L-plastin associated syndrome of immune deficiency and hematologic cytopenia.

BACKGROUND: LCP1 encodes L-plastin, an actin-bundling protein primarily expressed in hematopoietic cells. In mouse and fish models, LCP1 deficiency has been shown to result in hematologic and immune defects. OBJECTIVE: This study aimed to determine the nature of a human inborn error of immunity resulting from a novel genetic variant of LCP1. METHODS: We performed genetic, protein, and cellular analysis of PBMCs from a kindred with apparent autosomal dominant immune deficiency. We identified a candidate causal mutation in LCP1, which we evaluated by engineering the orthologous mutation in mice and Jurkat cells. RESULTS: A splice-site variant in LCP1 segregated with lymphopenia, neutropenia, and thrombocytopenia. The splicing defect resulted in at least 2 aberrant transcripts, producing an in-frame deletion of 24 nucleotides, and a frameshift deletion of exon 8. Cellular analysis of the kindred revealed a proportionate reduction of T and B cells and a mild expansion of transitional B cells. Similarly, mice carrying the orthologous genetic variant exhibited the same in-frame aberrant transcript, reduced expression Lcp1 and gene dose-dependent leukopenia, mild thrombocytopenia, and lymphopenia, with a significant reduction of T-cell populations. Functional analysis revealed that LCP1c740-1G>A confers a defect in platelet development and function with aberrant spreading on collagen. Immunologic analysis revealed defective actin organization in T cells, reduced migration of PBMCs from patients, splenocytes from mutant mice, and a mutant Jurkat cell line in response to CXCL12; impaired germinal center B-cell expansion after immunization; and reduced cytokinesis during T cell proliferation. CONCLUSIONS: We describe a unique human hematopoietic defect affecting neutrophils, lymphocytes, and platelets arising from partial LCP1 deficiency.

Rare SH2B3 coding variants in lupus patients impair B cell tolerance and predispose to autoimmunity.

Systemic lupus erythematosus (SLE) is a heterogeneous autoimmune disease with a clear genetic component. While most SLE patients carry rare gene variants in lupus risk genes, little is known about their contribution to disease pathogenesis. Amongst them, SH2B3-a negative regulator of cytokine and growth factor receptor signaling-harbors rare coding variants in over 5% of SLE patients. Here, we show that unlike the variant found exclusively in healthy controls, SH2B3 rare variants found in lupus patients are predominantly hypomorphic alleles, failing to suppress IFNGR signaling via JAK2-STAT1. The generation of two mouse lines carrying patients' variants revealed that SH2B3 is important in limiting the number of immature and transitional B cells. Furthermore, hypomorphic SH2B3 was shown to impair the negative selection of immature/transitional self-reactive B cells and accelerate autoimmunity in sensitized mice, at least in part due to increased IL-4R signaling and BAFF-R expression. This work identifies a previously unappreciated role for SH2B3 in human B cell tolerance and lupus risk.

Acquired Angioedema Associated with Lymphoproliferative Disorders.

Introduction: Acquired angioedema due to C1 esterase inhibitor deficiency (C1INH-AAE) is most associated with lymphoproliferative disorders (LPDs), particularly low-grade B-cell subtypes. The condition remains under-recognized with long diagnostic delays due to various challenges including a lack of awareness of the condition. Case Presentation: We discuss 4 cases of C1INH-AAE associated with low-grade B-cell LPDs, including various diagnostic and management challenges. As our cases illustrate, constitutional symptoms or overt manifestations of LPD at diagnosis are often absent. Hence, a comprehensive multimodal approach to screening for an underlying B-LPD is important when a diagnosis of acquired angioedema is made. Levels of complement C4, C1q, and C1INH are useful for diagnosing C1INH-AAE and for monitoring disease activity. Changes in these parameters may also indicate relapse of the underlying hematological malignancy. Treating the underlying disorder is important as this commonly leads to clinical improvement with decreased episodes of angioedema and normalization of complement studies. Conclusion: Awareness of C1INH-AAE can lead to an early diagnosis of hematological malignancies. The absence of constitutional symptoms emphasizes the need for a comprehensive multimodal approach to screening for LPD in C1INH-AAE. C4, C1INH level, and function are useful for monitoring disease activity.

DECTIN-1: A modifier protein in CTLA-4 haploinsufficiency.

