Denisovan, modern human and mouse TNFAIP3 alleles tune A20 phosphorylation and immunity.

Resisting and tolerating microbes are alternative strategies to survive infection, but little is known about the evolutionary mechanisms controlling this balance. Here genomic analyses of anatomically modern humans, extinct Denisovan hominins and mice revealed a TNFAIP3 allelic series with alterations in the encoded immune response inhibitor A20. Each TNFAIP3 allele encoded substitutions at non-catalytic residues of the ubiquitin protease OTU domain that diminished IκB kinase-dependent phosphorylation and activation of A20. Two TNFAIP3 alleles encoding A20 proteins with partial phosphorylation deficits seemed to be beneficial by increasing immunity without causing spontaneous inflammatory disease: A20 T108A;I207L, originating in Denisovans and introgressed in modern humans throughout Oceania, and A20 I325N, from an N-ethyl-N-nitrosourea (ENU)-mutagenized mouse strain. By contrast, a rare human TNFAIP3 allele encoding an A20 protein with 95% loss of phosphorylation, C243Y, caused spontaneous inflammatory disease in humans and mice. Analysis of the partial-phosphorylation A20 I325N allele in mice revealed diminished tolerance of bacterial lipopolysaccharide and poxvirus inoculation as tradeoffs for enhanced immunity.

A cell-based functional assay that accurately links genotype to phenotype in familial HLH.

Familial forms of the severe immunoregulatory disease hemophagocytic lymphohistiocytosis (HLH) arise from biallelic mutations in the PRF1, UNC13D, STXBP2, and STX11 genes. Early and accurate diagnosis of the disease is important to determine the most appropriate treatment option, including potentially curative stem cell transplantation. The diagnosis of familial HLH (FHL) is traditionally based on finding biallelic mutations in patients with HLH symptoms and reduced natural killer (NK)-cell cytotoxicity. However, patients often have a low NK-cell count or receive immunosuppressive therapies that may render the NK-cell cytotoxicity assay unreliable. Furthermore, to fully understand the nature of a disease it is critical to directly assess the effect of mutations on cellular function; this will help to avoid instances in which carriers of innocuous mutations may be recommended for invasive procedures including transplantation. To overcome this diagnostic problem, we have developed a rapid and robust method that takes advantage of the functional equivalence of the human and mouse orthologues of PRF1, UNC13D, STX11, and STXBP2 proteins. By knocking out endogenous mouse genes in CD8+ T cells and simultaneously replacing them with their mutated human orthologues, we can accurately assess the effect of mutations on cell function. The wide dynamic range of this novel system allowed us to understand the basis of, otherwise cryptic, cases of FHL or HLH and, in some instances, to demonstrate that previously reported mutations are unlikely to cause FHL. This novel approach provides valuable new information to enable more accurate diagnosis and treatment of patients with HLH or FHL who inherit mutations of undetermined pathogenicity.

Filaggrin-Associated Atopic Skin, Eye, Airways, and Gut Disease, Modifying the Presentation of X-Linked Reticular Pigmentary Disorder (XLPDR).

BACKGROUND: X-linked reticular pigmentary disorder (XLPDR) is a rare condition characterized by skin hyperpigmentation, ectodermal features, multiorgan inflammation, and recurrent infections. All probands identified to date share the same intronic hemizygous POLA1 hypomorphic variant (NM_001330360.2(POLA1):c.1393-354A > G) on the X chromosome. Previous studies have supported excessive type 1 interferon (IFN) inflammation and natural killer (NK) cell dysfunction in disease pathogenesis. Common null polymorphisms in filaggrin (FLG) gene underlie ichthyosis vulgaris and atopic predisposition. CASE: A 9-year-old boy born to non-consanguineous parents developed eczema with reticular skin hyperpigmentation in early infancy. He suffered recurrent chest infections with chronic cough, clubbing, and asthma, moderate allergic rhinoconjunctivitis with keratitis, multiple food allergies, and vomiting with growth failure. Imaging demonstrated bronchiectasis, while gastroscopy identified chronic eosinophilic gastroduodenitis. Interestingly, growth failure and bronchiectasis improved over time without specific treatment. METHODS: Whole-genome sequencing (WGS) using Illumina short-read sequencing was followed by both manual and orthogonal automated bioinformatic analyses for single-nucleotide variants, small insertions/deletions (indels), and larger copy number variations. NK cell cytotoxic function was assessed using 51Cr release and degranulation assays. The presence of an interferon signature was investigated using a panel of six interferon-stimulated genes (ISGs) by QPCR. RESULTS: WGS identified a de novo hemizygous intronic variant in POLA1 (NM_001330360.2(POLA1):c.1393-354A > G) giving a diagnosis of XLPDR, as well as a heterozygous nonsense FLG variant (NM_002016.2(FLG):c.441del, NP_0020.1:p.(Arg151Glyfs*43)). Compared to healthy controls, the IFN signature was elevated although the degree moderated over time with the improvement in his chest disease. NK cell functional studies showed normal cytotoxicity and degranulation. CONCLUSION: This patient had multiple atopic manifestations affecting eye, skin, chest, and gut, complicating the presentation of XLPDR. This highlights that common FLG polymorphisms should always be considered when assessing genotype-phenotype correlations of other genetic variation in patients with atopic symptoms. Additionally, while the patient exhibited an enhanced IFN signature, he does not have an NK cell defect, suggesting this may not be a constant feature of XLPDR.

