Human T-bet Governs Innate and Innate-like Adaptive IFN-γ Immunity against Mycobacteria.

Inborn errors of human interferon gamma (IFN-γ) immunity underlie mycobacterial disease. We report a patient with mycobacterial disease due to inherited deficiency of the transcription factor T-bet. The patient has extremely low counts of circulating Mycobacterium-reactive natural killer (NK), invariant NKT (iNKT), mucosal-associated invariant T (MAIT), and Vδ2+ γδ T lymphocytes, and of Mycobacterium-non reactive classic TH1 lymphocytes, with the residual populations of these cells also producing abnormally small amounts of IFN-γ. Other lymphocyte subsets develop normally but produce low levels of IFN-γ, with the exception of CD8+ αß T and non-classic CD4+ αß TH1∗ lymphocytes, which produce IFN-γ normally in response to mycobacterial antigens. Human T-bet deficiency thus underlies mycobacterial disease by preventing the development of innate (NK) and innate-like adaptive lymphocytes (iNKT, MAIT, and Vδ2+ γδ T cells) and IFN-γ production by them, with mycobacterium-specific, IFN-γ-producing, purely adaptive CD8+ αß T, and CD4+ αß TH1∗ cells unable to compensate for this deficit.

Dissecting human population variation in single-cell responses to SARS-CoV-2.

Humans display substantial interindividual clinical variability after SARS-CoV-2 infection1-3, the genetic and immunological basis of which has begun to be deciphered4. However, the extent and drivers of population differences in immune responses to SARS-CoV-2 remain unclear. Here we report single-cell RNA-sequencing data for peripheral blood mononuclear cells-from 222 healthy donors of diverse ancestries-that were stimulated with SARS-CoV-2 or influenza A virus. We show that SARS-CoV-2 induces weaker, but more heterogeneous, interferon-stimulated gene activity compared with influenza A virus, and a unique pro-inflammatory signature in myeloid cells. Transcriptional responses to viruses display marked population differences, primarily driven by changes in cell abundance including increased lymphoid differentiation associated with latent cytomegalovirus infection. Expression quantitative trait loci and mediation analyses reveal a broad effect of cell composition on population disparities in immune responses, with genetic variants exerting a strong effect on specific loci. Furthermore, we show that natural selection has increased population differences in immune responses, particularly for variants associated with SARS-CoV-2 response in East Asians, and document the cellular and molecular mechanisms by which Neanderthal introgression has altered immune functions, such as the response of myeloid cells to viruses. Finally, colocalization and transcriptome-wide association analyses reveal an overlap between the genetic basis of immune responses to SARS-CoV-2 and COVID-19 severity, providing insights into the factors contributing to current disparities in COVID-19 risk.

Human ancient DNA analyses reveal the high burden of tuberculosis in Europeans over the last 2,000 years.

Tuberculosis (TB), usually caused by Mycobacterium tuberculosis bacteria, is the first cause of death from an infectious disease at the worldwide scale, yet the mode and tempo of TB pressure on humans remain unknown. The recent discovery that homozygotes for the P1104A polymorphism of TYK2 are at higher risk to develop clinical forms of TB provided the first evidence of a common, monogenic predisposition to TB, offering a unique opportunity to inform on human co-evolution with a deadly pathogen. Here, we investigate the history of human exposure to TB by determining the evolutionary trajectory of the TYK2 P1104A variant in Europe, where TB is considered to be the deadliest documented infectious disease. Leveraging a large dataset of 1,013 ancient human genomes and using an approximate Bayesian computation approach, we find that the P1104A variant originated in the common ancestors of West Eurasians ∼30,000 years ago. Furthermore, we show that, following large-scale population movements of Anatolian Neolithic farmers and Eurasian steppe herders into Europe, P1104A has markedly fluctuated in frequency over the last 10,000 years of European history, with a dramatic decrease in frequency after the Bronze Age. Our analyses indicate that such a frequency drop is attributable to strong negative selection starting ∼2,000 years ago, with a relative fitness reduction on homozygotes of 20%, among the highest in the human genome. Together, our results provide genetic evidence that TB has imposed a heavy burden on European health over the last two millennia.

Genome-wide association study of resistance to Mycobacterium tuberculosis infection identifies a locus at 10q26.2 in three distinct populations.

