GIMAP5 deficiency reveals a mammalian ceramide-driven longevity assurance pathway.

Preserving cells in a functional, non-senescent state is a major goal for extending human healthspans. Model organisms reveal that longevity and senescence are genetically controlled, but how genes control longevity in different mammalian tissues is unknown. Here, we report a new human genetic disease that causes cell senescence, liver and immune dysfunction, and early mortality that results from deficiency of GIMAP5, an evolutionarily conserved GTPase selectively expressed in lymphocytes and endothelial cells. We show that GIMAP5 restricts the pathological accumulation of long-chain ceramides (CERs), thereby regulating longevity. GIMAP5 controls CER abundance by interacting with protein kinase CK2 (CK2), attenuating its ability to activate CER synthases. Inhibition of CK2 and CER synthase rescues GIMAP5-deficient T cells by preventing CER overaccumulation and cell deterioration. Thus, GIMAP5 controls longevity assurance pathways crucial for immune function and healthspan in mammals.

Serological responses to human virome define clinical outcomes of Italian patients infected with SARS-CoV-2.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for the pandemic respiratory infectious disease COVID-19. However, clinical manifestations and outcomes differ significantly among COVID-19 patients, ranging from asymptomatic to extremely severe, and it remains unclear what drives these disparities. Here, we studied 159 sequentially enrolled hospitalized patients with COVID-19-associated pneumonia from Brescia, Italy using the VirScan phage-display method to characterize circulating antibodies binding to 96,179 viral peptides encoded by 1,276 strains of human viruses. SARS-CoV-2 infection was associated with a marked increase in immune antibody repertoires against many known pathogenic and non-pathogenic human viruses. This antiviral antibody response was linked to longitudinal trajectories of disease severity and was further confirmed in additional 125 COVID-19 patients from the same geographical region in Northern Italy. By applying a machine-learning-based strategy, a viral exposure signature predictive of COVID-19-related disease severity linked to patient survival was developed and validated. These results provide a basis for understanding the role of memory B-cell repertoire to viral epitopes in COVID-19-related symptoms and suggest that a unique anti-viral antibody repertoire signature may be useful to define COVID-19 clinical severity.

Homozygous IL37 mutation associated with infantile inflammatory bowel disease.

Interleukin (IL)-37, an antiinflammatory IL-1 family cytokine, is a key suppressor of innate immunity. IL-37 signaling requires the heterodimeric IL-18R1 and IL-1R8 receptor, which is abundantly expressed in the gastrointestinal tract. Here we report a 4-mo-old male from a consanguineous family with a homozygous loss-of-function IL37 mutation. The patient presented with persistent diarrhea and was found to have infantile inflammatory bowel disease (I-IBD). Patient cells showed increased intracellular IL-37 expression and increased proinflammatory cytokine production. In cell lines, mutant IL-37 was not stably expressed or properly secreted and was thus unable to functionally suppress proinflammatory cytokine expression. Furthermore, induced pluripotent stem cell-derived macrophages from the patient revealed an activated macrophage phenotype, which is more prone to lipopolysaccharide and IL-1ß stimulation, resulting in hyperinflammatory tumor necrosis factor production. Insights from this patient will not only shed light on monogenic contributions of I-IBD but may also reveal the significance of the IL-18 and IL-37 axis in colonic homeostasis.

Human interleukin-2 receptor ß mutations associated with defects in immunity and peripheral tolerance.

Interleukin-2, which conveys essential signals for immunity, operates through a heterotrimeric receptor. Here we identify human interleukin-2 receptor (IL-2R) ß chain (IL2RB) gene defects as a cause of life-threatening immune dysregulation. We report three homozygous mutations in the IL2RB gene of eight individuals from four consanguineous families that cause disease by distinct mechanisms. Nearly all patients presented with autoantibodies, hypergammaglobulinemia, bowel inflammation, dermatological abnormalities, lymphadenopathy, and cytomegalovirus disease. Patient T lymphocytes lacked surface expression of IL-2Rß and were unable to respond to IL-2 stimulation. By contrast, natural killer cells retained partial IL-2Rß expression and function. IL-2Rß loss of function was recapitulated in a recombinant system in which IL2RB mutations caused reduced surface expression and IL-2 binding. Stem cell transplant ameliorated clinical symptoms in one patient; forced expression of wild-type IL-2Rß also increased the IL-2 responsiveness of patient T lymphocytes in vitro. Insights from these patients can inform the development of IL-2-based therapeutics for immunological diseases and cancer.

