Case report: De novo SAMD9L truncation causes neonatal-onset autoinflammatory syndrome which was successfully treated with hematopoietic stem cell transplantation.

During recent years, the identification of monogenic mutations that cause sterile inflammation has expanded the spectrum of autoinflammatory diseases, clinical disorders characterized by uncontrolled systemic and organ-specific inflammation that, in some cases, can mirror infectious conditions. Early studies support the concept of innate immune dysregulation with a predominance of myeloid effector cell dysregulation, particularly neutrophils and macrophages, in causing tissue inflammation. However, recent discoveries have shown a complex overlap of features of autoinflammation and/or immunodeficiency contributing to severe disease phenotypes. Here, we describe the first Argentine patient with a newly described frameshift mutation in SAMD9L c.2666delT/p.F889Sfs*2 presenting with a complex phenotypic overlap of CANDLE-like features and severe infection-induced cytopenia and immunodeficiency. The patient underwent a fully matched unrelated HSCT and has since been in inflammatory remission 5 years post-HSCT.

Distinct Immune Phenotypes and Cytokine Profiles in Children with Differing Severity of COVID-19.

BACKGROUND: Coronavirus disease 2019 (COVID-19) is usually mild and self-limited in children. However, a few Severe acute respiratory syndrome coronavirus 2 (SARS-CoV2) infections in children may progress to severe disease with respiratory distress or can result in a multisystem inflammatory syndrome (MIS-C) associated with COVID-19. The immune mechanisms for these differential clinical outcomes are largely unknown. METHODS: A prospective cohort study was performed to analyze the laboratory parameters, antibody response, immune phenotypes and cytokine profiles of 51 children with different clinical presentations of COVID-19. RESULTS: We found that the absolute lymphocyte counts gradually decreased with disease severity. Furthermore, SARS-CoV-2 IgG levels in the acute phase and convalescence were not significantly different in patients with different disease severity. A decrease in CD3 + , CD4 + and CD8 + T cells was observed as disease severity increased. Both CD4 + and CD8 + T cells were activated in children with COVID-19, but no difference in the percentage of HLADR + -expressing cells was detected across the severity groups. In contrast, MIS-C patients exhibited augmented exhausted effector memory CD8 + T cells. Interestingly, the cytokine profile in sera of moderate/severe and MIS-C patients revealed an increase in anti-inflammatory IL-1RA and a suppression of tumor necrosis factor-α, RANTES, eotaxin and PDGF-BB. MIS-C patients also exhibited augmented IL-1ß. CONCLUSIONS: We report distinct immune profiles dependent on severity in pediatric COVID-19 patients. Further investigation in a larger population will help unravel the immune mechanisms underlying pediatric COVID-19.

Pediatric inborn errors of immunity causing hemophagocytic lymphohistiocytosis: Case report and review of the literature.

Inborn errors of immunity are a group of genetic disorders caused by mutations that affect the development and/or function of several compartments of the immune system, predisposing patients to infections, autoimmunity, allergy and malignancies. In this regard, mutations that affect proteins involved in trafficking, priming, docking, or membrane fusion will impair the exocytosis of lytic granules of effector NK and cytotoxic T lymphocytes. This may predispose patients to hemophagocytic lymphohistiocytosis, a life-threatening immune disorder characterized by systemic lymphocyte and macrophage activation, and increased levels of cytokines, which lead to an uncontrolled hyperinflammation state and progressive multiorgan damage. In this review, we will describe a clinical case and recent advances in inborn errors of immunity predisposing to hemophagocytic lymphohistiocytosis. Summary sentence: Review of recent advances in inborn errors of immunity predisposing to hemophagocytic lymphohistiocytosis.

STAT5B restrains human B-cell differentiation to maintain humoral immune homeostasis.

