Importance of culturing primary lymphocytes at physiological oxygen levels

Abstract

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.