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Updated: Aug 10, 2026
Isolation and Activation of Murine Lymphocytes
Published on: October 30, 2016
Uptake of oligodeoxyribonucleotides by lymphoid cells is heterogeneous and inducible
A M Krieg1, F Gmelig-Meyling, M F Gourley
1Cellular Immunology Section, National Institute of Arthritis and Musculoskeletal and Skin Diseases, National Institutes of Health, Bethesda, MD 20892.
Insights
Oligonucleotide uptake in immune cells is variable and can be increased by cell culture and mitogens. Most taken-up oligonucleotides are found inside cells, not bound to the surface.
Area of Science:
- Immunology
- Molecular Biology
- Cell Biology
Background:
- Oligonucleotides are increasingly used in research and therapy.
- Understanding their cellular uptake is crucial for effective application.
- Previous studies show variable uptake, but mechanisms in lymphoid cells are not fully elucidated.
Purpose of the Study:
- To investigate oligonucleotide uptake in cultured murine lymphoid cells.
- To identify factors influencing uptake, including cell type and culture conditions.
- To determine the intracellular versus cell surface localization of oligonucleotides.
Main Methods:
- Use of radiolabeled and fluorescein-5-isothiocyanate (FITC)-labeled oligonucleotides.
- Analysis of lymphoid subpopulations (B cells, T cells [CD4+, CD8+, CD4-CD8-]) via flow cytometry.
- Assessment of uptake after varying culture periods and stimulation with mitogens.
- Acid glycine buffer wash to differentiate surface-bound from intracellular oligonucleotides.
Main Results:
- Oligonucleotide uptake is heterogeneous among fresh lymphoid cells (approx. 5%).
- Uptake significantly increases in B cells with prolonged culture (up to 10-fold).
- T-cell uptake is less efficient than B-cell uptake, with CD4-CD8- T cells showing higher percentages.
- Mitogen stimulation markedly enhances oligonucleotide uptake in respective T and B cells.
- The majority of oligonucleotides are located intracellularly, not on the cell surface.
Conclusions:
- Oligonucleotide uptake in cultured lymphoid cells is highly variable and inducible.
- B cells show greater inducibility of uptake compared to T cells.
- These findings are critical for designing in vitro experiments and planning in vivo antisense oligonucleotide therapies.
Abstract:
Oligonucleotide uptake was studied in cultured murine spleen and lymph node cells using internally radiolabeled and fluorescein-5-isothiocyanate (FITC)-labeled oligonucleotides. Lymphoid subpopulations were distinguished by flow cytometry and staining with antibodies to cell-surface molecules. Approximately 5% of fresh lymphoid cells take up substantial amounts of oligonucleotide. The percentage of B cells that take up oligonucleotide increased fivefold if cells were cultured for at least 24 hr prior to incubation with labeled oligonucleotides, and increased 10-fold if cells were precultured for 48 hr. T-cell uptake changed very little in culture. Cultured CD4+ and CD8+ T cells had similar oligonucleotide uptake that was less than one-third of that in cultured B cells, but CD4-CD8- T cells had a higher percentage of cells taking up oligonucleotide than did B cells. T- or B-cell mitogens caused markedly increased oligonucleotide uptake in T or B cells, respectively. Oligonucleotide uptake could be inhibited only partially with competitor DNA. To distinguish between cell membrane-bound and intracellular oligonucleotide, cells were washed in acid glycine buffer (which removes most surface oligonucleotide). This demonstrated that most of the oligonucleotide was intracellular. We conclude that oligonucleotide uptake is quite heterogeneous among cultured cells, and that this uptake is inducible by mitogens. These data may be important for the design and interpretation of in vitro experiments, and for the planning of in vivo therapy with antisense oligonucleotides.
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