The natural history of tuberculosis (TB) is characterized by a large inter-individual outcome variability after exposure to Mycobacterium tuberculosis. Specifically, some highly exposed individuals remain resistant to M. tuberculosis infection, as inferred by tuberculin skin test (TST) or interferon-gamma release assays (IGRAs). We performed a genome-wide association study of resistance to M. tuberculosis infection in an endemic region of Southern Vietnam. We enrolled household contacts (HHC) of pulmonary TB cases and compared subjects who were negative for both TST and IGRA (n = 185) with infected individuals (n = 353) who were either positive for both TST and IGRA or had a diagnosis of TB. We found a genome-wide significant locus on chromosome 10q26.2 with a cluster of variants associated with strong protection against M. tuberculosis infection (OR = 0.42, 95%CI 0.35-0.49, P = 3.71×10-8, for the genotyped variant rs17155120). The locus was replicated in a French multi-ethnic HHC cohort and a familial admixed cohort from a hyper-endemic area of South Africa, with an overall OR for rs17155120 estimated at 0.50 (95%CI 0.45-0.55, P = 1.26×10-9). The variants are located in intronic regions and upstream of C10orf90, a tumor suppressor gene which encodes an ubiquitin ligase activating the transcription factor p53. In silico analysis showed that the protective alleles were associated with a decreased expression in monocytes of the nearby gene ADAM12 which could lead to an enhanced response of Th17 lymphocytes. Our results reveal a novel locus controlling resistance to M. tuberculosis infection across different populations.

A genome-wide case-only test for the detection of digenic inheritance in human exomes.

Whole-exome sequencing (WES) has facilitated the discovery of genetic lesions underlying monogenic disorders. Incomplete penetrance and variable expressivity suggest a contribution of additional genetic lesions to clinical manifestations and outcome. Some monogenic disorders may therefore actually be digenic. However, only a few digenic disorders have been reported, all discovered by candidate gene approaches applied to at least one locus. We propose here a two-locus genome-wide test for detecting digenic inheritance in WES data. This approach uses the gene as the unit of analysis and tests all pairs of genes to detect pairwise gene × gene interactions underlying disease. It is a case-only method, which has several advantages over classic case-control tests, in particular by avoiding recruitment of controls. Our simulation studies based on real WES data identified two major sources of type I error inflation in this case-only test: linkage disequilibrium and population stratification. Both were corrected by specific procedures. Moreover, our case-only approach is more powerful than the corresponding case-control test for detecting digenic interactions in various population stratification scenarios. Finally, we confirmed the potential of our unbiased, genome-wide approach by successfully identifying a previously reported digenic lesion in patients with craniosynostosis. Our case-only test is a powerful and timely tool for detecting digenic inheritance in WES data from patients.

Homozygosity for TYK2 P1104A underlies tuberculosis in about 1% of patients in a cohort of European ancestry.

The human genetic basis of tuberculosis (TB) has long remained elusive. We recently reported a high level of enrichment in homozygosity for the common TYK2 P1104A variant in a heterogeneous cohort of patients with TB from non-European countries in which TB is endemic. This variant is homozygous in ∼1/600 Europeans and ∼1/5,000 people from other countries outside East Asia and sub-Saharan Africa. We report a study of this variant in the UK Biobank cohort. The frequency of P1104A homozygotes was much higher in patients with TB (6/620, 1%) than in controls (228/114,473, 0.2%), with an odds ratio (OR) adjusted for ancestry of 5.0 [95% confidence interval (CI): 1.96-10.31, P = 2 × 10-3]. Conversely, we did not observe enrichment for P1104A heterozygosity, or for TYK2 I684S or V362F homozygosity or heterozygosity. Moreover, it is unlikely that more than 10% of controls were infected with Mycobacterium tuberculosis, as 97% were of European genetic ancestry, born between 1939 and 1970, and resided in the United Kingdom. Had all of them been infected, the OR for developing TB upon infection would be higher. These findings suggest that homozygosity for TYK2 P1104A may account for ∼1% of TB cases in Europeans.

Developmental changes of upper airway dimensions in children.

BACKGROUND: Knowledge about airway dimensions during child growth is of paramount importance for pediatric clinical practice. Decisions about airway management in children are based on relatively limited, imprecise, or incomplete data about airway size. AIMS: The aim of this work was to determine the anatomical development and size of airway structures from birth to adolescence using high-resolution computed tomography scans and to study the correlation between airway measurements and biometric data. METHODS: We conducted a retrospective study of all high-resolution computed tomography scans including the respiratory tract, performed in our tertiary pediatric center (for reasons unrelated to airway symptoms) between June 1, 2016, and October 15, 2017, on children aged from 1 day to 14 years old. On each scan, 23 measurements of the larynx, trachea, and mainstem bronchi were performed. Patients were stratified into 16 groups according to their age. We calculated median value for each measurement in each group. Statistical models were calculated to explore correlation between measurements and age or weight. RESULTS: A total of 192 scans were included (127 boys/65 girls). The mean age was 7 years. The correlations between airway measurements and age or weight were always significant. The relationship between measurements and age was found to be suitably represented by a cubic polynomial equation suggesting that the airway has a rapid growth phase in the first 3 years, followed by a slow growth phase and a second rapid growth phase during adolescence. The most relevant biometric parameter was age concerning 21 of the measurements. CONCLUSION: This comprehensive anatomical database of upper airway dimensions provides important data in the field of pediatric airway anatomy, particularly relating to the cricoid. We demonstrated that laryngeal, tracheal, and bronchial parameters correlate better to age and have three different growth phases.

