Clinical utility of measuring CD4+ T follicular cells in patients with immune dysregulation.

Mechanistic studies of autoimmune disorders have identified circulating T follicular helper (cTfh) cells as drivers of autoimmunity. However, the quantification of cTfh cells is not yet used in clinical practice due to the lack of age-stratified normal ranges and the unknown sensitivity and specificity of this test for autoimmunity. We enrolled 238 healthy participants and 130 patients with common and rare disorders of autoimmunity or autoinflammation. Patients with infections, active malignancy, or any history of transplantation were excluded. In 238 healthy controls, median cTfh percentages (range 4.8%-6.2%) were comparable among age groups, sexes, races, and ethnicities, apart from a significantly lower percentages in children less than 1 year of age (median 2.1%, CI: 0.4%-6.8, p < 0.0001). Among 130 patients with over 40 immune regulatory disorders, a cTfh percentage exceeding 12% had 88% sensitivity and 94% specificity for differentiating disorders with adaptive immune cell dysregulation from those with predominantly innate cell defects. This threshold had a sensitivity of 86% and specificity of 100% for active autoimmunity and normalized with effective treatment. cTfh percentages exceeding 12% distinguish autoimmunity from autoinflammation, thereby differentiating two endotypes of immune dysregulation with overlapping symptoms and different therapies.

Clinical utility of measuring CD4 + T follicular cells in patients with immune dysregulation.

Mechanistic studies of autoimmune disorders have identified circulating T follicular helper (cTfh) cells as drivers of autoimmunity. However, the quantification of cTfh cells is not yet used in clinical practice due to the lack of age-stratified normal ranges and the unknown sensitivity and specificity of this test for autoimmunity. We enrolled 238 healthy participants and 130 patients with common and rare disorders of autoimmunity or autoinflammation. Patients with infections, active malignancy, or any history of transplantation were excluded. In 238 healthy controls, median cTfh percentages (range 4.8% - 6.2%) were comparable among age groups, sexes, races, and ethnicities, apart from a significantly lower percentages in children less than 1 year of age (median 2.1%, CI: 0.4% - 6.8, p< 0.0001). Among 130 patients with over 40 immune regulatory disorders, a cTfh percentage exceeding 12% had 88% sensitivity and 94% specificity for differentiating disorders with adaptive immune cell dysregulation from those with predominantly innate cell defects. This threshold had a sensitivity of 86% and specificity of 100% for active autoimmunity and normalized with effective treatment. cTfh percentages exceeding 12% distinguish autoimmunity from autoinflammation, thereby differentiating two endotypes of immune dysregulation with overlapping symptoms and different therapies.

Comparison of disease phenotypes and mechanistic insight on causal variants in patients with DADA2.

BACKGROUND: Deficiency of adenosine deaminase 2 (DADA2) results in heterogeneous manifestations including systemic vasculitis and red cell aplasia. The basis of different disease phenotypes remains incompletely defined. OBJECTIVE: We sought to further delineate disease phenotypes in DADA2 and define the mechanistic basis of ADA2 variants. METHODS: We analyzed the clinical features and ADA2 variants in 33 patients with DADA2. We compared the transcriptomic profile of 14 patients by bulk RNA sequencing. ADA2 variants were expressed experimentally to determine impact on protein production, trafficking, release, and enzymatic function. RESULTS: Transcriptomic analysis of PBMCs from DADA2 patients with the vasculitis phenotype or pure red cell aplasia phenotype exhibited similar upregulation of TNF, type I interferon, and type II interferon signaling pathways compared with healthy controls. These pathways were also activated in 3 asymptomatic individuals with DADA2. Analysis of ADA2 variants, including 7 novel variants, showed different mechanisms of functional disruption including (1) unstable transcript leading to RNA degradation; (2) impairment of ADA2 secretion because of retention in the endoplasmic reticulum; (3) normal expression and secretion of ADA2 that lacks enzymatic function; and (4) disruption of the N-terminal signal peptide leading to cytoplasmic localization of unglycosylated protein. CONCLUSIONS: Transcriptomic signatures of inflammation are observed in patients with different disease phenotypes, including some asymptomatic individuals. Disease-associated ADA2 variants affect protein function by multiple mechanisms, which may contribute to the clinical heterogeneity of DADA2.