Autosomal dominant loss-of-function (LoF) variants in cytotoxic T-lymphocyte associated protein 4 (CTLA4) cause immune dysregulation with autoimmunity, immunodeficiency and lymphoproliferation (IDAIL). Incomplete penetrance and variable expressivity are characteristic of IDAIL caused by CTLA-4 haploinsufficiency (CTLA-4h), pointing to a role for genetic modifiers. Here, we describe an IDAIL proband carrying a maternally inherited pathogenic CTLA4 variant and a paternally inherited rare LoF missense variant in CLEC7A, which encodes for the ß-glucan pattern recognition receptor DECTIN-1. The CLEC7A variant led to a loss of DECTIN-1 dimerization and surface expression. Notably, DECTIN-1 stimulation promoted human and mouse regulatory T cell (Treg) differentiation from naïve αß and γδ T cells, even in the absence of transforming growth factor-ß. Consistent with DECTIN-1's Treg-boosting ability, partial DECTIN-1 deficiency exacerbated the Treg defect conferred by CTL4-4h. DECTIN-1/CLEC7A emerges as a modifier gene in CTLA-4h, increasing expressivity of CTLA4 variants and acting in functional epistasis with CTLA-4 to maintain immune homeostasis and tolerance.

P2RY8 variants in lupus patients uncover a role for the receptor in immunological tolerance.

B cell self-tolerance is maintained through multiple checkpoints, including restraints on intracellular signaling and cell trafficking. P2RY8 is a receptor with established roles in germinal center (GC) B cell migration inhibition and growth regulation. Somatic P2RY8 variants are common in GC-derived B cell lymphomas. Here, we identify germline novel or rare P2RY8 missense variants in lupus kindreds or the related antiphospholipid syndrome, including a "de novo" variant in a child with severe nephritis. All variants decreased protein expression, F-actin abundance, and GPCR-RhoA signaling, and those with stronger effects increased AKT and ERK activity and cell migration. Remarkably, P2RY8 was reduced in B cell subsets from some SLE patients lacking P2RY8 gene variants. Low P2RY8 correlated with lupus nephritis and increased age-associated B cells and plasma cells. By contrast, P2RY8 overexpression in cells and mice restrained plasma cell development and reinforced negative selection of DNA-reactive developing B cells. These findings uncover a role of P2RY8 in immunological tolerance and lupus pathogenesis.

Skin manifestations of inborn errors of NF-κB.

More than 400 single gene defects have been identified as inborn errors of immunity, including many arising from genes encoding proteins that affect NF-κB activity. We summarise the skin phenotypes in this subset of disorders and provide an overview of pathogenic mechanisms. NF-κB acts cell-intrinsically in basal epithelial cells during differentiation of skin appendages, influences keratinocyte proliferation and survival, and both responses to and amplification of inflammation, particularly TNF. Skin phenotypes include ectodermal dysplasia, reduction and hyperproliferation of keratinocytes, and aberrant recruitment of inflammatory cells, which often occur in combination. Phenotypes conferred by these rare monogenic syndromes often resemble those observed with more common defects. This includes oral and perineal ulceration and pustular skin disease as occurs with Behcet's disease, hyperkeratosis with microabscess formation similar to psoriasis, and atopic dermatitis. Thus, these genotype-phenotype relations provide diagnostic clues for this subset of IEIs, and also provide insights into mechanisms of more common forms of skin disease.

Deletions in VANGL1 are a risk factor for antibody-mediated kidney disease.

We identify an intronic deletion in VANGL1 that predisposes to renal injury in high risk populations through a kidney-intrinsic process. Half of all SLE patients develop nephritis, yet the predisposing mechanisms to kidney damage remain poorly understood. There is limited evidence of genetic contribution to specific organ involvement in SLE.1,2 We identify a large deletion in intron 7 of Van Gogh Like 1 (VANGL1), which associates with nephritis in SLE patients. The same deletion occurs at increased frequency in an indigenous population (Tiwi Islanders) with 10-fold higher rates of kidney disease compared with non-indigenous populations. Vangl1 hemizygosity in mice results in spontaneous IgA and IgG deposition within the glomerular mesangium in the absence of autoimmune nephritis. Serum transfer into B cell-deficient Vangl1+/- mice results in mesangial IgG deposition indicating that Ig deposits occur in a kidney-intrinsic fashion in the absence of Vangl1. These results suggest that Vangl1 acts in the kidney to prevent Ig deposits and its deficiency may trigger nephritis in individuals with SLE.

Inborn Errors of Immunity and Their Phenocopies: CTLA4 and PD-1.

