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. 2023 Aug 11;2(1):kyad012. doi: 10.1093/discim/kyad012

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© The Author(s) 2023. Published by Oxford University Press on behalf of the British Society for Immunology.

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.

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Figure 3: — Assessing the proliferative capacity of CD4⁺ T cells in vitro. (A) Representative histograms depicting the proliferative capacity of CD4⁺ Tconv and Treg cells after 2.5 or 3.5 days of in vitro culture in the presence of 100 IU/ml IL-2 and TCR stimulation with αCD3/CD28 beads (1 bead: 3 T cells). The respective cell generation (G_n) are shown in each histogram. G₀ represents undivided CD4⁺ T cells. Data were generated from freshly isolated PBMCs from 14 healthy donors (n = 10 and n = 4 for day 2.5 and day 3.5, respectively). (B) Histograms depict the frequency (mean ± SEM) of CD4⁺ Tconv (red) and Treg (blue) cells at each generation of cell division after 2.5 (B) and 3.5 (C) days of stimulation with αCD3/CD28 beads and 100 IU/ml IL-2, followed by an additional 4 h of co-culture in the presence or absence of the respective CD56⁺ NK cell subset. The reported frequencies of proliferating CD4⁺ Tconvs and Tregs for each donor at each generation represent the average of the three co-culture conditions assessed in this study: (i) co-culture with CD56^br NK cells; (ii) co-culture with CD56^dim NK cells; and (iii) cultured alone in the absence of NK cells (background control to assess spontaneous cell death). The T cell proliferation rates were not affected by the different co-culture conditions, and were therefore averaged to increase the number of T cells assessed. CD4⁺ Tconvs (depicted by triangles) and Tregs (depicted by inverted triangles) isolated from the same donor are depicted using the same colour. (D) Scatter plots depict the distribution (mean ± SEM) of the observed DI for CD4⁺ Tconv and Treg cells after co-culture at 2.5 (left panel) or 3.5 (right panel) days of stimulation. (E) Same as for D, but depicting the PI of the cells. P values were calculated using two-tailed paired t-tests. DI denotes the average number of cell divisions undergone by a cell in the original population and PI denotes the average number of divisions among the cells that have divided at least once. n.s., P value not significant.