GRAIL Is Up-regulated in CD4+ CD25+ T Regulatory Cells and Is Sufficient for Conversion of T Cells to a Regulatory Phenotype

Abstract

GRAIL (gene related to anergy in lymphocytes) is an ubiquitin-protein isopeptide ligase (E3) ubiquitin ligase necessary for the induction of CD4+ T cell anergy in vivo. We have extended our previous studies to characterize the expression pattern of GRAIL in other murine CD4+ T cell types with a described anergic phenotype. These studies revealed that GRAIL expression is increased in naturally occurring (thymically derived) CD4+ CD25+ T regulatory cells (mRNA levels 10-fold higher than naive CD25– T cells). Further investigation demonstrated that CD25+ Foxp3+ antigen-specific T cells were induced after a “tolerizing-administration” of antigen and that GRAIL expression correlated with the CD25+ Foxp3+ antigen-specific subset. Lastly, using retroviral transduction, we demonstrated that forced expression of GRAIL in a T cell line was sufficient for conversion of these cells to a regulatory phenotype in the absence of detectable Foxp3. These data demonstrate that GRAIL is differentially expressed in naturally occurring and peripherally induced CD25+ T regulatory cells and that the expression of GRAIL is linked to their functional regulatory activity. GRAIL (gene related to anergy in lymphocytes) is an ubiquitin-protein isopeptide ligase (E3) ubiquitin ligase necessary for the induction of CD4+ T cell anergy in vivo. We have extended our previous studies to characterize the expression pattern of GRAIL in other murine CD4+ T cell types with a described anergic phenotype. These studies revealed that GRAIL expression is increased in naturally occurring (thymically derived) CD4+ CD25+ T regulatory cells (mRNA levels 10-fold higher than naive CD25– T cells). Further investigation demonstrated that CD25+ Foxp3+ antigen-specific T cells were induced after a “tolerizing-administration” of antigen and that GRAIL expression correlated with the CD25+ Foxp3+ antigen-specific subset. Lastly, using retroviral transduction, we demonstrated that forced expression of GRAIL in a T cell line was sufficient for conversion of these cells to a regulatory phenotype in the absence of detectable Foxp3. These data demonstrate that GRAIL is differentially expressed in naturally occurring and peripherally induced CD25+ T regulatory cells and that the expression of GRAIL is linked to their functional regulatory activity. CD4+CD25+ T cells, which comprise from 5 to 10% of peripheral blood CD4+ T cells in normal adult mice and humans, are a subset of regulatory CD4+T cells (CD25+ Tregs) 2The abbreviations used are: CD25+ Treg cell, CD4+ CD25+ T regulatory cell; IFN, interferon; IRES, internal ribosome entry site; GFP, green fluorescent protein; PE, phycoerythrin; GRAIL, gene related to anergy in lymphocytes; GITR, glucocorticoid-induced tumor necrosis factor receptor family-regulated gene; APC, allophycocyanin; QPCR, quantitative PCR; IL, interleukin; TGF, transforming growth factor. with an “anergic” phenotype in vitro. CD25+ Tregs have been shown to play a critical role in the prevention of organ-specific autoimmune disease (1Sakaguchi S. Sakaguchi N. Asano M. Itoh M. Toda M. J. Immunol. 1995; 155: 1151-1164Crossref PubMed Google Scholar). Mutations in the coding region of the transcription factor, Foxp3, result in aberrant development and function of CD25+ Treg cells in mice and humans, implicating Foxp3 as the master genetic regulator of CD25+ Treg cells (2Chatila T.A. Blaeser F. Ho N. Lederman H.M. Voulgaropoulos C. Helms C. Bowcock A.M. J. Clin. Investig. 2000; 106: R75-81Crossref PubMed Scopus (742) Google Scholar, 3Brunkow M.E. Jeffery E.W. Hjerrild K.A. Paeper B. Clark L.B. Yasayko S.A. Wilkinson J.E. Galas D. Ziegler S.F. Ramsdell F. Nat. Genet. 2001; 27: 68-73Crossref PubMed Scopus (2045) Google Scholar, 4Bennett C.L. Christie J. Ramsdell F. Brunkow M.E. Ferguson P.J. Whitesell L. Kelly T.E. Saulsbury F.T. Chance P.F. Ochs H.D. Nat. Genet. 