Homozygous DBF4 mutation as a cause of severe congenital neutropenia.

BACKGROUND: Severe congenital neutropenia presents with recurrent infections early in life as a result of 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, coimmunoprecipitation, 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 DBF4 variant 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 wild-type DBF4 into patient CD34+ cells rescued the promyelocyte differentiation arrest. CONCLUSION: Hypomorphic DBF4 mutation causes autosomal-recessive severe congenital neutropenia with syndromic features.

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.

A disease-associated missense mutation in CYP4F3 affects the metabolism of leukotriene B4 via disruption of electron transfer.

BACKGROUND: Cytochrome P450 4F3 (CYP4F3) is an ω-hydroxylase that oxidizes leukotriene B4 (LTB4), prostaglandins, and fatty acid epoxides. LTB4 is synthesized by leukocytes and acts as a chemoattractant for neutrophils, making it an essential component of the innate immune system. Recently, involvement of the LTB4 pathway was reported in various immunological disorders such as asthma, arthritis, and inflammatory bowel disease. We report a 26-year-old female with a complex immune phenotype, mainly marked by exhaustion, muscle weakness, and inflammation-related conditions. The molecular cause is unknown, and symptoms have been aggravating over the years. METHODS: Whole exome sequencing was performed and validated; flow cytometry and enzyme-linked immunosorbent assay were used to describe patient's phenotype. Function and impact of the mutation were investigated using molecular analysis: co-immunoprecipitation, western blot, and enzyme-linked immunosorbent assay. Capillary electrophoresis with ultraviolet detection was used to detect LTB4 and its metabolite and in silico modelling provided structural information. RESULTS: We present the first report of a patient with a heterozygous de novo missense mutation c.C1123 > G;p.L375V in CYP4F3 that severely impairs its activity by 50% (P < 0.0001), leading to reduced metabolization of the pro-inflammatory LTB4. Systemic LTB4 levels (1034.0 ± 75.9 pg/mL) are significantly increased compared with healthy subjects (305.6 ± 57.0 pg/mL, P < 0.001), and immune phenotyping shows increased total CD19+ CD27- naive B cells (25%) and decreased total CD19+ CD27+ IgD- switched memory B cells (19%). The mutant CYP4F3 protein is stable and binding with its electron donors POR and Cytb5 is unaffected (P > 0.9 for both co-immunoprecipitation with POR and Cytb5). In silico modelling of CYP4F3 in complex with POR and Cytb5 suggests that the loss of catalytic activity of the mutant CYP4F3 is explained by a disruption of an α-helix that is crucial for the electron shuffling between the electron carriers and CYP4F3. Interestingly, zileuton still inhibits ex vivo LTB4 production in patient's whole blood to 2% of control (P < 0.0001), while montelukast and fluticasone do not (99% and 114% of control, respectively). CONCLUSIONS: A point mutation in the catalytic domain of CYP4F3 is associated with high leukotriene B4 plasma levels and features of a more naive adaptive immune response. Our data provide evidence for the pathogenicity of the CYP4F3 variant as a cause for the observed clinical features in the patient. Inhibitors of the LTB4 pathway such as zileuton show promising effects in blocking LTB4 production and might be used as a future treatment strategy.

Astrocyte-targeted gene delivery of interleukin 2 specifically increases brain-resident regulatory T cell numbers and protects against pathological neuroinflammation.

The ability of immune-modulating biologics to prevent and reverse pathology has transformed recent clinical practice. Full utility in the neuroinflammation space, however, requires identification of both effective targets for local immune modulation and a delivery system capable of crossing the blood-brain barrier. The recent identification and characterization of a small population of regulatory T (Treg) cells resident in the brain presents one such potential therapeutic target. Here, we identified brain interleukin 2 (IL-2) levels as a limiting factor for brain-resident Treg cells. We developed a gene-delivery approach for astrocytes, with a small-molecule on-switch to allow temporal control, and enhanced production in reactive astrocytes to spatially direct delivery to inflammatory sites. Mice with brain-specific IL-2 delivery were protected in traumatic brain injury, stroke and multiple sclerosis models, without impacting the peripheral immune system. These results validate brain-specific IL-2 gene delivery as effective protection against neuroinflammation, and provide a versatile platform for delivery of diverse biologics to neuroinflammatory patients.

Treatment-Induced BAFF Expression and B Cell Biology in Multiple Sclerosis.

