T regulatory cell number and function: The autoimmune traits in liver diseases
Authors: Alessandra Fierabracci, Simona Cascioli, Sara Ceccarelli, Valerio Nobili
Journal: Journal of Hepatology (2012)

FOXP3+ 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 great interest a recent article published in this Journal by Peiseler et al. [[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 (143) Google Scholar], together with the accompanying editorial [[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], regarding the T regulatory cells (Tregs) in autoimmune hepatitis (AIH). Peiseler et al. found that CD4+CD25highCD127lowFOXP3+ Tregs in AIH are “fully functional and not reduced in frequency”. Longhi et al. [[3]Longhi M.S. Ma Y. Mieli-Vergani G. Vergani D. Regulatory T cells in autoimmune hepatitis.J Hepatol. 2012; 57: 932-933Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar], who had previously published that Tregs are functionally impaired in AIH, first argued against the methodology used for assessing the Tregs suppressor activity, since Peiseler et al. employed an “utterly non-physiological” suppressor/effector ratio. Peiseler et al. replied by stating that their methodology for staining proliferating cells by carboxyfluorescein succinimidyl ester (CFSE) is more appropriate than thymidine uptake and specifically stains the CD4+CD25− effector T cells. Another argument was raised regarding the choice of the Tregs labeling gate that resulted in a lower Tregs frequency compared with that reported by others [[4]Garg G. Tyler J.R. Yang J.H. Cutler A.J. 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: 4644-4653Crossref PubMed Scopus (135) Google Scholar]. The authors replied [[5]Peiseler M. Sebode M. Schramm C. Herkel J. Reply to: “T regulatory cell number and function: The autoimmune traits in liver diseases”.J Hepatol. 2012; 57: 1399-1400Abstract Full Text Full Text PDF Scopus (2) Google Scholar] by stating that the frequency of peripheral blood Tregs in adults reported by Garg et al. is “not vastly different from the frequencies reported by us”. Garg et al. [[4]Garg G. Tyler J.R. Yang J.H. Cutler A.J. 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: 4644-4653Crossref PubMed Scopus (135) Google Scholar] extrapolated that the Tregs number from CD4+CD25high-labeled cells, considering only the top 1% of CD25-staining CD4 T cells, as being estimated to be >98% CD127low. We believe that the comparison of Tregs frequencies is questionable and age-related, as demonstrated by our data. Peripheral blood cells from 4 healthy, 4 AIH, and 4 non-alcoholic steatohepatitis (NASH) children (age range: 10–14 years) were used to evaluate frequencies of CD4+CD25highCD127lowFOXP3+ Tregs (Fig. 1A–C). As reported in Fig. 1D, the number of CD25highCD127lowFOXP3+ Tregs within the CD4+ population is equal in all the groups of children. Our data confirms that the number of CD4+CD25highCD127low ‘True’ Tregs remains unchanged in liver disease with autoimmune traits, including AIH and NASH as already reported [1Peiseler 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 (143) Google Scholar, 6Wang P. Longhi M.S. Mieli-Vergani G. Vergani D. Ma Y. Multiple defects of the immunoregulatory system contribute to the development of autoimmune hepatitis.Hepatology. 2011; 54: 910A-911ACrossref PubMed Scopus (125) Google Scholar]. However, in children, the number of CD25highCD127lowFOXP3+ Tregs among CD4+ ranges from between 2.3% and 3.0%. In summary, all of these findings highlight the importance of a “consensus” in the standardization of protocols for evaluating the Tregs function in AIH and NASH. In addition, we would raise a discussion concerning some aspects related to non-alcoholic steatohepatitis (NASH). Peiseler et al. determined the intra-hepatic Tregs frequency by immunohistochemistry for FOXP3 in 8 NASH patients, in comparison to 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 (143) Google Scholar], and found a higher percentage of FOXP3+ cells in AIH patients when compared to NASH subjects, both in liver lobules and in portal tracts. Moreover, they determined that the FOXP3+ cells within the CD3+ population in the liver showed no statistical differences between AIH and NASH patients. No information about the diagnosis and severity of NASH was provided by the authors. Further, the usage of the modified histological activity index (mHAI) [[7]Ishak K. Baptista A. Bianch L. Callea F. De Groote J. Gudat F. et al.Histological grading and staging of chronic hepatitis.J Hepatol. 1995; 22: 696-699Abstract Full Text PDF PubMed Scopus (4055) Google Scholar], for quantifying the degree of liver inflammation, is not suitable for NASH patients who are histologically evaluated by using the NAFLD Clinical Research Network criteria [[8]Kleiner D.E. Brunt E.M. Van Natta M. Behling C. Contos M.J. Cummings O.W. et al.Design and validation of a histological scoring system for nonalcoholic fatty liver disease.Hepatology. 2005; 41: 1313-1321Crossref PubMed Scopus (6818) Google Scholar]. As liver-resident inflammatory cells are a key component in NAFLD, particularly in children [[9]De Vito R. Alisi A. Masotti A. Ceccarelli S. Panera N. Citti A. et al.Markers of activated inflammatory cells correlate with severity of liver damage in children with nonalcoholic fatty liver disease.Int J Mol Med. 2012; 30: 49-56PubMed Google Scholar], their role in the severity of NASH-associated necroinflammation and fibrosis requires particular attention. In fact, we demonstrated that CD3+ cells were significantly lower (p <0.01) in children with an NAFLD activity score (NAS) ⩾5 and fibrosis with respect to children with NAS <5. Furthermore, our preliminary data in 30 NAFLD children indicates that liver-resident CD25+ cells are rare in this condition (not shown). These findings oppose Peiseler et al. [[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 (143) Google Scholar] when stating about the presence of FOXP3+ in NASH. Despite several criticisms, Peiseler’s study and Longhi’s comments raise new questions and provide an experimental breakthrough regarding the study of the Tregs frequency and its function in inflammatory liver diseases. The role of the innate and adaptive immunity in NAFLD/NASH still remains an open and intriguing issue [[10]Söderberg C. Marmur J. Eckes K. Glaumann H. Sällberg M. Frelin L. et al.Microvesicular fat, inter cellular adhesion molecule-1 and regulatory T-lymphocytes are of importance for the inflammatory process in livers with non-alcoholic steatohepatitis.APMIS. 2011; 119: 412-420Crossref PubMed Scopus (28) Google Scholar]. The authors who have taken part in this study declared that they do not have anything to disclose regarding funding or conflict of interest with respect to this manuscript.