Publications by authors named "Simona Cascioli"

27 Publications

  • Page 1 of 1

Purification and Characterization of Murine MZ and T2-MZP Cells.

Methods Mol Biol 2021 ;2270:3-25

B Cell Physiopathology Unit, Immunology Research Area, Bambino Gesù Children Hospital, Rome, Italy.

The spleen is the second major reservoir of B cells in the adult. In the spleen, cells, generated in the bone marrow, are selected, mature, and become part of the peripheral B-cell pool. Murine spleen comprises several B-cell subsets representing various maturation stages and/or cell functions. The spleen is a complex lymphoid organ organized into two main structures with different functions: the red and white pulp. The red pulp is flowed with blood while the white pulp is organized in primary follicles, with a B-cell area composed of follicular B cells and a T-cell area surrounding a periarterial lymphatic sheath. The frontier between the red and white pulp is defined as the marginal zone (MZ) and contains the MZ B cells. Because B cells, localized in different areas, are characterized by distinct expression levels of B-cell receptor (BCR) and of other surface markers, splenic B-cell subsets can be easily identified and purified by flow cytometry analyses and fluorescence-activated cell sorting (FACS).Here, we will focus on MZ B cells and on their precursors, giving some experimental hints to identify, generate, and isolate these cells. We will combine the use of FACS analysis and confocal microscopy to visualize MZ B cells in cell suspensions and in tissue sections, respectively. We will also give some clues to analyze B-cell repertoire on isolated MZ-B cells.
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http://dx.doi.org/10.1007/978-1-0716-1237-8_1DOI Listing
April 2021

Different Innate and Adaptive Immune Responses to SARS-CoV-2 Infection of Asymptomatic, Mild, and Severe Cases.

Front Immunol 2020 16;11:610300. Epub 2020 Dec 16.

Department of Hematology/Oncology, Bambino Gesù Children's Hospital, IRCCS, Rome, Italy.

SARS-CoV-2 is a novel coronavirus, not encountered before by humans. The wide spectrum of clinical expression of SARS-CoV-2 illness suggests that individual immune responses to SARS-CoV-2 play a crucial role in determining the clinical course after first infection. Immunological studies have focused on patients with moderate to severe disease, demonstrating excessive inflammation in tissues and organ damage. In order to understand the basis of the protective immune response in COVID-19, we performed a longitudinal follow-up, flow-cytometric and serological analysis of innate and adaptive immunity in adults with a spectrum of clinical presentations: healthy SARS-CoV-2-negative contacts of COVID-19 cases; asymptomatic SARS-CoV-2-infected cases; patients with Mild COVID-19 disease and cases of Severe COVID-19 disease. Our data show that high frequency of NK cells and early and transient increase of specific IgA, IgM and, to a lower extent, IgG are associated with asymptomatic SARS-CoV-2 infection. By contrast, monocyte expansion and high and persistent levels of IgA and IgG, produced relatively late in the course of the infection, characterize severe disease. Modest increase of monocytes and different kinetics of antibodies are detected in mild COVID-19. The importance of innate NK cells and the short-lived antibody response of asymptomatic individuals and patients with mild disease suggest that only severe COVID-19 may result in protective memory established by the adaptive immune response.
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http://dx.doi.org/10.3389/fimmu.2020.610300DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7772470PMC
January 2021

The Interplay between CD27 and CD27 B Cells Ensures the Flexibility, Stability, and Resilience of Human B Cell Memory.

Cell Rep 2020 03;30(9):2963-2977.e6

Multifactorial Disease and Complex Phenotype Research Area, Bambino Gesù Children's Hospital, IRCSS, 00146 Rome, Italy.

Memory B cells (MBCs) epitomize the adaptation of the immune system to the environment. We identify two MBC subsets in peripheral blood, CD27 and CD27 MBCs, whose frequency changes with age. Heavy chain variable region (VH) usage, somatic mutation frequency replacement-to-silent ratio, and CDR3 property changes, reflecting consecutive selection of highly antigen-specific, low cross-reactive antibody variants, all demonstrate that CD27 and CD27 MBCs represent sequential MBC developmental stages, and stringent antigen-driven pressure selects CD27 into the CD27 MBC pool. Dynamics of human MBCs are exploited in pregnancy, when 50% of maternal MBCs are lost and CD27 MBCs transit to the more differentiated CD27 stage. In the postpartum period, the maternal MBC pool is replenished by the expansion of persistent CD27 clones. Thus, the stability and flexibility of human B cell memory is ensured by CD27 MBCs that expand and differentiate in response to change.
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http://dx.doi.org/10.1016/j.celrep.2020.02.022DOI Listing
March 2020

Lack of Gut Secretory Immunoglobulin A in Memory B-Cell Dysfunction-Associated Disorders: A Possible Gut-Spleen Axis.

Front Immunol 2019 8;10:2937. Epub 2020 Jan 8.

Department of Molecular Medicine, Sapienza University, Rome, Italy.

