well as cell-surface proteins that characterize T cells and other immune cells. A method for magnetic bead enrichment of antigen-stimulated T cells, based on their expression of CD154 and CD69, is also included. Coupling magnetic enrichment with highly multiparameter mass cytometry, this method enables the ability to dissect the frequency, phenotype, and function of antigen-specific T cells in greater detail than previously possible. Rare cell subsets can be examined, while minimizing run times on the CyTOF.
Subject(s)
Antigens , T-Lymphocytes , Flow Cytometry/methods , Immunologic Tests , Cytokines/metabolismABSTRACT
Understanding the immune responses to SARS-CoV-2 vaccination is critical to optimizing vaccination strategies for individuals with autoimmune diseases, such as systemic lupus erythematosus (SLE). Here, we comprehensively analyzed innate and adaptive immune responses in 19 patients with SLE receiving a complete 2-dose Pfizer-BioNTech mRNA vaccine (BNT162b2) regimen compared with a control cohort of 56 healthy control (HC) volunteers. Patients with SLE exhibited impaired neutralizing antibody production and antigen-specific CD4+ and CD8+ T cell responses relative to HC. Interestingly, antibody responses were only altered in patients with SLE treated with immunosuppressive therapies, whereas impairment of antigen-specific CD4+ and CD8+ T cell numbers was independent of medication. Patients with SLE also displayed reduced levels of circulating CXC motif chemokine ligands, CXCL9, CXCL10, CXCL11, and IFN-γ after secondary vaccination as well as downregulation of gene expression pathways indicative of compromised innate immune responses. Single-cell RNA-Seq analysis reveals that patients with SLE showed reduced levels of a vaccine-inducible monocyte population characterized by overexpression of IFN-response transcription factors. Thus, although 2 doses of BNT162b2 induced relatively robust immune responses in patients with SLE, our data demonstrate impairment of both innate and adaptive immune responses relative to HC, highlighting a need for population-specific vaccination studies.
Subject(s)
COVID-19 , Lupus Erythematosus, Systemic , Humans , BNT162 Vaccine , COVID-19 Vaccines , SARS-CoV-2 , COVID-19/prevention & control , VaccinationSubject(s)
Neoplasms , Humans , Biomarkers , Medical Oncology , Neoplasms/diagnosis , Neoplasms/therapy , Clinical Trials as TopicABSTRACT
Age is a major risk factor for severe coronavirus disease-2019 (COVID-19), yet the mechanisms responsible for this relationship have remained incompletely understood. To address this, we evaluated the impact of aging on host and viral dynamics in a prospective, multicenter cohort of 1,031 patients hospitalized for COVID-19, ranging from 18 to 96 years of age. We performed blood transcriptomics and nasal metatranscriptomics, and measured peripheral blood immune cell populations, inflammatory protein expression, anti-SARS-CoV-2 antibodies, and anti-interferon (IFN) autoantibodies. We found that older age correlated with an increased SARS-CoV-2 viral load at the time of admission, and with delayed viral clearance over 28 days. This contributed to an age-dependent increase in type I IFN gene expression in both the respiratory tract and blood. We also observed age-dependent transcriptional increases in peripheral blood IFN-γ, neutrophil degranulation, and Toll like receptor (TLR) signaling pathways, and decreases in T cell receptor (TCR) and B cell receptor signaling pathways. Over time, older adults exhibited a remarkably sustained induction of proinflammatory genes (e.g., CXCL6) and serum chemokines (e.g., CXCL9) compared to younger individuals, highlighting a striking age-dependent impairment in inflammation resolution. Augmented inflammatory signaling also involved the upper airway, where aging was associated with upregulation of TLR, IL17, type I IFN and IL1 pathways, and downregulation TCR and PD-1 signaling pathways. Metatranscriptomics revealed that the oldest adults exhibited disproportionate reactivation of herpes simplex virus and cytomegalovirus in the upper airway following hospitalization. Mass cytometry demonstrated that aging correlated with reduced naïve T and B cell populations, and increased monocytes and exhausted natural killer cells. Transcriptional and protein biomarkers of disease severity markedly differed with age, with the oldest adults exhibiting greater expression of TLR and inflammasome signaling genes, as well as proinflammatory proteins (e.g., IL6, CXCL8), in severe COVID-19 compared to mild/moderate disease. Anti-IFN autoantibody prevalence correlated with both age and disease severity. Taken together, this work profiles both host and microbe in the blood and airway to provide fresh insights into aging-related immune changes in a large cohort of vaccine-naïve COVID-19 patients. We observed age-dependent immune dysregulation at the transcriptional, protein and cellular levels, manifesting in an imbalance of inflammatory responses over the course of hospitalization, and suggesting potential new therapeutic targets.
