AN ISOLATED DONOR MHC-DERIVED PEPTIDE AND USES THEREOF
20170021001 ยท 2017-01-26
Inventors
Cpc classification
A61K39/001
HUMAN NECESSITIES
C12N5/0637
CHEMISTRY; METALLURGY
A61K40/11
HUMAN NECESSITIES
G01N2800/52
PHYSICS
G01N2800/245
PHYSICS
A61K40/418
HUMAN NECESSITIES
A61P37/06
HUMAN NECESSITIES
A61K2239/38
HUMAN NECESSITIES
International classification
A61K39/00
HUMAN NECESSITIES
Abstract
The invention relates to an peptide derived from a polymorphic region of donor MHC class II molecules which induces tolerance and thus prevents transplant rejection in a patient in need thereof. The invention relates to an isolated peptide of 15 or 16 amino acids long that comprises or consists of the amino acid sequence: REEYARFDSDVGEYR (SEQ ID NO: 1) or a function-conservative variant for use as drug. The invention relates to an in vitro method for determining whether a transplanted patient is tolerant, comprising a step of determining the presence of CD8.sup.+CD45RC.sup.low Tregs in a biological sample obtained from said transplanted patient, wherein the presence of CD8.sup.+CD45RC.sup.low Tregs is indicative of tolerance.
Claims
1. An isolated peptide derived from a MHC class II molecule, wherein the isolated peptide has a length of from 15 to 40 amino acids, and wherein the isolated peptide has an amino acid sequence REEYARFDSDVGEYR (SEQ ID NO: 1) or comprises the amino acid sequence: REEYARFDSDVGEYR (SEQ ID NO: 1), or a function-conservative variant of the isolated peptide, with the caveat that the isolated peptide is not: TABLE-US-00012 (SEQIDNO:2) NREEYARFDSDVGEYR; (SEQIDNO:29) QEEYVRFDSDVGEYR; (SEQIDNO:30) NREEFVRFDSDVGEFR; (SEQIDNO:31) REEFVRFDSDVGEFR; (SEQIDNO:32) HQEEYVRFDSDVGEYR; (SEQIDNO:33) HQEEYVRFDSDVGEYRA; or (SEQIDNO:34) HQEEYVRFDSDVGEYRAV.
2. (canceled)
3. The isolated peptide according to claim 16, wherein an amino acid sequence of the peptide differs from that of SEQ ID NO: 1 by 1, 2, 3 or 4 amino acids.
4. The isolated peptide according to claim 1, wherein the isolated peptide has an amino acid sequence selected from the group consisting of: TABLE-US-00013 (SEQIDNO:1) REEYARFDSDVGEYR (SEQIDNO:3) REEYARFDSDVGEFR; (SEQIDNO:4) REEYVRFDSDVGEYR; (SEQIDNO:5) QEEYARFDSDVGEYR; (SEQIDNO:6) REEYARFDSDVGVYR; (SEQIDNO:7) NREEYARFDSDVGEFR; (SEQIDNO:8) NREEYVRFDSDVGEYR; (SEQIDNO:9) NQEEYARFDSDVGEYR and (SEQIDNO:10) NREEYARFDSDVGVYR.
5. A nucleic acid sequence encoding a peptide derived from a MHC class II molecule, wherein the peptide has a length of from 15 to 40 amino acids, and wherein the peptide has an amino acid sequence: REEYARFDSDVGEYR (SEQ ID NO: 1) or comprises an amino acid sequence: REEYARFDSDVGEYR (SEQ ID NO: 1), or a function-conservative variant of the peptide, or an expression vector comprising the nucleic acid sequence, or a host cell comprising the expression vector.
6. A MHC/peptide multimer comprising a peptide sequence encoding multiple peptides, wherein at least one encoded peptide is derived from a MHC class II molecule and has a length of from 15 to 25 amino acids, and wherein the at least one encoded peptide has an amino acid sequence REEYARFDSDVGEYR (SEQ ID NO: 1) or comprises the amino acid sequence: REEYARFDSDVGEYR (SEQ ID NO: 1), or a function-conservative variant thereof.
7. (canceled)
8. An in vitro or ex vivo method for generating a population of CD8.sup.+CD45RC.sup.low Tregs, comprising a step of culturing a population of CD8.sup.+ Tregs with a culture medium comprising i) an isolated peptide derived from a MHC class II molecule, wherein the peptide has a length of from 15 to 25 amino acids, and wherein the peptide has an amino acid sequence REEYARFDSDVGEYR (SEQ ID NO: 1) or comprises the amino acid sequence: REEYARFDSDVGEYR (SEQ ID NO: 1) or ii) a function-conservative variant of the isolated peptide, wherein the step of culturing is performed in the presence of a population of plasmacytoid dendritic cells.
