Germinal Center Selection and Affinity Maturation Require Dynamic Regulation of mTORC1 Kinase

During antibody affinity maturation, germinal center (GC) B cells cycle between affinity-driven selection in the light zone (LZ) and proliferation and somatic hypermutation in the dark zone (DZ). Although selection of GC B cells is triggered by antigen-dependent signals delivered in the LZ, DZ proliferation occurs in the absence of such signals. We show that positive selection triggered by T cell help activates the mechanistic target of rapamycin complex 1 (mTORC1), which promotes the anabolic program that supports DZ proliferation. Blocking mTORC1 prior to growth prevented clonal expansion, whereas blockade after cells reached peak size had little to no effect. Conversely, constitutively active mTORC1 led to DZ enrichment but loss of competitiveness and impaired affinity maturation. Thus, mTORC1 activation is required for fueling B cells prior to DZ proliferation rather than for allowing cell-cycle progression itself and must be regulated dynamically during cyclic re-entry to ensure efficient affinity-based selection. During germinal center selection, signals from Tfh cells in the light zone dictate the extent of B cell proliferation in the dark zone. Ersching et al. (2017) show that Tfh help induces mTORC1 activation in light zone B cells, leading to cell growth that sustains the subsequent dark zone proliferative burst

Visualizing antibody affinity maturation in germinal centers

open12siAntibodies somatically mutate to attain high affinity in germinal centers (GCs). There, competition between B cell clones and among somatic mutants of each clone drives an increase in average affinity across the population. The extent to which higher-affinity cells eliminating competitors restricts clonal diversity is unknown. By combining multiphoton microscopy and sequencing, we show that tens to hundreds of distinct B cell clones seed each GC and that GCs lose clonal diversity at widely disparate rates. Furthermore, efficient affinity maturation can occur in the absence of homogenizing selection, ensuring that many clones can mature in parallel within the same GC. Our findings have implications for development of vaccines in which antibodies with nonimmunodominant specificities must be elicited, as is the case for HIV-1 and influenza.openTas J.M.J.; Mesin L.; Pasqual G.; Targ S.; Jacobsen J.T.; Mano Y.M.; Chen C.S.; Weill J.-C.; Reynaud C.-A.; Browne E.P.; Meyer-Hermann M.; Victora G.D.Tas, J. M. J.; Mesin, L.; Pasqual, G.; Targ, S.; Jacobsen, J. T.; Mano, Y. M.; Chen, C. S.; Weill, J. -C.; Reynaud, C. -A.; Browne, E. P.; Meyer-Hermann, M.; Victora, G. D

Use of 18F-2-Fluorodeoxyglucose to Label Antibody Fragments for Immuno-Positron Emission Tomography of Pancreatic Cancer

We generated 18F-labeled antibody fragments for positron emission tomography (PET) imaging using a sortase-mediated reaction to install a trans-cyclooctene-functionalized short peptide onto proteins of interest, followed by reaction with a tetrazine-labeled-18F-2-deoxyfluoroglucose (FDG). The method is rapid, robust, and site-specific (radiochemical yields > 25%, not decay corrected). The availability of 18F-2-deoxyfluoroglucose avoids the need for more complicated chemistries used to generate carbon–fluorine bonds. We demonstrate the utility of the method by detecting heterotopic pancreatic tumors in mice by PET, using anti-Class II MHC single domain antibodies. We correlate macroscopic PET images with microscopic two-photon visualization of the tumor. Our approach provides easy access to 18F-labeled antibodies and their fragments at a level of molecular specificity that complements conventional 18F-FDG imaging

Clonal replacement sustains long-lived germinal centers primed by respiratory viruses

Germinal centers (GCs) form in secondary lymphoid organs in response to infection and immunization and are the source of affinity-matured B cells. The duration of GC reactions spans a wide range, and long-lasting GCs (LLGCs) are potentially a source of highly mutated B cells. We show that rather than consisting of continuously evolving B cell clones, LLGCs elicited by influenza virus or SARS-CoV-2 infection in mice are sustained by progressive replacement of founder clones by naive-derived invader B cells that do not detectably bind viral antigens. Rare founder clones that resist replacement for long periods are enriched in clones with heavily mutated immunoglobulins, including some with very high affinity for antigen, that can be recalled by boosting. Our findings reveal underappreciated aspects of the biology of LLGCs generated by respiratory virus infection and identify clonal replacement as a potential constraint on the development of highly mutated antibodies within these structures

A framework for the development of a global standardised marine taxon reference image database (SMarTaR-ID) to support image-based analyses

Video and image data are regularly used in the field of benthic ecology to document biodiversity. However, their use is subject to a number of challenges, principally the identification of taxa within the images without associated physical specimens. The challenge of applying traditional taxonomic keys to the identification of fauna from images has led to the development of personal, group, or institution level reference image catalogues of operational taxonomic units (OTUs) or morphospecies. Lack of standardisation among these reference catalogues has led to problems with observer bias and the inability to combine datasets across studies. In addition, lack of a common reference standard is stifling efforts in the application of artificial intelligence to taxon identification. Using the North Atlantic deep sea as a case study, we propose a database structure to facilitate standardisation of morphospecies image catalogues between research groups and support future use in multiple front-end applications. We also propose a framework for coordination of international efforts to develop reference guides for the identification of marine species from images. The proposed structure maps to the Darwin Core standard to allow integration with existing databases. We suggest a management framework where high-level taxonomic groups are curated by a regional team, consisting of both end users and taxonomic experts. We identify a mechanism by which overall quality of data within a common reference guide could be raised over the next decade. Finally, we discuss the role of a common reference standard in advancing marine ecology and supporting sustainable use of this ecosystem

