IgG is the predominant antibody class generated during infections and used for the generation of therapeutic antibodies. Antibodies are mainly characterized in or generated from animal models that ...support particular infections, respond to particular antigens or allow the generation of hybridomas. Due to the availability of numerous transgenic mouse models and the ease of performing bioassays with human blood cells in vitro, most antibodies from species other than mice and humans are tested in vitro using human cells and/or in vivo using mice. In this process, it is expected, but not yet systematically documented, that IgG from these species interact with human or mouse IgG receptors (FcγRs). In this study, we undertook a systematic assessment of binding specificities of IgG from various species to the families of mouse and human FcγRs including their polymorphic variants. Our results document the specific binding patterns for each of these IgG (sub)classes, reveal possible caveats of antibody‐based immunoassays, and will be a useful reference for the transition from one animal model to preclinical mouse models or human cell‐based bioassays.
We undertook a systematic assessment of binding specificities of IgG from various species to mouse and human FcγRs including their polymorphic variants. Our data draw a comprehensive map of their interactions and reveal that most IgG interact with at least one, and mostly several, human and mouse FcγRs.
The major therapeutic goal for immune thrombocytopenic purpura (ITP) is to restore normal platelet counts using drugs to promote platelet production or by interfering with mechanisms responsible for ...platelet destruction. Eighty percent of patients with ITP possess anti-integrin αIIbβ3 IgG autoantibodies that cause platelet opsonization and phagocytosis. The spleen is considered the primary site of autoantibody production by autoreactive B cells and platelet destruction. The immediate failure in approximately 50% of patients to recover a normal platelet count after anti-CD20 rituximab-mediated B cell depletion and splenectomy suggests that autoreactive, rituximab-resistant, IgG-secreting B cells (IgG-SCs) reside in other anatomical compartments. We analyzed more than 3,300 single IgG-SCs from spleen, bone marrow, and/or blood of 27 patients with ITP, revealing high interindividual variability in affinity for αIIbβ3, with variations over 3 logs. IgG-SC dissemination and range of affinities were, however, similar for each patient. Longitudinal analysis of autoreactive IgG-SCs upon treatment with the anti-CD38 mAb daratumumab demonstrated variable outcomes, from complete remission to failure with persistence of high-affinity anti-αIIbβ3 IgG-SCs in the bone marrow. This study demonstrates the existence and dissemination of high-affinity autoreactive plasma cells in multiple anatomical compartments of patients with ITP that may cause the failure of current therapies.
Although the COVID‐19 pandemic peaked in March/April 2020 in France, the prevalence of infection is barely known. Using high‐throughput methods, we assessed herein the serological response against ...the severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) of 1847 participants working in three sites of an institution in Paris conurbation.
In May–July 2020, 11% (95% confidence interval CI: 9.7–12.6) of serums were positive for IgG against the SARS‐CoV‐2 N and S proteins, and 9.5% (95% CI: 8.2–11.0) were neutralizer in pseudo‐typed virus assays. The prevalence of seroconversion was 11.6% (95% CI: 10.2–13.2) when considering positivity in at least one assay. In 5% of RT‐qPCR positive individuals, no systemic IgGs were detected. Among immune individuals, 21% had been asymptomatic. Anosmia (loss of smell) and ageusia (loss of taste) occurred in 52% of the IgG‐positive individuals and in 3% of the negative ones. In contrast, 30% of the anosmia–ageusia cases were seronegative, suggesting that the true prevalence of infection may have reached 16.6%. In sera obtained 4–8 weeks after the first sampling, anti‐N and anti‐S IgG titers and neutralization activity in pseudo‐virus assay declined by 31%, 17%, and 53%, resulting thus in half‐life of 35, 87, and 28 days, respectively.
The population studied is representative of active workers in Paris. The short lifespan of the serological systemic responses suggests an underestimation of the true prevalence of infection.
In May 2020, 11% of workers at Institut Curie living in Paris conurbation were seropositive and 9.5% had detectable but short‐lived neutralizing antibodies of SARS‐CoV‐2. Some 21% of these neutralizing sera actually belong to asymptomatic individuals. Only 2% of the PCR‐detected infections had not been followed by humoral immune response.