A Variety of Alu-Mediated Copy Number Variations Can Underlie IL-12Rß1 Deficiency.

PURPOSE: Inborn errors of IFN-γ immunity underlie Mendelian susceptibility to mycobacterial disease (MSMD). Autosomal recessive complete IL-12Rß1 deficiency is the most frequent genetic etiology of MSMD. Only two of the 84 known mutations are copy number variations (CNVs), identified in two of the 213 IL-12Rß1-deficient patients and two of the 164 kindreds reported. These two CNVs are large deletions found in the heterozygous or homozygous state. We searched for novel families with IL-12Rß1 deficiency due to CNVs. METHODS: We studied six MSMD patients from five unrelated kindreds displaying adverse reactions to BCG vaccination. Three of the patients also presented systemic salmonellosis, two had mucocutaneous candidiasis, and one had disseminated histoplasmosis. We searched for CNVs and other variations by IL12RB1-targeted next-generation sequencing (NGS). RESULTS: We identified six new IL-12Rß1-deficient patients with a complete loss of IL-12Rß1 expression on phytohemagglutinin-activated T cells and/or EBV-transformed B cells. The cells of these patients did not respond to IL-12 and IL-23. Five different CNVs encompassing IL12RB1 (four deletions and one duplication) were identified in these patients by NGS coverage analysis, either in the homozygous state (n = 1) or in trans (n = 4) with a single-nucleotide variation (n = 3) or a small indel (n = 1). Seven of the nine mutations are novel. Interestingly, four of the five CNVs were predicted to be driven by nearby Alu elements, as well as the two previously reported large deletions. The IL12RB1 locus is actually enriched in Alu elements (44.7%), when compared with the rest of the genome (10.5%). CONCLUSION: The IL12RB1 locus is Alu-enriched and therefore prone to rearrangements at various positions. CNVs should be considered in the genetic diagnosis of IL-12Rß1 deficiency.

Genetic adaptation to pathogens and increased risk of inflammatory disorders in post-Neolithic Europe.

Ancient genomics can directly detect human genetic adaptation to environmental cues. However, it remains unclear how pathogens have exerted selective pressures on human genome diversity across different epochs and affected present-day inflammatory disease risk. Here, we use an ancestry-aware approximate Bayesian computation framework to estimate the nature, strength, and time of onset of selection acting on 2,879 ancient and modern European genomes from the last 10,000 years. We found that the bulk of genetic adaptation occurred after the start of the Bronze Age, <4,500 years ago, and was enriched in genes relating to host-pathogen interactions. Furthermore, we detected directional selection acting on specific leukocytic lineages and experimentally demonstrated that the strongest negatively selected candidate variant in immunity genes, lipopolysaccharide-binding protein (LBP) D283G, is hypomorphic. Finally, our analyses suggest that the risk of inflammatory disorders has increased in post-Neolithic Europeans, possibly because of antagonistic pleiotropy following genetic adaptation to pathogens.

The genetic and evolutionary determinants of COVID-19 susceptibility.

Devastating pandemics, such as that due to COVID-19, can provide strong testimony to our knowledge of the genetic and evolutionary determinants of infectious disease susceptibility and severity. One of the most remarkable aspects of such outbreaks is the stunning interindividual variability observed in the course of infection. In recent decades, enormous progress has been made in the field of the human genetics of infectious diseases, and an increasing number of human genetic factors have been reported to explain, to a great extent, the observed variability for a large number of infectious agents. However, our understanding of the cellular, molecular, and immunological mechanisms underlying such disparities between individuals and ethnic groups, remains very limited. Here, we discuss recent findings relating to human genetic predisposition to infectious disease, from an immunological or population genetic perspective, and show how these and other innovative approaches have been applied to deciphering the genetic basis of human susceptibility to COVID-19 and the severity of this disease. From an evolutionary perspective, we show how past demographic and selection events characterizing the history of our species, including admixture with archaic humans, such as Neanderthals, facilitated modern human adaptation to the threats imposed by ancient pathogens. In the context of emerging infectious diseases, these past episodes of genetic adaptation may contribute to some of the observed population differences in the outcome of SARS-CoV-2 infection and the severity of COVID-19 illness.