A Novel Case of IFNAR1 Deficiency Identified a Common Canonical Splice Site Variant in DOCK8 in Western Polynesia: The Importance of Validating Variants of Unknown Significance in Under-Represented Ancestries.

Advanced genomic technologies such as whole exome or whole genome sequencing have improved diagnoses and disease outcomes for individuals with genetic diseases. Yet, variants of unknown significance (VUS) require rigorous validation to establish disease causality or modification, or to exclude them from further analysis. Here, we describe a young individual of Polynesian ancestry who in the first 13 mo of life presented with SARS-CoV-2 pneumonia, severe enterovirus meningitis and adenovirus gastroenteritis, and severe adverse reaction to MMR vaccination. Genomic analysis identified a previously reported pathogenic homozygous variant in IFNAR1 (c.1156G > T, p.Glu386* LOF), which is common in Western Polynesia. Moreover, a new and putatively deleterious canonical splice site variant in DOCK8 was also found in homozygosity (c.3234 + 2T > C). This DOCK8 variant is common in Polynesians and other under-represented ancestries in large genomic databases. Despite in silico bioinformatic predictions, extensive in vitro and ex vivo analysis revealed the DOCK8 variant likely be neutral. Thus, our study reports a novel case of IFNAR1 deficiency, but also highlights the importance of functional validation of VUS, including those predicted to be deleterious, and the pressing need to expand our knowledge of the genomic architecture and landscape of under-represented populations and ancestries.

Pyrin variant E148Q potentiates inflammasome activation and the effect of pathogenic mutations in cis.

OBJECTIVE: The p.E148Q variant in pyrin is present in different populations at a frequency of up to 29%, and has been associated with diseases, including vasculitis and FMF. The pathogenicity of p.E148Q in FMF is unclear, even when observed in cis or in trans to a single, typically recessive, pathogenic mutation. We performed functional validation to determine whether p.E148Q increases the ability of pyrin to form an active inflammasome complex in cell lines. METHODS: We interrogated the Australian Autoinflammatory Disease RegistrY (AADRY) to find candidate inheritance patterns for the p.E148Q variant in pyrin. Different pyrin variant combinations were tested in HEK293T cells stably expressing the adaptor protein apoptosis-associated speck-like (ASC), which were analysed by flow cytometry to visualize inflammasome formation, with and without stimulation by Clostridioides difficile toxin B (TcdB). Inflammasome-dependent cytokine secretion was also quantified by ELISA of supernatants from THP-1 cells transduced with lentiviral expression vectors. RESULTS: In AADRY, we observed the p.E148Q allele in individuals with autoinflammatory diseases alone or in conjunction with other pyrin variants. Two FMF families harboured the allele p.E148Q-M694I in cis with dominant heritability. In vitro, p.E148Q pyrin could spontaneously potentiate inflammasome formation, with increased IL-1ß and IL-18 secretion. p.E148Q in cis to classical FMF mutations provided significant potentiation of inflammasome formation. CONCLUSION: The p.E148Q variant in pyrin potentiates inflammasome activation in vitro. In cis, this effect is additive to known pathogenic FMF mutations. In some families, this increased effect could explain why FMF segregates as an apparently dominant disease.

SAMD9L autoinflammatory or ataxia pancytopenia disease mutations activate cell-autonomous translational repression.