Spontaneous resolution of severe idiopathic T cell lymphopenia.

Potential etiologies of T-B+NK+ SCID include both hematopoietic defects and thymic aplasia. The management of patients with this phenotype, identified by newborn screen, may be unclear in the absence of a genetic diagnosis. We report an infant with lymphocyte flow cytometry consistent with T-B+NK+ SCID and reduced proliferative response to phytohemagglutinin. The patient had no genetic diagnosis after targeted panel and exome sequencing. The decision to trend laboratory values rather than move immediately to hematopoietic cell transplant was made given the absence of a genetic defect and the finding of a normal thymus on ultrasound. During the course of evaluation for transplant, the patient unexpectedly had normalization of T cell number and function. This case demonstrates a role for mediastinal ultrasound and the utility of trending laboratory values in patients with severe T cell lymphopenia but no genetic diagnosis, given the small but important possibility of spontaneous resolution.

Therapeutic options for CTLA-4 insufficiency.

BACKGROUND: Heterozygous germline mutations in cytotoxic T lymphocyte-associated antigen-4 (CTLA4) impair the immunomodulatory function of regulatory T cells. Affected individuals are prone to life-threatening autoimmune and lymphoproliferative complications. A number of therapeutic options are currently being used with variable effectiveness. OBJECTIVE: Our aim was to characterize the responsiveness of patients with CTLA-4 insufficiency to specific therapies and provide recommendations for the diagnostic workup and therapy at an organ-specific level. METHODS: Clinical features, laboratory findings, and response to treatment were reviewed retrospectively in an international cohort of 173 carriers of CTLA4 mutation. Patients were followed between 2014 and 2020 for a total of 2624 months from diagnosis. Clinical manifestations were grouped on the basis of organ-specific involvement. Medication use and response were recorded and evaluated. RESULTS: Among the 173 CTLA4 mutation carriers, 123 (71%) had been treated for immune complications. Abatacept, rituximab, sirolimus, and corticosteroids ameliorated disease severity, especially in cases of cytopenias and lymphocytic organ infiltration of the gut, lungs, and central nervous system. Immunoglobulin replacement was effective in prevention of infection. Only 4 of 16 patients (25%) with cytopenia who underwent splenectomy had a sustained clinical response. Cure was achieved with stem cell transplantation in 13 of 18 patients (72%). As a result of the aforementioned methods, organ-specific treatment pathways were developed. CONCLUSION: Systemic immunosuppressants and abatacept may provide partial control but require ongoing administration. Allogeneic hematopoietic stem cell transplantation offers a possible cure for patients with CTLA-4 insufficiency.

Multisystem inflammation and susceptibility to viral infections in human ZNFX1 deficiency.

BACKGROUND: Recognition of viral nucleic acids is one of the primary triggers for a type I interferon-mediated antiviral immune response. Inborn errors of type I interferon immunity can be associated with increased inflammation and/or increased susceptibility to viral infections as a result of dysbalanced interferon production. NFX1-type zinc finger-containing 1 (ZNFX1) is an interferon-stimulated double-stranded RNA sensor that restricts the replication of RNA viruses in mice. The role of ZNFX1 in the human immune response is not known. OBJECTIVE: We studied 15 patients from 8 families with an autosomal recessive immunodeficiency characterized by severe infections by both RNA and DNA viruses and virally triggered inflammatory episodes with hemophagocytic lymphohistiocytosis-like disease, early-onset seizures, and renal and lung disease. METHODS: Whole exome sequencing was performed on 13 patients from 8 families. We investigated the transcriptome, posttranscriptional regulation of interferon-stimulated genes (ISGs) and predisposition to viral infections in primary cells from patients and controls stimulated with synthetic double-stranded nucleic acids. RESULTS: Deleterious homozygous and compound heterozygous ZNFX1 variants were identified in all 13 patients. Stimulation of patient-derived primary cells with synthetic double-stranded nucleic acids was associated with a deregulated pattern of expression of ISGs and alterations in the half-life of the mRNA of ISGs and also associated with poorer clearance of viral infections by monocytes. CONCLUSION: ZNFX1 is an important regulator of the response to double-stranded nucleic acids stimuli following viral infections. ZNFX1 deficiency predisposes to severe viral infections and a multisystem inflammatory disease.

