Y. Akiyama, N. Shirasugi, O. Aramaki, K. Matsumoto, M. Shimazu et al., , 2002.

O. Aramaki, N. Shirasugi, Y. Akiyama, T. Takayama, M. Shimazu et al., Induction of operational tolerance and generation of regulatory cells after intratracheal delivery of alloantigen combined with nondepleting anti-CD4 monoclonal antibody, Transplantation, vol.76, pp.1305-1314, 2003.

C. Ballet, K. Renaudin, N. Degauque, H. L. Mai, F. Boë-ffard et al., Indirect CD4+ TH1 response, antidonor antibodies and diffuse C4d graft deposits in long-term recipients conditioned by donor antigens priming, Am. J. Transplant, vol.9, pp.697-708, 2009.

M. J. Bell, J. M. Burrows, R. Brennan, J. J. Miles, J. Tellam et al., The peptide length specificity of some HLA class I alleles is very broad and includes peptides of up to 25 amino acids in length, Mol. Immunol, vol.46, pp.1911-1917, 2009.

D. X. Beringer, F. S. Kleijwegt, F. Wiede, A. R. Van-der-slik, K. L. Loh et al., , 2015.

, T cell receptor reversed polarity recognition of a self-antigen major histocompatibility complex, Nat. Immunol, vol.16, pp.1153-1161

S. Bé-zie, E. Picarda, J. Ossart, L. Tesson, C. Usal et al., IL-34 is a Treg-specific cytokine and mediates transplant tolerance, J. Clin. Invest, vol.125, pp.3952-3964, 2015.

S. Bé-zie, E. Picarda, J. Ossart, B. Martinet, I. Anegon et al., Compensatory Regulatory Networks between CD8 T, B, and Myeloid Cells in Organ Transplantation Tolerance, J. Immunol, vol.195, pp.5805-5815, 2015.

S. Bé-zie, I. Anegon, and C. Guillonneau, Advances on CD8+ Treg Cells and Their Potential in Transplantation, Transplantation, vol.102, pp.1467-1478, 2018.

S. Bé-zie, B. Charreau, N. Vimond, J. Lasselin, N. Gé-rard et al., , 2019.

, Human CD8+ Tregs expressing a MHC-specific CAR display enhanced suppression of human skin rejection and GVHD in NSG mice, Blood Adv, vol.3, pp.3522-3538

S. Bé-zie, D. Meistermann, L. Boucault, S. Kilens, J. Zoppi et al.,

, Ex Vivo Expanded Human Non-Cytotoxic CD8 + CD45RC low/À Tregs Efficiently Delay Skin Graft Rejection and GVHD in Humanized Mice, Front. Immunol, vol.8, 2014.

M. L. Boytim, S. C. Lyu, R. Jung, A. M. Krensky, and C. Clayberger, , 1998.

, 4253 residues 65-79 of an HLA class II sequence: functional similarities but mechanistic differences with the immunosuppressive drug rapamycin, Cell Reports, vol.29, pp.2215-2222, 2019.

R. Buelow, P. Veyron, C. Clayberger, P. Pouletty, and J. L. Touraine, , 1995.

, Prolongation of skin allograft survival in mice following administration of ALLOTRAP, Transplantation, vol.59, pp.455-460

S. R. Burrows, J. Rossjohn, and J. Mccluskey, Have we cut ourselves too short in mapping CTL epitopes?, Trends Immunol, vol.27, pp.11-16, 2006.

K. F. Chan, B. S. Gully, S. Gras, D. X. Beringer, L. Kjer-nielsen et al., Divergent T-cell receptor recognition modes of a HLA-I, 2018.

, Nat. Commun, vol.9, p.1026

W. L. Delano, Unraveling hot spots in binding interfaces: progress and challenges, Curr. Opin. Struct. Biol, vol.12, pp.14-20, 2002.

P. Douillard, C. Vignes, R. Josien, E. Chiffoleau, J. M. Heslan et al., Reassessment of the role of CD8+ T cells in the induction of allograft tolerance by donor-specific blood transfusion, Eur. J. Immunol, vol.29, pp.1919-1924, 1999.

L. M. Ebert, Y. C. Liu, C. S. Clements, N. C. Robson, H. M. Jackson et al., A long, naturally presented immunodominant epitope from NY-ESO-1 tumor antigen: implications for cancer vaccine design, Cancer Res, vol.69, pp.1046-1054, 2009.

P. Emsley, B. Lohkamp, W. G. Scott, and K. Cowtan, Features and development of Coot, Acta Crystallogr. D Biol. Crystallogr, vol.66, pp.486-501, 2010.

P. R. Evans and G. N. Murshudov, How good are my data and what is the resolution?, Acta Crystallogr. D Biol. Crystallogr, vol.69, pp.1204-1214, 2013.