Sterile α motif domain-containing protein 9-like (SAMD9L) is encoded by a hallmark interferon-induced gene with a role in controlling virus replication that is not well understood. Here, we analyze SAMD9L function from the perspective of human mutations causing neonatal-onset severe autoinflammatory disease. Whole-genome sequencing of two children with leukocytoclastic panniculitis, basal ganglia calcifications, raised blood inflammatory markers, neutrophilia, anemia, thrombocytopaenia, and almost no B cells revealed heterozygous de novo SAMD9L mutations, p.Asn885Thrfs*6 and p.Lys878Serfs*13. These frameshift mutations truncate the SAMD9L protein within a domain a region of homology to the nucleotide-binding and oligomerization domain (NOD) of APAF1, ∼80 amino acids C-terminal to the Walker B motif. Single-cell analysis of human cells expressing green fluorescent protein (GFP)-SAMD9L fusion proteins revealed that enforced expression of wild-type SAMD9L repressed translation of red fluorescent protein messenger RNA and globally repressed endogenous protein translation, cell autonomously and in proportion to the level of GFP-SAMD9L in each cell. The children's truncating mutations dramatically exaggerated translational repression even at low levels of GFP-SAMD9L per cell, as did a missense Arg986Cys mutation reported recurrently as causing ataxia pancytopenia syndrome. Autoinflammatory disease associated with SAMD9L truncating mutations appears to result from an interferon-induced translational repressor whose activity goes unchecked by the loss of C-terminal domains that may normally sense virus infection.

The FOXP3 full-length isoform controls the lineage-stability of CD4+FOXP3+ regulatory T cells.

The transcription factor FOXP3 is essential for CD4+FOXP3+ regulatory T (Treg) cell development and function. Human FOXP3 exists in distinct isoforms and alterations in isoform expression is associated with inflammatory disease progression, however, the exact functions of FOXP3 isoforms remain poorly understood. Herein we used flow cytometry and RNA-sequencing to analyze subsets of Treg cells from two IPEX patients, and a healthy carrier, of a recently described FOXP3 mutation (c.305delT). This mutation is located in exon 2 and results in the loss of the full-length FOXP3 isoform. Treg cells lacking full-length FOXP3 are found at lower-than-expected frequencies. This loss cannot be explained solely by altered thymic output, changes in proliferation, peripheral induction of Treg cells, or apoptosis. Instead, fulllength FOXP3 control a distinct genetic program, involving the previously identified FOXP3 regulators ID3, BCL6 and eIF4E, that upholds Treg cell lineage stability, while it appears nonessential for Treg cell activation.

Hyper-IgE Syndrome due to an Elusive Novel Intronic Homozygous Variant in DOCK8.

Rare, biallelic loss-of-function mutations in DOCK8 result in a combined immune deficiency characterized by severe and recurrent cutaneous infections, eczema, allergies, and susceptibility to malignancy, as well as impaired humoral and cellular immunity and hyper-IgE. The advent of next-generation sequencing technologies has enabled the rapid molecular diagnosis of rare monogenic diseases, including inborn errors of immunity. These advances have resulted in the implementation of gene-guided treatments, such as hematopoietic stem cell transplant for DOCK8 deficiency. However, putative disease-causing variants revealed by next-generation sequencing need rigorous validation to demonstrate pathogenicity. Here, we report the eventual diagnosis of DOCK8 deficiency in a consanguineous family due to a novel homozygous intronic deletion variant that caused aberrant exon splicing and subsequent loss of expression of DOCK8 protein. Remarkably, the causative variant was not initially detected by clinical whole-genome sequencing but was subsequently identified and validated by combining advanced genomic analysis, RNA-seq, and flow cytometry. This case highlights the need to adopt multipronged confirmatory approaches to definitively solve complex genetic cases that result from variants outside protein-coding exons and conventional splice sites.

COVID-19 in children: I. Epidemiology, prevention and indirect impacts.

Children globally have been profoundly impacted by the coronavirus disease 2019 (COVID-19) pandemic. This review explores the direct and indirect public health impacts of COVID-19 on children. We discuss in detail the transmission dynamics, vaccination strategies and, importantly, the 'shadow pandemic', encompassing underappreciated indirect impacts of the pandemic on children. The indirect effects of COVID-19 will have a long-term impact beyond the immediate pandemic period. These include the mental health and wellbeing risks, disruption to family income and attendant stressors including increased family violence, delayed medical attention and the critical issue of prolonged loss of face-to-face learning in a normal school environment. Amplification of existing inequities and creation of new disadvantage are likely additional sequelae, with children from vulnerable families disproportionately affected. We emphasise the responsibility of paediatricians to advocate on behalf of this vulnerable group to ensure the longer-term effects of COVID-19 public health responses on the health and wellbeing of children are fully considered.