A Unique Heterozygous CARD11 Mutation Combines Pathogenic Features of Both Gain- and Loss-of-Function Patients in a Four-Generation Family.

CARD11 is a lymphocyte-specific scaffold molecule required for proper activation of B- and T-cells in response to antigen. Germline gain-of-function (GOF) mutations in the CARD11 gene cause a unique B cell lymphoproliferative disorder known as B cell Expansion with NF-κB and T cell Anergy (BENTA). In contrast, patients carrying loss-of-function (LOF), dominant negative (DN) CARD11 mutations present with severe atopic disease. Interestingly, both GOF and DN CARD11 variants cause primary immunodeficiency, with recurrent bacterial and viral infections, likely resulting from impaired adaptive immune responses. This report describes a unique four-generation family harboring a novel heterozygous germline indel mutation in CARD11 (c.701-713delinsT), leading to one altered amino acid and a deletion of 4 others (p.His234_Lys238delinsLeu). Strikingly, affected members exhibit both moderate B cell lymphocytosis and atopic dermatitis/allergies. Ectopic expression of this CARD11 variant stimulated constitutive NF-κB activity in T cell lines, similar to other BENTA patient mutations. However, unlike other GOF mutants, this variant significantly impeded the ability of wild-type CARD11 to induce NF-κB activation following antigen receptor ligation. Patient lymphocytes display marked intrinsic defects in B cell differentiation and reduced T cell responsiveness in vitro. Collectively, these data imply that a single heterozygous CARD11 mutation can convey both GOF and DN signaling effects, manifesting in a blended BENTA phenotype with atopic features. Our findings further emphasize the importance of balanced CARD11 signaling for normal immune responses.

Corrigendum: Germline hypomorphic CARD11 mutations in severe atopic disease.

This corrects the article DOI: 10.1038/ng.3898.

Germline hypomorphic CARD11 mutations in severe atopic disease.

Few monogenic causes for severe manifestations of common allergic diseases have been identified. Through next-generation sequencing on a cohort of patients with severe atopic dermatitis with and without comorbid infections, we found eight individuals, from four families, with novel heterozygous mutations in CARD11, which encodes a scaffolding protein involved in lymphocyte receptor signaling. Disease improved over time in most patients. Transfection of mutant CARD11 expression constructs into T cell lines demonstrated both loss-of-function and dominant-interfering activity upon antigen receptor-induced activation of nuclear factor-κB and mammalian target of rapamycin complex 1 (mTORC1). Patient T cells had similar defects, as well as low production of the cytokine interferon-γ (IFN-γ). The mTORC1 and IFN-γ production defects were partially rescued by supplementation with glutamine, which requires CARD11 for import into T cells. Our findings indicate that a single hypomorphic mutation in CARD11 can cause potentially correctable cellular defects that lead to atopic dermatitis.

Recurrent rhinovirus infections in a child with inherited MDA5 deficiency.

MDA5 is a cytosolic sensor of double-stranded RNA (ds)RNA including viral byproducts and intermediates. We studied a child with life-threatening, recurrent respiratory tract infections, caused by viruses including human rhinovirus (HRV), influenza virus, and respiratory syncytial virus (RSV). We identified in her a homozygous missense mutation in IFIH1 that encodes MDA5. Mutant MDA5 was expressed but did not recognize the synthetic MDA5 agonist/(ds)RNA mimic polyinosinic-polycytidylic acid. When overexpressed, mutant MDA5 failed to drive luciferase activity from the IFNB1 promoter or promoters containing ISRE or NF-κB sequence motifs. In respiratory epithelial cells or fibroblasts, wild-type but not knockdown of MDA5 restricted HRV infection while increasing IFN-stimulated gene expression and IFN-ß/λ. However, wild-type MDA5 did not restrict influenza virus or RSV replication. Moreover, nasal epithelial cells from the patient, or fibroblasts gene-edited to express mutant MDA5, showed increased replication of HRV but not influenza or RSV. Thus, human MDA5 deficiency is a novel inborn error of innate and/or intrinsic immunity that causes impaired (ds)RNA sensing, reduced IFN induction, and susceptibility to the common cold virus.

