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.

DOCK2-deficiency causes defects in anti-viral T cell responses and impaired control of herpes simplex virus infection.

The expanding number of rare immunodeficiency syndromes offers an opportunity to understand key genes that support immune defence against infectious diseases. However, analysis of these in patients is complicated by their treatments and co-morbid infections requiring the use of mouse models for detailed investigations. Here we develop a mouse model of DOCK2 immunodeficiency and demonstrate that these mice have delayed clearance of herpes simplex virus type 1 (HSV-1) infections. We also uncovered a critical, cell intrinsic role of DOCK2 in the priming of anti-viral CD8+ T cells and in particular their initial expansion, despite apparently normal early activation of these cells. When this defect was overcome by priming in vitro, DOCK2-deficient CD8+ T cells were surprisingly protective against HSV-1-disease, albeit not as effectively as wild type cells. These results shed light on a cellular deficiency that is likely to impact anti-viral immunity in DOCK2-deficient patients.

DOCK2-deficiency causes defects in anti-viral T cell responses and poor control of herpes simplex virus infection.

The expanding number of rare immunodeficiency syndromes offers an opportunity to understand key genes that support immune defence against infectious diseases. However, patients with these diseases are by definition rare. In addition, any analysis is complicated by treatments and co-morbid infections requiring the use of mouse models for detailed investigations. Here we develop a mouse model of DOCK2 immunodeficiency and demonstrate that these mice have delayed clearance of herpes simplex virus type 1 (HSV-1) infections. Further, we found that they have a critical, cell intrinsic role of DOCK2 in the clonal expansion of anti-viral CD8+ T cells despite normal early activation of these cells. Finally, while the major deficiency is in clonal expansion, the ability of primed and expanded DOCK2-deficient CD8+ T cells to protect against HSV-1-infection is also compromised. These results provide a contributing cause for the frequent and devastating viral infections seen in DOCK2-deficient patients and improve our understanding of anti-viral CD8+ T cell immunity.

Neutrophil extracellular traps and their histones promote Th17 cell differentiation directly via TLR2.

Neutrophils perform critical functions in the innate response to infection, including through the production of neutrophil extracellular traps (NETs) - web-like DNA structures which are extruded from neutrophils upon activation. Elevated levels of NETs have been linked to autoimmunity but this association is poorly understood. By contrast, IL-17 producing Th17 cells are a key player in various autoimmune diseases but are also crucial for immunity against fungal and bacterial infections. Here we show that NETs, through their protein component histones, directly activate T cells and specifically enhance Th17 cell differentiation. This modulatory role of neutrophils, NETs and their histones is mediated downstream of TLR2 in T cells, resulting in phosphorylation of STAT3. The innate stimulation of a specific adaptive immune cell subset provides an additional mechanism demonstrating a direct link between neutrophils, NETs and T cell autoimmunity.

Goat's milk allergy in a family following household sensitization to goat's milk soap.

Epicutaneous sensitization to food allergens is a recently recognised phenomenon. However, there is less understanding around necessary or sufficient cofactors that permit sensitization through this route. In this report, we describe the development of goat's milk allergy in members of a household with pre-existing atopic dermatitis, following use of a goat's milk soap marketed for sensitive skin. Soaps appear to be an effective vehicle for sensitization to food antigens and the risks are potentiated in those with compromised barrier function. Although such products may be marketed for atopic dermatitis, we would advocate for the avoidance of food-based skin care products in this patient group.

DOCK8 deficiency diminishes thymic T-regulatory cell development but not thymic deletion.

OBJECTIVE: To define the effect of DOCK8 deficiency on thymic tolerance in mice. METHODS: Thymocytes from wild-type (Dock8+/+ ) and DOCK8-deficient (Dock8pri/pri ) mice were examined by flow cytometry. Some mice had transgenic expression of the BCL2 anti-apoptotic protein in haemopoietic cells. Some mice expressed the transgenic 3A9 T-cell receptor (TCR), which triggers thymocyte deletion in mice also expressing hen egg lysozyme under the insulin promoter. RESULTS: In Dock8pr/pri mice, the proportion of thymocytes induced to acquire tolerance at the immature CCR7- stage was normal. Deletion of strongly self-reactive CD4+ thymocytes occurred efficiently in Dock8pri/pri mice in a TCR-transgenic model that requires self-antigen transfer from epithelial cells to bone marrow (BM)-derived antigen-presenting cells. Thymic Foxp3+ T-regulatory cells (TREG) and Helios+ Foxp3- TREG precursors were decreased in Dock8pri/pri mice, including when apoptosis was inhibited by BCL2 transgene expression. Dock8pri/pri thymic TREG expressed CD25 and CTLA-4 at normal levels. The results suggest that DOCK8 deficiency does not affect the function of BM-derived antigen-presenting cells in the thymus, the TCR self-reactivity threshold that activates tolerance mechanisms in thymocytes or the apoptotic deletion of these thymocytes. However, DOCK8 is required to prevent a subset of developing TREG cells from undergoing cell death via a mechanism that is distinct from apoptosis. CONCLUSION: DOCK8 deficiency diminishes TREG development in the thymus without compromising thymocyte deletion.