New insights into human immunity from ancient genomics.

Population genetic studies have clearly indicated that immunity and host defense are among the functions most frequently subject to natural selection, and increased our understanding of the biological relevance of the corresponding genes and their contribution to variable immune traits and diseases. Herein, we will focus on some recently studied forms of human adaptation to infectious agents, including hybridization with now-extinct hominins, such as Neanderthals and Denisovans, and admixture between modern human populations. These studies, which are partly enabled by the technological advances in the sequencing of DNA from ancient remains, provide new insight into the sources of immune response variation in contemporary humans, such as the recently reported link between Neanderthal heritage and susceptibility to severe COVID-19 disease. Furthermore, ancient DNA analyses, in both humans and pathogens, allow to measure the action of natural selection on immune genes across time and to reconstruct the impact of past epidemics on the evolution of human immunity.

Detection of homozygous and hemizygous complete or partial exon deletions by whole-exome sequencing.

The detection of copy number variations (CNVs) in whole-exome sequencing (WES) data is important, as CNVs may underlie a number of human genetic disorders. The recently developed HMZDelFinder algorithm can detect rare homozygous and hemizygous (HMZ) deletions in WES data more effectively than other widely used tools. Here, we present HMZDelFinder_opt, an approach that outperforms HMZDelFinder for the detection of HMZ deletions, including partial exon deletions in particular, in WES data from laboratory patient collections that were generated over time in different experimental conditions. We show that using an optimized reference control set of WES data, based on a PCA-derived Euclidean distance for coverage, strongly improves the detection of HMZ complete exon deletions both in real patients carrying validated disease-causing deletions and in simulated data. Furthermore, we develop a sliding window approach enabling HMZDelFinder_opt to identify HMZ partial deletions of exons that are undiscovered by HMZDelFinder. HMZDelFinder_opt is a timely and powerful approach for detecting HMZ deletions, particularly partial exon deletions, in WES data from inherently heterogeneous laboratory patient collections.

Inherited PD-1 deficiency underlies tuberculosis and autoimmunity in a child.

The pathophysiology of adverse events following programmed cell death protein 1 (PD-1) blockade, including tuberculosis (TB) and autoimmunity, remains poorly characterized. We studied a patient with inherited PD-1 deficiency and TB who died of pulmonary autoimmunity. The patient's leukocytes did not express PD-1 or respond to PD-1-mediated suppression. The patient's lymphocytes produced only small amounts of interferon (IFN)-γ upon mycobacterial stimuli, similarly to patients with inborn errors of IFN-γ production who are vulnerable to TB. This phenotype resulted from a combined depletion of Vδ2+ γδ T, mucosal-associated invariant T and CD56bright natural killer lymphocytes and dysfunction of other T lymphocyte subsets. Moreover, the patient displayed hepatosplenomegaly and an expansion of total, activated and RORγT+ CD4-CD8- double-negative αß T cells, similar to patients with STAT3 gain-of-function mutations who display lymphoproliferative autoimmunity. This phenotype resulted from excessive amounts of STAT3-activating cytokines interleukin (IL)-6 and IL-23 produced by activated T lymphocytes and monocytes, and the STAT3-dependent expression of RORγT by activated T lymphocytes. Our work highlights the indispensable role of human PD-1 in governing both antimycobacterial immunity and self-tolerance, while identifying potentially actionable molecular targets for the diagnostic and therapeutic management of TB and autoimmunity in patients on PD-1 blockade.

Inherited human IFN-γ deficiency underlies mycobacterial disease.

