Retinoic Acid Can Directly Promote TGF-β-Mediated Foxp3+ Treg Cell Conversion of Naive T Cells
2009; Cell Press; Volume: 30; Issue: 4 Linguagem: Inglês
10.1016/j.immuni.2009.03.008
ISSN1097-4180
AutoresDaniel Mucida, Karina Pino‐Lagos, Gisen Kim, Elizabeth C. Nowak, Micah J. Benson, Mitchell Kronenberg, Randolph J. Noelle, Hilde Cheroutre,
Tópico(s)Immunotherapy and Immune Responses
ResumoThe article by Hill et al., 2008Hill J.A. Hall J.A. Sun C.M. Cai Q. Ghyselinck N. Chambon P. Belkaid Y. Mathis D. Benoist C. Immunity. 2008; 29: 758-770Abstract Full Text Full Text PDF PubMed Scopus (261) Google Scholar, published in the November 14, 2008 issue of Immunity, describes a mechanism by which retinoic acid (RA) enhances TGF-β-induced Foxp3 expression. The authors propose that RA does not act directly on naive T cells during activation in culture but rather indirectly via negative regulation of an accompanying population of effector or memory CD4+CD44hi cells. They reasoned that the increased generation of Foxp3+ cells in response to RA in culture, as described previously (Coombes et al., 2007Coombes J.L. Siddiqui K.R. Arancibia-Carcamo C.V. Hall J. Sun C.M. Belkaid Y. Powrie F. J. Exp. Med. 2007; 204: 1757-1764Crossref PubMed Scopus (2036) Google Scholar, Elias et al., 2008Elias K.M. Laurence A. Davidson T.S. Stephens G. Kanno Y. Shevach E.M. O'Shea J.J. Blood. 2008; 111: 1013-1020Crossref PubMed Scopus (334) Google Scholar, Mucida et al., 2007Mucida D. Park Y. Kim G. Turovskaya O. Scott I. Kronenberg M. Cheroutre H. Science. 2007; 317: 256-260Crossref PubMed Scopus (1494) Google Scholar, Sun et al., 2007Sun C.M. Hall J.A. Blank R.B. Bouladoux N. Oukka M. Mora J.R. Belkaid Y. J. Exp. Med. 2007; 204: 1775-1785Crossref PubMed Scopus (1421) Google Scholar, Xiao et al., 2008Xiao S. Jin H. Korn T. Liu S.M. Oukka M. Lim B. Kuchroo V.K. J. Immunol. 2008; 181: 2277-2284PubMed Google Scholar), represented the lifting by RA of inhibition imparted by accompanying CD4+CD44hi T cells, rather than by direct or indirect effects of RA on the Foxp3 expression of the primed naive T cells themselves. In order to assess the effects of RA on naive T cells in the absence of accompanying CD4+CD44hi T cells, we sorted (CD4+CD25−CD44lo CD62L+) GFP− T cells (more than 99.7% purity) from Foxp3-eGFP reporter mice (Figure S1A available online) by flow cytometry. After 4 days of stimulation with anti-CD3 and anti-CD28, we stained CD4 cells with 7AAD to exclude dead cells; additionally, forward and side scatter (area versus width) was used to exclude doublets, and we evaluated Foxp3 expression via GFP staining. Addition of RA enhanced Foxp3 induction more than 50% by use of 1 or 10 ng/ml doses of TGF-β (Figure S1B). Because the sorting efficiency is not 100%, it is possible that extremely low numbers of “accompanying” memory or effector cells could still influence these results. To exclude this possibility, we used FACS-sorted CD4+CD25−CD44lo CD62L+ T cells, isolated from B7-1 and B7-2 double-deficient mice (Cd80−/−Cd86−/−), which even before sorting already contain less than 5% of memory or effector CD44hi cells (data not shown). RA also greatly enhanced Foxp3 induced by TGF-β in CD4+CD25−CD44lo CD62L+ naive T cells isolated from Cd80−/−Cd86−/− mice (Figure S1C). Moreover, we showed previously that RA is able to counterbalance the inhibitory effects of costimulation on TGF-β-mediated Foxp3 induction, with either CD4+CD25− or CD4+Foxp3− T cells (Benson et al., 2007Benson M.J. Pino-Lagos K. Rosemblatt M. Noelle R.J. J. Exp. Med. 2007; 204: 1765-1774Crossref PubMed Scopus (636) Google Scholar). To confirm these results, we used OTII TCR transgenic CD4+CD25−CD44lo CD62L+ cells sorted by flow cytometry and tested the effects of RA by using increasing doses of anti-CD28 stimulation. We found that RA markedly enhanced TGF-β-mediated Foxp3 induction on pure naive CD4+ T cells that were stimulated with anti-CD3 and various doses of anti-CD28 (Figure S1D). The enhanced Foxp3 expression mediated by RA is more pronounced on naive monoclonal OTII TCR transgenic T cells as compared to polyclonal T cells, consistent with a lesser frequency of “contaminating” memory T cells. Finally, because we showed previously that RA-mediated enhanced expression of Foxp3 is greatly reduced in the absence of IL-2 (Mucida et al., 2007Mucida D. Park Y. Kim G. Turovskaya O. Scott I. Kronenberg M. Cheroutre H. Science. 