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.

Isolation and characterisation of PR3-specific B cells and their immunoglobulin sequences.

BACKGROUND: PR3 autoantibodies are essential to the diagnosis and monitoring of granulomatosus with polyangiitis, but to date no PR3 autoantibody sequences have been published. OBJECTIVES: To identify and characterise PR3-specific B cells from the peripheral blood of patients with PR3 autoantibodies. METHODS: Peripheral blood mononuclear cells from seven patients with PR3 autoantibodies were stained with PR3. B cells that bound PR3 underwent single cell sorting, transcriptome sequencing, and their immunoglobulin sequences expressed as antibodies and tested for PR3-specificity by ELISA. RESULTS: We identified 19 PR3-specific B cells from only one PR3-seropositive patient at a low frequency (0.0075 % of B cells) in the peripheral blood. These were polyclonal, IgG+ and enriched for IgG4, lambda pairing, IGHJ6 gene usage, CDRH3 length, IGHE and CD71 expression. They demonstrated relatively low levels of somatic hypermutation and variably reduced PR3 binding when reverted to germline. CONCLUSIONS: Identifying PR3-specific B cells in the peripheral blood is possible but challenging and those we did identify exhibited features suggesting that PR3-self reactivity may occur early in B-cell development.

Structural and functional characterization of capsid binding by anti-AAV9 monoclonal antibodies from infants after SMA gene therapy.

Success in the treatment of infants with spinal muscular atrophy (SMA) underscores the potential of vectors based on adeno-associated virus (AAV). However, a major obstacle to the full realization of this potential is pre-existing natural and therapy-induced anti-capsid humoral immunity. Structure-guided capsid engineering is one possible approach to surmounting this challenge but necessitates an understanding of capsid-antibody interactions at high molecular resolution. Currently, only mouse-derived monoclonal antibodies (mAbs) are available to structurally map these interactions, which presupposes that mouse and human-derived antibodies are functionally equivalent. In this study, we have characterized the polyclonal antibody responses of infants following AAV9-mediated gene therapy for SMA and recovered 35 anti-capsid mAbs from the abundance of switched-memory B (smB) cells present in these infants. For 21 of these mAbs, seven from each of three infants, we have undertaken functional and structural analysis measuring neutralization, affinities, and binding patterns by cryoelectron microscopy (cryo-EM). Four distinct patterns were observed akin to those reported for mouse-derived mAbs, but with early evidence of differing binding pattern preference and underlying molecular interactions. This is the first human and largest series of anti-capsid mAbs to have been comprehensively characterized and will prove to be powerful tools for basic discovery and applied purposes.

STAT3 gain-of-function mutations connect leukemia with autoimmune disease by pathological NKG2Dhi CD8+ T cell dysregulation and accumulation.

The association between cancer and autoimmune disease is unexplained, exemplified by T cell large granular lymphocytic leukemia (T-LGL) where gain-of-function (GOF) somatic STAT3 mutations correlate with co-existing autoimmunity. To investigate whether these mutations are the cause or consequence of CD8+ T cell clonal expansions and autoimmunity, we analyzed patients and mice with germline STAT3 GOF mutations. STAT3 GOF mutations drove the accumulation of effector CD8+ T cell clones highly expressing NKG2D, the receptor for stress-induced MHC-class-I-related molecules. This subset also expressed genes for granzymes, perforin, interferon-γ, and Ccl5/Rantes and required NKG2D and the IL-15/IL-2 receptor IL2RB for maximal accumulation. Leukocyte-restricted STAT3 GOF was sufficient and CD8+ T cells were essential for lethal pathology in mice. These results demonstrate that STAT3 GOF mutations cause effector CD8+ T cell oligoclonal accumulation and that these rogue cells contribute to autoimmune pathology, supporting the hypothesis that somatic mutations in leukemia/lymphoma driver genes contribute to autoimmune disease.

Activation of the viral sensor oligoadenylate synthetase 2 (Oas2) prevents pregnancy-driven mammary cancer metastases.