COVID-19 in children. II: Pathogenesis, disease spectrum and management.

The global disruption of the COVID-19 pandemic has impacted the life of every child either directly or indirectly. This review explores the pathophysiology, immune response, clinical presentation and treatment of COVID-19 in children, summarising the most up-to-date data including recent developments regarding variants of concern. The acute infection with SARS-CoV-2 is generally mild in children, whilst the post-infectious manifestations, including paediatric inflammatory multisystem syndrome temporally associated with SARS-CoV-2 (PIMS-TS) and 'long COVID' in children, are more complex. Given that most research on COVID-19 has focused on adult cohorts and that clinical manifestations, treatment availability and impacts differ markedly in children, research that specifically examines COVID-19 in children needs to be prioritised.

Supporting Aboriginal and Torres Strait Islander Families to Stay Together from the Start (SAFeST Start): Urgent call to action to address crisis in infant removals.

Reducing the rate of over-representation of Aboriginal and Torres Strait Islander children in out-of-home care (OOHC) is a key Closing the Gap target committed to by all Australian governments. Current strategies are failing. The "gap" is widening, with the rate of Aboriginal and Torres Strait Islander children in OOHC at 30 June 2020 being 11 times that of non-Indigenous children. Approximately, one in five Aboriginal and Torres Strait Islander children entering OOHC each year are younger than one year. These figures represent compounding intergenerational trauma and institutional harm to Aboriginal and Torres Strait Islander families and communities. This article outlines systemic failures to address the needs of Aboriginal and Torres Strait Islander parents during pregnancy and following birth, causing cumulative harm and trauma to families, communities and cultures. Major reform to child and family notification and service systems, and significant investment to address this crisis, is urgently needed. The Family Matters Building Blocks and five elements of the Aboriginal and Torres Strait Islander Child Placement Principle (Prevention, Participation, Partnership, Placement and Connection) provide a transformative foundation to address historical, institutional, well-being and socioeconomic drivers of current catastrophic trajectories. The time for action is now.

Intrinsic Defects in B Cell Development and Differentiation, T Cell Exhaustion and Altered Unconventional T Cell Generation Characterize Human Adenosine Deaminase Type 2 Deficiency.

PURPOSE: Deficiency of adenosine deaminase type 2 (ADA2) (DADA2) is a rare inborn error of immunity caused by deleterious biallelic mutations in ADA2. Clinical manifestations are diverse, ranging from severe vasculopathy with lacunar strokes to immunodeficiency with viral infections, hypogammaglobulinemia and bone marrow failure. Limited data are available on the phenotype and function of leukocytes from DADA2 patients. The aim of this study was to perform in-depth immunophenotyping and functional analysis of the impact of DADA2 on human lymphocytes. METHODS: In-depth immunophenotyping and functional analyses were performed on ten patients with confirmed DADA2 and compared to heterozygous carriers of pathogenic ADA2 mutations and normal healthy controls. RESULTS: The median age of the patients was 10 years (mean 20.7 years, range 1-44 years). Four out of ten patients were on treatment with steroids and/or etanercept or other immunosuppressives. We confirmed a defect in terminal B cell differentiation in DADA2 and reveal a block in B cell development in the bone marrow at the pro-B to pre-B cell stage. We also show impaired differentiation of CD4+ and CD8+ memory T cells, accelerated exhaustion/senescence, and impaired survival and granzyme production by ADA2 deficient CD8+ T cells. Unconventional T cells (i.e. iNKT, MAIT, Vδ2+ γδT) were diminished whereas pro-inflammatory monocytes and CD56bright immature NK cells were increased. Expression of the IFN-induced lectin SIGLEC1 was increased on all monocyte subsets in DADA2 patients compared to healthy donors. Interestingly, the phenotype and function of lymphocytes from healthy heterozygous carriers were often intermediate to that of healthy donors and ADA2-deficient patients. CONCLUSION: Extended immunophenotyping in DADA2 patients shows a complex immunophenotype. Our findings provide insight into the cellular mechanisms underlying some of the complex and heterogenous clinical features of DADA2. More research is needed to design targeted therapy to prevent viral infections in these patients with excessive inflammation as the overarching phenotype.

Translational profiling of hypocretin neurons identifies candidate molecules for sleep regulation.

