title: Large-scale production of CD4+ T cells from HIV-1-infected donors after CD3/CD28 costimulation.
abstract: We describe a procedure for large-scale enrichment, growth, and harvesting CD4+ T cells. This method may be effective for HIV-1 immunotherapy, as the mode of stimulation, with anti-CD3 plus anti-CD28 coated beads (CD3/CD28 beads) induces a potent antiviral effect. PBMC were obtained by density gradient centrifugation of an apheresis product. Monocytes/macrophages were removed by incubating PBMC with beads coated with IgG. The cells were then magnetically depleted of B cells and CD8+ cells 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 system. After 14 days, the cell number increased an average of 37-fold, and cells were nearly 100% CD4+. Viral load, assessed by DNA PCR for HIV-1 gag, decreased >10-fold during culture in the absence of antiretroviral agents. Removal of CD3/CD28 beads from the cell suspension was accomplished by passing cells plus beads (3-30 x 10(9) cells in 2-12 L) over a MaxSep magnetic separator using gravity-driven flow. The cells were then concentrated to 300 ml in an automated centrifuge. This process allows safe and efficient growth of large numbers of CD4+ T cells from HIV-1+ donors.
authors.affiliation: Immune Cell Biology Program, Naval Medical Research Institute, Bethesda, MD 20889, USA.
authors.forename: B L
authors.initials: BL
authors.lastname: Levine
authors.forename: J
authors.initials: J
authors.lastname: Cotte
authors.forename: C C
authors.initials: CC
authors.lastname: Small
authors.forename: R G
authors.initials: RG
authors.lastname: Carroll
authors.forename: J L
authors.initials: JL
authors.lastname: Riley
authors.forename: W B
authors.initials: WB
authors.lastname: Bernstein
authors.forename: D E
authors.initials: DE
authors.lastname: Van Epps
authors.forename: R A
authors.initials: RA
authors.lastname: Hardwick
authors.forename: C H
authors.initials: CH
authors.lastname: June
mesh_terms: D019264:Adoptive Transfer / Q000379:methods*; D000818:Animals; D018106:CD28 Antigens / Q000276:immunology; D017252:CD3 Complex / Q000276:immunology; D015496:CD4-Positive T-Lymphocytes / Q000276:immunology / Q000473:pathology*; D002478:Cells, Cultured; D002499:Centrifugation, Density Gradient; D015658:HIV Infections / Q000276:immunology / Q000628:therapy*; D006801:Humans; D007163:Immunosorbent Techniques; D007937:Leukapheresis / Q000379:methods*; D051379:Mice
chemical_list: D018106:CD28 Antigens; D017252:CD3 Complex