MYCOBACTERIAL DISEASE DUE TO INHERITED IFN-G DEFICIENCY
Abstract
Background and Aims
Mendelian susceptibility to mycobacterial disease (MSMD) is characterized by severe infections by BCG vaccines and environmental mycobacteria in otherwise healthy individuals. Isolated or syndromic MSMD is caused by inborn errors of IFN-γ immunity, with mutations in 15 genes that control the production of, or the response to IFN-γ. Whereas a lot of mutations has been reported in IFNGR1 and IFNGR2 genes, mutations in the IFN-γ cytokine itself have not been reported.
Methods
By whole exome-sequencing, we set out to search for mutations in IFNG among patients with unexplained isolated or syndromic MSMD referred to our laboratory.
Results
We identified two Lebanese cousins who are both homozygous for a small deletion c.354_357del mutation in the IFNG gene causing a framesfhit p.T119Ifs4* which generates a premature stop codon. In overexpression system, the mutant allele encodes a truncated protein. T-saimiri lymphocytes had undetectable secretion of IFN-γ, which could be rescued by retrotransduction with WT-IFNG allele. Their primary lymphocytes failed to express and secrete detectable IFN-γ Moreover, IFNG has evolved under purifying selection unlike its two receptors, and suggest that IFNG can be less tolerant to heterozygous mutations than IFNGR1 and IFNGR2.
Conclusions
We thus identified and characterized a novel form of MSMD due to an autosomal recessive, complete IFN-g deficiency. This is the first characterization of a human deficiency in IFN-γ, the key cytokine necessary for the control of Mycobacteria and extends previous results performed in mice.
CLEAVAGE-RESISTANT RIPK1 MUTATIONS CAUSE AUTOINFLAMMATION
Abstract
Background and Aims
Receptor interacting protein kinase 1 (RIPK1) is a critical regulator of immune signaling pathways. Human RIPK1 deficiency has been associated with periodic fever, recurrent infections, arthritis and inflammatory bowel disease, and its pathogenesis has been attributed to attenuated NF-κB activity, dysregulated cell death pathways and increased inflammasome activity. Human RIPK1 is cleaved by caspase protease at p.D324 in vitro; however, the physiological relevance of this cleavage remains to be unclear.
Methods
By whole exome sequencing, we studied the genetic cause of seven individuals from three pedigrees with infantile-onset periodic fever and systemic lymphadenopathy, but without immunodeficiency.
Results
We identified novel heterozygous missense variants (p.D324N, p.D324H and p.D324Y) affecting the caspase cleavage site in affected individuals. In vitrostudies confirmed that these RIPK1 mutations are resistant to cleavage by caspase-6 and caspase-8. Patient derived PBMCs were hyper-responsive to LPS stimulation ex vivo. To further study the mechanism of this disease we generated a cleavage-resistant Rikp1D325A mutant mouse strain. Rikp1D325A/ D325Amice demonstrated embryonic lethality, due to vasculature formation. Embryonic lethality was rescued by combined loss of Casp8 and Ripk3 implicating cell death program in lethality. Rikp1D325A/ D325A and Rikp1D325A/+ MEFs and BMDMs demonstrated enhanced TNF-induced apoptosis and necroptosis. Importantly, similar to our patients heterozygous Rikp1D325A/+mice were viable and demonstrated hyper-responsiveness to inflammatory stimuli in vivo.
Conclusions
Our results reveal the importance of caspase-mediated RIPK1 cleavage for maintenance of inflammation in humans and mice. We designated this novel human disease as ‘Cleavage-resistant RIPK1-Induced Autoinflammatory syndrome’ (CRIA syndrome).
AN ESSENTIAL ROLE FOR THE ZN2+ TRANSPORTER ZIP7 IN B CELL DEVELOPMENT
Abstract
Background and Aims
Agammaglobulinaemia with absent B cells comprises diseases due to mutations in BTK, molecules signalling downstream of the BCR, and components of the BCR itself; in about 5% of cases the genetic cause remains unclear. Here we describe a novel human immunodeficiency, characterised by agammaglobulinaemia and B-lymphopenia, due to hypomorphic mutations in ZIP7, a ubiquitously expressed zinc transporter. whose absence leads to cell death through ER stress.
Methods
By whole exome sequencing, we identified biallelic hypomorphic mutations of ZIP7 in 6 patients affected by agammaglobulinaemia and B-lymphopenia. We introduced one of the identified mutations in mice using CRISPR/Cas9, obtaining a series of alleles around the targeted position, and performed detailed cellular and immunologic analyses.
