Fitc 2b4

Patient 355 (59-year old Male, infected with genotype 1a HCV, baseline viral load 467 000 IU/ml) received a prime vaccination of ChAd3-NSmut (2.5 × 10¹⁰ viral particles) and an MVA-NSmut (2 × 10⁸ plaque forming units) boost vaccination 8 weeks later. PBMC were collected 14 weeks post-boost vaccination for assessment of single cell gene expression (22 ). PBMC were thawed and prestained with a live/dead marker (Life Technologies, cat# L34976) and a PE-conjugated pentamer reagent (PE-labeled HCV NS31406–1415 (KLSALGINAV; HLA-A*0201)). The cells were then stained with the following antibodies: FITC 2B4 (BioLegend, cat# 329505), PerCP-eFluor 710 LAG3 (eBioscience, cat# 46–2239), PE-Cy7 CCR7 (BioLegend, cat# 329919), APC CD39 (BioLegend, cat# 328209), BV421 PD1 (BioLegend, cat# 329919), BV510 CD3 (BioLegend, cat# 317332), BV605 CD8A (BioLegend, cat# 301040), BV780 CD45RA (BioLegend, cat# 304140).
The relevant institutional review boards approved all human subject protocols, and all subjects provided written consent before enrollment.

Female C57BL/6 mice (The Jackson Laboratory), aged 7 weeks, were infected with 2 × 10⁵ plaque forming units (PFU) LCMV Armstrong intraperitoneally i.p. or 4 × 10⁶ PFU LCMV Clone 13 i.v. LCMV viruses were a generous gift from Dr. E John Wherry (University of Pennsylvania, Perelman School of Medicine). Peripheral blood was obtained from the mice at day 7 post infection (p.i.) and lymphocytes were enriched using LSM density centrifugation. Cells were prestained with a near-IR fixable live/dead marker (Life Technologies, cat# L34976) and an APC- conjugated dextramer reagent for gp33 (Immudex, cat# A2160-APC) according to manufacturer recommendations. The cells were then stained with the following antibodies: FITC 2B4 (BioLegend, cat# 133504), PerCP-Cy5.5 CD44 (BioLegend, cat# 103032), PE KLRG1 (BioLegend, cat# 138408), PE-Cy7 PD1 (BioLegend, cat# 135215), BV421 CD127 (BioLegend, cat# 135024), BV510 CD8A (BioLegend, cat# 100752).