9. An in vitro or ex vivo method for generating a population of CD8.sup.+CD45RC.sup.low Tregs, comprising a step of culturing a population of CD8+ Tregs with a culture medium comprising a MHC/peptide multimer comprising i) a peptide derived from a MHC class II molecule, wherein the peptide has a length of from 15 to 25 amino acids, and wherein the peptide has an amino acid sequence REEYARFDSDVGEYR (SEQ ID NO: 1) or comprises the amino acid sequence: REEYARFDSDVGEYR (SEQ ID NO: 1), or ii) a function-conservative variant of the peptide.
10. (canceled)
11. A method of inducing tolerance in a patient in need thereof, comprising administering to the patient a therapeutically effective amount of i) a peptide derived from a MHC class II molecule, wherein the peptide has a length of from 15 to 25 amino acids, and wherein the peptide has an amino acid sequence REEYARFDSDVGEYR (SEQ ID NO: 1) or comprises the amino acid sequence: TABLE-US-00014 (SEQIDNO:1) REEYARFDSDVGEYR, ii) a function-conservative variant of the peptide; or iii) a multimer comprising the peptide or the function-conservative variant of the peptide.
12. A method of preventing or reducing transplant rejection in a patient in need thereof in a patient in need thereof, comprising administering to the patient a therapeutically effective amount of i) a peptide derived from a MHC class II molecule, wherein the peptide has a length of from 15 to 25 amino acids, and wherein the peptide has an amino acid sequence REEYARFDSDVGEYR (SEQ ID NO: 1) or comprises the amino acid sequence: TABLE-US-00015 (SEQIDNO:1) REEYARFDSDVGEYR, ii) a function-conservative variant of the peptide; or iii) a multimer comprising the peptide or the function-conservative variant of the peptide.
13. The method of claim 11, wherein the peptide is a peptide of 15 or 16 amino acids in length that comprises or has an amino acid sequence: TABLE-US-00016 (SEQIDNO:2) NREEYARFDSDVGEYR, (SEQIDNO:3) REEYARFDSDVGEFR; (SEQIDNO:4) REEYVRFDSDVGEYR; (SEQIDNO:5) QEEYARFDSDVGEYR; (SEQIDNO:6) REEYARFDSDVGVYR; (SEQIDNO:7) NREEYARFDSDVGEFR; (SEQIDNO:8) NREEYVRFDSDVGEYR; (SEQIDNO:9) NQEEYARFDSDVGEYR; or (SEQIDNO:10) NREEYARFDSDVGVYR.
14. A pharmaceutical composition comprising a) a peptide of length of from 15 to 40 amino acids which is derived from a MHC class II molecule, wherein the isolated peptide comprises an amino acid sequence: REEYARFDSDVGEYR (SEQ ID NO: 1) or a function-conservative variant thereof, with the exclusion of the following peptides: TABLE-US-00017 (SEQIDNO:29) QEEYVRFDSDVGEYR; (SEQIDNO:30) NREEFVRFDSDVGEFR; (SEQIDNO:31) REEFVRFDSDVGEFR; (SEQIDNO:32) HQEEYVRFDSDVGEYR; (SEQIDNO:33) HQEEYVRFDSDVGEYRA; (SEQIDNO:34) HQEEYVRFDSDVGEYRAV; or b) an acid nucleic encoding the peptide; or c) an expression vector comprising the nucleic acid; or d) a host cell comprising the expression vector; or e) a MHC/peptide multimer comprising the peptide; or f) an antigen-presenting cell comprising a complex comprising an MHC molecule and the peptide; or g) a T lymphocyte that recognizes specifically the peptide; and a pharmaceutically acceptable carrier.
15. An in vitro method for determining whether a transplanted patient is tolerant, comprising a step of determining the presence of CD8+CD45RClow Tregs in a biological sample obtained from said transplanted patient, by contacting said biological sample with a binding partner capable of selectively interacting with CD8+CD45RClow Tregs, wherein the binding partner is a MHC/peptide multimer comprising i) a peptide derived from a MHC class II molecule, wherein the peptide has a length of from 15 to 25 amino acids, and wherein the peptide has an amino acid sequence REEYARFDSDVGEYR (SEQ ID NO: 1) or comprises the amino acid sequence: REEYARFDSDVGEYR (SEQ ID NO: 1), or ii) a function-conservative variant of the peptide, wherein the presence of CD8+CD45RClow Tregs is indicative of tolerance.
16. The isolated peptide according to claim 1, wherein the isolated peptide is 16 amino acids in length.
17. A T lymphocyte that recognizes specifically an isolated peptide derived from a MHC class II molecule, wherein the isolated peptide i) is 15 amino acids long and has an amino acid sequence TABLE-US-00018 (SEQIDNO:1) REEYARFDSDVGEYR; ii) is 16 amino acids long and comprises the amino acid sequence: REEYARFDSDVGEYR (SEQ ID NO: 1); or iii) is a function-conservative variant of i) or ii).
