Immunoglobulin-secreting Cells Research Articles

Monitoring free light chains in serum using mass spectrometry.

Serum immunoglobulin free light chains (FLC) are secreted into circulation by plasma cells as a by-product of immunoglobulin production. In a healthy individual the population of FLC is polyclonal as no single cell is secreting more FLC than the total immunoglobulin secreting cell population. In a person with a plasma cell dyscrasia, such as multiple myeloma (MM) or light chain amyloidosis (AL), a clonal population of plasma cells secretes a monoclonal light chain at a concentration above the normal polyclonal background. We recently showed that monoclonal immunoglobulin rapid accurate mass measurement (miRAMM) can be used to identify and quantify a monoclonal light chain (LC) in serum and urine above the polyclonal background. This was accomplished by reducing immunoglobulin disulfide bonds releasing the LC to be analyzed by microLC-ESI-Q-TOF mass spectrometry. Here we demonstrate that the methodology can also be applied to the detection and quantification of FLC by analyzing a non-reduced sample. Proof of concept experiments were performed using purified FLC spiked into normal serum to assess linearity and precision. In addition, a cohort of 27 patients with AL was analyzed and miRAMM was able to detect a monoclonal FLC in 23 of the 27 patients that had abnormal FLC values by immunonephelometry. The high resolution and high mass measurement accuracy provided by the mass spectrometry based methodology eliminates the need for κ/λ ratios as the method can quantitatively monitor the abundance of the κ and λ polyclonal background at the same time it measures the monoclonal FLC.

Early Thymus Involution--Manifestation of an Aging Program or a Program of Development?

"I see no physical reason why it should not have been possible for life to construct ageless individuals", said Carl von Weizsacker in 1979 at the Conference on DNA. An obvious biological reason for senescence may be the action of a built-in aging program. Many gerontologists believe that early thymic involution is an argument in favor of the existence of such a program. On the other hand, this involution may be a result of the program of development rather than aging. According to the concepts of noninfectious immunology, the immune system of vertebrates is also designed for immune surveillance over initial tumor development and for tissue-specific regulation of cell proliferation both in ontogenesis and during physiological and reparative regeneration of organs and tissues. Natural anti-tissue autoantibodies are the main effectors of such regulation. Therefore, the number of inherited genes of the variable part of immunoglobulin (V-genes) is not less than the number of all proliferative-competent cell types (~100). For the same reason, the maximal rate of growth, which is usually observed in the prepubertal period, coincides with the maximal thymus index and the maximal number of immunoglobulin-secreting cells as well as the minimal force of mortality during ontogeny. Thus, the circa-pubertal beginning of thymic involution is probably caused by the programmed deceleration of the growth rate in ontogeny, and not by the early manifestation of an aging program. This approach allows us to understand the mechanism of the well-known antitumor effect of the regeneration process of the organ homologous to the tumor, and hence we can try to use it in practical oncology.

Factors regulating immunoglobulin production by normal and disease-associated plasma cells.

Immunoglobulins are molecules produced by activated B cells and plasma cells in response to exposure to antigens. Upon antigen exposure, these molecules are secreted allowing the immune system to recognize and effectively respond to a myriad of pathogens. Immunoglobulin or antibody secreting cells are the mature form of B lymphocytes, which during their development undergo gene rearrangements and selection in the bone marrow ultimately leading to the generation of B cells, each expressing a single antigen-specific receptor/immunoglobulin molecule. Each individual immunoglobulin molecule has an affinity for a unique motif, or epitope, found on a given antigen. When presented with an antigen, activated B cells differentiate into either plasma cells (which secrete large amounts of antibody that is specific for the inducing antigen), or memory B cells (which are long-lived and elicit a stronger and faster response if the host is re-exposed to the same antigen). The secreted form of immunoglobulin, when bound to an antigen, serves as an effector molecule that directs other cells of the immune system to facilitate the neutralization of soluble antigen or the eradication of the antigen-expressing pathogen. This review will focus on the regulation of secreted immunoglobulin by long-lived normal or disease-associated plasma cells. Specifically, the focus will be on signaling and transcriptional events that regulate the development and homeostasis of long-lived immunoglobulin secreting plasma cells.

