We recently read with great interest the article by Tresoldi et al.1 in the September 2011 issue of Haematologica. Their study examined the stability of human CD4 regulatory T (Treg) cells exposed to rapamycin in vitro and in vivo, and concluded that rapamycin-expanded Treg cells maintained a stable phenotype. We believe that the data from this investigation are interesting but that they do not, however, provide adequate support for their conclusion.
In this study, it was shown that the frequency of FOXP3 cells within CD4CD25 T cells in the non-rapamycin group of patients declined more markedly than that in the rapamycin group after in vitro expansion. Accordingly, the rapamycin-expanded CD4CD25 T cells displayed a stronger suppressive activity and contained a smaller fraction of pro-inflammatory cytokine-producing T cells compared to expanded CD4CD25 T cells in the absence of rapamycin. These results may be due to the distinct effects of rapamycin on FOXP3 Treg cells and effector T cells, because rapamycin was shown to be able to selectively promote in vitro expansion of Treg cells while inhibiting proliferation of effector T cells.2,3 In this study, the initial population (CD4CD25 T cells) was contaminated with more than 30% effector T cells. Thus, we could not draw any conclusion about whether rapamycin has an intrinsic role in the stability of Treg cells. Furthermore, recent studies demonstrated that natural Treg cells were instable during in vitro culture and could convert to so-called exFOXP3 cells that produced pro-inflammatory cytokines.4–6 Therefore, the pro-inflammatory cytokines detected in the expanded T cells in this study can be from either preferentially expanded effector T cells or exFOXP3 cells converted from FOXP3+ Treg cells.
Additionally, the stability of Treg cells in vivo remains controversial. Several recent studies have reported that a fraction of FOXP3+ Treg cells can lose FOXP3 expression after in vivo transfer,7 especially in lymphopenic conditions.6,8,9 However, the study by Rubtsov et al.10 reported that FOXP3 Treg cells are notably stable under physiological and inflammatory conditions. In this study, Tresoldi and colleagues also tested the stability of Treg cells in vivo. However, the article reports that they only displayed the frequency of FOXP3 Treg cells after and not before in vivo transfer. Furthermore, it was unfortunate that they did not obtain data concerning the number of non-rapamycin expanded FOXP3 Treg cells recovered from the injected mice. It is, therefore, difficult to compare the stability of FOXP3 Treg cells before and after in vivo transfer in the rapamycin group, as well as those cultured with and without rapamycin. Therefore, we think that the data reported in the article cannot offer substantial support for the conclusion that rapamycin fixed the Treg cell phenotype.
- Tresoldi E, Dell’Albani I, Stabilini A, Jofra T, Valle A, Gagliani N. Stability of human rapamycin-expanded CD4+CD25+ T regulatory cells. Haematologica. 2011; 96(9):1357-65. PubMedhttps://doi.org/10.3324/haematol.2011.041483Google Scholar
- Strauss L, Whiteside TL, Knights A, Bergmann C, Knuth A, Zippelius A. Selective survival of naturally occurring human CD4(+)CD25(+)Foxp3(+) regulatory T cells cultured with rapamycin. J Immunol. 2007; 178(1):320-9. PubMedhttps://doi.org/10.4049/jimmunol.178.1.320Google Scholar
- Strauss L, Czystowska M, Szajnik M, Mandapathil M, Whiteside TL. Differential responses of human regulatory T cells (Treg) and effector T cells to rapamycin. Plos One. 2009; 4(6):e5994. PubMedhttps://doi.org/10.1371/journal.pone.0005994Google Scholar
- Hoffmann P, Boeld TJ, Eder R, Huehn J, Floess S, Wieczorek G. Loss of FOXP3 expression in natural human CD4(+)CD25(+) regulatory T cells upon repetitive in vitro stimulation. Eur J Immunol. 2009; 39(4):1088-97. PubMedhttps://doi.org/10.1002/eji.200838904Google Scholar
- d'Hennezel E, Yurchenko E, Sgouroudis E, Hay V, Piccirillo CA. Single-cell analysis of the human T regulatory population uncovers functional heterogeneity and instability within FOXP3+ cells. J Immunol. 2011; 186(12):6788-97. PubMedhttps://doi.org/10.4049/jimmunol.1100269Google Scholar
- Zhou XY, Bailey-Bucktrout SL, Jeker LT, Penaranda C, Martinez-Llordella M, Ashby M. Instability of the transcription factor Foxp3 leads to the generation of pathogenic memory T cells in vivo. Nat Immunol. 2009; 10(9):1000-7. PubMedhttps://doi.org/10.1038/ni.1774Google Scholar
- Hori S. Developmental plasticity of Foxp3(+) regulatory T cells. Curr Opin Immunol. 2010; 22(5):575-82. PubMedhttps://doi.org/10.1016/j.coi.2010.08.004Google Scholar
- Duarte JH, Zelenay S, Bergman ML, Martins AC, Demengeot J. Natural Treg cells spontaneously differentiate into pathogenic helper cells in lymphopenic conditions. Eur J Immunol. 2009; 39(4):948-55. PubMedhttps://doi.org/10.1002/eji.200839196Google Scholar
- Komatsu N, Mariotti-Ferrandiz ME, Wang Y, Malissen B, Waldmann H, Hori S. Heterogeneity of natural Foxp3(+) T cells: A committed regulatory T-cell lineage and an uncommitted minor population retaining plasticity. Proc Natl Acad Sci USA. 2009; 106(6):1903-8. PubMedhttps://doi.org/10.1073/pnas.0811556106Google Scholar
- Rubtsov YP, Niec RE, Josefowicz S, Li L, Darce J, Mathis D. Stability of the regulatory T cell lineage in vivo. Science. 2010; 24;329(5999):1667-71. Google Scholar