LARGE-SCALE PRODUCTION OF CD4-CELLS FROM HIV-1-INFECTED DONORS AFTER CD3( T)CD28 COSTIMULATION/

We describe a procedure for large-scale enrichment, growth, and harves ting CD4+ T cells, This method may be effective for HIV-1 immunotherap y, as the mode of stimulation, with anti-CD3 plus anti-CD28 coated bea ds (CD3/CD28 beads) induces a potent antiviral effect. PBMC were obtai ned by density gradient centrifugation of an apheresis product. Monocy tes/macrophages were removed by incubating PBMC with beads coated with IgG, The cells were then magnetically depleted of B cells and CD8+ ce lls with mouse anti-CD20 and anti-CD8 MAbs and sheep antimouse coated beads, The remaining cells were >80% CD4+ and were transferred to gas- permeable bags containing CD3/CD28 beads and cultured in a closed syst em. After 14 days, the cell number increased an average of 37-fold, an d cells were nearly 100% CD4+, Viral load, assessed by DNA PCR for HIV -1 gag, decreased >10-fold during culture in the absence of antiretrov iral agents, Removal of CD3/CD28 beads from the cell suspension was ac complished by passing cells plus beads (3-30 x 10(9) cells in 2-12 L) over a MaxSep(R) magnetic separator using gravity-driven flow, The cel ls were then concentrated to 300 mi in an automated centrifuge, This p rocess allows safe and efficient growth of large numbers of CD4+ T cel ls from HIV-1+ donors.