The significance of ANCA positivity in patients with inflammatory bowel disease.

Traditionally anti-neutrophil cytoplasmic antibodies (ANCA) are used to subtype patients with inflammatory bowel disease (IBD) and to predict primary sclerosing cholangitis (PSC). The clinical utility of this testing in the Australian context is not known. Our retrospective, cross-sectional study looked at the results of ANCA testing performed during routine clinical review and aimed to retrospectively review (1) the distribution of different ANCA subtypes for IBD patients, (2) the temporal change of ANCA status, and (3) the predictive value of ANCA for PSC. Sixty-four IBD patients attending our hospital gastroenterology clinic between 2012 and 2016 had at least one ANCA test requested. Surprisingly, >80% of the IBD patients in our cohort who underwent ANCA testing had a positive ANCA result and a significant proportion had positive PR3 antibodies. However, no specific ANCA pattern predicted a specific IBD subtype or clinical course. Pairing ANCA and anti-Saccharomyces cerevisiae (ASCA) did not add value in subtyping IBD for these patients. Our study suggests that there is little value in ordering an ANCA for patients with IBD.

Protection from EAE in DOCK8 mutant mice occurs despite increased Th17 cell frequencies in the periphery.

Mutation of Dedicator of cytokinesis 8 (DOCK8) has previously been reported to provide resistance to the Th17 cell dependent EAE in mice. Contrary to expectation, we observed an elevation of Th17 cells in two different DOCK8 mutant mouse strains in the steady state. This was specific for Th17 cells with no change in Th1 or Th2 cell populations. In vitro Th cell differentiation assays revealed that the elevated Th17 cell population was not due to a T cell intrinsic differentiation bias. Challenging these mutant mice in the EAE model, we confirmed a resistance to this autoimmune disease with Th17 cells remaining elevated systemically while cellular infiltration in the CNS was reduced. Infiltrating T cells lost the bias toward Th17 cells indicating a relative reduction of Th17 cells in the CNS and a Th17 cell specific migration disadvantage. Adoptive transfers of Th1 and Th17 cells in EAE-affected mice further supported the Th17 cell-specific migration defect, however, DOCK8-deficient Th17 cells expressed normal Th17 cell-specific CCR6 levels and migrated toward chemokine gradients in transwell assays. This study shows that resistance to EAE in DOCK8 mutant mice is achieved despite a systemic Th17 bias.

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.

DOCK8 Drives Src-Dependent NK Cell Effector Function.

Mutations in the dedicator of cytokinesis 8 (DOCK8) gene cause an autosomal recessive form of hyper-IgE syndrome, characterized by chronic immunodeficiency with persistent microbial infection and increased incidence of malignancy. These manifestations suggest a defect in cytotoxic lymphocyte function and immune surveillance. However, how DOCK8 regulates NK cell-driven immune responses remains unclear. In this article, we demonstrate that DOCK8 regulates NK cell cytotoxicity and cytokine production in response to target cell engagement or receptor ligation. Genetic ablation of DOCK8 in human NK cells attenuated cytokine transcription and secretion through inhibition of Src family kinase activation, particularly Lck, downstream of target cell engagement or NKp30 ligation. PMA/Ionomycin treatment of DOCK8-deficient NK cells rescued cytokine production, indicating a defect proximal to receptor ligation. Importantly, NK cells from DOCK8-deficient patients had attenuated production of IFN-γ and TNF-α upon NKp30 stimulation. Taken together, we reveal a novel molecular mechanism by which DOCK8 regulates NK cell-driven immunity.

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.

DOCK8 regulates signal transduction events to control immunity.

Genetic mutations in the gene encoding DOCK8 cause an autosomal recessive form of hyper immunoglobulin E syndrome (AR-HIES), referred to as DOCK8 deficiency. DOCK8 deficiency in humans results in the onset of combined immunodeficiency disease (CID), clinically associated with chronic infections with diverse microbial pathogens, and a predisposition to malignancy. It is now becoming clear that DOCK8 regulates the function of diverse immune cell sub-types, particularly lymphocytes, to drive both innate and adaptive immune responses. Early studies demonstrated that DOCK8 is required for lymphocyte survival, migration and immune synapse formation, which translates to poor pathogen control in the absence of DOCK8. However, more recent advances have pointed to a crucial role for DOCK8 in regulating the signal transduction events that control transcriptional activity, cytokine production and functional polarization of immune cells. Here, we summarize recent advances in our understanding of DOCK8 function, paying particular attention to an emerging role as a signaling intermediate to promote immune responses to diverse external stimuli.

Dedicator of cytokinesis 8-deficient CD4+ T cells are biased to a TH2 effector fate at the expense of TH1 and TH17 cells.