Noninvasive Imaging of Human Immune Responses in a Human Xenograft Model of Graft-Versus-Host Disease

The immune system plays a crucial role in many diseases. Activation or suppression of immunity is often related to clinical outcome. Methods to explore the dynamics of immune responses are important to elucidate their role in conditions characterized by inflammation, such as infectious disease, cancer, or autoimmunity. Immuno-PET is a noninvasive method by which disease and immune cell infiltration can be explored simultaneously. Using radiolabeled antibodies or fragments derived from them, it is possible to image disease-specific antigens and immune cell subsets. Methods: We developed a method to noninvasively image human immune responses in a relevant humanized mouse model. We generated a camelid-derived single-domain antibody specific for human class II major histocompatibility complex products and used it to noninvasively image human immune cell reconstitution in nonobese diabetic severe combined immune deficiency gamma-/- mice reconstituted with human fetal thymus, liver, and liver-derived hematopoietic stem cells (BLT mice). Results: We showed imaging of infiltrating immunocytes in BLT mice that spontaneously developed a graft-versus-host-like condition, characterized by alopecia and blepharitis. In diseased animals, we showed an increased PET signal in the liver, attributable to infiltration of activated class II major histocompatibility complex(+) T cells. Conclusion: Noninvasive imaging of immune infiltration and activation could thus be of importance for diagnosis and evaluation of treatment of graft-versus-host disease and holds promise for other diseases characterized by inflammation.</p

Cytotoxic <i>Escherichia coli</i> strains encoding colibactin isolated from immunocompromised mice with urosepsis and meningitis - Fig 7

<p><b>Histopathological lesions induced by <i>pks+ E</i>. <i>coli</i></b> (A) Moderate multifocal subacute suppurative pyelonephritis with tubular necrosis and intraluminal bacterial colonies. The renal architecture is multifocally disrupted by pyelonephritis that discretely extends from the capsular surface deep in to the medulla. The ectatic renal pelvis contains hemorrhage admixed with inflammatory cells. H&E Scale: 1mm. (B) Higher magnification of the affected renal tubule. Focally, ectatic tubule contained large numbers of bacteria admixed with cellular debris. The epithelial lining is completely lost with intact basement membrane. The adjacent tubular interstitium is infiltrated by granulocytes and mononuclear cells. H&E Scale: 20μm. (C) Mild focally extensive subacute necrohemorrhagic meningoencephalitis. The brain parenchyma and meninges are multifocally disrupted by hemorrhage and inflammatory infiltrates. H&E Scale: 200μm. (D) Lumbar spinal cord: Moderate multifocal subacute hemorrhagic meningiomyelitis with vasculitis and fibrin thrombi. The gray and white matter are disrupted by hemorrhage, vacuolations, granulocytes, and mononuclear cells. The meningeal blood vessels are multifocally affected by vasculitis and fibrin thrombi.</p

Cytotoxic <i>Escherichia coli</i> strains encoding colibactin isolated from immunocompromised mice with urosepsis and meningitis

<div><p>Immune-compromised mouse models allow for testing the preclinical efficacy of human cell transplantations and gene therapy strategies before moving forward to clinical trials. However, CRISPR/Cas9 gene editing of the <i>W</i>^<i>sh</i><i>/W</i>^<i>sh</i> mouse strain to create an immune-compromised model lacking function of <i>Rag2</i> and <i>Il2rγ</i> led to unexpected morbidity and mortality. This warranted an investigation to ascertain the cause and predisposing factors associated with the outbreak. Postmortem examination was performed on 15 moribund mice. The main lesions observed in these mice consisted of ascending urogenital tract infections, suppurative otitis media, pneumonia, myocarditis, and meningoencephalomyelitis. As <i>Escherichia coli</i> strains harboring polyketide synthase (<i>pks)</i> genomic island were recently isolated from laboratory mice, the tissue sections from the urogenital tract, heart, and middle ear were subjected to <i>E</i>. <i>coli</i> specific PNA-FISH assay that revealed discrete colonies of <i>E</i>. <i>coli</i> associated with the lesions. Microbiological examination and 16S rRNA sequencing confirmed <i>E</i>. coli-induced infection and septicemia in the affected mice. Further characterization by <i>clb</i> gene analysis and colibactin toxicity assays of the <i>pks+ E</i>. <i>coli</i> revealed colibactin-associated cytotoxicity. Rederivation of the transgenic mice using embryo transfer produced mice with an intestinal flora devoid of <i>pks+ E</i>. <i>coli</i>. Importantly, these barrier-maintained rederived mice have produced multiple litters without adverse health effects. This report is the first to describe acute morbidity and mortality associated with <i>pks+ E</i>. <i>coli</i> urosepsis and meningitis in immunocompromised mice, and highlights the importance of monitoring and exclusion of colibactin-producing <i>pks+ E</i>. <i>coli</i>.</p></div