Reconciling Mouse and Human Immunology at the Altar of Genetics.

Immunity to infection has been extensively studied in humans and mice bearing naturally occurring or experimentally introduced germline mutations. Mouse studies are sometimes neglected by human immunologists, on the basis that mice are not humans and the infections studied are experimental and not natural. Conversely, human studies are sometimes neglected by mouse immunologists, on the basis of the uncontrolled conditions of study and small numbers of patients. However, both sides would agree that the infectious phenotypes of patients with inborn errors of immunity often differ from those of the corresponding mutant mice. Why is that? We argue that this important question is best addressed by revisiting and reinterpreting the findings of both mouse and human studies from a genetic perspective. Greater caution is required for reverse-genetics studies than for forward-genetics studies, but genetic analysis is sufficiently strong to define the studies likely to stand the test of time. Genetically robust mouse and human studies can provide invaluable complementary insights into the mechanisms of immunity to infection common and specific to these two species.

Interfering with Interferons: A Critical Mechanism for Critical COVID-19 Pneumonia.

Infection with SARS-CoV-2 results in clinical outcomes ranging from silent or benign infection in most individuals to critical pneumonia and death in a few. Genetic studies in patients have established that critical cases can result from inborn errors of TLR3- or TLR7-dependent type I interferon immunity, or from preexisting autoantibodies neutralizing primarily IFN-α and/or IFN-ω. These findings are consistent with virological studies showing that multiple SARS-CoV-2 proteins interfere with pathways of induction of, or response to, type I interferons. They are also congruent with cellular studies and mouse models that found that type I interferons can limit SARS-CoV-2 replication in vitro and in vivo, while their absence or diminution unleashes viral growth. Collectively, these findings point to insufficient type I interferon during the first days of infection as a general mechanism underlying critical COVID-19 pneumonia, with implications for treatment and directions for future research.

From rare disorders of immunity to common determinants of infection: Following the mechanistic thread.

The immense interindividual clinical variability during any infection is a long-standing enigma. Inborn errors of IFN-γ and IFN-α/ß immunity underlying rare infections with weakly virulent mycobacteria and seasonal influenza virus have inspired studies of two common infections: tuberculosis and COVID-19. A TYK2 genotype impairing IFN-γ production accounts for about 1% of tuberculosis cases, and autoantibodies neutralizing IFN-α/ß account for about 15% of critical COVID-19 cases. The discovery of inborn errors and mechanisms underlying rare infections drove the identification of common monogenic or autoimmune determinants of related common infections. This "rare-to-common" genetic and mechanistic approach to infectious diseases may be of heuristic value.

The ouroboros of autoimmunity.

Human autoimmunity against elements conferring protective immunity can be symbolized by the 'ouroboros', a snake eating its own tail. Underlying infection is autoimmunity against three immunological targets: neutrophils, complement and cytokines. Autoantibodies against neutrophils can cause peripheral neutropenia underlying mild pyogenic bacterial infections. The pathogenic contribution of autoantibodies against molecules of the complement system is often unclear, but autoantibodies specific for C3 convertase can enhance its activity, lowering complement levels and underlying severe bacterial infections. Autoantibodies neutralizing granulocyte-macrophage colony-stimulating factor impair alveolar macrophages, thereby underlying pulmonary proteinosis and airborne infections, type I interferon viral diseases, type II interferon intra-macrophagic infections, interleukin-6 pyogenic bacterial diseases and interleukin-17A/F mucocutaneous candidiasis. Each of these five cytokine autoantibodies underlies a specific range of infectious diseases, phenocopying infections that occur in patients with the corresponding inborn errors. In this Review, we analyze this ouroboros of immunity against immunity and posit that it should be considered as a factor in patients with unexplained infection.

A Global Effort to Define the Human Genetics of Protective Immunity to SARS-CoV-2 Infection.

SARS-CoV-2 infection displays immense inter-individual clinical variability, ranging from silent infection to lethal disease. The role of human genetics in determining clinical response to the virus remains unclear. Studies of outliers-individuals remaining uninfected despite viral exposure and healthy young patients with life-threatening disease-present a unique opportunity to reveal human genetic determinants of infection and disease.

