Healthy Donors Exhibit a CD4 T Cell Repertoire Specific to the Immunogenic Human Hormone H2-Relaxin before Injection This information is current as of September 26, 2021. Aurélien Azam, Yann Gallais, Sergio Mallart, Stephane Illiano, Olivier Duclos, Catherine Prades and Bernard Maillère J Immunol published online 17 May 2019 http://www.jimmunol.org/content/early/2019/05/14/jimmun Downloaded from ol.1800856 Supplementary http://www.jimmunol.org/content/suppl/2019/05/14/jimmunol.180085 http://www.jimmunol.org/ Material 6.DCSupplemental Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! 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Published May 17, 2019, doi:10.4049/jimmunol.1800856 The Journal of Immunology Healthy Donors Exhibit a CD4 T Cell Repertoire Specific to the Immunogenic Human Hormone H2-Relaxin before Injection Aure´lien Azam,*,† Yann Gallais,† Sergio Mallart,‡ Stephane Illiano,x Olivier Duclos,‡ Catherine Prades,* and Bernard Maille`re† H2-relaxin (RLN2) is a two-chain peptide hormone structurally related to insulin with a therapeutic potential in multiple indica- tions. However, multiple injections of human RLN2 induced anti-RLN2 Abs in patients, hampering its clinical development. As T cell activation is required to produce Abs, we wondered whether T cells specific for RLN2 might be already present in the human blood before any injection. We therefore quantified the RLN2-specific T cell repertoire using PBMCs collected from healthy donors. CD4 T cells were stimulated in multiple replicates by weekly rounds of stimulation by dendritic cells loaded with RLN2, and their Downloaded from specificity was assessed by IFN-g ELISPOT. The number of specific T cell lines was used to estimate the frequency of circulating T cells. In vitro T cell response was demonstrated in 18 of the 23 healthy donors, leading to the generation of 70 independent RLN2-specific T cell lines. The mean frequency of RLN2-specific CD4 T cells was similar to that of T cells specific for known immunogenic therapeutic proteins. Using overlapping peptides, we identified multiple T cell epitopes hosted in the N-terminal parts of the a- and b-chains and common to multiple donors, in agreement with their capacity to bind to multiple HLA-DR molecules. Our results provide important clues to the immunogenicity of RLN2 and highlight the weak central immune tolerance http://www.jimmunol.org/ induced against this self-hormone. The Journal of Immunology, 2019, 202: 000–000. 2-relaxin (RLN2), a two-chain peptide hormone struc- are well tolerated, multiple injections of RLN2, as applied in turally related to insulin, is secreted by the corpus luteum systemic sclerosis, led to a high immunization rate (4, 5). After H and placenta during pregnancy (1). RLN2 promotes chronic s.c. administration, anti-RLN2 Abs were detected at growth of epithelial and stromal cells in the cervix and softens the 2 weeks in 43% of scleroderma subjects (4). These Abs were pelvic ligaments in preparation for parturition. RLN2 also contrib- nonneutralizing but were associated with increased serum con- utes to the cardiovascular changes that occur during pregnancy by centrations of recombinant RLN2, which might hamper drug ef- by guest on September 26, 2021 increasing cardiac output. In addition, RLN2 has anti-inflammatory, ficacy, owing to the bell-shaped curve effect observed in different angiogenic, and antifibrotic properties (1). Owing to its broad preclinical and clinical settings (4, 5). biological functions, recombinant RLN2 has been tested in Immunogenicity issues are frequently observed with replace- multiple acute clinical applications including cervical ripening (2) ment enzymes and therapeutic Abs (6) as a result of differences in and heart failure (3) and in chronic conditions in systemic scle- peptide sequence with endogenously produced counterparts. They rosis (4). Although a single dose or a reduced number of injections have also been encountered for some but not all human recombi- nant hormones and cytokines. Most diabetic patients develop anti– *Biologics Research, Sanofi Research and Development, 94400 Vitry sur Seine, human insulin Abs that are generally not neutralizing (7) but France; †Commissariat a` l’Energie Atomique et aux Energies Alternatives (CEA)- might prolong insulin lifetime (8). Altered batches of recombinant Saclay, Universite´ Paris-Saclay, Service d’Inge´nierie Mole´culaire des Prote´ines, 91191 Gif-sur-Yvette, France; ‡Integrated Drug Discovery, Sanofi Research and erythropoietin (EPO) were found to induce autoimmune symp- Development, 91380 Chilly Mazarin, France; and xCardiovascular Diseases and toms (9), whereas IFN-b, GM-CSF, and IL-2 were immunogenic