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. 2022 Jun 29;5:634. doi: 10.1038/s42003-022-03596-2

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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. 1 — a In vitro co-culture schema showing stimulation of sorted CFSE-labeled human memory CD8 T cell subsets with anti-CD3/CD28 magnetic beads (1:1 ratio). After overnight stimulation, the beads were removed using a plate magnet followed by incubation with CTV-labeled naïve CD8 T cells for 7 days (memory:naive ratio [1:1 ratio and 3:1 ratio]). CTV-labeled cells were examined for activated/memory phenotype. b Representative flow cytometry plots depicting gating strategy for live CTV-naïve CD8 T cells expressing CCR7^+/− CD45RO⁺ under three conditions: (T_N) alone, naïve co-cultured with unstimulated T_CM, or with stimulated T_CM. The gating strategy includes CTV^+/int subpopulations and excludes CTV^neg cells. This gating strategy has been used in the analyses to exclude CFSE^neg activated memory CD8 T cells which might overlap with the CTV^neg population. c Bar-graph showing percent of CTV-labeled naïve CD8 T cells that acquire activated/memory phenotype based on CCR7 and CD45RO cell surface expression in the presence of stimulated T_CM (blue box) at 1:1 ratio (T_CM:T_N). Naïve CD8 T cells were cultured alone or in the presence of unstimulated T_CM and were used as controls. Blue closed circles depict stimulation conditions, while open circles depict unstimulated conditions (n = 4–11 healthy donors). d Representative image (n = 1) showing cell surface markers expressed by CTV-labeled naïve CD8 T cells alone and in the presence of stimulated T_CM CD8 T cells using advanced imaging flow cytometry. e Representative flow cytometry plots and f Bar-graph showing the percentage of CTV-labeled naïve CD8 T cells that acquire an activated/memory phenotype in the presence of stimulated T_CM at 3:1 (n = 9) and 1:1 (n = 11) ratio (T_CM:T_N). g Histograms showing expression of CD95 (activation/memory marker) in both subpopulations CTV⁺ CD45RO^neg and CD45RO⁺ in the presence of CFSE-labeled activated T_CM CD8 T cells. h Bar-graph showing percent of cells expressing CD95 (n = 3) **P < 0.01 ****P < 0.0001. Unpaired nonparametric Mann-Whitney test was used. Data are presented as means ± SEM.