A global effort to dissect the human genetic basis of resistance to SARS-CoV-2 infection.

SARS-CoV-2 infections display tremendous interindividual variability, ranging from asymptomatic infections to life-threatening disease. Inborn errors of, and autoantibodies directed against, type I interferons (IFNs) account for about 20% of critical COVID-19 cases among SARS-CoV-2-infected individuals. By contrast, the genetic and immunological determinants of resistance to infection per se remain unknown. Following the discovery that autosomal recessive deficiency in the DARC chemokine receptor confers resistance to Plasmodium vivax, autosomal recessive deficiencies of chemokine receptor 5 (CCR5) and the enzyme FUT2 were shown to underlie resistance to HIV-1 and noroviruses, respectively. Along the same lines, we propose a strategy for identifying, recruiting, and genetically analyzing individuals who are naturally resistant to SARS-CoV-2 infection.

A variant in human AIOLOS impairs adaptive immunity by interfering with IKAROS.

In the present study, we report a human-inherited, impaired, adaptive immunity disorder, which predominantly manifested as a B cell differentiation defect, caused by a heterozygous IKZF3 missense variant, resulting in a glycine-to-arginine replacement within the DNA-binding domain of the encoded AIOLOS protein. Using mice that bear the corresponding variant and recapitulate the B and T cell phenotypes, we show that the mutant AIOLOS homodimers and AIOLOS-IKAROS heterodimers did not bind the canonical AIOLOS-IKAROS DNA sequence. In addition, homodimers and heterodimers containing one mutant AIOLOS bound to genomic regions lacking both canonical motifs. However, the removal of the dimerization capacity from mutant AIOLOS restored B cell development. Hence, the adaptive immunity defect is caused by the AIOLOS variant hijacking IKAROS function. Heterodimeric interference is a new mechanism of autosomal dominance that causes inborn errors of immunity by impairing protein function via the mutation of its heterodimeric partner.

Human TLRs and IL-1Rs in host defense: natural insights from evolutionary, epidemiological, and clinical genetics.

Toll-like receptors (TLRs) and interleukin-1 receptors (IL-1Rs) have TIR intracellular domains that engage two main signaling pathways, via the TIR-containing adaptors MyD88 (which is not used by TLR3) and TRIF (which is used only by TLR3 and TLR4). Extensive studies in inbred mice in various experimental settings have attributed key roles in immunity to TLR- and IL-1R-mediated responses, but what contribution do human TLRs and IL-1Rs actually make to host defense in the natural setting? Evolutionary genetic studies have shown that human intracellular TLRs have evolved under stronger purifying selection than surface-expressed TLRs, for which the frequency of missense and nonsense alleles is high in the general population. Epidemiological genetic studies have yet to provide convincing evidence of a major contribution of common variants of human TLRs, IL-1Rs, or their adaptors to host defense. Clinical genetic studies have revealed that rare mutations affecting the TLR3-TRIF pathway underlie herpes simplex virus encephalitis, whereas mutations in the TIR-MyD88 pathway underlie pyogenic bacterial diseases in childhood. A careful reconsideration of the contributions of TLRs and IL-1Rs to host defense in natura is required.

Human determinants of age-dependent patterns of death from infection.

Regardless of microbial virulence (i.e., the global infection-fatality ratio), age generally drives the prevalence of death from infection in unvaccinated humans. Four mortality patterns are recognized: the common U- and L-shaped curves of endemic infections and the unique W- and J-shaped curves of pandemic infections. We suggest that these patterns result from different sets of human genetic and immunological determinants. In this model, it is the interplay between (1) monogenic genotypes affecting immunity to primary infection that preferentially manifest early in life and related genotypes or their phenocopies, including auto-antibodies, which manifest later in life and (2) the occurrence and persistence of adaptive, acquired immunity to primary or cross-reactive infections, which shapes the age-dependent pattern of human deaths from infection.

Inborn Errors of RNA Lariat Metabolism in Humans with Brainstem Viral Infection.

