Integrating circulating T follicular memory cells and autoantibody repertoires for characterization of autoimmune disorders.

Introduction: Autoimmune diseases are heterogeneous and often lack specific or sensitive diagnostic tests. Increased percentages of CD4+CXCR5+PD1+ circulating T follicular helper (cTfh) cells and skewed distributions of cTfh subtypes have been associated with autoimmunity. However, cTfh cell percentages can normalize with immunomodulatory treatment despite persistent disease activity, indicating the need for identifying additional cellular and/or serologic features correlating with autoimmunity. Methods: The cohort included 50 controls and 56 patients with autoimmune cytopenias, gastrointestinal, pulmonary, and/or neurologic autoimmune disease. Flow cytometry was used to measure CD4+CXCR5+ T cell subsets expressing the chemokine receptors CXCR3 and/or CCR6: CXCR3+CCR6- Type 1, CXCR3-CCR6- Type 2, CXCR3+CCR6+ Type 1/17, and CXCR3- CCR6+ Type 17 T cells. IgG and IgA autoantibodies were quantified using a microarray featuring 1616 full-length, conformationally intact protein antigens. The 97.5th percentile in the control cohort defined normal limits for T cell subset percentages and total number (burden) of autoantibodies. Results: This study focused on CD4+CXCR5+ T cells because CXCR5 upregulation occurs after cognate T-B cell interactions characteristic of autoimmune diseases. We refer to these cells as circulating T follicular memory (cTfm) cells to acknowledge the dynamic nature of antigen-experienced CXCR5+ T cells, which encompass progenitors of cTfh or Tfh cells as well as early effector memory T cells that have not yet lost CXCR5. Compared to controls, 57.1% of patients had increased CXCR5+CXCR3+CCR6+ cTfm1/17 and 25% had increased CXCR5+CXCR3-CCR6+ cTfm17 cell percentages. Patients had significantly more diverse IgG and IgA autoantibodies than controls and 44.6% had an increased burden of autoantibodies of either isotype. Unsupervised autoantibody clustering identified three clusters of patients with IgG autoantibody profiles distinct from those of controls, enriched for patients with active autoimmunity and monogenic diseases. An increased percentage of cTfm17 cells was most closely associated with an increased burden of high-titer IgG and IgA autoantibodies. A composite measure integrating increased cTfm1/17, cTfm17, and high-titer IgG and/or IgA autoantibodies had 91.1% sensitivity and 90.9% specificity for identifying patients with autoimmunity. Percentages of cTfm1/17 and cTfm17 percentages and numbers of high-titer autoantibodies in patients receiving immunomodulatory treatment did not differ from those in untreated patients, thus suggesting that measurements of cTfm can complement measurements of other cellular markers affected by treatment. Conclusions: This study highlights two new approaches for assessing autoimmunity: measuring CD4+CXCR5+ cTfm subsets as well as total burden of autoantibodies. Our findings suggest that these approaches are particularly relevant to patients with rare autoimmune disorders for whom target antigens and prognosis are often unknown.

A homozygous stop-gain variant in ARHGAP42 is associated with childhood interstitial lung disease, systemic hypertension, and immunological findings.

ARHGAP42 encodes Rho GTPase activating protein 42 that belongs to a member of the GTPase Regulator Associated with Focal Adhesion Kinase (GRAF) family. ARHGAP42 is involved in blood pressure control by regulating vascular tone. Despite these findings, disorders of human variants in the coding part of ARHGAP42 have not been reported. Here, we describe an 8-year-old girl with childhood interstitial lung disease (chILD), systemic hypertension, and immunological findings who carries a homozygous stop-gain variant (c.469G>T, p.(Glu157Ter)) in the ARHGAP42 gene. The family history is notable for both parents with hypertension. Histopathological examination of the proband lung biopsy showed increased mural smooth muscle in small airways and alveolar septa, and concentric medial hypertrophy in pulmonary arteries. ARHGAP42 stop-gain variant in the proband leads to exon 5 skipping, and reduced ARHGAP42 levels, which was associated with enhanced RhoA and Cdc42 expression. This is the first report linking a homozygous stop-gain variant in ARHGAP42 with a chILD disorder, systemic hypertension, and immunological findings in human patient. Evidence of smooth muscle hypertrophy on lung biopsy and an increase in RhoA/ROCK signaling in patient cells suggests the potential mechanistic link between ARHGAP42 deficiency and the development of chILD disorder.

