Legendplex human th panel

To assess the quantities of secreted cytokines, the supernatant of a coculture of naive T cells and KCs was collected, and 13 cytokines were analyzed by the LEGENDplex Human Th Panel (BioLegend, San Diego, CA) according to the manufacturer’s protocol.

To detect the cytokine production by CD4⁺ T cells, 1 × 10⁵ CD4⁺ T cells were stimulated with anti-CD3/anti-CD28 beads (Thermo Fisher Scientific, Waltham, MA, USA) in the presence or absence of mitochondria at 100 μg/mL in a 96-well plate with a total of 200 μL X-VIVO 15 serum-free culture medium (Lonza, Walkersville, MD, USA) per well. After the treatment for 48 h, the supernatants were collected to examine the level of inflammatory cytokines (IL-2, IL-4, IL-5, IL-6, IL-9, IL-10, IL-13, IL-17F, IL-21, IL-22, IFN-γ, and TNF-α) using a LEGENDplex™ Human Th Panel (Biolegend, San Diego, CA, USA) by Gallios Flow Cytometer according to the manufacturer’s protocols.

PepMix pools containing 15-mer peptides overlapping by 10 amino acids from either SARS-CoV-2 spike S1 or S2, N/M/E protein domains were purchased from Alta Biosciences (University of Birmingham). T cell responses of postvaccination samples to the above peptide mixes were determined using a Human IFN-γ ELISpot PRO kit (Mabtech). Isolated PBMCs were thawed and rested overnight before assay in R10 (RPMI 1640 + 10% FCS + penicillin/streptomycin). Then, 2–3 × 10⁵ PBMCs were stimulated in duplicate with peptide mixes at 1 μg ml⁻¹ per peptide, anti-CD3 and CEFX cell stimulation mix (JPT) as a positive control or DMSO as a negative control for 16–18 h. Supernatants were collected and stored at −80 °C. After the development of plates according to the manufacturer’s instructions, plates were read using the BIOSYS Bioreader 5000. Mean spot counts in DMSO-treated negative control wells were deducted from the means to generate normalized spot counts for all other treated wells. Cutoff values were determined previously^{14 (link)}.
Cytokine concentrations in the ELISpot supernatants were assayed using a LEGENDplex Human Th Panel (BioLegend) according to the manufacturer’s instructions. Data were analyzed using the LEGENDplex Data Analysis Software Suite (BioLegend).