Levine B L, Cotte J, Small C C, Carroll R G, Riley J L, Bernstein W B, Van Epps D E, Hardwick R A, June C H
Immune Cell Biology Program, Naval Medical Research Institute, Bethesda, MD 20889, USA.
J Hematother. 1998 Oct;7(5):437-48. doi: 10.1089/scd.1.1998.7.437.
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.
我们描述了一种用于大规模富集、培养和收获CD4+ T细胞的方法。这种方法可能对HIV-1免疫疗法有效，因为用抗CD3加抗CD28包被的珠子（CD3/CD28珠子）进行刺激的方式可诱导强大的抗病毒效应。通过对单采血液成分进行密度梯度离心获得外周血单核细胞（PBMC）。通过将PBMC与IgG包被的珠子孵育来去除单核细胞/巨噬细胞。然后用小鼠抗CD20和抗CD8单克隆抗体以及羊抗小鼠包被的珠子通过磁性去除细胞中的B细胞和CD8+细胞。剩余细胞中CD4+细胞比例>80%，并将其转移至含有CD3/CD28珠子的透气袋中，在封闭系统中培养。14天后，细胞数量平均增加37倍，且细胞几乎100%为CD4+。在无抗逆转录病毒药物的培养过程中，通过针对HIV-1 gag的DNA聚合酶链反应（PCR）评估的病毒载量下降了>10倍。通过重力驱动使细胞加珠子（2 - 12 L中含3 - 30×10⁹个细胞）通过MaxSep磁性分离器，从而从细胞悬液中去除CD3/CD28珠子。然后在自动离心机中将细胞浓缩至300 ml。该过程可使来自HIV-1阳性供体的大量CD4+ T细胞安全且高效地生长。
