University of Rhode Island DigitalCommons@URI Institute for Immunology and Informatics Faculty Institute for Immunology and Informatics (iCubed) Publications 2008 Activation of natural regulatory T cells by IgG Fc- derived peptide "Tregitopes" Anne S. De Groot University of Rhode Island, [email protected] Leonard Moise University of Rhode Island, [email protected] See next page for additional authors Follow this and additional works at: https://digitalcommons.uri.edu/immunology_facpubs Terms of Use All rights reserved under copyright. Citation/Publisher Attribution De Groot, A. S., Moise, L., McMurry, J. A., Wambre, E., Van Overtvelt, L., Moingeon, P., Scott, D. W., & Martin, W. (2015). Activation of natural regulatory T cells by IgG Fc-derived peptide 'Tregitopes'. Blood, 112(8), 3303-3311. Available at: http://www.bloodjournal.org/content/112/8/3303 This Article is brought to you for free and open access by the Institute for Immunology and Informatics (iCubed) at DigitalCommons@URI. It has been accepted for inclusion in Institute for Immunology and Informatics Faculty Publications by an authorized administrator of DigitalCommons@URI. For more information, please contact [email protected]. Authors Anne S. De Groot, Leonard Moise, Julie A. McMurry, Erik Wambre, Laurence Van Overtvelt, Philippe Moingeon, David W. Scott, and William Martin This article is available at DigitalCommons@URI: https://digitalcommons.uri.edu/immunology_facpubs/74 From www.bloodjournal.org by guest on October 8, 2015. For personal use only. IMMUNOBIOLOGY Activation of natural regulatory T cells by IgG Fc–derived peptide “Tregitopes” Anne S. De Groot,1,2 Leonard Moise,1 Julie A. McMurry,1 Erik Wambre,3 Laurence Van Overtvelt,3 Philippe Moingeon,3 David W. Scott,4 and William Martin1 1EpiVax, Providence, RI; 2University of Rhode Island, Providence; 3Stallergenes, Anthony, France; and 4University of Maryland, Baltimore We have identified at least 2 highly pro- suppression of effector cytokine secre- for the immunosuppressive activity of miscuous major histocompatibility com- tion, reduced proliferation of effector IgG, such as with IVIG, may be related to plex class II T-cell epitopes in the Fc T cells, and caused an increase in cell the activity of regulatory T cells. In this fragment of IgG that are capable of specifi- surface markers associated with TRegs model, regulatory T-cell epitopes in IgG ؉ ؉ cally activating CD4 CD25HiFoxP3 natu- such as FoxP3. In vivo administration of activate a subset of nTRegs that tips the ral regulatory T cells (nTRegs). Coincuba- the murine homologue of the Fc region resulting immune response toward toler- tion of these regulatory T-cell epitopes or Tregitope resulted in suppression of im- ance rather than immunogenicity. (Blood. “Tregitopes” and antigens with periph- mune response to a known immunogen. 2008;112:3303-3311) eral blood mononuclear cells led to a These data suggest that one mechanism Introduction Induction of specific tolerance to self- or to foreign antigens is the We scanned the Fc region of IgG for natural TReg epitopes that goal of therapy for autoimmunity, transplant rejection, and allergy; may explain (1) tolerance to antibody variable regions and (2) the unresponsiveness is also desirable in the context of therapy with induction of tolerance to selected antigens after administration of potentially immunogenic autologous proteins (such as factor VIII) therapeutic immunoglobulins or Ig fusion proteins.7,8 Using periph- and nonautologous proteins (such as botulinum toxin). Until eral blood mononuclear cells (PBMCs) from individuals allergic to recently, therapeutic tolerance induction relied on broad-spectrum either house dust mite Dermatophagoides pteronyssinus (HDM) or interventions that resulted in widespread effects on immunity, to the major birch tree allergen, Bet v 1141-155, we evaluated the rather than on strategies directed toward restoring a balance effect of these IgG TReg epitopes (“Tregitopes”) in a standard 2-step between effector immune responses and regulatory immune re- “bystander suppression” assay. We explored whether the Tregi- sponses to a specific protein. topes induced aTReg to Bet v 1141-155 using HLA DR*1501 tetramers Natural means of controlling autoimmune responses (natural to the Bet v 1141-155 epitope. We also coadministered HDM lysate tolerance) and of inducing tolerance (adaptive tolerance) are and Tregitopes to HLA transgenic mice and observed suppression known to exist. For example, suppression of inflammation by of immune response to HDM as measured by whole-antibody ϩ ϩ CD4 CD25HiFoxP3 natural regulatory T cells (nTRegs)isan enzyme-linked immunosorbent assay (ELISA) and IL-4 enzyme- important mechanism of effector T-cell regulation, and may linked immunosorbent