GARP: a key receptor controlling FOXP3 in human regulatory T cells
M. Probst-Kepper
Junior Research Group for Xenotransplantation, Department of Visceral and Transplant Surgery, Hannover Medical School, Hannover, Germany
These authors contributed equally to this work.
Search for more papers by this authorR. Geffers
Mucosal Immunity Research Group, Helmholtz Centre for Infection Research, Braunschweig, Germany
These authors contributed equally to this work.
Search for more papers by this authorA. Kröger
Department of Molecular Biotechnology, Helmholtz Centre for Infection Research, Braunschweig, Germany
These authors contributed equally to this work.
Search for more papers by this authorN. Viegas
Department of Molecular Immunology, Helmholtz Centre for Infection Research, Braunschweig, Germany
Search for more papers by this authorH.-J. Hecht
Department of Structural Biology, Helmholtz Centre for Infection Research, Braunschweig, Germany
Search for more papers by this authorH. Lünsdorf
Department of Environmental Microbiology, Helmholtz Centre for Infection Research, Braunschweig, Germany
Search for more papers by this authorR. Roubin
Institut de Cancerologie de Marseille, Marseille, France
Search for more papers by this authorD. Moharregh-Khiabani
Junior Research Group for Xenotransplantation, Department of Visceral and Transplant Surgery, Hannover Medical School, Hannover, Germany
Search for more papers by this authorK. Wagner
Junior Research Group for Xenotransplantation, Department of Visceral and Transplant Surgery, Hannover Medical School, Hannover, Germany
Search for more papers by this authorF. Ocklenburg
Junior Research Group for Xenotransplantation, Department of Visceral and Transplant Surgery, Hannover Medical School, Hannover, Germany
Search for more papers by this authorA. Jeron
Mucosal Immunity Research Group, Helmholtz Centre for Infection Research, Braunschweig, Germany
Search for more papers by this authorH. Garritsen
Institute for Clinical Transfusion Medicine, Städtisches Klinikum Braunschweig gGmbH, Braunschweig, Germany
Search for more papers by this authorT.P. Arstila
Haartman Institute, Department of Immunology, University of Helsinki, Haartmaninkatu, Finland
Search for more papers by this authorE. Kekäläinen
Haartman Institute, Department of Immunology, University of Helsinki, Haartmaninkatu, Finland
Search for more papers by this authorR. Balling
Biological Systems Analysis, Helmholtz Centre for Infection Research, Inhoffenstraße, Braunschweig, Germany
Search for more papers by this authorH. Hauser
Department of Molecular Biotechnology, Helmholtz Centre for Infection Research, Braunschweig, Germany
Search for more papers by this authorCorresponding Author
J. Buer
Institute for Medical Microbiology, University Essen, Essen, Germany
Correspondence to: Jan BUER, Institute for Medical Microbiology, University Essen, Hufelandstr, 55, D-45122 Essen, Germany.Tel.: + 49 201 723-3500Fax: + 49 201 723-5602E-mail: [email protected]In memoriam of Dr. Hans-Jürgen HechtSearch for more papers by this authorS. Weiss
Department of Molecular Immunology, Helmholtz Centre for Infection Research, Braunschweig, Germany
Search for more papers by this authorM. Probst-Kepper
Junior Research Group for Xenotransplantation, Department of Visceral and Transplant Surgery, Hannover Medical School, Hannover, Germany
These authors contributed equally to this work.
Search for more papers by this authorR. Geffers
Mucosal Immunity Research Group, Helmholtz Centre for Infection Research, Braunschweig, Germany
These authors contributed equally to this work.
Search for more papers by this authorA. Kröger
Department of Molecular Biotechnology, Helmholtz Centre for Infection Research, Braunschweig, Germany
These authors contributed equally to this work.
