Helper T cell immunity in humans with inherited CD4 deficiency.

CD4+ T cells are vital for host defense and immune regulation. However, the fundamental role of CD4 itself remains enigmatic. We report seven patients aged 5-61 years from five families of four ancestries with autosomal recessive CD4 deficiency and a range of infections, including recalcitrant warts and Whipple's disease. All patients are homozygous for rare deleterious CD4 variants impacting expression of the canonical CD4 isoform. A shorter expressed isoform that interacts with LCK, but not HLA class II, is affected by only one variant. All patients lack CD4+ T cells and have increased numbers of TCRαß+CD4-CD8- T cells, which phenotypically and transcriptionally resemble conventional Th cells. Finally, patient CD4-CD8- αß T cells exhibit intact responses to HLA class II-restricted antigens and promote B cell differentiation in vitro. Thus, compensatory development of Th cells enables patients with inherited CD4 deficiency to acquire effective cellular and humoral immunity against an unexpectedly large range of pathogens. Nevertheless, CD4 is indispensable for protective immunity against at least human papillomaviruses and Trophyrema whipplei.

Genome-wide detection of human intronic AG-gain variants located between splicing branchpoints and canonical splice acceptor sites.

Human genetic variants that introduce an AG into the intronic region between the branchpoint (BP) and the canonical splice acceptor site (ACC) of protein-coding genes can disrupt pre-mRNA splicing. Using our genome-wide BP database, we delineated the BP-ACC segments of all human introns and found extreme depletion of AG/YAG in the [BP+8, ACC-4] high-risk region. We developed AGAIN as a genome-wide computational approach to systematically and precisely pinpoint intronic AG-gain variants within the BP-ACC regions. AGAIN identified 350 AG-gain variants from the Human Gene Mutation Database, all of which alter splicing and cause disease. Among them, 74% created new acceptor sites, whereas 31% resulted in complete exon skipping. AGAIN also predicts the protein-level products resulting from these two consequences. We performed AGAIN on our exome/genomes database of patients with severe infectious diseases but without known genetic etiology and identified a private homozygous intronic AG-gain variant in the antimycobacterial gene SPPL2A in a patient with mycobacterial disease. AGAIN also predicts a retention of six intronic nucleotides that encode an in-frame stop codon, turning AG-gain into stop-gain. This allele was then confirmed experimentally to lead to loss of function by disrupting splicing. We further showed that AG-gain variants inside the high-risk region led to misspliced products, while those outside the region did not, by two case studies in genes STAT1 and IRF7. We finally evaluated AGAIN on our 14 paired exome-RNAseq samples and found that 82% of AG-gain variants in high-risk regions showed evidence of missplicing. AGAIN is publicly available from https://hgidsoft.rockefeller.edu/AGAIN and https://github.com/casanova-lab/AGAIN.

Human MCTS1-dependent translation of JAK2 is essential for IFN-γ immunity to mycobacteria.

Human inherited disorders of interferon-gamma (IFN-γ) immunity underlie severe mycobacterial diseases. We report X-linked recessive MCTS1 deficiency in men with mycobacterial disease from kindreds of different ancestries (from China, Finland, Iran, and Saudi Arabia). Complete deficiency of this translation re-initiation factor impairs the translation of a subset of proteins, including the kinase JAK2 in all cell types tested, including T lymphocytes and phagocytes. JAK2 expression is sufficiently low to impair cellular responses to interleukin-23 (IL-23) and partially IL-12, but not other JAK2-dependent cytokines. Defective responses to IL-23 preferentially impair the production of IFN-γ by innate-like adaptive mucosal-associated invariant T cells (MAIT) and γδ T lymphocytes upon mycobacterial challenge. Surprisingly, the lack of MCTS1-dependent translation re-initiation and ribosome recycling seems to be otherwise physiologically redundant in these patients. These findings suggest that X-linked recessive human MCTS1 deficiency underlies isolated mycobacterial disease by impairing JAK2 translation in innate-like adaptive T lymphocytes, thereby impairing the IL-23-dependent induction of IFN-γ.

