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. 2021 Jan 26;11:602254. doi: 10.3389/fimmu.2020.602254

Figure 3.

Figure 3

Secreted gp96-Ig-S vaccine induces protein S specific CD8+ and CD4+ T cells in the spleen and lung tissue. Five days after the vaccination of C57Bl6 mice, splenocytes and lung cells were isolated from vaccinated and control mice (PBS) and in vitro restimulated with S1 and S2 overlapping peptides from SARS-CoV-2 protein in the presence of protein transport inhibitor, brefeldin A for the last 5 h of culture. After 20 h of culture, ICS was performed to quantify protein S-specific CD8+ and CD4+ T-cell responses. Cytokine expression in the presence of no peptides was considered background and it was subtracted from the responses measured from peptide pool stimulated samples for each individual mouse. (A, B) CD8+ T cells from spleen and lungs expressing IFNγ, TNFα, and IL-2 in response to S1 and S2 peptide pool; (C, D) CD4+ T cells from spleen and lungs expressing IFNγ, TNFα, and IL-2 in response to S1 and S2 peptide pool; (E) Representative dot plot of gated CD8+ T cells in lungs expressing indicated cytokines (IFNγ; IL-2, and TNFα) in vaccinated and non-vaccinated (PBS, control) HLA-A2 mice at day 5 (F) Proportion of antigen (protein S)-experienced CD8+ and CD4+ T cells isolated from spleen and lung tissue expressing IFNγ, TNFα, or IL-2 after o/n stimulation with S1 + S2 peptides. Pie charts corresponding to cytokine profiles of CD8+ and CD4+ T cells isolated from spleen and lung tissue; (G) Pie charts corresponding to cytokine profiles of CD8+ CD4+ T cells isolated from spleen and lung tissue after o/n stimulation with S1 + S2 peptides. Assessment of the mean proportion of cells making any combination of one to three cytokines (IFN-γ, TNFα, IL-2). Data represent at least two technical replicates with three to six independent biologic replicates per group. *p<0.05, ***p<0.001. Kruskal-Wallis ANOVA with Dunn’s multiple comparisons tests were applied. Asterisks (*) above or inside the column denote significant differences between indicated T cells producing cytokines in vaccine versus control (PBS) at 0.05 alpha level. ANOVA, analysis of variance; ICS, intracellular cytokine staining; IFN, interferon; IL, interleukin; PBS, phosphate-buffered saline; TNF, tumor necrosis factor.