Importance of culturing primary lymphocytes at physiological oxygen levels
Open accessAbstract
Although studies with primary lymphocytes are almost always conducted in CO 2 incubators maintained at atmospheric oxygen levels (atmosO 2 ; 20%), the physiological oxygen levels (physO 2 ; 5%) that cells encounter in vivo are 2–4 times lower. We show here that culturing primary T cells at atmosO 2 significantly alters the intracellular redox state (decreases intracellular glutathione, increases oxidized intracellular glutathione), whereas culturing at physO 2 maintains the intracellular redox environment (intracellular glutathione/oxidized intracellular glutathione) close to its in vivo status. Furthermore, we show that CD3/CD28-induced T cell proliferation (based on proliferation index and cell yield) is higher at atmosO 2 than at physO 2 . This apparently paradoxical finding, we suggest, may be explained by two additional findings with CD3/CD28-stimulated T cells: ( i ) the intracellular NO (iNO) levels are higher at physO 2 than at atmosO 2 ; and ( ii ) the peak expression of CD69 is significantly delayed and more sustained at physO 2 that at atmosO 2 . Because high levels of intracellular NO and sustained CD69 tend to down-regulate T cell responses in vivo , the lower proliferative T cell responses at physO 2 likely reflect the in vitro operation of the natural in vivo regulatory mechanisms. Thus, we suggest caution in culturing primary lymphocytes at atmosO 2 because the requisite adaptation to nonphysiological oxygen levels may seriously skew T cell responses, particularly after several days in culture.
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T cells activated at physiologic O2 tension (2-5% O2) have reduced proliferation and expansion following TCR stimulation.
“when T cells are activated in conditions of physiologic oxygen tension (2–5 % O2, ~2–5 kPa, or ~15–38 mm Hg), there are a number of changes in T cell dynamics. First, T cells activated at physiologic O2 tension have reduced proliferation and expansion following TCR stimulation.”
From Hypoxia and hypoxia-inducible factors as regulators of T cell development, differentiation, and function (2013) · cites this paper as [75]
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- Authors
- Atkuri, K. R., Herzenberg, L. A., Niemi, A.-K., Cowan, T., & Herzenberg, L. A.
- Affiliations
- Stanford Medicine (US)
- Venue
- Proceedings of the National Academy of Sciences
- Publisher
- National Academy of Sciences
- Published
- 2007-03-13
- Volume
- 104
- Issue
- 11
- Pages
- 4547-4552
- ISSN
- 0027-8424 (ISSN-L)
- 1091-6490
- PMC
- PMC1838638
- OpenAlex
- W1973393458
- Cited by
- 163
- References
- 38
- Citations by year
- 2026: 4 · 2025: 4 · 2024: 7 · 2023: 10 · 2022: 9 · 2021: 8 · 2020: 7 · 2019: 7 · 2018: 7 · 2017: 9 · 2016: 10 · 2015: 12 · 2014: 9 · 2013: 13 · 2012: 10
- FWCI
- 3.57
- Type
- journal article
- Language
- en
- Open access
- green
- www.ncbi.nlm.nih.gov
- Locations
- Proceedings of the National Academy of Sciences (publishedVersion)
- PubMed Central (publishedVersion)
- PubMed (publishedVersion)
- Funders
- National Institutes of Health
- School of Medicine, Stanford University
- National Institute of Allergy and Infectious Diseases (AI566223)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Redox biology and oxidative stress
- Life Sciences › Immunology and Microbiology › Immunology › T-cell and B-cell Immunology
- Health Sciences › Medicine › Physiology › Nitric Oxide and Endothelin Effects
- Keywords
- primary T cells, cellular redox state, glutathione, T cell proliferation, nitric oxide, CD69, anti-CD3/CD28 stimulation, T cell regulation
- MeSH terms
- CD69 Antigens, Antigens, Differentiation, T-Lymphocyte, Carbon Dioxide, Cell Differentiation, Cell Survival, Humans, Leukocytes, Mononuclear, Lymphocytes, Oxidation-Reduction, Oxygen, T-Lymphocytes, Antigens, CD, CD3 Complex, CD28 Antigens, Cell Culture Techniques, Lectins, C-Type, Cell Proliferation
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