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Background: Our recent publication "Inability of a monoclonal anti-light chain antibody to detect clonal plasma cells in a patient with multiple myeloma by multicolor flow cytometry," underlined the importance of choice of antibodies to detect cytoplasmic light chains. Our present study extends this finding for detection of surface immunoglobulin (SIg) light chains on clonal B-cells., Methods: Multicolor flow cytometry was used for analyzing bone marrow (BM) from a patient with a CD10-positive follicular lymphoma for infiltrating clonal B-cells., Results: In the BM aspirate, B cells could be identified expressing CD19, CD10, and high levels of CD20. No SIg light chain expression was found on this population of B cells employing monoclonal antibodies. Re-analysis using polyclonal antibodies against SIg light chains, revealed presence of lambda light chains on the CD10positive B-cells., Conclusions: These data illustrate when antibodies against SIg light chains are employed for B-cell clonality assessment, polyclonal antibodies are preferred over monoclonal antibodies., (© 2014 Wiley Periodicals, Inc.)

Background: Pulmonary disease is common in patients with common variable immunodeficiency disorders (CVID) and involves infections, chronic airway disease and interstitial lung disease. Chronic pulmonary disease is associated with excess morbidity and early mortality and therefore early detection and monitoring of progression is essential., Methods and Purpose: Thin slice CT scan and pulmonary function were used to determine the prevalence and spectrum of chronic (pre-clinical) pulmonary disease in adult CVID patients regardless of symptoms. CT Scans were scored for airway abnormalities (AD) and interstitial lung disease (ILD). Other CVID related complications and B and T lymphocyte subsets were analyzed to identify patients at risk for pulmonary disease., Results: Significant pulmonary abnormalities were detected in 24 of the 47 patients (51%) consisting of AD in 30% and ILD in 34% of cases. In only 7 (29%) of these 24 patients pulmonary function test proved abnormal. The presence of AD was correlated to (recurrent) lower respiratory tract infections despite IgG therapy. The presence of ILD was correlated to autoimmune disease and a reduction in the numbers of CD4 + T cells, naïve CD4 + T cells, naïve CD8 + T cells and memory B cells and lower IgG through levels over time., Conclusion: Preclinical signs of AD and ILD are common in CVID patients despite Ig therapy and do not correlate to pulmonary function testing. Patients at risk for ILD might be identified by the presence of autoimmunity or a deranged T cell pattern. Larger studies are needed to confirm these findings and to determine thresholds for the T lymphocyte subsets.

Objectives: 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is known to have toxic effects on the haematopoietic system in animals but epidemiological studies in humans have shown inconsistent results. In this cross-sectional study we investigated changes in peripheral blood cell counts and lymphocyte subsets among workers from a Dutch historical cohort occupationally exposed to chlorophenoxy herbicides and contaminants including TCDD., Methods: Forty-seven workers who had been exposed to high levels of TCDD in the past and 38 low-exposed workers were included in the current investigation. Complete blood counts and differential and major lymphocyte subsets were analysed. Current plasma levels of TCDD (TCDD(current)) were determined by high-resolution gas chromatography/isotope-dilution high resolution mass spectrometry. TCDD blood levels at the time of last exposure (TCDD(max)) were estimated using a one-compartment first order kinetic model., Results: Cell counts and lymphocyte subsets were similar between high- and low-exposed workers, except for a non-dose dependent increase in CD4/CD8 ratio among high-exposed workers. Interestingly, most lymphocyte subsets, in particular the B cell compartment, showed a decrease with increasing levels of both TCDD(current) and TCDD(max)., Conclusions: Overall, our study showed that plasma TCDD levels had no effect on white blood cell counts and major subsets. However, a non-significant decrease in most lymphocyte subsets was noted, with the strongest effect for B cells. The latter finding may suggest that dioxin exposure might have an adverse impact on the haematopoietic system and lends some support to B cell lymphoma induction by dioxin.

