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.

CART‐cells merge into the fast lane of cancer care

“…While lenti-and retroviral vectors are the methods of gene transfer in the represented studies, other methods such as electroporation or RNA-based methods can be employed [11,16]. Different cell culture manufacturing systems have been developed which may impact the final phenotypic composition of the CAR T-cell product [17][18][19]. As an example, our manufacturing system uses an anti-CD3/CD28 antibody-coated magnetic bead system which results in a final product capable of memory function and persistence [18].…”

“…Different cell culture manufacturing systems have been developed which may impact the final phenotypic composition of the CAR T-cell product [17][18][19]. As an example, our manufacturing system uses an anti-CD3/CD28 antibody-coated magnetic bead system which results in a final product capable of memory function and persistence [18]. Prior to CD19-CAR T-cell infusion, patients typically receive chemotherapy in an effort to induce further lymphodepletion and enhance CAR T-cell expansion and persistence in vivo [20].…”

Toward Synthetic Biology with Engineered T Cells: A Long Journey Just Begun

“…To culture T cells from patients with HIV infection, Levine developed beads with immobilized antibodies to the T cell receptor and CD28 to develop a system that for the first time provided robust growth of T cells from patients with HIV or cancer, and it also was scale independent: he could culture T cells in volumes that varied by a million-fold from 96-well microplates to liter-sized culture flasks (Levine et al, 1998). Bruce used this new culture system to conduct fundamental studies on the growth potential of human T cells (Weng et al, 1995(Weng et al, , 1996Levine et al, 1997;Palmer et al, 1997;Liu et al, 1999).…”

Molecular remission of CML after autotransplantation followed by adoptive transfer of costimulated autologous T cells

“…Magnetic beads with immobilized anti-CD3 (OKT3) and anti-CD28 (9.3) monoclonal antibodies were added at a 3:1 bead:CD3 þ cell ratio, and the cultures were maintained for up to 14 days prior to harvest and preparation for infusion, as described. 26,27 The cells were counted daily and fresh medium was added to maintain the cells at a density of 0.75-2 Â 10 6 /ml. IL-2 at 100 IU/ml was added, if necessary, to optimize the cell growth, but was not used on a routine basis.…”

“…After completion of cell culture, the magnetic beads were removed using a Baxter Fenwal Maxsep s magnetic cell separation device. 27 After removal of the beads, the cells were washed, concentrated and resuspended in 100-250 ml of Plasmalyte A containing 1% human serum albumin, using the Baxter Fenwal Harvester System.…”