scholarly journals Regulation of AID expression in the immune response

2007 ◽  
Vol 204 (5) ◽  
pp. 1145-1156 ◽  
Author(s):  
Elizabeth E. Crouch ◽  
Zhiyu Li ◽  
Makiko Takizawa ◽  
Stefan Fichtner-Feigl ◽  
Polyxeni Gourzi ◽  
...  
Keyword(s):  
Immune Response ◽  
B Cell ◽  
Fluorescent Protein ◽  
Regulatory Element ◽  
Yellow Fluorescent Protein ◽  
Affinity Maturation ◽  
Enzyme Activation ◽  
Mouse Strains ◽  
Artificial Chromosomes ◽  
Fluorescent Protein Reporter

The B cell–specific enzyme activation-induced cytidine deaminase (AID) has been shown to be essential for isotype switching and affinity maturation of antibody genes during the immune response. Conversely, AID activity has also been linked to autoimmunity and tumorigenesis. Determining how AID expression is regulated in vivo is therefore central to understanding its role in health and disease. Here we use phylogenetic footprinting and high-resolution histone acetylation mapping to accurately demarcate AID gene regulatory boundaries. Based on this strategy, we identify a novel, positive regulatory element required for AID transcription. Furthermore, we generate two AID indicator mouse strains using bacterial artificial chromosomes that faithfully recapitulate endogenous AID expression. The first strain uses a green fluorescent protein reporter to identify B cells that actively express AID during the immune response. In the second strain, AID transcription affects the permanent expression of a yellow fluorescent protein reporter in post–germinal center and terminally differentiated lymphocytes. We demonstrate the usefulness of these novel strains by resolving recent contradictory observations on AID expression during B cell ontogeny.

scholarly journals Detection on Surfaces and in Caco-2 Cells of Campylobacter jejuni Cells Transformed with New gfp, yfp, andcfp Marker Plasmids

2000 ◽  
Vol 66 (12) ◽  
pp. 5426-5436 ◽  
Author(s):  
William G. Miller ◽  
Anne H. Bates ◽  
Sharon T. Horn ◽  
Maria T. Brandl ◽  
Marian R. Wachtel ◽  
...  
Keyword(s):  
Reporter Gene ◽  
Fluorescent Protein ◽  
Yellow Fluorescent Protein ◽  
Promoter Sequence ◽  
Broth Culture ◽  
Single Strain ◽  
Shuttle Vectors ◽  
Chicken Skin ◽  
Green Fluorescent ◽  
Fluorescent Protein Reporter

ABSTRACT We have developed two sets of Campylobacter shuttle vectors containing either the gfp (green fluorescent protein), yfp (yellow fluorescent protein), orcfp (cyan fluorescent protein) reporter gene. In one set, the reporter gene is fused to a consensus Campylobacterpromoter sequence (Pc). The other set contains a pUC18 multicloning site upstream of the reporter gene, allowing the construction of transcriptional fusions using known promoters or random genomic fragments. C. jejuni cells transformed with the Pc fusion plasmids are strongly fluorescent and easily visualized on chicken skin, on plant tissue, and within infected Caco-2 cells. In each C. jejuni strain tested, these plasmids were maintained over several passages in the absence of antibiotic selection. Also, in many C. jejuni strains, >91% of the cells transformed with the Pc fusion plasmids remained fluorescent after several days. Experiments with yellow fluorescent and cyan fluorescent C. jejuni transformants suggest that aggregates containing two or more strains of C. jejuni may be present in an enrichment broth culture. Colonies arising from these aggregates would be heterologous in nature; therefore, isolation of a pure culture of C. jejuni, by selecting single colonies, from an environmental sample may not always yield a single strain.

Bim establishes the B-cell repertoire from early to late in the immune response

2020 ◽  
Author(s):  
Akiko Sugimoto-Ishige ◽  
Michishige Harada ◽  
Miho Tanaka ◽  
Tommy Terooatea ◽  
Yu Adachi ◽  
...  
Keyword(s):  
Immune Response ◽  
T Cell ◽  
B Cells ◽  
B Cell ◽  
Plasma Cells ◽  
Affinity Maturation ◽  
Late Response ◽  
Cell Repertoire ◽  
High Affinity ◽  
B Cell Repertoire

