The cDNA hits from the functional genetic screens inhibited activation in human primary T lymphocytes. (a) Human PBL were cultured with anti-CD3 and anti-CD28 for 3 days and then infected with the retroviral CRU5-GFP vector, whereby GFP was expressed from the constitutively active retroviral LTR promoter. Cells were stained with anti-CD3-APC, or with anti-CD4-PE and anti-CD8-APC antibodies and analyzed by FACS. The percentage of cells in each quadrant is shown. (b) Human primary T lymphocytes were infected with vector alone (CRU5-GFP and CRU5-IRES-GFP or CIG) or with the CIG vector expressing the Lck, PLCγ1, EDG1 and PAK2 hits. The infection rate was monitored by the percentage of GFP-positive cells (marked with M1). The geometric mean of GFP for cells in marker M1 was shown above the marker line. (c) Infected primary T lymphocytes were allowed to rest and then sorted to give rise to GFP-negative (open bars) and GFP-positive (filled bars) populations. Equal numbers of cells were cultured without stimulation, with anti-CD3 or anti-CD3 plus anti-CD28 antibodies, or with PMA plus ionomycin. Then, 40 h later the culture supernatants were harvested and assayed for IL-2 production by ELISA. Note the difference in the scales and the standard deviations with cells stimulated with anti-CD3 plus anti-CD28, or with PMA plus ionomycin (lower panels) compared to the upper panels.