Natural killer (NK) cell development is thought to occur in the bone marrow. Here we identify the transcription factor GATA-3 and CD127 (IL-7R alpha) as molecular markers of a pathway of mouse NK ...cell development that originates in the thymus. Thymus-derived CD127+ NK cells repopulated peripheral lymphoid organs, and their homeostasis was strictly dependent on GATA-3 and interleukin 7. The CD127+ NK cells had a distinct phenotype (CD11b(lo) CD16- CD69(hi) Ly49(lo)) and unusual functional attributes, including reduced cytotoxicity but considerable cytokine production. Those characteristics are reminiscent of human CD56(hi) CD16- NK cells, which we found expressed CD127 and had more GATA-3 expression than human CD56+ CD16+ NK cells. We propose that bone marrow and thymic NK cell pathways generate distinct mouse NK cells with properties similar to those of the two human CD56 NK cell subsets.
Omalizumab is an anti-IgE monoclonal antibody (mAb) approved for the treatment of severe asthma and chronic spontaneous urticaria. Use of omalizumab is associated with reported side effects ranging ...from local skin inflammation at the injection site to systemic anaphylaxis. To date, the mechanisms through which omalizumab induces adverse reactions are still unknown. Here, we demonstrated that immune complexes formed between omalizumab and IgE can induce both skin inflammation and anaphylaxis through engagement of IgG receptors (FcγRs) in FcγR-humanized mice. We further developed an Fc-engineered mutant version of omalizumab, and demonstrated that this mAb is equally potent as omalizumab at blocking IgE-mediated allergic reactions, but does not induce FcγR-dependent adverse reactions. Overall, our data indicate that omalizumab can induce skin inflammation and anaphylaxis by engaging FcγRs, and demonstrate that Fc-engineered versions of the mAb could be used to reduce such adverse reactions.
Mining the antibody repertoire of plasma cells and plasmablasts could enable the discovery of useful antibodies for therapeutic or research purposes
. We present a method for high-throughput, ...single-cell screening of IgG-secreting primary cells to characterize antibody binding to soluble and membrane-bound antigens. CelliGO is a droplet microfluidics system that combines high-throughput screening for IgG activity, using fluorescence-based in-droplet single-cell bioassays
, with sequencing of paired antibody V genes, using in-droplet single-cell barcoded reverse transcription. We analyzed IgG repertoire diversity, clonal expansion and somatic hypermutation in cells from mice immunized with a vaccine target, a multifunctional enzyme or a membrane-bound cancer target. Immunization with these antigens yielded 100-1,000 IgG sequences per mouse. We generated 77 recombinant antibodies from the identified sequences and found that 93% recognized the soluble antigen and 14% the membrane antigen. The platform also allowed recovery of ~450-900 IgG sequences from ~2,200 IgG-secreting activated human memory B cells, activated ex vivo, demonstrating its versatility.
Characterization of the Thymic IL-7 Niche in Vivo Alves, Nuno L.; Richard-Le Goff, Odile; Huntington, Nicholas D. ...
Proceedings of the National Academy of Sciences - PNAS,
02/2009, Letnik:
106, Številka:
5
Journal Article
Recenzirano
Odprti dostop
The thymus represents the "cradle" for T cell development, with thymic stroma providing multiple soluble and membrane cues to developing thymocytes. Although IL-7 is recognized as an essential factor ...for thymopoiesis, the "environmental niche" of thymic IL-7 activity remains poorly characterized in vivo. Using bacterial artificial chromosome transgenic mice in which YFP is under control of IL-7 promoter, we identify a subset of thymic epithelial cells (TECs) that co-express YFP and high levels of 117 transcripts ${\rm{IL -7}}^{{\rm{hi}}} $. ${\rm{IL - 7}}^{{\rm{hi}}} $ arise during early fetal development persist throughout life, and co-express homeostatic chemokines (Ccl19, Ccl25, Cxcl12) and cytokines (Il15) that are critical for normal thymopoiesis. In the adult thymus, ${\rm{IL - 7}}^{{\rm{hi}}} $ cells localize to the cortico-medullary junction and display traits of both cortical and medullary TECs. Interestingly, the frequency of ${\rm{IL - 7}}^{{\rm{hi}}} $ cells decreases with age, suggesting a mechanism for the age-related thymic involution that is associated with declining IL-7 levels. Our temporal-spatial analysis of IL-7-producing cells in the thymus in vivo suggests that thymic IL-7 levels are dynamically regulated under distinct physiological conditions. This IL-7 reporter mouse provides a valuable tool to further dissect the mechanisms that govern thymic IL-7 expression in vivo.