Background: B cells of patients with common variable immunodeficiency (CVID) disorders display impairment in production of immunoglobulin class-switched antibodies, which is possibly contributed to by defects in early B-cell activation. On resting B cells, B-cell receptors (BCRs) are organized in oligomers that are signaling inactive. Their triggering by cognate antigen causes the lateral reorganization of BCRs and associated proteins into signalosomes, resulting in BCR-activated calcium entry. In resting cells the B-cell surface antigen CD20 is associated with the BCR but dissociates on signalosome formation., Objective: We sought to determine whether CD20 dissociation from the BCR during early B-cell activation might contribute to the development of CVID disorders., Methods: We evaluated BCR signalosome formation, internalization, and signaling in primary B cells of pediatric patients with CVID disorders and healthy control subjects., Results: In many pediatric patients with CVID disorders, B cells exhibit significant deficits in BCR triggering-mediated calcium entry in the cytosol, which correlates with impaired plasmablast differentiation in vitro. These alterations did not originate from upregulation of CD22 or defects in calcium channels and did not involve gene mutations in phospholipase Cγ2 or Bruton tyrosine kinase. Instead, B cells from patients with CVID disorders exhibited reduced BCR dissociation from CD20. BCR or CD20 cross-linking induced less BCR internalization, and antibody-mediated CD20 triggering elicited less BCR downstream signaling, as measured based on secondary fluxes., Conclusions: We propose that CD20 dissociation from the BCR signalosome is pivotal to BCR-mediated calcium mobilization in the cytosol. Defects in CD20/BCR signalosome conformation might predispose to the spectrum of CVID disorders., (Copyright © 2011 American Academy of Allergy, Asthma & Immunology. Published by Mosby, Inc. All rights reserved.)

The diagnosis of common variable immunodeficiency (CVID) is reserved for patients who suffer from undefined B cell dysfunction. Division of the CVID population into subgroups enables research for underlying disease causes. We studied clinical features and lymphocyte characteristics in 38 children with CVID and compared them to 30 children with less severe antibody deficiencies (e.g. specific antibody deficiency combined with IgG subclass deficiency) and with 65 pediatric controls. Most pediatric immune phenotypes were comparable to adult CVID phenotypes, including a selective increase in newly formed B cells and a decrease in memory B cells and CD4(+) T cells. Eighteen percent of pediatric patients had a mutation in the TNFRSF13B gene, which requires further investigation. Finally, pediatric patients with decreased class-switched memory B cells had significantly more complications. A pediatric classification for CVID may enable prediction and early diagnosis of disease related complications and provide a framework for further etiologic research., (Copyright 2009 Elsevier Inc. All rights reserved.)

Some minor histocompatibility antigens (mHags) are expressed exclusively on patient hematopoietic and malignant cells, and this unique set of antigens enables specific targeting of hematological malignancies after human histocompatability leucocyte antigen (HLA)-matched allogeneic stem cell transplantation (allo-SCT). We report the first hematopoietic mHag presented by HLA class II (HLA-DQA1*05/B1*02) molecules to CD4(+) T cells. This antigen is encoded by a single-nucleotide polymorphism (SNP) in the B cell lineage-specific CD19 gene, which is an important target antigen for immunotherapy of most B cell malignancies. The CD19(L)-encoded antigen was identified using a novel and powerful genetic strategy in which zygosity-genotype correlation scanning was used as the key step for fine mapping the genetic locus defined by pairwise linkage analysis. This strategy was also applicable for genome-wide identification of a wide range of mHags. CD19(L)-specific CD4(+) T cells provided antigen-specific help for maturation of dendritic cells and for expansion of CD8(+) mHag-specific T cells. They also lysed CD19(L)-positive malignant cells, illustrating the potential therapeutic advantages of targeting this novel CD19(L)-derived HLA class II-restricted mHag. The currently available immunotherapy strategies enable the exploitation of these therapeutic effects within and beyond allo-SCT settings.

Cytotoxic T cells play an important role in graft-versus-host-disease (GvHD) and graft-versus-leukaemia/myeloma, which may occur in patients treated with an allogeneic stem cell transplantation (ASCT). Here, we describe the selection of a myeloma reactive CD4+ cytotoxic T cell-line (CTL) and two CD4+ clones from this CTL. The CTL was generated from the blood from a patient with multiple myeloma (MM) with graft versus myeloma/GvHD, following an ASCT. The CTL was stimulated using irradiated peripheral blood mononuclear cells and EBV transformed B cells from the myeloma patient (EBVp), both of which were obtained prior to ASCT. Both the CTL and the two T cell clones specifically lysed EBVp and secreted IFN-gamma after coculture with EBVp and autologous myeloma tumour cells in a class II restricted fashion. These results show that myeloma tumour cells and autologous B cells present a common polymorphic peptide that functions as a target for graft derived cytotoxic T cells. Identification of these proteins will give insight into the relationship between graft versus myeloma (GvM) and GvHD and may provide immunotherapeutical targets in the treatment of MM.