Human IRF1 governs macrophagic IFN-γ immunity to mycobacteria.

Inborn errors of human IFN-γ-dependent macrophagic immunity underlie mycobacterial diseases, whereas inborn errors of IFN-α/ß-dependent intrinsic immunity underlie viral diseases. Both types of IFNs induce the transcription factor IRF1. We describe unrelated children with inherited complete IRF1 deficiency and early-onset, multiple, life-threatening diseases caused by weakly virulent mycobacteria and related intramacrophagic pathogens. These children have no history of severe viral disease, despite exposure to many viruses, including SARS-CoV-2, which is life-threatening in individuals with impaired IFN-α/ß immunity. In leukocytes or fibroblasts stimulated in vitro, IRF1-dependent responses to IFN-γ are, both quantitatively and qualitatively, much stronger than those to IFN-α/ß. Moreover, IRF1-deficient mononuclear phagocytes do not control mycobacteria and related pathogens normally when stimulated with IFN-γ. By contrast, IFN-α/ß-dependent intrinsic immunity to nine viruses, including SARS-CoV-2, is almost normal in IRF1-deficient fibroblasts. Human IRF1 is essential for IFN-γ-dependent macrophagic immunity to mycobacteria, but largely redundant for IFN-α/ß-dependent antiviral immunity.

Anti-GM-CSF Neutralizing Autoantibodies in Colombian Patients with Disseminated Cryptococcosis.

BACKGROUND: Cryptococcosis is a potentially life-threatening fungal disease caused by encapsulated yeasts of the genus Cryptococcus, mostly C. neoformans or C. gattii. Cryptococcal meningitis is the most frequent clinical manifestation in humans. Neutralizing autoantibodies (auto-Abs) against granulocyte-macrophage colony-stimulating factor (GM-CSF) have recently been discovered in otherwise healthy adult patients with cryptococcal meningitis, mostly caused by C. gattii. We hypothesized that three Colombian patients with cryptococcal meningitis caused by C. neoformans in two of them would carry high plasma levels of neutralizing auto-Abs against GM-CSF. METHODS: We reviewed medical and laboratory records, performed immunological evaluations, and tested for anti-cytokine auto-Abs three previously healthy HIV-negative adults with disseminated cryptococcosis. RESULTS: Peripheral blood leukocyte subset levels and serum immunoglobulin concentrations were within the normal ranges. We detected high levels of neutralizing auto-Abs against GM-CSF in the plasma of all three patients. CONCLUSIONS: We report three Colombian patients with disseminated cryptococcosis associated with neutralizing auto-Abs against GM-CSF. Further studies should evaluate the genetic contribution to anti-GM-CSF autoantibody production and the role of the GM-CSF signaling pathway in the immune response to Cryptococcus spp.

Inborn errors of OAS-RNase L in SARS-CoV-2-related multisystem inflammatory syndrome in children.

Multisystem inflammatory syndrome in children (MIS-C) is a rare and severe condition that follows benign COVID-19. We report autosomal recessive deficiencies of OAS1, OAS2, or RNASEL in five unrelated children with MIS-C. The cytosolic double-stranded RNA (dsRNA)-sensing OAS1 and OAS2 generate 2'-5'-linked oligoadenylates (2-5A) that activate the single-stranded RNA-degrading ribonuclease L (RNase L). Monocytic cell lines and primary myeloid cells with OAS1, OAS2, or RNase L deficiencies produce excessive amounts of inflammatory cytokines upon dsRNA or severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) stimulation. Exogenous 2-5A suppresses cytokine production in OAS1-deficient but not RNase L-deficient cells. Cytokine production in RNase L-deficient cells is impaired by MDA5 or RIG-I deficiency and abolished by mitochondrial antiviral-signaling protein (MAVS) deficiency. Recessive OAS-RNase L deficiencies in these patients unleash the production of SARS-CoV-2-triggered, MAVS-mediated inflammatory cytokines by mononuclear phagocytes, thereby underlying MIS-C.