BACKGROUND: The interferon response can influence the primary and metastatic activity of breast cancers and can interact with checkpoint immunotherapy to modulate its effects. Using N-ethyl-N-nitrosourea mutagenesis, we found a mouse with an activating mutation in oligoadenylate synthetase 2 (Oas2), a sensor of viral double stranded RNA, that resulted in an interferon response and prevented lactation in otherwise healthy mice. METHODS: To determine if sole activation of Oas2 could alter the course of mammary cancer, we combined the Oas2 mutation with the MMTV-PyMT oncogene model of breast cancer and examined disease progression and the effects of checkpoint immunotherapy using Kaplan-Meier survival analysis with immunohistochemistry and flow cytometry. RESULTS: Oas2 mutation prevented pregnancy from increasing metastases to lung. Checkpoint immunotherapy with antibodies against programmed death-ligand 1 was more effective when the Oas2 mutation was present. CONCLUSIONS: These data establish OAS2 as a therapeutic target for agents designed to reduce metastases and increase the effectiveness of checkpoint immunotherapy.

Augmented neutralization of SARS-CoV-2 Omicron variant by boost vaccination and monoclonal antibodies.

Effective vaccines and monoclonal antibodies have been developed against coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). However, the appearance of virus variants with higher transmissibility and pathogenicity is a major concern because of their potential to escape vaccines and clinically approved SARS-CoV-2- antibodies. Here, we use flow cytometry-based binding and pseudotyped SARS-CoV-2 neutralization assays to determine the efficacy of boost immunization and therapeutic antibodies to neutralize the dominant Omicron variant. We provide compelling evidence that the third vaccination with BNT162b2 increases the amount of neutralizing serum antibodies against Delta and Omicron variants, albeit to a lower degree when compared to the parental Wuhan strain. Therefore, a third vaccination is warranted to increase titers of protective serum antibodies, especially in the case of the Omicron variant. We also found that most clinically approved and otherwise potent therapeutic antibodies against the Delta variant failed to recognize and neutralize the Omicron variant. In contrast, some antibodies under preclinical development potentially neutralized the Omicron variant. Our studies also support using a flow cytometry-based antibody binding assay to rapidly monitor therapeutic candidates and serum titers against emerging SARS-CoV-2 variants.

Uncontrolled CD21low age-associated and B1 B cell accumulation caused by failure of an EGR2/3 tolerance checkpoint.

CD21low age-associated or atypical memory B cells are autoantibody enriched and poised for plasma cell differentiation. These cells overaccumulate in chronic infections, autoimmune disease, and immunodeficiency, posing the question of what checkpoints normally oppose their accumulation. Here, we reveal a critical role for paralogous calcium-NFAT-regulated transcription factors EGR2 and EGR3 that are induced in self-reactive B cells. CD21low and B1 B cells lacking EGR2 and EGR3 accumulate and circulate in young mice in numbers 10- to 20-fold greater than normal and overexpress a large set of EGR2 ChIP-seq target genes, including known drivers of plasma cell differentiation. Most follicular B cells constitutively express Egr2 proportionally to surface IgM downregulation by self-antigens, and EGR2/3 deficiency abolishes this cardinal feature of B cell anergy. These results explain the cardinal features of B cell anergy, define a key transcriptional checkpoint repressing CD21low B cell formation, and inform how NFATC1 or EGR2 mutations promote B1 cell-derived chronic lymphocytic leukemias.

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.

Antigen-driven EGR2 expression is required for exhausted CD8+ T cell stability and maintenance.

Chronic stimulation of CD8+ T cells triggers exhaustion, a distinct differentiation state with diminished effector function. Exhausted cells exist in multiple differentiation states, from stem-like progenitors that are the key mediators of the response to checkpoint blockade, through to terminally exhausted cells. Due to its clinical relevance, there is substantial interest in defining the pathways that control differentiation and maintenance of these subsets. Here, we show that chronic antigen induces the anergy-associated transcription factor EGR2 selectively within progenitor exhausted cells in both chronic LCMV and tumours. EGR2 enables terminal exhaustion and stabilizes the exhausted transcriptional state by both direct EGR2-dependent control of key exhaustion-associated genes, and indirect maintenance of the exhausted epigenetic state. We show that EGR2 is a regulator of exhaustion that epigenetically and transcriptionally maintains the differentiation competency of progenitor exhausted cells.

Loss-of-function of Fbxo10, encoding a post-translational regulator of BCL2 in lymphomas, has no discernible effect on BCL2 or B lymphocyte accumulation in mice.

