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. 2021 Feb 9;10(2):353. doi: 10.3390/cells10020353

Figure 3.

Figure 3

Activated human Treg and CD137-transduced HEK293 (HEK) cells use CD137 to downregulate CD137L on APC cells. (A) Treg isolated from human peripheral blood mononuclear cells (PBMC) from healthy donors were activated with 100 ng/mL Phorbol 12-myristate 13-acetate (PMA) and 1 µg/mL ionomycin for 24 h and washed and cocultured with Carboxyfluorescein-succinimidyl-ester (CFSE)-labelled THP-1 or Raji cells for 1 h at a ratio of 2 Treg: 1 APC, with the addition of 5 µg/mL of isotype antibody (clone MOPC-21) or anti-CD137 antibody (clone BBK-2). (B,C) THP-1, Raji, and Daudi cells after coculture with transduced HEK cells. HEK WT, HEK293 cells expressing green fluorescent protein (GFP)-tagged full-length (HEK-FL-CD137-GFP), or HEK293 cells expressing GFP-tagged truncated CD137 (HEK-TR-CD137-GFP) cells were cocultured with APC for 1 h at a ratio of 10 HEK: 1 APC. (B) Surface CD137L levels. (C) GFP fluorescence of THP-1, Raji, and Daudi cells after the coculture. (D,E) Fixed isolated THP-1 cells were cocultured with isolated CD8+ T cells from PBMC of healthy donors at a ratio of 1 THP-1 cell: 10 CD8+ T cells, for 72 h, in the presence of 0.5 µg/mL anti-CD3 antibody (clone OKT3). (D) IFN-γ level, relative. (E) Surface CD25 level, relative. Cells were analysed by flow cytometry for surface CD137L levels after gating for CFSE+ cells (A), live and single CD45+ cells (B,C), or live and single cells (D,E). Graphs show means ± SD, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, one-way ANOVA with Dunnett multiple comparison test. Data are representative for at least 3 different donors (A,D,E) or at least three independent experiments (B,C).