B-1a B cells and gut secretory IgA (SIgA) are absent in asplenic mice. Human immunoglobulin M (IgM) memory B cells, which are functionally equivalent to mouse B-1a B cells, are reduced after splenectomy. To demonstrate whether IgM memory B cells are necessary for generating IgA-secreting plasma cells in the human gut. We studied intestinal SIgA in two disorders sharing the IgM memory B cell defect, namely asplenia, and common variable immune deficiency (CVID). Splenectomy was associated with reduced circulating IgM memory B cells and disappearance of intestinal IgA-secreting plasma cells. CVID patients with reduced circulating IgM memory B cells had a reduced frequency of gut IgA plasma cells and a disrupted film of SIgA on epithelial cells. Toll-like receptor 9 (TLR9) and transmembrane activator and calcium-modulator and cyclophilin ligand interactor (TACI) induced IgM memory B cell differentiation into IgA plasma cells . In the human gut, TACI-expressing IgM memory B cells were localized under the epithelial cell layer where the TACI ligand a proliferation inducing ligand (APRIL) was extremely abundant. Circulating IgM memory B cell depletion was associated with a defect of intestinal IgA-secreting plasma cells in asplenia and CVID. The observation that IgM memory B cells have a distinctive role in mucosal protection suggests the existence of a functional gut-spleen axis.
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http://dx.doi.org/10.3389/fimmu.2019.02937DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6960143PMC
November 2020

Atypical IgM on T cells predict relapse and steroid dependence in idiopathic nephrotic syndrome.

Kidney Int 2019 10 7;96(4):971-982. Epub 2019 May 7.

Laboratory of Nephrology, Department of Rare Diseases, Istituto di Ricovero e Cura a Carattere Scientifico (IRCCS) Ospedale Pediatrico Bambino Gesù, Rome, Italy; Division of Nephrology, Department of Pediatric Subspecialties, IRCCS Ospedale Pediatrico Bambino Gesù, Rome, Italy.

The clinical heterogeneity of idiopathic nephrotic syndrome in childhood may reflect different mechanisms of disease that are as yet unclear. Here, we evaluated the association between an atypical presence of IgM on the surface of T cells (T-cell IgM) and the response to steroid therapy in a total of 153 pediatric patients with idiopathic nephrotic syndrome in different phases of disease. At disease onset, T-cell IgM median levels were significantly elevated and predictive of risk of relapse in 47 patients. They were also significantly increased comparing 58 steroid-dependent to 8 infrequently relapsing and 14 frequently relapsing patients, especially during relapse, whereas they were within the normal range in 7 genetic steroid-resistant patients. T-cell IgM in vivo was not affected by the amount of total circulating IgM, nor by concomitant acute infections or oral immunosuppression. However, it was affected by rituximab treatment in 21 steroid-dependent patients. By in vitro experiments, elevated T-cell IgM was not influenced by total circulating IgM levels or by the presence of other circulating factors, and there was no distinctive antigen-specificity or atypical IgM polymerization. Rather, we found that increased T-cell IgM correlates with reduced IgM sialylation, which influences T-cell response to steroid inhibition and T-cell production of podocyte-damaging factors. Thus, the atypical presence of IgM on the surface of T cells may predispose a subset of steroid-sensitive pediatric patients with idiopathic nephrotic syndrome to a poor response to steroid therapy since disease onset.
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http://dx.doi.org/10.1016/j.kint.2019.04.006DOI Listing
October 2019

Dysregulated miR-155 and miR-125b Are Related to Impaired B-cell Responses in Down Syndrome.

Front Immunol 2018 20;9:2683. Epub 2018 Nov 20.

B cell Pathophysiology Unit, Immunology Research Area, Bambino Gesù Children's Hospital, IRCCS, Rome, Italy.

Children with Down Syndrome (DS) suffer from immune deficiency with a severe reduction in switched memory B cells (MBCs) and poor response to vaccination. Chromosome 21 (HSA21) encodes two microRNAs (miRs), miR-125b, and miR-155, that regulate B-cell responses. We studied B- and T- cell subpopulations in tonsils of DS and age-matched healthy donors (HD) and found that the germinal center (GC) reaction was impaired in DS. GC size, numbers of GC B cells and Follicular Helper T cells (T) expressing BCL6 cells were severely reduced. The expression of miR-155 and miR-125b was increased in tonsillar memory B cells and miR-125b was also higher than expected in plasma cells (PCs). Activation-induced cytidine deaminase (AID) protein, a miR-155 target, was significantly reduced in MBCs of DS patients. Increased expression of miR-155 was also observed . MiR-155 was significantly overexpressed in PBMCs activated with CpG, whereas miR-125b was constitutively higher than normal. The increase of miR-155 and its functional consequences were blocked by antagomiRs . Our data show that the expression of HSA21-encoded miR-155 and miR-125b is altered in B cells of DS individuals both and . Because of HSA21-encoded miRs may play a role also in DS-associated dementia and leukemia, our study suggests that antagomiRs may represent pharmacological tools useful for the treatment of DS.
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http://dx.doi.org/10.3389/fimmu.2018.02683DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6255899PMC
October 2019

B cell phenotype in pediatric idiopathic nephrotic syndrome.

Pediatr Nephrol 2019 01 28;34(1):177-181. Epub 2018 Sep 28.

Laboratory of Nephrology, Department of Rare Diseases, IRCCS Ospedale Pediatrico Bambino Gesù, Rome, Italy.

