Molecular understanding of oxygen‐tension and patient‐variability effects on ex vivo expanded T cells

Haddad, Hadar; Windgassen, Dirk; Ramsborg, Christopher G.; Paredes, Carlos J.; Papoutsakis, Eleftherios T.

doi:10.1002/bit.20166

Cited by 18 publications

(13 citation statements)

References 60 publications

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“…Consistent with this argument, a recent report by Haddad et al (15) showed that the expression of genes important in lymphocyte function (e.g., granzyme A) is increased in cells cultured at 5% oxygen, whereas the expression of genes involved in stress response, cell death, and cellular repair was elevated at 20% oxygen. Although these studies were conducted with PBMCs from hemachromatosis subjects, the findings point to differences in gene expression that are likely to occur in cultures of cells from healthy donors and are consistent with the differential responsiveness of lymphocytes that we observed at different oxygen levels.…”

Section: Discussionmentioning

confidence: 55%

Culturing at atmospheric oxygen levels impacts lymphocyte function

Atkuri

Herzenberg

2005

Proc. Natl. Acad. Sci. U.S.A.

104

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To determine whether culturing peripheral blood mononuclear cells at atmospheric oxygen levels skews responses in comparison with culturing lymphocytes at physiologic oxygen levels, we cultured peripheral blood mononuclear cells at 5%, 10%, and atmospheric (20%) gas-phase oxygen for 5 days. We found that incubator oxygen levels influenced lymphocyte proliferation stimulated by two commonly used stimuli: Con A and antibodies that crosslink surface CD3 and CD28 to mimic antigen presentation. In both cases, proliferation increased as gas-phase oxygen levels increased. In contrast, oxygen levels did not influence proliferation stimulated by phytohemagglutinin, another commonly used mitogen. Similarly, oxygen levels did not impact cell viability in unstimulated cultures. Thus, we conclude that the influence of oxygen levels on proliferation depends on the stimulus, and, most importantly from the standpoint of immune responses, culturing cells at atmospheric rather than physiologic oxygen levels results in significantly increased proliferation responses to the CD3͞CD28 crosslinking, a proliferation stimulus commonly used to mimic T cell antigen receptor signaling.CD3͞CD28 ͉ incubator ͉ T cell ͉ tissue culture P roliferation of lymphocytes in response to antigen or mitogen stimuli is a common measure of ex vivo lymphocyte function. Virtually all of our ideas about the autoimmune and protective mechanisms in which human lymphocytes participate are derived from studies based on this methodology. However, the CO 2 incubators in which nearly all of these studies have been conducted largely contain air to which a small percentage of CO 2 has been added. Thus, it is perhaps surprising that nearly all of what has been concluded to date about human lymphocyte function is based on evidence from cell-culture studies conducted at atmospheric oxygen levels (20% oxygen) that are well above the levels of oxygen to which cells are exposed in the human body.Determining the oxygen levels available to cells in the body is a difficult task. Blood has been shown to have a partial pressure of oxygen (pO 2 ) of 80-100 mmHg (1 mmHg ϭ 133 Pa) (1), which is equal to 10-12.5% O 2 . pO 2 levels in healthy tissue are considered to be in the range of 30-50 mmHg (2), which is equal to 3-6% O 2 . Thus, we estimate that standard culture conditions (20% oxygen) expose cells to Ϸ2-to 5-fold higher concentrations of oxygen than they would likely encounter in vivo.The disparity between the in vivo pO 2 and the pO 2 in incubators maintained at atmospheric oxygen levels clearly raises the question of whether current culture conditions are appropriate for the study of lymphocyte and other cell functions. In fact, several studies have already examined the effect of incubator oxygen levels on lymphocyte functions such as proliferation (3), cytolytic activity (4), cytokine production (4, 5), and antibody secretion (6). However, because the methods used and the findings obtained in these studies vary considerably, no clear picture has yet emerged as to whether ...

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Section: Discussionmentioning

confidence: 55%

Culturing at atmospheric oxygen levels impacts lymphocyte function

Atkuri

Herzenberg

2005

Proc. Natl. Acad. Sci. U.S.A.

104

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“…The mitogen induced PBMCs show increased intracellular nitric oxide levels and reduced intracellular glutathione levels at 20% oxygen [17, 18]. Haddad et al, showed the increase in expression of genes involved in cell death, cellular repair, and stress response in primary T-cells maintained at atmospheric O 2 levels indicating increased oxidative stress response [48]. Cultivation of rat liver sinusoidal endothelial cells (LSECs) at 5% O 2 , as opposed to 21% O 2 improved the survival of LSECs and scavenger receptor-mediated endocytic activity, reduced the production of the pro-inflammatory mediator, interleukin-6 and increased the production of the anti-inflammatory cytokine, interleukin-10 [49].…”

Section: Activation Of Redox Sensitive Inflammatory Signalingmentioning

confidence: 99%

Oxidative Stress Under Ambient and Physiological Oxygen Tension in Tissue Culture

2016

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Oxygen (O2) levels range from 2–9% in vivo. However, cell culture experiments are performed at atmospheric O2 levels (21%). Oxidative stress due to generation of reactive oxygen species (ROS) in cells cultured at higher than physiological levels is implicated in multitude of deleterious effects including DNA damage, genomic instability and senescence. In addition, oxidative stress activates redox sensitive transcription factors related to inflammatory signaling and apoptotic signaling. Furthermore, several chromatin-modifying enzymes are affected by ROS, potentially impacting epigenetic regulation of gene expression. While primary cells are cultured at lower O2 levels due to their inability to grow at higher O2, the immortalized cells, which display no such apparent growth difficulties, are typically cultured at 21% O2. This review will provide an overview of issues associated with increased oxygen levels in in vitro cell culture and point out the benefits of using lower levels of oxygen tension even for immortalized cells.

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“…Several studies indicate that incubator oxygen levels can modulate the metabolism (2, 4), gene expression (5,6), and function of primary cells (3,(7)(8)(9). However, the significance of these findings is obscured because the current notation for describing incubator oxygen levels is very confusing: atmospheric oxygen levels are typically referred to as ''normoxic,'' even though these levels are substantially above the levels animal cells normally encounter in vivo.…”

mentioning

confidence: 97%

Importance of culturing primary lymphocytes at physiological oxygen levels

Atkuri

Herzenberg²,

Niemi³

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

151

132

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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 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.glutathione ͉ low oxygen ͉ nitric oxide ͉ proliferation

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Molecular understanding of oxygen‐tension and patient‐variability effects on ex vivo expanded T cells

Cited by 18 publications

References 60 publications

Culturing at atmospheric oxygen levels impacts lymphocyte function

Culturing at atmospheric oxygen levels impacts lymphocyte function

Oxidative Stress Under Ambient and Physiological Oxygen Tension in Tissue Culture

Importance of culturing primary lymphocytes at physiological oxygen levels

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