18. The method of claim 12, wherein the peptide is a peptide of 15 or 16 amino acids in length that comprises or has amino acid sequence: TABLE-US-00019 (SEQIDNO:2) NREEYARFDSDVGEYR, (SEQIDNO:3) REEYARFDSDVGEFR; (SEQIDNO:4) REEYVRFDSDVGEYR; (SEQIDNO:5) QEEYARFDSDVGEYR; (SEQIDNO:6) REEYARFDSDVGVYR; (SEQIDNO:7) NREEYARFDSDVGEFR; (SEQIDNO:8) NREEYVRFDSDVGEYR; (SEQIDNO:9) NQEEYARFDSDVGEYR; or (SEQIDNO:10) NREEYARFDSDVGVYR.
Description
FIGURES
[0197]
[0198]
[0199]
[0200]
EXAMPLE
MHCII.SUP.+ Allopeptide Induce Tolerance
[0201] Material & Methods
[0202] Animals and Cardiac Transplantation Models:
[0203] Allotransplantations of heart were performed between whole MHC incompatible LEW.1W (RT1.A.sup.u as donors) and LEW.1A (RT1.A.sup.a as recipients) male rats as previously described (5). The experiments were approved by the regional ethical committee for animal experimentation.
[0204] Adenovirus-Mediated Gene Transfer:
[0205] The Ad encoding for the extracellular portion of mouse CD40 fused to the constant domains of human IgG1 (AdCD40Ig) and the recombinant non-coding adenovirus (Ad) Addl324, as well as the procedure of intragraft delivery, have been described previously (5). Briefly, adenoviral particles (2.Math.10.sup.10 infectious particles) were slowly injected at 3 points into the cardiac ventricular walls.
[0206] Peptides Libraries:
[0207] 16-mer overlapping peptide libraries with 4 aa lagging were designed to cover the entire polymorphic sequences of MHC-I RT1.Au (alpha 1, 2 and 3 domains), MHC-II RT1.B.sup.u (all domains) and MHC-II RT1.D.sup.u (alpha2 and beta1 domains) as previously published (17-19) and manufactured by GL Biochem Ltd (Shangai, China). Lyophilized peptides were dissolved in 0.4% sterile DMSO/sterile water and stored at 80 C. As control peptides, we used in vitro various allogeneic non activating peptides #7, 26 and 39 and in vivo, we used peptide #31.
[0208] Degenerated 9 to 15-mer overlapping peptides with 1 to 2 aa lagging were designed to cover the sequence of positively isolated 16-mer peptide and synthesized by GL Biochem Ltd (Shangai, China).
[0209] All peptides were shown to be >95% homogeneous by analytical reverse phase HPLC and aa sequences were confirmed. Peptides were diluted in complete RMPI-1640 at a concentration of 120 g/ml.
[0210] Cell Purification:
[0211] T cells were purified as previously described (6). Briefly, total splenocytes were depleted with a cocktail of anti- T cells (V65), anti-CD45RA cells (OX33), anti-CD161 NK cells (3.2.3) and anti-CD11b/c monocytes (OX42) using magnetic beads (Dynal). Enriched T cells were labeled with anti-CD45RC-biotin (OX22) and Strepavidin-PE-Cy7, anti-CD8-PE (OX8), anti-TCR-Alexa 647 (R73) and anti-CD25-FITC (OX39) mAbs. CD8.sup.+CD45RC.sup.low T cells and CD4.sup.+CD25.sup. T cells were sorted after gating of TCR.sup.+ cells with FACSAria (BD Biosciences, Mountain View, Calif.). Purity of sorted populations was greater than 99%.
[0212] Plasmacytoid dendritic cells (pDCs) were purified as previously described (6). Briefly, splenocytes recovered after collagenase digestion were further purified by negative depletion with anti-TCR (R73 and V65) T cells and anti-CD45RA (OX33) B cells mAbs. Enriched cells were labeled with anti-CD45R-PE (His24), anti-CD4-APC (OX35), anti-TCR-FITC (R73) and anti-CD45RA-FITC (OX33). pDCs, defined as CD45R and CD4 positive cells, were sorted after gating on FITC negative cells.
[0213] Mixed Lymphocyte Reaction:
[0214] For MLR coculture assays, pDCs from LEW.1A naive rats (1.2510.sup.4 cells), syngeneic CD8.sup.+CD40Ig Tregs (510.sup.4 cells) and 120 g/ml of allogeneic peptides were plated in triplicate in 200 l of complete RPMI-1640 medium in round-bottom 96 wells plates for 6 days at 37 C., 5% CO2. pDCs were matured with 0.5 M of CpG ODN 1826.
