Research ArticleIMMUNOLOGY

A facile technology for the high-throughput sequencing of the paired VH:VL and TCRβ:TCRα repertoires

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Science Advances  22 Apr 2020:
Vol. 6, no. 17, eaay9093
DOI: 10.1126/sciadv.aay9093
  • Fig. 1 A simple droplet system for NPS using cell lysate–resistant polymerase, RTX.

    (A) Varying numbers of HEK293 cells were spiked into 50 μl of RT-PCR solution containing detergent and subjected to RT-PCR using primers to amplify the housekeeping gene PGK1. Purified HEK293 RNA was used as a positive control. NTC, no template control. (B) Two laminar flow streams, one containing a cell suspension and the other comprising the reagents required for OE RT-PCR, are merged using a Y junction to form droplets that are immediately emulsified in a dispersing tube. OE RT-PCR is performed in the emulsion to create ca 850- or 550-bp amplicons of linked VH:VL or TCRβ:α amplicons, respectively.

  • Fig. 2 Functional analysis of TCRs encoded by peripheral blood TFH cells.

    (A) Sorting and gating strategy for follicular helper CD4+ T cells. CD3+CD4+CXCR5+CD45RA PD1++ TFH cells were isolated from PBMCs at day 7 post vaccination from a donor vaccinated with the Fluzone vaccine. (B) Histogram showing expression of the CD69 activation marker on Jurkat cells expressing exogenous TCRs from the vaccinated donor after 24 hours incubation in the presence or absence of autologous DCs and/or Fluzone 2011-2012 vaccine. Numbers of transfected cells are shown. HT-T-1 and HT-T-2, Jurkat cells transfected with TCRβ:α from peripheral TFH cells; RA14, CMV-specific TCRβ:α as a negative control. (C) CDR3 sequences and gene usages of the HT-T-1 clone.

  • Table 1 Summary of RTX-NPS results.

    Sample IDNumber of BCR/TCR clustersPairing accuracyCell inputDonor
    1VH:VL: 576193.8%3 × 104
    CD27+ B
    cells + 500
    ARH-77
    A
    1′VH:VL: 52603 × 104
    CD27+ B
    cells + 500
    ARH-77
    2VH:VL:
    21,801
    96.6%1.83 × 105
    CD27 B
    cells + 500
    ARH-77
    2′VH:VL:
    17,223
    1.83 × 105
    CD27 B
    cells + 500
    ARH-77
    3TCRβ:α:
    5528
    93.5%1.45 × 105
    pan T cells
    B
    3′TCRβ:α:
    6096
    1.45 × 105
    pan T cells
    4TCRβ:α:
    12,706
    93.2%3.62 × 105
    pan T cells
    A
    4′TCRβ:α:
    13,709
    3.62 × 105
    pan T cells
    5TCRβ:α:
    6991
    90.8%6.5 × 105
    PBMCs
    C
    5′TCRβ:α:
    7198
    6.5 × 105
    PBMCs +
    103 Jurkat
  • Table 2 Vaccine-binding or HA-binding affinities of immunoglobulin G antibodies identified by RTX-NPS from day 7 post-vaccination plasmablasts.

    Half-maximal effective concentration (EC50) values are reported as average ± SD (n ≥ 3). H3 A/Hong Kong/4801/2014 and B/Phuket/3073/2013 are components of the 2017-2018 Fluzone vaccine.

    Monoclonal antibody
    ID
    Vaccine or HA strainEC50 (nM)
    HT-AFluzone 2017-18Nonbinder*
    HT-BH3 A/
    Wisconsin/67/2005
    0.43 ± 0.22
    HT-BH3 A/New
    York/55/2004
    0.70 ± 0.36
    HT-CB/Florida/4/20061.18 ± 0.63
    HT-CB/Phuket/3073/20131.63 ± 0.32
    HT-DH3 A/
    Wisconsin/67/2005
    0.76 ± 0.42
    HT-DH3 A/New
    York/55/2004
    0.90 ± 0.42
    HT-DH3 A/Hong
    Kong/4801/2014
    7.64 ± 2.94
    HT-EFluzone 2017-180.57 ± 0.07

    *HT-A bound to the Fluzone 2017-18 vaccine weakly.

    Supplementary Materials

    • Supplementary Materials

      A facile technology for the high-throughput sequencing of the paired VH:VL and TCRβ:TCRα repertoires

      Hidetaka Tanno, Jonathan R. McDaniel, Christopher A. Stevens, William N. Voss, Jie Li, Russell Durrett, Jiwon Lee, Jimmy Gollihar, Yuri Tanno, George Delidakis, Arti Pothukuchy, Jared W. Ellefson, Jörg J. Goronzy, Jennifer A. Maynard, Andrew D. Ellington, Gregory C. Ippolito, George Georgiou

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      • Figs. S1 to S8
      • Tables S1 to S4
      • Legends for tables S5 to S9
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