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BackgroundRecent advances in highly-multiplexed tissue technologies and image analysis tools have enabled a more detailed investigation of the tumor microenvironment (TME) and its spatial features, including tertiary lymphoid structures (TLSs), at single-cell resolution. TLSs play a major part in antitumor immune responses, however, their role in antitumor immunity in ovarian cancer remains largely unexplored.MethodsIn this study, we generated a comprehensive single-cell spatial atlas of TLSs in ovarian cancer by extracting spatial topology information from in-situ highly-multiplexed cellular imaging using tissue cyclic immunofluorescence (CyCIF). Our analysis included 44 patients with high-grade serous ovarian cancer (HGSC) from the TOPACIO Phase II clinical trial. We combined spatial and phenotypic features from 302,545 single-cells with histopathology, targeted sequencing-based tumor molecular groups, and Nanostring gene expression data.ResultsWe find that TLSs are associated with a distinct TME composition and gene expression profile, characterized by elevated levels of the chemokines CCL19, CCL21, and CXCL13 correlating with the number of TLSs in the tumors. Using single-cell feature quantification and spatial mapping, we uncover enriched germinal center (GC) B cell infiltration and selective spatial attraction to follicular helper T and follicular regulatory T cells in the TLSs from chemo-exposed and BRCA1 mutated HGSCs. Importantly, spatial statistics reveal three main groups of cell-to-cell interactions; significantly enriched structural compartments of CD31+ cells, myeloid, and stromal cell types, homotypic cancer cell- and cancer cell to IBA1+ myeloid cell crosstalk, and enriched selective Tfh, Tfr, and Tfc communities with predominant Tfh - GC B cell interactions. Finally, we report spatiotemporal gradients of GC-B cell interactions during TLS maturation, with enriched non-GC B cell attraction towards the GC B cells in early TLSs, and avoidance patterns with selective GC B-cell communities in the TLSs with GCs.ConclusionsOur single-cell multi-omics analyses of TLSs showed evidence of active adaptive immunity with spatial and phenotypic variations among distinct clinical and molecular subtypes of HGSC. Overall, our findings provide new insights into the spatial biology of TLSs and have the potential to improve immunotherapeutic targeting of ovarian cancer.What is already known on this topicTLSs play a major part in antitumor immune responses, however, their exact role and mechanisms in antitumor immunity are widely unexplored.What this study addsOur results deepen the understanding of TLS biology including cell-cell interactions and shows how the presence of TLSs is characterized with a distinct TME composition and gene expression profile.How this study might affect research, practice or policyOverall, our findings provide new insights into the spatial biology of TLSs and have the potential to improve therapeutic options for ovarian cancer.

Though cryopreservation of cell fractions is widely used in flow cytometry studies, whole blood cryopreservation is more challenging due to the presence of erythrocytes and effects of fixatives commonly used for preservation. Here, we evaluated and compared head-to-head the performance of four commercial whole blood cryopreservation kits; (1) Cytodelics, (2) Stable-Lyse V2 and Stable-Store V2 (SLSS-V2), (3) Proteomic stabilizer (PROT-1), and (4) Transfix. We found that PROT-1, Transfix, and Cytodelics maintained the distribution of major leukocyte subsets-granulocytes, T cells, natural killer cells, and B cells, on a comparable level to unpreserved samples, despite the attenuation of fluorescence intensities in flow cytometric assays. Moreover, these three stabilizers also maintained the activated phenotypes of neutrophils upon stimulation with N-formylmethionyl-leucyl-phenylalanine and lipopolysaccharides. The upregulation of adhesion molecules (CD11b), Fc receptors (CD16), and granule proteins (CD66b), as well as the shedding of surface L-selectin (CD62L), was conserved most efficiently in PROT-1 and Cytodelics when compared to samples only treated with erythrocyte lysing. However, none of the stabilizers provided a reliable detection of CCR7 for accurate quantification of T cell maturation stages. We also evaluated the performance of Cytodelics in longitudinal clinical samples obtained from acute COVID-19 patients, where it allowed reliable detection of lymphopenia and granulocyte expansion. These results support the feasibility of whole blood cryopreservation for immunophenotyping by flow cytometry, particularly in longitudinal studies. In conclusion, the performance of different stabilizers is variable and therefore the choice of stabilizers should depend on cell type of interest, as well as antibody clones and experimental design of each study.

Background and Aim Single-cell RNA sequencing (scRNA-seq) is a powerful method utilising transcriptomic data for detailed characterisation of heterogeneous cell populations. The use of oligonucleotide-labelled antibodies for targeted proteomics addresses the shortcomings of the scRNA-seq-only based approach by improving detection of low expressing targets. However, optimisation of large antibody panels is challenging and depends on the availability of co-functioning oligonucleotide-labelled antibodies. Main Methods and Results We present here a simple adjustable oligonucleotide-antibody conjugation method which enables a desired level of oligo-conjugation per antibody. The mean labelling in the produced antibody batches varied from 1 to 6 oligos per antibody. In the scRNA-seq multimodal experiment, the highest sensitivity was seen with moderate antibody labelling as the high activation and/or labelling was detrimental to antibody performance. The conjugates were also tested for compatibility with the fixation and freeze storage protocols. The oligo-antibody signal was stable in fixed cells indicating the feasibility of a stain, fix, store, and analyse later type of workflow for multimodal scRNA-seq. Conclusions and Implications Optimised oligo-labelling will improve detection of weak protein targets in scRNA-seq multimodal experiments and reduce sequencing costs due to a more balanced amplification of different antibody signals in CITE-seq libraries. Furthermore, the use of a pre-stain, fix, run later protocol will allow for flexibility, facilitate sample pooling, and ease logistics in scRNA-seq multimodal experiments.

