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. 2022 Jun 16;13:3466. doi: 10.1038/s41467-022-31142-5

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© The Author(s) 2022

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. 3 — PBMCs collected from study participants (n = 10) pre-vaccination (day 0), day 28 post-vaccination (7 days post-boost), and day 105 post-vaccination were examined for ICOS, CD38, and other markers by fluorescence flow cytometry individually in one setting to avoid batch variation. A CD4⁺ T cells were gated from lymphocytes and defined as ICOS⁺CD38⁺ or ICOS^loCD38^lo. Percentages of CD4⁺ICOS⁺CD38⁺ T cells were quantified and data from each participant is connected by lines. B Mean fluorescence intensity (MFI) of CCR7 within CD4⁺ICOS⁺CD38⁺, CD8⁺ICOS⁺CD38⁺, CD4⁺ICOS^loCD38^lo, or CD8⁺ICOS^loCD38^lo T cells. C, D PBMCs were stimulated with PMA/ionomycin and examined for cytokine production. T cells defined as ICOS⁺CD38⁺ (red) or ICOS^loCD38^lo (blue) were examined for C IFN-γ or D TNF. Representative samples on the left and individual participants are shown on right. E Day 28 PBMC samples were stimulated with activation beads or T cells were sorted based on CD38 and ICOS expression. Sorted T cells were labeled with CellTrace Violet (CTV) to track and stimulated with T-cell (CD3)-depleted autologous PBMCs ± SARS-CoV-2 Spike peptide pool (+ peptide, blue and red). Sorted T-cell populations were analyzed after 48 h for IL-2, TNF, and Granzyme b in F. BCL6 expression in sorted T-cell populations, stimulated as in E. G Tfh markers CXCR5 and PD-1 compared on day 0 and 28 samples. H Unstimulated days 0 and 28 ICOS⁺CD38⁺ and ICOS^loCD38^lo T cells were analyzed for BCL6 expression (FMO; fluorescence-minus-one control for BCL6). I CXCR5 MFI within BCL6⁺ T cells. FMO control for BCL6 in ICOS^loCD38^lo T cells is shown by the blue dotted line, and by the red dotted line for ICOS⁺CD38⁺ T cells. B Significance was determined by paired two-way ANOVA mixed-effects model. E, F Repeated measures one-way ANOVA. C, D, G–I Two-way ANOVA with Sidak’s multiple comparisons test. Representative samples are shown and each data point represents an individual participant. All error bars ± SD. ns = not significant, *p < 0.5, **p < 0.005, ***p < 0.0005, ****p < 0.00005.