Ten Years of Newborn Screening for Severe Combined Immunodeficiency (SCID) in Massachusetts.

BACKGROUND: Massachusetts began newborn screening (NBS) for severe combined immunodeficiency (SCID) using measurement of T-cell receptor excision circles (TRECs) from dried blood spots. OBJECTIVE: We describe developments and outcomes from the first 10 years of this program (February 1, 2009, to January 31, 2019). METHODS: TREC values, diagnostic, and outcome data from all patients screened for SCID were evaluated. RESULTS: NBS of 720,038 infants prompted immunologic evaluation of 237 (0.03%). Of 237, 9 were diagnosed with SCID/leaky SCID (4% of referrals vs 0.001% general population). Another 7 were diagnosed with other combined immunodeficiencies, and 3 with athymia. SCID/leaky SCID incidence was approximately 1 in 80,000, whereas approximately 1 in 51,000 had severe T-cell lymphopenia for which definitive treatment was indicated. All patients with SCID/leaky SCID underwent hematopoietic cell transplant or gene therapy with 100% survival. One patient with athymia underwent successful thymus transplant. No known cases of SCID were missed. Compared with outcomes from the 10 years before SCID NBS, survival trended higher (9 of 9 vs 4 of 7), likely due to a lower rate of infection before treatment. CONCLUSIONS: Our data support a single NBS testing-and-referral algorithm for all gestational ages. Despite lower median TREC values in premature infants, the majority for all ages are well above the TREC cutoff and the algorithm, which selects urgent (undetectable TREC) and repeatedly abnormal TREC values, minimizes referral. We also found that low naïve T-cell percentage is associated with a higher risk of SCID/CID, demonstrating the utility of memory/naïve T-cell phenotyping as part of follow-up flow cytometry.

Distinct clinical and immunological features of SARS-CoV-2-induced multisystem inflammatory syndrome in children.

BACKGROUNDPediatric SARS-CoV-2 infection can be complicated by a dangerous hyperinflammatory condition termed multisystem inflammatory syndrome in children (MIS-C). The clinical and immunologic spectrum of MIS-C and its relationship to other inflammatory conditions of childhood have not been studied in detail.METHODSWe retrospectively studied confirmed cases of MIS-C at our institution from March to June 2020. The clinical characteristics, laboratory studies, and treatment response were collected. Data were compared with historic cohorts of Kawasaki disease (KD) and macrophage activation syndrome (MAS).RESULTSTwenty-eight patients fulfilled the case definition of MIS-C. Median age at presentation was 9 years (range: 1 month to 17 years); 50% of patients had preexisting conditions. All patients had laboratory confirmation of SARS-CoV-2 infection. Seventeen patients (61%) required intensive care, including 7 patients (25%) who required inotrope support. Seven patients (25%) met criteria for complete or incomplete KD, and coronary abnormalities were found in 6 cases. Lymphopenia, thrombocytopenia, and elevation in inflammatory markers, D-dimer, B-type natriuretic peptide, IL-6, and IL-10 levels were common but not ubiquitous. Cytopenias distinguished MIS-C from KD and the degree of hyperferritinemia and pattern of cytokine production differed between MIS-C and MAS. Immunomodulatory therapy given to patients with MIS-C included intravenous immune globulin (IVIG) (71%), corticosteroids (61%), and anakinra (18%). Clinical and laboratory improvement were observed in all cases, including 6 cases that did not require immunomodulatory therapy. No mortality was recorded in this cohort.CONCLUSIONMIS-C encompasses a broad phenotypic spectrum with clinical and laboratory features distinct from KD and MAS.FUNDINGThis work was supported by the National Institutes of Health, National Institute of Arthritis and Musculoskeletal and Skin Diseases; the National Institute of Allergy and Infectious Diseases; Rheumatology Research Foundation Investigator Awards and Medical Education Award; Boston Children's Hospital Faculty Career Development Awards; the McCance Family Foundation; and the Samara Jan Turkel Center.

