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1.
Front Immunol ; 12: 751869, 2021.
Article in English | MEDLINE | ID: covidwho-1634057

ABSTRACT

Background: Immunological characteristics of COVID-19 show pathological hyperinflammation associated with lymphopenia and dysfunctional T cell responses. These features provide a rationale for restoring functional T cell immunity in COVID-19 patients by adoptive transfer of SARS-CoV-2 specific T cells. Methods: To generate SARS-CoV-2 specific T cells, we isolated peripheral blood mononuclear cells from 7 COVID-19 recovered and 13 unexposed donors. Consequently, we stimulated cells with SARS-CoV-2 peptide mixtures covering spike, membrane and nucleocapsid proteins. Then, we culture expanded cells with IL-2 for 21 days. We assessed immunophenotypes, cytokine profiles, antigen specificity of the final cell products. Results: Our results show that SARS-CoV-2 specific T cells could be expanded in both COVID-19 recovered and unexposed groups. Immunophenotypes were similar in both groups showing CD4+ T cell dominance, but CD8+ and CD3+CD56+ T cells were also present. Antigen specificity was determined by ELISPOT, intracellular cytokine assay, and cytotoxicity assays. One out of 14 individuals who were previously unexposed to SARS-CoV-2 failed to show antigen specificity. Moreover, ex-vivo expanded SARS-CoV-2 specific T cells mainly consisted of central and effector memory subsets with reduced alloreactivity against HLA-unmatched cells suggesting the possibility for the development of third-party partial HLA-matching products. Discussion: In conclusion, our findings show that SARS-CoV-2 specific T cell can be readily expanded from both COVID-19 and unexposed individuals and can therefore be manufactured as a biopharmaceutical product to treat severe COVID-19 patients. One Sentence Summary: Ex-vivo expanded SARS-CoV-2 antigen specific T cells developed as third-party partial HLA-matching products may be a promising approach for treating severe COVID-19 patients that do not respond to previous treatment options.


Subject(s)
Adoptive Transfer , CD4-Positive T-Lymphocytes/transplantation , CD8-Positive T-Lymphocytes/transplantation , COVID-19/therapy , SARS-CoV-2/immunology , Adult , Antibodies, Viral/immunology , CD4-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/immunology , COVID-19/immunology , Cell- and Tissue-Based Therapy , Coronavirus Nucleocapsid Proteins/immunology , Epitopes, T-Lymphocyte/immunology , Female , Humans , Immunophenotyping , Leukocytes, Mononuclear/immunology , Male , Middle Aged , Phosphoproteins/immunology , Spike Glycoprotein, Coronavirus/immunology , Viral Matrix Proteins/immunology , Young Adult
2.
J Immunol ; 208(3): 685-696, 2022 02 01.
Article in English | MEDLINE | ID: covidwho-1604665

ABSTRACT

Immune response dysregulation plays a key role in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pathogenesis. In this study, we evaluated immune and endothelial blood cell profiles of patients with coronavirus disease 2019 (COVID-19) to determine critical differences between those with mild, moderate, or severe COVID-19 using spectral flow cytometry. We examined a suite of immune phenotypes, including monocytes, T cells, NK cells, B cells, endothelial cells, and neutrophils, alongside surface and intracellular markers of activation. Our results showed progressive lymphopenia and depletion of T cell subsets (CD3+, CD4+, and CD8+) in patients with severe disease and a significant increase in the CD56+CD14+Ki67+IFN-γ+ monocyte population in patients with moderate and severe COVID-19 that has not been previously described. Enhanced circulating endothelial cells (CD45-CD31+CD34+CD146+), circulating endothelial progenitors (CD45-CD31+CD34+/-CD146-), and neutrophils (CD11b+CD66b+) were coevaluated for COVID-19 severity. Spearman correlation analysis demonstrated the synergism among age, obesity, and hypertension with upregulated CD56+ monocytes, endothelial cells, and decreased T cells that lead to severe outcomes of SARS-CoV-2 infection. Circulating monocytes and endothelial cells may represent important cellular markers for monitoring postacute sequelae and impacts of SARS-CoV-2 infection during convalescence and for their role in immune host defense in high-risk adults after vaccination.


Subject(s)
COVID-19/immunology , Endothelial Cells/immunology , Monocytes/immunology , SARS-CoV-2 , Adolescent , Adult , Age Factors , Aged , Antibodies, Viral/biosynthesis , Antibodies, Viral/immunology , Biomarkers , CD56 Antigen/analysis , COVID-19/blood , COVID-19/epidemiology , Child , Comorbidity , Endothelial Cells/chemistry , Female , Flow Cytometry , Humans , Hypertension/epidemiology , Hypertension/immunology , Immunophenotyping , Lymphocyte Activation , Lymphocyte Subsets/immunology , Lymphopenia/etiology , Lymphopenia/immunology , Male , Middle Aged , Monocytes/chemistry , Neutrophils/immunology , Obesity/epidemiology , Obesity/immunology , Platelet Endothelial Cell Adhesion Molecule-1/analysis , SARS-CoV-2/immunology , Severity of Illness Index , Spike Glycoprotein, Coronavirus/immunology , Young Adult
3.
Front Immunol ; 12: 760249, 2021.
Article in English | MEDLINE | ID: covidwho-1581341