BACKGROUND: Lymphocyte differentiation is regulated by coordinated actions of cytokines and signaling pathways. IL-21 activates STAT1, STAT3, and STAT5 and is fundamental for the differentiation of human B cells into memory cells and antibody-secreting cells. While STAT1 is largely nonessential and STAT3 is critical for this process, the role of STAT5 is unknown. OBJECTIVES: This study sought to delineate unique roles of STAT5 in activation and differentiation of human naive and memory B cells. METHODS: STAT activation was assessed by phospho-flow cytometry cell sorting. Differential gene expression was determined by RNA-sequencing and quantitative PCR. The requirement for STAT5B in B-cell and CD4+ T-cell differentiation was assessed using CRISPR-mediated STAT5B deletion from B-cell lines and investigating primary lymphocytes from individuals with germline STAT5B mutations. RESULTS: IL-21 activated STAT5 and strongly induced SOCS3 in human naive, but not memory, B cells. Deletion of STAT5B in B-cell lines diminished IL-21-mediated SOCS3 induction. PBMCs from STAT5B-null individuals contained expanded populations of immunoglobulin class-switched B cells, CD21loTbet+ B cells, and follicular T helper cells. IL-21 induced greater differentiation of STAT5B-deficient B cells into plasmablasts in vitro than B cells from healthy donors, correlating with higher expression levels of transcription factors promoting plasma cell formation. CONCLUSIONS: These findings reveal novel roles for STAT5B in regulating IL-21-induced human B-cell differentiation. This is achieved by inducing SOCS3 to attenuate IL-21 signaling, and BCL6 to repress class switching and plasma cell generation. Thus, STAT5B is critical for restraining IL-21-mediated B-cell differentiation. These findings provide insights into mechanisms underpinning B-cell responses during primary and subsequent antigen encounter and explain autoimmunity and dysfunctional humoral immunity in STAT5B deficiency.

Chronic Hepatitis C Pathogenesis: Immune Response in the Liver Microenvironment and Peripheral Compartment.

Chronic hepatitis C (CHC) pathogenic mechanisms as well as the participation of the immune response in the generation of liver damage are still a topic of interest. Here, we evaluated immune cell populations and cytokines in the liver and peripheral blood (PB) to elucidate their role in CHC pathogenesis. B, CTL, Th, Treg, Th1, Th17, and NK cell localization and frequency were evaluated on liver biopsies by immunohistochemistry, while frequency, differentiation, and functional status on PB were evaluated by flow cytometry. TNF-α, IL-23, IFN-γ, IL-1ß, IL-6, IL-8, IL-17A, IL-21, IL-10, and TGF-ß expression levels were quantified in fresh liver biopsy by RT-qPCR and in plasma by CBA/ELISA. Liver CTL and Th1 at the lobular area inversely correlated with viral load (r = -0.469, p =0.003 and r = -0.384, p = 0.040). Treg correlated with CTL and Th1 at the lobular area (r = 0.784, p < 0.0001; r = 0.436, p = 0.013). Th17 correlated with hepatic IL-8 (r = 0.52, p < 0.05), and both were higher in advanced fibrosis cases (Th17 p = 0.0312, IL-8 p = 0.009). Hepatic cytokines were higher in severe hepatitis cases (IL-1ß p = 0.026, IL-23 p = 0.031, IL-8 p = 0.002, TGF-ß, p= 0.037). Peripheral NK (p = 0.008) and NK dim (p = 0.018) were diminished, while NK bright (p = 0.025) was elevated in patients vs. donors. Naïve Th (p = 0.011) and CTL (p = 0.0007) were decreased, while activated Th (p = 0.0007) and CTL (p = 0.0003) were increased. IFN-γ production and degranulation activity in NK and CTL were normal. Peripheral cytokines showed an altered profile vs. donors, particularly elevated IL-6 (p = 0.008) and TGF-ß (p = 0.041). Total hepatic CTLs favored damage. Treg could not prevent fibrogenesis triggered by Th17 and IL-8. Peripheral T-lymphocyte differentiation stage shift, elevated cytokine levels and NK-cell count decrease would contribute to global disease.

Immune Monitoring of Patients With Primary Immune Regulation Disorders Unravels Higher Frequencies of Follicular T Cells With Different Profiles That Associate With Alterations in B Cell Subsets.

