Reply to: “Regulatory T cells in autoimmune hepatitis”
Authors: Moritz Peiseler, Marcial Sebode, Christoph Schramm, Johannes Herkel
Journal: Journal of Hepatology (2012)

Regulatory T cells in autoimmune hepatitisJournal of HepatologyVol. 57Issue 4PreviewWe read with interest the paper by Peiseler et al., and the accompanying editorial by Oo and Adams, reporting that in patients with autoimmune hepatitis (AIH), regulatory T cells (T-regs) are ‘fully functional and not reduced in frequency’ [1]. These data contrast with our findings of T-reg impairment in AIH, especially during active disease [2–5]. This discrepancy is attributed by the authors of the paper and the editorial to their use of an ‘improved’ methodology to define ‘true’ T-regs, i.e. CD4+CD25highCD127low cells. Full-Text PDF Open AccessRegulatory T cells and autoimmune hepatitis: Defective cells or a hostile environment?Journal of HepatologyVol. 57Issue 1PreviewAutoimmunity occurs when genetically predisposed individuals are exposed to environmental factors that trigger immune responses to self-antigens. The immune system protects the host by recognising and removing invading pathogens and damaged cells and to do this efficiently it must be able to discriminate between infected or damaged cells and healthy self-tissue. This tolerance of self involves both central, thymic mechanisms and peripheral pathways involving regulatory T cells (Treg) (Fig. 1) [1]. Full-Text PDF Open AccessFOXP3+ regulatory T cells in autoimmune hepatitis are fully functional and not reduced in frequencyJournal of HepatologyVol. 57Issue 1PreviewThe pathogenesis of autoimmune hepatitis (AIH) is not understood, but it was suggested that AIH may be related to a numerical or functional impairment of CD4+CD25+FOXP3+ regulatory T cells (Treg), which are important mediators of immune tolerance to self-antigens. However, the role of Treg in AIH is not clear, since earlier studies reporting Treg impairment had used only CD25 as marker that cannot unambiguously distinguish Treg from activated effector T cells. Full-Text PDF Reply to: “T regulatory cell number and function: The autoimmune traits in liver diseases”Journal of HepatologyVol. 57Issue 6PreviewWe read with interest the comment of Fierabracci et al. on a recent discussion between Longhi et al. [1] and us [2] on the frequency of regulatory T cells (Treg) in autoimmune hepatitis (AIH). This debate was in connection with our recent publication demonstrating that, in contrast to previous reports, Treg frequencies in the blood of AIH patients are not decreased [3]. We gladly acknowledge that Fierabracci et al. basically confirm our finding of unreduced Treg numbers in AIH. Full-Text PDF Open Access We read with interest the comment by Longhi et al. on our paper on Tregs in autoimmune hepatitis (AIH) [[1]Peiseler M. Sebode M. Franke B. Wortmann F. Schwinge D. Quaas A. et al.FOXP3+ regulatory T cells in autoimmune hepatitis are fully functional and not reduced in frequency.J Hepatol. 2012; 57: 125-132Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar] and the accompanying editorial by Oo and Adams [[2]Oo Y.H. Adams D.H. Regulatory T cells and autoimmune hepatitis: defective cells or a hostile environment?.J Hepatol. 2012; 57: 6-8Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar]. The discrepant results by Longhi et al. and us on Treg function in autoimmune hepatitis (AIH) may originate from the use of different methods to purify and assess the suppressor activity of Tregs [3Buckner J.H. Mechanisms of impaired regulation by CD4(+)CD25(+)FOXP3(+) regulatory T cells in human autoimmune diseases.Nat Rev Immunol. 2010; 10: 849-859Crossref PubMed Scopus (612) Google Scholar, 4Sakaguchi S. Miyara M. Costantino C.M. Hafler D.A. FOXP3+ regulatory T cells in the human immune system.Nat Rev Immunol. 