Maxillary Swelling as the First Evidence of Multiple Myeloma.

Multiple myeloma is a malignant neoplasm of plasma cells characterized by proliferation of a single clone of abnormal immunoglobulin-secreting plasma cells. Since the amount of hemopoietic bone marrow is decreased in the maxilla, oral manifestations of multiple myeloma are less common in the maxilla than in the mandible. We report the case of 33-year-old Japanese man who presented with a mass in the right maxillary alveolar region. Computed tomography and magnetic resonance images showed a soft tissue mass in the right maxilla eroding the anterior and lateral walls of the maxillary sinus and extending into the buccal space. The biopsy results, imaging, and laboratory investigations led to the diagnosis of multiple myeloma. This case report suggests that oral surgeons and dentists should properly address oral manifestations as first indications of multiple myeloma.

Intestinal microbiota diversity and expression of pattern recognition receptors in newly weaned piglets

This study evaluated the gastrointestinal microbial diversity and the expression of pattern recognition receptors (PRRs) of the small intestine during the first week post-weaning in newly weaned piglets. Sixteen piglets were sacrificed on days 0, 1, 4, and 7 post-weaning. Luminal contents from the stomach, ileum, and colon were collected to determine the microbiota diversity; intestinal mucosa from the ileum was collected to assess mRNA expression of PRRs, including toll-like receptors (TLRs) and NOD-like receptors (NLRs); sections of ileum were examined immunohistochemically to assess the immunoglobulin-secreting cells. The results showed that the number of denaturing gradient gel electrophoresis (DGGE) bands from the ileum and colon contents were significantly reduced in the d 4 post-weaning group. Biodiversity indexes (Shannon–Wiener index, richness index, and evenness index) were significantly decreased in the ileum of weaning groups. These indexes decreased in the colon of the d 4 post-weaning group. No significant differences were obtained in the stomach. With the exception of TLR5, the mRNA expressions of TLR2, TLR4, and TLR7 increased post-weaning. The mRNA expressions of NOD1 and NOD2 were significantly affected in the d 4 post-weaning group, and there were no significant differences in the d 1 or d 7 post-weaning groups. Analysis of the immunoglobulin-secreting (IgA, IgG, and IgM) cells showed that the ratio of each immunoglobulin was significantly higher on d 7 than d 0. The results revealed that microbial diversity was lower in the ileum and on d 4 post-weaning. Weaning significantly affected the expression of intestinal PRRs mainly on d 1 and d 4 post-weaning. The expression of specific PRRs was triggered by weaning to recognize distinctive microbiota and promote the development and maturation of the intestinal mucosal immunity.

Expansion of CCR4+ activated T cells is associated with memory B cell reduction in DOCK8-deficient patients

Hyper-IgE syndrome (HIES) is a genetic disorder characterized by elevated IgE serum levels, mostly due to mutations in STAT3 or DOCK8. Despite clinical heterogeneity between the two forms of the disease, clinical manifestations may not be conclusive for diagnosis and immunological differences are still unclear. Herein, we performed a detailed characterization of the T- and B-cell compartments by flow cytometry in seven HIES patients with homozygous DOCK8 mutations and six patients presenting heterozygous STAT3 mutations. We observed that DOCK8-deficient patients showed a marked reduction of naive and recent thymic emigrant (RTE) T lymphocytes together with a relative increase of activated T cells, most of which co-expressed the chemokine receptor CCR4, a marker of Th2 polarization. Moreover, an extreme reduction of memory B cells was detected, despite a normal/increased proportion of immunoglobulin-secreting cells. These observations indicate that DOCK8-deficient patients display a distinctive immunophenotype which is characteristic of this form of HIES.