Background: A pathogenic role of B cells in non-genetic nephrotic syndrome has been suggested by the efficacy of rituximab, a B cell depleting antibody, in maintaining a prolonged remission. However, little information is available on B cell homeostasis in nephrotic syndrome patients.

Methods: We retrospectively analyzed by flow cytometry the distribution of different B cell subpopulations in 107 steroid-sensitive and in 6 genetic steroid-resistant nephrotic syndrome pediatric patients, compared with age- and sex-matched controls.

Results: Fifty-one steroid-sensitive patients at disease onset, before starting immunosuppression, presented significantly increased levels of total, transitional, memory, and switched memory B cells compared to controls. Oral immunosuppression strongly affected transitional and mature B cell levels in 27 patients in relapse and also in 29 patients in remission, whereas memory B cells were significantly higher compared to controls during relapse, despite the immunosuppressive treatment, and were normalized only in patients in remission. Children with genetic forms of steroid-resistant nephrotic syndrome presented no differences in B cell profile from controls.

Conclusions: Our study indicates that memory B cells, more than other B cell subsets, are increased and appear to be pathogenically relevant in steroid-sensitive nephrotic syndrome pediatric patients.
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http://dx.doi.org/10.1007/s00467-018-4095-zDOI Listing
January 2019

Spleen development is modulated by neonatal gut microbiota.

Immunol Lett 2018 07 30;199:1-15. Epub 2018 Apr 30.

B Cell Physiopathology Unit, Immunology Research Area, Bambino Gesù Children Hospital, Rome, Italy.

The full development of the mammalian immune system occurs after birth upon exposure to non self-antigens. The gut is the first site of bacterial colonization where it is crucial to create the appropriate microenvironment able to balance effector or tolerogenic responses to external stimuli. It is a well-established fact that at mucosal sites bacteria play a key role in developing the immune system but we ignore how colonising bacteria impact the maturation of the spleen. Here we addressed this issue. Taking advantage of the fact that milk SIgA regulates bacterial colonization of the newborn intestine, we generated immunocompetent mice born either from IgA pro-efficient or IgA deficient females. Having demonstrated that SIgA in maternal milk modulates neonatal gut microbiota by promoting an increased diversity of the colonizing species we also found that immunocompetent pups, not exposed to milk SIgA, fail to properly develop the FDC network and primary follicles in the spleen compromising the response to T-dependent antigens. The presence of a less diverse microbiota with a higher representation of pathogenic species leads to a fast replenishment of the marginal zone and the IgM plasma cell compartment of the spleen as well as IgA plasma cells in the gut.
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http://dx.doi.org/10.1016/j.imlet.2018.04.010DOI Listing
July 2018

Switched Memory B Cells Are Increased in Oligoarticular and Polyarticular Juvenile Idiopathic Arthritis and Their Change Over Time Is Related to Response to Tumor Necrosis Factor Inhibitors.

Arthritis Rheumatol 2018 04 25;70(4):606-615. Epub 2018 Mar 25.

Ospedale Pediatrico Bambino Gesù, IRCCS, Rome, Italy.

Objective: To investigate whether abnormalities in B cell subsets in patients with juvenile idiopathic arthritis (JIA) correlate with clinical features and response to treatment.

Methods: A total of 109 patients diagnosed as having oligoarticular JIA or polyarticular JIA were enrolled in the study. B cell subsets in peripheral blood and synovial fluid were analyzed by flow cytometry.

Results: Switched memory B cells were significantly increased in patients compared to age-matched healthy controls (P < 0.0001). When patients were divided according to age at onset of JIA, in patients with early-onset disease (presenting before age 6 years) the expansion in switched memory B cells was more pronounced than that in patients with late-onset disease and persisted throughout the disease course. In longitudinal studies, during methotrexate (MTX) treatment, regardless of the presence or absence of active disease, the number of switched memory B cells increased significantly (median change from baseline 36% [interquartile range {IQR} 15, 66]). During treatment with MTX plus tumor necrosis factor inhibitors (TNFi), in patients maintaining disease remission, the increase in switched memory B cells was significantly lower than that in patients who experienced active disease (median change from baseline 4% [IQR -6, 32] versus 41% [IQR 11, 73]; P = 0.004). The yearly rate of increases in switched memory B cells was 1.5% in healthy controls, 1.2% in patients who maintained remission during treatment with MTX plus TNFi, 4.7% in patients who experienced active disease during treatment with MTX plus TNFi, and ~4% in patients treated with MTX alone.

Conclusion: Switched memory B cells expand during the disease course at a faster rate in JIA patients than in healthy children. This increase is more evident in patients with early-onset JIA. TNFi treatment inhibits this increase in patients who achieve and maintain remission, but not in those with active disease.
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http://dx.doi.org/10.1002/art.40410DOI Listing
April 2018

Human B-cell memory is shaped by age- and tissue-specific T-independent and GC-dependent events.

Eur J Immunol 2017 02 14;47(2):327-344. Epub 2016 Dec 14.

B Cell Physiopathology Unit, Immunology Research Area, Ospedale Pediatrico Bambino, Gesù IRCSS, Roma, Italy.