Regulation of the anti-apoptotic BCL2 protein determines cell survival and is frequently abnormal in B cell lymphomas. An evolutionarily conserved post-translational mechanism for over-expression of BCL2 in human B cell lymphomas and the BCL2 paralogue CED-9 in Caenorhabditis elegans results from loss-of-function mutations in human FBXO10 and its C.elegans paralogue DRE-1, a BCL2/CED-9-binding subunit of the SKP-CULLIN-FBOX (SCF) ubiquitin ligase. Here, we tested the role of FBXO10 in BCL2 regulation by producing mice with two different CRISPR/Cas9-engineered Fbxo10 mutations: an Asp54Lys (E54K) missense mutation in the FBOX domain and a Cys55SerfsTer55 frameshift (fs) truncating mutation. Mice homozygous for either mutant allele were born at the expected Mendelian frequency and appeared normal in body weight and appearance as adults. Spleen B cells from homozygous mutant mice did not have increased BCL2 protein, nor were the numbers of mature B cells or germinal centre B cells increased as would be expected if BCL2 was increased. Other lymphocyte subsets that are also regulated by BCL2 levels also displayed no difference in frequency in homozygous Fbxo10 mutant mice. These results support one of two conclusions: either FBXO10 does not regulate BCL2 in mice, or it does so redundantly with other ubiquitin ligase complexes. Possible candidates for the latter include FBXO11 or ARTS-XIAP. The difference between the role of FBXO10 in regulating BCL2 protein levels in C. elegans and in human DLBCL, relative to single-gene deficient mouse leukocytes, should be further investigated.

NINJ1 mediates plasma membrane rupture during lytic cell death.

Plasma membrane rupture (PMR) is the final cataclysmic event in lytic cell death. PMR releases intracellular molecules known as damage-associated molecular patterns (DAMPs) that propagate the inflammatory response1-3. The underlying mechanism of PMR, however, is unknown. Here we show that the cell-surface NINJ1 protein4-8, which contains two transmembrane regions, has an essential role in the induction of PMR. A forward-genetic screen of randomly mutagenized mice linked NINJ1 to PMR. Ninj1-/- macrophages exhibited impaired PMR in response to diverse inducers of pyroptotic, necrotic and apoptotic cell death, and were unable to release numerous intracellular proteins including HMGB1 (a known DAMP) and LDH (a standard measure of PMR). Ninj1-/- macrophages died, but with a distinctive and persistent ballooned morphology, attributable to defective disintegration of bubble-like herniations. Ninj1-/- mice were more susceptible than wild-type mice to infection with Citrobacter rodentium, which suggests a role for PMR in anti-bacterial host defence. Mechanistically, NINJ1 used an evolutionarily conserved extracellular domain for oligomerization and subsequent PMR. The discovery of NINJ1 as a mediator of PMR overturns the long-held idea that cell death-related PMR is a passive event.

COVID-19, varying genetic resistance to viral disease and immune tolerance checkpoints.

Coronavirus disease 2019 (COVID-19) is a zoonosis like most of the great plagues sculpting human history, from smallpox to pandemic influenza and human immunodeficiency virus. When viruses jump into a new species the outcome of infection ranges from asymptomatic to lethal, historically ascribed to "genetic resistance to viral disease." People have exploited these differences for good and bad, for developing vaccines from cowpox and horsepox virus, controlling rabbit plagues with myxoma virus and introducing smallpox during colonization of America and Australia. Differences in resistance to viral disease are at the core of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) crisis, yet our understanding of the mechanisms in any interspecies leap falls short of the mark. Here I review how the two key parameters of viral disease are countered by fundamentally different genetic mechanisms for resistance: (1) virus transmission, countered primarily by activation of innate and adaptive immune responses; and (2) pathology, countered primarily by tolerance checkpoints to limit innate and adaptive immune responses. I discuss tolerance thresholds and the role of CD8 T cells to limit pathological immune responses, the problems posed by tolerant superspreaders and the signature coronavirus evasion strategy of eliciting only short-lived neutralizing antibody responses. Pinpointing and targeting the mechanisms responsible for varying pathology and short-lived antibody were beyond reach in previous zoonoses, but this time we are armed with genomic technologies and more knowledge of immune checkpoint genes. These known unknowns must now be tackled to solve the current COVID-19 crisis and the inevitable zoonoses to follow.

Lymphoma Driver Mutations in the Pathogenic Evolution of an Iconic Human Autoantibody.