ABSTRACT

Background: The humoral and cellular immune responses to SARS-COV-2 vaccination remain to be elucidated in hemodialysis (HD) patients and kidney transplant recipients (KTRs), considering their baseline immunosuppressed status. The aim of our study was to assess the associations of vaccine-induced antibody responses with circulating lymphocytes sub-populations and their respective patterns of alterations in maintenance HD patients and KTRs. Materials and Methods: We included 34 HD patients and 54 KTRs who received two doses of the mRNA-vaccine BNT162b2. Lymphocyte subpopulations were analyzed by flow cytometry before vaccination (T0), before the second vaccine dose (T1) and 2 weeks after the second dose (T2). The anti-SARS-CoV2 antibody response was assessed at T1 and at T2. Results: 31 HD patients (91.8%) and 16 KTRs (29.6%) became seropositive at T2. HD patients who became seropositive following the first dose displayed higher CD19+ B lymphocytes compared to their seronegative HD counterparts. A positive correlation was established between CD19+ B cells counts and antibody titers at all time-points in both groups (p < 0.001). KTRs showed higher naïve CD4+CD45RA+ T helper cells compared to HD patients at baseline and T2 whereas HD patients displayed higher memory CD45RO+ T cells compared to KTRs at T2. The naïve CD4+CD45RA to memory CD4+CD45RO+ T helper cells fraction was negatively associated with antibody production in both groups. Conclusions: Our study provides a potential conceptual framework for monitoring vaccination efficacy in HD patients and KTRs considering the correlation established between CD19+ B cells, generation of memory CD4+ T helper cells and anti SARS-CoV2 antibody response to vaccination.


Subject(s)
Antibody Formation/immunology , B-Lymphocytes/immunology , CD4-Positive T-Lymphocytes/immunology , Immunity, Humoral , Immunocompromised Host , Immunologic Memory , B-Lymphocytes/metabolism , Biomarkers , CD4-Positive T-Lymphocytes/metabolism , COVID-19/immunology , COVID-19/virology , COVID-19 Vaccines/immunology , Female , Humans , Immunophenotyping , Kidney Transplantation , Lymphocyte Count , Male , Renal Dialysis , SARS-CoV-2/immunology
4.
Front Immunol ; 12: 789735, 2021.
Article in English | MEDLINE | ID: covidwho-1581322

ABSTRACT

Background: The host immune response has a prominent role in the progression and outcome of SARS-CoV-2 infection. Lymphopenia has been described as an important feature of SARS-CoV-2 infection and has been associated with severe disease manifestation. Lymphocyte dysregulation and hyper-inflammation have been shown to be associated with a more severe clinical course; however, a T cell subpopulation whose dysfunction correlate with disease progression has yet to be identify. Methods: We performed an immuno-phenotypic analysis of T cell sub-populations in peripheral blood from patients affected by different severity of COVID-19 (n=60) and undergoing a different clinical evolution. Clinical severity was established based on a modified WHO score considering both ventilation support and respiratory capacity (PaO2/FiO2 ratio). The ability of circulating cells at baseline to predict the probability of clinical aggravation was explored through multivariate regression analyses. Results: The immuno-phenotypic analysis performed by multi-colour flow cytometry confirmed that patients suffering from severe COVID-19 harboured significantly reduced circulating T cell subsets, especially for CD4+ T, Th1, and regulatory T cells. Peripheral T cells also correlated with parameters associated with disease severity, i.e., PaO2/FiO2 ratio and inflammation markers. CD4+ T cell subsets showed an important significant association with clinical evolution, with patients presenting markedly decreased regulatory T cells at baseline having a significantly higher risk of aggravation. Importantly, the combination of gender and regulatory T cells allowed distinguishing between improved and worsened patients with an area under the ROC curve (AUC) of 82%. Conclusions: The present study demonstrates the association between CD4+ T cell dysregulation and COVID-19 severity and progression. Our results support the importance of analysing baseline regulatory T cell levels, since they were revealed able to predict the clinical worsening during hospitalization. Regulatory T cells assessment soon after hospital admission could thus allow a better clinical stratification and patient management.


