Intracellular Cytokine Stain for Antigen-Specific T Cell Responses

Intracellular cytokine staining to detect antigen-specific CD4+ and CD8+ T cells was performed with pools of overlapping 17-mer peptides from the nucleocapsid (N), membrane (M) and Spike (S) proteins of SARS-CoV-2 (USA-WA1/2020 strain) obtained from BEI Resources, NIAID, NIH. Lyophilized peptides were re-suspended in 10% DMSO and water and then pooled. Three pools were evaluated: 1) a combined N and M pool, 90-peptides, 2) S1 [S_1-668] pool, 94-peptides, 3) S2 [S_659-1273] pool, 87-peptides. One million PBMC were co-cultured with costimulatory antibodies directed against CD28 and CD49d along with the indicated pooled peptides at a final concentration of 1 µg/mL of each individual peptide, or alternatively with Phorbol 12-myristate 13-acetate (PMA) (InvivoGen) and Ionomycin (InvivoGen) as a positive control or a DMSO/peptide diluent-only negative control. Samples were incubated for 1 hour before the addition of Brefeldin A and monensin (both from BD Biosciences) and then incubated for an additional 5 h. Surface staining was performed followed by fixation in 1% paraformaldehyde, permeablization with a washing buffer supplemented with 0.1% w/v saponin (Sigma) and intracellular staining using fluorescently labeled antibodies directed against cytokine antigens. We used the following antibodies: CD45 Alexa Fluor 532 (clone HI30, Invitrogen), CD3 Alexa Fluor 700 (clone UCHT1, BioLegend), CD4 APC-Cy7 (clone OKT4, BioLegend), CD8 BV421 (clone RPA-T8, BioLegend), IFN-gamma FITC (clone B27, BD Biosciences), TNF-alpha PerCP-Cy5.5 (clone Mab11, BD Biosciences) and IL-2 APC (clone 5344.111, BD Biosciences). The panel also included Zombie NIR viability stain (BioLegend). All antibodies were used at pre-titrated optimal staining concentrations.

Samples were run on a Cytek Aurora spectral flow cytometer using SpectroFlo software (version 2.1.0, Cytek) and unmixed before final analysis using FlowJo software (version 10, BD Biosciences). The mean number of collected live singlet lymphocytes per sample was more than 140,000. Stimulation index was calculated as the frequency of live, singlet CD3+CD4+ (CD4 T cells) positive for any combination of IL-2 and IFN-gamma expression or live, singlet CD3+CD8+ (CD8 T cells) positive for any combination of IFN-gamma, IL-2 or TNF-alpha expression in the stimulated samples divided by the frequency of cytokine positive events in the negative vehicle-only control sample. Figures were prepared in Prism version 8 (GraphPad Software, Inc).