Expression of CD44v9-containing isoforms (CD44v9) on myeloma plasma cells correlates with unfavorable prognosis, suggesting that CD44 variant molecules are involved in the disease process. In this study, the presence of CD44v on B cell lines from different stages of development was analyzed by flow cytometry and a role in adhesion to stromal cells from different tissues was evaluated in in vitro binding assays. CD44v3, v6 and v9 isoforms were exclusively expressed on plasma cell lines and CD44v9 expression correlated with IL-6-dependent plasma cell growth. Binding studies using CD44 isoform- specific reagents showed that CD44v6 and CD44v9 were involved in binding to bone marrow stromal cells, but not to in vitro synthesized ECM or hyaluronic acid. CD44v9-mediated plasma cell binding resulted in a significant induction of IL-6 secretion by bone marrow stromal cells. Large differences in quantitative plasma cell binding to stromal cells from different tissues were observed. These, however, could not be related to a differential use of CD44v in these binding processes. The role of CD44v9 in adhesion induced IL-6 secretion and its preferential expression on IL-6-dependent plasma cell lines may explain the previously observed correlation between CD44v9 expression and adverse prognosis in multiple myeloma.

We show that major histocompatibility complex (MHC) class II molecules on B cells transit signals which regulate adhesion in a negative manner. Engagement of MHC class II molecules with antibodies results in detachment of B cells previously bound to interferon-gamma-activated human umbilical cord venous endothelial cells. This process depends on metabolic energy, active signaling and protein tyrosine kinase activity. The adhesion pathway influenced by this signaling event is neuraminidase sensitive. The anti-adhesive signaling program is activated in B cell lines with a mature phenotype, e.g. normal B cells from spleen and tonsil. In contrast, cell lines with a pre-B cell phenotype and normal B cells from peripheral blood are refractory to MHC class II-mediated regulation of adhesion. These results extend to neoplastic cells from patients with lymphoproliferative diseases representing different stages of B cell maturation. These results suggest that MHC class II-mediated signals regulate B cell adhesion in a developmentally programmed fashion; this might have implications for clinical behavior of B cell malignancies.

A possible role of intercellular adhesion molecule 1 (ICAM-1, CD54) in transmembrane signaling was investigated in B cells from the Burkitt lymphoma cell line MTLM4. Cross-linking of membrane IgM (mIgM) induced an increase in intracellular free Ca2+ as a result of the release from intracellular stores and an influx of extracellular Ca2+. When the B cells were incubated with limiting concentrations of anti-IgM, co-ligation of mIgM and CD54, but not CD19, resulted in an inhibition of the Ca2+ response. Separate cross-linking of mIgM and CD54 under these conditions, using isotype mismatched monoclonal antibodies (mAb), did not affect the mobilization of Ca2+. The CD54-mediated inhibition of the Ca2+ response was also observed in the absence of extracellular Ca2+. All CD54 mAb tested (F10.2, F10.3 and F7.11) interfered with mIgM signaling. The results presented in this report imply that CD54 is linked to intracellular signaling pathways and, via co-ligation with mIgM, interferes in the release of Ca2+ from intracellular stores.

Two monoclonal antibodies (MoAbs), F10.2 and F10.3, were selected for their ability to interfere in homotypic adhesion of human B cells. Precipitation studies and binding to intercellular adhesion molecule 1 (ICAM-1, CD54) cDNA transfected COS cells revealed that both MoAbs are directed against ICAM-1. The binding of MoAb F10.2 was inhibited by LB-2, a MoAb recognizing the NH2-terminal immunoglobulin-like domain of ICAM-1. This suggests that the epitope recognized by F10.2 is located on the first domain of the ICAM-1 molecule. Binding of the other MoAb, F10.3, was not inhibited by F10.2 nor by two other MoAbs mapping to the first domain of the ICAM-1 molecule. The ability of F10.3 to bind to ICAM-1 is influenced by glycosylation, suggesting that this epitope is located on one of the domains carrying possible glycosylation sites, i.e. domain 2, 3 or 4. The ICAM-1 epitopes recognized by F10.3 and LB-2 or F10.2 co-operated in homotypic adhesion of cells from the EBV cell line ML1. These results suggest that in addition to an epitope located on domain 1 of the ICAM-1 molecule, another epitope whose exposure can be regulated by glycosylation is involved in homotypic B-cell adhesion of cell line ML1.