Subject(s)
COVID-19/epidemiology , COVID-19/immunology , Hospitalization , Lymphocyte Count , SARS-CoV-2/immunology , T-Lymphocytes, Regulatory/immunology , Biomarkers , COVID-19/diagnosis , COVID-19/virology , COVID-19 Serological Testing , Cytokines/blood , Cytokines/metabolism , Disease Progression , Humans , Immunophenotyping , Inflammation Mediators/blood , Inflammation Mediators/metabolism , Prognosis , Public Health Surveillance , ROC Curve , Severity of Illness Index , T-Lymphocyte Subsets/immunology , T-Lymphocyte Subsets/metabolism , T-Lymphocytes, Regulatory/metabolism
5.
Methods Mol Biol ; 2386: 43-60, 2022.
Article in English | MEDLINE | ID: covidwho-1513947

ABSTRACT

A comprehensive study of the cellular components of the immune system demands both deep and broad immunophenotyping of numerous cell subsets in an effective and practical way. Novel full-spectrum technology reveals the complete emission spectrum of each dye maximizing the amount of information that can be obtained on a single sample regarding conventional flow cytometry and provide an expanded knowledge of biological processes. In this chapter, we describe a 37-color protocol that allows to identify more than 45 different cell populations on whole blood samples of SARS-CoV-2-infected patients.


Subject(s)
COVID-19 , Flow Cytometry , Immunophenotyping/methods , COVID-19/blood , Color , Humans , Immune System
6.
J Clin Invest ; 131(21)2021 11 01.
Article in English | MEDLINE | ID: covidwho-1495789

ABSTRACT

To explore how the immune system controls clearance of SARS-CoV-2, we used a single-cell, mass cytometry-based proteomics platform to profile the immune systems of 21 patients who had recovered from SARS-CoV-2 infection without need for admission to an intensive care unit or for mechanical ventilation. We focused on receptors involved in interactions between immune cells and virus-infected cells. We found that the diversity of receptor repertoires on natural killer (NK) cells was negatively correlated with the viral clearance rate. In addition, NK subsets expressing the receptor DNAM1 were increased in patients who more rapidly recovered from infection. Ex vivo functional studies revealed that NK subpopulations with high DNAM1 expression had cytolytic activities in response to target cell stimulation. We also found that SARS-CoV-2 infection induced the expression of CD155 and nectin-4, ligands of DNAM1 and its paired coinhibitory receptor TIGIT, which counterbalanced the cytolytic activities of NK cells. Collectively, our results link the cytolytic immune responses of NK cells to the clearance of SARS-CoV-2 and show that the DNAM1 pathway modulates host-pathogen interactions during SARS-CoV-2 infection.


Subject(s)
COVID-19/immunology , COVID-19/virology , Killer Cells, Natural/immunology , Receptors, Natural Killer Cell/immunology , SARS-CoV-2/immunology , Adolescent , Adult , Aged , Animals , Antigens, Differentiation, T-Lymphocyte/immunology , Cell Adhesion Molecules/immunology , Cohort Studies , Cytotoxicity, Immunologic , Female , Heterografts , Host Microbial Interactions/immunology , Humans , Immunophenotyping , In Vitro Techniques , Ligands , Male , Mice , Mice, SCID , Middle Aged , NK Cell Lectin-Like Receptor Subfamily D/immunology , Pandemics , Receptors, Immunologic/immunology , Receptors, Virus/immunology , Viral Load , Young Adult
7.
Proc Natl Acad Sci U S A ; 118(40)2021 10 05.
Article in English | MEDLINE | ID: covidwho-1493346

ABSTRACT

Since the outset of the COVID-19 pandemic, increasing evidence suggests that the innate immune responses play an important role in the disease development. A dysregulated inflammatory state has been proposed as a key driver of clinical complications in COVID-19, with a potential detrimental role of granulocytes. However, a comprehensive phenotypic description of circulating granulocytes in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-infected patients is lacking. In this study, we used high-dimensional flow cytometry for granulocyte immunophenotyping in peripheral blood collected from COVID-19 patients during acute and convalescent phases. Severe COVID-19 was associated with increased levels of both mature and immature neutrophils, and decreased counts of eosinophils and basophils. Distinct immunotypes were evident in COVID-19 patients, with altered expression of several receptors involved in activation, adhesion, and migration of granulocytes (e.g., CD62L, CD11a/b, CD69, CD63, CXCR4). Paired sampling revealed recovery and phenotypic restoration of the granulocytic signature in the convalescent phase. The identified granulocyte immunotypes correlated with distinct sets of soluble inflammatory markers, supporting pathophysiologic relevance. Furthermore, clinical features, including multiorgan dysfunction and respiratory function, could be predicted using combined laboratory measurements and immunophenotyping. This study provides a comprehensive granulocyte characterization in COVID-19 and reveals specific immunotypes with potential predictive value for key clinical features associated with COVID-19.