2001; 27: 20-21Crossref PubMed Scopus (2682) Google Scholar). The absence of Foxp3 protein is associated with immune dysregulation, polyendocrinopathy, enteropathy, X-linked syndrome in the majority of, but not all patients, suggesting that other genes may be important for CD25+ Treg function (5Gavin M.A. Torgerson T.R. Houston E. Deroos P. Ho W.Y. Stray-Pedersen A. Ocheltree E.L. Greenberg P.D. Ochs H.D. Rudensky A.Y. Proc. Natl. Acad. Sci. U. S. A. 2006; Google Scholar). Furthermore, there is increasing evidence that antigen-specific CD25+ Tregs can be induced by antigen exposure in a non-inflammatory context and play an essential role in modulating adaptive immune responses, and clearly defining the similarities and differences between naturally occurring and induced CD25+ Treg cells is a high priority for making inroads into potential therapeutics for multiple disease states (6Thorstenson K.M. Khoruts A. J. Immunol. 2001; 167: 188-195Crossref PubMed Scopus (378) Google Scholar, 7Kretschmer K. Apostolou I. Hawiger D. Khazaie K. Nussenzweig M.C. von Boehmer H. Nat. Immunol. 2005; 6: 1219-1227Crossref PubMed Scopus (1040) Google Scholar, 8Apostolou I. Von Boehmer H. J. Exp. Med. 2004; 199: 1401-1408Crossref PubMed Scopus (604) Google Scholar). Characterization of cells with intrinsic anergic properties (termed recessive tolerance) was formally undertaken in the late 1980s after the Schwartz laboratory described a model system in vitro for induction of anergic T cell clones that, following engagement of their T cell receptor, required new protein and mRNA synthesis (reviewed in Ref. 9Schwartz R.H. Mueller D.L. Jenkins M.K. Quill H. Cold Spring Harbor Symp. Quant. Biol. 1989; 54: 605-610Crossref PubMed Google Scholar). This model system has led to numerous studies to identify “anergy factors.” Our published work using this in vitro T cell anergy paradigm demonstrated the expression of a novel E3 ubiquitin ligase GRAIL, in T cell clones under anergy-inducing conditions (10Anandasabapathy N. Ford G. Bloom D. Holness C. Seroogy C. Skrenta H. Hollenhurst M. Fathman C.G. Soares L. Immunity. 2003; 18: 1-20Abstract Full Text Full Text PDF PubMed Scopus (242) Google Scholar). The development of immune unresponsiveness in vivo to neoantigens predates the development of the in vitro T cell clone model by over 20 years (11Dresser D.W. Nature. 1961; 191: 1169-1171Crossref PubMed Scopus (94) Google Scholar). An intriguing observation in the early 1970s suggested that there is development de novo of a suppressor population when neoantigen is presented in a non-inflammatory context. Our recent published work demonstrated a correlation between CD4+ T cell anergy in vitro and in vivo and GRAIL expression (12Seroogy C.M. Soares L. Ranheim E.A. Su L. Holness C. Bloom D. Fathman C.G. J. Immunol. 2004; 173: 79-85Crossref PubMed Scopus (89) Google Scholar) that is complemented by studies from Heissmeyer et al. (13Heissmeyer V. Macian F. Im S.H. Varma R. Feske S. Venuprasad K. Gu H. Liu Y.C. Dustin M.L. Rao A. Nat. Immunol. 2004; 5: 255-265Crossref PubMed Scopus (448) Google Scholar). These findings, coupled with several recent reports demonstrating an induction of CD25+ Treg cells in vivo after antigen presentation to naive CD4+ T cells in a tolerizing fashion, led us to pursue the role of GRAIL- and antigen-induced tolerance in vivo (6Thorstenson K.M. Khoruts A. J. Immunol. 2001; 167: 188-195Crossref PubMed Scopus (378) Google Scholar, 7Kretschmer K. Apostolou I. Hawiger D. Khazaie K. Nussenzweig M.C. von Boehmer H. Nat. Immunol. 2005; 6: 1219-1227Crossref PubMed Scopus (1040) Google Scholar, 8Apostolou I. Von Boehmer H. J. Exp. Med. 2004; 199: 1401-1408Crossref PubMed Scopus (604) Google Scholar). In the current study, we investigated the mRNA and protein levels of GRAIL in naturally occurring and induced CD25+ Treg cells. We demonstrated that both naturally occurring CD25+ Treg cells and antigen-specific CD25+ Foxp3+ CD4+ T cells induced in vivo after receiving antigen in a tolerizing fashion express GRAIL as a bulk population. Furthermore, retroviral-mediated forced expression of GRAIL in an antigen-specific CD4+ T cell line was sufficient for conversion of the transduced cells to a suppressor cell phenotype. These data suggest that GRAIL, along with and separable from Foxp3, might be involved in differentiation and biological function of CD4+ T cells into a regulatory phenotype. Animals and Adoptive Transfer—BALB/c mice were obtained from Harlan-Sprague (Madison, WI). DO11.10 and DO11.10 Rag2–/– mice (14Murphy K.M. Heimberger A.B. Loh D.Y. Science. 