Switched and IgM memory B cells execute different and noninterchangeable functions. We studied memory B cells in children of different ages, in peripheral blood and spleen and compared them with those of children born asplenic or unable to build germinal centers. We show that, whereas switched memory B cells are mostly generated in the germinal centers at all ages, IgM memory B cells can be distinct in three types with different developmental history. Innate IgM memory B cells, the largest pool in infants, are generated in the spleen by a germinal center-independent mechanism. With age, if the spleen is present and germinal centers are functional, innate IgM memory B cells are remodelled and accumulate somatic mutations. The third type of IgM memory B cell is a by-product of the germinal center reaction. Our data suggest that the B-cell memory developmental program is implemented during the first 5-6 years of life.
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http://dx.doi.org/10.1002/eji.201646642DOI Listing
February 2017

B-cell activation with CD40L or CpG measures the function of B-cell subsets and identifies specific defects in immunodeficient patients.

Eur J Immunol 2017 01 25;47(1):131-143. Epub 2016 Nov 25.

B Cell Physiopathology Unit, Immunology Research Area, Ospedale Pediatrico Bambino Gesù IRCSS, Roma, Italy.

Around 65% of primary immunodeficiencies are antibody deficiencies. Functional tests are useful tools to study B-cell functions in vitro. However, no accepted guidelines for performing and evaluating functional tests have been issued yet. Here, we report our experience on the study of B-cell functions in infancy and throughout childhood. We show that T-independent stimulation with CpG measures proliferation and differentiation potential of memory B cells. Switched memory B cells respond better than IgM memory B cells. On the other hand, CD40L, a T-dependent stimulus, does not induce plasma cell differentiation, but causes proliferation of naïve and memory B cells. During childhood, the production of plasmablasts in response to CpG increases with age mirroring the development of memory B cells. The response to CD40L does not change with age. In patients with selective IgA deficiency (SIgAD), we observed that switched memory B cells are reduced due to the absence of IgA memory B cells. In agreement, IgA plasma cells are not generated in response to CpG. Unexpectedly, B cells from SIgAD patients show a reduced proliferative response to CD40L. Our results demonstrate that functional tests are an important tool to assess the functions of the humoral immune system.
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http://dx.doi.org/10.1002/eji.201646574DOI Listing
January 2017

B Cell Reconstitution after Rituximab Treatment in Idiopathic Nephrotic Syndrome.

J Am Soc Nephrol 2016 06 13;27(6):1811-22. Epub 2015 Nov 13.

Division of Nephrology and Dialysis.

The pathogenesis of nephrotic syndrome is unclear. However, the efficacy of rituximab, a B cell-depleting antibody, in nephrotic syndrome suggests a pathogenic role of B cells. In this retrospective study, we determined by flow cytometry levels of B and T cell subpopulations before and after rituximab infusion in 28 pediatric patients with frequently relapsing or steroid-dependent nephrotic syndrome. At baseline, patients had lower median percentages of transitional and mature B cells than age-matched healthy controls (P<0.001). Rituximab induced full depletion of B cells (<1% of lymphocytes). At 1 year, most patients exhibited complete total and mature B cell recovery, whereas memory B cell subsets remained significantly depleted. Total T cell concentration did not change with rituximab, whereas the CD4(+)/CD8(+) T cell ratio tended to increase. Fourteen patients relapsed within 24 months, with a median follow-up of 11.2 months (interquartile range, 8-17.7 months). We observed no difference at baseline between nonrelapsing and relapsing patients in several clinical parameters and cell subset concentrations. Reconstitution of all memory B cell subpopulations, number of immunosuppressive drugs, and dose of tacrolimus during the last 4 months of follow-up were predictive of relapse in univariate Cox regression analysis. However, only delayed reconstitution of switched memory B cells, independent of immunosuppressive treatment, was protective against relapse in multivariate (P<0.01) and receiver operator characteristic (P<0.01 for percentage of lymphocytes; P=0.02 for absolute count) analyses. Evaluation of switched memory B cell recovery after rituximab may be useful for predicting relapse in patients with nephrotic syndrome.
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http://dx.doi.org/10.1681/ASN.2015050523DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4884116PMC
June 2016

Combined immunodeficiency due to JAK3 mutation in a child presenting with skin granuloma.

J Allergy Clin Immunol 2016 Mar 3;137(3):948-51.e5. Epub 2015 Nov 3.

University Department of Pediatrics, DPUO, Unit of Immune and Infectious Diseases, Bambino Gesù Children's Hospital, Rome, Italy. Electronic address:

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http://dx.doi.org/10.1016/j.jaci.2015.09.017DOI Listing
March 2016

B-cell hyperfunction in children with immune thrombocytopenic purpura persists after splenectomy.

Pediatr Res 2016 Feb 22;79(2):262-70. Epub 2015 Oct 22.

Immunology and Pharmacotherapy Research Area, Bambino Gesù Children's Hospital IRCCS, Rome, Italy.

Background: Immune thrombocytopenic purpura (ITP) is characterized by reduced platelet count secondary to immune-mediated destruction, this results in an increased bleeding risk. Autoantibodies binding to platelets tag them for premature destruction in the spleen. For this reason, splenectomy is often performed as treatment of chronic forms of disease that are resistant to pharmacological therapy.