Search for more papers by this authorN. Viegas
Department of Molecular Immunology, Helmholtz Centre for Infection Research, Braunschweig, Germany
Search for more papers by this authorH.-J. Hecht
Department of Structural Biology, Helmholtz Centre for Infection Research, Braunschweig, Germany
Search for more papers by this authorH. Lünsdorf
Department of Environmental Microbiology, Helmholtz Centre for Infection Research, Braunschweig, Germany
Search for more papers by this authorR. Roubin
Institut de Cancerologie de Marseille, Marseille, France
Search for more papers by this authorD. Moharregh-Khiabani
Junior Research Group for Xenotransplantation, Department of Visceral and Transplant Surgery, Hannover Medical School, Hannover, Germany
Search for more papers by this authorK. Wagner
Junior Research Group for Xenotransplantation, Department of Visceral and Transplant Surgery, Hannover Medical School, Hannover, Germany
Search for more papers by this authorF. Ocklenburg
Junior Research Group for Xenotransplantation, Department of Visceral and Transplant Surgery, Hannover Medical School, Hannover, Germany
Search for more papers by this authorA. Jeron
Mucosal Immunity Research Group, Helmholtz Centre for Infection Research, Braunschweig, Germany
Search for more papers by this authorH. Garritsen
Institute for Clinical Transfusion Medicine, Städtisches Klinikum Braunschweig gGmbH, Braunschweig, Germany
Search for more papers by this authorT.P. Arstila
Haartman Institute, Department of Immunology, University of Helsinki, Haartmaninkatu, Finland
Search for more papers by this authorE. Kekäläinen
Haartman Institute, Department of Immunology, University of Helsinki, Haartmaninkatu, Finland
Search for more papers by this authorR. Balling
Biological Systems Analysis, Helmholtz Centre for Infection Research, Inhoffenstraße, Braunschweig, Germany
Search for more papers by this authorH. Hauser
Department of Molecular Biotechnology, Helmholtz Centre for Infection Research, Braunschweig, Germany
Search for more papers by this authorCorresponding Author
J. Buer
Institute for Medical Microbiology, University Essen, Essen, Germany
Correspondence to: Jan BUER, Institute for Medical Microbiology, University Essen, Hufelandstr, 55, D-45122 Essen, Germany.Tel.: + 49 201 723-3500Fax: + 49 201 723-5602E-mail: [email protected]In memoriam of Dr. Hans-Jürgen HechtSearch for more papers by this authorS. Weiss
Department of Molecular Immunology, Helmholtz Centre for Infection Research, Braunschweig, Germany
Search for more papers by this authorAbstract
Recent evidence suggests that regulatory pathways might control sustained high levels of FOXP3 in regulatory CD4+CD25hi T (Treg) cells. Based on transcriptional profiling of ex vivo activated Treg and helper CD4+CD25− T (Th) cells we have identified GARP (glycoprotein-A repetitions predominant), LGALS3 (lectin, galactoside-binding, soluble, 3) and LGMN (legumain) as novel genes implicated in human Treg cell function, which are induced upon T-cell receptor stimulation. Retroviral overexpression of GARP in antigen-specific Th cells leads to an efficient and stable re-programming of an effector T cell towards a regulatory T cell, which involves up-regulation of FOXP3, LGALS3, LGMN and other Treg-associated markers. In contrast, overexpression of LGALS3 and LGMN enhance FOXP3 and GARP expression, but only partially induced a regulatory phenotype. Lentiviral down-regulation of GARP in Treg cells significantly impaired the suppressor function and was associated with down-regulation of FOXP3. Moreover, down-regulation of FOXP3 resulted in similar phenotypic changes and down-regulation of GARP. This provides compelling evidence for a GARP-FOXP3 positive feedback loop and provides a rational molecular basis for the known difference between natural and transforming growth factor-β induced Treg cells as we show here that the latter do not up-regulate GARP. In summary, we have identified GARP as a key receptor controlling FOXP3 in Treg cells following T-cell activation in a positive feedback loop assisted by LGALS3 and LGMN, which represents a promising new system for the therapeutic manipulation of T cells in human disease.
Supporting Information
Fig. S1 Alignment of human GARP with the structure of the ectodomain of human TLR3. (A) Comparison of the ectodomain of TLR3 (pdb-id: 2A0Z) and GARP, conserved sequences are indicated by blue boxes, strictly conserved residues are highlighted white-on-red, and similar residues indicated by red characters. Secondary structure elements of the TLR3 structure are indicated by arrows (β strand), coils (helices) and TT (turns). (B) Ribbon diagram of a hypothetical GARP dimmer model, based on the dimerization proposed for TLR3. The prominent loops at 296–308 and 421–432 of GARP (▾) could have similar functions for dimerization as proposed for TLR3. Putative glycolysation sites are indicated with space-filling representations.
Fig. S2 GARP induced transcriptional control. (A) Analysis of LGMN, UBD, IL1R2, and GARP mRNA by semi-quantitative RT-PCR in Th cells transduced with GARP, FOXP3, LGMN, LGALS3, and GFP as described in Figs 1–4. cDNA was tested in threefold dilutions starting with 1:3 using RPS9 as housekeeping control. (B) Western blot analysis of LGMN protein expression in the same cells as in (A).