Expanded genetic insight and clinical experience of DNMT1-complex disorder.

OBJECTIVE: To report novel causal mutations, expanded clinical phenotypes, and clinical management of DNA methyltransferase 1 (DNMT1)-complex disorder. METHODS: Neurophysiologic testing, imaging, and genetic findings were summarized in clinical context for 5 cases with DNMT1-complex disorder. RESULTS: We identified 2 novel DNMT1 mutations (p.E510K and p.P1546A) by whole-exome sequencing (WES). Case 1 (p.E510K) presented with childhood ataxia, treatment-refractory seizures, and rapid cognitive decline in his 50s. Case 2 also had childhood onset and presented with seizures, language regression, hearing loss, narcolepsy with cataplexy symptoms, optic atrophy, sensory neuropathy, and hypogammaglobulinemia requiring IV immunoglobulin. Case 2 (p.P1546A) was identified with a de novo and the first mutation residing outside the targeting sequence domain. Case 3 (p.A570V) had paralytic asymmetric onset attacks triggered by emotionality and lasting sometimes for weeks. Neuropsychological testing showed executive dysfunction localizing to frontosubcortical and frontoparietal structures. He gradually developed left predominant brain atrophy. MRI showed T2 hyperintense lesions that enhanced on T1 postgadolinium images, and brain PET showed hypometabolism in atrophied regions. Case 4 (p.T497P) underwent left cochlear implant, resulting in significant hearing improvements at all tested frequencies (250-6,000 Hz). Case 5 (p.Y511H) had profound gait ataxia with posterior column atrophy of the spinal cord and abnormal evoked potentials primarily affecting the fasciculus gracilis. CONCLUSIONS: Broader application of WES further expands genotype-phenotype correlations of DNMT1-complex disorder. Two mutations are identified with early childhood onsets. The expanded new phenotypes include asymmetric brain hemiatrophy with parenchymal gadolinium enhancement, spinal cord atrophy, prolonged cataplectic spells, and hypogammaglobulinemia. Hearing loss treatment by cochlear implantation is helpful and should be considered.

Acetaminophen Inhibits the Neutrophil Oxidative Burst: Implications for Diagnostic Testing.

BACKGROUND: Chronic granulomatous disease is a primary immunodeficiency characterized by recurrent bacterial and fungal infections, granuloma formation, and inflammatory disease. Impaired neutrophil oxidative function is an essential diagnostic criterion. In vitro exposure of neutrophils to acetaminophen, a commonly used over-the-counter medication, has been associated with reduced neutrophil oxidative function. The clinical implications of acetaminophen intake for dihydrorhodamine (DHR) testing remain unknown. OBJECTIVE: To evaluate the effect of in vivo administration of therapeutic doses of acetaminophen on DHR diagnostic testing. METHODS: We performed DHR testing in 15 healthy adults before and after administering a single dose of acetaminophen. We retrospectively reviewed 195 DHR test results from hospitalized patients who had received acetaminophen, nonsteroidal anti-inflammatory drug, or corticosteroid before testing. RESULTS: DHR testing result was abnormal in 100% (n = 15) of healthy adults 2 hours after acetaminophen intake. We identified 195 instances of DHR testing less than or equal to 72 hours after acetaminophen ingestion in hospitalized patients who did not have chronic granulomatous disease. DHR results were abnormal in 43 of 195 cases (22.1%). Frequency of false-positive testing was increased in patients who received acetaminophen within 24 hours of testing, and in patients who received more than 1 dose of acetaminophen. Nonsteroidal anti-inflammatory drug and corticosteroid intakes were not associated with abnormal DHR result. CONCLUSIONS: Patients treated with acetaminophen have decreased neutrophil oxidative burst as measured by DHR testing. To avoid falsely abnormal testing for chronic granulomatous disease, patients should be advised to avoid acetaminophen for at least 24 hours before DHR testing.

