FoxP3 mRNA Expression in Regulatory T Cells from Patients with Tuberculosis

Abstract

We read with great interest the article by Guyot-Revol and colleagues (1) assessing the role of T regulatory cells (Tregs) in tuberculosis (TB) pathogenesis. Guyot-Revol and coworkers found that in patients with TB, percentages of CD4 CD25 T cells and levels of FoxP3 mRNA expression in peripheral blood mononuclear cells were both significantly higher in comparison with controls. To better comprehend Tregs’ mechanism of action, the authors investigated whether the increased levels of FoxP3 mRNA that they had observed were effectively caused by increased gene expression, or were simply the result of increasedCD4 CD25high frequency.FoxP3mRNAinCD4 CD25 T cells was compared between patients with TB and control subjects. As no quantitative difference was observed, the authors suggest that the greater levels of FoxP3 mRNA are simply an effect of increases in Treg frequency rather than a result of an up-regulated gene expression. The transcription factor FoxP3 is the most specific molecular marker for Tregs available to date, and its correct evaluation is crucial (2–4). In the analysis of Treg dynamics, an assessment of what happens in the frequency and level of FoxP3 mRNA expression at the single cell level is mandatory. The recent literature demonstrates a strong correlation between levels of CD25 expression and the frequency of FoxP3-positive cells in healthy donors (4), and suggests that to correctly evaluate FoxP3 expression in Tregs, mRNA should be quantified in CD4 CD25 rather than in CD4 CD25 T cells (5). We are currently performing a prospective study to evaluate Tregs in patients with TB. Analysis of CD4 CD25 percentage on CD4 T cells at TB diagnosis showed similar levels of Tregs among patients with TB and controls (median percentage: 2.1% [IQR, 1.8–6.6] vs. 1.9% [IQR, 1.5–2.2]). Unlike Guyot-Revol and coworkers, we evaluated FoxP3 mRNA expression by real-time polymerase chain reaction in CD4 CD25 T cells, purified using an EPICSALTRACell Sorter (Beckman-Coulter; purity 93.6%). A considerable increase of FoxP3 mRNA expression in patients with TB with respect to control subjects was clearly observed (median: 39.1 [IQR, 5.1–109] vs. 1.6 [IQR, 0.8–2.8]; p 0.0062). These data implicate FoxP3 gene overexpression as a probable mechanism involving Tregs in the pathogenesis of TB, and suggest a possible role of FoxP3 gene up-regulation in the outcome of chronic infections. Our results underscore the importance of using the correct methodology in evaluating Treg cell dynamics, and prove that an accurate evaluation of FoxP3 mRNA expression in Treg cells may lead to the identification of previously unknown mechanisms of Treg cell involvement in disease pathogenesis.