Funding Information: We thank the nurses of the Transplantation Unit of Helsinki University Central Hospital, HiLife Flow Cytometry Unit of University of Helsinki, and Tamás Bazsinka for technical support. This study has been funded by Emil Aaltonen Foundation, Finnish Medical Foundation, Foundation for Pediatric Research, Biomedicum Helsinki Foundation, Roche Research grant, the MD PhD program of the University of Helsinki, and the Competitive State Research Financing of Responsibility Area of Helsinki University Hospital (TYH2019307). Publisher Copyright: © 2022 The Authors. European Journal of Immunology published by Wiley-VCH GmbH. Long-term T-cell memory is dependent on the maintenance of memory T cells in the lymphoid tissues, and at the surface interfaces that provide entry routes for pathogens. However, much of the current information on human T-cell memory is based on analyzing circulating T cells. Here, we have studied the distribution and age-related changes of memory T-cell subsets in samples from blood, mesenteric LNs, spleen, and ileum, obtained from donors ranging in age from 5 days to 67 years of age. Our data show that the main reservoir of polyclonal naive cells is found in the LNs, and the resting memory subsets capable of self-renewal are also prominent there. In contrast, nondividing but functionally active memory subsets dominate the spleen, and especially the ileum. In general, the replacement of naive cells with memory subsets continues throughout our period of observation, with no apparent plateau. In conclusion, the analysis of lymphoid and nonlymphoid tissues reveals a dynamic pattern of changes distinct to each tissue, and with substantial differences between CD4+ and CD8+ compartments.

Highlights • Female subjects seek SARS-CoV-2 testing more frequently than male subjects. • The positivity rate for SARS-CoV-2 was significantly higher in tested men in Finland. • The proportion of SARS-CoV-2-positive young adults seemed to increase in late May., Objectives The aim was to characterise age- and sex-specific severe acute respiratory syndrome coronavirus disease-2 (SARS-CoV-2) RT-PCR sampling frequency and positivity rate in Greater Helsinki area in Finland during February–June 2020. We also describe the laboratory capacity building for these diagnostics. Methods Laboratory registry data for altogether 80,791 specimens from 70,517 individuals was analysed. The data included the date of sampling, sex, age and the SARS-CoV-2 RT-PCR test result on specimens collected between 1 February and 15 June 2020. Results Altogether, 4057/80,791 (5.0%) of the specimens were positive and 3915/70,517 (5.6%) of the individuals were found positive. In all, 37% of specimens were from male and 67% from female subjects. While the number of positive cases was similar in male and female subjects, the positivity rate was significantly higher in male subjects: 7.5% of male and 4.4% of female subjects tested positive. The highest incidence/100,000 was observed in those aged ≥80 years. The proportion of young adults in positive cases increased in late May 2020. Large dips in testing frequency were observed during every weekend and also during public holidays. Conclusions Our data suggest that men pursue SARS-CoV-2 testing less frequently than women. Consequently, a subset of coronavirus disease-2019 infections in men may have gone undetected. People sought testing less frequently on weekends and public holidays, and this may also lead to missing of positive cases. The proportion of young adults in positive cases increased towards the end of the study period, which may suggest their returning back to social behaviour with an increased risk of infection.

Lymphocyte responses to mitogens constitute a key part of the diagnostics of combined immunodeficiency (CID). Currently, mostly radioactive thymidine incorporation and carboxyfluorescein diacetate succinimidyl ester (CFSE) dilution methods are used. Flow-cytometric Assay for Specific Cell-mediated Immune-response in Activated whole blood (FASCIA) has been put forth as an easy-to-perform option for the measurement of lymphocyte responses with the advantage of recognising different lymphocyte subtypes and avoiding the use of radioactive reagents. Our aim was to analyse retrospectively the usefulness of FASCIA in the diagnostics of CID. We included all lymphocyte stimulation tests done with FASCIA in HUSLAB (Helsinki, Finland) between February 2015 and September 2018 in our analysis. The cohort was divided into two groups by the patients’ final diagnoses: CID (n = 30) or non-CID (n = 159). We evaluated the stimulation responses with a combined FASCIA-score (the average of all mitogen responses). The FASCIA-score was significantly lower among the CID group compared to the other patients (p = 0.002) and in the ROC-analysis the AUC was 0.75 (p p = 0.001). Immunosuppressive medication affected the FASCIA-result significantly and needs to be considered when evaluating results. In conclusion, FASCIA can reliably detect the CID patients from a population tested for suspected immunodeficiency in the absence of immunosuppressive medication. It emerges as a method with many benefits compared to tests requiring radioactive reagents or the complicated CFSE staining.