ABSTRACT
Top-down-mass spectrometry (MS)-based proteomics has emerged as a premier technology to examine proteins at the proteoform level, enabling characterization of genetic mutations, alternative splicing, and post-translational modifications. However, significant challenges that remain in top-down proteomics include the analysis of large proteoforms and the sensitivity required to examine proteoforms from minimal amounts of sample. To address these challenges, we have developed a new method termed "small-scale serial Size Exclusion Chromatography" (s3SEC), which incorporates a small-scale protein extraction (1 mg of tissue) and serial SEC without postfractionation sample handling, coupled with online high sensitivity capillary reversed-phase liquid chromatography tandem MS (RPLC-MS/MS) for analysis of large proteoforms. The s3SEC-RPLC-MS/MS method significantly enhanced the sensitivity and reduced the proteome complexity across the fractions, enabling the detection of high MW proteoforms previously undetected in one-dimensional (1D)-RPLC analysis. Importantly, we observed a drastic improvement in the signal intensity of high MW proteoforms in early fractions when using the s3SEC-RPLC method. Moreover, we demonstrate that this s3SEC-RPLC-MS/MS method also allows the analysis of lower MW proteoforms in subsequent fractions without significant alteration in proteoform abundance and equivalent or improved fragmentation efficiency to that of the 1D-RPLC approach. Although this study focuses on the use of cardiac tissue, the s3SEC-RPLC-MS/MS method could be broadly applicable to other systems with limited sample inputs.
ABSTRACT
A variety of commercial platforms are available for the simultaneous detection of multiple cytokines and associated proteins, often employing Ab pairs to capture and detect target proteins. In this study, we comprehensively evaluated the performance of three distinct platforms: the fluorescent bead-based Luminex assay, the proximity extension-based Olink assay, and a novel proximity ligation assay platform known as Alamar NULISAseq. These assessments were conducted on human serum samples from the National Institutes of Health IMPACC study, with a focus on three essential performance metrics: detectability, correlation, and differential expression. Our results reveal several key findings. First, the Alamar platform demonstrated the highest overall detectability, followed by Olink and then Luminex. Second, the correlation of protein measurements between the Alamar and Olink platforms tended to be stronger than the correlation of either of these platforms with Luminex. Third, we observed that detectability differences across the platforms often translated to differences in differential expression findings, although high detectability did not guarantee the ability to identify meaningful biological differences. Our study provides valuable insights into the comparative performance of these assays, enhancing our understanding of their strengths and limitations when assessing complex biological samples, as exemplified by the sera from this COVID-19 cohort.
Subject(s)
COVID-19 , Humans , COVID-19/diagnosis , Immunoassay/methods , Cytokines/metabolism , Serum/metabolismABSTRACT
As part of a symposium, current and former directors of Immune Monitoring cores and investigative oncologists presented insights into the past, present and future of immune assessment. Dr. Gnjatic presented a classification of immune monitoring technologies ranging from universally applicable to experimental protocols, while emphasizing the need for assay harmonization. Dr. Obeng discussed physiologic differences among CD8 T cells that align with anti-tumor responses. Dr. Lyerly presented the Soldano Ferrone lecture, commemorating the passionate tumor immunologist who inspired many, and covered a timeline of monitoring technology development and its importance to immuno-oncology. Dr. Sonabend presented recent achievements in glioblastoma treatment, accentuating the range of monitoring techniques that allowed him to refine patient selection for clinical trials. Dr. Guevara-Patiño focused on hypoxia within the tumor environment and stressed that T cell viability is not to be confused with functionality. Dr. Butterfield accentuated monitoring of dendritic cell metabolic (dys)function as a determinant for tumor vaccine success. Lectures were interspersed with select abstract presentations. To summarize the concepts, Dr. Maecker from Stanford led an informative forum discussion, pointing towards the future of immune monitoring. Immune monitoring continues to be a guiding light towards effective immunotherapeutic strategies.
ABSTRACT
The glycosylation of IgG plays a critical role during human severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, activating immune cells and inducing cytokine production. However, the role of IgM N-glycosylation has not been studied during human acute viral infection. The analysis of IgM N-glycosylation from healthy controls and hospitalized coronavirus disease 2019 (COVID-19) patients reveals increased high-mannose and sialylation that correlates with COVID-19 severity. These trends are confirmed within SARS-CoV-2-specific immunoglobulin N-glycan profiles. Moreover, the degree of total IgM mannosylation and sialylation correlate significantly with markers of disease severity. We link the changes of IgM N-glycosylation with the expression of Golgi glycosyltransferases. Lastly, we observe antigen-specific IgM antibody-dependent complement deposition is elevated in severe COVID-19 patients and modulated by exoglycosidase digestion. Taken together, this work links the IgM N-glycosylation with COVID-19 severity and highlights the need to understand IgM glycosylation and downstream immune function during human disease.