Hypocretin (orexin; Hcrt)-containing neurons of the hypothalamus are essential for the normal regulation of sleep and wake behaviors and have been implicated in feeding, anxiety, depression, and reward. The absence of these neurons causes narcolepsy in humans and model organisms. However, little is known about the molecular phenotype of these cells; previous attempts at comprehensive profiling had only limited sensitivity or were inaccurate. We generated a Hcrt translating ribosome affinity purification (bacTRAP) line for comprehensive translational profiling of all ribosome-bound transcripts in these neurons in vivo. From this profile, we identified >6000 transcripts detectably expressed above background and 188 transcripts that are highly enriched in these neurons, including all known markers of the cells. Blinded analysis of in situ hybridization databases suggests that ~60% of these are expressed in a Hcrt marker-like pattern. Fifteen of these were confirmed with double labeling and microscopy, including the transcription factor Lhx9. Ablation of this gene results in a >30% loss specifically of Hcrt neurons, without a general disruption of hypothalamic development. Polysomnography and activity monitoring revealed a profound hypersomnolence in these mice. These data provide an in-depth and accurate profile of Hcrt neuron gene expression and suggest that Lhx9 may be important for specification or survival of a subset of these cells.

The Role of ZEB2 in Human CD8 T Lymphocytes: Clinical and Cellular Immune Profiling in Mowat-Wilson Syndrome.

The Zeb2 gene encodes a transcription factor (ZEB2) that acts as an important immune mediator in mice, where it is expressed in early-activated effector CD8 T cells, and limits effector differentiation. Zeb2 homozygous knockout mice have deficits in CD8 T cells and NK cells. Mowat-Wilson syndrome (MWS) is a rare genetic disease resulting from heterozygous mutations in ZEB2 causing disease by haploinsufficiency. Whether ZEB2 exhibits similar expression patterns in human CD8 T cells is unknown, and MWS patients have not been comprehensively studied to identify changes in CD8 lymphocytes and NK cells, or manifestations of immunodeficiency. By using transcriptomic assessment, we demonstrated that ZEB2 is expressed in early-activated effector CD8 T cells of healthy human volunteers following vaccinia inoculation and found evidence of a role for TGFß-1/SMAD signaling in these cells. A broad immunological assessment of six genetically diagnosed MWS patients identified two patients with a history of recurrent sinopulmonary infections, one of whom had recurrent oral candidiasis, one with lymphopenia, two with thrombocytopenia and three with detectable anti-nuclear antibodies. Immunoglobulin levels, including functional antibody responses to protein and polysaccharide vaccination, were normal. The MWS patients had a significantly lower CD8 T cell subset as % of lymphocytes, compared to healthy controls (median 16.4% vs. 25%, p = 0.0048), and resulting increased CD4:CD8 ratio (2.6 vs. 1.8; p = 0.038). CD8 T cells responded normally to mitogen stimulation in vitro and memory CD8 T cells exhibited normal proportions of subsets with important tissue-specific homing markers and cytotoxic effector molecules. There was a trend towards a decrease in the CD8 T effector memory subset (3.3% vs. 5.9%; p = 0.19). NK cell subsets were normal. This is the first evidence that ZEB2 is expressed in early-activated human effector CD8 T cells, and that haploinsufficiency of ZEB2 in MWS patients had a slight effect on immune function, skewing T cells away from CD8 differentiation. To date there is insufficient evidence to support an immunodeficiency occurring in MWS patients.

Mental health and well-being of Aboriginal and Torres Strait Islander peoples in Australia during COVID-19.

In May 2020, an independent working party was convened to determine the mental health and well-being needs of Aboriginal and Torres Strait Islander peoples in Australia, in response to COVID-19. Thirty Aboriginal and Torres Strait Islander leaders and allies worked together in a two-month virtual collaboration process. Here, we provide the working party's five key recommendations and highlight the evidence supporting these proposals. Aboriginal and Torres Strait Islander self-determination and governance must be prioritised to manage the COVID-19 recovery in Aboriginal and Torres Strait Islander communities. To mitigate long-term social and economic impacts of COVID-19 to Australian society, the historical underinvestment in Aboriginal and Torres Strait Islander peoples must be reconciled. Equitable, needs-based funding is required to support strengths-based, place-based initiatives that address the determinants of health. This includes workforce and infrastructure development and effective evaluation. There is a clear, informed pathway to health and healing for Aboriginal and Torres Strait Islander peoples being enacted by Aboriginal and Torres Strait Islander leadership and community organisations; it remains to be seen how these recommendations will be implemented.

The Clinical Immunogenomics Research Consortium Australasia (CIRCA): a Distributed Network Model for Genomic Healthcare Delivery.