The ability of thymic histopathology to predict the long-term impact of thymectomy in non-thymomatous myasthenia gravis (NTMG) is mainly uncharted. We applied digital pathology to quantitatively characterize differences of thymic histology between early-onset (EOMG) and late-onset MG (LOMG) and to investigate the role of thymic changes for thymectomy outcomes in MG. We analyzed 83 thymic H&E slides from thymectomized NTMG patients, of which 69 had EOMG and 14 LOMG, using digital pathology open-access software QuPath. We compared the results to the retrospectively assessed clinical outcome at two years after thymectomy and at the last follow-up visit where complete stable remission and minimal use of medication were primary outcomes. The automated annotation pipeline was an effective and reliable way to analyze thymic H&E samples compared to manual annotation with mean intraclass correlation of 0.80. The ratio of thymic tissue to stroma and fat was increased in EOMG compared to LOMG (p = 8.7e-07), whereas no difference was observed in the ratio of medulla to cortex between these subtypes. AChRAb seropositivity correlated with the number of ectopic germinal centers (eGC; p = 0.00067) but not with other histological areas. Patients with an increased number of eGCs had better post-thymectomy outcomes at two years after thymectomy (p = 0.0035) and at the last follow-up (p = 0.0267). ROC analysis showed that eGC area predicts thymectomy outcome in EOMG with an AUC of 0.79. Digital pathology can thus help in providing a predictive tool to the clinician, the eGC number, to guide the post-thymectomy treatment decisions in EOMG patients.

Background Patients with haematological malignancies have an increased susceptibility for COVID-19 and higher mortality. They may also have prolonged symptoms and viral shedding. Clinical trials have not specifically addressed the management of this patient group. We present a lymphoma patient with COVID-19 who was treated with remdesivir, and a literature review of similar cases. Methods SARS-CoV-2 RT-PCR, virus culture and whole-genome sequencing were performed from nasopharyngeal swabs and antibody testing from serum. In addition, SARS-CoV-2 nucleocapsid antigen was tested from serum. Medline was searched for reported cases of lymphoma and COVID-19 treated with remdesivir. Results The patient was undergoing lymphoma treatment including chemotherapy, rituximab and prednisolone. After diagnosis of COVID-19, broad-spectrum antibiotics were administered due to neutropenia and fever. After 20 d of fever with no signs of co-infection, remdesivir was initiated with rapid response. The treatment was continued for 4 d. Serum SARS-CoV-2 antibody tests were negative 20, 30 and 66 d from symptom onset. Before starting remdesivir, the SARS-CoV-2 PCR and virus culture from the nasopharynx and serum antigen test were positive. From earlier reports, we identified a total of eleven cases of lymphoma and COVID-19 treated with remdesivir accompanied by other antivirals and anti-inflammatory agents. Conclusions As shown in this and earlier reports on lymphoma patients, the clinical course of COVID-19 may be protracted and a humoral immune response may remain absent. In addition, optimal management remains undecided. The presented patient responded well to a short course of remdesivir.

The Ikaros family transcription factors regulate lymphocyte development. Loss-of-function variants in

Almost all patients with autoimmune polyendocrine syndrome type 1 (APS-1) have neutralizing antibodies against type 1 interferons (IFN), important mediators of antiviral defense. Recently, neutralizing anti-IFN antibodies were shown to be a risk factor of severe COVID-19. Here we show in a cohort of 44 patients with APS-1 that higher titers of neutralizing anti-IFN alpha 4 antibodies are associated with a higher and earlier incidence of VZV reactivation (herpes zoster). The patients also present with uncommonly severe clinical sequelae of herpetic infections. APS-1 patients had decreased humoral immune responses to varicella zoster virus, but cellular responses were comparable to healthy controls. These results suggest that blocking the type I interferon pathway in patients with APS-1 patients leads to a clinically significant immune deficiency, and susceptibility to herpesviruses should be taken into account when treating patients with APS-1.

The IKAROS family transcription factors regulate lymphocyte development. Loss-of-function variants in IKZF1 cause primary immunodeficiency, but IKAROS family members IKZF2 and IKZF3 have not yet been associated with immunodeficiency yet. Here, we describe a pedigree with a heterozygous truncating variant in IKZF2, encoding the translational activator and repressor HELIOS which is highly expressed in regulatory T cells and effector T cells, particularly of the CD8+ T cell lineage. Protein-protein interaction analysis revealed that the variant abolished HELIOS dimerizations as well as binding to members of the Mi-2/NuRD chromatin remodeling complex. Patients carrying the IKZF2 variant presented with a combined immunodeficiency phenotype characterized by recurrent upper respiratory infections, thrush and mucosal ulcers, as well as chronic lymphadenopathy. With extensive immunophenotyping, functional assays, and transcriptional analysis we show that reduced HELIOS expression was associated with chronic T cell activation and increased production of pro-inflammatory cytokines both in effector and regulatory T cells. Lymph node histology from patients indicated dysregulated germinal center reactions. Moreover, affected individuals displayed profoundly reduced circulating MAIT cell numbers. In summary, we show that this novel loss-of-function variant in HELIOS leads to an immunodeficiency with signs of immune overactivation.One sentence summaryTruncating variant of HELIOS causes immunodeficiency with signs of immune overactivation.

Mitigation of the ongoing coronavirus disease 2019 (COVID-19) pandemic requires reliable and accessible laboratory diagnostic services. In this study, the performance of one laboratory-developed test (LDT) and two commercial tests, cobas SARS-CoV-2 (Roche) and Amplidiag COVID-19 (Mobidiag), were evaluated for the detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA in respiratory specimens. A total of 183 specimens collected from suspected COVID-19 patients were studied with all three methods to compare their performance. In relation to the reference standard, which was established as the result obtained by two of the three studied methods, the positive percent agreement was highest for the cobas test (100%), followed by the Amplidiag test and the LDT (98.9%). The negative percent agreement was lowest for the cobas test (89.4%), followed by the Amplidiag test (98.8%), and the highest value was obtained for the LDT (100%). The dilution series of positive specimens, however, suggests significantly higher sensitivity for the cobas assay in comparison with the other two assays, and the low negative percent agreement value may be due to the same reason. In general, all tested assays performed adequately. Clinical laboratories need to be prepared for uninterrupted high-throughput testing during the coming months to mitigate the pandemic. To ensure no interruption, it is critical that clinical laboratories maintain several simultaneous platforms in their SARS-CoV-2 nucleic acid testing.