Subject(s)
COVID-19 , Humans , Glycosylation , SARS-CoV-2 , Glycosyltransferases , Complement System Proteins , Immunoglobulin MABSTRACT
A variety of commercial platforms are available for the simultaneous detection of multiple cytokines and associated proteins, often employing antibody pairs to capture and detect target proteins. In this study, we comprehensively evaluated the performance of three distinct platforms: the fluorescent bead-based Luminex assay, the proximity extension-based Olink assay, and a novel proximity ligation assay platform known as Alamar NULISAseq. These assessments were conducted on serum samples from the NIH IMPACC study, with a focus on three essential performance metrics: detectability, correlation, and differential expression. Our results reveal several key findings. Firstly, the Alamar platform demonstrated the highest overall detectability, followed by Olink and then Luminex. Secondly, the correlation of protein measurements between the Alamar and Olink platforms tended to be stronger than the correlation of either of these platforms with Luminex. Thirdly, we observed that detectability differences across the platforms often translated to differences in differential expression findings, although high detectability did not guarantee the ability to identify meaningful biological differences. Our study provides valuable insights into the comparative performance of these assays, enhancing our understanding of their strengths and limitations when assessing complex biological samples, as exemplified by the sera from this COVID-19 cohort.
ABSTRACT
Respiratory viruses such as influenza are encountered multiple times through infection and/or vaccination and thus have the potential to shape immune cell phenotypes over time. In particular, memory T cell compartments may be affected, as both CD4+ and CD8+ T cell responses likely contribute to viral control. In this study, we assessed immune phenotypes using cytometry by time of flight in the peripheral blood of 22 humans with acute respiratory illness and 22 age-matched noninfected controls. In younger infected individuals (1-19 y of age), we found decreased B and NK cell frequencies and a shift toward more effector-like CD4+ and CD8+ T cell phenotypes, compared with young healthy controls. Significant differences between noninfected and infected older individuals (30-74 y of age) were not seen. We also observed a decrease in naive CD4+ T cells and CD27+CD8+ T cells as well as an increase in effector memory CD8+ T cells and NKT cells in noninfected individuals with age. When cell frequencies were regressed against age for infected versus noninfected subjects, significant differences in trends with age were observed for multiple cell types. These included B cells and various subsets of CD4+ and CD8+ T cells. We conclude that acute respiratory illness drives T cell differentiation and decreases circulating B cell frequencies preferentially in young compared with older individuals.
Subject(s)
CD8-Positive T-Lymphocytes , Influenza, Human , Humans , Cell Differentiation , Lymphocyte Activation , B-LymphocytesABSTRACT
The clinical trajectory of COVID-19 may be influenced by previous responses to heterologous viruses. We examined the relationship of Abs against different viruses to clinical trajectory groups from the National Institutes of Health IMPACC (Immunophenotyping Assessment in a COVID-19 Cohort) study of hospitalized COVID-19 patients. Whereas initial Ab titers to SARS-CoV-2 tended to be higher with increasing severity (excluding fatal disease), those to seasonal coronaviruses trended in the opposite direction. Initial Ab titers to influenza and parainfluenza viruses also tended to be lower with increasing severity. However, no significant relationship was observed for Abs to other viruses, including measles, CMV, EBV, and respiratory syncytial virus. We hypothesize that some individuals may produce lower or less durable Ab responses to respiratory viruses generally (reflected in lower baseline titers in our study), and that this may carry over into poorer outcomes for COVID-19 (despite high initial SARS-CoV-2 titers). We further looked at longitudinal changes in Ab responses to heterologous viruses, but found little change during the course of acute COVID-19 infection. We saw significant trends with age for Ab levels to many of these viruses, but no difference in longitudinal SARS-CoV-2 titers for those with high versus low seasonal coronavirus titers. We detected no difference in longitudinal SARS-CoV-2 titers for CMV seropositive versus seronegative patients, although there was an overrepresentation of CMV seropositives among the IMPACC cohort, compared with expected frequencies in the United States population. Our results both reinforce findings from other studies and suggest (to our knowledge) new relationships between the response to SARS-CoV-2 and Abs to heterologous viruses.