The Clinical Immunogenomics Research Consortium Australasia (CIRCA) crowdsources expertise in medicine, genomics, data science, and fundamental biology to diagnose and treat patients with rare inborn errors of immunity. This distributed network model operates free of geographic borders and allows rapid progression through the full research/translation/clinical management pipeline, from initial gene variant discovery, through functional validation, and on to precision mechanism-based treatment of patients throughout Australia and New Zealand. The model is scalable and applicable to other rare diseases where clinical experience and scientific know-how are limited, and enables efficient delivery of genomics for all.

Diversity of XMEN Disease: Description of 2 Novel Variants and Analysis of the Lymphocyte Phenotype.

Variants in MAGT1 have been identified as the cause of an immune deficiency termed X-linked immunodeficiency with magnesium defect, Epstein-Barr virus (EBV) infection and neoplasia (XMEN) disease. Here, we describe 2 cases of XMEN disease due to novel mutations in MAGT1, one of whom presented with classical features of XMEN disease and another who presented with a novel phenotype including probable CNS vasculitis, HHV-8 negative multicentric Castelman disease and severe molluscum contagiosum, thus highlighting the clinical diversity that may be seen in this condition. Peripheral blood immunophenotyping of these 2 patients, together with an additional 4 XMEN patients, revealed reduced NKG2D expression, impaired CD28 expression on CD8+ T cells, CD4+ T cell lymphopenia, an inverted CD4:CD8 ratio and decreased memory B cells. In addition, we showed for the first time alterations to the CD8+ T cell memory compartment, reduced CD56hi NK cells, MAIT and iNKT cells, as well as compromised differentiation of naïve CD4+ T cells into IL-21-producing Tfh-type cells in vitro. Both patients were treated with supplemental magnesium with limited benefit. However, one patient has undergone allogeneic haematopoietic stem cell transplant, with full donor chimerism and immune reconstitution. These results expand our understanding of the clinical and immunological phenotype in XMEN disease, adding to the current literature, which we further discuss here.

Diagnosis and management of severe combined immunodeficiency in Australia and New Zealand.

This consensus document outlines the recommendations from the Australasian Society of Clinical Immunology and Allergy Transplantation and Primary Immunodeficiency group for the diagnosis and management of patients with severe combined immunodeficiency. It also provides a proposed framework for the early investigation, management and supportive care prior to haematopoietic stem cell transplantation.

Activating PIK3CD mutations impair human cytotoxic lymphocyte differentiation and function and EBV immunity.

BACKGROUND: Germline gain-of function (GOF) mutations in PIK3CD, encoding the catalytic p110δ subunit of phosphoinositide 3-kinase (PI3K), result in hyperactivation of the PI3K-AKT-mechanistic target of rapamycin pathway and underlie a novel inborn error of immunity. Affected subjects exhibit perturbed humoral and cellular immunity, manifesting as recurrent infections, autoimmunity, hepatosplenomegaly, uncontrolled EBV and/or cytomegalovirus infection, and increased incidence of B-cell lymphoproliferation, lymphoma, or both. Mechanisms underlying disease pathogenesis remain unknown. OBJECTIVE: Understanding the cellular and molecular mechanisms underpinning inefficient surveillance of EBV-infected B cells is required to understand disease in patients with PIK3CD GOF mutations, identify key molecules required for cell-mediated immunity against EBV, and develop immunotherapeutic interventions for the treatment of this and other EBV-opathies. METHODS: We studied the consequences of PIK3CD GOF mutations on the generation, differentiation, and function of CD8+ T cells and natural killer (NK) cells, which are implicated in host defense against infection with herpesviruses, including EBV. RESULTS: PIK3CD GOF total and EBV-specific CD8+ T cells were skewed toward an effector phenotype, with exaggerated expression of markers associated with premature immunosenescence/exhaustion and increased susceptibility to reactivation-induced cell death. These findings were recapitulated in a novel mouse model of PI3K GOF mutations. NK cells in patients with PIK3CD GOF mutations also exhibited perturbed expression of differentiation-associated molecules. Both CD8+ T and NK cells had reduced capacity to kill EBV-infected B cells. PIK3CD GOF B cells had increased expression of CD48, programmed death ligand 1/2, and CD70. CONCLUSIONS: PIK3CD GOF mutations aberrantly induce exhaustion, senescence, or both and impair cytotoxicity of CD8+ T and NK cells. These defects might contribute to clinical features of affected subjects, such as impaired immunity to herpesviruses and tumor surveillance.