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Response to Comment on "Cutting Edge: Human CD4^–CD8^– Thymocytes Express FOXP3 in the Absence of a TCR"

Heli Tuovinen^*, Eliisa Kekäläinen^*, Laura H. Rossi^*, Juha Puntila and T. Petteri Arstila^*

^* Haartman Institute, Department of Immunology, University of Helsinki, Helsinki, Finland Department of Surgery, Hospital for Children and Adolescents, Helsinki University Hospital, Helsinki, Finland

We thank Battaglia and colleagues (1) for their interest in our work (2). However, contrary to their claim, CD3 is not a fully reliable surrogate for β TCR on thymocytes, and surface CD3 is not always associated with a TCR. Most notably, early thymocytes express low levels of surface CD3 together with pre-TCR before TCR locus rearrangement (3). We did not detect pre-TCR on FOXP3⁺ DN thymocytes, but the intensity of staining with the noncommercial anti-preT mAb was low and establishing the status of pre-TCR expression was not the focus of our study.

As regards the anti-β TCR mAb, we are not sure what Battaglia et al. mean by "scarcely efficient reagent" (1). We are not aware of any particular problems with the widely used WT31 clone, and we did not find it unduly difficult to assign both FOXP3⁺ and FOXP3^– thymocytes to TCR^–, TCR^dim, and TCR^high subsets (Fig. 1 in Ref.2). Perhaps Battaglia et al. refer to the known propensity of WT31 to cross-react with an epitope formed by TCR and CD3 (4), but we fail to see how in the present context this could be a source of error. Arguing against the functional expression of an β TCR is also the fact that the FOXP3⁺ DN thymocytes did not express CD5 or CD69, molecules up-regulated in response to TCR signaling (2).

Apart from the divergence of using anti-CD3 instead of anti-TCR mAb, Battaglia et al. (1) found a much higher frequency of FOXP3⁺ cells within the DN population than we did (2), their mean value (6.1%) lying altogether outside our range of observations (0.1–3.8%). This discrepancy also suggests either patient-related differences or different methodology. We note that Battaglia and colleagues (5) have used the PCH101 anti-FOXP3 mAb, recently suggested to bind nonspecifically to activated T cells (6). Thymocytes at different developmental stages are heterogeneous in terms of metabolic activity and cell cycle status, and it is not known whether some of them display similar nonspecific binding. This raises the possibility that some fraction of the FOXP3⁺ DN cells observed by Battaglia et al. might in fact not express FOXP3.

Finally, we wonder why Battaglia et al. conclude that FOXP3 expression is restricted to CD3^dim cells (legend to Fig. 1 in Ref. 1), when the representative figure itself seems to indicate that some of the FOXP3⁺ DN thymocytes are indeed CD3^– (1). A plausible synthesis of the two data sets (1, 2) might thus be that the FOXP3⁺ DN thymocytes can be divided into CD3^–TCR^–, CD3^dimpre-TCR⁺, and CD3^dimTCR^dim populations. These stages would also form a logical and theoretically consistent progression toward the FOXP3⁺TCR^high thymocytes found in the CD4⁺CD8⁺ population. Our conclusion remains unchanged: the up-regulation of FOXP3 in human thymocytes precedes TCR expression.

References

1. Battaglia, A., A. Evoli, G. Scambia, A. Fattorossi. 2008. Comment on "Cutting Edge: human CD4-CD8- thymocytes express FOXP3 in the absence of a TCR. J. Immunol. 181: 857-858. [Free Full Text]
2. Tuovinen, H., E. Kekäläinen, L. H. Rossi, J. Puntila, T. P. Arstila. 2008. Cutting Edge: human CD4-CD8- thymocytes express FOXP3 in the absence of a TCR. J. Immunol. 180: 3651-3654. [Abstract/Free Full Text]
3. Trigueros, C., A. R. Ramiro, Y. R. Carrasco, V. G. de Yebenes, J. P. Albar, M. L. Toribio. 1998. Identification of a late stage of small noncycling pT- pre-T cells as immediate precursors of T cell receptor /β⁺ thymocytes. J. Exp. Med. 188: 1401-1412. [Abstract/Free Full Text]
4. van de Griend, R. J., J. Borst, W. J. Tax, R. L. Bolhuis. 1988. Functional reactivity of WT31 monoclonal antibody with T cell receptor- expressing CD3⁺4^–8^– T cells. J. Immunol. 140: 1107-1110. [Abstract]
5. Fattorossi, A., A. Battaglia, A. Buzzonetti, G. Minicuci, R. Riso, L. Peri, G. Scambia, A. Evoli. 2008. Thymopoiesis, regulatory T cells, and TCRVβ expression in thymoma with and without myasthenia gravis, and modulatory effects of steroid therapy. J. Clin. Immunol. 28: 194-206. [Medline]
6. Tran, D. Q., E. M. Shevach. 2008. Anti-human FOXP3 mAb PCH101 stains activated human naive T cells nonspecifically. Blood 111: 464-466. [Free Full Text]

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