Methods: We studied 30 patients with ITP and compared them with age-matched controls.

Results: We show that B cells of patients with chronic ITP are intrinsically hyperreactive, producing more than normal IgG in vivo and plasma cells in vitro. In normal individuals after splenectomy, a significant depletion of memory B cells is observed, associated with loss of reactivity to CpG oligodeoxynucleotide and consequent inability to form antibody-producing cells. In Enzyme-Linked ImmunoSpot Methods, we compared three splenectomized ITP patients relapsing after surgery, 30 healthy controls, and 37 individuals splenectomized for trauma, spherocytosis, thalassemia, nonhematological tumor, and other diseases.

Conclusions: We confirmed that B cells of ITP patients remain hyperreactive in vitro and form high numbers of antibody-producing cells after splenectomy. Thus, chronic ITP may be associated with intrinsic B-cell hyperfunction, leading to the production of antibodies with multiple specificities including that against platelets.
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http://dx.doi.org/10.1038/pr.2015.211DOI Listing
February 2016

Longitudinal Evaluation of Immune Reconstitution and B-cell Function After Hematopoietic Cell Transplantation for Primary Immunodeficiency.

J Clin Immunol 2015 May 15;35(4):373-83. Epub 2015 Apr 15.

University Department of Pediatrics, DPUO, Unit of Immune and Infectious Diseases, Bambino Gesù Children's Hospital, Rome, Italy.

Purpose: Hematopoietic cell transplantation (HCT) provides a curative therapy for severe forms of primary immunodeficiencies (PID). While the timing and extent of T-cell reconstitution following transplant for PID has been studied in depth, less is known about the kinetics of B-cell development and long-term restoration of humoral functions, which been often reported to be suboptimal after HCT.

Methods: We studied longitudinally B-cell development and function in a cohort of 13 PID patients transplanted between 1997 and 2010, with a follow-up ranging from 0.7 to 15 years. Flow cytometric analysis of naïve and antigen-experienced B-cell subsets and in vitro functional responses to CpG were compared with data from healthy children and correlated with the degree of B-cell chimerism and in vivo antibody production.

Results: We found that total memory B-cells count remained below normal levels for the first 2 years of follow up and progressively normalized. Switched memory B-cells (CD19+CD27+IgD-IgM-) were restored early and better than IgM memory B-cells (CD19+CD27+IgD+IgM+), which remained significantly reduced long-term. The recovery of memory B-cells correlated with good in vivo humoral function and normalization of CpG-response. A complete B-cell reconstitution was usually associated with donor B-cells chimerism and pre-transplant conditioning. Donor source and the underlying genetic defect represented also important variables.

Conclusion: Monitoring of phenotypic and functional changes on B-cells following HCT may prove clinically relevant to tailor patients' care. In particular the analysis of IgM memory and switched memory B-cells in addition to in vitro B-cells stimulation are recommended before Ig replacement therapy (IgRT) discontinuation.
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http://dx.doi.org/10.1007/s10875-015-0154-4DOI Listing
May 2015

The possible implication of the S250C variant of the autoimmune regulator protein in a patient with autoimmunity and immunodeficiency: in silico analysis suggests a molecular pathogenic mechanism for the variant.

Gene 2014 Oct 25;549(2):286-94. Epub 2014 Jul 25.

Immunology and Pharmacotherapy Area, Bambino Gesù Children's Hospital IRCCS, Piazza S. Onofrio 4, 00165 Rome, Italy. Electronic address:

Autoimmunity can develop from an often undetermined interplay of genetic and environmental factors. Rare forms of autoimmune conditions may also result from single gene mutations as for autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy, an autosomal recessive disease associated with mutated forms of the autoimmune regulator gene. It was proposed that genetic variability in the autoimmune regulator locus, in particular heterozygous loss-of-function mutations, might favor the development of organ-specific autoimmunity by affecting the presentation of self-antigens in the thymus. Indeed, heterozygous mutations of the autoimmune regulator gene were reported in patients with organ-specific autoimmunity. Also, in primary immunodeficiencies, a breakdown in central/peripheral tolerance frequently produces association with autoimmunity. The causative link may involve a common genetic background and several gene defects have been identified as putative culprits. We report a unique patient, a 14 year old male from Lazio region, affected by common variable immunodeficiency associated with autoimmune manifestations (alopecia, onychodystrophy) and heterozygote for the S250C variant located in the SAND domain of the autoimmune regulator gene protein. To our knowledge this is the first report of the S250C variant in a patient bearing this unusual combination of autoimmunity and immunodeficiency. To obtain insights into the possible molecular effects of the S250C variant, we have carried out an in silico analysis of the SAND domain structure of the autoimmune regulator protein. In particular, homology modeling has allowed us to observe that the cysteine introduced by the S250C variant is surrounded by cationic residues, and by means of molecular dynamics simulations together with pKa calculations, we have shown that these residues remain stably proximal to cysteine-250 lowering its pKa and thus conferring high chemical reactivity to the mutated residue. We propose that the enhanced reactivity of cysteine-250, which is likely to impair the protein function but probably insufficient to produce alone a phenotype as a heterozygous S250C variant due to compensation mechanisms, might become manifest when combined with other genetic/environmental factors. These results can provide the rationale for the patient's unusual phenotype, shedding new light into the pathogenesis of the clinical association of autoimmunity and immunodeficiency.
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http://dx.doi.org/10.1016/j.gene.2014.07.064DOI Listing
October 2014

Inhibition of B-cell proliferation and antibody production by mesenchymal stromal cells is mediated by T cells.

