Low oxygen tension and autologous plasma enhance T-cell proliferation and CD49d expression density in serum-free media

Haddad, Hadar; Papoutsakis, E. T.

doi:10.1080/146532401317248045

Cited by 10 publications

(12 citation statements)

References 41 publications

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“…Similarly, Carswell et al (13) and Haddad and Papoutsakis (14) found that PHA and CD3͞CD28 crosslinking in the presence of IL-2 (in the culture medium) induce higher proliferation at 5% than at 20% oxygen. The reasons underlying the conflict between our findings and the findings reported in these studies are unclear.…”

Section: Discussionmentioning

confidence: 99%

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: 99%

Culturing at atmospheric oxygen levels impacts lymphocyte function

Atkuri

Herzenberg

2005

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

104

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“…Direct measurements in murine lymphoid organs indicate a range of 0.5-4.5% pO 2 (Caldwell et al, 2001). We previously reported that activation and expansion of human T cells under 5% pO 2 increases expansion by 6 -13-fold compared to cells cultured at the traditional 20% pO 2 (Carswell et al, 2000;Haddad and Papoutsakis, 2001). Reduced pO 2 resulted in additional phenotypic changes, such as changes in CD49d and CD25 (a subunit of interleukin-2 receptor) expression, cytotoxicity, and cell metabolism (Carswell et al, 2000;Haddad and Papoutsakis, 2001).…”

Section: Introductionmentioning

confidence: 97%

“…We previously reported that activation and expansion of human T cells under 5% pO 2 increases expansion by 6 -13-fold compared to cells cultured at the traditional 20% pO 2 (Carswell et al, 2000;Haddad and Papoutsakis, 2001). Reduced pO 2 resulted in additional phenotypic changes, such as changes in CD49d and CD25 (a subunit of interleukin-2 receptor) expression, cytotoxicity, and cell metabolism (Carswell et al, 2000;Haddad and Papoutsakis, 2001). A broader understanding of the effects of pO 2 on the transcriptional program of the cells may offer new insights into the properties of ex vivo expanded T cells, and how such properties might be enhanced by improved expansion protocols.…”

Section: Introductionmentioning

confidence: 99%

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

Haddad

Windgassen

Ramsborg

et al. 2004

Biotech & Bioengineering

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Immunotherapy with ex vivo cultured T cells depends on a large supply of biologically active cells. Understanding the effects of culture parameters is essential for improving the proliferation and efficacy of the expanded cells. Low oxygen tension (5% pO(2)) was previously reported to improve T-cell expansion and alter cellular phenotypic characteristics compared to T cells cultured at 20% pO(2). Here we report the use of DNA-array based transcriptional analysis coupled with protein-level analysis to provide molecular insights into pO(2) and patient-variability effects on expanded primary human T cells. Analysis of seven blood samples showed that reduced pO(2) results in higher expression of genes important in lymphocyte biology, immune function, and cell-cycle progression. 20% pO(2) resulted in higher expression of genes involved in stress response, cell death, and cellular repair. Expression of granzyme A (gzmA) was found to be significantly regulated by oxygen tension with cells at 5% pO(2) having greater gzmA expression than at 20% pO(2). Protein-level analysis of gzmA was consistent with transcriptional analysis. Granzyme K (gzmK) was coexpressed with gzmA, whereas Granzyme B (gzmB) expression was found to precede the expression of both gzmA and gzmK in 15-day cultures. Temporal gene expression patterns for seven blood samples demonstrate that most genes are expressed by all patient samples in similar temporal patterns. However, several patient-specific gene clusters were identified, and one cluster was found to correlate well with cell proliferation and may potentially be used to predict patient-specific T-cell expansion.

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“…Thirty nylon arrays (Human Atlas 1.2, CLONTECH) containing 1,191 singly spotted genes, and 12 plastic arrays (Atlas Plastic Human 8K, CLONTECH), containing over 8,300 doubly spotted genes, were used to analyze samples of primary human T cells cultured at 5% or 20% oxygen tension (pO 2 ) in serum-free medium (13). Gene-expression analysis was performed pairwise by using samples of 20% vs. 5% pO 2 cultures initiated with the same donor cells.…”

Section: Methodsmentioning

confidence: 99%

A segmental nearest neighbor normalization and gene identification method gives superior results for DNA-array analysis

Yang

Haddad

Tomas

et al. 2003

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

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An intuitive normalization and gene identification method is proposed. After segmentation of the entire expression range into intensity intervals, the mean and standard deviation of the logarithm of expression ratios are calculated for each interval using the nearest neighbor genes. Genes with high differential expression are excluded from these calculations. For glass arrays, normalization is performed for each interval by using the mean of the logarithm of expression ratios in the interval. For nylon͞plastic membranes, the average of the means of the logarithm of ratios across the intervals of higher intensities is used for normalization.

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Low oxygen tension and autologous plasma enhance T-cell proliferation and CD49d expression density in serum-free media

Cited by 10 publications

References 41 publications

Culturing at atmospheric oxygen levels impacts lymphocyte function

Culturing at atmospheric oxygen levels impacts lymphocyte function

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

A segmental nearest neighbor normalization and gene identification method gives superior results for DNA-array analysis

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