Mouse antibody of IgM class is prone to non-enzymatic cleavage between CH1 and CH2 domains

Abstract IgM is a multivalent antibody which evolved as a first line defense of adaptive immunity. It consists of heavy and light chains assembled into a complex oligomer. In mouse serum there are two forms of IgM, a full-length and a truncated one. The latter contains μ’ chain, which lacks a variable region. Although μ’ chain was discovered many years ago, its origin has not yet been elucidated. Our results indicate that μ’ chain is generated from a full-length heavy chain by non-enzymatic cleavage of the protein backbone. The cleavage occurred specifically after Asn209 and is prevented by mutating this residue into any other amino acid. The process requires the presence of other proteins, preferentially with an acidic isoelectric point, and is facilitated by neutral or alkaline pH. This unique characteristic of the investigated phenomenon distinguishes it from other, already described, Asn-dependent protein reactions. A single IgM molecule is able to bind up to 12 epitopes via its antigen binding fragments (Fabs). The cleavage at Asn209 generates truncated IgM molecules and free Fabs, resulting in a reduced IgM valence and probably affecting IgM functionality in vivo

A distinct role for B1b lymphocytes in T cell-independent immunity

Pathogenesis of infectious disease is not only determined by the virulence of the microbe but also by the immune status of the host. Vaccination is the most effective means to control infectious diseases. A hallmark of the adaptive immune system is the generation of B cell memory, which provides a long-lasting protective antibody response that is central to the concept of vaccination. Recent studies revealed a distinct function for B1b lymphocytes, a minor subset of mature B cells that closely resembles that of memory B cells in a number of aspects. In contrast to the development of conventional B cell memory, which requires the formation of germinal centers and T cells, the development of B1b cell-mediated long-lasting antibody responses occurs independent of T cell help. T cell-independent (TI) antigens are important virulence factors expressed by a number of bacterial pathogens, including those associated with biological threats. TI antigens cannot be processed and presented to T cells and therefore are known to possess restricted T cell-dependent (TD) immunogenicity. Nevertheless, specific recognition of TI antigens by B1b cells and the highly protective antibody responses mounted by them clearly indicate a crucial role for this subset of B cells. Understanding the mechanisms of long-term immunity conferred by B1b cells may lead to improved vaccine efficacy for a variety of TI antigens

Functional dissection of the cytoplasmic subregions of the interleukin-5 receptor α chain in growth and immunoglobulin G1 switch recombination of B cells

The interleukin-5 receptor α chain (IL-5Rα) is known to regulate the development and function of B cells and eosinophils. Although the functions of IL-5Rα cytoplasmic domain subregions have been studied extensively using cultured cell lines, this approach has limitations when studying the functions of distinct primary B-cell subpopulations and their responsiveness to IL-5. In the present study, we generated mice on an IL-5Rα null background, each expressing a mutant form of an IL-5Rα transgene ligated to a µ enhancer and VH promoter, either lacking the cytoplasmic DC3 region or substituting two proline residues for alanine (ApvA) in the membrane-proximal ppvp motif of the cytoplasmic domain. The ppvp motif, which mediates activation of JAK2/STAT5 and Btk, also contributes to c-fos, c-jun and c-myc expression. IL-5Rα null mutant mice showed impaired B-1-cell development, reduced serum immunoglobulin G3 (IgG3) and IgM, no IL-5-induced enhancement of B-cell proliferation and IL-5-induced switch recombination from the µ gene to γ1 gene; these were not recovered following the expression of the ApvA mutant. In contrast, absence of the DC3 region affected the IL-5-induced switch recombination from the µ to the γ1 gene and B-1-cell development, while IL-5-induced proliferation and IgM production were at levels similar to those of B cells expressing wild-type IL-5Rα transgene. The results clearly indicated that the ppvp motif and the DC3 region of IL-5Rα played distinct roles in B-cell proliferation and differentiation. Thus, this present approach offers new insights into the functions of the cytoplasmic subregions of IL-5Rα, in particular its carboxy-terminal region

Purification and Immune Phenotyping of B-1 Cells from Body Cavities of Mice.