1990; 250: 1720-1723Crossref PubMed Scopus (1653) Google Scholar) were bred under specific pathogen-free conditions at the University of Wisconsin animal facility (Madison, WI). Foxp3-IRES-GFP knock-in mice were a generous gift from Dr. Talal Chatila (UCLA, Los Angeles, CA). The Animal Care Committee at the University of Wisconsin approved all experimental protocols involving the use of mice. The adoptive transfer of DO11.10 (DO11) T cells into BALB/c mice and subsequent immunization followed a previously described protocol (12Seroogy C.M. Soares L. Ranheim E.A. Su L. Holness C. Bloom D. Fathman C.G. J. Immunol. 2004; 173: 79-85Crossref PubMed Scopus (89) Google Scholar). Flow Cytometry—Single cell suspensions were stained using the following antibodies according to the manufacturer's protocol: KJ1.26 PE (Caltag, Burlingame, CA); CD4 Cy7APC (RM-5), Foxp3 PE (FJK-165) (both from eBioscience, San Diego, CA); CD25 APC (PC61), CTLA4 PE (UC10-4B9), CD103 PE (M290) (all from Pharmingen); GITR PE (FAB5241P, R+D, Minneapolis, MN). Acquisition was done on a LSR II (BD Biosciences), and sorting was done on a Vantage SE equipped with a FACS-DiVa digital upgrade. Analysis was done using FlowJo software (Treestar, San Carlos, CA). Cells—An OVA323–339-specific T cell line was derived from a DO11 T cell receptor transgenic mouse by serially stimulating the cells with 0.4 μg/ml OVA323–339 peptide and irradiated APCs from a BALB/c mouse every 8–14 days in supplemented RPMI 1640 as described previously (12Seroogy C.M. Soares L. Ranheim E.A. Su L. Holness C. Bloom D. Fathman C.G. J. Immunol. 2004; 173: 79-85Crossref PubMed Scopus (89) Google Scholar). Retrovirus Production and T cell Transduction—Ecotropic retrovirus was produced using Phoenix-E cells and a modified CaPO4 precipitation transfection protocol as described previously (15Costa G.L. Benson J.M. Seroogy C.M. Achacoso P. Fathman C.G. Nolan G.P. J. Immunol. 2000; PubMed Scopus Google Scholar). The DO11.10 T cell line was transduced after by with 5 μg/ml and murine as described previously Seroogy C.M. G. Fathman C.G. R.H. J. Clin. Immunol. Full Text Full Text PDF PubMed Scopus Google Scholar). Rag2–/– were used for of OVA323–339-specific naive CD4 T cells by the suppressor 5 were with μg/ml and μg/ml μg/ml as described previously J. V. Ford V. Fathman C.G. K. J. Immunol. 2001; 167: PubMed Scopus Google Scholar). and were at a of in were at for with of the peptide suppressor cells were in a with 0.4 μg/ml OVA323–339 and irradiated BALB/c at a of were at for with of The of DO11 cell line was by the following DO11 T cells with naive CD25– DO11 T DO11 T cells with naive CD25– DO11 T cells the naive DO11 were with an of DO11 T cells DO11 T cells in a and with 0.4 μg/ml OVA323–339 and 5 irradiated BALB/c were and with for an from transduced DO11 T cells were after with and for the of using an obtained from (Madison, WI). in the of were with of for and with an of and were as previously (10Anandasabapathy N. Ford G. Bloom D. Holness C. Seroogy C. Skrenta H. Hollenhurst M. Fathman C.G. Soares L. Immunity. 2003; 18: 1-20Abstract Full Text Full Text PDF PubMed Scopus (242) Google Scholar). CD25+ T cells were using according to the manufacturer's from DO11 mice were stained with in a followed by several and with cells were using a and was by CD4+ was on and to as described previously L. Seroogy C. Skrenta H. N. P. E. Fathman C.G. Nat. Immunol. 2004; 5: PubMed Scopus Google Scholar). The was with a the of murine GRAIL L. Seroogy C. Skrenta H. N. P. E. Fathman C.G. Nat. Immunol. 