CD55 Deficiency, Early-Onset Protein-Losing Enteropathy, and Thrombosis.

BACKGROUND: Studies of monogenic gastrointestinal diseases have revealed molecular pathways critical to gut homeostasis and enabled the development of targeted therapies. METHODS: We studied 11 patients with abdominal pain and diarrhea caused by early-onset protein-losing enteropathy with primary intestinal lymphangiectasia, edema due to hypoproteinemia, malabsorption, and less frequently, bowel inflammation, recurrent infections, and angiopathic thromboembolic disease; the disorder followed an autosomal recessive pattern of inheritance. Whole-exome sequencing was performed to identify gene variants. We evaluated the function of CD55 in patients' cells, which we confirmed by means of exogenous induction of expression of CD55. RESULTS: We identified homozygous loss-of-function mutations in the gene encoding CD55 (decay-accelerating factor), which lead to loss of protein expression. Patients' T lymphocytes showed increased complement activation causing surface deposition of complement and the generation of soluble C5a. Costimulatory function and cytokine modulation by CD55 were defective. Genetic reconstitution of CD55 or treatment with a complement-inhibitory therapeutic antibody reversed abnormal complement activation. CONCLUSIONS: CD55 deficiency with hyperactivation of complement, angiopathic thrombosis, and protein-losing enteropathy (the CHAPLE syndrome) is caused by abnormal complement activation due to biallelic loss-of-function mutations in CD55. (Funded by the National Institute of Allergy and Infectious Diseases and others.).

Combined immunodeficiency and Epstein-Barr virus-induced B cell malignancy in humans with inherited CD70 deficiency.

In this study, we describe four patients from two unrelated families of different ethnicities with a primary immunodeficiency, predominantly manifesting as susceptibility to Epstein-Barr virus (EBV)-related diseases. Three patients presented with EBV-associated Hodgkin's lymphoma and hypogammaglobulinemia; one also had severe varicella infection. The fourth had viral encephalitis during infancy. Homozygous frameshift or in-frame deletions in CD70 in these patients abolished either CD70 surface expression or binding to its cognate receptor CD27. Blood lymphocyte numbers were normal, but the proportions of memory B cells and EBV-specific effector memory CD8+ T cells were reduced. Furthermore, although T cell proliferation was normal, in vitro-generated EBV-specific cytotoxic T cell activity was reduced because of CD70 deficiency. This reflected impaired activation by, rather than effects during killing of, EBV-transformed B cells. Notably, expression of 2B4 and NKG2D, receptors implicated in controlling EBV infection, on memory CD8+ T cells from CD70-deficient individuals was reduced, consistent with their impaired killing of EBV-infected cells. Thus, autosomal recessive CD70 deficiency is a novel cause of combined immunodeficiency and EBV-associated diseases, reminiscent of inherited CD27 deficiency. Overall, human CD70-CD27 interactions therefore play a nonredundant role in T and B cell-mediated immunity, especially for protection against EBV and humoral immunity.

AUTOIMMUNE DISEASE. Patients with LRBA deficiency show CTLA4 loss and immune dysregulation responsive to abatacept therapy.