An emerging role for endothelial barrier support therapy for congenital disorders of glycosylation.

Congenital disorders of glycosylation (CDGs) are clinically heterogeneous disorders defined by a decreased ability to modify biomolecules with oligosaccharides. Critical disruptions in protein recognition, interaction, binding, and anchoring lead to broad physiological effects. Patients present with endocrinopathy, immunodeficiency, hepatopathy, coagulopathy, and neurodevelopmental impairment. Patients may experience mortality/morbidity associated with shock physiology that is frequently culture negative and poorly responsive to standard care. Oedema, pleural and pericardial effusions, ascites, proteinuria, and protein-losing enteropathy are observed with an exaggerated inflammatory response. The negative serum protein steady state results from several mechanisms including reduced hepatic synthesis and secretion, increased consumption, and extravasation. Disruption of the glycocalyx, a layer of glycosylated proteins that lines the endothelium preventing thrombosis and extravasation, is a suspected cause of endothelial dysfunction in CDG patients. We performed a retrospective review of CDG patients admitted to our institution with acute illness over the past 2 years. Longitudinal clinical and laboratory data collected during the sick and well states were assessed for biomarkers of inflammation and efficacy of interventions. Six patients representing 4 CDG subtypes and 14 hospitalisations were identified. Acute D-dimer elevation, proteinuria, decreased serum total protein levels, coagulation proteins, and albumin were observed with acute illness. Infusion of fresh frozen plasma, and in some cases protein C concentrate, was associated with clinical and biomarker improvement. This was notable with intra-patient comparison of treated vs untreated courses. Use of endothelial barrier support therapy may reduce endothelial permeability by restoring both regulatory serum protein homeostasis and supporting the glycocalyx and is likely a critical component of care for this population.

Long-Term Outcome of Peanut Oral Immunotherapy Facilitated Initially by Omalizumab.

BACKGROUND: We successfully used omalizumab to facilitate peanut oral immunotherapy (OIT) in children with reactivity to ≤50mg peanut protein and with high peanut IgE (median, 229 kU/L). OBJECTIVE: We report on long-term OIT outcomes in these patients, including dosing changes, adverse events, peanut immunoglobulin changes, and quality of life (QoL). METHODS: Patients were followed for up to 72 months (67 months of maintenance). Outcomes were collected on peanut dose amount, form, and frequency, as well as adverse events, (QoL), and laboratory studies. RESULTS: Of 13 patients initially enrolled, 7 patients (54%) continued on peanut OIT through month 72; 6 (46%) discontinued therapy because of adverse reactions. Maintenance peanut protein dose varied between 500 and 3500mg. Most patients consumed different peanut-containing products. All patients experienced at least 1 adverse event, and 1 patient developed eosinophilic esophagitis. Peanut-IgE, Arah1-IgE and Arah2-IgE, peanut-SPT, peanut-IgE:IgE ratio, and Arah2-IgE:Arah2-IgG4 ratio decreased on OIT. Peanut-IgG4, Arah1-IgG4, and Arah2-IgG4 initially increased on OIT and then decreased, though not falling to baseline levels. In patients who stopped OIT, there was a trend for reversal of these biomarker changes. Higher peanut-IgE and Arah2-IgE at study month 12 were associated with discontinuation. Patient and parent QoL improved from baseline, even in patients who discontinued OIT. CONCLUSIONS: Although adjunctive omalizumab allowed for faster and successful desensitization in patients with high peanut-IgE, almost half of patients discontinued OIT within 72 months because of reactions. Patients who stopped therapy had higher month 12 peanut-IgE and Arah2-IgE. It is possible that these patients might benefit from longer omalizumab administration.

Acquired Cold-Induced Urticaria in Pediatric Patients: A 22-Year Experience in a Tertiary Care Center (1996-2017).