L. Flippe, S. Bé-zie, I. Anegon, and C. Guillonneau, Future prospects for CD8 + regulatory T cells in immune tolerance, Immunol. Rev. Published on, 2019.
URL : https://hal.archives-ouvertes.fr/inserm-02341615

A. Fuchs, M. Gliwi-nski, N. Grageda, R. Spiering, A. K. Abbas et al., Minimum Information about T Regulatory Cells: A Step toward Reproducibility and Standardization, Front. Immunol, vol.8, p.1844, 2018.
URL : https://hal.archives-ouvertes.fr/inserm-02157859

C. Guillonneau, M. Hill, F. Hubert, E. Chiffoleau, C. Hervé et al., CD40Ig treatment results in allograft acceptance mediated by CD8CD45RC T cells, IFN-gamma, and indoleamine 2,3-dioxygenase, J. Clin. Invest, vol.117, pp.1096-1106, 2007.

C. Guillot, C. Guillonneau, P. Mathieu, C. A. Gerdes, S. Mé-noret et al., Prolonged blockade of CD40-CD40 ligand interactions by gene transfer of CD40Ig results in long-term heart allograft survival and donor-specific hyporesponsiveness, but does not prevent chronic rejection, J. Immunol, vol.168, pp.1600-1609, 2002.

C. Hassan, E. Chabrol, L. Jahn, M. G. Kester, A. H. De-ru et al., Naturally processed non-canonical HLA-A*02:01 presented peptides, J. Biol. Chem, vol.290, pp.2593-2603, 2015.

T. M. Josephs, E. J. Grant, and S. Gras, Molecular challenges imposed by MHC-I restricted long epitopes on T cell immunity, Biol. Chem, vol.398, pp.1027-1036, 2017.

W. Kabsch, XDS. Acta Crystallogr. D Biol. Crystallogr, vol.66, pp.125-132, 2010.

J. Liu, Z. Liu, P. Witkowski, G. Vlad, J. S. Manavalan et al., Rat CD8+, 2004.

, FOXP3+ T suppressor cells mediate tolerance to allogeneic heart transplants, inducing PIR-B in APC and rendering the graft invulnerable to rejection, Transpl. Immunol, vol.13, pp.239-247

R. Mallone, S. A. Kochik, E. M. Laughlin, V. H. Gersuk, H. Reijonen et al., Differential recognition and activation thresholds in human autoreactive GAD-specific T-cells, Diabetes, vol.53, pp.971-977, 2004.

R. Marcé-n, Immunosuppressive drugs in kidney transplantation: impact on patient survival, and incidence of cardiovascular disease, malignancy and infection, Drugs, vol.69, pp.2227-2243, 2009.

E. L. Masteller, M. R. Warner, W. Ferlin, V. Judkowski, D. Wilson et al., Peptide-MHC class II dimers as therapeutics to modulate antigen-specific T cell responses in autoimmune diabetes, J. Immunol, vol.171, pp.5587-5595, 2003.

E. L. Masteller, Q. Tang, and J. A. Bluestone, Antigen-specific regulatory T cells-ex vivo expansion and therapeutic potential, Semin. Immunol, vol.18, pp.103-110, 2006.

H. Meier-kriesche, S. Li, R. W. Gruessner, J. J. Fung, R. T. Bustami et al., Immunosuppression: evolution in practice and trends, Am. J. Transplant, vol.6, pp.1111-1131, 1994.

B. Murphy, K. S. Kim, R. Buelow, M. H. Sayegh, and W. W. Hancock, , 1997.

, Synthetic MHC class I peptide prolongs cardiac survival and attenuates transplant arteriosclerosis in the Lewis->Fischer 344 model of chronic allograft rejection, Transplantation, vol.64, pp.14-19

B. Murphy, C. C. Magee, S. I. Alexander, A. M. Waaga, H. W. Snoeck et al., Inhibition of allorecognition by a human class II MHC-derived peptide through the induction of apoptosis, J. Clin. Invest, vol.103, pp.859-867, 1999.

B. Murphy, J. Yu, Q. Jiao, M. Lin, T. Chitnis et al., A novel mechanism for the immunomodulatory functions of class II MHC-derived peptides, J. Am. Soc. Nephrol, vol.14, pp.1053-1065, 2003.

V. Oling, K. Geubtner, J. Ilonen, and H. Reijonen, A low antigen dose selectively promotes expansion of high-avidity autoreactive T cells with distinct phenotypic characteristics: a study of human autoreactive CD4+T cells specific for GAD65, Autoimmunity, vol.43, pp.573-582, 2010.

E. Picarda, I. Anegon, and C. Guillonneau, T-cell receptor specificity of CD8(+) Tregs in allotransplantation, Immunotherapy, vol.3, pp.35-37, 2011.