Viruses that are typically benign sometimes invade the brainstem in otherwise healthy children. We report bi-allelic DBR1 mutations in unrelated patients from different ethnicities, each of whom had brainstem infection due to herpes simplex virus 1 (HSV1), influenza virus, or norovirus. DBR1 encodes the only known RNA lariat debranching enzyme. We show that DBR1 expression is ubiquitous, but strongest in the spinal cord and brainstem. We also show that all DBR1 mutant alleles are severely hypomorphic, in terms of expression and function. The fibroblasts of DBR1-mutated patients contain higher RNA lariat levels than control cells, this difference becoming even more marked during HSV1 infection. Finally, we show that the patients' fibroblasts are highly susceptible to HSV1. RNA lariat accumulation and viral susceptibility are rescued by wild-type DBR1. Autosomal recessive, partial DBR1 deficiency underlies viral infection of the brainstem in humans through the disruption of tissue-specific and cell-intrinsic immunity to viruses.

Systemic Human ILC Precursors Provide a Substrate for Tissue ILC Differentiation.

Innate lymphoid cells (ILCs) represent innate versions of T helper and cytotoxic T cells that differentiate from committed ILC precursors (ILCPs). How ILCPs give rise to mature tissue-resident ILCs remains unclear. Here, we identify circulating and tissue ILCPs in humans that fail to express the transcription factors and cytokine outputs of mature ILCs but have these signature loci in an epigenetically poised configuration. Human ILCPs robustly generate all ILC subsets in vitro and in vivo. While human ILCPs express low levels of retinoic acid receptor (RAR)-related orphan receptor C (RORC) transcripts, these cells are found in RORC-deficient patients and retain potential for EOMES+ natural killer (NK) cells, interferon gamma-positive (IFN-γ+) ILC1s, interleukin (IL)-13+ ILC2s, and for IL-22+, but not for IL-17A+ ILC3s. Our results support a model of tissue ILC differentiation ("ILC-poiesis"), whereby diverse ILC subsets are generated in situ from systemically distributed ILCPs in response to local environmental signals.

Human Adaptive Immunity Rescues an Inborn Error of Innate Immunity.

The molecular basis of the incomplete penetrance of monogenic disorders is unclear. We describe here eight related individuals with autosomal recessive TIRAP deficiency. Life-threatening staphylococcal disease occurred during childhood in the proband, but not in the other seven homozygotes. Responses to all Toll-like receptor 1/2 (TLR1/2), TLR2/6, and TLR4 agonists were impaired in the fibroblasts and leukocytes of all TIRAP-deficient individuals. However, the whole-blood response to the TLR2/6 agonist staphylococcal lipoteichoic acid (LTA) was abolished only in the index case individual, the only family member lacking LTA-specific antibodies (Abs). This defective response was reversed in the patient, but not in interleukin-1 receptor-associated kinase 4 (IRAK-4)-deficient individuals, by anti-LTA monoclonal antibody (mAb). Anti-LTA mAb also rescued the macrophage response in mice lacking TIRAP, but not TLR2 or MyD88. Thus, acquired anti-LTA Abs rescue TLR2-dependent immunity to staphylococcal LTA in individuals with inherited TIRAP deficiency, accounting for incomplete penetrance. Combined TIRAP and anti-LTA Ab deficiencies underlie staphylococcal disease in this patient.

An essential role for the Zn2+ transporter ZIP7 in B cell development.

Despite the known importance of zinc for human immunity, molecular insights into its roles have remained limited. Here we report a novel autosomal recessive disease characterized by absent B cells, agammaglobulinemia and early onset infections in five unrelated families. The immunodeficiency results from hypomorphic mutations of SLC39A7, which encodes the endoplasmic reticulum-to-cytoplasm zinc transporter ZIP7. Using CRISPR-Cas9 mutagenesis we have precisely modeled ZIP7 deficiency in mice. Homozygosity for a null allele caused embryonic death, but hypomorphic alleles reproduced the block in B cell development seen in patients. B cells from mutant mice exhibited a diminished concentration of cytoplasmic free zinc, increased phosphatase activity and decreased phosphorylation of signaling molecules downstream of the pre-B cell and B cell receptors. Our findings highlight a specific role for cytosolic Zn2+ in modulating B cell receptor signal strength and positive selection.

