Jun Wang, Hans Ulrich Scherer and Rene E. M. Toes
Department of Rheumatology, Leiden University Medical Center, Leiden, The Netherlands.

To The Editor

We read with interest the article “Induction of FOXP3 expression in naïve human CD4⁺FOXP3^- T cells by T cell receptor stimulation is TGFβ-dependent but does not confer a regulatory phenotype” by Tran et al (1). The authors analyzed FOXP3 expression in TCR-stimulated human naïve CD4⁺CD25^-CD127⁺CD45RA⁺ T cells in the presence/absence of TGF-β by two different mAbs recognizing different epitopes of the protein FOXP3. Although the final conclusions reached by the authors confirm previous studies describing that the expression of FOXP3 is not confined to CD4⁺CD25⁺ Treg cells in humans, the authors’ data suggest that the anti-FOXP3 mAb PCH101 (eBioscience) is non-specific for human FOXP3. As PCH101 has been and still is widely used to visualize FOXP3 expression in the human system at the cellular level, it implies that the conclusions on FOXP3 expression and FOXP3-expressing cells presented by numerous studies should be interpreted with caution. For this reason, we feel it is important to address the issue concerning the specificity of PCH101 in more detail.

To compare the specificity between PCH101 and another anti-FOXP3 mAb 236A/E7 used by the authors (1), we isolated CD4⁺CD25^- by FACS-sorting. Similar to Tran et al, these cells were subsequently stimulated by anti-CD3/28 and IL-2 in the presence or absence of TGF-β. After 5 days, cells were stained for FOXP3 expression using the fixation/permeabilization kit of eBioscience. In line with previous findings (2, 3), equivalent results were obtained as cells positive for 236A/E7 are positive for PCH101 and vice versa. These data indicate that the results obtained by PCH101 staining do not differ from those obtained by using other FOXP3-specific antibodies. Because the discrepancy between our results and those presented by Tran et al could be a consequence of as yet unknown technicalities (4), it is advised to use more than one anti-FOXP3 antibody in further studies. This will allow the most accurate visualization of FOXP3 expression under various conditions and will control for a possible nonspecific activity of PCH101 as well as exclude a possible underestimation of the number of FOXP3-expressing cells due to a low efficiency of other anti-FOXP3 antibodies.

Figures

Figure 1. Comparison between anti-FOXP3 antibodies PCH101 and 236A/E7.
FACS-sorted CD4⁺CD25^- T cells were activated with plate-bound anti-CD3 (5 μg/ml), soluble anti-CD28 (1 μg/ml) and IL-2 (100 U/ml) in the presence (lower panel) or absence (upper panel) of TGF-β (1 ng/ml). After 5 days of activation, cells were intracellular co-stained with the FITC-labeled anti-FOXP3 antibody PCH101 and the APC-conjugated anti-FOXP3 Antibody 236A/E7. (A) Cells positive for 236A/E7 are also positive for PCH101. Similar results were obtained when PCH101 positive cells were first gated (data not shown). (B) Correlation between PCH101 and 236A/E7 staining. Cells detected by staining positive for either anti-FOXP3-antibody are also stained positive for the other FOXP3-antibody, indicating FOXP3-positive cells are detected to a similar extent by both antibodies. The quadrant was based on the isotype control antibody. Similar results were obtained with CD4⁺CD25⁺⁺ T cells (data not shown).

References

1. Tran DQ, Ramsey H, Shevach EM. Induction of FOXP3 expression in naive human CD4⁺FOXP3^- T cells by T cell receptor stimulation is TGFβ-dependent but does not confer a regulatory phenotype. Blood. July 20, 2007; DOI 10.1182/blood-2007-06-094656.
   
   
2. Wang J, Ioan-Facsinay A, van der Voort EI, et al. Transient expression of FOXP3 in human activated nonregulatory CD4⁺ T cells. Eur J Immunol. 2007; 37: 129-138.
   
   
3. Allan SE, Crome SQ, Crellin NK, et al. Activation-induced FOXP3 in human T effector cells does not suppress proliferation or cytokine production. Int Immunol. 2007; 19: 345-354.
   
   
4. Dejaco C, Duftner C, Schirmer M. Analysis of FOXP3 protein expression in human CD4⁺CD25⁺ regulatory T cells at the single-cell level. Eur J Immunol. 2006; 36: 245-246.

Disclosures

The authors have no financial conflicts of interest.

Footnotes

Correspondence: Dr. Rene E. M. Toes
Department of Rheumatology C1-R, Leiden University Medical Center, Albinusdreef 2, 2300 RC, Leiden, The Netherlands.
Telephone: +31-71-5261946; Fax: +31-71-5266752
Email: R.E.M.Toes@lumc.nl

Authorship: J.W. performed research, analyzed data, and wrote the paper; H.U.S performed research, analyzed data, and contributed to the writing of the paper; R.E.M.T. analyzed data and wrote the paper.