Stem Cells Dev 2015 Jan;24(1):93-103

1 Immunology Research Area, Ospedale Pediatrico Bambino Gesù, IRCSS , Roma, Italy .

Bone marrow (BM)-derived mesenchymal stromal cells (MSCs), endowed with immunosuppressive and anti-inflammatory properties, represent a promising tool in immunoregulatory and regenerative cell therapy. Clarifying the interactions between MSCs and B-lymphocytes may be crucial for designing innovative MSC-based strategies in conditions in which B cells play a role, including systemic lupus erythematosus (SLE) and rejection of kidney transplantation. In this study, we show that, both in healthy subjects and in patients, in vitro B-cell proliferation, plasma-cell differentiation, and antibody production are inhibited by BM-derived MSCs when peripheral blood lymphocytes are stimulated with CpG, but not when sorted B cells are cultured with MSCs+CpG. Inhibition is restored in CpG+MSC cocultures when sorted T cells are added to sorted B cells, suggesting that this effect is mediated by T cells, with both CD4(+) and CD8(+) cells playing a role. Moreover, cell-cell contact between MSCs and T cells, but not between MSCs and B cells, is necessary to inhibit B-cell proliferation. Thus, the presence of functional T cells, as well as cell-cell contact between MSCs and T cells, are crucial for B-cell inhibition. This information can be relevant for implementing MSC-based therapeutic immune modulation in patients in whom T-cell function is impaired.
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http://dx.doi.org/10.1089/scd.2014.0155DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4273193PMC
January 2015

Purification and immunophenotypic characterization of murine MZ and T2-MZP cells.

Methods Mol Biol 2014 ;1190:3-16

Immunology Unit, Research Center Ospedale Pediatrico Bambino Gesù, IRCSS, Piazza S. Onofrio 4, 00165, Rome, Italy,

B cells are generated every day in the bone marrow, but only a small fraction integrates the peripheral B-cell pool. In the murine spleen, we can find several B-cell subsets representing various maturation stages and/or cell functions. The spleen is a complex lymphoid organ organized in two main structures with different functions: the red and white pulp. The red pulp is flowed with blood while the white pulp is organized in primary follicles, with a B-cell area composed of follicular B cells and a T-cell area surrounding a periarterial lymphatic sheath. The frontier between the red and white pulp is defined as the marginal zone and contains the marginal zone B cells. Because B cells, localized in different areas, are characterized by distinct expression levels of B-cell receptor (BCR) and other surface markers, splenic B-cell subsets can be easily identified and purified by flow cytometry analyses and cell sorting (FACS).Here, we will focus on marginal zone B cells and their precursors giving some experimental hints to identify, generate, and isolate these cells. We will combine the use of FACS analysis and confocal microscopy to visualize marginal zone B cells in cell suspension and tissue sections, respectively.
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http://dx.doi.org/10.1007/978-1-4939-1161-5_1DOI Listing
February 2015

Evaluating B-cells: from bone marrow precursors to antibody-producing cells.

Methods Mol Biol 2013 ;1032:45-57

Research Center Ospedale Pediatrico Bambino Gesù (IRCCS), Rome, Italy.

Lymphocyte characterization is primarily based on the differential expression of surface markers. In this context, flow-cytometry analysis (FACS) is an exceptional technique that not only allows the identification of B-cell subsets, but can also be used to evaluate cell function, activation, and division. Here, we will combine the use of FACS analysis and ELISA techniques to identify murine bone marrow and peripheral B-cell subsets. The main function of B cells, derived through a multistage differentiation process from precursor cells, is to produce antibodies. This task is performed by terminally differentiated B cells called antibody-secreting cells (ASC) present at mucosal sites, in the bone marrow and in the spleen. The number and specificity of ASC can be measured by Enzyme-linked immunosorbent spot (ELISPOT) assay, a variation of the enzyme-linked immunosorbent assay (ELISA) used to quantify serum immunoglobulins.
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http://dx.doi.org/10.1007/978-1-62703-496-8_4DOI Listing
March 2014

Immunodeficiency in Vici syndrome: a heterogeneous phenotype.

Am J Med Genet A 2012 Feb 30;158A(2):434-9. Epub 2011 Sep 30.

Division of Immunology and Infectious Disease, University Hospital Department of Pediatrics, University of Rome Tor Vergata and Children's Hospital Bambino Gesù, Rome, Italy.