A1.41 Effect of multiple cycles of B-cell depletion therapy in patients with rheumatoid arthritis on serological evidence of plasmablast activation, serum baff levels and autoantibody specificity

Background and ObjectivesRituximab induces clinical remission in a majority of seropositive patients with Rheumatoid arthritis (RA), however, all patients eventually relapse. Although B cell return precedes clinical relapse absolute B...

B-cell memory and primary immune deficiencies

The purpose of this study is to describe recent advances in our understanding of the role of interleukin-21 (IL-21) in B-cell maturation, and how defects in IL-21 receptor (IL-21R) signalling pathways (IL-21R/γc/JAK3/STAT3) are related to primary immune deficiencies. Abnormal signalling through IL-21R/γc/JAK3/STAT3 pathway has been related to decreased specific antibody responses following vaccination, and to increased susceptibility to encapsulated bacterial infections. This is manifested in the hyper-IgE syndrome, X-linked and JAK3-related severe combined immunodeficiency (SCID) and loss-of-function mutations in the IL-21R gene. Common variable immunodeficiency is associated with impaired in-vitro development of peripheral blood mononuclear cells or purified B-cells into memory or CD38 B-cells following addition of IL-21. IL-21 is a key cytokine in development of B-cells into immunoglobulin-secreting cells. Abnormal signalling through the IL-21R/γc/JAK3/STAT3 pathway leads to defective humoral immune responses to both T-dependent and T-independent antigens and impairs the establishment of long-lasting B-cell memory. Studies involving patients with hyper-IgE syndrome demonstrated the nonredundant role of STAT3 in B-cell production of high-affinity specific antibodies, while total serum immunoglobulins could be maintained through STAT3-independent activation of AID (activation-induced cytidine-deaminase). IL-21 related defects may also be associated with reduced natural killer (NK)-cell cytotoxicity and TH17 cytokine production, indicating that abnormalities in the IL-21-IL-21R pathway have profound effects on crucial immune responses.

Human T follicular helper (Tfh) cells and disease

The generation of protective antibodies by B cells following natural infection or vaccination requires 'help' from CD4(+) T cells. T follicular helper (Tfh) cells are the specialized CD4(+) T cell subset that has evolved the appropriate mechanisms to induce the activation and differentiation of B cells into immunoglobulin (Ig) secreting cells. As such, appropriate control of Tfh cell generation and function is essential to human health as overactivation is likely to result in autoimmunity, whereas underactivation is often associated with immunodeficiency. Furthermore, an understanding of the regulation of these cells may be invaluable to improved vaccine development strategies. Traditionally Tfh cells have been identified by their anatomical location in secondary lymphoid tissues, which has hindered the study of these cells in humans as access to these tissues is often not feasible. However, recent studies have identified the circulating counterparts to tissue Tfh cells and with this has come a wealth of knowledge gained from the study of these cells in human disease. Here we review some of the recent developments on the role of human Tfh cells in health and disease.

Close relationship between immunoglobulin secreting‐cells and Epstein–Barr virus reservoir in patients infected with HIV

Individuals infected with HIV have higher circulating Epstein-Barr virus (EBV) DNA load compared to healthy carriers. This study investigated whether level of spontaneous immunoglobulin secreting cells, one of the major hallmarks of HIV infection, is associated with an increase of EBV DNA load in PBMCs and the spontaneous EBV lytic cycle ex vivo in patients infected with HIV. Spontaneous virus production by cells infected with EBV and EBV DNA loads in PBMCs from which CD8(+) T-cells were removed were measured in 20 HIV-aviremic and 14 HIV-viremic patients. The number of circulating immunoglobulin-secreting cells (Ig-SCs) and CD8(+) T-lymphocyte activation were also investigated. Patients with detectable HIV RNA in plasma exhibited higher spontaneous ex vivo EBV secretion and higher levels of EBV DNA in PBMCs than their aviremic counterparts. In the two groups observed, a positive correlation was found between PBMCs EBV DNA viral load and Ig-SCs, CD38(bright) expression on CD8(+) T-cells and EBV DNA load in cell culture supernatants. These findings suggest that B-cell polyclonal activation and B-cell terminal differentiation into Ig-SCs may fuel EBV DNA reservoir and promote EBV production ex vivo in patients infected with HIV.

