Elevated levels of enteric IgA in an unimmunised mouse model of Hyper IgM syndrome derived from gut-associated secondary lymph organs even in the absence of germinal centres

Introduction. Patients with Human Hyper IgM syndromes (HIGM) developed pulmonary and gastrointestinal infections since infancy and most patients have mutations in the CD40 ligand (CD40L) gene. Most HIGM patients compared to healthy subjects have higher/similar IgM and lower IgG, and IgA serum concentrations but gut antibody concentrations are unknown. CD40L on activated T-cells interacts with CD40 on B-cells, essential for the formation of germinal centres (GCs) inside secondary lymphoid organs (SLOs), where high-affinity antibodies, long-lived antibody-secreting plasma cells, and memory B-cells, are produced. C57BL6-CD40 ligand deficient mice (C57BL6-cd40l−/−), are a model of HIGM, because serum immunoglobulin concentrations parallel levels observed in HIGM patients and have higher faecal IgA concentrations. In mice, TGFβ and other cytokines induce IgA production. Aims. To compare and evaluate B-cell populations and IgA-producing plasma cells in peritoneal lavage, non-gut-associated SLOs, spleen/inguinal lymph nodes (ILN), and gut-associated SLOs, mesenteric lymph nodes (MLN)/Peyer´s patches (PP) of unimmunised C57BL6-cd40l−/− and C57BL6-wild-type (WT) mice. Material and methods. Peritoneal lavages, spleens, ILN, MLN, and PP from 8-10 weeks old C57BL6-cd40l−/− and WT mice, were obtained. Organ cryosections were analysed by immunofluorescence and B-cell populations and IgA-positive plasma cell suspensions by flow cytometry. Results. In unimmunised WT mice, GCs were only observed in the gut-associated SLOs, but GCs were absent in all C57BL6-cd40l−/− SLOs. PP and MLN of C57BL6-cd40l−/− mice exhibited a significantly higher number of IgA-producing cells than WT mice. In the spleen and ILN of C57BL6-cd40l−/− mice IgA-producing cells significantly decreased, while IgM-positive plasma cells increased. C57BL6-cd40l−/− B-1 cells were more abundant in all analysed SLOs, whereas in WT mice most B-1 cells were contained within the peritoneal cavity. C57BL6-cd40l−/− B-cells in MLN expressed a higher TGFβ receptor-1 than WT mice. Mouse strains small intestine microvilli (MV), have a similar frequency of IgA-positive cells. Discussion. Together our results confirm the role of PP and MLN as gut inductive sites, whose characteristic features are to initiate an IgA preferential immune response production in these anatomical sites even in the absence of GCs. IgA antibodies play a pivotal role in neutralizing, eliminating, and regulating potential pathogens and microorganisms in the gut.CONACYT-Mexico, scholarship 780260 was awarded to FH-C. TE-G and L-FR were recipients of CONACYT Grant 254994, and 21102, respectively

Elevated levels of enteric IgA in an unimmunised mouse model of Hyper IgM syndrome derived from gut-associated secondary lymph organs even in the absence of germinal centres