Persistence of immune memory in humans is a crucial yet poorly understood aspect of immunology. Here we have studied the effect of Puumala hantavirus infection on unrelated, pre-existing immune memory by studying T cell- and antibody responses against toxoid vaccine antigens of diphtheria, tetanus and pertussis in a cohort of 45 patients. We found that tetanus- and pertussis-specific IgG concentrations elevate during acute Puumala virus infection. Increase in vaccine IgG was associated with proliferation of heterologous T cells. Interestingly, increases in tetanus-specific IgG persisted a year after the infection while pertussis-specific IgG declined rapidly; a difference in IgG kinetics resembling the difference seen after vaccination against tetanus and pertussis. These results suggest that persistence of immune memory is facilitated by heterologous boosting of old memory during memory formation against newly encountered antigens. They also show that different toxoid antigens may be treated differently. Our study gives new insight into how immune memory formation may alter pre-existing immune memory, and also shows that heterologous immunity may have an impact on vaccination outcomes. (C) 2021 Elsevier Ltd. All rights reserved.

Finland has had a low incidence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV2) infections as compared to most European countries. Here we report the origins and turnover of SARS-CoV-2 lineages circulating in Finland in 2020. SARS-CoV-2 introduced to Finland in January 2020 and spread rapidly across southern Finland during spring. We observed rapid turnover among Finnish lineages during this period. Clade 20C became the most prevalent among sequenced cases and was replaced by other strains in fall 2020. Bayesian phylogeographic reconstructions suggested 42 independent introductions into Finland during spring 2020, mainly from Italy, Austria, and Spain, which might have been the source for a third of cases. The investigations of the original introductions of SARS-CoV-2 to Finland during the early stages of the pandemic and of the subsequent lineage dynamics could be utilized to assess the role of transboundary movements and effects of early intervention and public health measures.

ImportanceUnderstanding the false negative rates of SARS-CoV-2 RT-PCR testing is pivotal for the management of the COVID-19 pandemic and it has practical implications for patient management in healthcare facilities.ObjectiveTo determine the real-life clinical sensitivity of SARS-CoV-2 RT-PCR testing.DesignA retrospective study on case series from 4 March – 15 April 2020.SettingA population-based study conducted in primary and tertiary care in the Helsinki Capital Region, Finland.ParticipantsAdults who were clinically suspected of SARS-CoV-2 infection and underwent SARS-CoV-2 RT-PCR testing, and who had sufficient data for grading of clinical suspicion of COVID-19 in their medical records were eligible. All 1,194 inpatients admitted to COVID-19 cohort wards during the study period were included. The outpatient cohort of 1,814 individuals was sampled from epidemiological line lists by systematic quasi-random sampling. Altogether 83 eligible outpatients (4.6%) and 3 inpatients (0.3%) were excluded due to insufficient data for grading of clinical suspicion.ExposuresHigh clinical suspicion for COVID-19 was used as the reference standard for the RT-PCR test. Patients were considered to have high clinical suspicion of COVID-19 if the physician in charge recorded the suspicion on clinical grounds, or the patient fulfilled specifically defined clinical and exposure criteria.Main measuresSensitivity of SARS-CoV-2 RT-PCR by using manually curated clinical characteristics as the gold standard.ResultsThe study population included 1,814 outpatients (mean [SD] age, 45.4 [17.2] years; 69.1% women) and 1,194 inpatients (mean [SD] age, 63.2 [18.3] years; 45.2% women). The sensitivity (95% CI) for laboratory confirmed cases, i.e. repeatedly tested patients were as follows: 85.7% (81.5–89.1%) inpatients; 95.5% (92.2–97.5%) outpatients, 89.9% (88.2–92.1%) all. When also patients that were graded as high suspicion but never tested positive were included in the denominator, the following sensitivity values (95% CI) were observed: 67.5% (62.9–71.9%) inpatients; 34.9% (31.4–38.5%) outpatients; 47.3% (44.4–50.3%) all.Conclusions and relevanceThe clinical sensitivity of SARS-CoV-2 RT-PCR testing was only moderate at best. The relatively high false negative rates of SARS-CoV-2 RT-PCR testing need to be accounted for in clinical decision making, epidemiological interpretations and when using RT-PCR as a reference for other tests.Key PointsQuestionWhat is the clinical sensitivity of SARS-CoV-2 RT-PCR test?FindingsIn this population-based retrospective study on medical records of 1,814 outpatients and 1,194 inpatients, the clinical sensitivity of SARS-CoV-2 RT-PCR was 47.3–89.9%.MeaningThe false negative rates of SARS-CoV-2 RT-PCR testing need to be accounted for in clinical decision making, epidemiological interpretations and when using RT-PCR as a reference for other tests.