2010; 10: 490-500Crossref PubMed Scopus (1831) Google Scholar]. Therefore, we will reply to the criticism by comparing the methodologies used by the Kings College group and by us, and explain why we chose the methodology as we did. To determine Treg function, Longhi et al. used the 3H-thymidine assay. First, Treg were purified with magnetic beads to enrich CD25+ CD4+ T cells; indeed, >95% of these cells were CD25+. These CD25+ cells were then co-cultured together with CD25− CD4+ T cells at a ratio of 1:8 for several days, after which 3H-thymidine was added to determine proliferation of the cell mixture. We also enriched CD25+ CD4+ T cells with magnetic beads, and found that >95% of these cells expressed CD25. However, in humans, CD25 alone cannot be used as a marker for Treg cells, since, in human blood, up to 30% of the CD4+ T cells can express CD25, but only 1–2% of these are functional suppressors [[4]Sakaguchi S. Miyara M. Costantino C.M. Hafler D.A. FOXP3+ regulatory T cells in the human immune system.Nat Rev Immunol. 2010; 10: 490-500Crossref PubMed Scopus (1831) Google Scholar]. In contrast to Longhi et al., we therefore further characterised the CD25+ cells by staining Foxp3 and CD127 (Fig. 2A of our manuscript), demonstrating that the majority of these cells (80–90%) were indeed Treg. More importantly, we also characterised the CD25− effector population by showing that more than 90% of cells were non-Tregs (Fig. 2A). We then determined Treg function with the CFSE dilution assay, in which only CD25− CD4+ effector T cells were labelled with CFSE and subsequently co-cultured together with the unlabelled CD25+ Treg fraction. The great advantage of the CFSE assay over the 3H-thymidine assay is that it is able to specifically evaluate the proliferation of the CD25− CD4+ responder T cell population [5Venken K. Thewissen M. Hellings N. Somers V. Hensen K. Rummens J.L. et al.A CFSE based assay for measuring CD4+CD25+ regulatory T cell mediated suppression of auto-antigen specific and polyclonal T cell responses.J Immunol Methods. 2007; 322: 1-11Crossref PubMed Scopus (118) Google Scholar, 6Boks M.A. Zwaginga J.J. van Ham S.M. ten Brinke A. An optimized CFSE-based T-cell suppression assay to evaluate the suppressive capacity of regulatory T-cells induced by human tolerogenic dendritic cells.Scand J Immunol. 2010; 72: 158-168Crossref PubMed Scopus (21) Google Scholar, 7Schneider A. Buckner J.H. Assessment of suppressive capacity by human regulatory T cells using a reproducible, bi-directional CFSE-based in vitro assay.Methods Mol Biol. 2011; 707: 233-241Crossref PubMed Scopus (15) Google Scholar]. The 3H-thymidine assay, in contrast, cannot distinguish which cells in the co-culture have incorporated 3H-thymidine, which can result in false estimates of Treg suppression [[7]Schneider A. Buckner J.H. Assessment of suppressive capacity by human regulatory T cells using a reproducible, bi-directional CFSE-based in vitro assay.Methods Mol Biol. 2011; 707: 233-241Crossref PubMed Scopus (15) Google Scholar]. In addition, unlike the CFSE assay, 3H-thymidine assays can only give a snapshot of proliferation for the period of time during which 3H-thymidine was present in the culture [[7]Schneider A. Buckner J.H. Assessment of suppressive capacity by human regulatory T cells using a reproducible, bi-directional CFSE-based in vitro assay.Methods Mol Biol. 2011; 707: 233-241Crossref PubMed Scopus (15) Google Scholar]. Moreover, unlike murine Treg cells, human Treg cells have a tendency to proliferate after activation [[4]Sakaguchi S. Miyara M. Costantino C.M. Hafler D.A. FOXP3+ regulatory T cells in the human immune system.Nat Rev Immunol. 