Subject(s)
COVID-19/immunology , Granulocytes/immunology , COVID-19/blood , COVID-19/diagnosis , COVID-19/physiopathology , Granulocytes/cytology , Humans , Immunity, Innate , Immunophenotyping , Leukocyte Count , Lung/physiopathology , Models, Biological , Organ Dysfunction Scores , SARS-CoV-2 , Severity of Illness Index
8.
Proc Natl Acad Sci U S A ; 118(40)2021 10 05.
Article in English | MEDLINE | ID: covidwho-1434221

ABSTRACT

Since the outset of the COVID-19 pandemic, increasing evidence suggests that the innate immune responses play an important role in the disease development. A dysregulated inflammatory state has been proposed as a key driver of clinical complications in COVID-19, with a potential detrimental role of granulocytes. However, a comprehensive phenotypic description of circulating granulocytes in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-infected patients is lacking. In this study, we used high-dimensional flow cytometry for granulocyte immunophenotyping in peripheral blood collected from COVID-19 patients during acute and convalescent phases. Severe COVID-19 was associated with increased levels of both mature and immature neutrophils, and decreased counts of eosinophils and basophils. Distinct immunotypes were evident in COVID-19 patients, with altered expression of several receptors involved in activation, adhesion, and migration of granulocytes (e.g., CD62L, CD11a/b, CD69, CD63, CXCR4). Paired sampling revealed recovery and phenotypic restoration of the granulocytic signature in the convalescent phase. The identified granulocyte immunotypes correlated with distinct sets of soluble inflammatory markers, supporting pathophysiologic relevance. Furthermore, clinical features, including multiorgan dysfunction and respiratory function, could be predicted using combined laboratory measurements and immunophenotyping. This study provides a comprehensive granulocyte characterization in COVID-19 and reveals specific immunotypes with potential predictive value for key clinical features associated with COVID-19.


Subject(s)
COVID-19/immunology , Granulocytes/immunology , COVID-19/blood , COVID-19/diagnosis , COVID-19/physiopathology , Granulocytes/cytology , Humans , Immunity, Innate , Immunophenotyping , Leukocyte Count , Lung/physiopathology , Models, Biological , Organ Dysfunction Scores , SARS-CoV-2 , Severity of Illness Index
9.
PLoS Pathog ; 17(9): e1009804, 2021 09.
Article in English | MEDLINE | ID: covidwho-1416909

ABSTRACT

Prior studies have demonstrated that immunologic dysfunction underpins severe illness in COVID-19 patients, but have lacked an in-depth analysis of the immunologic drivers of death in the most critically ill patients. We performed immunophenotyping of viral antigen-specific and unconventional T cell responses, neutralizing antibodies, and serum proteins in critically ill patients with SARS-CoV-2 infection, using influenza infection, SARS-CoV-2-convalescent health care workers, and healthy adults as controls. We identify mucosal-associated invariant T (MAIT) cell activation as an independent and significant predictor of death in COVID-19 (HR = 5.92, 95% CI = 2.49-14.1). MAIT cell activation correlates with several other mortality-associated immunologic measures including broad activation of CD8+ T cells and non-Vδ2 γδT cells, and elevated levels of cytokines and chemokines, including GM-CSF, CXCL10, CCL2, and IL-6. MAIT cell activation is also a predictor of disease severity in influenza (ECMO/death HR = 4.43, 95% CI = 1.08-18.2). Single-cell RNA-sequencing reveals a shift from focused IFNα-driven signals in COVID-19 ICU patients who survive to broad pro-inflammatory responses in fatal COVID-19 -a feature not observed in severe influenza. We conclude that fatal COVID-19 infection is driven by uncoordinated inflammatory responses that drive a hierarchy of T cell activation, elements of which can serve as prognostic indicators and potential targets for immune intervention.


Subject(s)
COVID-19/immunology , COVID-19/mortality , Antibodies, Neutralizing/immunology , Antibodies, Viral/immunology , Antigens, CD/immunology , Antigens, Differentiation, T-Lymphocyte/immunology , B-Lymphocytes/immunology , Biomarkers/blood , Blood Proteins/metabolism , Cohort Studies , Critical Illness/mortality , Female , Humans , Immunophenotyping , Influenza, Human/immunology , Lectins, C-Type/immunology , Lymphocyte Activation , Male , Middle Aged , Mucosal-Associated Invariant T Cells/immunology , Patient Acuity
10.
Am J Clin Pathol ; 156(4): 634-643, 2021 Sep 08.
Article in English | MEDLINE | ID: covidwho-1408305

ABSTRACT

OBJECTIVES: Myelodysplastic/myeloproliferative neoplasms (MDS/MPN) are a group of rare and heterogeneous hematopoietic disorders that frequently present a diagnostic challenge. Here we present our institutional experience with next-generation sequencing (NGS), together with morphologic, flow cytometric, and cytogenetic evaluation, in the diagnosis of MDS/MPN, with particular emphasis on MDS/MPN unclassifiable (MPN-U). METHODS: We evaluated the morphologic, flow cytometric, cytogenetic, and molecular characteristics of all MDS/MPN cases that underwent NGS at our institution between April 2016 and February 2019. RESULTS: Thirty-seven cases of MDS/MPN were identified, including 14 cases of MDS/MPN-U. Ninety-seven percent harbored mutations and immunophenotypic aberrancies (36/37), while only 38% had cytogenetic abnormalities (12/32). The MDS/MPN-U group had the highest rate of myeloblast phenotypic abnormalities and had a high mutation rate of approximately 2.7 mutated genes per case, most commonly in JAK2, SRSF2, and ASXL1. CONCLUSIONS: No single ancillary study was abnormal in every case, but all cases had at least one abnormal finding, demonstrating the usefulness of a multiparameter approach to the diagnosis of MDS/MPN. Although a few specific mutations were found exclusively in MDS/MPN-U and JAK2 mutations were most prevalent, larger studies are needed to determine whether MDS/MPN-U has a mutational "fingerprint," which may aid in diagnosis and targeted therapy.


