Dynamics of Virus-specific CD8+ T cells in the Human Nasal Cavity
Dynamics of Virus-specific CD8+ T cells in the Human Nasal Cavity
Freshly isolated nasal cells and PBMCs were rested in culture medium (AIM-V supplemented with 2% AB serum) for 2 hours at 37 °C. Following the resting period, half of the cells were stimulated with anti-CD3/CD28 Dynabeads at a 1:1 cell-to-bead ratio, while the remaining half were left unstimulated as controls. Cells were cultured overnight in 96-well U-bottom tissue culture plates under standard incubation conditions (37 °C, 5% CO2). After 24 hours, both nasal and blood T cells were washed with PBS and subsequently stained with surface markers (NIR, CD3, CD4, CD8, CD45RO) and sorted using FACS (BD FACSAria III). Specifically, NIR-CD3+CD4-CD8+CD45RO+ cells were sorted from each sample for the single-cell RNA sequencing experiment. The sorted cells were labeled with distinct Hash Tag Oligonucleotides (HTOs) for multiplexing.  Single-cell suspensions were loaded onto the 10x Genomics Chromium Controller for partitioning into Gel Bead-In-Emulsion (GEMs) using the Chromium Next GEM Single Cell 5' Kit v2 (10x Genomics). Reverse transcription was performed within the GEMs, followed by amplification and library construction. HTO libraries were constructed following the TotalSeq™ protocol.  Reverse transcription was performed within the GEMs, followed by amplification and library construction. HTO libraries were constructed following the TotalSeq™ protocol. 