Importance of culturing primary lymphocytes at physiological oxygen levels

doi:10.1073/pnas.0611732104

Importance of culturing primary lymphocytes at physiological oxygen levels

Departments of *Genetics and
^§Pathology, Stanford University School of Medicine, Stanford, CA 94305

Contributed by Leonard A. Herzenberg, January 24, 2007 (received for review December 21, 2006)

Abstract

Although studies with primary lymphocytes are almost always conducted in CO₂ incubators maintained at atmospheric oxygen levels (atmosO₂; 20%), the physiological oxygen levels (physO₂; 5%) that cells encounter in vivo are 2–4 times lower. We show here that culturing primary T cells at atmosO₂ significantly alters the intracellular redox state (decreases intracellular glutathione, increases oxidized intracellular glutathione), whereas culturing at physO₂ 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 atmosO₂ than at physO₂. 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 physO₂ than at atmosO₂; and (ii) the peak expression of CD69 is significantly delayed and more sustained at physO₂ that at atmosO₂. 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 physO₂ likely reflect the in vitro operation of the natural in vivo regulatory mechanisms. Thus, we suggest caution in culturing primary lymphocytes at atmosO₂ because the requisite adaptation to nonphysiological oxygen levels may seriously skew T cell responses, particularly after several days in culture.

Footnotes

^†To whom correspondence may be addressed at:
B007 Beckman Center, Stanford University School of Medicine, Stanford, CA 94305-5318.
E-mail: atkuri{at}stanford.edu
^‡To whom correspondence may be addressed. E-mail: lenherz{at}darwin.stanford.edu
Author contributions: K.R.A. and Leonore A. Herzenberg designed research; K.R.A., A.-K.N., and T.C. performed research; K.R.A., Leonard A. Herzenberg, and Leonore A. Herzenberg analyzed data; and K.R.A., Leonard A. Herzenberg, and Leonore A. Herzenberg wrote the paper.
The authors declare no conflict of interest.
Abbreviations:

atmosO2,

atmospheric oxygen level;

CFDA-SE,

5-(and 6)-carboxyfluorescein diacetate, succinimidyl ester;

DAF-FM,

4-amino-5-methyamino-2′,7′-difluorofluorescein diacetate;

iGSH,

intracellular glutathione;

iGSSG,

oxidized intracellular glutathione;

iNO,

intracellular NO;

iROS,

intracellular reactive oxygen species;

NAC,

N-acetylcysteine;

PBMC,

peripheral blood mononuclear cell;

physO2,

physiological oxygen levels.