Respiratory viral infections in otherwise healthy humans with inherited IRF7 deficiency.

Autosomal recessive IRF7 deficiency was previously reported in three patients with single critical influenza or COVID-19 pneumonia episodes. The patients' fibroblasts and plasmacytoid dendritic cells produced no detectable type I and III IFNs, except IFN-ß. Having discovered four new patients, we describe the genetic, immunological, and clinical features of seven IRF7-deficient patients from six families and five ancestries. Five were homozygous and two were compound heterozygous for IRF7 variants. Patients typically had one episode of pulmonary viral disease. Age at onset was surprisingly broad, from 6 mo to 50 yr (mean age 29 yr). The respiratory viruses implicated included SARS-CoV-2, influenza virus, respiratory syncytial virus, and adenovirus. Serological analyses indicated previous infections with many common viruses. Cellular analyses revealed strong antiviral immunity and expanded populations of influenza- and SARS-CoV-2-specific memory CD4+ and CD8+ T cells. IRF7-deficient individuals are prone to viral infections of the respiratory tract but are otherwise healthy, potentially due to residual IFN-ß and compensatory adaptive immunity.

Recessive inborn errors of type I IFN immunity in children with COVID-19 pneumonia.

Recessive or dominant inborn errors of type I interferon (IFN) immunity can underlie critical COVID-19 pneumonia in unvaccinated adults. The risk of COVID-19 pneumonia in unvaccinated children, which is much lower than in unvaccinated adults, remains unexplained. In an international cohort of 112 children (<16 yr old) hospitalized for COVID-19 pneumonia, we report 12 children (10.7%) aged 1.5-13 yr with critical (7 children), severe (3), and moderate (2) pneumonia and 4 of the 15 known clinically recessive and biochemically complete inborn errors of type I IFN immunity: X-linked recessive TLR7 deficiency (7 children) and autosomal recessive IFNAR1 (1), STAT2 (1), or TYK2 (3) deficiencies. Fibroblasts deficient for IFNAR1, STAT2, or TYK2 are highly vulnerable to SARS-CoV-2. These 15 deficiencies were not found in 1,224 children and adults with benign SARS-CoV-2 infection without pneumonia (P = 1.2 × 10-11) and with overlapping age, sex, consanguinity, and ethnicity characteristics. Recessive complete deficiencies of type I IFN immunity may underlie ∼10% of hospitalizations for COVID-19 pneumonia in children.

X-linked recessive TLR7 deficiency in ~1% of men under 60 years old with life-threatening COVID-19.

Autosomal inborn errors of type I IFN immunity and autoantibodies against these cytokines underlie at least 10% of critical COVID-19 pneumonia cases. We report very rare, biochemically deleterious X-linked TLR7 variants in 16 unrelated male individuals aged 7 to 71 years (mean: 36.7 years) from a cohort of 1,202 male patients aged 0.5 to 99 years (mean: 52.9 years) with unexplained critical COVID-19 pneumonia. None of the 331 asymptomatically or mildly infected male individuals aged 1.3 to 102 years (mean: 38.7 years) tested carry such TLR7 variants (p = 3.5 × 10-5). The phenotypes of five hemizygous relatives of index cases infected with SARS-CoV-2 include asymptomatic or mild infection (n=2, 5 and 38 years), or moderate (n=1, 5 years), severe (n=1, 27 years), or critical (n=1, 29 years) pneumonia. Two boys (aged 7 and 12 years) from a cohort of 262 male patients with severe COVID-19 pneumonia (mean: 51.0 years) are hemizygous for a deleterious TLR7 variant. The cumulative allele frequency for deleterious TLR7 variants in the male general population is < 6.5x10-4 We also show that blood B cell lines and myeloid cell subsets from the patients do not respond to TLR7 stimulation, a phenotype rescued by wild-type TLR7 The patients' blood plasmacytoid dendritic cells (pDCs) produce low levels of type I IFNs in response to SARS-CoV-2. Overall, X-linked recessive TLR7 deficiency is a highly penetrant genetic etiology of critical COVID-19 pneumonia, in about 1.8% of male patients below the age of 60 years. Human TLR7 and pDCs are essential for protective type I IFN immunity against SARS-CoV-2 in the respiratory tract.