E. Picarda, S. Bé-zie, V. Venturi, K. Echasserieau, E. Mé-rieau et al., MHC-derived allopeptide activates TCR-biased CD8+ Tregs and suppresses organ rejection, J. Clin. Invest, vol.124, pp.2497-2512, 2014.
URL : https://hal.archives-ouvertes.fr/inserm-02167782

E. Picarda, S. Bé-zie, L. Boucault, E. Autrusseau, S. Kilens et al., Transient antibody targeting of CD45RC induces transplant tolerance and potent antigen-specific regulatory T cells, JCI Insight, vol.2, p.90088, 2017.
URL : https://hal.archives-ouvertes.fr/hal-01833115

S. J. Powis, L. L. Young, E. Joly, P. J. Barker, L. Richardson et al., The rat cim effect: TAP allele-dependent changes in a class I MHC anchor motif and evidence against C-terminal trimming of peptides in the ER, Immunity, vol.4, pp.159-165, 1996.

R. J. Read, Pushing the boundaries of molecular replacement with maximum likelihood, Acta Crystallogr. D Biol. Crystallogr, vol.57, pp.1373-1382, 2001.

M. J. Rist, A. Theodossis, N. P. Croft, M. A. Neller, A. Welland et al., HLA peptide length preferences control CD8+ T cell responses, J. Immunol, vol.191, pp.561-571, 2013.

M. G. Rudolph, J. Stevens, J. A. Speir, J. Trowsdale, G. W. Butcher et al., Crystal structures of two rat MHC class Ia (RT1-A) molecules that are associated differentially with peptide transporter alleles TAP-A and TAP-B, J. Mol. Biol, vol.324, pp.975-990, 2002.

P. Sagoo, N. Ali, G. Garg, F. O. Nestle, R. I. Lechler et al., Human regulatory T cells with alloantigen specificity are more potent inhibitors of alloimmune skin graft damage than polyclonal regulatory T cells, Acta Crystallogr. D Biol. Crystallogr, vol.3, pp.368-380, 2011.

J. A. Speir, J. Stevens, E. Joly, G. W. Butcher, and I. A. Wilson, Two different, highly exposed, bulged structures for an unusually long peptide bound to rat MHC class I RT1-Aa, Immunity, vol.14, pp.81-92, 2001.

J. Stevens, K. H. Wiesm?-uller, P. J. Barker, P. Walden, G. W. Butcher et al., Efficient generation of major histocompatibility complex class I-peptide complexes using synthetic peptide libraries, J. Biol. Chem, vol.273, pp.2874-2884, 1998.

J. Stevens, K. H. Wiesm?-uller, P. Walden, J. , and E. , Peptide length preferences for rat and mouse MHC class I molecules using random peptide libraries, Eur. J. Immunol, vol.28, pp.1272-1279, 1998.

Q. Tang and F. Vincenti, Transplant trials with Tregs: perils and promises, J. Clin. Invest, vol.127, pp.2505-2512, 2017.

C. J. Thorpe, D. S. Moss, S. J. Powis, J. C. Howard, G. W. Butcher et al., An analysis of the antigen binding site of RT1.Aa suggests an allele-specific motif, Immunogenetics, vol.41, pp.329-331, 1995.

H. Ueta, Y. Kitazawa, Y. Sawanobori, T. Ueno, S. Ueha et al., Single blood transfusion induces the production of donor-specific alloantibodies and regulatory T cells mainly in the spleen, Int. Immunol, vol.30, pp.53-67, 2018.

C. Vignes, E. Chiffoleau, P. Douillard, R. Josien, H. Pêche et al., Anti-TCR-specific DNA vaccination demonstrates a role for a CD8+ T cell clone in the induction of allograft tolerance by donor-specific blood transfusion, J. Immunol, vol.165, pp.96-101, 2000.

K. L. Womer, C. C. Magee, N. Najafian, J. P. Vella, E. L. Milford et al., A pilot study on the immunological effects of oral administration of donor major histocompatibility complex class II peptides in renal transplant recipients, Clin. Transplant, vol.22, pp.754-759, 2008.