Although fingolimod and interferon-ß are two mechanistically different multiple sclerosis (MS) treatments, they both induce B cell activating factor (BAFF) and shift the B cell pool towards a regulatory phenotype. However, whether there is a shared mechanism between both treatments in how they influence the B cell compartment remains elusive. In this study, we collected a cross-sectional study population of 112 MS patients (41 untreated, 42 interferon-ß, 29 fingolimod) and determined B cell subsets, cell-surface and RNA expression of BAFF-receptor (BAFF-R) and transmembrane activator and cyclophilin ligand interactor (TACI) as well as plasma and/or RNA levels of BAFF, BAFF splice forms and interleukin-10 (IL-10) and -35 (IL-35). We added an in vitro B cell culture with four stimulus conditions (Medium, CpG, BAFF and CpG+BAFF) for untreated and interferon-ß treated patients including measurement of intracellular IL-10 levels. Our flow experiments showed that interferon-ß and fingolimod induced BAFF protein and mRNA expression (P ≤ 3.15 x 10-4) without disproportional change in the antagonizing splice form. Protein BAFF correlated with an increase in transitional B cells (P = 5.70 x 10-6), decrease in switched B cells (P = 3.29 x 10-4), and reduction in B cell-surface BAFF-R expression (P = 2.70 x 10-10), both on TACI-positive and -negative cells. TACI and BAFF-R RNA levels remained unaltered. RNA, plasma and in vitro experiments demonstrated that BAFF was not associated with increased IL-10 and IL-35 levels. In conclusion, treatment-induced BAFF correlates with a shift towards transitional B cells which are enriched for cells with an immunoregulatory function. However, BAFF does not directly influence the expression of the immunoregulatory cytokines IL-10 and IL-35. Furthermore, the post-translational mechanism of BAFF-induced BAFF-R cell surface loss was TACI-independent. These observations put the failure of pharmaceutical anti-BAFF strategies in perspective and provide insights for targeted B cell therapies.

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.

Microglia Require CD4 T Cells to Complete the Fetal-to-Adult Transition.

The brain is a site of relative immune privilege. Although CD4 T cells have been reported in the central nervous system, their presence in the healthy brain remains controversial, and their function remains largely unknown. We used a combination of imaging, single cell, and surgical approaches to identify a CD69+ CD4 T cell population in both the mouse and human brain, distinct from circulating CD4 T cells. The brain-resident population was derived through in situ differentiation from activated circulatory cells and was shaped by self-antigen and the peripheral microbiome. Single-cell sequencing revealed that in the absence of murine CD4 T cells, resident microglia remained suspended between the fetal and adult states. This maturation defect resulted in excess immature neuronal synapses and behavioral abnormalities. These results illuminate a role for CD4 T cells in brain development and a potential interconnected dynamic between the evolution of the immunological and neurological systems. VIDEO ABSTRACT.

Defective Sec61α1 underlies a novel cause of autosomal dominant severe congenital neutropenia.

BACKGROUND: The molecular cause of severe congenital neutropenia (SCN) is unknown in 30% to 50% of patients. SEC61A1 encodes the α-subunit of the Sec61 complex, which governs endoplasmic reticulum protein transport and passive calcium leakage. Recently, mutations in SEC61A1 were reported to be pathogenic in common variable immunodeficiency and glomerulocystic kidney disease. OBJECTIVE: Our aim was to expand the spectrum of SEC61A1-mediated disease to include autosomal dominant SCN. METHODS: Whole exome sequencing findings were validated, and reported mutations were compared by Western blotting, Ca2+ flux assays, differentiation of transduced HL-60 cells, in vitro differentiation of primary CD34 cells, quantitative PCR for unfolded protein response (UPR) genes, and single-cell RNA sequencing on whole bone marrow. RESULTS: We identified a novel de novo missense mutation in SEC61A1 (c.A275G;p.Q92R) in a patient with SCN who was born to nonconsanguineous Belgian parents. The mutation results in diminished protein expression, disturbed protein translocation, and an increase in calcium leakage from the endoplasmic reticulum. In vitro differentiation of CD34+ cells recapitulated the patient's clinical arrest in granulopoiesis. The impact of Q92R-Sec61α1 on neutrophil maturation was validated by using HL-60 cells, in which transduction reduced differentiation into CD11b+CD16+ cells. A potential mechanism for this defect is the uncontrolled initiation of the unfolded protein stress response, with single-cell analysis of primary bone marrow revealing perturbed UPR in myeloid precursors and in vitro differentiation of primary CD34+ cells revealing upregulation of CCAAT/enhancer-binding protein homologous protein and immunoglobulin heavy chain binding protein UPR-response genes. CONCLUSION: Specific mutations in SEC61A1 cause SCN through dysregulation of the UPR.