Inherited GATA2 Deficiency Is Dominant by Haploinsufficiency and Displays Incomplete Clinical Penetrance.

PURPOSE: Germline heterozygous mutations of GATA2 underlie a variety of hematological and clinical phenotypes. The genetic, immunological, and clinical features of GATA2-deficient patients with mycobacterial diseases in the familial context remain largely unknown. METHODS: We enrolled 15 GATA2 index cases referred for mycobacterial disease. We describe their genetic and clinical features including their relatives. RESULTS: We identified 12 heterozygous GATA2 mutations, two of which had not been reported. Eight of these mutations were loss-of-function, and four were hypomorphic. None was dominant-negative in vitro, and the GATA2 locus was found to be subject to purifying selection, strongly suggesting a mechanism of haploinsufficiency. Three relatives of index cases had mycobacterial disease and were also heterozygous, resulting in 18 patients in total. Mycobacterial infection was the first clinical manifestation in 11 patients, at a mean age of 22.5 years (range: 12 to 42 years). Most patients also suffered from other infections, monocytopenia, or myelodysplasia. Strikingly, the clinical penetrance was incomplete (32.9% by age 40 years), as 16 heterozygous relatives aged between 6 and 78 years, including 4 older than 60 years, were completely asymptomatic. CONCLUSION: Clinical penetrance for mycobacterial disease was found to be similar to other GATA2 deficiency-related manifestations. These observations suggest that other mechanisms contribute to the phenotypic expression of GATA2 deficiency. A diagnosis of autosomal dominant GATA2 deficiency should be considered in patients with mycobacterial infections and/or other GATA2 deficiency-related phenotypes at any age in life. Moreover, all direct relatives should be genotyped at the GATA2 locus.

Inborn errors of type I IFN immunity in patients with life-threatening COVID-19.

Clinical outcome upon infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) ranges from silent infection to lethal coronavirus disease 2019 (COVID-19). We have found an enrichment in rare variants predicted to be loss-of-function (LOF) at the 13 human loci known to govern Toll-like receptor 3 (TLR3)- and interferon regulatory factor 7 (IRF7)-dependent type I interferon (IFN) immunity to influenza virus in 659 patients with life-threatening COVID-19 pneumonia relative to 534 subjects with asymptomatic or benign infection. By testing these and other rare variants at these 13 loci, we experimentally defined LOF variants underlying autosomal-recessive or autosomal-dominant deficiencies in 23 patients (3.5%) 17 to 77 years of age. We show that human fibroblasts with mutations affecting this circuit are vulnerable to SARS-CoV-2. Inborn errors of TLR3- and IRF7-dependent type I IFN immunity can underlie life-threatening COVID-19 pneumonia in patients with no prior severe infection.

Defective formation of IgA memory B cells, Th1 and Th17 cells in symptomatic patients with selective IgA deficiency.