Primary immune regulation disorders lead to autoimmunity, allergy and inflammatory conditions due to defects in the immune homeostasis affecting different T, B and NK cell subsets. To improve our understanding of these conditions, in this work we analyzed the T and B cell compartments of 15 PID patients with dysregulation, including 3 patients with STAT1 GOF mutation, 7 patients with CVID with dysregulation, 3 patients with mutations in CTLA4, 1 patient with CD25 mutation and 1 patient with STAT5b mutation and compared them with healthy donors and with CVID patients without dysregulation. CD4+ and CD8+ T cells from the patients exhibited a significant decreased frequency of naïve and regulatory T cells with increased frequencies of activated cells, central memory CD4+ T cells, effector memory CD8+ T cells and terminal effector CD8+ T cells. Patients also exhibited a significantly increased frequency of circulating CD4+ follicular helper T cells, with altered frequencies of cTfh cell subsets. Such cTfh cells were skewed toward cTfh1 cells in STAT1 GOF, CTLA4, and CVID patients, while the STAT5b deficient patient presented a skew toward cTfh17 cells. These alterations confirmed the existence of an imbalance in the cTfh1/cTfh17 ratio in these diseases. In addition, we unraveled a marked dysregulation in the B cell compartment, characterized by a prevalence of transitional and naïve B cells in STAT1 GOF and CVID patients, and of switched-memory B cells and plasmablast cells in the STAT5b deficient patient. Moreover, we observed a significant positive correlation between the frequencies cTfh17 cells and switched-memory B cells and between the frequency of switched-memory B cells and the serum IgG. Therefore, primary immunodeficiencies with dysregulation are characterized by a skew toward an activated/memory phenotype within the CD4+ and CD8+ T cell compartment, accompanied by abnormal frequencies of Tregs, cTfh, and their cTfh1 and cTfh17 subsets that likely impact on B cell help for antibody production, which likely contributes to their autoimmune and inflammatory conditions. Therefore, assessment of these alterations by flow cytometry constitutes a simple and straightforward manner to improve diagnosis of these complex clinical entities that may impact early diagnosis and patients' treatment. Also, our findings unravel phenotypic alterations that might be associated, at least in part, with some of the clinical manifestations observed in these patients.

Dysregulatory syndromes: the role of signal transducers and activators of transcription.

PURPOSE OF REVIEW: A comparative description of dysregulatory syndromes with mutations in signal transducer and activator of transcription (STAT) genes. RECENT FINDINGS: STAT 1, 3 and 5b loss of function (LOF) and gain of function (GOF) mutations are a heterogeneous group of genetic disorders that range from immunodeficiency (ID) to autoimmune disease (AID), depending on the underlying signalling pathway defect. Between them, there are clear overlapping and differences in clinical presentation and laboratory findings. SUMMARY: Dysregulatory syndromes due to LOF and GOF mutations in STAT1, 3 and 5b are a particular group of primary immunodeficiencies (PIDs) in which AID may be the predominant finding in addition to infections susceptibility. STAT1 GOF mutations were described as the major cause of chronic mucocutaneous candidiasis, while activating STAT3 mutations result in early-onset multiorgan autoimmunity and ID. Human STAT5b deficiency is a rare disease that also involves ID and severe growth failure. In recent years, the identification of the genes involved in these disorders allowed to differentiate these overlapping syndromes in order to choose the most effective therapeutic options.

Primary Immunodeficiencies Unravel the Role of IL-2/CD25/STAT5b in Human Natural Killer Cell Maturation.

Natural killer (NK) cells play a pivotal role during immunity against viruses and circumstantial evidence also indicates that they can protect the host against developing tumors. Peripheral blood NK cells comprise CD56brightCD16lo/- cells that constitutively express CD25 (IL-2Rα) and CD56dimCD16hi cells that express CD25 upon activation. Using NK cells from two patients, one with a primary immunodeficiency characterized by a homozygous mutation in CD25 (born in year 2007 and studied since she was 3 years old) and one with a homozygous mutation in STAT5b (born in year 1992 and studied since she was 10 years old), we observed that the absence of IL-2 signaling through CD25 promotes the accumulation of CD56brightCD16high NK cells, and that CD56brightCD16lo, CD56brightCD16high, and CD56dimCD16high NK cells of this patient exhibited higher content of perforin and granzyme B, and proliferation capacity, compared to healthy donors. Also, CD56bright and CD56dim NK cells of this patient exhibited a reduced IFN-γ production in response to cytokine stimulation and increased degranulation against K562 cells. Also, the CD25-deficient patient presented a lower frequency of terminally differentiated NK cells in the CD56dimCD16hi NK subpopulation compared to the HD (assessed by CD57 and CD94 expression). Remarkably, CD56dimCD16high NK cells from both patients exhibited notoriously higher expression of CD62L compared to HD, suggesting that in the absence of IL-2 signaling through CD25 and STAT5b, NK cells fail to properly downregulate CD62L during their transition from CD56brightCD16lo/- to CD56dimCD16hi cells. Thus, we provide the first demonstration about the in vivo requirement of the integrity of the IL-2/CD25/STAT5b axis for proper human NK cell maturation.