Results
Mice bearing homozygous null mutation of ZIP7 could not be obtained, implying embryonic lethality. In contrast, animals with hypomorphic ZIP7 function were viable and lacked B cells, phenocopying affected patients. We found reduced levels of cytoplasmic zinc in primary B cell lines derived from mutant mice. Late-pre and immature B cells lacked signatures of ER stress, but showed increased phosphatase activity, reduced signalling downstream of the BCR and failure to progress through the transitional stages to fully mature B cells.
Conclusions
We identify a novel and indispensable role of ZIP7 in B cell development. Intact ZIP7 is necessary to regulate subcellular Zn distribution, reducing phosphatase activity and allowing signalling downstream of the nascent BCR. B cell development is highly dependent on the integrity of BCR signalling; this is the first study describing the role of zinc in this process.
IMPAIRED CONTROL OF MULTIPLE VIRUS INFECTIONS IN A FAMILY WITH COMPLETE IRF9 DEFICIENCY
Abstract
Background and Aims
IRF9 is an integral transcription factor in mediating type-I interferon antiviral-response, as part of ISGF3. Our aim is to report a family where several members showed a striking susceptibility to viral-infection and IRF9-deficient.
Methods
The index patient is a 10yo boy, born at term from healthy, consanguineous-parents. From the first-year of life, he suffered severe viral-infections (bronchiolitis-RSV, disseminated-chickenpox, EV-encephalitis, influenza-A/B, dengue-fever and zika-virus disease). Immunological study showed a mild CD4+T and B-lymphopaenia and hypogammaglobulinaemia. Another remarkable feature is the rapidity with which septic-shock episodes develop.
Results
Genetic analysis revealed a homozygous splicing mutation in IRF9 gene [c.577+1G>T (NM_006084)], which was confirmed in homozygosity in the proband and his sister, and in heterozygosity in both parents. Skipping of exon-5 generated a frameshift and a premature stop codon (p.Glu166LeufsTer80). No expression of IRF9 was observed in: unstimulated or IFNα-stimulated patient-PBMCs and 293T-cells transfected with c.577+1G>T IRF9-construct. After IFNα stimulation or RSV-infection of primary fibroblasts, initial steps of signalling cascade were conserved in IRF9-/-, but no induction of ISGs (MX,IFIT3,ISG15) was observed. In cell lines where IRF9 expression was targeted by CRISPR/Cas9-technology, an impaired induction of ISGs after IFNα treatment was noted and decreased cellular control of viral-replication. In contrast, transfection of patient-cells with wt, but not mutant, IRF9 enhanced their ability to control the replication of various viruses.
Conclusions
The marked susceptibility to virus-infections associated with IRF9-deficiency emphasises that children presenting with unexpectedly severe consequences after either viral infection or vaccination (live-attenuated) should be evaluated bearing in mind the possibility of immune defects affecting IFN-system.
POLYMERASE DELTA DEFICIENCY CAUSES SYNDROMIC IMMUNODEFICIENCY WITH REPLICATIVE STRESS
Abstract
Background and Aims
Polymerase δ is essential for eukaryotic genome maintenance and synthesizes DNA at the leading and the lagging strand. The polymerase δ complex is a heterotetramer comprising the catalytic subunit POLD1 and the accessory subunits POLD2, POLD3 and POLD4. To date, no germline biallelic mutations affecting this complex have been reported in humans.
Methods
The reported patients were genetically diagnosed by exome sequencing or target panel sequencing. The cellular phenotype of primary patient cells was defined using a combination of flow cytometry, immunoblotting and immunofluorescence analysis to assess cell proliferation, cell cycle progression, the activation of DNA repair pathways and the dynamics of replication fork progression. Furthermore, we used overexpression systems to assess mutant stability and interactions as well as recombinant expression of polymerase δ for in vitro assessment of the polymerase enzymatic activity.
Results
Cells from polymerase d-deficient patients showed impaired cell cycle progression and replication-associated DNA lesions, reversible upon overexpression of polymerase δ. The mutations affected the stability and interactions within the polymerase δ complex or its intrinsic polymerase activity.
Conclusions
In patients from two independent pedigrees, we here identify a novel syndrome with reduced functionality of the polymerase d complex caused by germline biallelic mutations in POLD1 or POLD2, as the underlying etiology of a previously unknown, autosomal-recessive syndrome combining replicative stress, neurodevelopmental abnormalities and immunodeficiency. Our discovery of human polymerase δ deficiency identifies the central role of this complex in prevention of replication-related DNA lesions with particular relevance to adaptive immunity.