Peer reviewed: TrueIntroduction: Patients with Human Hyper IgM syndromes (HIGM) developed pulmonary and gastrointestinal infections since infancy and most patients have mutations in the CD40 ligand (CD40L) gene. Most HIGM patients compared to healthy subjects have higher/similar IgM and lower IgG, and IgA serum concentrations but gut antibody concentrations are unknown. CD40L on activated T-cells interacts with CD40 on B-cells, essential for the formation of germinal centres (GCs) inside secondary lymphoid organs (SLOs), where high-affinity antibodies, long-lived antibody-secreting plasma cells, and memory B-cells, are produced. C57BL6-CD40 ligand deficient mice (C57BL6-cd40l −/−), are a model of HIGM, because serum immunoglobulin concentrations parallel levels observed in HIGM patients and have higher faecal IgA concentrations. In mice, TGFβ and other cytokines induce IgA production. Aims: To compare and evaluate B-cell populations and IgA-producing plasma cells in peritoneal lavage, non-gut-associated SLOs, spleen/inguinal lymph nodes (ILN), and gut-associated SLOs, mesenteric lymph nodes (MLN)/Peyer´s patches (PP) of unimmunised C57BL6-cd40l −/− and C57BL6-wild-type (WT) mice. Material and methods: Peritoneal lavages, spleens, ILN, MLN, and PP from 8-10 weeks old C57BL6-cd40l −/− and WT mice, were obtained. Organ cryosections were analysed by immunofluorescence and B-cell populations and IgA-positive plasma cell suspensions by flow cytometry. Results: In unimmunised WT mice, GCs were only observed in the gut-associated SLOs, but GCs were absent in all C57BL6-cd40l −/− SLOs. PP and MLN of C57BL6-cd40l −/− mice exhibited a significantly higher number of IgA-producing cells than WT mice. In the spleen and ILN of C57BL6-cd40l− /− mice IgA-producing cells significantly decreased, while IgM-positive plasma cells increased. C57BL6-cd40l −/− B-1 cells were more abundant in all analysed SLOs, whereas in WT mice most B-1 cells were contained within the peritoneal cavity. C57BL6-cd40l −/− B-cells in MLN expressed a higher TGFβ receptor-1 than WT mice. Mouse strains small intestine microvilli (MV), have a similar frequency of IgA-positive cells. Discussion: Together our results confirm the role of PP and MLN as gut inductive sites, whose characteristic features are to initiate an IgA preferential immune response production in these anatomical sites even in the absence of GCs. IgA antibodies play a pivotal role in neutralising, eliminating, and regulating potential pathogens and microorganisms in the gut

A Low Cost Antibody Signal Enhancer Improves Immunolabeling in Cell Culture, Primate Brain and Human Cancer Biopsy

The use of antibodies to identify neuronal receptors, neurotransmitters, cytoskeletal elements or pathologic protein aggregates, ion channels, adhesion molecules or other cell-type specific markers, is common practice in neuroscience. Antibody detection systems are often based on confocal, epifluorescence or brightfield microscopy. Three types of technical issues can interfere with immunolabeling: low abundance of the target protein, low specific affinity of the antibody and/or signal background sometimes related to tissue fixation. Here, giving tribute to Professor Miledis mentorship, we propose the application of an antibody signal enhancer (ASE) solution based on glycine, hydrogen peroxide and a detergent mix as a simple, low cost, protocol variation that significantly and specifically improves the signal to noise ratio during immunostaining experiments. We describe three new settings in which ASE improves the detection of a variety of antibodies applied on long-time stored non-human primate brain sections, cell culture monolayers and on squamous carcinomas retrieved from cervical cancer patients. The significant improvement of ASE over optimized immunohistochemical protocols used in clinical practice (i.e. cancer detection) combined with its simplicity and low cost makes it an attractive method for biomedical applications. This article is part of a Special Issue entitled: Honoring Ricardo Miledi - outstanding neuroscientist of XX-XXI centuries. (C) 2020 IBRO. Published by Elsevier Ltd. All rights reserved.Funding Agencies|Unidad de Imagenologia IFC-UNAM; CONACYT-Mexico (Postdoctoral) [264069, 295473, 308515]</p

Recycling of memory B cells between germinal center and lymph node subcapsular sinus supports affinity maturation to antigenic drift.

Infection or vaccination leads to the development of germinal centers (GC) where B cells evolve high affinity antigen receptors, eventually producing antibody-forming plasma cells or memory B cells. Here we follow the migratory pathways of B cells emerging from germinal centers (BEM) and find that many BEM cells migrate into the lymph node subcapsular sinus (SCS) guided by sphingosine-1-phosphate (S1P). From the SCS, BEM cells may exit the lymph node to enter distant tissues, while some BEM cells interact with and take up antigen from SCS macrophages, followed by CCL21-guided return towards the GC. Disruption of local CCL21 gradients inhibits the recycling of BEM cells and results in less efficient adaption to antigenic variation. Our findings thus suggest that the recycling of antigen variant-specific BEM cells and transport of antigen back to GC may support affinity maturation to antigenic drift