OBJECTIVE: Selective IgA deficiency (sIgAD) is the most common primary immunodeficiency in Western countries. Patients can suffer from recurrent infections and autoimmune diseases because of a largely unknown aetiology. To increase insights into the pathophysiology of the disease, we studied memory B and T cells and cytokine concentrations in peripheral blood. METHODS: We analysed 30 sIgAD patients (12 children, 18 adults) through detailed phenotyping of peripheral B-cell, CD8+ T-cell and CD4+ T-cell subsets, sequence analysis of IGA and IGG transcripts, in vitro B-cell activation and blood cytokine measurements. RESULTS: All patients had significantly decreased numbers of T-cell-dependent (TD; CD27+) and T-cell-independent (TI; CD27-) IgA memory B cells and increased CD21low B-cell numbers. IgM+IgD- memory B cells were decreased in children and normal in adult patients. IGA and IGG transcripts contained normal SHM levels. In sIgAD children, IGA transcripts more frequently used IGA2 than controls (58.5% vs. 25.1%), but not in adult patients. B-cell activation after in vitro stimulation was normal. However, adult sIgAD patients exhibited increased blood levels of TGF-ß1, BAFF and APRIL, whereas they had decreased Th1 and Th17 cell numbers. CONCLUSION: Impaired IgA memory formation in sIgAD patients is not due to a B-cell activation defect. Instead, decreased Th1 and Th17 cell numbers and high blood levels of BAFF, APRIL and TGF-ß1 might reflect disturbed regulation of IgA responses in vivo.These insights into B-cell extrinsic immune defects suggest the need for a broader immunological focus on genomics and functional analyses to unravel the pathogenesis of sIgAD.

Early-Onset Invasive Infection Due to Corynespora cassiicola Associated with Compound Heterozygous CARD9 Mutations in a Colombian Patient.

PURPOSE: CARD9 deficiency is an inborn error of immunity that predisposes otherwise healthy humans to mucocutaneous and invasive fungal infections, mostly caused by Candida, but also by dermatophytes, Aspergillus, and other fungi. Phaeohyphomycosis are an emerging group of fungal infections caused by dematiaceous fungi (phaeohyphomycetes) and are being increasingly identified in patients with CARD9 deficiency. The Corynespora genus belongs to phaeohyphomycetes and only one adult patient with CARD9 deficiency has been reported to suffer from invasive disease caused by C. cassiicola. We identified a Colombian child with an early-onset, deep, and destructive mucocutaneous infection due to C. cassiicola and we searched for mutations in CARD9. METHODS: We reviewed the medical records and immunological findings in the patient. Microbiologic tests and biopsies were performed. Whole-exome sequencing (WES) was made and Sanger sequencing was used to confirm the CARD9 mutations in the patient and her family. Finally, CARD9 protein expression was evaluated in peripheral blood mononuclear cells (PBMC) by western blotting. RESULTS: The patient was affected by a large, indurated, foul-smelling, and verrucous ulcerated lesion on the left side of the face with extensive necrosis and crusting, due to a C. cassiicola infectious disease. WES led to the identification of compound heterozygous mutations in the patient consisting of the previously reported p.Q289* nonsense (c.865C > T, exon 6) mutation, and a novel deletion (c.23_29del; p.Asp8Alafs10*) leading to a frameshift and a premature stop codon in exon 2. CARD9 protein expression was absent in peripheral blood mononuclear cells from the patient. CONCLUSION: We describe here compound heterozygous loss-of-expression mutations in CARD9 leading to severe deep and destructive mucocutaneous phaeohyphomycosis due to C. cassiicola in a Colombian child.

T-cell defects in patients with ARPC1B germline mutations account for combined immunodeficiency.

ARPC1B is a key factor for the assembly and maintenance of the ARP2/3 complex that is involved in actin branching from an existing filament. Germline biallelic mutations in ARPC1B have been recently described in 6 patients with clinical features of combined immunodeficiency (CID), whose neutrophils and platelets but not T lymphocytes were studied. We hypothesized that ARPC1B deficiency may also lead to cytoskeleton and functional defects in T cells. We have identified biallelic mutations in ARPC1B in 6 unrelated patients with early onset disease characterized by severe infections, autoimmune manifestations, and thrombocytopenia. Immunological features included T-cell lymphopenia, low numbers of naïve T cells, and hyper-immunoglobulin E. Alteration in ARPC1B protein structure led to absent/low expression by flow cytometry and confocal microscopy. This molecular defect was associated with the inability of patient-derived T cells to extend an actin-rich lamellipodia upon T-cell receptor (TCR) stimulation and to assemble an immunological synapse. ARPC1B-deficient T cells additionally displayed impaired TCR-mediated proliferation and SDF1-α-directed migration. Gene transfer of ARPC1B in patients' T cells using a lentiviral vector restored both ARPC1B expression and T-cell proliferation in vitro. In 2 of the patients, in vivo somatic reversion restored ARPC1B expression in a fraction of lymphocytes and was associated with a skewed TCR repertoire. In 1 revertant patient, memory CD8+ T cells expressing normal levels of ARPC1B displayed improved T-cell migration. Inherited ARPC1B deficiency therefore alters T-cell cytoskeletal dynamics and functions, contributing to the clinical features of CID.

