- Email: [email protected]
Reply to: “Regulatory T cells in autoimmune hepatitis”
JOURNAL OF HEPATOLOGY In conclusion, we would urge the authors to be more cautious in the interpretation of their and published observations and we agree with the editorial comment that improved methodologies will help better understand the role of T-regs in disease.
Conflict of interest The authors declare that they do not have anything to disclose regarding funding or conflict of interest with respect to this manuscript.
Financial support M.S.L. is supported by a Clinician Scientist Fellowship from the Medical Research Council, UK. References [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;57:125–132. [2] Longhi MS, Ma Y, Bogdanos DP, Cheeseman P, Mieli-Vergani G, Vergani D. Impairment of CD4(+)CD25(+) regulatory T-cells in autoimmune liver disease. J Hepatol 2004;41:31–37. [3] Longhi MS, Ma Y, Mitry RR, Bogdanos DP, Heneghan M, Cheeseman P, et al. Effect of CD4+ CD25+ regulatory T-cells on CD8 T-cell function in patients with autoimmune hepatitis. J Autoimmun 2005;25:63–71.
[4] Longhi MS, Hussain MJ, Mitry RR, Arora SK, Mieli-Vergani G, Vergani D, et al. Functional study of CD4+CD25+ regulatory T cells in health and autoimmune hepatitis. J Immunol 2006;176:4484–4491. [5] Longhi MS, Mitry RR, Samyn M, Scalori A, Hussain MJ, Quaglia A, et al. Vigorous activation of monocytes in juvenile autoimmune liver disease escapes the control of regulatory T-cells. Hepatology 2009;50:130–142. [6] Wang P, Longhi MS, Mieli-Vergani G, Vergani D, Ma Y. Multiple defects of the immunoregulatory system contribute to the development of autoimmune hepatitis. Hepatology 2011;54:910A–911A. [7] Lobo Yeo A, Alviggi L, Mieli-Vergani G, Portmann B, Mowat AP, Vergani D. Preferential activation of helper/inducer T lymphocytes in children with autoimmune chronic active hepatitis. Clin Exp Immunol 1987;67:95–104. [8] Lobo Yeo A, Mieli-Vergani G, Mowat AP, Vergani D. Soluble interleukin 2 receptors in autoimmune chronic active hepatitis. Gut 1990;31:690–693. [9] Ferri S, Longhi MS, De Molo C, Lalanne C, Muratori P, Granito A, et al. A multifaceted imbalance of T cells with regulatory function characterizes type 1 autoimmune hepatitis. Hepatology 2010;52:999–1007. [10] Garg G, Tyler JR, Yang JH, Cutler AJ, Downes K, Pekalski M, et al. Type 1 diabetes-associated IL2RA variation lowers IL-2 signaling and contributes to diminished CD4+CD25+ regulatory T cell function. J Immunol 2012;188: 4644–4653.
Maria Serena Longhi Yun Ma Giorgina Mieli-Vergani ⇑ Diego Vergani Institute of Liver Studies, King’s College London School of Medicine at King’s College Hospital, Denmark Hill, London SE5 9RS, UK ⇑ Corresponding author. Tel.: +44 203 2993305; fax: +44 203 2994224 E-mail address: [email protected]
Reply to: ‘‘Regulatory T cells in autoimmune hepatitis’’ To the Editor: We read with interest the comment by Longhi et al. on our paper on Tregs in autoimmune hepatitis (AIH) [1] and the accompanying editorial by Oo and Adams [2]. 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 [3,4]. 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 3 H-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]. 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 [5–7]. 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]. 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]. Moreover, unlike murine Treg cells, human Treg cells have a tendency to proliferate after activation [4], 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] 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 [5–7], can be reasonably performed with the CFSE assay using ratios in a range between 2:1 and 1:4.
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Letters to the Editor 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]; 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], 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.
Conflict of interest The authors declared that they do not have anything to disclose regarding funding or conflict of interest with respect to this manuscript. References
[2] Oo YH, Adams DH. Regulatory T cells and autoimmune hepatitis: defective cells or a hostile environment? J Hepatol 2012;57:6–8. [3] Buckner JH. Mechanisms of impaired regulation by CD4(+)CD25(+)FOXP3(+) regulatory T cells in human autoimmune diseases. Nat Rev Immunol 2010;10:849–859. [4] Sakaguchi S, Miyara M, Costantino CM, Hafler DA. FOXP3+ regulatory T cells in the human immune system. Nat Rev Immunol 2010;10:490–500. [5] Venken K, Thewissen M, Hellings N, Somers V, Hensen K, Rummens JL, 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;322:1–11. [6] Boks MA, Zwaginga JJ, van Ham SM, ten Brinke A. An optimized CFSE-based Tcell suppression assay to evaluate the suppressive capacity of regulatory Tcells induced by human tolerogenic dendritic cells. Scand J Immunol 2010;72:158–168. [7] Schneider A, Buckner JH. Assessment of suppressive capacity by human regulatory T cells using a reproducible, bi-directional CFSE-based in vitro assay. Methods Mol Biol 2011;707:233–241. [8] Garg G, Tyler JR, Yang JH, Cutler AJ, 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;188:4644–4653.
Moritz Peiseler Marcial Sebode ⇑ Christoph Schramm ⇑ Johannes Herkel Department of Medicine I, University Medical Centre Hamburg-Eppendorf, 20246 Hamburg, Germany ⇑ Corresponding authors. E-mail addresses: [email protected], [email protected]
[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;57:125–132.
