Author: Bancroft, Tara; DeBuysscher, Blair L.; Weidle, Connor; Schwartz, Allison; Wall, Abigail; Gray, Matthew D.; Feng, Junli; Steach, Holly R.; Fitzpatrick, Kristin S.; Gewe, Mesfin M.; Skog, Patrick D.; Doyle-Cooper, Colleen; Ota, Takayuki; Strong, Roland K.; Nemazee, David; Pancera, Marie; Stamatatos, Leonidas; McGuire, Andrew T.; Taylor, Justin J.
Title: Detection and activation of HIV broadly neutralizing antibody precursor B cells using anti-idiotypes Document date: 2019_10_7
ID: 63yvpuqx_14_0
Snippet: The moderate similarity of IB2 + BCR sequences to iglb12 led us to consider whether there were biological mechanisms selecting against CDRH3s that more closely matched iglb12. Specifically, we hypothesized that the iglb12 heavy chain was autoreactive, resulting in deletion of similar sequences from the B cell repertoire. Autoreactivity of iglb12 has not been reported, and conflicting reports of autoreactivity have been reported for the mature for.....
Document: The moderate similarity of IB2 + BCR sequences to iglb12 led us to consider whether there were biological mechanisms selecting against CDRH3s that more closely matched iglb12. Specifically, we hypothesized that the iglb12 heavy chain was autoreactive, resulting in deletion of similar sequences from the B cell repertoire. Autoreactivity of iglb12 has not been reported, and conflicting reports of autoreactivity have been reported for the mature form of b12. One study reported that mature b12 exhibited autoreactivity using an in vitro assay (Haynes et al., 2005; Ota et al., 2013) , however B cell development in transgenic mice expressing this antibody was normal (Ota et al., Figure 4 . Identification and analysis of human B cells able to bind anti-iglb12 idiotypes. (A) Detection of live CD19 + CD3 − CD14 − CD16 − B cells from human PBMCs that bound IB2-PE and IB3-APC tetramers with or without enrichment using anti-PE and anti-APC microbeads before flow cytometry. PE594 and APC755 tetramers containing isotype control antibodies were included in these experiments to exclude B cells specific for the PE, APC, streptavidin, and conserved portions of the anti-idiotypes. The displayed plots were derived from ∼400,000 unfractionated PBMCs or ∼400,000 PEand APC-enriched cells derived from 200 million PBMCs and representative of three individuals in three independent experiments. The percentages of single and double positive B cells in the enriched and unenriched fractions from one individual are shown on the plots. (B) The frequency of heavy chains using V H 1-3 within the population of IB2 + B cells, IB3 + B cells, and a population of control B cells that was not selected based upon antigen binding are displayed for three individuals. (C) The frequency of total V H 1-3 + , IB2 + V H 1-3 + , and IB3 + V H 1-3 + within the entire B cell repertoire is displayed for three individuals. (D) The frequency of V K 3-20/V K 3D-20 usage among 120 IB2 + V H 1-3 + BCRs and 10 IB3 + V H 1-3 + BCRs using kappa light chains pooled from three individuals are compared with 259 V H 1-3 + BCRs using kappa light chains from a control dataset derived from naive B cells . (E) Quantitation of the percent BSA between IB2 and the segments derived from V H 1-3, D H 2-21, J H 6, and N nucleotide additions within iglb12 heavy chain CDRH3 from the crystal structures displayed in Fig. 2 . (F) D H 2-21 usage among 228 IB2 + V H 1-3 + BCRs pooled from three individuals are compared with 467 V H 1-3 + BCRs from a control dataset derived from naive B cells . (G) Comparison of CDRH3 similarity from 228 IB2 + V H 1-3 + BCRs compared with the CDRH3 of iglb12 using pairwise alignment. The P values (*, P < 0.05; **, P < 0.01; ***, P < 0.001) in B and C were determined using an unpaired two-tailed Student's t test, and the P values in D were determined by Fisher's exact test. 2013). As a preliminary assessment of potential autoreactivity, we measured iglb12 binding to human HEp-2 cells using immunofluorescence. This approach revealed that iglb12 bound moderately to HEp-2 cells compared with 4E10 (Fig. 5 , A and B), an HIV-specific antibody previously shown to exhibit binding in this assay (Haynes et al., 2005; Verkoczy et al., 2010) . The iglb12 staining was similar to the mature form of b12, both of which were brighter than that of 10E8 (Fig. 5 , A and B), an antibody that was previously shown to be nonreactive in this assay (Haynes et al., 2005; Verkoczy et al., 2010) . Together, these
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