Subject(s)
Myelodysplastic-Myeloproliferative Diseases/diagnosis , Myeloproliferative Disorders/diagnosis , Adult , Aged , Aged, 80 and over , Cytogenetics , Female , Flow Cytometry , Granulocyte Precursor Cells/pathology , High-Throughput Nucleotide Sequencing , Humans , Immunophenotyping , Male , Middle Aged , Mutation , Mutation Rate , Myelodysplastic-Myeloproliferative Diseases/classification , Myelodysplastic-Myeloproliferative Diseases/genetics , Myelodysplastic-Myeloproliferative Diseases/pathology , Myeloproliferative Disorders/classification , Myeloproliferative Disorders/genetics , Myeloproliferative Disorders/pathology , Sequence Analysis, DNA , Young Adult
11.
J Exp Med ; 218(4)2021 04 05.
Article in English | MEDLINE | ID: covidwho-1387677

ABSTRACT

SARS-CoV-2 is responsible for an ongoing pandemic that has affected millions of individuals around the globe. To gain further understanding of the immune response in recovered individuals, we measured T cell responses in paired samples obtained an average of 1.3 and 6.1 mo after infection from 41 individuals. The data indicate that recovered individuals show persistent polyfunctional SARS-CoV-2 antigen-specific memory that could contribute to rapid recall responses. Recovered individuals also show enduring alterations in relative overall numbers of CD4+ and CD8+ memory T cells, including expression of activation/exhaustion markers, and cell division.


Subject(s)
COVID-19/immunology , COVID-19/virology , Host-Pathogen Interactions/immunology , Immunity, Cellular , SARS-CoV-2/immunology , Adult , Aged , Antigens, Viral/immunology , Biomarkers , Female , Humans , Immunophenotyping , Lymphocyte Count , Male , Middle Aged , T-Cell Antigen Receptor Specificity , T-Lymphocyte Subsets/immunology , T-Lymphocyte Subsets/metabolism , Young Adult
12.
Sci Immunol ; 6(62)2021 08 10.
Article in English | MEDLINE | ID: covidwho-1352519

ABSTRACT

To understand how a protective immune response against SARS-CoV-2 develops over time, we integrated phenotypic, transcriptional and repertoire analyses on PBMCs from mild and severe COVID-19 patients during and after infection, and compared them to healthy donors (HD). A type I IFN-response signature marked all the immune populations from severe patients during the infection. Humoral immunity was dominated by IgG production primarily against the RBD and N proteins, with neutralizing antibody titers increasing post infection and with disease severity. Memory B cells, including an atypical FCRL5+ T-BET+ memory subset, increased during the infection, especially in patients with mild disease. A significant reduction of effector memory, CD8+ T cells frequency characterized patients with severe disease. Despite such impairment, we observed robust clonal expansion of CD8+ T lymphocytes, while CD4+ T cells were less expanded and skewed toward TCM and TH2-like phenotypes. MAIT cells were also expanded, but only in patients with mild disease. Terminally differentiated CD8+ GZMB+ effector cells were clonally expanded both during the infection and post-infection, while CD8+ GZMK+ lymphocytes were more expanded post-infection and represented bona fide memory precursor effector cells. TCR repertoire analysis revealed that only highly proliferating T cell clonotypes, which included SARS-CoV-2-specific cells, were maintained post-infection and shared between the CD8+ GZMB+ and GZMK+ subsets. Overall, this study describes the development of immunity against SARS-CoV-2 and identifies an effector CD8+ T cell population with memory precursor-like features.


Subject(s)
COVID-19/genetics , COVID-19/immunology , Host-Pathogen Interactions/immunology , Immunophenotyping , SARS-CoV-2/immunology , Transcriptome , Adult , Aged , Antibodies, Viral/immunology , B-Lymphocytes/immunology , B-Lymphocytes/metabolism , Biomarkers , COVID-19/virology , Cell Plasticity/genetics , Cell Plasticity/immunology , Clonal Evolution/immunology , Female , Gene Expression Profiling , Humans , Immunoglobulin Isotypes/immunology , Immunologic Memory , Leukocytes, Mononuclear/immunology , Leukocytes, Mononuclear/metabolism , Lymphocyte Count , Male , Middle Aged , T-Lymphocyte Subsets/immunology , T-Lymphocyte Subsets/metabolism
13.
Sci Immunol ; 6(62)2021 08 10.
Article in English | MEDLINE | ID: covidwho-1352518

