AutoSpill is a principled framework that simplifies the analysis of multichromatic flow cytometry data.

Compensating in flow cytometry is an unavoidable challenge in the data analysis of fluorescence-based flow cytometry. Even the advent of spectral cytometry cannot circumvent the spillover problem, with spectral unmixing an intrinsic part of such systems. The calculation of spillover coefficients from single-color controls has remained essentially unchanged since its inception, and is increasingly limited in its ability to deal with high-parameter flow cytometry. Here, we present AutoSpill, an alternative method for calculating spillover coefficients. The approach combines automated gating of cells, calculation of an initial spillover matrix based on robust linear regression, and iterative refinement to reduce error. Moreover, autofluorescence can be compensated out, by processing it as an endogenous dye in an unstained control. AutoSpill uses single-color controls and is compatible with common flow cytometry software. AutoSpill allows simpler and more robust workflows, while reducing the magnitude of compensation errors in high-parameter flow cytometry

Increased IL-10-producing regulatory T cells are characteristic of severe cases of COVID-19.

OBJECTIVES: The pandemic spread of the coronavirus SARS-CoV-2 is due, in part, to the immunological properties of the host-virus interaction. The clinical presentation varies from individual to individual, with asymptomatic carriers, mild-to-moderate-presenting patients and severely affected patients. Variation in immune response to SARS-CoV-2 may underlie this clinical variation. METHODS: Using a high-dimensional systems immunology platform, we have analysed the peripheral blood compartment of 6 healthy individuals, 23 mild-to-moderate and 20 severe COVID-19 patients. RESULTS: We identify distinct immunological signatures in the peripheral blood of the mild-to-moderate and severe COVID-19 patients, including T-cell lymphopenia, more consistent with peripheral hypo- than hyper-immune activation. Unique to the severe COVID-19 cases was a large increase in the proportion of IL-10-secreting regulatory T cells, a lineage known to possess anti-inflammatory properties in the lung. CONCLUSION: As IL-10-secreting regulatory T cells are known to possess anti-inflammatory properties in the lung, their proportional increase could contribute to a more severe COVID-19 phenotype. We openly provide annotated data (https://flowrepository.org/experiments/2713) with clinical correlates as a systems immunology resource for the COVID-19 research community

Common variable immunodeficiency in two kindreds with heterogeneous phenotypes caused by novel heterozygous NFKB1 mutations

NFKB1 haploinsufficiengcy was first described in 2015 in three families with common variable immunodeficiency (CVID), presenting heterogeneously with symptoms of increased infectious susceptibility, skin lesions, malignant lymphoproliferation and autoimmunity. The described mutations all led to a rapid degradation of the mutant protein, resulting in a p50 haploinsufficient state. Since then, more than 50 other mutations have been reported, located throughout different domains of NFKB1 with the majority situated in the N-terminal Rel homology domain (RHD). The clinical spectrum has also expanded with possible disease manifestations in almost any organ system. In silico prediction tools are often used to estimate the pathogenicity of NFKB1 variants but to prove causality between disease and genetic findings, further downstream functional validation is required. In this report, we studied 2 families with CVID and two novel variants in NFKB1 (c.1638-2A>G and c.787G>C). Both mutations affected mRNA and/or protein expression of NFKB1 and resulted in excessive NLRP3 inflammasome activation in patient macrophages and upregulated interferon stimulated gene expression. Protein-protein interaction analysis demonstrated a loss of interaction with NFKB1 interaction partners for the p.V263L mutation. In conclusion, we proved pathogenicity of two novel variants in NFKB1 in two families with CVID characterized by variable and incomplete penetrance.Peer reviewe

Common variable immunodeficiency in two kindreds with heterogeneous phenotypes caused by novel heterozygous NFKB1 mutations

