Efficient retroviral vector - mediated gene transfer into primary human chronic lymphocytic leukemia (CLL) cells after short term in vitro culture

Michael J. Robertson, Larry Cripe, Kristen Ganjoo, Lynette Timmons, Kenneth Cornelia

Research output: Contribution to journalArticlepeer-review


CLL is incurable by conventional therapy and more effective treatments are required. One novel therapeutic approach is vaccination of patients with their autologous CLL cells to stimulate antitumor immune responses in vivo. Nevertheless, primary CLL cells are known to be poorly immunogenic. Retrovirus-mediated gene transfer has been used to introduce immunostimulatory molecules into primary tumor cells to enhance their immunogenicity. We tested several stimuli for their ability to induce in vitro proliferation of CLL cells and susceptibility to retrovirus-mediated gene transfer. Mononuclear cells were isolated from the peripheral blood of patients with typical CD 19+/CD5+ CLL and plated in 5 day tritiated thymidine incorporation assays. Of 24 different stimulatory conditions tested, the combination of Staph. aureus Cowan (SAC) and IL-2 most consistently induced proliferation of CLL cells (not shown). Either S AC ( 10 ng/ml) or IL-2 ( 1000 pM) alone also stimulated proliferation, but the combination of SAC plus IL-2 was better than SAC alone (P<0.0005) or IL-2 alone (P<0.01). CLL cells stimulated with SAC plus IL-2 were transduced in the presence of CH-296 fibronectin using the MFG-eGFP retroviral vector (Bierhuizen et al. Blood 1997; 90: 3304) encoding the green fluorescence protein (GFP). Expression of OFF was analyzed by flow cytometry 48 hours after transduction by gating on viable CD19+/ CD5+ cells. A time course experiment using 3 CLL samples showed that transduction efficiency was higher (up to 8%) on day 7 than on days 5 or 1 of culture. CLL cells from 4 separate samples were transduced for 4 hours on days 7 and 8 of culture in SAC plus IL-2. GFP was expressed by 4 ±0.2% (mean ±SE) of all viable CD19+/CD5+ CLL cells after transduction. A subpopulation ( 19 ±3%) of cultured CLL cells appeared to be more activated based on high forward and side scattering properties. Transduction efficiency was better for these large cells: a median of 16% (range, 14 to 31%) expressed GFP. These results indicate that primary human CLL cells can be induced to proliferate in vitro and can subsequently be successfully transduced using retroviral vectors. Modifications of culture conditions to yield a greater proportion of activated CLL cells may lead to higher gene transfer efficiency.

Original languageEnglish (US)
Pages (from-to)282b
Issue number11 PART II
StatePublished - Dec 1 2000

ASJC Scopus subject areas

  • Biochemistry
  • Immunology
  • Hematology
  • Cell Biology

Fingerprint Dive into the research topics of 'Efficient retroviral vector - mediated gene transfer into primary human chronic lymphocytic leukemia (CLL) cells after short term in vitro culture'. Together they form a unique fingerprint.

Cite this