Published descriptions of the neuropathological features of COVID-19 patients have been controversial, ranging from only modest or no pathology to severe hypoxic and hemorrhagic phenotypes, thrombotic complications, acute disseminated encephalomyelitis-like changes, and encephalitis and meningitis. Here, we describe the neuropathological findings of four COVID-19-positive patients autopsied at the Helsinki University Hospital during the spring of 2020. While three of the patients (age range 63-90) exhibited merely mild to moderate hypoxia-associated changes, one 38-year-old subject with obesity, diabetes (type 2), Parkinson's disease and a very severe clinical course was found to have severe ischemic injury, abundant microhemorrhages and enlarged perivascular spaces most pronounced in the white matter and deep gray matter. The pattern of ischemic changes suggested a defect in microcirculation. In addition, a few small perivascular white matter lesions, with macrophages engulfing myelin, were found. No signs of encephalitis or meningitis were detected in any of the patients. When conducting RT-PCR and immunohistochemical analyses of brain tissue, we could not demonstrate in any of the patients marked injury or presence of SARS-CoV2 in the olfactory epithelium, olfactory bulbs or brain areas responsible for respiratory control. In conclusion, our small autopsy series demonstrates various hypoxia-associated neuropathological features in COVID-19 patients, but no evidence of neurotropism or meningitis/encephalitis.

Transcriptome analysis at a single-cell level with single-cell RNA sequencing (scRNA-seq) is a powerful method for detailed characterization of heterogeneous cell populations. Recent developments have enabled parallel analysis of both transcript and protein levels by using antibodies conjugated to barcoded oligonucleotides. These antibodies enable protein levels to be converted into nucleotide format, allowing the sequencing-based detection of both modalities at single-cell level. Here we present a simple and reliable method for conjugation of oligonucleotides with antibodies and a protocol for their use in single-cell transcriptome sequencing.

Summary Severe disease of SARS-CoV2 is characterized by vigorous inflammatory responses in the lung, often with a sudden onset after 5-7 days of stable disease. Efforts to modulate this hyperinflammation and the associated acute respiratory distress syndrome, rely on the unraveling of the immune cell interactions and cytokines that drive such responses. Given that every patient is captured at different stages of infection, longitudinal monitoring of the immune response is critical and systems-level analyses required to capture cellular interactions. Here we report on a systems-level blood immunomonitoring study of 37 adult patients diagnosed with COVID-19 and followed with up to 14 blood samples from acute to recovery phases of the disease. We describe an IFNγ – Eosinophil axis activated prior to lung hyperinflammation and changes in cell-cell coregulation during different stages of the disease. We also map an immune trajectory during recovery that is shared among patients with severe COVID-19., Graphical Abstract, Highlights Immunomonitoring from acute to recovery phase COVID-19 An IFNg - Eosinophil axis precede lung hyperinflammation Basophils modulate SARS-CoV2 IgG responses A shared trajectory of immunological recovery in COVID-19, Immune dysregulation plays a pivotal role during severe COVID-19. Rodriguez et al present a systems-level, longitudinal study of 37 COVID-19 patients monitored from acute to recovery phases of disease. This study reveals cell-protein dependencies and co-regulated features, as well as a shared immunological trajectory during recovery.

Mitigation of the ongoing coronavirus disease 2019 (COVID-19) pandemic requires reliable and accessible laboratory diagnostic services. In this study, the performance of one laboratory-developed test (LDT) and two commercial tests, cobas SARS-CoV-2 (Roche) and Amplidiag COVID-19 (Mobidiag), were evaluated for the detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA in respiratory specimens. A total of 183 specimens collected from suspected COVID-19 patients were studied with all three methods to compare their performance. In relation to the reference standard, which was established as the result obtained by two of the three studied methods, the positive percent agreement was highest for the cobas test (100%), followed by the Amplidiag test and the LDT (98.9%). The negative percent agreement was lowest for the cobas test (89.4%), followed by the Amplidiag test (98.8%), and the highest value was obtained for the LDT (100%). The dilution series of positive specimens, however, suggests significantly higher sensitivity for the cobas assay in comparison with the other two assays, and the low negative percent agreement value may be due to the same reason. In general, all tested assays performed adequately. Clinical laboratories need to be prepared for uninterrupted high-throughput testing during the coming months to mitigate the pandemic. To ensure no interruption, it is critical that clinical laboratories maintain several simultaneous platforms in their SARS-CoV-2 nucleic acid testing.

Highlights • Microneutralisation test (MNT) was carried out for 62 COVID-19 patients. • Results from six commercial SARS-CoV-2 immunoassays were compared to MNT. • Performance of different immunoassays were variable., There is an urgent need for reliable high-throughput serological assays for the management of the ongoing COVID-19 pandemic. Preferably, the performance of serological tests for a novel virus should be determined with clinical specimens against a gold standard, i.e. virus neutralisation. We compared the performance of six commercial immunoassays for the detection of SARS-COV-2 IgG, IgA and IgM antibodies, including four automated assays [Abbott SARS-COV-2 IgG (CE marked), Diasorin Liaison® SARS-COV-2 S1/S2 IgG (research use only, RUO), and Euroimmun SARS-COV-2 IgG and IgA (CE marked)], and two rapid lateral flow (immunocromatographic) tests [Acro Biotech 2019-nCoV IgG/IgM (CE marked) and Xiamen Biotime Biotechnology SARS-COV-2 IgG/IgM (CE marked)] with a microneutralisation test (MNT). Two specimen panels from serum samples sent to Helsinki University Hospital Laboratory (HUSLAB) were compiled: the patient panel (N=70) included sera from PCR confirmed COVID-19 patients, and the negative panel (N=81) included sera sent for screening of autoimmune diseases and respiratory virus antibodies in 2018 and 2019. The MNT was carried out for all COVID-19 samples (70 serum samples, 62 individuals) and for 53 samples from the negative panel. Forty-one out of 62 COVID-19 patients showed neutralising antibodies.The specificity and sensitivity values of the commercial tests against MNT, respectively, were as follows: 95.1 %/80.5 % (Abbott Architect SARS-CoV-2 IgG), 94.9 %/43.8 % (Diasorin Liaison SARS-CoV-2 IgG; RUO), 68.3 %/87.8 % (Euroimmun SARS-CoV-2 IgA), 86.6 %/70.7 % (Euroimmun SARS-CoV-2 IgG), 74.4 %/56.1 % (Acro 2019-nCoV IgG), 69.5 %/46.3 % (Acro 2019-nCoV IgM), 97.5 %/71.9 % (Xiamen Biotime SARS-CoV-2 IgG), and 88.8 %/81.3 % (Xiamen Biotime SARS-CoV-2 IgM). This study shows variable performance values. Laboratories should carefully consider their testing process, such as a two-tier approach, in order to optimize the overall performance of SARS- CoV-2 serodiagnostics.