Abstract In T cell-dependent antibody responses, some of the activated B cells differentiate along extrafollicular pathways into low-affinity memory and plasma cells, whereas others are involved in subsequent germinal center (GC) formation in follicular pathways, in which somatic hypermutation and affinity maturation occur. The present study demonstrated that Bim, a proapoptotic BH3-only member of the Bcl-2 family, contributes to the establishment of the B-cell repertoire from early to late stages of immune responses to T cell-dependent antigens. Extrafollicular plasma cells grew in the spleen during the early immune response, but their numbers rapidly declined with the appearance of GC-derived progeny in wild-type mice. By contrast, conditional Bim deficiency in B cells resulted in expansion of extrafollicular IgG1+ antibody-forming cells (AFCs) and this expansion was sustained during the late response, which hampered the formation of GC-derived high-affinity plasma cells in the spleen. Approximately 10% of AFCs in mutant mice contained mutated VH genes; thus, Bim deficiency appears not to impede the selection of high-affinity AFC precursor cells. These results suggest that Bim contributes to the replacement of low-affinity antibody by high-affinity antibody as the immune response progresses.

scholarly journals Immune response to vaccine candidates based on different types of nanoscaffolded RBD domain of the SARS-CoV-2 spike protein

2020 ◽  
Author(s):  
Duško Lainšček ◽  
Tina Fink ◽  
Vida Forstnerič ◽  
Iva Hafner-Bratkovič ◽  
Sara Orehek ◽  
...  
Keyword(s):  
Immune Response ◽  
B Cell ◽  
Neutralizing Antibodies ◽  
Affinity Maturation ◽  
T Cell Response ◽  
Spike Protein ◽  
Cytotoxic Lymphocytes ◽  
Protein Antigens ◽  
Vaccine Platform ◽  
Suitable Target

AbstractEffective and safe vaccines against SARS-CoV-2 are highly desirable to prevent casualties and societal cost caused by Covid-19 pandemic. The receptor binding domain (RBD) of the surface-exposed spike protein of SARS-CoV-2 represents a suitable target for the induction of neutralizing antibodies upon vaccination. Small protein antigens typically induce weak immune response while particles measuring tens of nanometers are efficiently presented to B cell follicles and subsequently to follicular germinal center B cells in draining lymph nodes, where B cell proliferation and affinity maturation occurs. Here we prepared and analyzed the response to several DNA vaccines based on genetic fusions of RBD to four different scaffolding domains, namely to the foldon peptide, ferritin, lumazine synthase and β-annulus peptide, presenting from 6 to 60 copies of the RBD on each particle. Scaffolding strongly augmented the immune response with production of neutralizing antibodies and T cell response including cytotoxic lymphocytes in mice upon immunization with DNA plasmids. The most potent response was observed for the 24-residue β-annulus peptide scaffold that forms large soluble assemblies, that has the advantage of low immunogenicity in comparison to larger scaffolds. Our results support the advancement of this vaccine platform towards clinical trials.

scholarly journals T-dependent B cell responses toPlasmodiuminduce antibodies that form a high-avidity multivalent complex with the circumsporozoite protein

2017 ◽  
Author(s):  
Camilla R. Fisher ◽  
Henry J. Sutton ◽  
Joe A. Kaczmarski ◽  
Hayley A. McNamara ◽  
Ben Clifton ◽  
...  
Keyword(s):  
Immune Response ◽  
T Cell ◽  
B Cell ◽  
Affinity Maturation ◽  
Circumsporozoite Protein ◽  
Titration Calorimetry ◽  
High Avidity ◽  
Repeat Region ◽  
X Ray ◽  
X Ray Crystallography