2010; 10: 490-500Crossref PubMed Scopus (1831) Google Scholar], making the distinction of those cells that actually proliferated in the culture even more problematic. We thus would like to refer to Sakaguchi et al. [[4]Sakaguchi S. Miyara M. Costantino C.M. Hafler D.A. FOXP3+ regulatory T cells in the human immune system.Nat Rev Immunol. 2010; 10: 490-500Crossref PubMed Scopus (1831) Google Scholar] who state that ‘this indicates that thymidine uptake by cultured Treg and effector cells is not appropriate to assess suppressive activity of human Treg cells’, and instead ‘propose that it is more accurate to assess dilution of CFSE in effector T cells to determine the percentage and number of proliferating cells’. Another point addressed by Longhi et al. is that we cultured Treg cells and effector cells at a ratio of 1:1, instead of 1:8 as they did, arguing that this ratio is ‘utterly non-physiological’. We do agree that this assay is non-physiological, as basically any in vitro assay. However, the aim of the assay was not to determine the Treg physiology, but to assess their suppressive potential, which, as others have shown [5Venken K. Thewissen M. Hellings N. Somers V. Hensen K. Rummens J.L. et al.A CFSE based assay for measuring CD4+CD25+ regulatory T cell mediated suppression of auto-antigen specific and polyclonal T cell responses.J Immunol Methods. 2007; 322: 1-11Crossref PubMed Scopus (118) Google Scholar, 6Boks M.A. Zwaginga J.J. van Ham S.M. ten Brinke A. An optimized CFSE-based T-cell suppression assay to evaluate the suppressive capacity of regulatory T-cells induced by human tolerogenic dendritic cells.Scand J Immunol. 2010; 72: 158-168Crossref PubMed Scopus (21) Google Scholar, 7Schneider A. Buckner J.H. Assessment of suppressive capacity by human regulatory T cells using a reproducible, bi-directional CFSE-based in vitro assay.Methods Mol Biol. 2011; 707: 233-241Crossref PubMed Scopus (15) Google Scholar], can be reasonably performed with the CFSE assay using ratios in a range between 2:1 and 1:4. Longhi et al. further criticise our gating strategy and claim that the Treg frequencies in peripheral blood reported by us were vastly lower than those reported in the literature [[8]Garg G. Tyler J.R. Yang J.H. Cutler A.J. Downes K. Pekalski M. et al.Type I diabetes-associated IL2RA variation lowers IL-2 signaling and contributes to diminished CD4+CD25+ regulatory T cell function.J Immunol. 2012; 188: 4644-4653Crossref PubMed Scopus (145) Google Scholar]; however, in the paper they refer to, we find a gating strategy to determine CD25(high) cells (see Fig. 1 in that paper) that is very similar to that used by us (see our Fig. 1A). Moreover, it is actually stated in this paper that only ‘the top 1% of CD25-staining CD4 T cells’ contain Treg cells [[8]Garg G. Tyler J.R. Yang J.H. Cutler A.J. Downes K. Pekalski M. et al.Type I diabetes-associated IL2RA variation lowers IL-2 signaling and contributes to diminished CD4+CD25+ regulatory T cell function.J Immunol. 2012; 188: 4644-4653Crossref PubMed Scopus (145) Google Scholar], a number not vastly different from the frequencies reported by us. Longhi et al. also refer to a conference abstract, in which they have reported that Treg cell numbers are similar in AIH patients and healthy subjects; we apologise for having cited only previous papers of this group, which had reported impaired Treg frequencies in AIH. Longhi et al. quote us having attributed the discrepancies between previous findings and our report to the use of ‘improved methodology’ to define ‘true Treg’. We would like to emphasise that we did not use the quoted terms in our paper. Nevertheless, we believe that the methodology used by us was appropriate and that the conclusions drawn from these experiments are valid. The authors declared that they do not have anything to disclose regarding funding or conflict of interest with respect to this manuscript.