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(C) LCDR3 length distribution and amino acid frequencies for kappa light chains. (TIFF) Click here for more data file.(437K, tiff) S8 FigPercent heavy chain identity of public PBs that are a part of one of the four pubCDR3 convergent organizations. mean weighty chain variable section usage for each participant. (B) Divergence from your mean weighty chain variable section utilization by participant age. (C) Divergence from your mean light chain variable segment utilization for each participant. (D) Divergence from your mean light chain variable segment utilization by participant age.(TIFF) pone.0247253.s004.tiff (576K) GUID:?E6F62AD6-03F6-4C82-8563-7EB28BD1FC68 S4 Fig: Proportion of unique clonotypes is not significantly increased in younger participants (= 0.054). (TIFF) pone.0247253.s005.tiff (52K) GUID:?FD42F86C-984C-4216-9F3E-D7E72BC4D8E9 S5 Fig: Preferential V(D)J segment joining of heavy chain sequences from 17 influenza vaccinated individuals. (A) Heatmap of preferential association between V and J gene segments. (B) Heatmap of preferential association between J and D gene segments. (C) Heatmap of preferential association between V and D gene segments. Yellow corresponds to low pairing A-674563 rate of recurrence, while dark blue corresponds to the highest observed pairing rate of recurrence.(TIFF) pone.0247253.s006.tiff (263K) GUID:?D4227BDA-BE54-4FEB-BE5A-350B02111A36 S6 Fig: Preferential VJ segment joining of light chain sequences from 17 influenza vaccinated individuals. (A) Heatmap of preferential association between V and J gene segments for lambda light chains. (B) Heatmap of preferential association between V and J gene segments or kappa light chains. Yellow corresponds to low pairing rate of recurrence, while dark blue corresponds to the highest observed pairing rate of recurrence.(TIFF) pone.0247253.s007.tiff (191K) GUID:?F6C14577-CC0A-4CE8-9E35-D68D4878F599 S7 Fig: CDR3 characteristics of heavy and light chain amino acid sequences. (A) HCDR3 size distribution and amino acid frequencies. (B) LCDR3 size distribution and amino acid frequencies for lambda light chains. (C) LCDR3 size distribution and amino acid frequencies for kappa A-674563 light chains.(TIFF) pone.0247253.s008.tiff (437K) GUID:?7438D117-4949-43C5-84ED-36E4331FF612 S8 Fig: Percent weighty chain identity of general public PBs that are a part of one of the four pubCDR3 convergent organizations. General public PBs are contrasted with non-public PBs belonging to the same weighty chain variable section, based on (A) weighty chain variable section nucleotide identity, and (B) HCDR3 peptide identity.(TIFF) pone.0247253.s009.tiff (217K) GUID:?25EF060B-A485-4D5D-9785-0FDE60CA012C S1 A-674563 Table: PB response to influenza vaccination. (DOCX) pone.0247253.s010.docx (15K) GUID:?1A5F0BE9-405D-42B2-86B1-5FB4A9FBEB36 S2 Table: List of the three most expanded clonotypes for each donor. Colors correspond to those use in Fig 3.(DOCX) pone.0247253.s011.docx (17K) GUID:?29974054-3DEA-4A40-91A7-F93B69A9BB42 S3 Table: List of convergent (general public) clonotypes for each donor. Colors correspond to those use in Fig 5.(DOCX) pone.0247253.s012.docx (16K) GUID:?D59FA783-690B-4807-953A-5545A3C00947 S4 Table: Key resources. (DOCX) pone.0247253.s013.docx (20K) A-674563 GUID:?5114E546-8583-415B-9D1C-D6CF8CD2711A S1 Text: Strain abbreviations. (DOCX) pone.0247253.s014.docx (13K) GUID:?52A128BB-F19D-4C4B-B775-850EF57E1F96 Attachment: Submitted filename: differentiation to stimulate memory space B cells by incubating with 500 ng/mL R848 (Invivogen, San Diego, CA, USA) and 5 ng/mL rIL-2 (R&D, Minneapolis, MN, USA) for 7C9 days at 37C in 5% CO2 as previously described [20]. Total and rHA-specific Rabbit Polyclonal to CCR5 (phospho-Ser349) IgG large quantity of the conditioned medium supernantants were assessed by ELISA starting at a 1:5 dilution, and the rate of recurrence of B cells was assessed by CD19 surface labeling and circulation cytometry. For circulation cytometry analysis, human being PBMCs (~5 x 106 live cells) were treated as previously explained [20]. In brief, PBMCs were first treated with Fc receptor obstructing answer (Biolegend, Dedham, MA, USA) then stained for 30 min on snow using titrated quantities of fluorescently conjugated mAbs. After completion of surface labeling, cells were washed extensively with staining buffer prior to fixation with 1.6% paraformaldehyde in staining buffer for 15 min at RT. Following fixation, cells were pelleted by centrifugation at 400xfor 5 min, resuspended in staining buffer and managed at 4?C protected from light until acquisition. Data acquisition was performed using the BD FACSARIA Fusion and analysis performed using FlowJo (FlowJo LLC, Ashland, OR, USA). Payment values were founded prior to acquisition using appropriate single stain controls. PBs were defined as CD3/CD14neg CD19+, CD27+, CD38++ cells as previously described [26, 27]. Flow cytometric analysis and cell sorting were performed on BD FACSAria Fusion using the BD A-674563 FACSDiva Software. IgG-enriched PBs were isolated by gating for CD19+CD20low/?CD27+CD38highIgA?IgM?IgD?CD3?CD14? live cells. The PBs were single-cell sorted into 384-well PCR plates made up of lysis buffer (10 mM Tris-HCl, pH 8.0), 2 mM dNTPs (New England Biolabs), 2 mM DTT (Sigma-Aldrich), 0.2 mg/mL BSA (New England Biolabs), 0.01% IGEPAL-630 (Sigma-Aldrich) and 0.5 unit/l of RiboLock RNase Inhibitor (ThermoFisher Scientific). Single-cell sorted plates were stored at ?80C until use for paired Ig heavy and light chain sequencing using Atrecas Immune Repertoire Capture? technology. IgG sequencing Reverse transcription, PCR, barcode assignment, sequence assembly, V(D)J assignment, and identification of mutations were performed around the single-cell sorted samples as previously.