Pathogenic autoantibodies arise in many autoimmune diseases, but it is not understood how the cells making them evade immune checkpoints. Here, single-cell multi-omics analysis demonstrates a shared mechanism with lymphoid malignancy in the formation of public rheumatoid factor autoantibodies responsible for mixed cryoglobulinemic vasculitis. By combining single-cell DNA and RNA sequencing with serum antibody peptide sequencing and antibody synthesis, rare circulating B lymphocytes making pathogenic autoantibodies were found to comprise clonal trees accumulating mutations. Lymphoma driver mutations in genes regulating B cell proliferation and V(D)J mutation (CARD11, TNFAIP3, CCND3, ID3, BTG2, and KLHL6) were present in rogue B cells producing the pathogenic autoantibody. Antibody V(D)J mutations conferred pathogenicity by causing the antigen-bound autoantibodies to undergo phase transition to insoluble aggregates at lower temperatures. These results reveal a pre-neoplastic stage in human lymphomagenesis and a cascade of somatic mutations leading to an iconic pathogenic autoantibody.

Preponderance of CTLA4 Variation Associated With Autosomal Dominant Immune Dysregulation in the MYPPPY Motif.

One of the primary targets of immune checkpoint inhibition is the negative immune regulatory molecule CTLA-4. Immune-related adverse events are commonly observed following CTLA-4 inhibition in melanoma treatment, and a spectrum of these conditions are also observed in individuals with germline haploinsufficiency of CTLA4. Here we describe a heterozygous de novo missense variant of CTLA4 in a young girl with childhood-onset autoimmune hepatitis and polyarthritis, the latter responding to treatment with CTLA-4-Ig fusion protein. This variant lay within the highly conserved MYPPPY motif of CTLA-4: a critical structural determinant of ligand binding, which is also bound by the anti-CTLA-4 monoclonal antibody ipilimumab. Within the spectrum of CTLA4 variants reported, missense variants in the MYPPPY motif were overrepresented when compared to variants within a control population, highlighting the physiological importance of this motif in both the genetic and pharmacological regulation of autoimmunity and anti-tumor immunity.

High-throughput targeted long-read single cell sequencing reveals the clonal and transcriptional landscape of lymphocytes.

High-throughput single-cell RNA sequencing is a powerful technique but only generates short reads from one end of a cDNA template, limiting the reconstruction of highly diverse sequences such as antigen receptors. To overcome this limitation, we combined targeted capture and long-read sequencing of T-cell-receptor (TCR) and B-cell-receptor (BCR) mRNA transcripts with short-read transcriptome profiling of barcoded single-cell libraries generated by droplet-based partitioning. We show that Repertoire and Gene Expression by Sequencing (RAGE-Seq) can generate accurate full-length antigen receptor sequences at nucleotide resolution, infer B-cell clonal evolution and identify alternatively spliced BCR transcripts. We apply RAGE-Seq to 7138 cells sampled from the primary tumor and draining lymph node of a breast cancer patient to track transcriptome profiles of expanded lymphocyte clones across tissues. Our results demonstrate that RAGE-Seq is a powerful method for tracking the clonal evolution from large numbers of lymphocytes applicable to the study of immunity, autoimmunity and cancer.

CARD11 is dispensable for homeostatic responses and suppressive activity of peripherally induced FOXP3+ regulatory T cells.

FOXP3+ regulatory T (Treg) cells are essential for immunological tolerance and immune homeostasis. Despite a great deal of interest in modulating their number and function for the treatment of autoimmune disease or cancer, the precise mechanisms that control the homeostasis of Treg cells remain unclear. We report a new ENU-induced mutant mouse, lack of costimulation (loco), with atopic dermatitis and Treg cell deficiency typical of Card11 loss-of-function mutants. Three distinct single nucleotide variants were found in the Card11 introns 2, 10 and 20 that cause the loss of CARD11 expression in these mutant mice. These mutations caused the loss of thymic-derived, Neuropilin-1+ (NRP1+ ) Treg cells in neonatal and adult loco mice; however, residual peripherally induced NRP1- Treg cells remained. These peripherally generated Treg cells could be expanded in vivo by the administration of IL-2:anti-IL-2 complexes, indicating that this key homeostatic signaling axis remained intact in CARD11-deficient Treg cells. Furthermore, these expanded Treg cells could mediate near-normal suppression of activated, conventional CD4+ T cells, suggesting that CARD11 is dispensable for Treg cell function. In addition to shedding light on the requirements for CARD11 in Treg cell homeostasis and function, these data reveal novel noncoding Card11 loss-of-function mutations that impair the expression of this critical immune-regulatory protein.

IRF2 transcriptionally induces GSDMD expression for pyroptosis.