The exact function of receptors for C3b (CR1), which are present on B lymphocytes, is not clear. The present studies were performed to determine the influence of heterologous anti-CR1 on the production of IgM, IgG, and IgA by human peripheral B lymphocytes in vitro. Anti-CR1, raised in rabbits by immunization with purified erythrocyte CR1, was rendered immunospecific and was converted to F(ab')2 and Fab' by enzyme digestion. Pre-immune F(ab')2 and Fab' served as controls. Normal human blood T lymphocytes and T cell-depleted mononuclear cells were cultured for 6 days in RPMI medium and 10% heated fetal calf serum with a low dose (7 micrograms/ml) of pokeweed mitogen (PWM) alone or with PWM and F(ab')2-anti-CR1. In control experiments the effects of F(ab')2-anti-CR1 without PWM and of pre-immune F(ab')2, Fab'-anti-CR1 and pre-immune Fab' in combination with PWM were also tested. When PWM and F(ab')2-anti-CR1 were present simultaneously, 4.3 X 10(3), 2.5 X 10(3), and 1.8 X 10(3) ng/ml of IgM, IgG, and IgA were synthesized, respectively, in cultures containing 4.5 X 10(5) mononuclear blood cells. All other combinations resulted in synthesis of less than 250 ng/ml of each class of Ig. The presence of purified CR1 in the cultures containing PWM and F(ab')2-anti-CR1 caused a dose-dependent decrease of Ig synthesis. By culturing B cells isolated by E-C3b rosetting with T cells and monocytes, the anti-CR1 responding population was demonstrated to be mainly in the CR1-rich B cell fraction. Fab'-anti-CR1 at high doses was also able to stimulate Ig production in the presence of low concentrations of PWM. These data suggest that triggering of CR1 on B cells results in modulation of antibody production and that triggering of CR1 with more than one antibody is required for an optimal response.

In this study the efficiencies of a monoclonal anti-idiotopic (Id) antibody (anti-Id498) and of various preparations of nominal antigen in the induction of an antigen-specific human B cell response in vitro were compared. Anti-Id498 recognizes a recurrent, binding site-related Id present on IgM antibodies with specificity for N-acetyl-D-glucosamine, the immunodominant group of streptococcal group A carbohydrate (A-CHO). We have previously shown that anti-Id498 induces IgM anti-A-CHO secretion from B cells of donors that possess Id-498+ antibodies in their serum. A-CHO was presented to B cells either in soluble or insoluble form, i.e. coupled to beads or as intact bacteria. Purified blood B cell populations from three Id+ healthy donors with high numbers of circulating anti-A-CHO B cells were used and antibody-producing B cells are enumerated in a single-cell assay (spot ELISA). The data show that anti-Id498 was superior in the induction of IgM anti-A-CHO-secreting B cells in two donors (factor 4.6 and 13.5 as compared to the most efficient antigenic stimulation). In the third donor antigen stimulation was slightly more efficient than anti-Id but only with Sepharose-bound A-CHO and not with soluble A-CHO or intact bacteria. The increase of specific B cells induced after stimulation with anti-Id498 could be abolished after addition of autologous T cells in two donors. On the contrary, an enhancement of the specific response was observed after addition of autologous T cells in antigen-stimulated cultures. Neither suppression nor enhancement were induced by addition of irradiated T cells.

With the use of the fluorescent Ca++ indicator Quin-2, we have measured changes in intracellular calcium levels in human B cells in response to anti-Ig antibodies, to Staphylococcus aureus (Staph) or to protein A. Cells of an Epstein-Barr virus-transformed mu lambda-carrying B-cell line, AZU-1, increased free cytosolic calcium after addition of anti-mu or anti-lambda antibodies; F (ab')2 fragments with anti-mu specificity were equally effective. Fab fragments of sheep anti-Ig antibodies only induced a rise in calcium levels after addition of a second anti-sheep Ig antiserum. Cross-linking of non-Ig surface determinants did not influence calcium homeostasis. The calcium channel blockers verapamil (100 microM), nifedipine (20 microM), and LaCl3 (200 microM) inhibited the anti-mu-induced calcium influx. Peripheral blood B cells reacted in essentially the same way in response to anti-mu antibodies. The B cell mitogens protein A and Staph also induced a rise in intracellular calcium. These observations indicate that Ca++ may play a role as a messenger in the activation of human B cells via surface Ig.

Peripheral B lymphocytes of five CLL patients were tested in a radioimmunoassay to determine the density of the C3b receptor (CR1) and the cells were assayed for their ability to mature into IgM secreting cells after in-vitro culture with a combination of Pokeweed Mitogen (PWM) and antibodies directed against CR1. Despite the presence of normal amounts of CR1 on the leukemic B cells, crosslinking of these receptors by anti-CR1 antibodies stimulated only a fraction of the leukemic cell population to differentiate into IgM secreting cells. These results add to the partial functional impairment of CLL-B cells.