ABSTRACT

The IMmunoPhenotyping Assessment in a COVID-19 Cohort (IMPACC) is a prospective longitudinal study designed to enroll 1000 hospitalized patients with COVID-19 (NCT04378777). IMPACC collects detailed clinical, laboratory and radiographic data along with longitudinal biologic sampling of blood and respiratory secretions for in depth testing. Clinical and lab data are integrated to identify immunologic, virologic, proteomic, metabolomic and genomic features of COVID-19-related susceptibility, severity and disease progression. The goals of IMPACC are to better understand the contributions of pathogen dynamics and host immune responses to the severity and course of COVID-19 and to generate hypotheses for identification of biomarkers and effective therapeutics, including optimal timing of such interventions. In this report we summarize the IMPACC study design and protocols including clinical criteria and recruitment, multi-site standardized sample collection and processing, virologic and immunologic assays, harmonization of assay protocols, high-level analyses and the data sharing plans.


Subject(s)
Biomarkers , COVID-19/immunology , COVID-19/virology , Immunophenotyping , SARS-CoV-2/immunology , Biomarkers/blood , COVID-19/diagnosis , COVID-19/epidemiology , Computational Biology/methods , Data Analysis , Gene Expression Profiling , Hospitalization , Humans , Longitudinal Studies , Molecular Diagnostic Techniques/methods , Prospective Studies , Proteomics/methods , United States/epidemiology
14.
Adv Sci (Weinh) ; 8(18): e2100323, 2021 09.
Article in English | MEDLINE | ID: covidwho-1316190

ABSTRACT

Blood cell analysis is a major pillar of biomedical research and healthcare. These analyses are performed in central laboratories. Rapid shipment from collection site to the central laboratories is currently needed because cells and biomarkers degrade rapidly. The dried blood spot from a fingerstick allows the preservation of cellular molecules for months but entire cells are never recovered. Here leucocyte elution is optimized from dried blood spots. Flow cytometry and mRNA expression profiling are used to analyze the recovered cells. 50-70% of the leucocytes that are dried on a polyester solid support via elution after shaking the support with buffer are recovered. While red blood cells lyse upon drying, it is found that the majority of leucocytes are preserved. Leucocytes have an altered structure that is improved by adding fixative in the elution buffer. Leucocytes are permeabilized, allowing an easy staining of all cellular compartments. Common immunophenotyping and mRNAs are preserved. The ability of a new biomarker (CD169) to discriminate between patients with and without Severe Acute Respiratory Syndrome induced by Coronavirus 2 (SARS-CoV-2) infections is also preserved. Leucocytes from blood can be dried, shipped, and/or stored for at least 1 month, then recovered for a wide variety of analyses, potentially facilitating biomedical applications worldwide.


Subject(s)
Communicable Diseases/diagnosis , Diagnostic Tests, Routine/methods , Dried Blood Spot Testing/methods , Hematology/methods , Immunophenotyping/methods , Antibodies, Viral/blood , Biomarkers/blood , Blood Specimen Collection/methods , COVID-19/diagnosis , Cell Separation/methods , Communicable Diseases/virology , Erythrocytes/virology , Flow Cytometry/methods , Humans , Leukocytes/virology , RNA, Messenger/blood , SARS-CoV-2/genetics
15.
J Immunol ; 207(2): 720-734, 2021 07 15.
Article in English | MEDLINE | ID: covidwho-1311404

ABSTRACT

Most shared resource flow cytometry facilities do not permit analysis of radioactive samples. We are investigating low-dose molecular targeted radionuclide therapy (MTRT) as an immunomodulator in combination with in situ tumor vaccines and need to analyze radioactive samples from MTRT-treated mice using flow cytometry. Further, the sudden shutdown of core facilities in response to the COVID-19 pandemic has created an unprecedented work stoppage. In these and other research settings, a robust and reliable means of cryopreservation of immune samples is required. We evaluated different fixation and cryopreservation protocols of disaggregated tumor cells with the aim of identifying a protocol for subsequent flow cytometry of the thawed sample, which most accurately reflects the flow cytometric analysis of the tumor immune microenvironment of a freshly disaggregated and analyzed sample. Cohorts of C57BL/6 mice bearing B78 melanoma tumors were evaluated using dual lymphoid and myeloid immunophenotyping panels involving fixation and cryopreservation at three distinct points during the workflow. Results demonstrate that freezing samples after all staining and fixation are completed most accurately matches the results from noncryopreserved equivalent samples. We observed that cryopreservation of living, unfixed cells introduces a nonuniform alteration to PD1 expression. We confirm the utility of our cryopreservation protocol by comparing tumors treated with in situ tumor vaccines, analyzing both fresh and cryopreserved tumor samples with similar results. Last, we use this cryopreservation protocol with radioactive specimens to demonstrate potentially beneficial effector cell changes to the tumor immune microenvironment following administration of a novel MTRT in a dose- and time-dependent manner.