Partial growth hormone insensitivity and dysregulatory immune disease associated with de novo germline activating STAT3 mutations.

Germinal heterozygous activating STAT3 mutations represent a novel monogenic defect associated with multi-organ autoimmune disease and, in some cases, severe growth retardation. By using whole-exome sequencing, we identified two novel STAT3 mutations, p.E616del and p.C426R, in two unrelated pediatric patients with IGF-I deficiency and immune dysregulation. The functional analyses showed that both variants were gain-of-function (GOF), although they were not constitutively phosphorylated. They presented differences in their dephosphorylation kinetics and transcriptional activities under interleukin-6 stimulation. Both variants increased their transcriptional activities in response to growth hormone (GH) treatment. Nonetheless, STAT5b transcriptional activity was diminished in the presence of STAT3 GOF variants, suggesting a disruptive role of STAT3 GOF variants in the GH signaling pathway. This study highlights the broad clinical spectrum of patients presenting activating STAT3 mutations and explores the underlying molecular pathway responsible for this condition, suggesting that different mutations may drive increased activity by slightly different mechanisms.

Chronic hepatitis C liver microenvironment: role of the Th17/Treg interplay related to fibrogenesis.

The role of the different lymphocyte populations in liver microenvironment of chronic hepatitis C (CHC) patients is still matter of debate. Since Th17 and Treg have opposite functions, their balance could affect disease progression. The aim was to explore liver microenvironment and its peripheral blood counterpart in adult CHC patients. CD4+ lymphocytes were predominant in the liver, with high Foxp3+ but low IL-17A+ frequency. IL-17A+ lymphocytes and IL-17A+/Foxp3+ ratio displayed association with advanced fibrosis (p = 0.0130; p = 0.0236, respectively), while Foxp3+ lymphocytes and IL-10 expression level inversely correlated with fibrosis severity (p = 0.0381, p = 0.0398, respectively). TGF-ß/IL-6 ratio correlated with IL-17A+/Foxp3+ ratio (p = 0.0036, r = 0.5944) and with IL-17A+ lymphocytes (p = 0.0093; r = 0.5203). TNF-α and TGF-ß were associated with hepatitis severity (p = 0.0409, p = 0.0321). Peripheral blood lymphocyte frequency was not associated with liver damage. There are functionally different immune cell populations actively involved in liver damage, but the liver cytokine milieu actually drives the pathogenesis. The intrahepatic Foxp3+ lymphocytes predominance beside the low IL-17A+ lymphocytes frequency, delineate a skewed IL-17A+/Foxp3+ balance towards Foxp3+ lymphocytes. However, the IL-17A+ lymphocytes association with advanced fibrosis denotes their role in the pathogenesis. Therefore, the interplay between Th17 and Treg conditions liver fibrogenesis.

Long-term follow-up of STAT5B deficiency in three argentinian patients: clinical and immunological features.

UNLABELLED: The signal transducer and activator of transcription (STAT) family of proteins regulate gene transcription in response to a variety of cytokines. STAT5B, in particular, plays an important role in T cells, where it is a key mediator of interleukin-2 (IL-2) induced responses. STAT5B deficiency causes a rare autosomal recessive disorder reported in only a handful of individuals. There are currently ten published cases of STAT5B deficiency, four of which are Argentinians. AIM: This is a report of more than 10 years follow up of the clinical and immunological features of three Argentinian STAT5B deficient patients. CONCLUSION: More than a decade of follow-up demonstrates that STAT5B deficiency is associated with various clinical pathologies that cause significant morbidity. Early diagnosis is critical for the prevention and improvement of clinical outcomes for STAT5B deficient patients.

Human natural killer cell maturation defect supports in vivo CD56(bright) to CD56(dim) lineage development.