Candidate genes of Waldenström's macroglobulinemia: current evidence and research.

Waldenström’s macroglobulinemia (WM) is a relatively uncommon, indolent malignancy of immunoglobulin M-producing B cells. The World Health Organization classifies it as a lymphoplasmacytic lymphoma and patients typically present with anemia, hepatosplenomegaly and diffuse lymphadenopathies. Historically, the genetic characterization of the disease has been hampered by the relatively low proliferative rate of WM cells, thus making karyotyping challenging. The use of novel technologies such as fluorescence in situ hybridization, gene array, and whole genome sequencing has contributed greatly to establishing candidate genes in the pathophysiology of WM and to identifying potential treatment targets, such as L265P MYD88. The discovery of microRNAs and the recognition of epigenetics as a major modulatory mechanism of oncogene expression and/or oncosuppressor silencing have aided in further understanding the pathogenesis of WM. Once thought to closely resemble multiple myeloma, a cancer of terminally differentiated, immunoglobulin-secreting plasma cells, WM appears to genetically cluster with other indolent B-cell lymphomas such as chronic lymphocytic leukemia/small cell lymphoma. The relative high incidence of familial cases of WM and other B-cell malignancies has been helpful in identifying high-risk gene candidates. In this review, we focus on the established genes involved in the pathogenesis of WM, with special emphasis on the key role of derangement of the nuclear factor kappa B signaling pathway and epigenetic mechanisms.

N-acetylcysteine modulation of JAK2 and JAK3 results in decreased STAT3 activation in human B lymphocytes (P1473)

Abstract Activation of STAT3 is crucial for human B lymphocyte differentiation and inhibition of its phosphorylation in CD40-activated B lymphocytes results in impairment of in vitro generation of immunoglobulin-secreting cells. Conversely, redox homeostasis plays a role in B lymphocyte maturation. Thus, the presence of antioxidants can potentially modulate their outcome. We showed that N-acetylcysteine (NAC), an antioxidant molecule, can inhibit STAT3 phosphorylation, which is paralleled with strong inhibition of immunoglobulins secretion. Western analysis reveals that NAC-treatment of CD40-activated human B lymphocytes, in the presence of IL-2, IL-4 and IL-10, causes a reduced phosphorylation of JAK2 and JAK3, which are important activators of STAT3. Furthermore, this inhibition appears to be dose-dependant. Our results indicate that other antioxidant molecule such as α-tocopherol and trolox do not affect these two kinases phosphorylation. Other kinases or their inhibitors associated to STAT3 phosphorylation do not seem affected by the presence of antioxidant in the media. In conclusion, these observations reveal that STAT3 phosphorylation down regulation in NAC conditions is due to its association with JAK2 and JAK3.

Introduction: the unfolded protein response’s role in disease pathophysiology

Introduction: the unfolded protein response’s role in disease pathophysiology

Sockeye salmon retain immunoglobulin-secreting plasma cells throughout their spawning journey and post-spawning

Antibody-producing plasma cells are a major source of protective immunity in vertebrates, including salmon. During the spawning phase, salmon undergo drastic, hormonally driven changes in their physiology, including elevated levels of cortisol, which are known to suppress the immune system. As adult fish need to survive their long journey to the spawning grounds, we hypothesized that humoral immunity, in the form of IgM-secreting plasma cells, remains functional until post-spawning. This was investigated by measuring changes in membrane and secreted immunoglobulin heavy chain mu and Pax5 transcripts in spleen and kidney from migrating sockeye salmon, using real-time qPCR. As an additional measurement, the abundance of developing B, mature B, and plasma cells was determined in spawning fish, using flow cytometry. Immune tissue samples were collected from fish from the Kenai River drainage and Main Bay, Prince William Sound. Our results reveal that spawning fish express high levels of secreted heavy chain mu transcripts in their spleen and anterior kidney throughout the spawning journey. Furthermore, we show that IgM-secreting PCs (HCmu++/Pax5−) remain abundant in anterior kidney and spleen of post-spawning sockeye salmon, with a concomitant loss in developing B cells (HCmu−/Pax5+). This suggests that successful spawners retain their PCs throughout the spawning journey and post-spawning.