BACKGROUND: Acquired cold-induced urticaria (ACU) has not been well evaluated in pediatrics. OBJECTIVE: To further evaluate the presentation of ACU in children and associated risk of anaphylaxis. METHODS: A retrospective chart review was performed in children 18 years or younger diagnosed with ACU at Boston Children's Hospital (US, Northeast) from 1996 to 2017. RESULTS: A total of 415 patients with ACU were identified, aged 4 months to 18.3 years at the time of diagnosis, with similar male:female distribution. Most patients had a history of atopic disease (78.3%), and 25.8% had other urticaria. Around two-third of patients experienced only localized cold-induced symptoms (grade 1), whereas 14.0% had diffuse cutaneous symptoms (grade 2) as the most severe reaction, and 18.6% experienced anaphylaxis (grade 3). Swimming triggered 77.6% of grade 3 reactions, whereas the rest were secondary to ingestion of cold food or beverages, or cold air or cold water exposure. Seven percent of subjects had more than 1 episode of anaphylaxis. Cold stimulation test (CST) was performed in 61.7% of patients, and the result was positive in 69.9% of those tested. Positive CST result was significantly associated with increased risk of anaphylaxis. There was a 11.7% rate of anaphylaxis among patients with negative CST result. Disease resolution at any point in the study period was documented in 8.9% of patients and was associated with a negative history of anaphylaxis. CONCLUSIONS: In the largest study to date on ACU, grade 3 reactions occurred in about a fifth of patients. Positive CST result was associated with a higher risk for anaphylaxis from ACU. Epinephrine prescription and patient/family counseling about risk factors for grade 3 reactions are recommended.

A twenty-two-year experience with Hymenoptera venom immunotherapy in a US pediatric tertiary care center 1996-2018.

BACKGROUND: The rate of systemic reactions (SRs) to venom immunotherapy (VIT) in children has not been well evaluated. OBJECTIVE: To evaluate the rate of SRs to VIT in pediatric patients age 5 to 18 years who were treated with a standard protocol. METHODS: A retrospective chart review was conducted to identify patients who received VIT at Boston Children's Hospital from 1996 through 2018. Information on venom testing, severity of reaction to insect field sting, and SRs to VIT were retrieved. RESULTS: A total of 78 patients were included. Most had moderate to severe reactions to insect sting before VIT. The rate of SRs was 0.2% of injection visits, occurring in 9% of patients. The SRs from VIT were mild (mostly grade 1 and some grade 2), and no grades 3, 4, or 5 reactions were seen. Male sex was a significant risk factor for moderate to severe reactions to insect sting. Positive testing to vespinae was seen in 98.7% of patients, and none had exclusive sensitivity to honeybee. The severity of the initial, pre-VIT insect sting reactions in our patients did not correlate with the occurrence of SRs from VIT. Twenty-seven percent of the patients were subsequently stung while on VIT. Only 1 patient (5%) had a mild SR, while all others had only local or no reaction at all. CONCLUSION: In the largest US study evaluating the safety of VIT in children, SRs to VIT were mild, and none required epinephrine. Male sex was significantly associated with higher risk of moderate to severe reactions to insect sting. Larger multicenter studies are needed to further evaluate the rate of SRs to VIT in pediatric patients.

Patients with CD3G mutations reveal a role for human CD3γ in Treg diversity and suppressive function.

Integrity of the T-cell receptor/CD3 complex is crucial for positive and negative selection of T cells in the thymus and for effector and regulatory functions of peripheral T lymphocytes. In humans, CD3D, CD3E, and CD3Z gene defects are a cause of severe immune deficiency and present early in life with increased susceptibility to infections. By contrast, CD3G mutations lead to milder phenotypes, mainly characterized by autoimmunity. However, the role of CD3γ in establishing and maintaining immune tolerance has not been elucidated. In this manuscript, we aimed to investigate abnormalities of T-cell repertoire and function in patients with genetic defects in CD3G associated with autoimmunity. High throughput sequencing was used to study composition and diversity of the T-cell receptor ß (TRB) repertoire in regulatory T cells (Tregs), conventional CD4+ (Tconv), and CD8+ T cells from 6 patients with CD3G mutations and healthy controls. Treg function was assessed by studying its ability to suppress proliferation of Tconv cells. Treg cells of patients with CD3G defects had reduced diversity, increased clonality, and reduced suppressive function. The TRB repertoire of Tconv cells from patients with CD3G deficiency was enriched for hydrophobic amino acids at positions 6 and 7 of the CDR3, a biomarker of self-reactivity. These data demonstrate that the T-cell repertoire of patients with CD3G mutations is characterized by a molecular signature that may contribute to the increased rate of autoimmunity associated with this condition.