Gasdermin-D (GSDMD) is cleaved by caspase-1, caspase-4, and caspase-11 in response to canonical and noncanonical inflammasome activation. Upon cleavage, GSDMD oligomerizes and forms plasma membrane pores, resulting in interleukin-1ß (IL-1ß) secretion, pyroptotic cell death, and inflammatory pathologies, including periodic fever syndromes and septic shock-a plague on modern medicine. Here, we showed that IRF2, a member of the interferon regulatory factor (IRF) family of transcription factors, was essential for the transcriptional activation of GSDMD. A forward genetic screen with N-ethyl-N-nitrosourea (ENU)-mutagenized mice linked IRF2 to inflammasome signaling. GSDMD expression was substantially attenuated in IRF2-deficient macrophages, endothelial cells, and multiple tissues, which corresponded with reduced IL-1ß secretion and inhibited pyroptosis. Mechanistically, IRF2 bound to a previously uncharacterized but unique site within the GSDMD promoter to directly drive GSDMD transcription for the execution of pyroptosis. Disruption of this single IRF2-binding site abolished signaling by both the canonical and noncanonical inflammasomes. Together, our data illuminate a key transcriptional mechanism for expression of the gene encoding GSDMD, a critical mediator of inflammatory pathologies.

Deletion of self-reactive CCR7- thymocytes in the absence of MHC expression on thymic epithelial cells.

The selection of αß T cells in the thymus is punctuated by checkpoints at which thymocytes differentiate or undergo apoptosis. Wave 1 deletion is defined as apoptosis within nascent αß T-cell antigen receptor (TCR)-signalled thymocytes that lack CCR7 expression. The antigen-presenting cell (APC) types that mediate wave 1 deletion are unclear. To measure wave 1 deletion, we compared the frequencies of TCRß + CD5 + Helios + CCR7- cells in nascent thymocyte cohorts in mice with normal or defective apoptosis. This thymocyte population is small in mice lacking major histocompatibility complex (MHC) expression. The scale of wave 1 deletion was increased by transgenic expression of the self-reactive Yae62 TCRß chain, was almost halved when haemopoietic APCs lacked MHC expression and, surprisingly, was unchanged when epithelial cells lacked MHC expression. These findings demonstrate efficiency, and some redundancy, in the APC types that mediate wave 1 deletion in the normal mouse thymus.

DNA Hypermethylation Encroachment at CpG Island Borders in Cancer Is Predisposed by H3K4 Monomethylation Patterns.

Promoter CpG islands are typically unmethylated in normal cells, but in cancer a proportion are subject to hypermethylation. Using methylome sequencing we identified CpG islands that display partial methylation encroachment across the 5' or 3' CpG island borders. CpG island methylation encroachment is widespread in prostate and breast cancer and commonly associates with gene suppression. We show that the pattern of H3K4me1 at CpG island borders in normal cells predicts the different modes of cancer CpG island hypermethylation. Notably, genetic manipulation of Kmt2d results in concordant alterations in H3K4me1 levels and CpG island border DNA methylation encroachment. Our findings suggest a role for H3K4me1 in the demarcation of CpG island methylation borders in normal cells, which become eroded in cancer.

The Ubiquitin Ligase Adaptor NDFIP1 Selectively Enforces a CD8+ T Cell Tolerance Checkpoint to High-Dose Antigen.

Escape from peripheral tolerance checkpoints that control cytotoxic CD8+ T cells is important for cancer immunotherapy and autoimmunity, but pathways enforcing these checkpoints are mostly uncharted. We reveal that the HECT-type ubiquitin ligase activator, NDFIP1, enforces a cell-intrinsic CD8+ T cell checkpoint that desensitizes TCR signaling during in vivo exposure to high antigen levels. Ndfip1-deficient OT-I CD8+ T cells responding to high exogenous tolerogenic antigen doses that normally induce anergy aberrantly expanded and differentiated into effector cells that could precipitate autoimmune diabetes in RIP-OVAhi mice. In contrast, NDFIP1 was dispensable for peripheral deletion to low-dose exogenous or pancreatic islet-derived antigen and had little impact upon effector responses to Listeria or acute LCMV infection. These data provide evidence that NDFIP1 mediates a CD8+ T cell tolerance checkpoint, with a different mechanism to CD4+ T cells, and indicates that CD8+ T cell deletion and anergy are molecularly separable checkpoints.