Antibody-screening methods to detect severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) need to be validated. We evaluated SARS-CoV-2 IgG and IgA ELISAs in conjunction with the EUROLabworkstation (Euroimmun, Lübeck, Germany). Overall specificities were 91.9% and 73.0% for IgG and IgA ELISAs, respectively. Of 39 coronavirus disease patients, 13 were IgG and IgA positive and 11 IgA alone at sampling. IgGs and IgAs were respectively detected at a median of 12 and 11 days after symptom onset.

Transcriptome analysis at a single-cell level with single-cell RNA sequencing (scRNA-seq) is a powerful method for detailed characterization of heterogeneous cell populations. Recent developments have enabled parallel analysis of both transcript and protein levels by using antibodies conjugated to barcoded oligonucleotides. These antibodies enable protein levels to be converted into nucleotide format, allowing the sequencing-based detection of both modalities at single-cell level. Here we present a simple and reliable method for conjugation of oligonucleotides with antibodies and a protocol for their use in single-cell transcriptome sequencing.

Human adaptive-like “memory” CD56dimCD16+ NK cells in peripheral blood from cytomegalovirus-seropositive individuals have been extensively investigated in recent years and are currently explored as a treatment strategy for hematological cancers. However, treatment of solid tumors remains limited due to insufficient NK cell tumor infiltration, and it is unknown whether large expansions of adaptive-like NK cells that are equipped for tissue-residency and tumor-homing exist in peripheral tissues. Here, we show that human lung and blood contains adaptive-like CD56brightCD16− NK cells with hallmarks of tissue-residency, including expression of CD49a. Expansions of adaptive-like lung tissue-resident (tr)NK cells were found to be present independently of adaptive-like CD56dimCD16+ NK cells and to be hyperresponsive towards target cells. Together, our data demonstrate that phenotypically, functionally, and developmentally distinct subsets of adaptive-like NK cells exist in human lung and blood. Given their tissue-related character and hyperresponsiveness, human lung adaptive-like trNK cells might represent a suitable alternative for therapies targeting solid tumors.

Publisher Copyright: © 2021 Background: Isolation precautions are essential prevent spread of COVID-19 infection but may have a negative impact on inpatient care. The impact of these measures on non-COVID-19 patients remains largely unexplored. Aim: This study aimed to investigate diagnostic and treatment delays related to isolation precautions, the associated patient outcome, and the predisposing risk factors for delays. Methods: This observational study was conducted in seven Helsinki region hospitals during the first wave of the COVID-19 pandemic in Finland. The study used data on all non-COVID-19 inpatients, who were initially isolated due to suspected COVID-19, to estimate whether isolation precautions resulted in diagnostic or treatment delays. Results: Out of 683 non-COVID-19 patients, 33 (4.8%) had delays related to isolation precautions. Clinical condition deteriorated non-fatally in seven (1.0%) patients. The following events were associated with an increased risk of treatment or a diagnostic delay: more than three ward transfers (P = 0.025); referral to an incorrect speciality in the emergency department (P = 0.004); more than three SARS-CoV-2 RT-PCR tests performed (P = 0.022); and where cancer was the final diagnosis (P = 0.018). In contrast, lower respiratory tract symptoms (P = 0.013) decreased the risk. Conclusions: The use of isolation precautions for patients who did not have COVID-19 had minor negative effects on patient outcomes. The present study underlines the importance of targeting diagnostic efforts to patients with unspecified symptoms and to those with a negative SARS-CoV-2 test result. Thorough investigations to achieve an accurate diagnosis improves the prognosis of patients and facilitates appropriate targeting of hospital resources.

This case series investigates the frequency and type of SARS-CoV-2 nasopharyngeal test complications in Helsinki, Finland.

Autoimmune regulator's (AIRE) best characterized role is in the generation immunological tolerance, but it is also involved in many other processes such as spermatogenesis. Loss-of-function mutations in AIRE cause a disease called autoimmune polyendocrinopathy, candidiasis and ectodermal dystrophy (APECED; also called autoimmune polyendocrinopathy syndrome type 1, APS-1) that is dominated by various autoimmune manifestations, mainly endocrinopathies. Both patients with APECED and Aire(-/-) mice suffer from varying levels of infertility, but it is not clear if it is a result of an autoimmune tissue damage or more of a developmental defect. In this study, we wanted to resolve whether or not the reduced fertility of Aire(-/-) mice is dependent on the adaptive immune system and therefore a manifestation of autoimmunity in these mice. We generated lymphopenic mice without Aire expression that were devoid of the autoimmune manifestations previously reported in immunocompetent Aire(-/-) mice. These Aire(-/-) Rag1(-/-) mice regained full fertility. This confirms that the development of infertility in Aire(-/-) mice requires a functional adaptive immune system. We also show that only the male Aire(-/-) mice are subfertile, whereas Aire(-/-) females produce litters normally. Moreover, the male subfertility can be adoptively transferred with lymphocytes from Aire(-/-) donor mice to previously fertile lymphopenic Aire(-/-) recipients. Our data show that subfertility in Aire(-/-) mice is dependent on a functional adaptive immune system thus confirming its autoimmune aetiology.