Effectiveness of a social robot, "Paro," in a VA long-term care setting.

Interest in animal assisted interventions (AAI) has grown over the years, but acceptance of AAI by the clinical and research community has been hampered by safety, hygiene, and logistical concerns. Advances in the field of social robotics have provided a promising route to deliver AAI while avoiding these aforementioned obstacles. Although there has been promising initial research on social robotics in older adults, to date there has been no such research conducted with a veteran population. The present pilot study followed 23 veteran residents of a Veterans Affairs (VA) geropsychiatric long-term care facility over the span of approximately a year and a half. It was found that use of Paro, a social robot, resulted in increased observed positive affective and behavioral indicators, with concomitant decreases observed in negative affective and behavioral indicators. The authors concluded that Paro is likely an effective nonpharmacological approach for managing dementia-related mood and behavior problems with veterans in VA long term care facilities. They additionally observed that Paro is best presented to residents who are relatively calm and approachable, as opposed to actively exhibiting behavior or mood problems. Future research directions are discussed in light of both the positive results noted and the inherent limitations of our pilot study. (PsycINFO Database Record

The Heterogeneity of Oral Immunotherapy Clinical Trials: Implications and Future Directions.

Food allergy is a potentially life-threatening disease which affects up to 8% of children and 2-3% of adults. Increasing food allergy prevalence poses a major public health concern. Induction of desensitization to food allergens through oral immunotherapy (OIT) is an expanding area of study encompassing peanut, egg, milk, and other food allergens. OIT consists of administering incremental doses of food allergen to food-allergic patients, to induce a state of desensitization. Safety, tolerability, and efficacy all remain ongoing concerns. Clinical trials for oral immunotherapy have encompassed many variations, including differences in dosage sizes and frequency, duration of build-up, type of allergen used, patient characteristics, and adjuvant therapies. Consequently, studies have also shown variation in rates of adverse effects, and successful desensitization. Here, we provide an overview of the key studies and discuss the implications of this heterogeneity. While desensitization is successful in the majority of patients, only a minority appear to develop sustained unresponsiveness even after years of therapy. Much larger and longitudinal studies using more homogenous protocols are needed in order to evaluate the clinical applicability of OIT, its long-term effectiveness, and effect on quality of life. The role of adjunctive therapies, including omalizumab and probiotics, requires further evaluation.

Recurrent viral infections associated with a homozygous CORO1A mutation that disrupts oligomerization and cytoskeletal association.

BACKGROUND: Coronin-1A (CORO1A) is a regulator of actin dynamics important for T-cell homeostasis. CORO1A deficiency causes T(-)B(+) natural killer-positive severe combined immunodeficiency or T-cell lymphopenia with severe viral infections. However, because all known human mutations in CORO1A abrogate protein expression, the role of the protein's functional domains in host immunity is unknown. OBJECTIVE: We sought to identify the cause of the primary immunodeficiency in 2 young adult siblings with a history of disseminated varicella, cutaneous warts, and CD4(+) T-cell lymphopenia. METHODS: We performed immunologic, genetic, and biochemical studies in the patients, family members, and healthy control subjects. RESULTS: Both patients had CD4(+) T-cell lymphopenia and decreased lymphocyte proliferation to mitogens. IgG, IgM, IgA, and specific antibody responses were normal. Whole-genome sequencing identified a homozygous frameshift mutation in CORO1A disrupting the last 2 C-terminal domains by replacing 61 amino acids with a novel 91-amino-acid sequence. The CORO1A(S401fs) mutant was expressed in the patients' lymphocytes at a level comparable with that of wild-type CORO1A in normal lymphocytes but did not oligomerize and had impaired cytoskeletal association. CORO1A(S401fs) was associated with increased filamentous actin accumulation in T cells, severely defective thymic output, and impaired T-cell survival but normal calcium flux and cytotoxicity, demonstrating the importance of CORO1A oligomerization and subcellular localization in T-cell homeostasis. CONCLUSIONS: We describe a truncating mutation in CORO1A that permits protein expression and survival into young adulthood. Our studies demonstrate the importance of intact CORO1A C-terminal domains in thymic egress and T-cell survival, as well as in defense against viral pathogens.