HLA-E-restricted CD8+ T Lymphocytes Efficiently Control Mycobacterium tuberculosis and HIV-1 Coinfection.

We investigated the contribution of human leukocyte antigen A2 (HLA-A2) and HLA-E-restricted CD8+ T cells in patients with Mycobacterium tuberculosis and human immunodeficiency virus 1 (HIV-1) coinfection. 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, owing to the inability of the virus to downmodulate their expression. Therefore, antigen-specific HLA-E-restricted CD8+ T cells could play a protective role in Mycobacterium tuberculosis and HIV-1 coinfection. 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 were evaluated ex vivo in patients with active tuberculosis and concomitant HIV-1 infection. HIV-1 and Mycobacterium tuberculosis coinfection 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 coinfection, as measured by tetramer staining, but displayed a terminally differentiated and exhausted phenotype that was rescued in vitro by anti-PD-1 (programmed cell death protein 1) monoclonal antibody. Together, these results indicate that HLA-E-restricted and Mycobacterium tuberculosis-specific CD8+ T cells in patients with Mycobacterium tuberculosis/HIV-1 coinfection have an exhausted phenotype and fail to expand in vitro in response to antigen stimulation, which can be restored by blocking the PD-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-1ß 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-1ß as a pivotal cytokine. CONCLUSIONS: NFIL3 mutation can sensitise for arthritis development, in mice and humans, and rewires the innate immune system for IL-1ß over-production.

Genetic Architecture of Adaptive Immune System Identifies Key Immune Regulators.

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.

Detailed characterization of human Mycobacterium tuberculosis specific HLA-E restricted CD8+ T cells.

HLA-E presented antigens are interesting targets for vaccination given HLA-Es' essentially monomorphic nature. We have shown previously that Mycobacterium tuberculosis (Mtb) peptides are presented by HLA-E to CD8+ effector T cells, but the precise phenotype and functional capacity of these cells remains poorly characterized. We have developed and utilized in this study a new protocol combining HLA-E tetramer with intracellular staining for cytokines, transcription factors and cytotoxic molecules to characterize these cells in depth. We confirm in this study the significantly increased ex vivo frequency of Mtb-peptide/HLA-E-TM+ CD8+ T cells in the circulation of patients with active tuberculosis (TB). HLA-E restricted CD8+ T cells from TB patients produced more IL-13 than cells from controls or subjects with latent tuberculosis infection (LTBI). Compared to total CD8+ T cells, HLA-E restricted cells produced more IFNγ, IL-4, IL-10, and granulysin but less granzyme-A. Moreover, compared to "classical" Mtb specific HLA-A2 restricted CD8+ T cells, HLA-E restricted CD8+ T cells produced less TNFα and perforin, but more IL-4. In conclusion, HLA-E restricted- Mtb specific cells can produce Th2 cytokines directly.

Human CD8 T lymphocytes recognize Mycobacterium tuberculosis antigens presented by HLA-E during active tuberculosis and express type 2 cytokines.

CD8 T cells contribute to protective immunity against Mycobacterium tuberculosis. In humans, M. tuberculosis reactive CD8 T cells typically recognize peptides associated to classical MHC class Ia molecules, but little information is available on CD8 T cells recognizing M. tuberculosis Ags presented by nonclassical MHC class Ib molecules. We show here that CD8 T cells from tuberculosis (TB) patients recognize HLA-E-binding M. tuberculosis peptides in a CD3/TCR αß mediated and CD8-dependent manner, and represent an additional type of effector cells playing a role in immune response to M. tuberculosis during active infection. HLA-E-restricted recognition of M. tuberculosis peptides is detectable by a significant enhanced ex vivo frequency of tetramer-specific circulating CD8 T cells during active TB. These CD8 T cells produce type 2 cytokines upon antigenic in vitro stimulation, help B cells for Ab production, and mediate limited TRAIL-dependent cytolytic and microbicidal activity toward M. tuberculosis infected target cells. Our results, together with the finding that HLA-E/M. tuberculosis peptide specific CD8 T cells are detected in TB patients with or without HIV coinfection, suggest that this is a new human T-cell population that participates in immune response in TB.