2004; 5: PubMed Scopus Google Scholar). The was and with and as a was using and the system were on cells transduced with murine GRAIL were used as a from cell was using according to the manufacturer's and with was using an according to the manufacturer's were with green on an using the following murine GRAIL and and murine Foxp3 and the gene was are as and and and T cells transduced with murine Foxp3 were used to for of and of from knock-in mice were and cell suspensions were CD25+ cells were using and cells were to to for at were the in with with and with were in with in for at followed by for at and cells were with for at and were stained with and were with on and with a and software for University of GRAIL mRNA in CD25+ T have previously that GRAIL is expressed in anergic CD4+ T cells using in vitro and in vivo model (10Anandasabapathy N. Ford G. Bloom D. Holness C. Seroogy C. Skrenta H. Hollenhurst M. Fathman C.G. Soares L. Immunity. 2003; 18: 1-20Abstract Full Text Full Text PDF PubMed Scopus (242) Google Scholar, C.M. Soares L. Ranheim E.A. Su L. Holness C. Bloom D. Fathman C.G. J. Immunol. 2004; 173: 79-85Crossref PubMed Scopus (89) Google Scholar). Furthermore, GRAIL function was to be a necessary for the induction of an anergic phenotype in CD4+ T cells in vivo (12Seroogy C.M. Soares L. Ranheim E.A. Su L. Holness C. Bloom D. Fathman C.G. J. Immunol. 2004; 173: 79-85Crossref PubMed Scopus (89) Google Scholar). on these we to the expression of GRAIL in other anergic CD4+ T cells. this we that both GRAIL mRNA and protein are expressed in naturally occurring CD25+ the GRAIL expression was 10-fold higher in the CD25+ population than the CD25– naive CD4+ T cells a we were to demonstrate at the protein that GRAIL was expressed in the CD25+ Treg subset of the mRNA data that GRAIL and Foxp3 are expressed in the cell, was We of transgenic mice that to the region of the Foxp3 that as a of Foxp3 protein of the CD25+ T cells from these mice with the GRAIL revealed expression of Foxp3 and GRAIL the cell The of GRAIL is as previously demonstrated GRAIL, which is in is in the in a These data suggested that GRAIL, in to Foxp3, is differentially expressed in CD25+ Treg cells and may be involved in CD25+ Treg function GRAIL with the in have previously shown that GRAIL is expressed in antigen-specific anergic T cells induced following antigen in a tolerizing fashion in vivo (12Seroogy C.M. Soares L. Ranheim E.A. Su L. Holness C. Bloom D. Fathman C.G. J. Immunol. 2004; 173: 79-85Crossref PubMed Scopus (89) Google and in this study, we have demonstrated that GRAIL is expressed in naturally occurring CD25+ Treg cells is evidence demonstrating that CD25+ antigen-specific Treg cells can be induced from naive CD4+ CD25– T cells following of antigen under non-inflammatory conditions to the used in the adoptive transfer tolerance model of our previous studies that demonstrated that GRAIL was necessary for the development of the CD4+ T cell anergic phenotype in vivo (6Thorstenson K.M. Khoruts A. J. Immunol. 2001; 167: 188-195Crossref PubMed Scopus (378) Google Scholar). GRAIL expression in antigen-specific anergic T cells with CD25 we used the in vivo adoptive transfer model described previously to CD25 and GRAIL expression (12Seroogy C.M. Soares L. Ranheim E.A. Su L. Holness C. Bloom D. Fathman C.G. J. Immunol. 2004; 173: 79-85Crossref PubMed Scopus (89) Google Scholar). In our CD25+ antigen-specific CD4+ T cells were induced and by on after antigen in a tolerizing fashion of DO11 naive T cells from a Rag2–/– a and antigen demonstrated of CD25 expression T cell receptor transgenic T cells from mice on a Rag2–/– naturally occurring CD25+ Treg cells, we that this population of CD4+ CD25+ T cells was induced by antigen under non-inflammatory These data are with the published work by et al. (6Thorstenson K.M. Khoruts A. J. Immunol. 2001; 167: 188-195Crossref PubMed Scopus (378) Google with CD25+ antigen-specific cells between days and after antigen in a tolerizing We our studies and that the CD25+ antigen-specific CD4+ T cells at days after of antigen in a tolerizing fashion not which is with data suggesting that CD25+ Treg cells not of antigen for I. Von Boehmer H. J. Exp. Med. 2004; 199: 1401-1408Crossref PubMed Scopus (604) Google Scholar). characterize the of GRAIL and CD25 the cells were days after the tolerizing into CD25+ CD25– of GRAIL by revealed that GRAIL was expressed in the T cell subset the genetic for cells is Foxp3, and we for this gene as a for induced CD25+ Treg cells R. Yasayko S.A. Ramsdell F. Nat. Immunol. 2003; PubMed Scopus Google Scholar, M.A. Rudensky A.Y. Nat. Immunol. 