CD4+CD25� regulatory T-cells (TR) have emerged aspotent regulators of immune responses. The ability togenerate and expand antigen-specific TR may providetargeted immunotherapy for autoimmune diseases andtransplant. In a previous report, we have shown thatactivation of human CD4+CD25� results in expression ofFoxP3 and CD25, as well as, acquisition of a cell contactdependent, cytokine independent regulatory activity by aportion of the original CD4+CD25� T-cells. These TR can begenerated by activation on peptide antigen and subse-quently behave in an antigen-specific manner. First, weinduced CD4+CD25� T-cells to become regulatory with aforeign peptide, Hemaglutanin, HA (306-319), however,recently we have induced TR with a diabetes autoantigen,GAD65 (555-567). In both systems selection of antigen-specific regulatory T-cells is dependent on MHC class IItetramers. Antigen activation of CD4+CD25� cells results intwo populations of cells that are CD25, Tetramer+ andTetramer�, with only the Tetramer cells suppressingantigen-specifically. These induced TR behave antigen-specifically in that they need to be activated on cognateantigen in order to be suppressive, but once activated theyare antigen nonspecific. Thus, suggesting that antigen-specific TR may be able to control the response tounidentified autoantigens once activated on a knownautoantigen. The mechanism by which these cells suppressis unknown, however it does not appear that respondercells undergo cytolysis. Notably, GAD-specific TR can begenerated from both normal and diabetic subjects. Recentresults show that these cells can be expanded at least 50-fold and retain suppressive ability, suggesting that thesecells have the potential to be useful therapeutically.Supported by NIH grant DK072457, The Irvington Instituteand Dana Foundation for Immunological Research, and TheJuvenile Diabetes Research Foundation.

doi:10.1016/j.clim.2006.04.201

OR.46. Comparative Analysis of FOXP3 in TargetOrgan and Circulation of Rheumatic Patients.Duojia Cao,1 Sukanya Raghavan,1 Mona Widhe,1

Ann-Kristin Ulfgren,1 Giovanna Roncador,2 Alison Banham,3

Vivianne Malmstrom,1 Tina Trollmo.1 1Unit ofRheumatology, Karolinska Institutet, Stockholm, Sweden;2Biotechnology program, Centro Nacional deInvestigaciones Oncologicas, Madrid, Spain; 3Dept. ofClinical Laboratory Sciences, John Radcliffe Hospital,Oxford, United Kingdom.

FOXP3 is still the only specific marker for naturalregulatory T-cells and in a naive host it correlates withthe level of expression of surface-bound CD25, however inan antigen experienced host both activated and regulatoryT-cells express CD25. In this study rheumatic patients withchronic joint inflammation were recruited, and both theinflamed site and peripheral blood were analyzed.Our data using flow cytometry and RT PCR demonstratedthat FOXP3 expression was most abundant in theCD25brightCD4+ population independent of site of sam-pling. Interestingly, joint fluid derived CD25-CD4 T-cells

expressed low but distinct FOXP3+ levels as compared totheir counterpart in circulation. We also detected FOXP3+cells in the synovial tissue of patients with rheumaticdisease using immunohistochemistry and a monoclonalantibody against scurfin. The FOXP3+ cells were foundmainly in T-cell rich areas of the tissue and were confinednot only to CD25+ but were also found among CD25� T-cells, indicating that at a chronically active site of inflam-mation CD25 could be down regulated on the natural regu-latory T-cells, alternatively FOXP3+CD25� cells exist. Inconclusion, at the site of inflammation in patients withrheumatic disease, FOXP3+ regulatory T-cells exist both inthe inflamed joint fluid and tissue. Also, the new methodallows frequency determinations of such cells during thedifferent phases of disease progression and in response totreatments.

doi:10.1016/j.clim.2006.04.202

OR.47. Characterization of FOXP3+CD4+CD25+Regulatory T-Cells from Mesenteric Lymph Nodes inHuman Ulcerative Colitis (UC).Konstantinos Papadakis,1 Masayuki Saruta,1 Qi Yu,1 AlisonBanham.2 1Gastroenterology, Cedars-Sinai Medical Center/UCLA, Los Angeles, CA; 2Nuffield Department of ClinicalLaboratory Sciences, University of Oxford, Oxford, UnitedKingdom.

Background and Aims: Treg cells are able to control thedevelopment of experimental colitis. The aim of our studywas to determine the presence and functional character-istics of Treg cells isolated from Mesenteric lymph nodes(MLN) in human UC. Results: MLN from human UC coloncontain a subset of CD4+CD25+ T-cells with phenotypiccharacteristics of Treg cells (expression of CCR4, CD122,HLA-DR, GITR and intracellular CTLA4). FoxP3+ Cells weredetected by IHC in the MLN medulla dispersed in the T-cellrich areas and occasionally in the follicles. CD4+CD25+ cellsexpress FoxP3 mRNA and protein and potently suppress theproliferation of autologous MLN CD4+ T-cells. In contrast toperipheral blood Treg from healthy donors, the CD4+CD25hiand CD4+CD25int MLN T-cell subsets exhibit potent sup-pressor activity in vitro. The suppressor function ofCD4+CD25+FoxP3+ cells is not mediated through TGF-betaor IL-10, whereas addition of exogenous IL-2 restores theproliferation of responder CD4+ MLN T-cells. In addition totheir ability to suppress the proliferation of CD4+CD25�cells, CD4+CD25+ cells potently suppressed the secretion ofIL-2, IL-5, IL-13 and IFN-g by CD4+CD25� effector T-cells.Conclusions: CD4+CD25+ T-cells isolated from human co-lonic MLN in UC display typical features of Treg cells andpossess potent suppressor activity in vitro in spite ofpersistent mucosal inflammation. These data suggest thattheir suppressor activity is likely compromised in vivo andfinding ways to restore their suppressive activity isdesirable in order to achieve long-term tolerance in thegut.

doi:10.1016/j.clim.2006.04.203

Abstracts S21