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. 2023 Aug 11;13:13074. doi: 10.1038/s41598-023-40346-8

Figure 4.

Figure 4

Effect of CCL8 on immune cells and MSCs. (A) T cells, B cells, and NK cells (1 × 105 cells/well) were added to the upper wells of transwell plates. CCL8 was added to the lower wells at 30 or 100 ng/ml. The number of cells that migrated to the lower wells over 2 h was counted using a flow cytometer. (B) MSCs (2 × 104 cells/well) were added to the upper wells of transwell plates. CCL8 was added to the lower wells at 30 or 100 ng/ml. The number of migrating MSCs was counted as described in the “Materials and methods”. (C) T cells isolated from the spleen of MRL.Faslpr mice were activated with concanavalin A (Con A, 1 μg/ml) in the presence or absence of recombinant CCL8 for 72 h. IFN-γ level in the medium was determined by ELISA. (D) MSCs were pretreated with recombinant CCL8 at 30–300 ng/ml for 24 h. T cells (1 × 105 cells/well) were co-cultured with MSCs (1 × 104 cells/well) in the presence of anti-CD3 and anti-CD28 antibodies (1 μg/ml each) for 72 h. IFN-γ level in the medium was determined by ELISA. (E) MSCs were treated with recombinant CCL8 (300 ng/ml). The levels of mRNAs of the indicated soluble factors were determined by RT-qPCR. (F) MSCs were pretreated with recombinant CCL8 (300 ng/ml; CCL8-MSCs). CCL8-MSCs were transfected with negative-control (neg), IL-10, IDO, TGF-β1, or iNOS siRNA. T cells (1 × 105 cells/well) were co-cultured with MSCs (0.1 × 105 cells/well) for 72 h. Anti-CD3 and anti-CD28 antibodies (1 μg/ml each) were added to activate T cells. IFN-γ level in the medium was determined by ELISA. RQ, Relative quantitation. UN, Untreated. n = 3 in all panels. *p < 0.01.