Oral tolerance correlates with high levels of lymphocyte activity

Oral tolerance is defined as an inhibition of specific immune responsiveness to a previously ingested antigen. Paradoxically, we found an increased lymphocyte activity in tolerant mice alongside the specific inhibition. Orally-tolerant mice presented higher number of immunoglobulin secreting cells (ISC) in spleen and bone marrow; showed a greater variety of Ig classes being produced: IgM and IgA in the spleen and IgG and IgM in the bone marrow. ISC from immunized mice produced mainly IgG. Despite having the same number of regulatory and activated T cells in the spleen after immunization, these cells appeared earlier in tolerant mice, right after the primary immunization. Also, tolerant mice showed a prompt expression of regulatory cytokines (TGF-β and IL-10) and a transient expression of effector cytokines (IL-2 and IFN-γ). Thus, in addition to an inhibited specific responsiveness, orally-tolerant mice displayed an early and widespread mobilization of activated and regulatory lymphocytes.

Clinical, molecular, and cellular immunologic findings in patients with SP110-associated veno-occlusive disease with immunodeficiency syndrome

Mutations in the SP110 gene result in infantile onset of the autosomal recessive primary immunodeficiency disease veno-occlusive disease with immunodeficiency syndrome (VODI), which is characterized by hypogammaglobulinemia, T-cell dysfunction, and a high frequency of hepatic veno-occlusive disease. We sought to further characterize the clinical features, B-lineage cellular immunologic findings, and molecular pathogenesis of this disorder in 9 patients with new diagnoses, including 4 novel mutations from families of Italian, Hispanic, and Arabic ethnic origin. Methods used include clinical review; Sanger DNA sequencing of the SP110 gene; determination of transfected mutant protein function by using immunofluorescent studies in Hep-2 cells; quantitation of B-cell subsets by means of flow cytometry; assessments of B-cell function after stimulation with CD40 ligand, IL-21, or both; and differential gene expression array studies of EBV-transformed B cells. We confirm the major diagnostic criteria and the clinical utility of SP110 mutation testing for the diagnosis of VODI. Analysis of 4 new alleles confirms that VODI is caused by reduced functional SP110 protein levels. Detailed B-cell immunophenotyping demonstrated that Sp110 deficiency compromises the ability of human B cells to respond to T cell-dependent stimuli and differentiate into immunoglobulin-secreting cells invitro. Expression microarray studies have identified pathways involved in B-lymphocyte differentiation and macrophage function. These studies show that a range of mutations in SP110 that cause decreased SP110 protein levels and impaired late B-cell differentiation cause VODI and that the condition is not restricted to the Lebanese population.

Localized and peripheral autoantibodies: evidence of humoral autoimmunity in patients with inclusion body myositis (159.21)

Abstract Inclusion body myositis (IBM) belongs to a group of muscle diseases known as the inflammatory myopathies. The disease is viewed to be T cell mediated. However, emerging data suggests that B cells may play a role in IBM. To further the understanding of IBM immunopathology and explore the role of the humoral autoimmune response in IBM, we searched for autoantibodies both in the periphery and at the site of tissue damage. Peripheral autoantibodies that recognized muscle-derived cells were demonstrated in IBM serum using both immunohistochemistry and flowcytometry. To focus on the site of tissue injury, we examined the immunoglobulin-secreting plasma cells that infiltrate IBM muscle. A series of single, tissue-associated plasma cells were isolated from IBM muscle. Recombinant immunoglobulins (rIgG) produced from these cells were used as molecular tools to search for autoantigens. Flow cytometry and immunoblotting revealed that these tissue-derived rIgGs recognized human muscle tissue-associated antigens. To identify the target of the rIgGs, a multi-step protein separation scheme and mass spectrometry were employed. Desmin, a major intermediate filament protein expressed in muscle, was identified as a candidate autoantigen for one IBM rIgG. Specificity to desmin was confirmed with a solid-phase assay. Collectively, these data indicate that IBM autoimmunity includes a humoral component.