Mutations in the LRBA gene (encoding the lipopolysaccharide-responsive and beige-like anchor protein) cause a syndrome of autoimmunity, lymphoproliferation, and humoral immune deficiency. The biological role of LRBA in immunologic disease is unknown. We found that patients with LRBA deficiency manifested a dramatic and sustained improvement in response to abatacept, a CTLA4 (cytotoxic T lymphocyte antigen-4)-immunoglobulin fusion drug. Clinical responses and homology of LRBA to proteins controlling intracellular trafficking led us to hypothesize that it regulates CTLA4, a potent inhibitory immune receptor. We found that LRBA colocalized with CTLA4 in endosomal vesicles and that LRBA deficiency or knockdown increased CTLA4 turnover, which resulted in reduced levels of CTLA4 protein in FoxP3(+) regulatory and activated conventional T cells. In LRBA-deficient cells, inhibition of lysosome degradation with chloroquine prevented CTLA4 loss. These findings elucidate a mechanism for CTLA4 trafficking and control of immune responses and suggest therapies for diseases involving the CTLA4 pathway.

Dual proteolytic pathways govern glycolysis and immune competence.

Proteasomes and lysosomes constitute the major cellular systems that catabolize proteins to recycle free amino acids for energy and new protein synthesis. Tripeptidyl peptidase II (TPPII) is a large cytosolic proteolytic complex that functions in tandem with the proteasome-ubiquitin protein degradation pathway. We found that autosomal recessive TPP2 mutations cause recurrent infections, autoimmunity, and neurodevelopmental delay in humans. We show that a major function of TPPII in mammalian cells is to maintain amino acid levels and that TPPII-deficient cells compensate by increasing lysosome number and proteolytic activity. However, the overabundant lysosomes derange cellular metabolism by consuming the key glycolytic enzyme hexokinase-2 through chaperone-mediated autophagy. This reduces glycolysis and impairs the production of effector cytokines, including IFN-γ and IL-1ß. Thus, TPPII controls the balance between intracellular amino acid availability, lysosome number, and glycolysis, which is vital for adaptive and innate immunity and neurodevelopmental health.

DOCK8 regulates lymphocyte shape integrity for skin antiviral immunity.

DOCK8 mutations result in an inherited combined immunodeficiency characterized by increased susceptibility to skin and other infections. We show that when DOCK8-deficient T and NK cells migrate through confined spaces, they develop cell shape and nuclear deformation abnormalities that do not impair chemotaxis but contribute to a distinct form of catastrophic cell death we term cytothripsis. Such defects arise during lymphocyte migration in collagen-dense tissues when DOCK8, through CDC42 and p21-activated kinase (PAK), is unavailable to coordinate cytoskeletal structures. Cytothripsis of DOCK8-deficient cells prevents the generation of long-lived skin-resident memory CD8 T cells, which in turn impairs control of herpesvirus skin infections. Our results establish that DOCK8-regulated shape integrity of lymphocytes prevents cytothripsis and promotes antiviral immunity in the skin.

Somatic reversion in dedicator of cytokinesis 8 immunodeficiency modulates disease phenotype.

BACKGROUND: Autosomal recessive loss-of-function mutations in dedicator of cytokinesis 8 (DOCK8) cause a combined immunodeficiency characterized by atopy, recurrent infections, and cancer susceptibility. A genotype-phenotype explanation for the variable disease expression is lacking. OBJECTIVE: We investigated whether reversions contributed to the variable disease expression. METHODS: Patients followed at the National Institutes of Health's Clinical Center were studied. We performed detailed genetic analyses and intracellular flow cytometry to detect DOCK8 protein expression within lymphocyte subsets. RESULTS: We identified 17 of 34 DOCK8-deficient patients who had germline mutations with variable degrees of reversion caused by somatic repair. Somatic repair of the DOCK8 mutations resulted from second-site mutation, original-site mutation, gene conversion, and intragenic crossover. Higher degrees of reversion were associated with recombination-mediated repair. DOCK8 expression was restored primarily within antigen-experienced T cells or natural killer cells but less so in naive T or B cells. Several patients exhibited multiple different repair events. Patients who had reversions were older and had less severe allergic disease, although infection susceptibility persisted. No patients were cured without hematopoietic cell transplantation. CONCLUSIONS: In patients with DOCK8 deficiency, only certain combinations of germline mutations supported secondary somatic repair. Those patients had an ameliorated disease course with longer survival but still had fatal complications or required hematopoietic cell transplantation. These observations support the concept that some DOCK8-immunodeficient patients have mutable mosaic genomes that can modulate disease phenotype over time.

