CD21- CD27- Atypical B Cells in a Pediatric Cohort Study: An Extensive Single Center Flow Cytometric Analysis.

Atypical B cells (atBCs) are a distinct B-cell population and represent approximately 5% of B cells in peripheral blood (PB) of healthy adult individuals. However, in adults these cells are expanded in conditions of chronic infections, inflammation, primary immunodeficiencies, autoimmune diseases, and aging. Their immunophenotype is characterized by the lack of CD21 expression and the hallmark human memory B-cell marker CD27. In this study, we investigated the immunophenotype of atBCs in different pediatric pathological conditions and correlated their expansion with the children's clinical diagnosis. We were able to retrospectively evaluate 1,571 consecutive PB samples, corresponding to 1,180 pediatric patients, by using a 9-color flow-cytometric panel. The results, compared with a pediatric healthy cohort, confirmed an expansion of atBCs in patient samples with percentages greater than 5% of total B cells. Four subpopulations with different expressions of IgM and IgD were discriminated: IgM+IgD+, IgM+-only, IgD+-only, and IgM-IgD-. IgG+ atBCs were predominant in the IgM- IgD- subpopulation. Moreover, the study highlighted some features of atBCs, such as a low CD38 expression, a heterogeneity of CD24, a high expression of CD19 and a large cell size. We also demonstrated that an increase of atBCs in a pediatric cohort is correlated with immunodeficiencies, autoimmune, inflammatory, and hematological disorders, consistent with previous studies mainly performed in adults. Furthermore, our flow cytometric clustering analysis corroborated the recent hypothesis of an alternative B origin for atBCs.

Comprehensive phenotyping of human peripheral blood B lymphocytes in pathological conditions.

Several diseases are associated with alterations of the B-cell compartment. Knowing how to correctly identify by flow cytometry the distribution of B-cell populations in the peripheral blood is important to help in the early diagnosis. In the accompanying article we describe how to identify the different B-cell subsets in the peripheral blood of healthy donors. Here we show a few examples of diseases that cause dysregulation of the B-cell compartment.

Comprehensive phenotyping of human peripheral blood B lymphocytes in healthy conditions.

The B cell compartment provides innate and adaptive immune defenses against pathogens. Different B cell subsets, reflecting the maturation stages of B cells, have noninterchangeable functions and roles in innate and adaptive immune responses. In this review, we provide an overview of the B cell subsets present in peripheral blood of healthy individuals. A specific gating strategy is also described to clearly and univocally identify B cell subsets based on the their phenotypic traits by flow cytometric analysis.

Immune Response of Neonates Born to Mothers Infected With SARS-CoV-2.

Importance: Although several studies have provided information on short-term clinical outcomes in children with perinatal exposure to SARS-CoV-2, data on the immune response in the first months of life among newborns exposed to the virus in utero are lacking. Objective: To characterize systemic and mucosal antibody production during the first 2 months of life among infants who were born to mothers infected with SARS-CoV-2. Design, Setting, and Participants: This prospective cohort study enrolled 28 pregnant women who tested positive for SARS-CoV-2 infection and who gave birth at Policlinico Umberto I in Rome, Italy, from November 2020 to May 2021, and their newborns. Maternal and neonatal systemic immune responses were investigated by detecting spike-specific antibodies in serum, and the mucosal immune response was assessed by measuring specific antibodies in maternal breastmilk and infant saliva 48 hours after delivery and 2 months later. Exposures: Maternal infection with SARS-CoV-2 in late pregnancy. Main Outcomes and Measures: The systemic immune response was evaluated by the detection of SARS-CoV-2 IgG and IgA antibodies and receptor binding domain-specific IgM antibodies in maternal and neonatal serum. The mucosal immune response was assessed by measuring spike-specific antibodies in breastmilk and in infant saliva, and the presence of antigen-antibody spike IgA immune complexes was investigated in breastmilk samples. All antibodies were detected using an enzyme-linked immunosorbent assay. Results: In total, 28 mother-infant dyads (mean [SD] maternal age, 31.8 [6.4] years; mean [SD] gestational age, 38.1 [2.3] weeks; 18 [60%] male infants) were enrolled at delivery, and 21 dyads completed the study at 2 months' follow-up. Because maternal infection was recent in all cases, transplacental transfer of virus spike-specific IgG antibodies occurred in only 1 infant. One case of potential vertical transmission and 1 case of horizontal infection were observed. Virus spike protein-specific salivary IgA antibodies were significantly increased (P = .01) in infants fed breastmilk (0.99 arbitrary units [AU]; IQR, 0.39-1.68 AU) vs infants fed an exclusive formula diet (0.16 AU; IQR, 0.02-0.83 AU). Maternal milk contained IgA spike immune complexes at 48 hours (0.53 AU; IQR, 0.25-0.39 AU) and at 2 months (0.09 AU; IQR, 0.03-0.17 AU) and may have functioned as specific stimuli for the infant mucosal immune response. Conclusions and Relevance: In this cohort study, SARS-CoV-2 spike-specific IgA antibodies were detected in infant saliva, which may partly explain why newborns are resistant to SARS-CoV-2 infection. Mothers infected in the peripartum period appear to not only passively protect the newborn via breastmilk secretory IgA but also actively stimulate and train the neonatal immune system via breastmilk immune complexes.