Mutations in the Autoimmune Regulator (AIRE) gene disrupt thymic T cell development and negative selection, leading to the recessively inherited polyendocrine autoimmune disease APS-1 (autoimmune polyendocrine syndrome type 1). The patients also have a functional defect in the FOXP3+ regulatory T cell population, but its origin is unclear. Here we have used T cell receptor sequencing to analyze the clonal relationship of major CD4+ T cell subsets in 3 patients and 3 healthy controls. The naive regulatory T cells showed little overlap with helper T cell subsets, supporting divergence in the thymus. The activated/memory regulatory T cell subset displayed more sharing with helper T cells, but was mainly recruited from the naive regulatory T cell population. These clonal patterns were very similar in both patients and controls. However, naive regulatory T cells isolated from the patients had a significantly longer T cell receptor complementarity determining region 3 than any other population, suggesting failure of thymic selection. These data indicate that the peripheral differentiation of regulatory T cells in APS-1 patients is not different from that in healthy controls. Rather, the patients’ naive regulatory T cells may have an intrinsic defect imprinted already in the thymus. This article is protected by copyright. All rights reserved.

Human innate lymphoid cells have been described to exist in different organs, with functional deregulation of these cells contributing to several disease states. Here, we performed the first detailed characterization of the phenotype, tissue-residency properties, and functionality of ILC1s, ILC2s, and ILC3s in the human adult and fetal liver. In addition, we investigated changes in the ILC compartment in liver fibrosis. A unique composition of tissue-resident ILCs was observed in nonfibrotic livers as compared with that in mucosal tissues, with NKp44(-) ILC3s accounting for the majority of total intrahepatic ILCs. The frequency of ILC2s, representing a small fraction of ILCs in nonfibrotic livers, increased in liver fibrosis and correlated directly with the severity of the disease. Notably, intrahepatic ILC2s secreted the profibrotic cytokine IL-13 when exposed to IL-33 and thymic stromal lymphopoetin (TSLP); these cytokines were produced by hepatocytes, hepatic stellate cells (HSCs), and Kupffer cells in response to TLR-3 stimulation. In summary, the present results provide the first detailed characterization of intrahepatic ILCs in human adult and fetal liver. The results indicate a role for ILC2s in human liver fibrosis, implying that targeting ILC2s might be a novel therapeutic strategy for its treatment

Autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED) is a rare human autoimmune disorder caused by mutations in the AIRE (autoimmune regulator) gene. Loss of AIRE disrupts thymic negative selection and gives rise to impaired cytotoxic and regulatory T cell populations. To date, CD4(+) T helper (Th) cells remain little studied. This study aims to elucidate their role in APECED pathogenesis.Th cells were explored in ten APECED patients and ten healthy controls using cell culture assays, multiparameter flow cytometry, and transcriptome analysis.The proportions of effector/memory populations were increased while the fraction of naive cells was diminished. The naive population was abnormally activated, with an increased number of cells expressing characteristic Th1, Th2, and Th17 cytokines. No clear deviation to any Th subclass was observed, but transcriptome analysis suggested abnormalities in the Th1 cytokine interferon gamma (IFN-γ) pathway and flow cytometry showed that INF-γ had the highest expression. The augmented INF-γ signaling may promote the function of the putative pathogenic CD8(+) cytotoxic population in the patients. In addition, the frequency of CD4(+) recent thymic emigrants (RTEs) was decreased in the patients, and RTEs also contained cytokine-producing cells at an increased frequency.These data reveal abnormalities in the Th population and suggest that they may in part be traced to premature activation already in the thymus.

In contrast to the extensive knowledge about human natural killer (NK) cells in peripheral blood, relatively little is known about NK cells in the human lung. Knowledge about the composition, differentiation, and function of human lung NK cells is critical to better understand their role in diseases affecting the lung, including asthma, chronic obstructive pulmonary disease, infections, and cancer.We sought to analyze and compare the phenotypic and functional characteristics of NK cells in the human lung and peripheral blood at the single-cell level.NK cells in human lung tissue and matched peripheral blood from 132 subjects were analyzed by using 16-color flow cytometry and confocal microscopy.CD56Here, we characterize the major NK cell populations in the human lung. Our data suggest a model in which the majority of NK cells in the human lung dynamically move between blood and the lung rather than residing in the lung as bona fide tissue-resident CD69

Transcriptional regulator autoimmune regulator (AIRE) controls thymic negative selection but it is also expressed in secondary lymphoid organs. The relative contribution of AIRE's central and peripheral function to the maintenance of tolerance is unclear. We transferred mature lymphocytes from Aire(-/-) or wild-type donors to Aire(+/+) lymphopenic recipients, which allowed us to gauge the autoreactivity inherent in the cells originating in an Aire(-/-) thymus. In the ensuing lymphopenia-induced proliferation (LIP), the recipients of cells from Aire(-/-) showed definite T cell hyperproliferation and developed autoantibodies at a higher frequency than the recipients of wild-type cells. However, neither of the recipient groups developed clinical symptoms, and pathological tissue infiltrates were also absent. The recipients of Aire(-/-) cells showed hyperproliferation and increased accumulation of regulatory T cells (Tregs), especially in tissues susceptible to inflammation triggered by LIP. These data are consistent with the view that T cells developing in the absence of Aire are autoreactive. However, overt autoimmunity was prevented, most likely by the suppressive function of Treg cells in the Aire-sufficient recipients. Our results support the importance of the peripheral AIRE expression in the maintenance of immunological tolerance.