Elucidating links between genotype and phenotype in patients with rare inborn errors of immunity (IEIs) provides insights into mechanisms of immune regulation. In many autosomal dominant IEIs, however, variation in expressivity and penetrance result in complex genotype-phenotype relations, while some autosomal recessive IEIs are so rare that it is difficult to draw firm conclusions. Phenocopies arise when an environmental or non-genetic factor replicates a phenotype conferred by a specific genotype. Phenocopies can result from therapeutic antibodies or autoantibodies that target a protein to replicate aspects of the phenotype conferred by mutations in the gene encoding the same protein. Here, we consider IEIs arising from rare genetic variants in CTLA4 and PDCD1 and compare clinical and laboratory manifestations arising as drug-induced phenocopies (immune related adverse events, IRAEs) in cancer patients treated with immune checkpoint inhibitors (ICI) and identify outstanding questions regarding mechanism of disease.

Retrospective single-centre analysis of diagnostic approach to adult-onset haemophagocytic lymphohistiocytosis.

BACKGROUND: Haemophagocytic lymphohistiocytosis (HLH) is a rare disorder with a poor prognosis characterised by substantial immune activation leading to end-organ failure. In childhood, genetic defects that impair cytotoxic function of natural killer cells and T cells. (HLH) are often identified. In adults, clinical manifestations are similar to those observed in children but the aetiology is often unclear. AIMS: To evaluate whether poor prognosis for adult HLH is in part due to lack of awareness of the disorder, which results in incomplete investigation and failure to implement timely treatment. METHODS: We performed a retrospective case series of adult-onset HLH in a tertiary hospital in Australia. We evaluated clinical characteristics, treatment and outcome, and related these to application of standard diagnostic criteria for HLH. RESULTS: In our centre, incomplete assessment of HLH criteria was common. Serum ferritin was the criterion most commonly assessed. Hyperferritinaemia ≥10 000 µg/L was highly sensitive in detecting patients with adult-onset HLH; however, the majority of patients who had hyperferritinaemia ≥10 000 µg/L did not have adult-onset HLH. CONCLUSION: The present study highlights the importance of comprehensive application of diagnostic criteria to improve accuracy and timelines of the diagnosis of adult onset HLH.

A Dual-Antigen Enzyme-Linked Immunosorbent Assay Allows the Assessment of Severe Acute Respiratory Syndrome Coronavirus 2 Antibody Seroprevalence in a Low-Transmission Setting.

Estimates of seroprevalence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antibodies have been hampered by inadequate assay sensitivity and specificity. Using an enzyme-linked immunosorbent assay-based approach that combines data about immunoglobulin G responses to both the nucleocapsid and spike receptor binding domain antigens, we show that excellent sensitivity and specificity can be achieved. We used this assay to assess the frequency of virus-specific antibodies in a cohort of elective surgery patients in Australia and estimated seroprevalence in Australia to be 0.28% (95% Confidence Interval, 0-1.15%). These data confirm the low level of transmission of SARS-CoV-2 in Australia before July 2020 and validate the specificity of our assay.

A Decade Experience on Severe Combined Immunodeficiency Phenotype in Oman, Bridging to Newborn Screening.

Introduction: Severe combined immunodeficiency (SCID) results from various monogenic defects that impair immune function and brings on early severe and life-threatening infections. The main stay of treatment for SCID is hematopoietic stem cell transplant (HSCT) with near normal survival at 5 years for an early transplant done at or before the age of 3.5 months of life and the patient is maintained free of infections. Although overall rare, it constitutes a major burden on affected children, their families and on the health system especially in communities with a high rate of consanguinity where incidence and prevalence of recessive inborn errors of immunity (IEI) are expected to be high. Method: Here, we report the clinical, immunological, and molecular findings in 36 children diagnosed with SCID from a single tertiary center in Oman for the last decade. Results: We observed a median annual incidence rate of 4.5 per 100,000 Omani live births, and 91.7% of affected children were born to consanguineous parents. Twenty-three children (63.9%) fulfilled the criteria for typical SCID. The median age at onset, diagnosis and diagnostic delay were 54, 135, and 68 days, respectively. The most common clinical manifestations were pneumonia, septicemia, and chronic diarrhea. Eleven children (30.6%) have received hematopoietic stem cell transplant (HSCT) with a survival rate of 73%. The most frequent genetic cause of SCID in this cohort (n = 36) was (RAG-1), encoding for recombination activating gene (n = 5, 13.9%). Similarly, Major histocompatibility complex type II deficiency accounted for (n = 5, 13.9%) of our cohort. Conclusion: Our report broadens the knowledge of clinical and molecular manifestations in children with SCID in the region and highlights the need to initiate newborn based screening program (NBS) program.

STAT3 regulates cytotoxicity of human CD57+ CD4+ T cells in blood and lymphoid follicles.