Subject(s)
Cryopreservation/methods , Flow Cytometry/methods , Leukocytes, Mononuclear/immunology , Melanoma, Experimental/pathology , Myeloid Cells/immunology , Animals , CD4-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/immunology , Cell Line, Tumor , Immunophenotyping/methods , Mice , Mice, Inbred C57BL , Natural Killer T-Cells/immunology , Pandemics , Signal Transduction/immunology , Tumor Microenvironment/immunology
16.
J Infect Dis ; 224(2): 229-240, 2021 07 15.
Article in English | MEDLINE | ID: covidwho-1310926

ABSTRACT

BACKGROUND: Etiopathogenesis of the clinical variability of the coronavirus disease 2019 (COVID-19) remains mostly unknown. In this study, we investigate the role of killer cell immunoglobulin-like receptor (KIR)/human leukocyte antigen class-I (HLA-I) interactions in the susceptibility and severity of COVID-19. METHODS: We performed KIR and HLA-I genotyping and natural killer cell (NKc) receptors immunophenotyping in 201 symptomatic patients and 210 noninfected controls. RESULTS: The NKcs with a distinctive immunophenotype, suggestive of recent activation (KIR2DS4low CD16low CD226low CD56high TIGIThigh NKG2Ahigh), expanded in patients with severe COVID-19. This was associated with a higher frequency of the functional A-telomeric activating KIR2DS4 in severe versus mild and/or moderate patients and controls (83.7%, 55.7% and 36.2%, P < 7.7 × 10-9). In patients with mild and/or moderate infection, HLA-B*15:01 was associated with higher frequencies of activating B-telomeric KIR3DS1 compared with patients with other HLA-B*15 subtypes and noninfected controls (90.9%, 42.9%, and 47.3%; P < .002; Pc = 0.022). This strongly suggests that HLA-B*15:01 specifically presenting severe acute respiratory syndrome coronavirus 2 peptides could form a neoligand interacting with KIR3DS1. Likewise, a putative neoligand for KIR2DS4 could arise from other HLA-I molecules presenting severe acute respiratory syndrome coronavirus 2 peptides expressed on infected an/or activated lung antigen-presenting cells. CONCLUSIONS: Our results support a crucial role of NKcs in the clinical variability of COVID-19 with specific KIR/ligand interactions associated with disease severity.


Subject(s)
COVID-19/genetics , Genetic Predisposition to Disease/genetics , Receptors, KIR/genetics , Aged , COVID-19/immunology , COVID-19/pathology , Cross-Sectional Studies , Female , Genotype , HLA Antigens/genetics , HLA Antigens/metabolism , Humans , Immunophenotyping , Killer Cells, Natural/metabolism , Male , Middle Aged , Prospective Studies , Receptors, KIR/metabolism , SARS-CoV-2 , Severity of Illness Index
17.
Nat Commun ; 12(1): 4117, 2021 07 05.
Article in English | MEDLINE | ID: covidwho-1297301

ABSTRACT

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


Subject(s)
COVID-19/complications , COVID-19/immunology , Cytokine Release Syndrome/complications , Monocytes/pathology , Neutrophil Activation , Aged , Antigen-Presenting Cells/immunology , COVID-19/blood , COVID-19/virology , Case-Control Studies , Cytokine Release Syndrome/blood , Cytokine Release Syndrome/pathology , Cytokine Release Syndrome/virology , Cytokines/blood , Extracellular Traps/metabolism , Female , Histocompatibility Antigens Class II/metabolism , Humans , Immunophenotyping , Male , Middle Aged , SARS-CoV-2/physiology , Severity of Illness Index
18.
J Infect Dis ; 224(1): 39-48, 2021 07 02.
Article in English | MEDLINE | ID: covidwho-1294730

ABSTRACT

BACKGROUND: Understanding the memory T-cell response to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is crucial for assessing the longevity of protective immunity after SARS-CoV-2 infection or coronavirus disease 2019 (COVID-19) vaccination. However, the longitudinal memory T-cell response up to 8 months post-symptom onset (PSO) according to the severity of illness is unknown. METHODS: We analyzed peripheral blood mononuclear cells (PBMCs) from healthy volunteers or patients with COVID-19 who experienced asymptomatic, mild, or severe illness at 2, 5, and 8 months PSO. SARS-CoV-2 spike, nucleocapsid, and membrane protein-stimulated PBMCs were subjected to flow cytometry analysis. RESULTS: A total of 24 patients (7 asymptomatic, 9 with mild disease, and 8 with severe disease) and 6 healthy volunteers were analyzed. SARS-CoV-2-specific OX40+CD137+CD4+ T cells and CD69+CD137+CD8+ T cells persisted at 8 months PSO. Also, antigen-specific cytokine-producing or polyfunctional CD4+ T cells were maintained for up to 8 months PSO. Memory CD4+ T-cell responses tended to be greater in patients who had severe illness than in those with mild or asymptomatic disease. CONCLUSIONS: Memory response to SARS-CoV-2, based on the frequency and functionality, persists for 8 months PSO. Further investigations involving its longevity and protective effect from reinfection are warranted.