2003; PubMed Scopus Google Scholar). expression by and for protein expression revealed that Foxp3 was expressed in the CD25+ suggesting that this population induced CD25+ Treg cells. of GRAIL in a T for to a in a a to the role of GRAIL in the suppressor cell we GRAIL in an CD4+ T cell line by retroviral of this cell line in vitro required of the with in to with antigen APC to the necessary for the of T cells M.A. Rudensky A.Y. Nat. Immunol. 2003; PubMed Scopus Google Scholar). the DO11 T cell line demonstrated in to antigen APC when with the transduced cell line the protein DO11 T cells cells the cell line was with DO11 data not of the DO11 T cell line and naive CD25– CD4+ T cells from a DO11.10 mice with the APC OVA323–339 peptide demonstrated of the naive cells when with naive cells and when with with DO11 cells, The suppressor of the T cell line was with that of naturally occurring CD25+ Treg cells from DO11 mice using a previously described system J. V. Ford V. Fathman C.G. K. J. Immunol. 2001; 167: PubMed Scopus Google Scholar). The T cell line naive CD25– CD4+ T cell as than the naturally occurring CD25+ Treg cells when at a in this system with previous of higher on the the of naive CD4+ CD25– T cells experimental The of in a in the in cells with naturally occurring CD25+ Treg cells when with a in the the DO11 T cell of in a in the in the system when naturally occurring CD25+ Treg cells were used as suppressor cells when with a when using the DO11 T cell line as We the in of the naive T cells between naturally occurring CD25+ Treg cells and DO11 T cell line to expression of GRAIL on data not The by DO11 T cells was demonstrated by of naive CD25– CD4+ T cells at cell We the role of cell for in the DO11 T cells using a This demonstrated that was the cells were in when with by a DO11 T of GITR, and and from Foxp3 in the potential for suppressor function of the DO11 T cells, receptor expression and was for that have been suggested to be important for suppressor function of naturally occurring CD25+ Treg cells S. J. Sakaguchi N. Sakaguchi S. J. Exp. Med. 2000; PubMed Scopus Google Scholar, K. A. I. A. N. H. J. Immunol. 2004; PubMed Scopus Google Scholar). Analysis of demonstrated detectable levels produced by the cell and levels in the from DO11 T cells of naturally occurring CD25+ Treg cells and T cells Foxp3 have increased levels of M.A. Rudensky A.Y. Nat. Immunol. 2003; PubMed Scopus Google our data suggest that the in the DO11 T cells not the T cell line of we that the levels of in the were higher in the DO11 T cell line recent by et al. P. G. R.H. Nat. Immunol. 2004; 5: PubMed Scopus Google Scholar) has described the development of a suppressor T cell that Foxp3 and evidence that not with suppressor The that has been associated with suppressor T cells is of the from DO11 T cells demonstrated increased levels of when with the DO11 T cell line Flow of of CD25+ Treg cells revealed an expression to naturally occurring CD25+ Treg cells and T cells, with the of CD103 P. G. R.H. Nat. Immunol. 2004; 5: PubMed Scopus Google Scholar). The T cells levels of GITR, and We our T cell line for Foxp3 protein by and detectable Foxp3 protein we a DO11 T cell line expression by Dr. A. for GRAIL mRNA by and of GRAIL mRNA in these cells suppressor as previously published not These data suggest that GRAIL Foxp3 expression by is sufficient but not necessary for regulatory T cell function in vitro. The functional of naive CD4+ T cells following engagement with antigen in a non-inflammatory in vivo has been to of several in peripheral In this engagement was to result in The anergic phenotype has in to a of tolerance a in of for evidence has suggested that these cells of antigen to this of induced and in the absence of this phenotype is C. D.L. L. Schwartz R.H. J. Immunol. 2001; 167: PubMed Scopus Google Scholar, K.A. R. A. Khoruts A. Jenkins M.K. J. Immunol. Google Scholar). recent studies have to suggest that exposure to antigen in a non-inflammatory in vivo can to the induction of CD25+ Treg cells from CD4+ T cells (6Thorstenson K.M. Khoruts A. J. Immunol. 2001; 167: 188-195Crossref PubMed Scopus (378) Google Scholar, 7Kretschmer K. Apostolou I. Hawiger D. Khazaie K. Nussenzweig M.C. von Boehmer H. Nat. Immunol. 