Kinetics of Cytokine Gene Expression in Human CD4+ and CD8+ T‐Lymphocyte Subsets Using Quantitative Real‐Time PCR

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Abstract
The time kinetics of five cytokines [interleukin‐2 (IL‐2), IL‐5, interferon‐γ (IFN‐γ), granulocyte macrophage‐colony stimulating factor (GM‐CSF) and tumour necrosis factor‐α (TNF‐α)] and one cytotoxic effector protein (granzyme B) was analysed by real‐time quantitative polymerase chain reaction (PCR) following in vitro stimulation of human CD4 and CD8 T lymphocytes. Two stimuli were used, a mitogen [phytohemagglutinin (PHA)] and a recall antigen [purified protein derivative (PPD)]. The pattern of cytokine mRNA expression was found to be dependent on the T‐cell subset and stimulus used. A wide interindividual variability in the cytokine gene expression pattern was demonstrated. Two expression patterns were observed. A bell‐shaped expression profile was seen for most cytokines upon PHA activation in both subsets and PPD‐activated CD4 T cells, whereas a biphasic/multiphasic expression pattern was noted in CD8 T cells upon PPD stimulation. For most cytokines, the time to induction was within 30 min of activation, and maximum accumulation seemed to be obtained after 4–8 h of activation. A sustained high level could, however, be noticed for up to 24 h. Granzyme B gene expression was also induced within 30 min of activation but showed a continuous gradual increase and late maximal accumulation (48–72 h). The findings of the present study are of importance when designing studies using the cytokine gene expression profile as a marker for antigen‐specific T lymphocytes. It might be recommended that cytokine gene expression (IL‐2, IL‐5 and IFN‐γ) should be measured after 4–8 h of specific activation but also up to 24 h of stimulation is acceptable. Granzyme B should preferentially be measured after 48–72 h of activation.