AbstractThe repeat region of thePlasmodium falciparumcircumsporozoite protein (CSP) is a major vaccine antigen because it can be targeted by parasite neutralizing antibodies; however, little is known about this interaction. We used isothermal titration calorimetry, X-ray crystallography and mutagenesis-validated modeling to analyze the binding of a murine neutralizing antibody toPlasmodium falciparumCSP. Strikingly, we found that the repeat region of CSP is bound by multiple antibodies. This repeating pattern allows multiple weak interactions of single FABdomains to accumulate and yield a complex with a dissociation constant in the low nM range. Because the CSP protein can potentially cross-link multiple B cell receptors (BCRs) we hypothesized that the B cell response might be T cell independent. However, while there was a modest response in mice deficient in T cell help, the bulk of the response was T cell dependent. By sequencing the BCRs of CSP-repeat specific B cells in inbred mice we found that these cells underwent somatic hypermutation and affinity maturation indicative of a T-dependent response. Last, we found that the BCR repertoire of responding B cells was limited suggesting that the structural simplicity of the repeat may limit the breadth of the immune response.Author SummaryVaccines aim to protect by inducing the immune system to make molecules called antibodies that can recognize molecules on the surface of invading pathogens. In the case of malaria, our most advanced vaccine candidates aim to promote the production of antibodies that recognize the circumsporozoite protein (CSP) molecule on the surface of the invasive parasite stage called the sporozoite. In this report we use X-ray crystallography to determine the structure of CSP-binding antibodies at the atomic level. We use other techniques such as isothermal titration calorimetry and structural modeling to examine how this antibody interacts with the CSP molecule. Strikingly, we found that each CSP molecule could bind 6 antibodies. This finding has implications for the immune response and may explain why high titers of antibody are needed for protection. Moreover, because the structure of the CSP repeat is quite simple we determined that the number of different kinds of antibodies that could bind this molecule are quite small. However a high avidity interaction between those antibodies and CSP can result from a process called affinity maturation that allows the body to learn how to make improved antibodies specific for pathogen molecules. These data show that while it is challenging for the immune system to recognize and neutralize CSP, it should be possible to generate viable vaccines targeting this molecule.

scholarly journals B-cell enforced expression of the mouse ortholog of MYD88L265P is responsible for Waldenström-like B-cell lymphoma

2019 ◽  
Author(s):  
Catherine Ouk ◽  
Lilian Roland ◽  
Alexis Saintamand ◽  
Nathalie Gachard ◽  
Morgane Thomas ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
Fluorescent Protein ◽  
Cell Lymphoma ◽  
Yellow Fluorescent Protein ◽  
B Cell Lymphoma ◽  
Immunoglobulin M ◽  
Transcriptomic Signature ◽  
Old Mice ◽  
Conditional Transgenic Mouse

AbstractHere, we created a conditional transgenic mouse model with insertion of a sequence coding for both MYD88L252P and the Yellow Fluorescent Protein (YFP) into the rosa26-locus. B-cell specific induction of the transgene constantly led to spleen enlargement with expansion of YFP positive B-cells in 8-12 month-old mice, with a moderate B-cell proliferation increase. Being clonal or oligoclonal, these B-cells exhibited a marked morphological and immunophenotypic lymphoplasmocytic aspect with a plasma cell transcriptomic signature and a serum immunoglobulin M peak. Therefore, continuous activation of MYD88 in mice can lead on its own to a lymphoma close to Waldenström Macroglobulinemia.Key pointB-cell specific enforced expression of MYD88L252P leads to a clonal indolent lymphoplasmocytic B-cell lymphoma with a serum IgM peak.

scholarly journals The Problem of Antigen Affinity Discrimination in B-Cell Immunology

2013 ◽  
Vol 2013 ◽  
pp. 1-18 ◽  
Author(s):  
Subhadip Raychaudhuri
Keyword(s):  
Immune Response ◽  
B Cell ◽  
Immune Cells ◽  
Molecular Mechanisms ◽  
Vaccine Development ◽  
Single Cells ◽  
Affinity Maturation ◽  
Adaptive Immune ◽  
Adaptive Immune Cells ◽  
Cell Affinity

B and T lymphocytes activate the humoral and cellular arms of the adaptive immune system. The adaptive strategy works because receptors of adaptive immune cells can mount an immune response based on their affinity for antigens. Thus, affinity discrimination is central to adaptive immunity and has important biomedical ramifications. Due to its intricate connection to the affinity maturation process, affinity discrimination has a special significance in B-cell-mediated immune response. The role of affinity-matured high-affinity antibodies is increasingly recognized in vaccine development. In this paper, we discuss the recent progress made in mathematical and computational studies to explore the cellular and molecular mechanisms of B-cell affinity discrimination. Formation of B-cell receptor (BCR) oligomers and BCR-lipid rafts, upon antigenic stimulation, emerge to be key factors in B-cell affinity discrimination (at the level of single cells). It also provides a new way of thinking about kinetic proofreading and serial triggering, concepts that have been widely utilized to understand affinity discrimination in adaptive immune cells. Potential future applications of mathematical and computational modeling of affinity discrimination are discussed in the context of autoimmune disorders and vaccine design.

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