W. Zang, M. , and B. , Peptide-mediated immunosuppression, Am. J. Ther, vol.12, pp.592-599, 2005.

. Picarda, Human PBMCs were isolated by Ficoll-Paque density-gradient centrifugation at 2000 rpm for 20 min at room temperature without brake. Remaining red blood cells and platelets were removed using 5 min incubation with a hypotonic solution and centrifugation at 1000 rpm for 10 min at 4 C. For pDC and T cell sorting, B cells, monocytes and NK cells were magnetically depleted (Dynabeads, Invitrogen) by using anti-CD19 (clone: HBI19, eBiociences), anti-CD14 (clone: M5E2, eBiociences) and anti-CD16 (Clone: 3G8, purified) mAbs respectively, Enriched PBMCs were stained with anti-CD45RC-FITC (clone: MT2, IQ-Products), anti-CD8a-PE-Cy7 (clone: RPT8, eBiociences) and anti-Nrp1-PE (clone: u21-1283, BD Biosciences) for sorting of CD8a + CD45RC low Tregs and Neurophilin-1 + pDCs. Enriched PBMCs were stained with anti-CD3-PeCy7 (clone SK7, BD Biosciences), anti-CD4-PerCP-Cy5.5 (clone: RPA-T4, BD Biosciences) and anti-CD25-APC-Cy7 (clone: M-A251, BD Biosciences) mAbs for sorting of CD4 + CD25 -Teff cells. APCs were obtained from PBMCs by either magnetically depleting T cells with an anti-CD3 (OKT3 purified, 5mg/ml) mAb, 2014.

, 1A rats, 5 3 10 4 CD8 + CD45RC low Tregs from tolerant > 120 days CD40Ig-treated recipients and 120 mg/ml of individual allogeneic peptides were plated in triplicate in RPMI-1640 medium supplemented with 10% FCS and 0.5mM of CpG ODN 1826 in round-bottom 96 wells plates for 6 days at 37 C, 5% CO2. The CD25 activation marker was then analyzed by flow cytometry, Peptide stimulation assay Rat 1.25 3 10 4 pDCs from naive LEW

, CpG ODN 2006 (0.5 mM) and the different synthesized peptides (120 mg/ml) at a ratio 4:1 of Tregs:pDCs for 5 days. CD8 + CD45RC low Treg activation was analyzed based on expression of CD69 and CD25 markers. As negative control and in order to normalize the results, an irrelevant peptide was used (ALIAPV-HAV). Dapi was used as viability marker. As a positive control, CD8 + CD45RC low Tregs were stimulated with anti-CD3 (OKT3, 1 mg/ml) and anti-CD28 mAbs (clone: CD28.2; 1mg/ml). Results were analyzed using the FACS Canto II cytometer, + CD45RC low Treg cells and autologous pDCs from the same healthy HLA-A2 + donor were co-cultured in serum-free Texmacs medium (Miltenyi Biotec) supplemented with IL-2 (25 U/ml, Proleukin, Novartis)

. Emsley, Refolding was performed in a cold solution (4 C) containing: 3M urea, 10 mM Tris-HCl pH 8, 2 mM Na-EDTA, 400 mM L-arginine-HCl, 0.5 mM oxidized glutathione, and 5 mM reduced glutathione. Each pMHC was then purified using a series of anion exchange chromatography columns. Crystals of the RT1.A a /Bu31-10 complex were grown by the vapor-diffusion method in a hanging-drop configuration at 20 C, at a concentration of 10 mg/mL in 0.1 M Bis-Tris-Propane pH 6.6, 28% PEG 8000 and 0.2 M Mg 2 SO 4 . Crystals were directly flash frozen in liquid nitrogen and data were collected on the MX2 beamline at the Australian Synchrotron, Clayton using the ADSC-Quantum 315r CCD detector (at 100K), 10 4 CFSE-labeled CD4 + CD25 -T cells from naive LEW.1A rats and 1.25 3 10 4 allogeneic pDCs from naive donor LEW.1W animals were plated in triplicate for 6 days in supplemented RPMI-1640 medium in round-bottom 96 wells plates in presence of 5 3 10 4 facssorted CD8 + CD45RC low Tregs from tolerant > 120 days CD40Ig-treated recipients, 2001.

, The fluorescent dye Sypro orange was used to monitor the protein unfolding, using a Real Time Detection system (Corbett RotorGene 3000) originally designed for PCR. Each pMHC complex was tested twice at two different concentrations (5 and 10 mM) in duplicate in 10 mM Tris-HCl pH8, 150 mM NaCl. Each pMHC was heated from 25 to 95 C, and the fluorescence intensity measured (excitation at 530 nm, and emission at 555 nm). The Tm, or thermal melting point, Thermal stability assay Stability of the three peptide-RT1.A a complexes was assessed using a thermal shift assay

, QUANTIFICATION AND STATISTICAL ANALYSIS For the peptide activation test, a non-parametric Wilcoxon signed-rank test, comparing column median to a hypothetical value of 1.0, was done. Statistical significance for the phenotype of activated cells, cytokine expression and proliferation assay was evaluated by a one-tailed Mann Whitney t test. Allo-antibody titration and suppression assay with range of doses were analyzed by Two Way Anova Matched test and Bonferroni post-test. Graft survival was analyzed by Kaplan-Meier log-rank test

, DATA AND CODE AVAILABILITY Data Resources Crystal structure files for the RT1.Aa/Bu31-10 crystal structure have been deposited in the PDB under accession number 6NF7