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Journal of Hepatology 2012 vol. 57 j 921–934
Conflict of interest The authors declare that they do not have anything to disclose regarding funding or conflict of interest with respect to this manuscript.
Financial support M.S.L. is supported by a Clinician Scientist Fellowship from the Medical Research Council, UK. References [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;57:125–132. [2] Longhi MS, Ma Y, Bogdanos DP, Cheeseman P, Mieli-Vergani G, Vergani D. Impairment of CD4(+)CD25(+) regulatory T-cells in autoimmune liver disease. J Hepatol 2004;41:31–37. [3] Longhi MS, Ma Y, Mitry RR, Bogdanos DP, Heneghan M, Cheeseman P, et al. Effect of CD4+ CD25+ regulatory T-cells on CD8 T-cell function in patients with autoimmune hepatitis. J Autoimmun 2005;25:63–71.
[4] Longhi MS, Hussain MJ, Mitry RR, Arora SK, Mieli-Vergani G, Vergani D, et al. Functional study of CD4+CD25+ regulatory T cells in health and autoimmune hepatitis. J Immunol 2006;176:4484–4491. [5] Longhi MS, Mitry RR, Samyn M, Scalori A, Hussain MJ, Quaglia A, et al. Vigorous activation of monocytes in juvenile autoimmune liver disease escapes the control of regulatory T-cells. Hepatology 2009;50:130–142. [6] Wang P, Longhi MS, Mieli-Vergani G, Vergani D, Ma Y. Multiple defects of the immunoregulatory system contribute to the development of autoimmune hepatitis. Hepatology 2011;54:910A–911A. [7] Lobo Yeo A, Alviggi L, Mieli-Vergani G, Portmann B, Mowat AP, Vergani D. Preferential activation of helper/inducer T lymphocytes in children with autoimmune chronic active hepatitis. Clin Exp Immunol 1987;67:95–104. [8] Lobo Yeo A, Mieli-Vergani G, Mowat AP, Vergani D. Soluble interleukin 2 receptors in autoimmune chronic active hepatitis. Gut 1990;31:690–693. [9] Ferri S, Longhi MS, De Molo C, Lalanne C, Muratori P, Granito A, et al. A multifaceted imbalance of T cells with regulatory function characterizes type 1 autoimmune hepatitis. Hepatology 2010;52:999–1007. [10] Garg G, Tyler JR, Yang JH, Cutler AJ, Downes K, Pekalski M, et al. Type 1 diabetes-associated IL2RA variation lowers IL-2 signaling and contributes to diminished CD4+CD25+ regulatory T cell function. J Immunol 2012;188: 4644–4653.
Maria Serena Longhi Yun Ma Giorgina Mieli-Vergani ⇑ Diego Vergani Institute of Liver Studies, King’s College London School of Medicine at King’s College Hospital, Denmark Hill, London SE5 9RS, UK ⇑ Corresponding author. Tel.: +44 203 2993305; fax: +44 203 2994224 E-mail address: [email protected]
Reply to: ‘‘Regulatory T cells in autoimmune hepatitis’’ To the Editor: We read with interest the comment by Longhi et al. on our paper on Tregs in autoimmune hepatitis (AIH) [1] and the accompanying editorial by Oo and Adams [2]. 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 [3,4]. 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 3 H-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]. 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 [5–7]. 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]. 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]. Moreover, unlike murine Treg cells, human Treg cells have a tendency to proliferate after activation [4], 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] 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 [5–7], can be reasonably performed with the CFSE assay using ratios in a range between 2:1 and 1:4.
Journal of Hepatology 2012 vol. 57 j 921–934
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Letters to the Editor 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]; 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], 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.
Conflict of interest The authors declared that they do not have anything to disclose regarding funding or conflict of interest with respect to this manuscript. References
[2] Oo YH, Adams DH. Regulatory T cells and autoimmune hepatitis: defective cells or a hostile environment? J Hepatol 2012;57:6–8. [3] Buckner JH. Mechanisms of impaired regulation by CD4(+)CD25(+)FOXP3(+) regulatory T cells in human autoimmune diseases. Nat Rev Immunol 2010;10:849–859. [4] Sakaguchi S, Miyara M, Costantino CM, Hafler DA. FOXP3+ regulatory T cells in the human immune system. Nat Rev Immunol 2010;10:490–500. [5] Venken K, Thewissen M, Hellings N, Somers V, Hensen K, Rummens JL, 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;322:1–11. [6] Boks MA, Zwaginga JJ, van Ham SM, ten Brinke A. An optimized CFSE-based Tcell suppression assay to evaluate the suppressive capacity of regulatory Tcells induced by human tolerogenic dendritic cells. Scand J Immunol 2010;72:158–168. [7] Schneider A, Buckner JH. Assessment of suppressive capacity by human regulatory T cells using a reproducible, bi-directional CFSE-based in vitro assay. Methods Mol Biol 2011;707:233–241. [8] Garg G, Tyler JR, Yang JH, Cutler AJ, 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;188:4644–4653.
Moritz Peiseler Marcial Sebode ⇑ Christoph Schramm ⇑ Johannes Herkel Department of Medicine I, University Medical Centre Hamburg-Eppendorf, 20246 Hamburg, Germany ⇑ Corresponding authors. E-mail addresses: [email protected], [email protected]
[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;57:125–132.
934
Journal of Hepatology 2012 vol. 57 j 921–934