Engineered crystallizable fragment (Fc) regions of antibody domains, which assume a unique and unprecedented asymmetric structure within the homodimeric Fc polypeptide, enable completely selective ...binding to the complement component C1q and activation of complement via the classical pathway without any concomitant engagement of the Fcγ receptor (FcγR). We used the engineered Fc domains to demonstrate in vitro and in mouse models that for therapeutic antibodies, complement-dependent cell-mediated cytotoxicity (CDCC) and complement-dependent cell-mediated phagocytosis (CDCP) by immunological effector molecules mediated the clearance of target cells with kinetics and efficacy comparable to those of the FcγR-dependent effector functions that are much better studied, while they circumvented certain adverse reactions associated with FcγR engagement. Collectively, our data highlight the importance of CDCC and CDCP in monoclonal-antibody function and provide an experimental approach for delineating the effect of complement-dependent effector-cell engagement in various therapeutic settings.
Transcription factors orchestrate T-lineage differentiation in the thymus. One critical checkpoint involves Notch1 signaling that instructs T-cell commitment at the expense of the B-lineage program. ...While GATA-3 is required for T-cell specification, its mechanism of action is poorly understood. We show that GATA-3 works in concert with Notch1 to commit thymic progenitors to the T-cell lineage via 2 distinct pathways. First, GATA-3 orchestrates a transcriptional “repertoire” that is required for thymocyte maturation up to and beyond the pro–T-cell stage. Second, GATA-3 critically suppresses a latent B-cell potential in pro–T cells. As such, GATA-3 is essential to sealing in Notch-induced T-cell fate in early thymocyte precursors by promoting T-cell identity through the repression of alternative developmental options.
•Gata3 is critical for the transition of “double-negative” (DN) thymocyte DN1 to DN2.•Gata3 represses a latent B-cell potential in DN thymocytes.
TSEs (transmissible spongiform encephalopathies) are neurodegenerative diseases caused by pathogenic isoforms (PrPSc) of the host-encoded PrPc (cellular prion protein). After consumption of ...contaminated food, PrPSc deposits rapidly accumulate in lymphoid tissues before invasion of the CNS (central nervous system). However, the mechanisms of prion spreading from the periphery to the nervous system are still unclear. In the present study, we investigated the role of DCs (dendritic cells) in the spreading of prion infection to neuronal cells. First, we determined that BMDCs (bone-marrow-derived DCs) rapidly uptake PrPSc after exposure to infected brain homogenate. Next, we observed a progressive catabolism of the internalized prion aggregates. Similar experiments performed with BMDCs isolated from KO (knockout) mice or mice overexpressing PrP (tga20) indicate that both PrPSc uptake and catabolism are independent of PrPc expression in these cells. Finally, using co-cultures of prion-loaded BMDCs and cerebellar neurons, we characterized the transfer of the prion protein and the resulting infection of the neuronal cultures. Interestingly, the transfer of PrPSc was triggered by direct cell-cell contact. As a consequence, BMDCs retained the prion protein when cultured alone, and no transfer to the recipient neurons was observed when a filter separated the two cultures or when neurons were exposed to the BMDC-conditioned medium. Additionally, fixed BMDCs also failed to transfer prion infectivity to neurons, suggesting an active transport of prion aggregates, in accordance with a role of TNTs (tunnelling nanotubes) observed in the co-cultures.
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