Autosomal recessive phosphoglucomutase 3 (PGM3) mutations link glycosylation defects to atopy, immune deficiency, autoimmunity, and neurocognitive impairment.

BACKGROUND: Identifying genetic syndromes that lead to significant atopic disease can open new pathways for investigation and intervention in allergy. OBJECTIVE: We sought to define a genetic syndrome of severe atopy, increased serum IgE levels, immune deficiency, autoimmunity, and motor and neurocognitive impairment. METHODS: Eight patients from 2 families with similar syndromic features were studied. Thorough clinical evaluations, including brain magnetic resonance imaging and sensory evoked potentials, were performed. Peripheral lymphocyte flow cytometry, antibody responses, and T-cell cytokine production were measured. Whole-exome sequencing was performed to identify disease-causing mutations. Immunoblotting, quantitative RT-PCR, enzymatic assays, nucleotide sugar, and sugar phosphate analyses, along with matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry of glycans, were used to determine the molecular consequences of the mutations. RESULTS: Marked atopy and autoimmunity were associated with increased T(H)2 and T(H)17 cytokine production by CD4(+) T cells. Bacterial and viral infection susceptibility were noted along with T-cell lymphopenia, particularly of CD8(+) T cells, and reduced memory B-cell numbers. Apparent brain hypomyelination resulted in markedly delayed evoked potentials and likely contributed to neurologic abnormalities. Disease segregated with novel autosomal recessive mutations in a single gene, phosphoglucomutase 3 (PGM3). Although PGM3 protein expression was variably diminished, impaired function was demonstrated by decreased enzyme activity and reduced uridine diphosphate-N-acetyl-D-glucosamine, along with decreased O- and N-linked protein glycosylation in patients' cells. These results define a new congenital disorder of glycosylation. CONCLUSIONS: Autosomal recessive hypomorphic PGM3 mutations underlie a disorder of severe atopy, immune deficiency, autoimmunity, intellectual disability, and hypomyelination.

Mg2+ regulates cytotoxic functions of NK and CD8 T cells in chronic EBV infection through NKG2D.

The magnesium transporter 1 (MAGT1) is a critical regulator of basal intracellular free magnesium (Mg(2+)) concentrations. Individuals with genetic deficiencies in MAGT1 have high levels of Epstein-Barr virus (EBV) and a predisposition to lymphoma. We show that decreased intracellular free Mg(2+) causes defective expression of the natural killer activating receptor NKG2D in natural killer (NK) and CD8(+) T cells and impairs cytolytic responses against EBV. Notably, magnesium supplementation in MAGT1-deficient patients restores intracellular free Mg(2+) and NKG2D while concurrently reducing EBV-infected cells in vivo, demonstrating a link between NKG2D cytolytic activity and EBV antiviral immunity in humans. Moreover, these findings reveal a specific molecular function of free basal intracellular Mg(2+) in eukaryotic cells.

Congenital B cell lymphocytosis explained by novel germline CARD11 mutations.

Nuclear factor-κB (NF-κB) controls genes involved in normal lymphocyte functions, but constitutive NF-κB activation is often associated with B cell malignancy. Using high-throughput whole transcriptome sequencing, we investigated a unique family with hereditary polyclonal B cell lymphocytosis. We found a novel germline heterozygous missense mutation (E127G) in affected patients in the gene encoding CARD11, a scaffolding protein required for antigen receptor (AgR)-induced NF-κB activation in both B and T lymphocytes. We subsequently identified a second germline mutation (G116S) in an unrelated, phenotypically similar patient, confirming mutations in CARD11 drive disease. Like somatic, gain-of-function CARD11 mutations described in B cell lymphoma, these germline CARD11 mutants spontaneously aggregate and drive constitutive NF-κB activation. However, these CARD11 mutants rendered patient T cells less responsive to AgR-induced activation. By reexamining this rare genetic disorder first reported four decades ago, our findings provide new insight into why activating CARD11 mutations may induce B cell expansion and preferentially predispose to B cell malignancy without dramatically perturbing T cell homeostasis.