Vici syndrome is a rare congenital multisystem disorder characterized by agenesis of the corpus callosum, hypotonia, developmental delay, hypopigmentation, cataract, cardiomyopathy, and immunological abnormalities. Recurrent infections, mainly affecting the respiratory tract, have been reported in the majority of cases, representing an important risk factor for morbidity and mortality. The immunological phenotype of patients is extremely variable, ranging from a combined immunodeficiency to nearly normal immunity. We report on a new patient with Vici syndrome, in whom we have extensively investigated immunological features. Despite a mild impairment of the cellular compartment, a defect of humoral immunity was found, requiring treatment with intravenous immunoglobulin. A wider knowledge of immune system abnormalities of Vici syndrome will help to plan strategies for treatment and prevention of infections, such as immunoglobulin replacement and antimicrobial prophylaxis, resulting in improved survival rates.
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http://dx.doi.org/10.1002/ajmg.a.34244DOI Listing
February 2012

Rituximab in a childhood-onset idiopathic refractory chronic inflammatory demyelinating polyneuropathy.

Eur J Paediatr Neurol 2012 May 7;16(3):301-3. Epub 2011 Sep 7.

Unit of Neuromuscular and Neurodegenerative Diseases, Department of Neurosciences, IRCCS Bambino Gesù Pediatric Children's Hospital, P.zza S. Onofrio 4, 00165 Rome, Italy.

Childhood-onset chronic inflammatory demyelinating polyneuropathy (CIDP) are generally responsive to conventional immunosuppressant treatments. However about 20% of patients may be refractory to several treatments and the disease has poor outcome. Rituximab is becoming a promising treatment in selected adult cases of severe CIDPs. We report the effectiveness of Rituximab in a refractory childhood-onset CIDP and we suggest this treatment as an effective choice in unresponsive childhood CIDP.
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http://dx.doi.org/10.1016/j.ejpn.2011.08.002DOI Listing
May 2012

Switched memory B cells maintain specific memory independently of serum antibodies: the hepatitis B example.

Eur J Immunol 2011 Jun 25;41(6):1800-8. Epub 2011 May 25.

Research Center Ospedale Pediatrico Bambino Gesù, IRCSS, Laboratory of Flow-Cytometry and B Cell Development, Rome, Italy.

The immunogenicity of a vaccine is conventionally measured through the level of serum Abs early after immunization, but to ensure protection specific Abs should be maintained long after primary vaccination. For hepatitis B, protective levels often decline over time, but breakthrough infections do not seem to occur. The aim of this study was to demonstrate whether, after hepatitis B vaccination, B-cell memory persists even when serum Abs decline. We compared the frequency of anti-hepatitis-specific memory B cells that remain in the blood of 99 children five years after priming with Infanrix -hexa (GlaxoSmithKline) (n=34) or with Hexavac (Sanofi Pasteur MSD) (n=65). These two vaccines differ in their ability to generate protective levels of IgG. Children with serum Abs under the protective level, <10 mIU/mL, received a booster dose of hepatitis B vaccine, and memory B cells and serum Abs were measured 2 wk later. We found that specific memory B cells had a similar frequency in all children independently of primary vaccine. Booster injection resulted in the increase of memory B cell frequencies (from 11.3 in 10(6) cells to 28.2 in 10(6) cells, p<0.01) and serum Abs (geometric mean concentration, GMC from 2.9 to 284 mIU/mL), demonstrating that circulating memory B cells effectively respond to Ag challenge even when specific Abs fall under the protective threshold.
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http://dx.doi.org/10.1002/eji.201041187DOI Listing
June 2011

Pharmacological inhibition of TLR9 activation blocks autoantibody production in human B cells from SLE patients.

Rheumatology (Oxford) 2010 Dec 25;49(12):2281-9. Epub 2010 Aug 25.

Cytometry and B-cell Development Unit, Research Center, Department of Nephrology and Urology, Children Hospital Bambino Gesù, and Department of Public Health and Cell Biology, University of Rome Tor Vergata, Rome, Italy.

Objectives: Toll-like receptor 9 (TLR9), which recognizes hypomethylated DNA [cytosine-phosphate-guanine (CpG)], plays a role in the maintenance of serological memory and has been recently implicated in the pathogenesis of SLE. We previously reported that in vitro TLR9 triggers memory B-cell differentiation into antibody-producing cells, and that the MyD88-inhibitor ST2825 blocks TLR9-induced plasma cell (PC) generation. Here, we investigated whether memory B cells produce autoantibodies in SLE patients with active disease or in clinical remission, and whether ST2825 could inhibit PC generation in SLE patients.

Methods: Peripheral blood mononuclear cells from 10 SLE patients in clinical remission and 2 with active SLE were cultured in the presence of CpG with or without ST2825. Phenotypical analysis of CpG-stimulated cells was performed by flow cytometry. Supernatants were collected to measure antibody production by ELISA and to detect autoantibodies by IF.

Results: CpG-induced TLR9 stimulation caused autoantibody secretion in patients with active disease and in the majority of patients in clinical remission. Inhibition of MyD88 completely blocked the de novo generation of PCs and the secretion of autoantibodies.

Conclusions: Autoreactive B cells persist in SLE patients during disease remission in the circulating B-cell memory pool. TLR9-dependent activation of memory B cells by pathogens could be one of the mechanisms triggering relapses in SLE. Compounds targeting the TLR/MyD88 pathway may be used as novel therapeutic tools to treat acute disease and to prevent relapses in SLE patients.
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http://dx.doi.org/10.1093/rheumatology/keq226DOI Listing
December 2010

CpG drives human transitional B cells to terminal differentiation and production of natural antibodies.