Two populations of human natural killer (NK) cells can be identified in peripheral blood. The majority are CD3(-)CD56(dim) cells while the minority exhibits a CD3(-)CD56(bright) phenotype. In vitro evidence indicates that CD56(bright) cells are precursors of CD56(dim) cells, but in vivo evidence is lacking. Here, we studied NK cells from a patient that suffered from a melanoma and opportunistic fungal infection during childhood. The patient exhibited a stable phenotype characterized by a reduction in the frequency of peripheral blood CD3(-)CD56(dim) NK cells, accompanied by an overt increase in the frequency and absolute number of CD3(-)CD56(bright) cells. These NK cells exhibited similar expression of perforin, CD57 and CD158, the major activating receptors CD16, NKp46, NKG2D, DNAM-1, and 2B4, as well as the inhibitory receptor CD94/NKG2A, on both CD56(bright) and CD56(dim) NK cells as healthy controls. Also, both NK cell subpopulations produced IFN-γ upon stimulation with cytokines, and CD3(-)CD56(dim) NK cells degranulated in response to cytokines or K562 cells. However, upon stimulation with cytokines, a substantial fraction of CD56(dim) cells failed to up-regulate CD57 and CD158, showed a reduction in the percentage of CD16(+) cells, and CD56(bright) cells did not down-regulate CD62L, suggesting that CD56(dim) cells could not acquire a terminally differentiated phenotype and that CD56(bright) cells exhibit a maturation defect that might result in a potential altered migration pattern. These observations, support the notion that NK cells of this patient display a maturation/activation defect that precludes the generation of mature NK cells at a normal rate accompanied by CD56(dim) NK cells that cannot completely acquire a terminally differentiated phenotype. Thus, our results provide evidence that support the concept that in vivo CD56(bright) NK cells differentiate into CD56(dim) NK cells, and contribute to further understand human NK cell ontogeny.

A novel missense mutation in the SH2 domain of the STAT5B gene results in a transcriptionally inactive STAT5b associated with severe IGF-I deficiency, immune dysfunction, and lack of pulmonary disease.

CONTEXT: Signal transducer and activator of transcription 5b (STAT5b) deficiency, first reported in a patient who carried a p.Ala630Pro missense mutation in the Src homology 2 (SH2) domain, results in a rare clinical condition of GH insensitivity (GHI), IGF-I deficiency (IGFD), and severe immune dysregulation manifesting as progressive worsening of pulmonary function. PATIENT: The new patient presented with severe cutaneous eczema, episodic infections in the first years of life, and autoimmune thyroiditis. Immunological evaluation revealed T lymphopenia, but severe pulmonary symptoms were notably absent. She concomitantly exhibited pronounced growth failure, reaching an adult height of 124.7 cm [-5.90 SD score (SDS)]. Endocrine evaluations (normal provocative GH tests; low serum IGF-I, -3.7 SDS, and IGF-binding protein-3, -4.5 SDS) were consistent with GHI and IGFD. RESULTS: Analysis of the STAT5B gene revealed a novel homozygous missense mutation, p.Phe646Ser, located within the ßD' strand of the SH2 domain. Reconstitution studies demonstrated expression of the p.Phe646Ser variant was less robust than wild type but, in contrast to the previously described STAT5B p.Ala630Pro SH2 mutation, could be phosphorylated in response to GH and interferon-γ. The phosphorylated p.Phe646Ser, however, could not drive transcription. CONCLUSION: A novel STAT5B p.Phe646Ser mutation has been identified in a patient with clinical characteristics of STAT5b deficiency. Only the second STAT5B missense mutation identified, its lack of transcriptional activities despite GH-induced phosphorylation, confirms the crucial role of STAT5b for regulating the expression of IGF1 and provides insights into the importance of the SH2 ßD' strand for full STAT5b transcriptional activities. Whether the phosphorylated p.Phe646Ser variant retained functions that prevented pulmonary distress remains unresolved.