Why spawning salmon return to their natal stream: The immunological imprinting hypothesis

The immune system of salmonids is remarkably similar to that of mammals, including the presence of B and T lymphocytes and a highly diverse antibody repertoire. However, fish lack bone marrow. Instead, development of immune cells takes place in the anterior kidney, which also houses long-lived, immunoglobulin-secreting plasma cells (LLPCs). These LLPCs should protect adult salmon against pathogens that are encountered upon return to their natal grounds for spawning. Here I present a hypothesis which links immune memory in the form of LLPCs to chemical imprinting and the highly accurate return rates of adults to their natal streams.

Pathogen-Specific Circulating Plasmablasts in Patients with Pneumonia

Lower respiratory tract infections (LRTI) are the leading cause of death world-wide, with Streptococcus pneumoniae (Pnc) as the most prevalent pathogen. Local immune mechanisms appear central to protection against the disease, yet they are poorly characterized. Infections at other, non-respiratory mucosal sites are associated with a transient circulation of mucosa-originating lymphocytes from the mucosal site to blood and back to the mucosa. The present study explored whether pathogen-specific plasmablasts appear in the circulation also in patients with infection of the lower respiratory tract. 16 patients with bacteremic Pnc pneumonia and 14 healthy volunteers were explored for circulating plasmablasts secreting antibodies against their own pathogenic Pnc strain isolated in blood cultures (patients) or against several pathogenic strains from pneumonia patients (14 controls) or a mixture of nine different purified pneumococcal polysaccharides (8 controls). Both patients and volunteers were studied for all plasmablasts. The cells were identified with ELISPOT as Pnc-specific antibody-secreting cells (ASC) and as all immunoglobulin-secreting cells (ISC). High numbers of circulating Pnc-specific ASC were found in the acute phase of the disease in all patients with pneumonia (median 97 ASC/106 PBMC), but in none of the controls. IgG isotype predominated in 9/16 patients. The numbers of ISC were significantly higher in the patients than in the healthy controls, yet Pnc-specific ASC only accounted for 0.7% of all the patients' ISC.The present study is the first to show that antigen-specific plasmablasts appear in the circulation in pneumonia, suggesting that pulmonary lypmhocytes recirculate in humans. Assessing these cells provides a novel tool for studying immune response to antigens encountered at the LRT.

Differentiation of Chronic Lymphocytic Leukemia B Cells into Immunoglobulin Secreting Cells Decreases LEF-1 Expression

Lymphocyte enhancer binding factor 1 (LEF-1) plays a crucial role in B lineage development and is only expressed in B cell precursors as B cell differentiation into mature B and plasma cells silences its expression. Chronic lymphocytic leukemia (CLL) cells aberrantly express LEF-1 and its expression is required for cellular survival. We hypothesized that modification of the differentiation status of CLL cells would result in loss of LEF-1 expression and eliminate the survival advantage provided by its aberrant expression. In this study, we first established a methodology that induces CLL cells to differentiate into immunoglobulin (Ig) secreting cells (ISC) using the TLR9 agonist, CpG, together with cytokines (CpG/c). CpG/c stimulation resulted in dramatic CLL cell phenotypic and morphologic changes, expression of cytoplasmic Ig, and secretion of light chain restricted Ig. CpG/c stimulation also resulted in decreased CLL cell LEF-1 expression and increased Blimp-1 expression, which is crucial for plasma cell differentiation. Further, Wnt pathway activation and cellular survival were impaired in differentiated CLL cells compared to undifferentiated CLL cells. These data support the notion that CLL can differentiate into ISC and that this triggers decreased leukemic cell survival secondary to the down regulation of LEF-1 and decreased Wnt pathway activation.