Mendelian susceptibility to mycobacterial disease (MSMD) is characterized by a selective predisposition to clinical disease caused by the Bacille Calmette-Guérin (BCG) vaccine and environmental mycobacteria. The known genetic etiologies of MSMD are inborn errors of IFN-γ immunity due to mutations of 15 genes controlling the production of or response to IFN-γ. Since the first MSMD-causing mutations were reported in 1996, biallelic mutations in the genes encoding IFN-γ receptor 1 (IFN-γR1) and IFN-γR2 have been reported in many patients of diverse ancestries. Surprisingly, mutations of the gene encoding the IFN-γ cytokine itself have not been reported, raising the remote possibility that there might be other agonists of the IFN-γ receptor. We describe 2 Lebanese cousins with MSMD, living in Kuwait, who are both homozygous for a small deletion within the IFNG gene (c.354_357del), causing a frameshift that generates a premature stop codon (p.T119Ifs4*). The mutant allele is loss of expression and loss of function. We also show that the patients' herpesvirus Saimiri-immortalized T lymphocytes did not produce IFN-γ, a phenotype that can be rescued by retrotransduction with WT IFNG cDNA. The blood T and NK lymphocytes from these patients also failed to produce and secrete detectable amounts of IFN-γ. Finally, we show that human IFNG has evolved under stronger negative selection than IFNGR1 or IFNGR2, suggesting that it is less tolerant to heterozygous deleterious mutations than IFNGR1 or IFNGR2. This may account for the rarity of patients with autosomal-recessive, complete IFN-γ deficiency relative to patients with complete IFN-γR1 and IFN-γR2 deficiencies.

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.

Autoantibodies against type I IFNs in patients with life-threatening COVID-19.

Interindividual clinical variability in the course of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is vast. We report that at least 101 of 987 patients with life-threatening coronavirus disease 2019 (COVID-19) pneumonia had neutralizing immunoglobulin G (IgG) autoantibodies (auto-Abs) against interferon-ω (IFN-ω) (13 patients), against the 13 types of IFN-α (36), or against both (52) at the onset of critical disease; a few also had auto-Abs against the other three type I IFNs. The auto-Abs neutralize the ability of the corresponding type I IFNs to block SARS-CoV-2 infection in vitro. These auto-Abs were not found in 663 individuals with asymptomatic or mild SARS-CoV-2 infection and were present in only 4 of 1227 healthy individuals. Patients with auto-Abs were aged 25 to 87 years and 95 of the 101 were men. A B cell autoimmune phenocopy of inborn errors of type I IFN immunity accounts for life-threatening COVID-19 pneumonia in at least 2.6% of women and 12.5% of men.

Severe influenza pneumonitis in children with inherited TLR3 deficiency.

Autosomal recessive IRF7 and IRF9 deficiencies impair type I and III IFN immunity and underlie severe influenza pneumonitis. We report three unrelated children with influenza A virus (IAV) infection manifesting as acute respiratory distress syndrome (IAV-ARDS), heterozygous for rare TLR3 variants (P554S in two patients and P680L in the third) causing autosomal dominant (AD) TLR3 deficiency. AD TLR3 deficiency can underlie herpes simplex virus-1 (HSV-1) encephalitis (HSE) by impairing cortical neuron-intrinsic type I IFN immunity to HSV-1. TLR3-mutated leukocytes produce normal levels of IFNs in response to IAV. In contrast, TLR3-mutated fibroblasts produce lower levels of IFN-ß and -λ, and display enhanced viral susceptibility, upon IAV infection. Moreover, the patients' iPSC-derived pulmonary epithelial cells (PECs) are susceptible to IAV. Treatment with IFN-α2b or IFN-λ1 rescues this phenotype. AD TLR3 deficiency may thus underlie IAV-ARDS by impairing TLR3-dependent, type I and/or III IFN-mediated, PEC-intrinsic immunity. Its clinical penetrance is incomplete for both IAV-ARDS and HSE, consistent with their typically sporadic nature.

Human SNORA31 variations impair cortical neuron-intrinsic immunity to HSV-1 and underlie herpes simplex encephalitis.

Herpes simplex virus-1 (HSV-1) encephalitis (HSE) is typically sporadic. Inborn errors of TLR3- and DBR1-mediated central nervous system cell-intrinsic immunity can account for forebrain and brainstem HSE, respectively. We report five unrelated patients with forebrain HSE, each heterozygous for one of four rare variants of SNORA31, encoding a small nucleolar RNA of the H/ACA class that are predicted to direct the isomerization of uridine residues to pseudouridine in small nuclear RNA and ribosomal RNA. We show that CRISPR/Cas9-introduced bi- and monoallelic SNORA31 deletions render human pluripotent stem cell (hPSC)-derived cortical neurons susceptible to HSV-1. Accordingly, SNORA31-mutated patient hPSC-derived cortical neurons are susceptible to HSV-1, like those from TLR3- or STAT1-deficient patients. Exogenous interferon (IFN)-ß renders SNORA31- and TLR3- but not STAT1-mutated neurons resistant to HSV-1. Finally, transcriptome analysis of SNORA31-mutated neurons revealed normal responses to TLR3 and IFN-α/ß stimulation but abnormal responses to HSV-1. Human SNORA31 thus controls central nervous system neuron-intrinsic immunity to HSV-1 by a distinctive mechanism.