Immunoglobulins in the treatment of COVID-19 infection: Proceed with caution!

The COVID-19 pandemic is one of the greatest infectious challenges in recent history. Presently, few treatment options exist and the availability of effective vaccines is at least one year away. There is an urgent need to find currently available, effective therapies in the treatment of patients with COVID-19 infection. In this review, we compare and contrast the use of intravenous immunoglobulin and hyperimmune globulin in the treatment of COVID-19 infection.

Genotype and functional correlates of disease phenotype in deficiency of adenosine deaminase 2 (DADA2).

BACKGROUND: Deficiency of adenosine deaminase 2 (DADA2) is a syndrome with pleiotropic manifestations including vasculitis and hematologic compromise. A systematic definition of the relationship between adenosine deaminase 2 (ADA2) mutations and clinical phenotype remains unavailable. OBJECTIVE: We sought to test whether the impact of ADA2 mutations on enzyme function correlates with clinical presentation. METHODS: Patients with DADA2 with severe hematologic manifestations were compared with vasculitis-predominant patients. Enzymatic activity was assessed using expression constructs reflecting all 53 missense, nonsense, insertion, and deletion genotypes from 152 patients across the DADA2 spectrum. RESULTS: We identified patients with DADA2 presenting with pure red cell aplasia (n = 5) or bone marrow failure (BMF, n = 10) syndrome. Most patients did not exhibit features of vasculitis. Recurrent infection, hepatosplenomegaly, and gingivitis were common in patients with BMF, of whom half died from infection. Unlike patients with DADA2 with vasculitis, patients with pure red cell aplasia and BMF proved largely refractory to TNF inhibitors. ADA2 variants associated with vasculitis predominantly reflected missense mutations with at least 3% residual enzymatic activity. In contrast, pure red cell aplasia and BMF were associated with missense mutations with minimal residual enzyme activity, nonsense variants, and insertions/deletions resulting in complete loss of function. CONCLUSIONS: Functional interrogation of ADA2 mutations reveals an association of subtotal function loss with vasculitis, typically responsive to TNF blockade, whereas more extensive loss is observed in hematologic disease, which may be refractory to treatment. These findings establish a genotype-phenotype spectrum in DADA2.

Immunodeficiency and EBV-induced lymphoproliferation caused by 4-1BB deficiency.

BACKGROUND: The tumor TNF receptor family member 4-1BB (CD137) is encoded by TNFRSF9 and expressed on activated T cells. 4-1BB provides a costimulatory signal that enhances CD8+ T-cell survival, cytotoxicity, and mitochondrial activity, thereby promoting immunity against viruses and tumors. The ligand for 4-1BB is expressed on antigen-presenting cells and EBV-transformed B cells. OBJECTIVE: We investigated the genetic basis of recurrent sinopulmonary infections, persistent EBV viremia, and EBV-induced lymphoproliferation in 2 unrelated patients. METHODS: Whole-exome sequencing, immunoblotting, immunophenotyping, and in vitro assays of lymphocyte and mitochondrial function were performed. RESULTS: The 2 patients shared a homozygous G109S missense mutation in 4-1BB that abolished protein expression and ligand binding. The patients' CD8+ T cells had reduced proliferation, impaired expression of IFN-γ and perforin, and diminished cytotoxicity against allogeneic and HLA-matched EBV-B cells. Mitochondrial biogenesis, membrane potential, and function were significantly reduced in the patients' activated T cells. An inhibitory antibody against 4-1BB recapitulated the patients' defective CD8+ T-cell activation and cytotoxicity against EBV-infected B cells in vitro. CONCLUSION: This novel immunodeficiency demonstrates the critical role of 4-1BB costimulation in host immunity against EBV infection.

