5A8 cells, a J-Lat cell line responsive to TCR stimulation and CsA inhibition.

A, generation of 5A8 cells. B, LTR-GFP reporter activity of Jordan J-Lat 6.3 and 5A8 cells in response to different stimuli in the presence or absence of CsA. Cells were treated with DMSO (white, light gray, black) or 500 nM CsA (dark gray) and then left untreated (white) or stimulated with platebound anti-CD3 (10 µg/ml) and soluble anti-CD28 antibodies (2 µg/ml) (light and dark gray) or 10 ng/ml TNF-α (black) for 24 h, followed by FACS analysis to determine the percentage of GFP-positive cells (y-axis). Values are mean ± SEM from three experiments. Crosslinking of TCR induced latent HIV reporter activity in 5A8 cells that was partially inhibited by CsA. These responses were not observed in Jordan J-Lat 6.3 cells. C, TCR-crosslinking-dependent induction of IL-2 in Jurkat and 5A8 cells. Cells were treated with DMSO (left and middle columns) or 500 nM CsA (right column) and incubated with Brefeldin A alone or together with anti-CD3 and anti-CD28 antibodies prepared as above for 16 h. Cells were collected, stained with anti-human IL-2 antibody conjugated to allophycocyanin (APC), and analyzed by FACS. 5A8 cells produced more IL-2 than their parental Jurkat cells, probably because they were selected by their enriched expression of TCR complexes. CsA abolished IL-2 production but only partially inhibited LTR-driven GFP reporter activity.