NFKB1 haploinsufficiengcy was first described in 2015 in three families with common variable immunodeficiency (CVID), presenting heterogeneously with symptoms of increased infectious susceptibility, skin lesions, malignant lymphoproliferation and autoimmunity. The described mutations all led to a rapid degradation of the mutant protein, resulting in a p50 haploinsufficient state. Since then, more than 50 other mutations have been reported, located throughout different domains of NFKB1 with the majority situated in the N-terminal Rel homology domain (RHD). The clinical spectrum has also expanded with possible disease manifestations in almost any organ system. In silico prediction tools are often used to estimate the pathogenicity of NFKB1 variants but to prove causality between disease and genetic findings, further downstream functional validation is required. In this report, we studied 2 families with CVID and two novel variants in NFKB1 (c.1638-2A>G and c.787G>C). Both mutations affected mRNA and/or protein expression of NFKB1 and resulted in excessive NLRP3 inflammasome activation in patient macrophages and upregulated interferon stimulated gene expression. Protein-protein interaction analysis demonstrated a loss of interaction with NFKB1 interaction partners for the p.V263L mutation. In conclusion, we proved pathogenicity of two novel variants in NFKB1 in two families with CVID characterized by variable and incomplete penetrance.Peer reviewe

Homozygous DBF4 mutation as a cause for severe congenital neutropenia

Background Severe congenital neutropenia presents with recurrent infections early in life due to arrested granulopoiesis. Multiple genetic defects are known to block granulocyte differentiation, however a genetic cause remains unknown in approximately 40% of cases. Objective We aimed to characterize a patient with severe congenital neutropenia and syndromic features without a genetic diagnosis. METHODS: Whole exome sequencing results were validated using flow cytometry, Western blotting, co-immunoprecipitation, quantitative PCR, cell cycle and proliferation analysis of lymphocytes and fibroblasts and granulocytic differentiation of primary CD34+ and HL-60 cells. Results We identified a homozygous missense mutation in DBF4 in a patient with mild extra-uterine growth retardation, facial dysmorphism and severe congenital neutropenia. DBF4 is the regulatory subunit of the CDC7 kinase, together known as DBF4-dependent kinase (DDK), the complex essential for DNA replication initiation. The variant allele demonstrated impaired ability to bind CDC7, resulting in decreased DDK-mediated phosphorylation, defective S phase entry and progression and impaired differentiation of granulocytes associated with activation of the p53-p21 pathway. The introduction of WT DBF4 into patient CD34+ cells rescued the promyelocyte differentiation arrest. Conclusion Hypomorphic DBF4 mutation causes autosomal recessive severe congenital neutropenia with syndromic features

HLA-E-Restricted CD8+ T Lymphocytes Efficiently Control Mycobacterium tuberculosis and HIV-1 Co-Infection

RATIONALE: We have investigated the contribution of HLA-A2 and HLA-E-restricted CD8+ T cells in patients with Mycobacterium tuberculosis and HIV-1 co-infection. OBJECTIVE: HIV-1 downregulates HLA-A, -B and -C molecules in infected cells thus influencing recognition by HLA class I-restricted CD8+ T cells but not by HLA-E restricted CD8+ T cells due to the inability of the virus to downmodulate their expression. Therefore antigen-specific HLA-E-restricted CD8+ T cells could play a protective response in Mycobacterium tuberculosis and HIV-1 co-infection. METHODS: HLA-E- and HLA-A2-restricted Mycobacterium tuberculosis-specific CD8+ T cells were tested in vitro for cytotoxic and microbicidal activities and their frequencies and phenotypes evaluated ex vivo in patients with active tuberculosis and concomitant HIV-1 infection. MEASUREMENTS AND MAIN RESULTS: HIV-1 and Mycobacterium tuberculosis co-infection caused downmodulation of HLA-A2 expression in human monocyte-derived, macrophages associated with resistance to lysis by HLA-A2-restricted CD8+ T cells and failure to restrict the growth of intracellular Mycobacterium tuberculosis. Conversely, HLA-E surface expression and HLA-E-restricted cytolytic and microbicidal CD8 responses were not affected. HLA-E-restricted and Mycobacterium tuberculosis-specific CD8+ T cells were expanded in the circulation of patients with Mycobacterium tuberculosis/HIV-1 co-infection, as measured by tetramer staining, but displayed a terminally-differentiated and exhausted phenotype which was rescued in vitro by anti-programmed death-1 monoclonal antibody. CONCLUSIONS: HLA-E-restricted and Mycobacterium tuberculosis-specific CD8+ T cells in patients with Mycobacterium tuberculosis/HIV-1 co-infection have an exhausted phenotype and fail to expand in vitro in response to antigen stimulation, which can be restored by blocking programmed death-1 pathway using the specific monoclonal antibody Nivolumab