2007; 317: 256-260Crossref PubMed Scopus (1494) Google Scholar), we investigated the effects of RA on naive T cells with various doses of exogenous IL-2. Although IL-2-deficient mice develop inflammatory disorders, Il2−/−Cd80−/−Cd86−/− mice are healthy and, more importantly, they do not contain T regulatory cells. At steady state, ∼99% of all CD4+ T cells isolated from Il2−/−Cd80−/−Cd86−/− mice are naive (data not shown). The CD4+ T cells were further sorted by flow cytometry so that highly purified naive CD4+CD25−CD44lo CD62L+ cells (more than 99.9% purity) were obtained. The sorted naive CD4+ Il2−/−Cd80−/−Cd86−/− T cells were tested for TGF-β-induced Foxp3 expression in the presence of increasing doses of IL-2 and anti-CD3 and anti-CD28 coated beads, with or without RA. The data showed that 1 nM RA distinctly enhanced TGF-β (1 ng/ml)-mediated Foxp3 induction in pure naive CD4+ T cells at all doses of IL-2 examined (Figures S1E and S1F). Strikingly, although the expression of Foxp3 was much reduced, RA enhanced TGF-β-mediated Foxp3 induction not only in the absence of memory or effector T cells but also in the absence of IL-2. These data demonstrate that RA mediates enhanced TGF-β-induced Foxp3 expression upon activation of pure naive T cells in the absence of accompanying CD4+CD44hi T cells. In addition, we confirmed, as Hill et al., 2008Hill J.A. Hall J.A. Sun C.M. Cai Q. Ghyselinck N. Chambon P. Belkaid Y. Mathis D. Benoist C. Immunity. 2008; 29: 758-770Abstract Full Text Full Text PDF PubMed Scopus (261) Google Scholar proposed, that RA also efficiently counteracts inhibitory effects of CD44hi T cells on Foxp3 induction (data not shown), which indicates that RA is able to enhance Foxp3 expression both, via effects directly on the primed naive T cells as well as indirectly via inhibitory effects on accompanying CD4+CD44hi T cells. There is no doubt that the new findings by Hill et al., 2008Hill J.A. Hall J.A. Sun C.M. Cai Q. Ghyselinck N. Chambon P. Belkaid Y. Mathis D. Benoist C. Immunity. 2008; 29: 758-770Abstract Full Text Full Text PDF PubMed Scopus (261) Google Scholar add an important new pathway by which RA can enhance Foxp3 induction, which had been suggested previously (Elias et al., 2008Elias K.M. Laurence A. Davidson T.S. Stephens G. Kanno Y. Shevach E.M. O'Shea J.J. Blood. 2008; 111: 1013-1020Crossref PubMed Scopus (334) Google Scholar, Mucida et al., 2007Mucida D. Park Y. Kim G. Turovskaya O. Scott I. Kronenberg M. Cheroutre H. Science. 2007; 317: 256-260Crossref PubMed Scopus (1494) Google Scholar, Xiao et al., 2008Xiao S. Jin H. Korn T. Liu S.M. Oukka M. Lim B. Kuchroo V.K. J. Immunol. 2008; 181: 2277-2284PubMed Google Scholar). Nevertheless, published data, together with the data presented here, disagree with the central statement proposed by Hill et al., 2008Hill J.A. Hall J.A. Sun C.M. Cai Q. Ghyselinck N. Chambon P. Belkaid Y. Mathis D. Benoist C. Immunity. 2008; 29: 758-770Abstract Full Text Full Text PDF PubMed Scopus (261) Google Scholar that the enhanced expression of TGF-β driven Foxp3 mediated by RA is an indirect effect that requires suppression of accompanying CD4+CD44hi T cells rather than via direct or indirect effects on the primed T cells themselves. Under physiological conditions, naive T cells may be exposed to cytokines and effector or memory cells, and hence it is likely that during priming of naive T cells, both mechanisms of RA-mediated enhanced TGF-β driven Foxp3 expression in the primed T cells will synergize in vivo. Therefore, elucidating and understanding both processes by which RA affects naive and already differentiated T cells is important and may lead to the identification of possible targets for therapeutic interventions to treat various inflammatory and autoimmune diseases. Download .pdf (.17 MB) Help with pdf files Document S1. One Figure Response to Letter from Mucida et al.Hill et al.ImmunityApril 17, 2009In BriefWe thank our colleagues for their interest in our recent paper, which showed that retinoic acid (RA), binding to the RARα receptor, influences TGFβ-induced Foxp3 expression through an indirect manner, either by repressing IL-6Rα or by dampening the production of inhibitory cytokines (IFN-γ, IL-4, and IL-21). The source of these cytokines, in our culture conditions, was CD44hi memory and/or effector phenotype CD4+ T cells. Several other papers have appeared on the topic, investigating the responsiveness of isolated naive cells to RA, a question of importance in understanding the molecular mechanisms that link RA to FoxP3 expression. Full-Text PDF Open Archive
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