α cd3e fitc cd3 12

Of each animal, 50μl of heparin-blood was used for trucount analysis with the non-centrifugation PerFix-NC kit (Beckman Coulter). Cells were first stained extracellular with α-CD4-APC (02, Sino Biological), α-CD8a-eFluor450 (OKT8, eBioscience), and α-CD14-PE (Tük4; Thermo Fisher) for 15 minutes at RT. Cells were then fixated with 5μl Fixative Reagent for 15 minutes after which 300μl Permeabilizing reagent was added. The subsequent intracellular staining consisted of α-CD3e-FITC (CD3-12, Biorad) and α-CD79a-APC/eFluor780 (eBioscience). After 15 minutes incubation at RT, 3ml of Final reagent was added to each sample. To decrease measurement time, samples were spun down for 5 minutes at 500x g and 2.8ml suspension was removed. The pellet was resuspended in the remaining volume and 50μl of Precision Count beads (Biolegend) was added to each sample to calculate the absolute number of cells. Samples were measured on a FACSymphony A3 (BD) and analysed using FlowJo™ Software V10.6.2 (BD). An example of the gating strategy is present in Supplemental Figure 1.

In total, 1–3 million lymphocytes were stimulated with virus at MOI 1 for 24 h or H2N2 peptide pools for 8 h. For the last 6 hours of stimulation, Brefeldin A (Golgiplug, BD) was added to the cells, followed by storage o/n at 4 °C. The next day, cells were washed twice with FACS buffer (2 mM EDTA, 0.5% BSA in PBS) and extracellular staining was performed in 100 µl FACS buffer with live-dead aqua (Invitrogen), α-CD4-APC (02, Sino Biological) and α-CD8a-eFluor450 (OKT8, eBioscience) for 30 mins at 4 °C. After washing, cells were fixated and permeabilised with Foxp3/Transcription factor staining buffer set (eBioscience) according to the manufacturers protocol. Cells were then stained intracellularly with α-CD3e-FITC (CD3–12, Biorad), α-CD79a-APC/eFluor780 (eBioscience), and α-IFNγ-PE (CC302, Bioconnect) for 30 mins at 4 °C. After washing twice, the pellet was resuspended in FACS buffer and measured on a LSR Fortessa X-20 (BD). Data were analyzed using FlowJo^TM Software V10 (BD) and an example of the gating strategy is presented in Supplementary Fig. 4a.