Nylon wool-purified T cells (Tn) of two patients with chronic lymphocytic leukemia of the B cell type were phenotyped and tested in various assays for antigen-specific T helper (Th), T suppressor effector (Tse), T suppressor precursor (Tsp), and T suppressor inducer (Tsi) function. Antigen-specific Th as well as Tsi activity could be effectively generated. Although phenotypically CD8+ T cells, carrying the receptor for the Fc part of IgG, were present in mononuclear blood cells and Tn fractions, no antigen-specific Tse cell activity could be induced. In addition, Tsp cells were found to be functionally absent. These findings are discussed in relation to a tumor-induced limited heterogeneity within the T suppressor (Ts) cell compartment.

Peripheral blood lymphocytes (PBL) from 14 patients with chronic lymphocytic leukaemia (CLL) were stimulated with pokeweed mitogen (PWM), formalinized Staphylococcus aureus (Sta) and a combination of both mitogens. The leukaemic B cells were characterized by rosetting techniques (using mouse erythrocytes and complement coated erythrocytes) and immunofluorescence for membrane bound immunoglobulin (mIg). No clear correlation between phenotype and the reactivity with PWM could be found. Results of stimulation with Sta however, indicate that lymphocytes carrying membrane bound IgM (mIgM) and IgD (mIgD) and the receptor of the third complement component (C3R) can be induced to differentiate into immunoglobulin (Ig) containing cells. Addition of PWM to these cultures often enhanced this response. Some leukaemic B cells are able to differentiate after challenge with the appropriate stimulus.

This study provides evidence that leukaemic B cells are able to proliferate and differentiate into plasmablasts. The Ig produced was monoclonal in nature and this was shown by gel electrophoresis.

The monoclonal B cells present in the blood of three patients with Waldenstrøm's macroglobulinaemia were analysed according to phenotype and function. As a marker for the monoclonal nature of the detected immunoglobulin (Ig) molecules, the kappa/lambda-ratio (two patients) or the presence of idiotypic (Id) determinants (one patient) were used. With double immunofluorescence it was shown that the neoplastic blood B cells were heterogeneous in respect to maturation stage. In addition to B cells carrying membrane bound IgM and IgD (mIgM+/IgD+), both mIgM+/IgD- and cytoplasmic IgM+ blastoid cells could be detected in the blood of the patients. This heterogeneity was also reflected in the responses of the monoclonal B cells upon stimulation with mitogens and T cell factors. B blastoid cells and IgM secretion could be induced after in vitro culture in the presence of Staphylococcus aureus, Pokeweed mitogen or a combination of both mitogens. It was shown that also T cell factors were effective in inducing monoclonal B cell differentiation.

In this study the idiotype-positive B cells of one patient with smouldering multiple myeloma (IgG kappa) and of one patient with multiple myeloma (IgG lambda) were analysed phenotypically and functionally. As regards the expression of B cell-associated differentiation antigens and size distribution, the idiotype-positive B cells resembled normal IgG-bearing blood B cells. In functional studies the lymphocytes were cultured in vitro with Staphylococcus aureus, pokeweed mitogen, T-cell factors, or combinations of these. After culture, proliferation and differentiation of the idiotype-positive B cells were measured by autoradiography, an idiotype-specific ELISA, and a spot ELISA. The results show that idiotype-positive B cells of both patients are able to proliferate after stimulation in vitro. In contrast to their normal counterparts, however, almost no increase in the amount of secreted idiotype IgG could be induced. This suggests that the idiotype-positive blood B cells have lost some of their ability to respond to exogenous stimuli.

Human B cell subpopulations can be distinguished by the expression of B cell-associated antigens. mAb directed against these structures allow the isolation and subsequent functional analysis of such subsets. Blood B cells from healthy individuals were separated on basis of the expression of the antigens recognized by the mAb HB4 and FMC7. The B cells present in the thus obtained populations were analyzed for their ability to secrete IgM and IgG after stimulation in vitro with polyclonal B cell activators (PWM, Staphylococcus aureus Cowan I), antigens (tetanus toxoid, type 4 pneumococcal polysaccharides), and soluble B cell differentiation factors. The results suggest that B cells capable of Ig and anti-tetanus toxoid or anti-type 4 pneumococcal polysaccharide antibody production after in vitro culture are localized in a relative small B cell subpopulation carrying the FMC7 determinant but lacking HB4. This holds true for both the IgM- and IgG-secreting B cells. These data further suggest that the majority of B cells found in the blood and which can be characterized as being surface IgM+/IgD+ HB4+ are immature cells unable to respond to differentiation-inducing signals.