CONGENITAL MUTATION IN JAK1 UNCOVERS NOVEL TRANSCRIPTIONAL AND REGULATORY PROCESSES
Abstract
Background and Aims
Hastened by new technologies, the study of monogenic immune disease is beginning to resolve unusual phenotypes, genetic complexities and intricate immunologic mechanisms. Here, we describe a patient with severe, early-onset immunodysregulatory disease marked by membranous nephropathy, eosinophilic enteritis, widespread dermatitis and growth disturbances. Whole-exome sequencing uncovered a de novo mutation (S703I) in JAK1, which mediates signal transduction from a remarkably broad range of cytokines by phosphorylation of STAT proteins.
Methods
We utilized genomic, molecular and multi-parametric immunological tools to probe the role of S703I JAK1in vitroand ex vivoin order to investigate clinical dysfunction in vivo.
Results
In vitro, ectopic expression with S703I JAK1 increased basal and cytokine-induced STAT signaling. Surprisingly, S703I could increase signaling independent of its own catalytic activity, indicating that S703I trans-activates partnering JAKs. Next, CyTOF analysis of patient blood revealed upregulated STAT phosphorylation profiles unique to each immune cell. When cytokine-stimulated ex vivo, patient leukocytes exhibited heightened response along some cytokine pathways but not others. Furthering this complexity, patient cells demonstrated non-canonical STAT phosphorylation, suggesting that S703I JAK1 is promiscuous as well as hyperactive. Next, digital droplet PCR and single-cell-RNA-sequencing uncovered genetic mosaicism, monoallelic expression of JAK1, and mutant-cell-enriched interferon signatures. Lastly, JAK inhibitors were evaluated for personalized therapy. After demonstration of efficacy ex vivoand in vitro, tofacitinib was initiated and clinical disease resolved rapidly.
Conclusions
This study of a novel gain-of-function JAK1mutation uncovered novel regulatory and transcriptomic processes governing basic JAK1 function, and reveals the clinical consequences of JAK1 dysfunction.
DOCK8 EXPRESSION IN T CELLS IS ESSENTIAL FOR LFA-1 ACTIVATION AND IN VIVO GENERATION OF GERMINAL CENTER B CELLS
Abstract
Background and Aims
Patients with Dedicator of Cytokinesis 8 (DOCK8) deficiency have recurrent infections and have impaired responses to vaccines, the mechanisms underlying this impairment are incompletely understood. T follicular helper (Tfh) cell migration into germinal centers (GC) is essential for the generation of GC B cells and antibody responses to T dependent (TD) antigens. Tfh cells enter GCs through interactions between LFA-1 on Tfh cells and ICAMs on B cells.
Methods
Dock8-/- and Cd4-CreTg/Dock8flox/flox mice were immunized in the hock with TNP-KLH. Tfh and GC B cell in the draining LNs were enumerated by flow cytometry. Adhesion of activated T cells to immobilized ICAM-1 was measured at a flow rate of 0.75 dynes/cm2. PBMCs from patients and controls were activated with anti-CD3, and activated LFA-1 content was determined by binding of the m24 antibody.
Results
DOCK8 deficient mice and mice with a selective DOCK8 deficiency in T cells generated a poor IgG antibody response to TD antigens and have impaired GC formation. While these mice have normal numbers of Tfh cells, they have very low numbers of GC B cells. This is despite normal conjugate formation between DOCK8 deficient T cells and WT B cells, and normal ability of DOCK8 deficient Tfh cells to drive B cell differentiation in vitro. Following TCR/CD3 ligation, T cells from both DOCK8 deficient patients and mice either failed to activate LFA-1 or had impaired binding to ICAM-1.
Conclusions
DOCK8 expression in T cells is important for LFA-1 activation, normal development of GC B cells, and TD antibody responses.
IMMUNODEFICIENCY AND EPSTEIN BARR VIRUS INDUCED LYMPHOPROLIFERATION CAUSED BY 4-1BB DEFICIENCY
Abstract
Background and Aims
The tumor necrosis family factor receptor (TNFR) family member 4-1BB (CD137) is encoded by TNFRSF9 and expressed on activated T cells. 4-1BB provides a co-stimulatory signal that enhances CD8+ T cell survival, cytotoxicity, and mitochondrial activity, thereby promoting immunity against viruses and tumors. The ligand for 4-1BB (4-1BBL) is expressed on antigen-presenting cells and Epstein-Barr virus (EBV) transformed B cells. We investigated the genetic basis of recurrent sino-pulmonary infections, persistent EBV viremia, and EBV-induced lymphoproliferation in two unrelated patients.