BACKGROUND: Dedicator of cytokinesis 8 (DOCK8) deficiency is a combined immunodeficiency caused by autosomal recessive loss-of-function mutations in DOCK8. This disorder is characterized by recurrent cutaneous infections, increased serum IgE levels, and severe atopic disease, including food-induced anaphylaxis. However, the contribution of defects in CD4+ T cells to disease pathogenesis in these patients has not been thoroughly investigated. OBJECTIVE: We sought to investigate the phenotype and function of DOCK8-deficient CD4+ T cells to determine (1) intrinsic and extrinsic CD4+ T-cell defects and (2) how defects account for the clinical features of DOCK8 deficiency. METHODS: We performed in-depth analysis of the CD4+ T-cell compartment of DOCK8-deficient patients. We enumerated subsets of CD4+ T helper cells and assessed cytokine production and transcription factor expression. Finally, we determined the levels of IgE specific for staple foods and house dust mite allergens in DOCK8-deficient patients and healthy control subjects. RESULTS: DOCK8-deficient memory CD4+ T cells were biased toward a TH2 type, and this was at the expense of TH1 and TH17 cells. In vitro polarization of DOCK8-deficient naive CD4+ T cells revealed the TH2 bias and TH17 defect to be T-cell intrinsic. Examination of allergen-specific IgE revealed plasma IgE from DOCK8-deficient patients is directed against staple food antigens but not house dust mites. CONCLUSION: Investigations into the DOCK8-deficient CD4+ T cells provided an explanation for some of the clinical features of this disorder: the TH2 bias is likely to contribute to atopic disease, whereas defects in TH1 and TH17 cells compromise antiviral and antifungal immunity, respectively, explaining the infectious susceptibility of DOCK8-deficient patients.

Rituximab in autoimmune diseases.

Rituximab is a monoclonal antibody that depletes B cells from the circulation. It was originally used to treat lymphoma but is increasingly used for the treatment of autoimmune diseases. Rituximab was found to be effective in randomised controlled trials for rheumatoid arthritis, granulomatosis with polyangiitis and other antineutrophil cytoplasmic antibody-associated vasculitides. However, evidence of efficacy is very limited for many other autoimmune conditions. Before starting rituximab, it is important to check the patient's baseline immunoglobulins and immunisation status. Patients should also be screened for latent infections and other contraindications.

A DOCK8-WIP-WASp complex links T cell receptors to the actin cytoskeleton.

Wiskott-Aldrich syndrome (WAS) is associated with mutations in the WAS protein (WASp), which plays a critical role in the initiation of T cell receptor-driven (TCR-driven) actin polymerization. The clinical phenotype of WAS includes susceptibility to infection, allergy, autoimmunity, and malignancy and overlaps with the symptoms of dedicator of cytokinesis 8 (DOCK8) deficiency, suggesting that the 2 syndromes share common pathogenic mechanisms. Here, we demonstrated that the WASp-interacting protein (WIP) bridges DOCK8 to WASp and actin in T cells. We determined that the guanine nucleotide exchange factor activity of DOCK8 is essential for the integrity of the subcortical actin cytoskeleton as well as for TCR-driven WASp activation, F-actin assembly, immune synapse formation, actin foci formation, mechanotransduction, T cell transendothelial migration, and homing to lymph nodes, all of which also depend on WASp. These results indicate that DOCK8 and WASp are in the same signaling pathway that links TCRs to the actin cytoskeleton in TCR-driven actin assembly. Further, they provide an explanation for similarities in the clinical phenotypes of WAS and DOCK8 deficiency.

'Epinephrine-resistant' angioedema.

A man in his 60s was brought to the emergency department, with airway compromise and dysarthria due to a grossly enlarged tongue. As he was on a current course of antibiotics, he was treated for a likely antibiotic-associated allergic reaction. However, as he failed to improve with intramuscular and nebulised epinephrine, another cause of his symptoms was sought. Further discussion revealed a history of chronic lymphocytic leukaemia (CLL), which had recently relapsed. Investigations were ordered to confirm that the symptoms were due to acquired angioedema, and the patient was managed for this diagnosis based on the presence of an undetectable C4 level. This diagnosis was later confirmed when the results of specialist tests became available. The patient was treated for his relapsed CLL with good effect, and has had no further episodes of angioedema and an improvement in the level of his C1 esterase protein level and function.

Delayed control of herpes simplex virus infection and impaired CD4(+) T-cell migration to the skin in mouse models of DOCK8 deficiency.

DOCK8 deficiency in humans and mice leads to multiple defects in immune cell numbers and function. Patients with this immunodeficiency have a high morbidity and mortality, and are distinguished by chronic cutaneous viral infections, including those caused by herpes simplex virus (HSV). The underlying mechanism of the specific susceptibility to these chronic cutaneous viral infections is currently unknown, largely because the effect of DOCK8 deficiency has not been studied in suitable models. A better understanding of these mechanisms is required to underpin the development of more specific therapies. Here we show that DOCK8-deficient mice have poor control of primary cutaneous herpes simplex lesions and this is associated with increased virus loads. Furthermore, DOCK8-deficient mice showed a lack of CD4(+) T-cell infiltration into HSV-infected skin.