NFIL3 mutations alter immune homeostasis and sensitise for arthritis pathology

OBJECTIVES: NFIL3 is a key immunological transcription factor, with knockout mice studies identifying functional roles in multiple immune cell types. Despite the importance of NFIL3, little is known about its function in humans. METHODS: Here, we characterised a kindred of two monozygotic twin girls with juvenile idiopathic arthritis at the genetic and immunological level, using whole exome sequencing, single cell sequencing and flow cytometry. Parallel studies were performed in a mouse model. RESULTS: The patients inherited a novel p.M170I in NFIL3 from each of the parents. The mutant form of NFIL3 demonstrated reduced stability in vitro. The potential contribution of this mutation to arthritis susceptibility was demonstrated through a preclinical model, where Nfil3-deficient mice upregulated IL-1β production, with more severe arthritis symptoms on disease induction. Single cell sequencing of patient blood quantified the transcriptional dysfunctions present across the peripheral immune system, converging on IL-1β as a pivotal cytokine. CONCLUSIONS: NFIL3 mutation can sensitise for arthritis development, in mice and humans, and rewires the innate immune system for IL-1β over-production.status: publishe

NFIL3 mutations alter immune homeostasis and sensitise for arthritis pathology

Objectives NFIL3 is a key immunological transcription factor, with knockout mice studies identifying functional roles in multiple immune cell types. Despite the importance of NFIL3, little is known about its function in humans. Methods Here, we characterised a kindred of two monozygotic twin girls with juvenile idiopathic arthritis at the genetic and immunological level, using whole exome sequencing, single cell sequencing and flow cytometry. Parallel studies were performed in a mouse model. Results The patients inherited a novel p.M170I in NFIL3 from each of the parents. The mutant form of NFIL3 demonstrated reduced stability in vitro. The potential contribution of this mutation to arthritis susceptibility was demonstrated through a preclinical model, where Nfil3-deficient mice upregulated IL-1β production, with more severe arthritis symptoms on disease induction. Single cell sequencing of patient blood quantified the transcriptional dysfunctions present across the peripheral immune system, converging on IL-1β as a pivotal cytokine. Conclusions NFIL3 mutation can sensitise for arthritis development, in mice and humans, and rewires the innate immune system for IL-1β over-production

Genetic Architecture of Adaptive Immune System Identifies Key Immune Regulators

Summary: The immune system is highly diverse, but characterization of its genetic architecture has lagged behind the vast progress made by genome-wide association studies (GWASs) of emergent diseases. Our GWAS for 54 functionally relevant phenotypes of the adaptive immune system in 489 healthy individuals identifies eight genome-wide significant associations explaining 6%–20% of variance. Coding and splicing variants in PTPRC and COMMD10 are involved in memory T cell differentiation. Genetic variation controlling disease-relevant T helper cell subsets includes RICTOR and STON2 associated with Th2 and Th17, respectively, and the interferon-lambda locus controlling regulatory T cell proliferation. Early and memory B cell differentiation stages are associated with variation in LARP1B and SP4. Finally, the latrophilin family member ADGRL2 correlates with baseline pro-inflammatory interleukin-6 levels. Suggestive associations reveal mechanisms of autoimmune disease associations, in particular related to pro-inflammatory cytokine production. Pinpointing these key human immune regulators offers attractive therapeutic perspectives. : Lagou et al. identify genetic factors explaining interindividual variation in composition of the adaptive immune system. Factors pinpoint key human immune regulators controlling B and T cell differentiation and levels of disease-relevant T helper and regulatory cells. These findings shed light on mechanisms of autoimmune disease and offer therapeutic perspectives. Keywords: adaptive immune system, immune phenotype, genetics, association, genome-wide association, autoimmunity, susceptibilit