J Immunol 2008 Jan;180(2):800-8

Laboratory of B-cell Development Research Center, Department of Medical and Surgical Neonatology, Children Hospital Bambino Gesù, and Department of Clinical Medicine, University of Rome La Sapienza, Italy.

The receptor TLR9, recognizing unmethylated bacterial DNA (CpG), is expressed by B cells and plays a role in the maintenance of serological memory. Little is known about the response of B cells stimulated with CpG alone, without additional cytokines. In this study, we show for the first time the phenotypic modification, changes in gene expression, and functional events downstream to TLR9 stimulation in human B cell subsets. In addition, we demonstrate that upon CpG stimulation, IgM memory B cells differentiate into plasma cells producing IgM Abs directed against the capsular polysaccharides of Streptococcus pneumoniae. This novel finding proves that IgM memory is the B cell compartment responsible for the defense against encapsulated bacteria. We also show that cord blood transitional B cells, corresponding to new bone marrow emigrants, respond to CpG. Upon TLR9 engagement, they de novo express AID and Blimp-1, genes necessary for hypersomatic mutation, class-switch recombination, and plasma cell differentiation and produce Abs with anti-pneumococcal specificity. Transitional B cells, isolated from cord blood, have not been exposed to pneumococcus in vivo. In addition, it is known that Ag binding through the BCR causes apoptotic cell death at this stage of development. Therefore, the ability of transitional B cells to sense bacterial DNA through TLR9 represents a tool to rapidly build up the repertoire of natural Abs necessary for our first-line defense at birth.
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http://dx.doi.org/10.4049/jimmunol.180.2.800DOI Listing
January 2008

Human Mena protein, a serex-defined antigen overexpressed in breast cancer eliciting both humoral and CD8+ T-cell immune response.

Int J Cancer 2004 May;109(6):909-18

Laboratory of Experimental Chemotherapy, Regina Elena Cancer Institute, Rome, Italy.

Screening of a cDNA expression library from a primary breast tumor with the autologous patient serum led to the isolation of 6 cDNA clones corresponding to 3 different genes, including a novel gene that maps to chromosome 1 and encodes the human homologue of mouse Mena (hMena, cDNA clone RMNY-BR-55), a protein of the Ena/VASP family involved in the regulation of cell motility and adhesion. A cancer-restricted antibody response against hMena was demonstrated, since 18/93 cancer patient sera, the majority (10/52) from breast cancer, showed anti-hMena-specific IgG, while no antibodies were present in healthy donors. When hMena protein expression was analyzed by Western blot and immunohistochemistry, the antigen was overexpressed in the majority of breast cancer cell lines and in 75% of primary breast tumor lesions evaluated. Furthermore, when HLA-A2-restricted peptides from the hMena sequence were used to stimulate CD8+ T cells, an hMena-specific response was found in 9 out of 12 HLA-A2+ breast cancer patients. In 4 patients, this cell-mediated immune response was concomitant with antibody response to hMena. Furthermore, an hMena-specific T-cell line was established from an HLA-A2+ breast cancer patient whose primary tumor lesion overexpressed the hMena protein. The present findings highlight the emerging role that overexpression of cytoskeleton regulatory components may have in the induction of a specific antitumor immune response.
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http://dx.doi.org/10.1002/ijc.20094DOI Listing
May 2004

Polyclonal Antibodies Against gp185HER2 Peptides: Their Putative Role in the Identification of a Particular HER2 Status in Patients With Breast Cancer.

J Immunother (1991) 2001 May;24(3):221-231

Laboratory of Pathophysiology, CRS Regina Elena Cancer Institute; Laboratory of Pathology, Regina Elena Cancer Institute; Laboratory of Biochemistry, CRS Regina Elena Cancer Institute; and Department of Experimental Medicine and Pathology, University of Rome "La Sapienza", Rome, Italy.

SUMMARY: The HER2 oncogene and its relative oncoprotein, gp185HER2, a transmembrane glycoprotein belonging to the epidermal growth factor receptor family, are overexpressed in a wide range of solid tumors including breast and ovarian cancer. In patients with breast cancer, both humoral and cell-mediated HER2 immune responses have been found as well as in some patients with gp185HER2 nonoverexpressing tumors. To establish whether peptide sequences identified as HLA-A2-restricted T-cell epitopes are expressed in breast tumor cell lines and tissues, we produced and characterized by different methodologic approaches polyclonal antibodies raised against four gp185HER2 peptides. Two of the antibodies recognized peptides eluted from the HLA-A2 groove of the mDAmB231 breast cancer cell line expressing a basal level of gp185HER2. Paraffin-embedded primary and metastatic breast tumors were specifically immunostained by all four reagents, thereby showing an overlapping reactivity. When this immunoreactivity was compared with that obtained using two different monoclonal antibodies, in 105 breast primary tumors and 36 corresponding lymph node metastases, we identified a subset of tumors that were negative with anti-gp185HER2 monoclonal antibodies and positive with the four antipeptide antibodies. Our novel observations provide in vivo evidence of the complexity involved in evaluating HER2 expression, and open a new path for understanding the biologic significance of HER2 status in breast tumors.
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May 2001
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