Flow Based Enumeration of Plasmablasts in Peripheral Blood After Vaccination as a Novel Diagnostic Marker for Assessing Antibody Responses in Patients with Hypogammaglobulinaemia

Computer modelling & simulatio

Defects in memory B-cell and plasma cell subsets expressing different immunoglobulin-subclasses in patients with CVID and immunoglobulin subclass deficiencies

Background: Predominantly antibody deficiencies (PADs) are the most prevalent primary immunodeficiencies, but their B-cell defects and underlying genetic alterations remain largely unknown. Objective: We investigated patients with PADs for the distribution of 41 blood B-cell and plasma cell (PC) subsets, including subsets defined by expression of distinct immunoglobulin heavy chain subclasses. Methods: Blood samples from 139 patients with PADs, 61 patients with common variable immunodeficiency (CVID), 68 patients with selective IgA deficiency (IgAdef), 10 patients with IgG subclass deficiency with IgA deficiency, and 223 age matched control subjects were studied by using flow cytometry with EuroFlow immunoglobulin isotype staining. Patients were classified according to their B-cell and PC immune profile, and the obtained patient clusters were correlated with clinical manifestations of PADs. Results: Decreased counts of blood PCs, memory B cells (MB Cs), or both expressing distinct IgA and IgG subclasses were identified in all patients with PADs. In patients with IgAdef, B-cell defects were mainly restricted to surface membrane (sm)IgA(+) PCs and MBCs, with 2 clear subgroups showing strongly decreased numbers of smIgA(+) PCs with mild versus severe smIgA(+) MBC defects and higher frequencies of nonrespiratory tract infections, autoimmunity, and affected family members. Patients with IgG subclass deficiency with IgA deficiency and those with CVID showed defects in both smIgA(+) and smIgG(+) MBCs and PCs. Reduced numbers of switched PCs were systematically found in patients with CVID (absent in 98%), with 6 different defective MBC (and clinical) profiles: (1) profound decrease in MBC numbers; (2) defective CD27(+) MBCs with almost normal IgG(3)(+) MBCs; (3) absence of switched MBCs; and (4) presence of both unswitched and switched MBCs without and; (5) with IgG(2)(+) MBCs; and (6) with IgA(1)(+) MBCs. Conclusion: Distinct PAD defective B-cell patterns were identified that are associated with unique clinical profiles

The EuroFlow PID Orientation Tube for Flow Cytometric Diagnostic Screening of Primary Immunodeficiencies of the Lymphoid System

In the rapidly evolving field of primary immunodeficiencies (PID), the EuroFlow consortium decided to develop a PID orientation and screening tube that facilitates fast, standardized, and validated immunophenotypic diagnosis of lymphoid PID, and allows full exchange of data between centers. Our aim was to develop a tool that would be universal for all lymphoid PIDs and offer high sensitivity to identify a lymphoid PID (without a need for specificity to diagnose particular PID) and to guide and prioritize further diagnostic modalities and clinical management. The tube composition has been defined in a stepwise manner through several cycles of design-testing-evaluation-redesign in a multicenter setting. Equally important appeared to be the standardized pre-analytical procedures (sample preparation and instrument setup), analytical procedures (immunostaining and data acquisition), the software analysis (a multidimensional view based on a reference database in Infinicyt software), and data interpretation. This standardized EuroFlow concept has been tested on 250 healthy controls and 99 PID patients with defined genetic defects. In addition, an application of new EuroFlow software tools with multidimensional pattern recognition was designed with inclusion of maturation pathways in multidimensional patterns (APS plots). The major advantage of the EuroFlow approach is that data can be fully exchanged between different laboratories in any country of the world, which is especially of interest for the PID field, with generally low numbers of cases per center

Defects in memory B-cell and plasma cell subsets expressing different immunoglobulin-subclasses in patients with CVID and immunoglobulin subclass deficiencies

Background: Predominantly antibody deficiencies (PADs) are the most prevalent primary immunodeficiencies, but their B-cell defects and underlying genetic alterations remain largely unknown. Objective: We investigated patients with PADs for the distribution of 41 blood B-cell and plasma cell (PC) subsets, including subsets defined by expression of distinct immunoglobulin heavy chain subclasses. Methods: Blood samples from 139 patients with PADs, 61 patients with common variable immunodeficiency (CVID), 68 patients with selective IgA deficiency (IgAdef), 10 patients with IgG subclass deficiency with IgA deficiency, and 223 agematched control subjects were studied by using flow cytometry with EuroFlow immunoglobulin isotype staining. Patients were classified according to their B-cell and PC immune profile, and the obtained patient clusters were correlated with clinical manifestations of PADs. Results: Decreased counts of blood PCs, memory B cells (MBCs), or both expressing distinct IgA and IgG subclasses were identified in all patients with PADs. In patients with IgAdef, B-cell defects were mainly restricted to surface membrane (sm)IgA1 PCs and MBCs, with 2 clear subgroups showing strongly decreased numbers of smIgA1 PCs with mild versus severe smIgA1 MBC defects and higher frequencies of nonrespiratory tract infections, autoimmunity, and affected family members. Patients with IgG subclass deficiency with IgA deficiency and those with CVID showed defects in both smIgA1 and smIgG1 MBCs and PCs. Reduced numbers of switched PCs were systematically found in patients with CVID (absent in 98%), with 6 different defective MBC (and clinical) profiles: (1) profound decrease in MBC numbers; (2) defective CD271 MBCs with almost normal IgG3 1 MBCs; (3) absence of switched MBCs; and (4) presence of both unswitched and switched MBCs without and; (5) with IgG2 1 MBCs; and (6) with IgA