B-1 cells are fetal-origin B lymphocytes with unique developmental and functional characteristics that can generate natural, polyreactive antibodies with important functions in tissue homeostasis and immune defense. While B-1 cell frequencies in bone marrow and secondary lymphoid tissues are low, relative high frequencies exist within peritoneal and pleural cavities of mice, including both CD5+ and CD5- B-1 cells. These cells represent B-1 reservoirs that, when activated, migrate to lymphoid tissues to secrete antibodies and/or cytokines. Here, we outline efficient methods for the extraction and magnetic isolation of CD5+ B-1 cells from the peritoneal and pleural cavities as well as the separation and phenotypic characterization of CD5+ and CD5- B-1 cells by flow cytometry

The Eritrean Military/National Service Programme: Slavery and the notion of persecution in refugee status determination

Despite the overwhelming evidence of human rights violations within the Eritrean Military/National Service Programme (“MNSP”), adjudication of asylum applications made by Eritreans remains a challenge. Narrow interpretations of “slavery” have created obstacles for protection under the 1951 Convention Relating to the Status of Refugees (“1951 Refugee Convention”). This article discusses MST and Others, the latest Country Guidance case on Eritrea issued by the UK Upper Tribunal Immigration and Asylum Chamber (“UTIAC”), and also the lead case E-5022/2017 of the Swiss Federal Administrative Court (“FAC”), which to a large extent replicated the UTIAC’s approach. The article focuses on how “slavery,” “servitude” and “forced labour” under article 4 of the European Convention on Human Rights (“ECHR”) have been interpreted in the British and Swiss case-law. While both, the British and the Swiss Courts, had recourse to the European Court of Human Rights’ (“ECtHR”) interpretation of article 4(1) ECHR (the right not to be subjected to slavery or servitude), they refused the applicability of international criminal law notions to this provision, and thus to the concept of “persecution” in article 1A(2) of the 1951 Refugee Convention. In doing so, the UTIAC and the FAC set unreasonable requirements to satisfy article 4(1) ECHR. Due to the very limited case-law pertaining to slavery by the ECtHR, the ECHR does not offer an appropriate framework for examining asylum applications of victims of slavery. It is therefore suggested that slavery cases are considered against a wider legal framework, which involves the examination of concepts developed by international criminal law (“ICL”). ICL has indeed developed a significant body of jurisprudence on the interpretation of the international law concept of “slavery” and its application to contemporary situations. The article contrasts the British and Swiss Courts’ position to develop an interpretative approach that connects different areas of international law, including not only international refugee law and international human rights law (“IHRL”), but also ICL. If applied in line with the principle of systemic integration and according to the overall purposes of the 1951 Refugee Convention, this approach would yield consistent results. Ultimately, this article seeks to assist asylum decision-makers and practitioners in the interpretation and application of the refugee definition to asylum applications of persons from Eritrea

The role of B-1 cells in inflammation

B-1 lymphocytes exhibit unique phenotypic, ontogenic, and functional characteristics that differ from the conventional B-2 cells. B-1 cells spontaneously secrete germline-like, repertoire skewed polyreactive natural antibody, which acts as a first line of defense by neutralizing a wide range of pathogens before launching of the adaptive immune response. Immunomodulatory molecules, such as interleukin-10 (IL-10), adenosine, granulocyte-macrophage colony-stimulating factor (GM-CSF), IL-3, and IL-35 are also produced by B-1 cells in the presence or absence of stimulation, which regulate acute and chronic inflammatory diseases. Considerable progress has been made during the past three decades since the discovery of B-1 cells, which has not only improved our understanding of their phenotypic and ontogenic uniqueness but also their role in various inflammatory diseases including influenza, pneumonia, sepsis, atherosclerosis, inflammatory bowel disease (IBD), autoimmunity, obesity and diabetes mellitus. Recent identification of human B-1 cells widens the scope of this field, leading to novel innovations that can be implemented from bench to bedside. Among the vast number of studies on B-1 cells, we have carried out a literature review highlighting current trends in the study of B-1 cell involvement during inflammation, which may result in a paradigm shift towards sustainable therapeutics in various inflammatory diseases