Metabolism, Sanofi Research and Development, 91380 Chilly Mazarin, France in many patients (6). Thus, multiple lines of evidence indicate that ORCIDs: 0000-0001-7680-4679 (A.A.); 0000-0003-1846-8683 (S.M.); 0000-0002- recombinant proteins with a complete human sequence could be 0477-1721 (O.D.). immunogenic. Received for publication June 18, 2018. Accepted for publication April 11, 2019. CD4 T cells contribute to humoral responses, including anti-drug This work was supported by Sanofi and CEA. This work was also supported by the Ab responses, by providing cognate signals and cytokines for the Laboratory of Excellence in Research on Medication and Therapeutic Innovation (to B.M.). B lymphocytes to differentiate into Ab-secreting plasma cells A.A. and Y.G. designed and performed the research, analyzed data, and wrote the (10, 11). CD4 T cells are selected by self-peptides presented paper; S.M., S.I., O.D., and C.P. designed the research and analyzed data; and B.M. by HLA class II molecules in the thymus. A large number of the designed the research, analyzed data, and wrote the paper. autoreactive CD4 T cells are deleted by this process, thus ensuring Address correspondence and reprint requests to Dr. Bernard Maille`re, Commissariat a` immune tolerance to many self-proteins. Multiple studies indicate l’Energie Atomique et aux Energies Alternatives (CEA), CEA-Saclay, Universite´ de Paris-Saclay, Service d’Inge´nierie Mole´culaire des Prote´ines Bat 152, Gif sur Yvette that the amplitude of the CD4 T cell response to foreign Ags (12–15) Cedex 91191, France. E-mail address: [email protected] and therapeutic proteins (16, 17) relies on the size of the naive T cell The online version of this article contains supplemental material. repertoire resulting from thymic selection. A large naive T cell Abbreviations used in this article: CEA, Commissariat a` l’Energie Atomique et aux repertoire is indeed found for immunogenic peptides (12, 14, 15) Energies Alternatives; DC, dendritic cell; EPO, erythropoietin; KLH, keyhole limpet and proteins (13, 16, 17), whereas in the absence of pre-existing hemocyanin; rh, recombinant human; RLN2, H2-relaxin. Ag-specific CD4 T cells, Ag injection did not lead to any immune Copyright Ó 2019 by The American Association of Immunologists, Inc. 0022-1767/19/$37.50 responses, even in the presence of adjuvant (18). www.jimmunol.org/cgi/doi/10.4049/jimmunol.1800856 2 RLN2-SPECIFIC CD4 T CELLS FROM HEALTHY DONORS To shed light on the immunogenicity of RLN2, we quantified molecules with an appropriate biotinylated peptide and serial dilutions the RLN2-specific CD4 T cell repertoire in healthy donors and of competitor peptides. After 24–72 h of incubation and pH neutraliza- identified the CD4 T cell epitopes that contribute to its immuno- tion, samples were applied to 96-well MaxiSorp ELISA plates (Invitrogen) previously coated with 10 mg/ml L243 or B7/21. Bound biotinylated genicity. A large T cell repertoire was found to be supported by peptide was detected by addition of streptavidin–alkaline phosphatase T cell epitopes hosted by both chains. conjugate (GE Healthcare, Buc, France) and 4-methylumbelliferyl phosphate substrate (Sigma-Aldrich). Emitted fluorescence was measured at 450 nm upon excitation at 365 nm. Sequences of the biotinylated reporter peptides Materials and Methods and IC50 values of their unlabeled forms (reference peptides) were the Proteins and peptides following: HA306–318 (PKYVKQNTLKLAT) for DRB1*0101 (3 nM), DRB1*0401 (40 nM), DRB1*1101 (20 nM), DRB1*0701 (30 nM), and Keyhole limpet hemocyanin (KLH) was purchased from Thermo Fisher DRB5*0101 (15 nM); A3 152–166 (EAEQLRAYLDGTGVE) for DRB1*1501 Scientific (Brebie`res, France). RLN2 (UniProtKB, ID: P04090) was (2 nM); MT 2–16 (AKTIAYDEEARRGLE) for DRB1*0301 (16 nM); obtained from American Peptide Company (Sunnyvale, CA), and overlapping peptides (Table I) were synthesized by peptides&elephants (Hennigsdorf, Germany). Generation of RLN2-specific T cell lines PBMCs were purified from the blood of anonymous healthy donors who gave informed consent (Etablissement Franc¸ais du Sang, Rungis, France). Monocyte-derived dendritic cells (DCs) were generated from plastic- adherent cells of PBMCs after 5 d of culture in AIM-V medium (Invitrogen, Villebon-sur-Yvette, France) supplemented with 1000 U/ml Downloaded from recombinant human (rh) IL-4 and rhGM-CSF (both from R&D Sys- tems, Lille, France). CD4 T cells were isolated from autologous nonadherent PBMCs by positive selection using magnetic labeling with anti-CD4 mAbs conjugated to magnetic microbeads followed by magnetic cell sorting, as recommended by the manufacturer (Miltenyi Biotec, Bergisch Gladbach, Germany) (16, 17).