The pathogenetic mechanisms of organ-specific autoimmune diseases remain obscured by the complexity of the genetic and environmental factors participating in the breakdown of tolerance. A unique opportunity to study the pathogenesis of human autoimmunity is provided by autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED), a rare inherited autoimmune disease caused by mutations in Autoimmune Regulator (AIRE) gene. Loss of AIRE function disrupts the deletion of autoreactive T cells and impairs the suppressive function of regulatory T (Treg) cells. Here we show by multiparameter flow cytometry that in healthy controls the peripheral naive Treg cell subset forms a slowly dividing, persistent reservoir of recent thymic emigrants (RTEs). In APECED patients the RTE Treg cells show accelerated turnover and shift to the activated pool and the RTE reservoir is depleted. Moreover, the activated Treg cell population in the patients expresses significantly less Forkhead box protein P3 (FOXP3) than in the healthy controls, consistent with the impairment of peripheral activation. Our results indicate that in addition to their thymic effects, loss-of-function mutations in AIRE disrupt the peripheral homeostasis and activation of Treg cells. This may synergize with failed negative selection to cause APECED.

During T cell development both alleles of the T cell receptor (TCR) alpha locus are rearranged. As a result, a sizeable proportion of T cells can express two distinct TCRs, but the functional significance of this phenomenon remains controversial. Studies on transgenic mice with two TCRs have focused on the risk of immunopathology that such cells may pose, while some have suggested that most dual-specific T cells are nonfunctional or even protective. We tracked the fate and TCR repertoire of single- and dual-specific T cells within a normal polyclonal population undergoing lymphopenia-induced proliferation, a setting which has been shown to cause immunopathology and autoimmunity. After the expansion the repertoire of dual-specific T cells had become highly biased, with both prominent clonal expansions and the complete disappearance of other clones. Our results suggest that the normal repertoire of dual-specific T cells contains both nonfunctional cells and a small, 5% fraction of clones which display a much higher than average affinity to antigens normally tolerated as harmless. This heterogeneity may also help in reconciling some of the earlier, conflicting results.

The timing of thymic regulatory T (Treg) cell commitment remains unclear. Specifically, there is disagreement as to whether the CD4(+)CD8(+) FOXP3(+) thymocytes are precursors of mature CD4(+) FOXP3(+) Treg cells, or an independent Treg cell lineage. We reasoned that precursors should be more susceptible to apoptosis than mature Treg cells, and tested this by growth factor removal and anti-CD3 stimulation. Both treatments resulted in an increase of CD4(+) FOXP3(+) thymocytes, whereas the frequency of CD4(+)CD8(+) FOXP3(+) thymocytes decreased significantly. These changes were accompanied by an increase of annexin(+) apoptotic cells. Both of these FOXP3(+) subsets expressed higher levels of Bcl-2 and BIM than other thymocytes, and while in our setting expression of BIM seemed to predispose the cells to apoptosis, Bcl-2 had no apparent protective effect. These results indicate that CD4(+)CD8(+) FOXP3(+) thymocytes are more susceptible to apoptosis than mature CD4(+) FOXP3(+) Treg cells. This is consistent with the view that they are still immature and thus likely to represent a precursor population.

Recent evidence suggests that regulatory pathways might control sustained high levels of FOXP3 in regulatory CD4(+)CD25(hi) T (T(reg)) cells. Based on transcriptional profiling of ex vivo activated T(reg) and helper CD4(+)CD25(-) T (T(h)) cells we have identified GARP (glycoprotein-A repetitions predominant), LGALS3 (lectin, galactoside-binding, soluble, 3) and LGMN (legumain) as novel genes implicated in human T(reg) cell function, which are induced upon T-cell receptor stimulation. Retroviral overexpression of GARP in antigen-specific T(h) cells leads to an efficient and stable re-programming of an effector T cell towards a regulatory T cell, which involves up-regulation of FOXP3, LGALS3, LGMN and other T(reg)-associated markers. In contrast, overexpression of LGALS3 and LGMN enhance FOXP3 and GARP expression, but only partially induced a regulatory phenotype. Lentiviral down-regulation of GARP in T(reg) cells significantly impaired the suppressor function and was associated with down-regulation of FOXP3. Moreover, down-regulation of FOXP3 resulted in similar phenotypic changes and down-regulation of GARP. This provides compelling evidence for a GARP-FOXP3 positive feedback loop and provides a rational molecular basis for the known difference between natural and transforming growth factor-beta induced T(reg) cells as we show here that the latter do not up-regulate GARP. In summary, we have identified GARP as a key receptor controlling FOXP3 in T(reg) cells following T-cell activation in a positive feedback loop assisted by LGALS3 and LGMN, which represents a promising new system for the therapeutic manipulation of T cells in human disease.