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

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 64 adults with a spectrum of clinical presentations: 28 healthy SARS-CoV-2-negative contacts of COVID-19 cases; 20 asymptomatic SARS-CoV-2-infected cases; eight patients with Mild COVID-19 disease and eight 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.

Prolonged Impairment of Immunological Memory After Anti-CD20 Treatment in Pediatric Idiopathic Nephrotic Syndrome.

Anti-CD20 therapy is effective in idiopathic nephrotic syndrome (INS). However, transient or sustained hypogammaglobulinemia predisposing to an increased risk of infectious diseases can follow treatment in some patients. We analyzed the long-term effects of anti-CD20 therapy on immunological memory in 27 frequently-relapsing/steroid-dependent INS pediatric patients after more than 4 years from the first and at least 2 years from the last anti-CD20 infusion. Twenty-one INS children, never treated with anti-CD20 and under an intense oral immunosuppression with prednisone, mycophenolate mofetil, and calcineurin inhibitors were also included as control group. Levels of circulating B-cell subpopulations, total serum immunoglobulins and IgG and memory B cells directed against hepatitis B virus (HBV) and tetanus were determined and correlated with clinical characteristics. Nine patients never relapsed after more than 2 years from the last anti-CD20 administration (5 after the first, 3 after the second, and 1 after the fifth infusion). At last follow-up, most patients showed a complete recovery and normalization of total (27/27), transitional (27/27), and mature-naïve B cells (25/27). However, a sustained and significant reduction of total memory (20/27) and switched memory (21/27) B cells was found in most patients. 11/27 patients showed hypogammaglobulinemia at last follow-up and, among these, four presented with a severe hypogammaglobulinemia (IgG < 160 mg/dl). In contrast, no patient in the control group developed a severe hypogammaglobulinemia. Age at the time of first anti-CD20 administration was positively associated with IgG levels at last follow-up (p = 0.008); accordingly, younger patients had an increased risk of hypogammaglobulinemia (p = 0.006). Furthermore, severe hypogammaglobulinemia and delayed switched memory B-cell reconstitution were more frequent in non-relapsing patients. Reduced IgG levels against HBV and tetanus were observed at baseline and further declined at last follow-up. Antigen-specific memory B-cells were induced by re-immunization, but specific IgG titers remained low. In conclusion, anti-CD20 therapy can be disease-modifying in some INS patients. However, a prolonged impairment of immunological memory occurs frequently, independently from the number of anti-CD20 infusions, particularly in younger patients. Re-immunization may be necessary in these patients.

Distinct gut microbiota profile in antiretroviral therapy-treated perinatally HIV-infected patients associated with cardiac and inflammatory biomarkers.