A subset of human follicular helper T cells (TFH) cells expresses CD57 for which no distinct function has been identified. We show that CD57+ TFH cells are universally PD-1hi, but compared to their CD57- PD-1hi counterparts, express little IL-21 or IL-10 among others. Instead, CD57 expression on TFH cells marks cytotoxicity transcriptional signatures that translate into only a weak cytotoxic phenotype. Similarly, circulating PD-1+ CD57+ CD4+ T cells make less cytokine than their CD57- PD-1+ counterparts, but have a prominent cytotoxic phenotype. By analysis of responses to STAT3-dependent cytokines and cells from patients with gain- or loss-of-function STAT3 mutations, we show that CD4+ T cell cytotoxicity is STAT3-dependent. TFH formation also requires STAT3, but paradoxically, once formed, PD-1hi cells become unresponsive to STAT3. These findings suggest that changes in blood and germinal center cytotoxicity might be affected by changes in STAT3 signaling, or modulation of PD-1 by therapy.

Comparison of enzyme-linked immunosorbent assay and rapid chemiluminescent analyser in the detection of myeloperoxidase and proteinase 3 autoantibodies.

Antibodies to myeloperoxidase (MPO) and proteinase 3 (PR3) are vital in the diagnosis and management of ANCA-associated vasculitis. A chemiluminescent immunoassay (CLIA; Quanta Flash) provides MPO and PR3 antibody results in 30 minutes, which is much faster than enzyme-linked immunosorbent assay (ELISA). We compared the performance of ELISA (Orgentec) and CLIA (Quanta Flash) for MPO and PR3 antibody quantitation on 303 samples, comprising 196 consecutive samples received in a single diagnostic laboratory over a 3 month period, and 107 samples collected from 42 known vasculitis patients over a 40 month period. We observed a correlation between both methods using spearman correlation coefficients (MPO, rs = 0.63, p < 0.01; PR3, rs = 0.69, p < 0.01). There was agreement between both methods in determining a positive or negative result. In the vasculitis cohort, CLIA performed well at clinically important stages of disease; diagnosis (eight samples all positive by both assays) and disease relapse (correlation for both MPO and PR3 antibody quantitation rs = 0.84, p = 0.03 and rs = 0.78, p < 0.01, respectively). Three samples were discordant at clinical relapse, testing positive by CLIA, including one high positive associated with relapse requiring a change in treatment. In summary, CLIA appears to be at least as accurate as ELISA for measurement of MPO and PR3 antibodies.

Unique and shared signaling pathways cooperate to regulate the differentiation of human CD4+ T cells into distinct effector subsets.

Naive CD4(+) T cells differentiate into specific effector subsets-Th1, Th2, Th17, and T follicular helper (Tfh)-that provide immunity against pathogen infection. The signaling pathways involved in generating these effector cells are partially known. However, the effects of mutations underlying human primary immunodeficiencies on these processes, and how they compromise specific immune responses, remain unresolved. By studying individuals with mutations in key signaling pathways, we identified nonredundant pathways regulating human CD4(+) T cell differentiation in vitro. IL12Rß1/TYK2 and IFN-γR/STAT1 function in a feed-forward loop to induce Th1 cells, whereas IL-21/IL-21R/STAT3 signaling is required for Th17, Tfh, and IL-10-secreting cells. IL12Rß1/TYK2 and NEMO are also required for Th17 induction. Strikingly, gain-of-function STAT1 mutations recapitulated the impact of dominant-negative STAT3 mutations on Tfh and Th17 cells, revealing a putative inhibitory effect of hypermorphic STAT1 over STAT3. These findings provide mechanistic insight into the requirements for human T cell effector function, and explain clinical manifestations of these immunodeficient conditions. Furthermore, they identify molecules that could be targeted to modulate CD4(+) T cell effector function in the settings of infection, vaccination, or immune dysregulation.

Monogenic mutations differentially affect the quantity and quality of T follicular helper cells in patients with human primary immunodeficiencies.

