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. 2021 Oct 5;12:5839. doi: 10.1038/s41467-021-26137-7

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© The Author(s) 2021

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 4 — Intracellular cytokine staining (ICS) was performed to detect cytokine-producing T cells to the indicated peptide pools in HIV-negative (HIV−, n = 12) and HIV-positive individuals (HIV+, n = 11). a Representative flow cytometric plots for the identification of antigen-specific CD4 T cells based on double expression (CD154⁺IFN-γ⁺, CD154⁺IL-2⁺, and CD154⁺TNF-α⁺) following 6-h stimulation with media alone (control) or overlapping SARS-CoV-2 peptides against Spike pool 1 and 2 (Spike), Nucleoprotein (N), and Membrane protein (M) directly ex vivo. b Frequency of aggregated CD4 T-cell responses (CD154⁺IFN-γ⁺, CD154⁺IL-2⁺, and CD154⁺TNF-α⁺) against Spike, M/N or combined (Spike and M/N) peptide pools (HIV−, n = 12; HIV+, n = 11). Error bars represent SEM. c Pie charts representing the relative proportions of Spike, M/N, or total (combined Spike and M/N) CD4 T-cell responses for one (gray), two (green) or three (dark blue) cytokines, and pie arcs denoting IFN-γ, TNF-α and IL-2. d Representative flow cytometric plots for the identification of antigen-specific CD8 T cells based on the expression of (IFN-γ⁺, TNF-α⁺, and IL-2⁺) against the specified peptide pools or media alone (control). e Proportion of aggregated CD8 T-cell responses against Spike, M/N or combined (Spike and M/N) responses in HIV− (n = 12) and HIV+ (n = 11) donors. Error bars represent SEM. f Pie charts representing the relative proportions of Spike, M/N and combined CD8 T-cell responses for one (gray), two (green) or three (dark blue) cytokines, and pie arcs showing IFN-γ, TNF-α and IL-2. g Comparison of the frequencies of summed SARS-CoV-2-specific CD4 and CD8 T-cell responses against Spike in n = 12 HIV− donors (p = 0.0004) and n = 11 HIV+ (p = 0.001) and M/N proteins (HIV−, p = 0.042; HIV+, p = 0.0009). Error bars represent SEM. h Correlation between the frequency of total SARS-CoV-2-specific CD4 T cells and overall T-cell responses detected by IFN-γ ELISpot responses or i ID50 neutralization titer (log10) in HIV-negative (n = 12) and HIV-positive (n = 11) individuals. The non-parametric Spearman test was used for correlation analysis (two-tailed); p values for individual correlation analysis within groups, HIV− (green) or HIV+ (red) or combined correlation analysis (black) are presented. Significance determined by two-tailed Mann−Whitney U test or Wilcoxon matched-pairs signed rank test, *p < 0.05, **p < 0.01, ***p < 0.001. SPICE was used for polyfunctional analysis.