Johanne T. Jacobsen

Visualizing antibody affinity maturation in germinal centers

Diversity reigns in antibody responses During the course of an immune response, B cells specific for an invading pathogen divide. The antibodies they produce increase in affinity via somatic mutation in specialized lymph node structures called germinal centers. Tas et al. used multiphoton microscopy and sequencing to determine how different B cell clones compete with one another within mouse germinal centers. Multiple B cell clones can seed individual germinal centers, and germinal centers lose diversity at disparate rates. Such heterogeneity suggests that manipulating minor clonal populations to gain an advantage during vaccination may one day be possible. Science, this issue p. 1048 Germinal center B cells are clonally diverse, and the antibodies these cells express mature at different rates. Antibodies 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.

The use of (18)F-2-fluorodeoxyglucose (FDG) to label antibody fragments for immuno-PET 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.

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.

The cellular mechanism by which complementary Id + and anti-Id antibodies communicate: T cells integrated into idiotypic regulation

The V region antigenic determinants (idiotopes (Ids)) of antibodies (Abs) have been suggested to be involved in regulating the immune system. Certain diseases such as diabetes mellitus have recently been associated with a disequilibrium between Id+ and anti‐Id Abs. However, it is unknown how Abs carrying complementary idiotypes (that is, Id+ and anti‐Id Abs) regulate each other at the level of B and T cells. In this study, we show that B lymphoma cells genetically equipped with anti‐Id BCR V regions receive a signal when exposed to Id+Ig. Moreover, they become × 104 more efficient at presenting exogenous Id+ Ab to CD4+ T cells in vitro. Activated Id‐specific T cells in turn regulated the Id‐specific B lymphoma cells. Similar results were obtained in vivo in a surrogate model in which an Id‐peptide was incorporated genetically into the C‐region of a recombinant Ab that targeted IgD on B cells. The findings suggest that conventional T–B collaboration can explain communication between complementary Id+ and anti‐Id Ab at the cellular level. A model is suggested that integrates present and previous data on B‐cell regulation by Id‐specific T cells.

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

Summary 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. Graphical Abstract Figure. No Caption available. HighlightsPositive selection of GC B cells triggers mTORC1 activation prior to clonal expansionmTORC1 is required for biomass accumulation but dispensable for cell‐cycle progressionDeregulated mTORC1 promotes DZ phenotype and leads to loss of GC competitivenessRapamycin‐resistant mouse strain is used for cell‐intrinsic mTORC1 inhibition &NA; 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.

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 γ−/− 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.

Naive Idiotope-Specific B and T Cells Collaborate Efficiently in the Absence of Dendritic Cells

Anti-idiotope (anti-Id) Abs have a role in therapy against B cell lymphomas, as inhibitors of pathogenic autoantibodies, and as surrogate Ags for immunization. Despite these observations, the mechanism by which Id+ Ig generates anti-Id Abs is essentially unknown. To address this issue, we generated a double knock-in mouse that expresses V regions of a somatically mutated anti-Id mAb with intermediate affinity (affinity constant [Ka] = 0.77 × 107 M−1) for the myeloma protein M315. The anti-Id mice have normal peripheral B cell populations, and allelic exclusion is efficient. Anti-Id B cells from BCR knock-in mice, together with Id-specific CD4+ T cells from previously established TCR-transgenic mice, enabled us to study Id-specific T cell–B cell collaboration by dilution of transferred cells into syngeneic BALB/c recipients. We show that previously unstimulated (naive) Id-specific B and T cells collaborate efficiently in vivo, even at low frequencies and in the presence of low amounts of Id+ Ig, resulting in germinal center formation, plasma cell development, and secretion of isotype-switched anti-Id Abs. We further demonstrate that Id-specific T cell–B cell collaboration occurs readily in the absence of adjuvant and is not dependent on Id-presentation by dendritic cells. The results underscore the potency of anti-Id B cells in MHC class II–restricted presentation of Id+ Ig and suggest that Id-specific T cell–B cell collaboration is of physiological relevance.

B-cell tolerance to the B-cell receptor variable regions

An enormous number of B cells with different B‐cell receptors (BCRs) are continuously produced in the bone marrow. BCRs are further diversified during the germinal center reaction. Due to extensive recirculation, B cells with mutually binding BCR are likely to meet in lymphoid organs. We have addressed possible outcomes of such an encounter in vitro. B lymphoma cells were transfected with complementary BCR, one transfectant expressing an Idiotype+ (Id+) BCR and the other an anti‐Id BCR. To exclude confounding effects of secreted Ig, the transfected B lymphoma cells only expressed membrane IgD. Coincubation of paired Id+/anti‐Id lymphoma cells results in conjugate formation, signaling, activation of Caspase 3/7, and apoptosis of at least one of the two cells in the pair. Our data provide suggestive evidence for a mechanism whereby the B‐cell compartment is partly purged of B cells with complementary BCRs.

Cytotoxic Escherichia coli strains encoding colibactin isolated from immunocompromised mice with urosepsis and meningitis

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 Wsh/Wsh mouse strain to create an immune-compromised model lacking function of Rag2 and Il2rγ 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 Escherichia coli strains harboring polyketide synthase (pks) genomic island were recently isolated from laboratory mice, the tissue sections from the urogenital tract, heart, and middle ear were subjected to E. coli specific PNA-FISH assay that revealed discrete colonies of E. coli associated with the lesions. Microbiological examination and 16S rRNA sequencing confirmed E. coli-induced infection and septicemia in the affected mice. Further characterization by clb gene analysis and colibactin toxicity assays of the pks+ E. coli revealed colibactin-associated cytotoxicity. Rederivation of the transgenic mice using embryo transfer produced mice with an intestinal flora devoid of pks+ E. coli. 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 pks+ E. coli urosepsis and meningitis in immunocompromised mice, and highlights the importance of monitoring and exclusion of colibactin-producing pks+ E. coli.

One-step generation of monoclonal B cell receptor mice capable of isotype switching and somatic hypermutation

We developed a method for rapid generation of B cell receptor (BCR) monoclonal mice expressing prerearranged Igh and Igk chains monoallelically from the Igh locus by CRISPR-Cas9 injection into fertilized oocytes. B cells from these mice undergo somatic hypermutation (SHM), class switch recombination (CSR), and affinity-based selection in germinal centers. This method combines the practicality of BCR transgenes with the ability to study Ig SHM, CSR, and affinity maturation.