Aqua live dead dye

Twenty-four hours after TCR mRNA transfection, the T cells were stained with antibodies for CD3, CD8 and TCRVβ (BD Biosciences, San Jose, CA) of the respective TCR and analyzed by a FACSVerse flow cytometer (BD Biosciences, San Jose, CA). Dead cells were excluded by LIVE/DEAD Aqua dye (Life Technologies, Carlsbad, CA). The data was analyzed with the FlowJo V10.0.7 software (Tree Star, Ashland, OR). The NS3 H4 and F8 TCRs, and the NS5A 69 and 19 TCRs were analyzed. Redirected T cells were thereafter co-cultured with gt1a NS3_1073-1081 peptide- or gt1b NS5A_1992-2000 peptide-loaded T2 cells overnight. IFN-γ secretion into supernatants was quantified using the IFN-γ ELISA kit (Mabtech, Nacka Strand, Sweden).
Antiviral efficacy in the respective anti-HCV TCRs were measured as previously described30 (link) in co-culture assays with Huh7^A2HCV^Rep cells that contains a stably replicating HCV reporter replicon. The replicon-driven luciferase activity was measured using the ONE-Glo Luciferase Assay System (Promega, Madison, WI). Data is presented as percentage of relative light unit (RLU) reduction compared to untreated Huh7^A2HCV^Rep cells alone (without co-culture). RLU values of non-luciferase cells (transfected T cells alone) were subtracted.

GICs and PBMCs were stained for CD3-PE-Cy5.5 (eBioscience), CD4-BV711, CD8-PerCP, CD25-BV605, CD45RA-BV570, CCR7-BV650, CD39-BV421, PD-1-APC-Cy, BTLA-PE, Tim-3-PE-Cy7 (all Biolegend), LAG3 (Enzo Life Sciences, Lörrach, Germany), CTLA4-PE-CF594 (BD) and Live/dead-Aqua dye (Life technologies, Carlsbad, CA) or with isotype controls. Cells were fixed and permeabilized, followed by ICS using Foxp3-FITC (eBioscience) and Ki67-Alexa Fluor 700 (BD) and measured on an LSR Fortessa (BD).

Cell suspensions were stained for viability using LIVE/DEAD Aqua dye (Life Technologies). Cells were washed and resuspended in 50 μl of blocking buffer containing TruStain FcX PLUS (anti-mouse CD16/32, clone S17011E) antibody diluted in PBS and incubated in the dark for 15 min on ice. Conjugated anti-mouse antibodies CD45 (30-F11), CD9 (eBioKMC8), CD29 (HMβ1-1), CD31 (390), and Integrin α 7 (334908), Sca-1 (D7) were used to stain cell surface markers for 30 min on ice. Finally, cells were fixed with 50 μl of eBioscience IC Fixation Buffer (Thermo Fisher Scientific) for 5 min and acquired using a 5-laser LSR II flow cytometer (BD Biosciences) with BD FACSDiva software. Data were analyzed with FlowJo v10.6.2 (Becton, Dickinson and Company).

The following antibodies were used: CD44-APC Cy7 (clone IM7), CD62L-PE (clone MEL-14), CD8β-PE Cy7 (clone YTS156.7.7), IFN-γ-AF488 (clone XMG1.2), TNF-PerCP Cy5.5 (clone MP6-XT22), Granzyme B-BV421 (clone GB11), Tbet-PE (clone 4B10), CD45.1-AF647 (clone A20), and CD45.2-PE (clone 104) (all from BioLegend). Live/dead aqua dye was from Life Technologies. For analysis of intracellular cytokines, memory phenotype OT-1 T cells or ex vivo LN cells from tumor recipient mice were restimulated with N4 or T4 peptides at concentrations described in figures, in the presence of Brefeldin A (MilliporeSigma) for 4 hours. Cells were labeled with surface and live/dead stains prior to fixation/permeabilization in FoxP3 buffer (eBioscience). In glucose uptake experiments, T cells were cultured in the presence of 50 μM 2-NBDG for 1 hour and then washed 3 times in PBS prior to FACS analysis. Samples were acquired using an LSRII flow cytometer (BD), and data were analyzed using FlowJo software (TreeStar).

Levels of VRC01 were measured as described in [27 (link)]. Levels of rhesus Rh-α₄β₇ antibody in macaque plasma were measured using the α₄β₇-expressing human T cell line HuT-78 (NIH AIDS Reagent Program, Division of AIDS, NIAID, NIH: HuT 78 from Dr. Robert Gallo) in a flow cytometry-based assay as described in [16 (link), 22 (link)] using the standard curve method. Briefly, HuT 78 cells were first incubated for 2–3 days in complete RPMI 1640 media containing 100 nM retinoic acid to increase the surface expression of α₄β₇. Cells (150,000/condition) were stained with LIVE/DEAD Aqua dye (Thermo Fisher Scientific, Waltham, MA) for live/dead discrimination, incubated for 30min at 4°C with the plasma to be tested (1:10 diluted in PBS) obtained from macaques from the VRC01-α₄β₇ treatment group before (baseline, BL) and up to 6 weeks after treatment. Cells were then washed and incubated for 30 min at 4°C with anti-rhesus IgG1 (NHP Resource Center, antibody 7H11, in house biotinylated with EZ-link NHS-biotin (Thermo Fisher Scientific) following the manufacturer’s instructions), washed again and resuspended in neutravidin-PE (Thermo Fisher Scientific) for 20 min at 4°C. PE fluorescence was analyzed on a flow cytometer. For the standard curve, baseline plasma was pooled and spiked with serial dilutions of Rh-α₄β₇ (2,500 μg/ml– 0 μg/ml).

TRuC or CAR expression on cells was analyzed by flow cytometry. Live Dead Aqua dye (Thermo Fisher, Waltham, MA) was used according to the manufacturer’s instructions to determine live cells. TRuC or CAR surface expression was detected with a goat anti-mouse F(ab’)₂ biotin antibody (Invitrogen, Carlsbad, CA) followed by a secondary streptavidin-PE antibody (BD Biosciences, San Jose, CA). For T cell profiling the following antibodies were used: anti-CD3 (UCHT1), anti-CD8 (SK1), anti-CD4 (RPA-T4), and appropriate isotype controls (BD Biosciences, San Jose, CA). Samples were analyzed using the BD LSR Fortessa X-20 cell analyzer (BD Biosciences, San Jose, CA). Data analysis was performed with the FlowJo software (Treestar Inc, Ashland, OR).