Methods
Whole exome sequencing, immunoblotting, immunophenotyping, and in vitro assays of lymphocyte and mitochondrial function were performed.
Results
The two patients shared a homozygous G109S missense mutation in 4-1BB that abolished protein expression and ligand binding. Patient 1 presented with EBV driven haemophagocytic lymphohistiocytosis. Patient 2 presented with relapsing EBV associated lymphomas. The patients’ CD8+ T cells had reduced proliferation, impaired expression of interferon-γ (IFN-γ) and perforin, and diminished cytotoxicity against allogeneic and HLA matched EBV-B cells. Mitochondrial biogenesis, membrane potential, and function were significantly reduced in the patients’ activated T cells. An inhibitory antibody against 4-1BB recapitulated the patients’ defective CD8+ T cell activation and cytotoxicity against EBV-infected B cells in vitro.
Conclusions
This novel immunodeficiency demonstrates the critical role of 4-1BB co-stimulation in host immunity against EBV infection.
GAIN-OF-FUNCTION CEBPE MUTATION CAUSES NON-CANONICAL AUTOINFLAMMATORY INFLAMMASOMOPATHY
Abstract
Background and Aims
C/EBPε is a transcription factor involved in late myeloid lineage differentiation and cellular function previously known to cause neutrophil-specific granule deficiency. To molecularly characterize the effects of C/EBPε transcription factor gene’s p.Arg219His mutation in an autoinflammatory and immunodeficiency syndrome.
Methods
Genetic analysis, proteomics, genome-wide transcriptional profiling by RNAsequencing, ChIP-sequencing and assessment of the inflammasome function of primary macrophages were performed.
Results
Studies revealed a novel mechanism of genome-wide gain-of-function that dysregulated transcription of 464 genes. Mechanisms involved dysregulated non-canonical inflammasome activation due to decreased association with transcriptional repressors leading to increased chromatin occupancy and considerable changes in transcriptional activity, including increased expression of NLRP3 and constitutively expressed caspase-5 in macrophages.
Conclusions
We describe a novel autoinflammatory disease with defective neutrophil function caused by homozygous p.Arg219His mutation in transcription factor CEBPE. Mutated CEBPE acts as a regulator of both the inflammasome and interferome, and the Arg219His mutation causes the first human monogenic neomorphic and non-canonical inflammasomopathy/immunodeficiency. The same mutation causes both LOF and GOF, i.e. there is variation-of-function (VOF). This widely dysregulated transcription is likely not unique for C/EBPε. Similar multiomics approaches should be utilized when studying other transcription factor-associated diseases.
HUMAN DEF6 DEFICIENCY CAUSES AN IMMUNODEFICIENCY SYNDROME WITH SYSTEMIC AUTOIMMUNITY AND ABERRANT CTLA-4 DYNAMICS
Abstract
Background and Aims
DEF6 is a guanine nucleotide exchange factor acting downstream of T-cell receptor stimulation. While murine studies suggest a role in immune homeostasis and possibly in the onset of autoimmunity, DEF6 deficiency has not yet been attributed with human disease.
Methods
In three patients from two unrelated kindreds with systemic autoimmune manifestations including lymphocytic organ infiltrates, splenomegaly, bowel inflammation, autoimmune hemolytic anemia, and intermittent lymphopenic episodes, we applied homozygosity mapping and whole-exome sequencing to identify biallelic missense variants in DEF6. We investigated immune cell functions and employed cell biological and biochemical assays to characterize functional DEF6 deficiency in T cells.
Results
We identified impaired regulation of the immune checkpoint protein CTLA-4 as mechanistic consequence underlying human DEF6 deficiency, causing autoimmune presentation. Patient T cells exhibited impaired regulation of CTLA-4 vesicular trafficking associated with reduced CTLA-4 surface availability. Cycling defects were replicated in DEF6-knockout cell lines. We identified the small GTPase and endosomal recycling marker RAB11 as a novel interactor and in vitro substrate of DEF6, and found disrupted binding of mutant DEF6 to RAB11. RAB11+CTLA-4+ recycling vesicles were absent in DEF6-mutated cells. CTLA-4-Ig treatment was initiated for one of the patients, achieving sustained remission.
Conclusions
We uncovered DEF6 as player in human immune homeostasis ensuring availability of the checkpoint protein CTLA-4 at T-cell surfaces, with potential significance for autoimmune and/or cancer therapy.