Fig. 4: QW9-specific, B53-restricted CTL isolation and functional examination.
a PBMCs from a B53+ individual (viral load <50 RNA copies/mL, CD4 count = 1003, antiretroviral therapy-naive) were stimulated with QW9 peptide for 6 days and stained with an QW9-B53 tetramer. Expanded cells were subjected to single-cell FACS sorting by using QW9-B53 tetramers. b QW9-B53 specific CD8+ T cell, Clone 3, were expanded and tested for lysis of autologous B cells loaded with irrelevant, QW9 and QW9_S3T at an effector cell/target cell ratio of 1:1. Each peptide was tested with 8 technical replicates. Two-tailed unpaired t test and 95% confidence interval were used for P-value calculation, ****p ≤ 0.0001. The Error bars represent SD. c The C3 TCR was cloned into a lentivirus and used to transduce a TCR null Jurkat cell line. C3 TCR expression was confirmed by flow cytometry as evidenced by binding to QW9-B53-APC tetramer staining (red cloud) in comparison to mock transduced cells (blue cloud). X-axis: QW9-B57-PE conjugate, Y-axis: QW9-B53-APC conjugate. d Representative steady state SPR measurements for C3 interacting with QW9-B53 and QW9_S3T-B53. C3 TCR flowed through a QW9-B53 and QW9_S3T-B53 coated chip (the red curve represents QW9-B53, and the blue curve represents QW9_S3T-B53; duplicate number, n = 2). e, f Representative kinetic state SPR measurements for C3 interacting with QW9-B53 (panel e) and QW9_S3T-B53 (panel f) at 25 °C. 10 serial dilutions of concentrated TCR were injected at 50 μl/min throughout the experiment, with a contact time of 120 s and a dissociation time of 300 s (duplicate number, n = 2). Each binding response was fit to a 1:1 binding model, and the kinetic parameters were calculated using the BiaEval software, with kon, koff and the Rmax fit globally.