Calm in the midst of cytokine storm: a collaborative approach to the diagnosis and treatment of hemophagocytic lymphohistiocytosis and macrophage activation syndrome.

BACKGROUND: Hemophagocytic lymphohistiocytosis (HLH) and macrophage activation syndrome (MAS) were historically thought to be distinct entities, often managed in isolation. In fact, these conditions are closely related. A collaborative approach, which incorporates expertise from subspecialties that previously treated HLH/MAS independently, is needed. We leveraged quality improvement (QI) techniques in the form of an Evidence-Based Guideline (EBG) to build consensus across disciplines on the diagnosis and treatment of HLH/MAS. METHODS: A multidisciplinary work group was convened that met monthly to develop the HLH/MAS EBG. Literature review and expert opinion were used to develop a management strategy for HLH/MAS. The EBG was implemented, and quality metrics were selected to monitor outcomes. RESULTS: An HLH/MAS clinical team was formed with representatives from subspecialties involved in the care of patients with HLH/MAS. Broad entry criteria for the HLH/MAS EBG were established and included fever and ferritin ≥500 ng/mL. The rheumatology team was identified as the "gate-keeper," charged with overseeing the diagnostic evaluation recommended in the EBG. First-line medications were recommended based on the acuity of illness and risk of concurrent infection. Quality metrics to be tracked prospectively based on time to initiation of treatment and clinical response were selected. CONCLUSION: HLH/MAS are increasingly considered to be a spectrum of related conditions, and joint management across subspecialties could improve patient outcomes. Our experience in creating a multidisciplinary approach to HLH/MAS management can serve as a model for care at other institutions.

Combined immunodeficiency with EBV positive B cell lymphoma and epidermodysplasia verruciformis due to a novel homozygous mutation in RASGRP1.

RASGRP1 is a guanine-nucleotide-exchange factor essential for MAP-kinase mediated signaling in lymphocytes. We report the second case of RASGRP1 deficiency in a patient with a homozygous nonsense mutation in the catalytic domain of the protein. The patient had epidermodysplasia verruciformis, suggesting a clinically important intrinsic T cell function defect. Like the previously described patient, our proband also presented with CD4+ T cell lymphopenia, impaired T cell proliferation to mitogens and antigens, reduced NK cell function, and EBV-associated lymphoma. The severity of the disease and the development of EBV lymphoma in both patients suggest that hematopoietic stem cell transplantation should be performed rapidly in patients with RASGRP1 deficiency.

Rituximab Desensitization in Pediatric Patients: Results of a Case Series.

Rituximab is a monoclonal antibody (mAb) primarily used to treat oncologic and autoinflammatory conditions. Although hypersensitivity reactions (HSRs) and desensitization protocols to mAbs have been well described in adults, the experience in the pediatric population is very limited. We sought to determine the safety and efficacy of desensitization to rituximab in the pediatric population at our institution. We retrospectively reviewed the experience with HSRs and desensitization to rituximab during a 5-year period in our tertiary care pediatric center, including reaction evaluation, premedication regimens, and desensitization procedures and protocols. A total of 17 desensitizations to rituximab were performed in three patients. A 14-year-old patient underwent successful desensitization to rituximab using a published adult protocol without incident. Two younger patients (ages 7 years and 23 months) experienced significant reactions during initial desensitization attempts. Therefore, we designed a modified desensitization protocol to rituximab, with particular attention to the rate of infusion as mg/kg/h. This new patient weight-based protocol was successfully used in a total of 13 desensitizations in these two patients. Desensitization to rituximab was a safe and effective procedure in our pediatric population. We present a new patient weight-based desensitization protocol for pediatric patients who develop HSRs to rituximab, with particular usefulness for younger pediatric patients and potential utility in pediatric patients with HSRs to other mAbs.