BACKGROUND: Follicular helper T (TFH) cells underpin T cell-dependent humoral immunity and the success of most vaccines. TFH cells also contribute to human immune disorders, such as autoimmunity, immunodeficiency, and malignancy. Understanding the molecular requirements for the generation and function of TFH cells will provide strategies for targeting these cells to modulate their behavior in the setting of these immunologic abnormalities. OBJECTIVE: We sought to determine the signaling pathways and cellular interactions required for the development and function of TFH cells in human subjects. METHODS: Human primary immunodeficiencies (PIDs) resulting from monogenic mutations provide a unique opportunity to assess the requirement for particular molecules in regulating human lymphocyte function. Circulating follicular helper T (cTFH) cell subsets, memory B cells, and serum immunoglobulin levels were quantified and functionally assessed in healthy control subjects, as well as in patients with PIDs resulting from mutations in STAT3, STAT1, TYK2, IL21, IL21R, IL10R, IFNGR1/2, IL12RB1, CD40LG, NEMO, ICOS, or BTK. RESULTS: Loss-of-function (LOF) mutations in STAT3, IL10R, CD40LG, NEMO, ICOS, or BTK reduced cTFH cell frequencies. STAT3 and IL21/R LOF and STAT1 gain-of-function mutations skewed cTFH cell differentiation toward a phenotype characterized by overexpression of IFN-γ and programmed death 1. IFN-γ inhibited cTFH cell function in vitro and in vivo, as corroborated by hypergammaglobulinemia in patients with IFNGR1/2, STAT1, and IL12RB1 LOF mutations. CONCLUSION: Specific mutations affect the quantity and quality of cTFH cells, highlighting the need to assess TFH cells in patients by using multiple criteria, including phenotype and function. Furthermore, IFN-γ functions in vivo to restrain TFH cell-induced B-cell differentiation. These findings shed new light on TFH cell biology and the integrated signaling pathways required for their generation, maintenance, and effector function and explain the compromised humoral immunity seen in patients with some PIDs.

A deleterious RNF43 germline mutation in a severely affected serrated polyposis kindred.

We report a germline nonsense mutation within the extracellular domain of the RING finger ubiquitin ligase RNF43, segregating with a severe form of serrated polyposis within a kindred. The finding provides evidence that inherited RNF43 mutations define a familial cancer syndrome.

Naive and memory human B cells have distinct requirements for STAT3 activation to differentiate into antibody-secreting plasma cells.

Long-lived antibody memory is mediated by the combined effects of long-lived plasma cells (PCs) and memory B cells generated in response to T cell-dependent antigens (Ags). IL-10 and IL-21 can activate multiple signaling pathways, including STAT1, STAT3, and STAT5; ERK; PI3K/Akt, and potently promote human B cell differentiation. We previously showed that loss-of-function mutations in STAT3, but not STAT1, abrogate IL-10- and IL-21-mediated differentiation of human naive B cells into plasmablasts. We report here that, in contrast to naive B cells, STAT3-deficient memory B cells responded to these STAT3-activating cytokines, differentiating into plasmablasts and secreting high levels of IgM, IgG, and IgA, as well as Ag-specific IgG. This was associated with the induction of the molecular machinery necessary for PC formation. Mutations in IL21R, however, abolished IL-21-induced responses of both naive and memory human B cells and compromised memory B cell formation in vivo. These findings reveal a key role for IL-21R/STAT3 signaling in regulating human B cell function. Furthermore, our results indicate that the threshold of STAT3 activation required for differentiation is lower in memory compared with naive B cells, thereby identifying an intrinsic difference in the mechanism underlying differentiation of naive versus memory B cells.

IL-21 signalling via STAT3 primes human naive B cells to respond to IL-2 to enhance their differentiation into plasmablasts.

B-cell responses are guided by the integration of signals through the B-cell receptor (BCR), CD40, and cytokine receptors. The common γ chain (γc)-binding cytokine interleukin (IL)-21 drives humoral immune responses via STAT3-dependent induction of transcription factors required for plasma cell generation. We investigated additional mechanisms by which IL-21/STAT3 signaling modulates human B-cell responses by studying patients with STAT3 mutations. IL-21 strongly induced CD25 (IL-2Rα) in normal, but not STAT3-deficient, CD40L-stimulated naïve B cells. Chromatin immunoprecipitation confirmed IL2RA as a direct target of STAT3. IL-21-induced CD25 expression was also impaired on B cells from patients with IL2RG or IL21R mutations, confirming a requirement for intact IL-21R signaling in this process. IL-2 increased plasmablast generation and immunoglobulin secretion from normal, but not CD25-deficient, naïve B cells stimulated with CD40L/IL-21. IL-2 and IL-21 were produced by T follicular helper cells, and neutralizing both cytokines abolished the B-cell helper capacity of these cells. Our results demonstrate that IL-21, via STAT3, sensitizes B cells to the stimulatory effects of IL-2. Thus, IL-2 may play an adjunctive role in IL-21-induced B-cell differentiation. Lack of this secondary effect of IL-21 may amplify the humoral immunodeficiency in patients with mutations in STAT3, IL2RG, or IL21R due to impaired responsiveness to IL-21.