Method of identifying compounds capable of activating ISL production

A method for identifying a compound capable of activating Immunodeficiency-virus Suppressing Lymphokine (ISL) production in human blood lymphocytes, said method comprising:

• a) isolating PBMC from healthy donors by Ficoll-gradient separation, isolating CD8+ cells from the PBMC by magnetic cell sorting to obtain a purified CD8+ cell preparation, testing the purity of the preparation by FACS analysis, and selecting the preparation having a content of approximately 95% CD8+ cells and 5% non-CD8+ contaminants to ensure adequate stimulation of the CD8+ cells;
• b) culturing
• 1) the CD8+ cell preparation in a cell culture medium containing a substance to be tested for induction of expression of ISL activity at a concentration of 180 U/ml of cell culture medium, removing the cell culture medium after three days, and
• 2) thereafter culturing the CD8+ cell preparation in a cell culture medium containing IL-2 at a concentration of 180 U/ml of cell culture medium for three days followed by centrifugation of the cell culture medium at 1,000 rpm to obtain a supernatant and to remove the CD8+ cell preparation, and sterile filtering and aliquoting the supernatant;
• c) detecting ISL expression induced by said compound in a sample comprising the supernatant or the CD8+ cell preparation;
• d) further identifying ISL-activating activity of said compound in an HIV-inhibition assay, said assay comprising
• 1) incubating the supernatant of step b2) with CD8+-depleted PBMC in culture;
• 2) infecting the CD8+-depleted PBMC in culture with HIV-1;
• 3) thereafter culturing the CD8+-depleted PBMC;
• and
• 4) quantitating inhibition of HIV-1 replication;
• and
• e) using a combination of said ISL expression of step c) and said inhibition of HIV-1 replication of step d) to indicate that said compound is capable of activating ISL production in human blood lymphocytes.