Glycosylation-Dependent IFN-γR Partitioning in Lipid and Actin Nanodomains Is Critical for JAK Activation.

Understanding how membrane nanoscale organization controls transmembrane receptors signaling activity remains a challenge. We studied interferon-γ receptor (IFN-γR) signaling in fibroblasts from homozygous patients with a T168N mutation in IFNGR2. By adding a neo-N-glycan on IFN-γR2 subunit, this mutation blocks IFN-γ activity by unknown mechanisms. We show that the lateral diffusion of IFN-γR2 is confined by sphingolipid/cholesterol nanodomains. In contrast, the IFN-γR2 T168N mutant diffusion is confined by distinct actin nanodomains where conformational changes required for Janus-activated tyrosine kinase/signal transducer and activator of transcription (JAK/STAT) activation by IFN-γ could not occur. Removing IFN-γR2 T168N-bound galectins restored lateral diffusion in lipid nanodomains and JAK/STAT signaling in patient cells, whereas adding galectins impaired these processes in control cells. These experiments prove the critical role of dynamic receptor interactions with actin and lipid nanodomains and reveal a new function for receptor glycosylation and galectins. Our study establishes the physiological relevance of membrane nanodomains in the control of transmembrane receptor signaling in vivo. VIDEO ABSTRACT.

Parsing the Interferon Transcriptional Network and Its Disease Associations.

Type 1 interferon (IFN) is a key mediator of organismal responses to pathogens, eliciting prototypical "interferon signature genes" that encode antiviral and inflammatory mediators. For a global view of IFN signatures and regulatory pathways, we performed gene expression and chromatin analyses of the IFN-induced response across a range of immunocyte lineages. These distinguished ISGs by cell-type specificity, kinetics, and sensitivity to tonic IFN and revealed underlying changes in chromatin configuration. We combined 1,398 human and mouse datasets to computationally infer ISG modules and their regulators, validated by genetic analysis in both species. Some ISGs are controlled by Stat1/2 and Irf9 and the ISRE DNA motif, but others appeared dependent on non-canonical factors. This regulatory framework helped to interpret JAK1 blockade pharmacology, different clusters being affected under tonic or IFN-stimulated conditions, and the IFN signatures previously associated with human diseases, revealing unrecognized subtleties in disease footprints, as affected by human ancestry.

Disruption of an antimycobacterial circuit between dendritic and helper T cells in human SPPL2a deficiency.

Human inborn errors of IFN-γ immunity underlie mycobacterial diseases. We describe patients with Mycobacterium bovis (BCG) disease who are homozygous for loss-of-function mutations of SPPL2A. This gene encodes a transmembrane protease that degrades the N-terminal fragment (NTF) of CD74 (HLA invariant chain) in antigen-presenting cells. The CD74 NTF therefore accumulates in the HLA class II+ myeloid and lymphoid cells of SPPL2a-deficient patients. This toxic fragment selectively depletes IL-12- and IL-23-producing CD1c+ conventional dendritic cells (cDC2s) and their circulating progenitors. Moreover, SPPL2a-deficient memory TH1* cells selectively fail to produce IFN-γ when stimulated with mycobacterial antigens in vitro. Finally, Sppl2a-/- mice lack cDC2s, have CD4+ T cells that produce small amounts of IFN-γ after BCG infection, and are highly susceptible to infection with BCG or Mycobacterium tuberculosis. These findings suggest that inherited SPPL2a deficiency in humans underlies mycobacterial disease by decreasing the numbers of cDC2s and impairing IFN-γ production by mycobacterium-specific memory TH1* cells.

Disentangling inborn and acquired immunity in human twins.

The human geneticist Archibald Garrod noted in 1931 that, "It is, of necessity, no easy matter to distinguish between immunity which is inborn and that which has been acquired" (The Inborn Factors in Disease). In this issue of Cell, Brodin et al. show that the heritability of blood counts rapidly decreases with age for the lymphoid subsets responsible for adaptive immunity, unlike cells from other hematopoietic lineages.