Terminally differentiated memory T cells are increased in patients with common variable immunodeficiency and selective IgA deficiency

Introduction : Previous studies showed that several lymphocyte abnormalities seen in the most frequent symptomatic immunoglobulin deficiency, common variable immunodeficiency (CVID), were also observed in a genetically related asymptomatic disorder – selective IgA deficiency (IgAD). In this study we searched for abnormalities in the differentiation stages of T cells as well as for similarities of these abnormalities in CVID and IgAD patients. Material and methods : Using flow cytometry in 80 patients with IgAD, 48 patients with CVID, and 80 control persons we determined T-lymphocyte subsets: both CD4 and CD8 were divided into the naïve CD45RO–CD27 + , early differentiated CD45RO + CD27 + , late differentiated CD45RO + CD27– and fully differentiated effector CD45RO–CD27– memory T cells, as well as Treg cells, defined as CD4 + CD25highCD127low T cells. Results : An increase of CD4 + and CD8 + late differentiated memory cells was observed comparing CVID patients to controls, as well as comparing IgAD patients to controls. In CVID patients an increase of CD4 + early differentiated memory cells, a decrease of CD8 + intermediate memory cells, and CD4 + and CD8 + naïve cells were found as well. The abnormalities in IgAD patients might be explained by higher CMV seropositivity observed in our IgAD. We confirmed the repeatedly published decrease of Treg cells in CVID patients, while Treg cells in IgAD patients were increased compared to controls. Conclusions : Our results show T-cell activation not only in CVID, but also in IgAD patients. The increase in IgAD patients may be influenced by a more frequent CMV infection in our group of IgAD patients

B cell subsets reconstitution and immunoglobulin levels in children and adolescents with B non-Hodgkin lymphoma after treatment with single anti CD20 agent dose included in chemotherapeutic protocols: single center experience and review of the literature

Background. RTX, an anti-CD20 monoclonal antibody, added to chemotherapy has proven to be effective in children and adolescents with high-grade, high-risk and matured non-Hodgkin lymphoma. RTX leads to prompt CD19+ B lymphocyte depletion. However, despite preserved immunoglobulin production by long-lived plasmablasts after treatment, patients remain at risk of prolonged hypogammaglobulinemia. Further, there are few general guidelines for immunology laboratories and clinical feature monitoring after B cell-targeted therapies. The aim of this paper is to describe B cell reconstitution and immunoglobulin levels after pediatric B-NHL protocols, that included a single RTX dose and to review the literature. Methods. A retrospective single-center study on the impact of a single RTX dose included in a chemotherapeutic pediatric B Non-Hodgkin Lymphoma (B-NHL) treatment protocols. Immunology laboratory and clinical features were evaluated over an eight hundred days follow-up (FU) period, after completing B-NHL treatment. Results. Nineteen patients (fifteen Burkitt lymphoma, three Diffuse large B cell lymphoma, and one Marginal zone B cell lymphoma) fulfilled the inclusion criteria. Initiation of B cell subset reconstitution occurred a median of three months after B-NHL treatment. Naïve and transitional B cells declined over the FU in contrast to the marginal zone and the switched memory B cell increase. The percentage of patients with IgG, IgA, and IgM hypogammaglobulinemia declined consistently over the FU. Prolonged IgG hypogammaglobulinemia was detectable in 9%, IgM in 13%, and IgA in 25%. All revaccinated patients responded to protein-based vaccines by specific IgG antibody production increase. Following antibiotic prophylaxes, none of the patients with hypogammaglobulinemia manifested with either a severe or opportunistic infection course. Conclusion. The addition of a single RTX dose to the chemotherapeutic treatment protocols was not shown to increase the risk of developing secondary antibody deficiency in B-NHL pediatric patients. Observed prolonged hypogammaglobulinemia remained clinically silent. However interdisciplinary agreement on regular long-term immunology FU after anti-CD20 agent treatment is required

Chapter Flow Based Enumeration of Plasmablasts in Peripheral Blood After Vaccination as a Novel Diagnostic Marker for Assessing Antibody Responses in Patients with Hypogammaglobulinaemia

Computer modelling & simulatio

TNFR2 expression is a hallmark of human memory B cells with suppressive function

Tumor Necrosis Factor Receptor 2 (TNFR2) expression is increasingly being linked to tolerogenic immune reactions and cells with suppressor function including a subset of T-regulatory cells. B-regulatory cells play an important role in control of T-cell responses and inflammation. Recently, we described TNFR2 as a marker for IL-10-producing B cells, a hallmark of this cell subset. Here, we demonstrate that proliferation of T cells is reduced in the presence of TNFR2 positive human memory B cells generated with TLR9 ligand, while TNFR2- and TNFR2+CD27- B cells display costimulatory activity. Our data further reveal that IL-10 secretion is characteristic of IgM+ naïve and memory B cells but suppressive activity is not restricted to IL-10: (i) the inhibitory effect of TNFR2+ switched memory B cells was comparable to that exerted by TNFR2+ IgM+ memory B cells although IL-10 secretion levels in the cocultures were lower; (ii) supernatants from TNFR2+ memory B cells failed to suppress T-cell proliferation. Based on our findings, we propose that formation of Breg is a specific characteristic of human memory B cells undergoing terminal differentiation. Our data further corroborate that TNFR2 represents a viable marker for identification of memory B cells with regulatory function