Functional Signatures of Human CD4 and CD8 T Cell Responses to Mycobacterium tuberculosis.

With 1.4 million deaths and 8.7 million new cases in 2011, tuberculosis (TB) remains a global health care problem and together with HIV and Malaria represents one of the three infectious diseases world-wide. Control of the global TB epidemic has been impaired by the lack of an effective vaccine, by the emergence of drug-resistant forms of Mycobacterium tuberculosis (Mtb) and by the lack of sensitive and rapid diagnostics. It is estimated, by epidemiological reports, that one third of the world's population is latently infected with Mtb, but the majority of infected individuals develop long-lived protective immunity, which controls and contains Mtb in a T cell-dependent manner. Development of TB disease results from interactions among the environment, the host, and the pathogen, and known risk factors include HIV co-infection, immunodeficiency, diabetes mellitus, overcrowding, malnutrition, and general poverty; therefore, an effective T cell response determines whether the infection resolves or develops into clinically evident disease. Consequently, there is great interest in determining which T cells subsets mediate anti-mycobacterial immunity, delineating their effector functions. On the other hand, many aspects remain unsolved in understanding why some individuals are protected from Mtb infection while others go on to develop disease. Several studies have demonstrated that CD4(+) T cells are involved in protection against Mtb, as supported by the evidence that CD4(+) T cell depletion is responsible for Mtb reactivation in HIV-infected individuals. There are many subsets of CD4(+) T cells, such as T-helper 1 (Th1), Th2, Th17, and regulatory T cells (Tregs), and all these subsets co-operate or interfere with each other to control infection; the dominant subset may differ between active and latent Mtb infection cases. Mtb-specific-CD4(+) Th1 cell response is considered to have a protective role for the ability to produce cytokines such as IFN-γ or TNF-α that contribute to the recruitment and activation of innate immune cells, like monocytes and granulocytes. Thus, while other antigen (Ag)-specific T cells such as CD8(+) T cells, natural killer (NK) cells, γδ T cells, and CD1-restricted T cells can also produce IFN-γ during Mtb infection, they cannot compensate for the lack of CD4(+) T cells. The detection of Ag-specific cytokine production by intracellular cytokine staining (ICS) and the use of flow cytometry techniques are a common routine that supports the studies aimed at focusing the role of the immune system in infectious diseases. Flow cytometry permits to evaluate simultaneously the presence of different cytokines that can delineate different subsets of cells as having "multifunctional/polyfunctional" profile. It has been proposed that polyfunctional T cells, are associated with protective immunity toward Mtb, in particular it has been highlighted that the number of Mtb-specific T cells producing a combination of IFN-γ, IL-2, and/or TNF-α may be correlated with the mycobacterial load, while other studies have associated the presence of this particular functional profile as marker of TB disease activity. Although the role of CD8 T cells in TB is less clear than CD4 T cells, they are generally considered to contribute to optimal immunity and protection. CD8 T cells possess a number of anti-microbial effector mechanisms that are less prominent or absent in CD4 Th1 and Th17 T cells. The interest in studying CD8 T cells that are either MHC-class Ia or MHC-class Ib-restricted, has gained more attention. These studies include the role of HLA-E-restricted cells, lung mucosal-associated invariant T-cells (MAIT), and CD1-restricted cells. Nevertheless, the knowledge about the role of CD8(+) T cells in Mtb infection is relatively new and recent studies have delineated that CD8 T cells, which display a functional profile termed "multifunctional," can be a better marker of protection in TB than CD4(+) T cells. Their effector mechanisms could contribute to control Mtb infection, as upon activation, CD8 T cells release cytokines or cytotoxic molecules, which cause apoptosis of target cells. Taken together, the balance of the immune response in the control of infection and possibly bacterial eradication is important in understanding whether the host immune response will be appropriate in contrasting the infection or not, and, consequently, the inability of the immune response, will determine the dissemination and the transmission of bacilli to new subjects. In conclusion, the recent highlights on the role of different functional signatures of T cell subsets in the immune response toward Mtb infection will be discerned in this review, in order to summarize what is known about the immune response in human TB. In particular, we will discuss the role of CD4 and CD8 T cells in contrasting the advance of the intracellular pathogen in already infected people or the progression to active disease in subjects with latent infection. All the information will be aimed at increasing the knowledge of this complex disease in order to improve diagnosis, prognosis, drug treatment, and vaccination.