Subject(s)
COVID-19/immunology , COVID-19/virology , Host-Pathogen Interactions/immunology , Immunologic Memory , SARS-CoV-2/immunology , T-Lymphocyte Subsets/immunology , Adult , Aged , Antigens, Viral , Biomarkers , COVID-19/diagnosis , COVID-19/epidemiology , Case-Control Studies , Cytokines/metabolism , Disease Management , Epitopes, T-Lymphocyte/immunology , Female , Humans , Immunity, Cellular , Immunophenotyping , Leukocytes, Mononuclear/immunology , Leukocytes, Mononuclear/metabolism , Longitudinal Studies , Male , Middle Aged , Severity of Illness Index , Symptom Assessment , T-Lymphocyte Subsets/metabolism , Time Factors
19.
Mol Cells ; 44(6): 401-407, 2021 Jun 30.
Article in English | MEDLINE | ID: covidwho-1268430

ABSTRACT

Infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes coronavirus disease 2019 (COVID-19), which is an ongoing pandemic disease. SARS-CoV-2-specific CD4+ and CD8+ T-cell responses have been detected and characterized not only in COVID-19 patients and convalescents, but also unexposed individuals. Here, we review the phenotypes and functions of SARS-CoV-2-specific T cells in COVID-19 patients and the relationships between SARS-CoV-2-specific T-cell responses and COVID-19 severity. In addition, we describe the phenotypes and functions of SARS-CoV-2-specific memory T cells after recovery from COVID-19 and discuss the presence of SARS-CoV-2-reactive T cells in unexposed individuals and SARS-CoV-2-specific T-cell responses elicited by COVID-19 vaccines. A better understanding of T-cell responses is important for effective control of the current COVID-19 pandemic.


Subject(s)
Antibodies, Neutralizing/biosynthesis , CD4-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/immunology , COVID-19/immunology , Immunity, Cellular , SARS-CoV-2/pathogenicity , Antibodies, Viral/biosynthesis , CD4-Positive T-Lymphocytes/classification , CD4-Positive T-Lymphocytes/virology , CD8-Positive T-Lymphocytes/classification , CD8-Positive T-Lymphocytes/virology , COVID-19/pathology , COVID-19/prevention & control , COVID-19/virology , COVID-19 Vaccines/administration & dosage , Convalescence , Cytokines/biosynthesis , Cytotoxicity, Immunologic , Host-Pathogen Interactions/immunology , Humans , Immunity, Humoral , Immunologic Memory , Immunophenotyping , SARS-CoV-2/immunology , Severity of Illness Index
20.
Front Immunol ; 12: 665329, 2021.
Article in English | MEDLINE | ID: covidwho-1268251

ABSTRACT

Infection by novel coronavirus SARS-CoV-2 causes different presentations of COVID-19 and some patients may progress to a critical, fatal form of the disease that requires their admission to ICU and invasive mechanical ventilation. In order to predict in advance which patients could be more susceptible to develop a critical form of COVID-19, it is essential to define the most adequate biomarkers. In this study, we analyzed several parameters related to the cellular immune response in blood samples from 109 patients with different presentations of COVID-19 who were recruited in Hospitals and Primary Healthcare Centers in Madrid, Spain, during the first pandemic peak between April and June 2020. Hospitalized patients with the most severe forms of COVID-19 showed a potent inflammatory response that was not translated into an efficient immune response. Despite the high levels of effector cytotoxic cell populations such as NK, NKT and CD8+ T cells, they displayed immune exhaustion markers and poor cytotoxic functionality against target cells infected with pseudotyped SARS-CoV-2 or cells lacking MHC class I molecules. Moreover, patients with critical COVID-19 showed low levels of the highly cytotoxic TCRγδ+ CD8+ T cell subpopulation. Conversely, CD4 count was greatly reduced in association to high levels of Tregs, low plasma IL-2 and impaired Th1 differentiation. The relative importance of these immunological parameters to predict COVID-19 severity was analyzed by Random Forest algorithm and we concluded that the most important features were related to an efficient cytotoxic response. Therefore, efforts to fight against SARS-CoV-2 infection should be focused not only to decrease the disproportionate inflammatory response, but also to elicit an efficient cytotoxic response against the infected cells and to reduce viral replication.


Subject(s)
COVID-19/epidemiology , COVID-19/immunology , Cytotoxicity, Immunologic , Intensive Care Units , Leukocytes, Mononuclear/immunology , Patient Admission/statistics & numerical data , SARS-CoV-2/immunology , Adult , Aged , Aged, 80 and over , Antibodies, Viral/blood , Antibodies, Viral/immunology , Biomarkers , COVID-19/diagnosis , COVID-19/virology , Comorbidity , Cytokines/metabolism , Female , Humans , Immunophenotyping , Leukocytes, Mononuclear/metabolism , Male , Middle Aged , Severity of Illness Index , T-Lymphocyte Subsets/immunology , T-Lymphocyte Subsets/metabolism
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