spot (ELISpot). Further studies need to be represent one of the critical forms of autoregulatory response to performed, but these Tregitopes may provide an explanation for the self-antigens. Upon antigen-specific activation through their TCR, limited immunogenicity of Fc fusion proteins, the expansion of ϩ nTRegs are able to suppress bystander effector T-cell responses to CD4 CD25Hi regulatory T cells after administration of therapeutic unrelated antigens by contact-dependent and -independent mecha- IVIG,8 and the observed effect of immunoglobulin therapy on autoimmune diseases and other medical conditions. nisms. Adaptive TReg (aTReg) induction is one outcome of a T-regulatory immune response, and sustained tolerance (to grafts, to allergens, and to autologous proteins) probably requires the existence of aTRegs with the same antigen specificity as the 1-3 Methods self-reactive T cells. Adaptive TRegs are also known as induced TRegs (iTRegs). However, despite extensive efforts and with few Computational epitope mapping 4,5 exceptions, the antigen specificity of nTRegs is still unknown. To determine whether TReg epitopes exist in immunoglobulin G, we used Natural TRegs may also control immune responses to autologous proteins to which central tolerance may not exist. For example, it the EpiMatrix and ClustiMer epitope-mapping algorithms (EpiVax) to scan the complete amino acid sequence of human IgG sequences derived from has been suggested that T cells need to be rendered tolerant to the the human IgG germ-line heavy and light chain sequences (GenBank variable regions of antibodies that have undergone somatic hyper- accession J00228 and J00241, respectively9). The EpiMatrix system is a 6 mutation. To date, no natural TRegs that respond to IgG epitopes suite of epitope-mapping tools (including EpiMatrix, ClustiMer, and have been identified nor have adaptive TRegs to hypervariable IgG BlastiMer) that has been validated over the course of more than a decade, regions been identified. both in vitro and in vivo (for example, see De Groot et al10 and Koita et Submitted February 12, 2008; accepted June 14, 2008. Prepublished online as The publication costs of this article were defrayed in part by page charge Blood First Edition paper, July 25, 2008; DOI 10.1182/blood-2008-02-138073. payment. Therefore, and solely to indicate this fact, this article is hereby marked ‘‘advertisement’’ in accordance with 18 USC section 1734. An Inside Blood analysis of this article appears at the front of this issue. © 2008 by The American Society of Hematology BLOOD, 15 OCTOBER 2008 ⅐ VOLUME 112, NUMBER 8 3303 From www.bloodjournal.org by guest on October 8, 2015. For personal use only. 3304 DE GROOT et al BLOOD, 15 OCTOBER 2008 ⅐ VOLUME 112, NUMBER 8 al11). For this evaluation of IgG sequences, we used EpiMatrix to identify Suppression assays 9-mer peptides likely to bind to at least 1 of 8 common class II alleles HDM lysate. To test for direct (adaptive) suppression of immune response, (DRB1*0101, *0301, *0401, *0701, *0801, *1101, *1301, and *1501).10 sorted and unsorted PBMCs were plated at 5 ϫ 106 Then, using the ClustiMer algorithm, we mapped the EpiMatrix motif cells/well in a 12-well tissue culture dish (Corning, Corning, NY) and stimulated with 2 ␮g/mL matches (for these 8 alleles) along the length of IgG and calculated the D pteronyssinus (HDM) lysate (Greer Labs, Lenoir, NC), HDM lysate with density of motifs for the panel of 8 HLA alleles. Epitope-dense regions, or 10 ␮g/mL Tregitopes, or no peptide and placed in a 37°C incubator with 5% clusters of HLA-binding potential, are usually highly immunogenic in vitro CO and in vivo.12,13 2. After 24 hours, 10 U/mL IL-2 and 20 ng/mL IL-7 (R&D Systems, Minneapolis, MN) were added to each of the wells. The cells were fed every 2 days by half media replacement containing the same concentration of Peptide synthesis cytokines. On day 8, all the PBMCs were collected, washed, and ␮ Peptides were synthesized using solid-phase Fmoc chemistry. Automated restimulated with 2 g/mL HDM lysate. On day 8, all the PBMCs were ␮ ␥ production was carried out on Gilson, Applied Biosystems, and Advanced collected, washed, and restimulated with 2 g/mL HDM lysate in IFN- or Chemtech peptide synthesizers, initially at SynPep (Dublin, CA) and IL-4 ELISpot kits (Mabtech, Nacka Strand, Sweden) according to manufac- subsequently at New England Peptide (Gardner, MA). Tregitope 289 was turer’s instructions. Culture supernatants were analyzed by Searchlight amidated on the C-terminus. Peptides were purified via reverse phase multiplex ELISA assay (Pierce, Woburn, MA). high-performance liquid chromatography (HPLC) to more than 80% purity C3d peptides. Suppression was also tested with a second antigenic and characterized