Evaluación clínica e inmunológica de 90 pacientes con deficiencia selectiva de inmunoglobulina A / A clinical and immunological evaluation of 90 patients with selective IgA deficiency

Introducción.La deficiencia selectiva de IgA se comporta como una enfermedad heterogénea, (asintomática o sintomática con infecciones recurrentes respiratorias o gastrointestinales, enfermedades atópicas y autoinmunes), caracterizada por presentar IgA de 7mg/dl o menos en pacientes de 4 años de edad o mas. Entre 1990 y 1999 se registraron en nuestro servicio 176 inmunodeficiencias primarias; la inmunodeficiencia selectiva de IgA representó el 61 por ciento. Objetivo. Describir las distintas formas de presentación clínica de la deficiencia selectiva de IgA y buscar un patrón inmunológico que permita diferenciarlas. Población, material y métodos. Se evaluaron inmunológicamente 90 pacientes con diagnóstico de inmunodeficiencia selectiva de IgA. Se excluyeron 18 pacientes por falta de seguimiento

Enfermedad de Kawasaki. Evaluación inmunológica de 26 casos / Kawasaki disease. Immunological evaluation of 26 cases

La enfermedad de Kawasaki (EK) es una vasculitis aguda febril de la infancia, caracterizada por mútiples signos clínicos y bioquímicos de la inflamación con especial compromiso del corazón. La activación de linfocitos y monocitos/macrófagos y sus produtos solubles secretados, citoquinas , juegan un papel central en la patogénesis de la enfermadad. En este estudio realizamos estudios de laboratorio inmunológico en 26 pacientes con EK. En 22 pacientes en etapa aguda medimos niveles séricos de IgG, IgA, IgM, fracciones del complemento C3 y C4 por Nefelometría, sin encontrar un patrón constante y los anticuerpos FAN y ANCA por imunoflorescencia indirecta fueron negativos. En células mononucleares periféricas de 25 pacientes en etapa aguda encontramos porcentajes variables de CD3, CD4, CD8, CD20, CD56 y DR mediante tinción específica y análisis por cimetría de flujo. El porcentaje de CD25 estuvo elevado en 17/25 casos. Los níveles séricos de TNFalfa medidos poe ELISA en 12 pacientes , fueron bajos. Evaluamos citoquinas intracelulares TNAalfa, IL1beta, IL2, e IFNgamaen células mononucleares periféricas en 15 pacientes en etapas agudas, subagudas y de covalecencia, utilizando tinción específica y análisis por cimetría de flujo, sin encontrar un perfil característico. Dos pacientes mostraron porcentajes elevados de TNFalfa e IL1beta en monocitos durante la etapa de covalecencia, ambos presentaron secuelas coronarias. Es necesario realizar investigación adicioal acerca de este hallazgo. En conclusión, la evaluación inmunulógica mostró un perfil heterógeneo y no se encontró ningún factor de laboratorio en la etapa aguda que sea predictivo de compromiso cardivascular.

Primer estudio multicéntrico realizado por el Grupo Rioplatense de Citometría de Flujo: control de calidad en las mediciones de subpoblaciones de linfocitos T / First multicentric study by Grupo Rioplatense de Citometría de Flujo : quality control of lymphocyte subsets

El control de calidad se efectuó sobre los valores obtenidos, relativos y absolutos, de linfocitos T y de sus subpoblaciones CD4+ y CD8+ en muestras de sangre de pacientes infectados con el virus de la inmunodeficiencia humana (HIV). El estudio incluyó dieciocho centros: diez utilizaron citómetros de flujo de Becton Dickinson, tres de Coulter y 5 de Ortho que representan a 17 laboratorios de Argentina y a uno de Uruguay. Los siguientes programas se utilizaron para analizar los datos : SimulSET, Paint a Gate (Becton Dickinson), Profile II, XL System (Coulter), ImmunoCount Trio y Combo Cytoron (Ortho). Se obtuvieron muestras de sangre periférica en horas de la mañana (8 a 10 hs) de 10 voluntarios normales (por serología y hemograma) y de 10 pacientes HIV positivos con valores previos de CD4 que variaron entre 200-350 células por microlito y fueron procesadas dentro de las 12 horas. Cada centro obtuvo los valores relativos con el procedimiento técnico habitual y el de los valores absolutos utilizando el hemograma propio. Además, en un contador hematológico Cell-Dyn 3500 se obtuvo para cada muestra el hemograma correspondiente considerado de referencia. Los valores absolutos medios, obtenidos en cada centro con el hemograma propio, para los linfocitos T y los de sus subpoblaciones fueron significativamente diferentes. No hubo diferencias significativas para los valores porcentuales entre los diferentes centros ni para los valores absolutos obtenidos con el hemograma de referencia. Concluimos que las diferencias en los valores absolutos de los linfocitos T y sus subpoblaciones dependen del recuento hematológico empleado.