2005; 6: 1219-1227Crossref PubMed Scopus (1040) Google Scholar, 8Apostolou I. Von Boehmer H. J. Exp. Med. 2004; 199: 1401-1408Crossref PubMed Scopus (604) Google Scholar). The from et al. (6Thorstenson K.M. Khoruts A. J. Immunol. 2001; 167: 188-195Crossref PubMed Scopus (378) Google Scholar) the induction of CD4+ CD25+ antigen-specific T cells when antigen was in a non-inflammatory context. The functional of these cells in vitro suppressor (6Thorstenson K.M. Khoruts A. J. Immunol. 2001; 167: 188-195Crossref PubMed Scopus (378) Google Scholar). Our the that the anergic phenotype in CD4+ T cells in vivo in to a neoantigen under non-inflammatory conditions is to the of a suppressor phenotype by the anergic CD4+ T cells. This is the induction of Foxp3 expression in the T cells as as their to be used in functional suppressor as regulatory T cells. the E3 ubiquitin GRAIL, with these CD25+ antigen-specific regulatory T cells. in to Foxp3, GRAIL may be gene involved in CD25+ Treg cell and by separable from to be sufficient to regulatory function to T cells that over express Our and the observation by et al. (6Thorstenson K.M. Khoruts A. J. Immunol. 2001; 167: 188-195Crossref PubMed Scopus (378) Google Scholar) by demonstrating that these induced CD25+ Treg cells are and express of naturally occurring CD25+ Treg cells, Foxp3 and In our high antigen not result in a in of CD4+ CD25+ antigen-specific T cells between the We these data with the antigen Treg cell induction published by et al. (6Thorstenson K.M. Khoruts A. J. Immunol. 2001; 167: 188-195Crossref PubMed Scopus (378) Google Scholar) with induction of CD4+ CD25+ antigen-specific cells, but potential be that might have been using a peptide that was making the observation that non-inflammatory exposure to antigen can result in suppressor T cells may have of the conditions that this be for of multiple disease states that result from and adaptive immune we have that the GRAIL mRNA levels were higher in the induced population when with the naturally occurring CD25+ Treg cell population This that GRAIL may be in CD25+ Treg cells, that GRAIL is differentially expressed between naturally occurring and induced CD25+ Treg cells. This observation is in to that of Foxp3 expression that not to be differentially with the of CD25+ Treg cells R. D. M. Brunkow M.E. Ziegler S.F. Ramsdell F. J. Immunol. 2001; 167: PubMed Scopus Google Scholar). is that GRAIL is differentially between naturally occurring CD25+ Treg cells and induced CD25+ Treg cells, an intriguing is the and functional differences between these important T cell are in our laboratory to the expression of GRAIL after CD25+ Treg cell and of the differences and similarities between naturally occurring and induced CD25+ Treg cells. The suppressor of DO11 T cells similarities with previously described of naturally occurring CD25+ Treg cells and T cells with expression of Foxp3 in that the is and is when cell is and receptor expression to with all of these cells the expression of and GITR as as increased of has been on the role of and in the suppressor function of CD25+ Treg cells. and of these has been demonstrated to be essential for suppressor function H. M. J. Immunol. 2004; PubMed Scopus Google Scholar). is in to identify the of which GRAIL suppressor activity. Our at have not the suppressor and are with published with naturally occurring CD25+ Treg cells H. M. J. Immunol. 2004; PubMed Scopus Google Scholar, A.M. M. H. J. Exp. Med. PubMed Scopus Google Scholar). studies have suggested that is important for of CD25+ Treg cells and of Foxp3 expression in vivo and that the of not to be M. Rudensky A.Y. J. Exp. Med. 2005; PubMed Scopus Google Scholar). is that exposure of naive CD25– T cells to in vitro has been demonstrated to these cells to CD25+ Treg cells on functional and of Foxp3. potential for the of of Foxp3 in our GRAIL T cells may be a a related to naive cells previously T cells, the used as our T cells. to be the exposure of naive CD25– T cells to at the of in of GRAIL in to Foxp3. In these for studies the role of GRAIL in CD25+ Treg cell to Foxp3, and to GRAIL protein as a in We and other of the University of Wisconsin Flow for their