FIG. 6.

Differential suppressive activity of fractions after HiTrap heparan sulfate chromatography of supernatant of 4-h anti-CD3-stimulated HIV-1-specific CTL clone 15160-D75. A 5-ml heparin column (Pharmacia) was loaded with 10 ml of supernatant and washed with 20 ml of PBS. The heparin-unbound fraction was filtered and concentrated to 100 μl sequentially through Centricon membranes with exclusion sizes of 100, 50, 8, and 3 kDa. The heparin-bound fraction was then obtained by washing the heparin column with 20 ml of 2 M NaCl in PBS, followed by the above-described size exclusion centrifugation steps. The supernatants on top of the Centricon membranes were concentrated to 100 μl, washed twice with a 200-fold times volume of PBS, and tested for activity. The controls include the buffer conditions.