Fig. S3 GARP induces genes of the extended Treg-signature. (A) Semi-quantitative RT-PCR analysis of IL7R, CPE, RYR-1 and HPGD in Th cells as in Figs 1–4 under resting conditions (no stim.) and 3 days after stimulation with anti-CD3 and 100 U/ml IL-2 as described in Fig. S1. (B) Quantitative real-time RT-PCR analysis of GARP and FOXP3 in Th cells as in (A) tested under resting conditions and 3 days after stimulation as in (A). Relative mRNA expression of ThGFP cells was arbitrarily set as 1.
Fig. S4 Characterization of the Treg cell line used in this study and for siRNA experiments. (A) This alloantigen-reactive Treg cells line (TregTHU) used, has been established and characterized in detail recently [15]. These cells constitutively express known Treg-markers, FOXP3, LGALS3, and CD25 are shown as selected examples. For comparison T cells derived from sorted CD4+CD25− Th cells were stimulated with the same allogeneic EBV B cells and IL-2 in the presence of vehicle or 10 ng/ml TGF-β1. Analysis was done at day 7 after stimulation. (B) The same cells as in (A) were stimulated with anti-CD3/-CD28 DynalBeads (Invitrogen) and IL-2 for 6 hrs in the presence of 10 μg/ml brefeldin and tested for intracellular IL-2 and FOXP3 expression. Allo-antigen specific Th cells served as control. (C) Lentiviral transduction efficacy of the Treg cells as in (A) is similar as compared to Th cells. (D) The hallmark feature of Treg cells, anergy and suppressor function, are preserved over a period of six months of in vitro expansion of the same Treg cells as in (A) and Figs 1–5.
Fig. S5 GARP is not up-regulated in TGF-β1-induced Treg cells. (A) Sorted CD4+CD25− Th cells were stimulated with anti-CD3/-CD28/IL-2 without (Th), in the presence of solvent (Thvehicle), and 10 ng/ml human TGF-β1 (ThTGF-β1) as in Fig. S4. Expression of GARP and FOXP3 mRNA was assessed at day 6 by real-time RT-PCR as in Fig. 1. (B) TGF-β1-induced Treg cells as in (A) were tested for proliferation and suppressor function (upper panel) and proliferation with exogenous IL-2 (lower panel); bkg. ∇ background proliferation, stim. ∇ T-cell proliferation induced by irradiated allogeneic EBV B cells. Proliferation was assessed at day 3 by measuring incorporation of H3-thymidin (cpm).
Fig. S6 Anergy and suppressor function in alloantigen-specific Th cells transduced with GARP following cryopreservation. (A) Treg cells (Treg cell lines MPO and HG [15]) and Th cells as in Figs 1–4 were stimulated for proliferation using irradiated EBV B cells in the absence (stim.) or presence of IL-2 (stim.+IL-2) 1 week after thawing cryopreserved cells; bkg. ∇ background proliferation. Proliferation was assessed at day 3 by measuring incorporation of H3-thymidin (cpm). Treg and Th cells as in (A) were tested for suppressor function of alloantigen-stimulated parental Th cells at a ratio of 1:1. Percent inhibition of Th cell proliferation by the addition of retrovirally engineered Th cells is indicated, setting addition of wilt-type Th cells (Th/Th) as 100%. Proliferation was assessed at day 3.
Fig. S7 Effects of S6A-mutant LGALS3 on gene expression. (A) Th cells transduced with either wild-type (ThLGALS3) or mutant (ThLGALS3S6A) were analysed for FOXP3 and CD25 expression at day 3 following stimulation with anti-CD3 and 100 U/ml IL-2 compared to ThGFP cells. (B) Quantitative real-time RT-PCR analysis of GARP, LGALS3, LGMN and KLF-2 expression of the same cells as in (A). Th cells were tested under resting conditions and 3 days after stimulation with anti-CD3 and 100 U/ml IL-2. Relative mRNA expression of ThGFP cells was arbitrarily set as 1.
Table S1 Differential gene expression of resting and activated CD4+CD25hi Treg versus CD4+CD25− Th cells analysed ex vivo as detected by array analysis
Table S2 Differential gene expression Treg cells, ThGARP and ThFOXP3 compared to ThGFP cells
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