Broad-spectrum antibodies against self-antigens and cytokines in RAG deficiency.

Patients with mutations of the recombination-activating genes (RAG) present with diverse clinical phenotypes, including severe combined immune deficiency (SCID), autoimmunity, and inflammation. However, the incidence and extent of immune dysregulation in RAG-dependent immunodeficiency have not been studied in detail. Here, we have demonstrated that patients with hypomorphic RAG mutations, especially those with delayed-onset combined immune deficiency and granulomatous/autoimmune manifestations (CID-G/AI), produce a broad spectrum of autoantibodies. Neutralizing anti-IFN-α or anti-IFN-ω antibodies were present at detectable levels in patients with CID-G/AI who had a history of severe viral infections. As this autoantibody profile is not observed in a wide range of other primary immunodeficiencies, we hypothesized that recurrent or chronic viral infections may precipitate or aggravate immune dysregulation in RAG-deficient hosts. We repeatedly challenged Rag1S723C/S723C mice, which serve as a model of leaky SCID, with agonists of the virus-recognizing receptors TLR3/MDA5, TLR7/-8, and TLR9 and found that this treatment elicits autoantibody production. Altogether, our data demonstrate that immune dysregulation is an integral aspect of RAG-associated immunodeficiency and indicate that environmental triggers may modulate the phenotypic expression of autoimmune manifestations.

Leucine-rich repeat containing 8A (LRRC8A) is essential for T lymphocyte development and function.

Lrrc8a is a ubiquitously expressed gene that encodes a leucine-rich repeat (LRR)-containing protein detected at higher levels on the surface of thymocytes than on other immune cells. We generated Lrrc8a(-/-) mice to investigate the role of LRRC8A in lymphocyte development and function. Lrrc8a(-/-) mice had increased prenatal and postnatal mortality, growth retardation, and multiple tissue abnormalities. Lrrc8a(-/-) mice displayed a modest block in B cell development but intact intrinsic B cell function. In contrast, both Lrrc8a(-/-) mice and Lrrc8a(-/-)→Rag2(-/-) bone marrow chimeras exhibited a severe cell-intrinsic block in early thymic development, with decreased proliferation and increased apoptosis of thymocytes, and impaired peripheral T cell function. Thymic epithelial cells expressed an LRRC8A ligand that was critical for double-negative to double-positive thymocyte differentiation and survival in vitro. LRRC8A constitutively associated with the GRB2-GAB2 complex and lymphocyte-specific protein tyrosine kinase (LCK) in thymocytes. LRRC8A ligation activated AKT via the LCK-ZAP-70-GAB2-PI3K pathway, and AKT phosphorylation was markedly reduced in the thymus of Lrrc8a(-/-) mice. These findings reveal an essential role for LRRC8A in T cell development, survival, and function.

Disruption of MHC class II-restricted antigen presentation by vaccinia virus.

Vaccinia virus (VV), currently used in humans as a live vaccine for smallpox, can interfere with host immunity via several discrete mechanisms. In this study, the effect of VV on MHC class II-mediated Ag presentation was investigated. Following VV infection, the ability of professional and nonprofessional APC to present Ag and peptides to CD4+ T cells was impaired. Viral inhibition of class II Ag presentation could be detected within 1 h, with diminished T cell responses dependent upon the duration of APC infection and virus titer. Exposure of APC to replication-deficient virus also diminished class II Ag presentation. Virus infection of APC perturbed Ag presentation by newly synthesized and recycling class II molecules, with disruptions in both exogenous and cytoplasmic Ag presentation. Virus-driven expression of an endogenous Ag, failed to restore T cell responsiveness specific for this Ag in the context of MHC class II molecules. Yet, both class II protein steady-state and cell surface expression were not altered by VV. Biochemical and functional analysis revealed that VV infection directly interfered with ligand binding to class II molecules. Together, these observations suggest that disruption of MHC class II-mediated Ag presentation may be one of multiple strategies VV has evolved to escape host immune surveillance.