A serpin shapes the extracellular environment to prevent influenza A virus maturation.

Interferon-stimulated genes (ISGs) act in concert to provide a tight barrier against viruses. Recent studies have shed light on the contribution of individual ISG effectors to the antiviral state, but most have examined those acting on early, intracellular stages of the viral life cycle. Here, we applied an image-based screen to identify ISGs inhibiting late stages of influenza A virus (IAV) infection. We unraveled a directly antiviral function for the gene SERPINE1, encoding plasminogen activator inhibitor 1 (PAI-1). By targeting extracellular airway proteases, PAI-1 inhibits IAV glycoprotein cleavage, thereby reducing infectivity of progeny viruses. This was biologically relevant for IAV restriction in vivo. Further, partial PAI-1 deficiency, attributable to a polymorphism in human SERPINE1, conferred increased susceptibility to IAV in vitro. Together, our findings reveal that manipulating the extracellular environment to inhibit the last step in a virus life cycle is an important mechanism of the antiviral response.

Role of IL-27 in Epstein-Barr virus infection revealed by IL-27RA deficiency.

Epstein-Barr virus (EBV) infection can engender severe B cell lymphoproliferative diseases1,2. The primary infection is often asymptomatic or causes infectious mononucleosis (IM), a self-limiting lymphoproliferative disorder3. Selective vulnerability to EBV has been reported in association with inherited mutations impairing T cell immunity to EBV4. Here we report biallelic loss-of-function variants in IL27RA that underlie an acute and severe primary EBV infection with a nevertheless favourable outcome requiring a minimal treatment. One mutant allele (rs201107107) was enriched in the Finnish population (minor allele frequency = 0.0068) and carried a high risk of severe infectious mononucleosis when homozygous. IL27RA encodes the IL-27 receptor alpha subunit5,6. In the absence of IL-27RA, phosphorylation of STAT1 and STAT3 by IL-27 is abolished in T cells. In in vitro studies, IL-27 exerts a synergistic effect on T-cell-receptor-dependent T cell proliferation7 that is deficient in cells from the patients, leading to impaired expansion of potent anti-EBV effector cytotoxic CD8+ T cells. IL-27 is produced by EBV-infected B lymphocytes and an IL-27RA-IL-27 autocrine loop is required for the maintenance of EBV-transformed B cells. This potentially explains the eventual favourable outcome of the EBV-induced viral disease in patients with IL-27RA deficiency. Furthermore, we identified neutralizing anti-IL-27 autoantibodies in most individuals who developed sporadic infectious mononucleosis and chronic EBV infection. These results demonstrate the critical role of IL-27RA-IL-27 in immunity to EBV, but also the hijacking of this defence by EBV to promote the expansion of infected transformed B cells.

TMEFF1 is a neuron-specific restriction factor for herpes simplex virus.

The brain is highly sensitive to damage caused by infection and inflammation1,2. Herpes simplex virus 1 (HSV-1) is a neurotropic virus and the cause of herpes simplex encephalitis3. It is unknown whether neuron-specific antiviral factors control virus replication to prevent infection and excessive inflammatory responses, hence protecting the brain. Here we identify TMEFF1 as an HSV-1 restriction factor using genome-wide CRISPR screening. TMEFF1 is expressed specifically in neurons of the central nervous system and is not regulated by type I interferon, the best-known innate antiviral system controlling virus infections. Depletion of TMEFF1 in stem-cell-derived human neurons led to elevated viral replication and neuronal death following HSV-1 infection. TMEFF1 blocked the HSV-1 replication cycle at the level of viral entry through interactions with nectin-1 and non-muscle myosin heavy chains IIA and IIB, which are core proteins in virus-cell binding and virus-cell fusion, respectively4-6. Notably, Tmeff1-/- mice exhibited increased susceptibility to HSV-1 infection in the brain but not in the periphery. Within the brain, elevated viral load was observed specifically in neurons. Our study identifies TMEFF1 as a neuron-specific restriction factor essential for prevention of HSV-1 replication in the central nervous system.