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. 2023 Oct 9;25(10):674–687. doi: 10.22074/CELLJ.2023.2001712.1304

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Fig.3 — Expansion of T and NK cells and evaluation of markers of activation, exhaustion, and immunophenotype of T cells in aAPC activation method. A. Schematic illustration of the experiment for T cell activation with aAPC cells, anti-CD3/CD28 antibodies, and K562 cell line and cell expansion and staining. B. Fold expansion of activated T cells with aAPC cells, anti-CD3/CD28 antibodies (Ab), and K562 cell line as a negative control. Cell expansion was assessed from day 3 to day 7, where a 22-fold expansion was observed on day 4. C. Cells were stained with anti-CD56 and anti-CD3 antibodies to determine T cell and NK cell populations. The aAPC cells expanded T cells (CD3⁺ CD56⁻) similarly to the Ab-activated method and in the two groups with no significant variation in the expansion of the NKT population (CD3⁺ CD56⁺). K562 cell line was not suitable for T cell expansion, but it was able to expand NK cells (CD3⁻ CD56⁺). D. Analysis of T cells to determine the CD4/CD8 ratio showed that CD8⁺ T cells were expanded at a higher level in the aAPC-activated method and CD4⁺ T cells in the Ab-activated method. E. To assess CD25 and CD69 activation markers, cells were stained and measured by flow cytometry. The data showed no difference between the expression of CD25⁺ CD69⁺ dual activation markers in aAPC- and Ab-activated methods, but the expression of CD25⁺ CD69⁻ marker increased in Ab-activated method. F. Representative flow cytometry and quantification data for memory phenotype analysis of T cells on day 8. The aAPC activated method increased the differentiation of T cells into TEM cells, and the Ab-activated method increased the differentiation of T cells into TNL cells. G. The expression of PD-1 and TIM-3 exhaustion markers in different methods of activation measured by flow cytometry showed that T cell exhaustion markers were not significant in either groups (n=3). TNL; Naïve-like T cell, TCM; Central memory T cell, TEM; Effector memory T cell, TEMRA; CD45RA⁺ effector memory T cell, ns; Not significant, *; P<0.05, ***; P<0.001, ****; P<0.0001, NK; Natural killer, NKT; Natural killer T, and aAPC; Artificial antigen presenting cell.