Inherited p40phox deficiency differs from classic chronic granulomatous disease.

Biallelic loss-of-function (LOF) mutations of the NCF4 gene, encoding the p40phox subunit of the phagocyte NADPH oxidase, have been described in only 1 patient. We report on 24 p40phox-deficient patients from 12 additional families in 8 countries. These patients display 8 different in-frame or out-of-frame mutations of NCF4 that are homozygous in 11 of the families and compound heterozygous in another. When overexpressed in NB4 neutrophil-like cells and EBV-transformed B cells in vitro, the mutant alleles were found to be LOF, with the exception of the p.R58C and c.120_134del alleles, which were hypomorphic. Particle-induced NADPH oxidase activity was severely impaired in the patients' neutrophils, whereas PMA-induced dihydrorhodamine-1,2,3 (DHR) oxidation, which is widely used as a diagnostic test for chronic granulomatous disease (CGD), was normal or mildly impaired in the patients. Moreover, the NADPH oxidase activity of EBV-transformed B cells was also severely impaired, whereas that of mononuclear phagocytes was normal. Finally, the killing of Candida albicans and Aspergillus fumigatus hyphae by neutrophils was conserved in these patients, unlike in patients with CGD. The patients suffer from hyperinflammation and peripheral infections, but they do not have any of the invasive bacterial or fungal infections seen in CGD. Inherited p40phox deficiency underlies a distinctive condition, resembling a mild, atypical form of CGD.

A purely quantitative form of partial recessive IFN-γR2 deficiency caused by mutations of the initiation or second codon.

Mendelian susceptibility to mycobacterial disease (MSMD) is characterized by clinical disease caused by weakly virulent mycobacteria, such as environmental mycobacteria and Bacillus Calmette-Guérin vaccines, in otherwise healthy individuals. All known genetic etiologies disrupt interferon (IFN)-γ immunity. Germline bi-allelic mutations of IFNGR2 can underlie partial or complete forms of IFN-γ receptor 2 (IFN-γR2) deficiency. Patients with partial IFN-γR2 deficiency express a dysfunctional molecule on the cell surface. We studied three patients with MSMD from two unrelated kindreds from Turkey (P1, P2) and India (P3), by whole-exome sequencing. P1 and P2 are homozygous for a mutation of the initiation codon(c.1A>G) of IFNGR2, whereas P3 is homozygous for a mutation of the second codon (c.4delC). Overexpressed mutant alleles produce small amounts of full-length IFN-γR2 resulting in an impaired, but not abolished, response to IFN-γ. Moreover, SV40-fibroblasts of P1 and P2 responded weakly to IFN-γ, and Epstein Barr virus-transformed B cells had a barely detectable response to IFN-γ. Studies in patients' primary T cells and monocyte-derived macrophages yielded similar results. The residual expression of IFN-γR2 protein of normal molecular weight and function is due to the initiation of translation between the second and ninth non-AUG codons. We thus describe mutations of the first and second codons of IFNGR2, which define a new form of partial recessive IFN-γR2 deficiency. Residual levels of IFN-γ signaling were very low, accounting for the more severe clinical phenotype of these patients with residual expression levels of normally functional surface receptors than of patients with partial recessive IFN-γR2 deficiency due to surface-expressed dysfunctional receptors, whose residual levels of IFN-γ signaling were higher.