OBJECTIVE: Persistent inflammation and higher risk to develop cardiovascular diseases still represent a major complication for HIV-infected patients despite effective antiretroviral therapy (ART). We investigated the correlation between the gut microbiota profile, markers of inflammation, vascular endothelial activation (VEA) and microbial translocation (MT) in perinatally HIV-infected patients (PHIV) under ART. DESIGN: Cross-sectional study including 61 ART-treated PHIV (age range 3-30 years old) and 71 age-matched healthy controls. Blood and stool sample were collected at the same time and analyzed for gut microbiota composition and plasma biomarkers. METHODS: Gut microbiota composition was determined by 16S rRNA targeted-metagenomics. Soluble markers of MT, inflammation and VEA were quantified by ELISA or Luminex assay. Markers of immune activation were analyzed by flow cytometry on CD4 and CD8T cells. RESULTS: We identified two distinct gut microbiota profiles (groups A and B) among PHIV. No different clinical parameters (age, sex, ethnicity, clinical class), dietary and sexual habits were found between the groups. The group A showed a relative dominance of Akkermansia muciniphila, whereas gut microbiota of group B was characterized by a higher biodiversity. The analysis of soluble markers revealed a significantly higher level of soluble E-selectine (P = 0.0296), intercellular adhesion molecule-1 (P = 0.0028), vascular adhesion molecule-1 (P = 0.0230), IL-6 (P = 0.0247) and soluble CD14 (P = 0.0142) in group A compared with group B. CONCLUSION: Distinctive gut microbiota profiles are differently associated with inflammation, microbial translocation and VEA. Future studies are needed to understand the role of A. muciniphila and risk to develop cardiovascular diseases in PHIV.

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

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.

Timing of HAART defines the integrity of memory B cells and the longevity of humoral responses in HIV-1 vertically-infected children.

HIV-1 infection induces a progressive disruption of the B cell compartment impairing long-term immune responses to routine immunizations. Depletion of specific memory B cell pools occurs during the 1st stages of the infection and cannot be reestablished by antiretroviral treatment. We reasoned that an early control of viral replication through treatment could preserve the normal development of the memory B cell compartment and responses to routine childhood vaccines. Accordingly, we evaluated the effects of different highly-active antiretroviral therapy (HAART) schedules in 70 HIV-1 vertically-infected pediatric subjects by B cell phenotypic analyses, antigen-specific B cell enzyme-linked immunosorbent spot (ELISpot) and ELISA for common vaccination and HIV-1 antigens. Initiation of HAART within the 1st year of life permits the normal development and maintenance of the memory B cell compartment. On the contrary, memory B cells from patients treated later in time are remarkably reduced and their function is compromised regardless of viral control. A cause for concern is that both late-treated HIV-1 controllers and noncontrollers loose protective antibody titers against common vaccination antigens. Timing of HAART initiation is the major factor predicting the longevity of B cell responses in vaccinated HIV-1-infected children.

Post-natal ontogenesis of the T-cell receptor CD4 and CD8 Vbeta repertoire and immune function in children with DiGeorge syndrome.

DiGeorge syndrome (DGS) is a congenital disorder characterized by typical facial features, hypoparatyroidism, conotruncal cardiac defects and thymic hypoplasia. Although there are some reports addressing lymphocytes counts and function in DGS children over time, few data have been reported on the T-cell receptor V beta (TCRBV) repertoire in relation to disease progression. The aim of this study was to evaluate the degree and nature of immunodeficiency and to investigate a possible correlation to clinical findings. We used third complementary region (CDR3) size spectratyping as a tool for monitoring T-cell repertoire diversity in 7 DGS's children. The rate of thymic output, the phenotype and function of peripheral T-cells and the humoral immunity were also investigated. At baseline a profound alteration of the TCR repertoire was noted, mainly in the CD8+ T-cells, in DGS patients when compared to a control group. Furthermore, analysis of thymic output showed a significant decrease in TCR rearrangement excision circles (TRECs) levels in the patient group. Immunoglobulin abnormalities were also detected. The observed TCR repertoire alterations, although not statistically significant, may suggest an increased susceptibility to infections. A parallel increase in the TCR repertoire diversity and clinical improvement occurred during the follow-up. Our results confirm that the extent of immunodeficiency is highly variable and could improve through childhood, and indicate that TCR repertoire may be a useful marker to clinically monitor thymic function in this primary immunodeficiency.