[0215] For direct MLR suppressive assays, sorted CFSE-labeled CD4.sup.+CD25.sup. T cells from LEW.1A origin (510.sup.4 cells) and allogeneic pDCs isolated from donor LEW.1W animals (1.2510.sup.4 cells) were plated in triplicate for 6 days in a final volume of 200 l of completed RPMI-1640 medium in round-bottom 96 wells plates with FACS-sorted freshly purified naive CD8.sup.+CD45RC.sup.low Treg cells, peptide-expanded CD8.sup.+CD40Ig Treg cells (510.sup.4 cells), Du51 tetramer.sup.+ or .sup. CD8.sup.+CD40Ig Tregs. For indirect MLR suppressive assay, splenocytes isolated from donor LEW.1W animals were frozen-thawed to induce apoptosis. Apoptotic cells were then cultured overnight with pDCs isolated from recipient LEW.1A animals (8:1 ratio) at 0.6510.sup.6 pDCs/mL. Alloantigens-loaded pDCs were finally washed and plated as described before for the direct MLR.
[0216] For anti-CD3/anti-CD28 stimulations, round-bottom 96 wells plates were coated with anti-CD3 (1 g/mL, BD Pharmingen) and anti-CD28 (10 g/mL) mAbs for 1 hour at 37 C., 5% CO2, then washed and 5.Math.10.sup.4 CD8.sup.+CD40Ig Treg cells were added to each well in 200 l completed RPMI-1640 for 1, 2, 3 and 6 days.
[0217] Proliferation of CFSE-labeled naive CD4.sup.+CD25.sup. T cells and phenotype of CD8.sup.+CD45RC.sup.low Tregs were analyzed by flow cytometry on a FACSCanto II cytometer (BD Biosciences, Mountain View, Calif.) after gating on TCR.sup.+CD4.sup.+ cells or TCR.sup.+CD8.sup.+ cells among live cells (DAPI negative).
[0218] Extracellular and Intracellular Staining:
[0219] For extracellular staining, cells were stained with the following mAbs: anti-TCR (R73, Alexa Fluor 647-conjugated), anti-CD8 (OX8, PE-Cy7-conjugated, ebiosciences), anti-CD4 (W3.25, PE-Cy7-conjugated), anti-CD45RC (OX22, FITC-conjugated), anti-CD28 (JJ319, biotin-labeled), anti-CD71 (OX26, biotin-labeled), anti-mouse Vb11 (KT11, biotin-labeled, AbD Serotec) anti-CD25 (OX39, biotin-labeled) and anti-MHC-II (OX6, biotin-labeled).
[0220] For intracellular staining, cells were then stained for Foxp3 (biotin-labeled, ebiosciences) using BD cytofix/cytoperm kit (BD Biosciences) according to the manufacturer's instructions.
[0221] All biotinylated mAbs were visualized using Streptavidin-PerCP.Cy5.5 (BD Biosciences). A FACSCanto II cytofluorimeter (BD Biosciences, Mountain View, Calif.) was used to measure fluorescence, and data were analyzed using FlowJo software (Tree Star, Inc. USA, version 7.6.5). Cells were first gated by their morphology and dead cells excluded by selecting DAPI negative viable cells.
[0222] Cytokine Assays:
[0223] IFN, IL-10 were measured in coculture supernatants using ELISA from BD Biosciences OptEIA, IL-12 and TGF using ELISA from Invitrogen and R&D System respectively.
[0224] Production of Biotinylated RT1-A.sup.a-Peptide Complexes:
[0225] Briefly, the heavy chain RT1-A.sup.a and the 2microglobuline (2m) were cloned in pET24 and produced in Escherichia coli BL21-DE3. Recombinant proteins were produced as inclusion bodies, dissolved in 8M urea and refolded in vitro as previously described for human HLA-A2/peptide complexes (61). RT1-A.sup.a, 2m and peptide Du51 were refolded in 0.4M L-arginine, 0.1M Tris pH8, 2 mM EDTA, 5 mM reduced glutathione, and 0.5 mM oxydated glutathione for 5 days at 4 C. The solution was then concentrated and the buffer changed on amicon membrane 10 Kd (Millipore, Bedford, Mass.). Folded MHC/peptide complexes were biotinylated with the BirA enzyme (Avidity, Denvers Colo.) for 5 h at 30 C. and desalted on Hiprep 26/10 desalting column (GE Healthcare). MHC/peptide complexes were then purified by anion exchange Q-Sepharose chromatography. Biotinylation was tested by tetramerization with streptavidin (Sigma Aldrich) at a molar ratio of 4:1.
[0226] Tetramerization and Staining:
[0227] Tetramerization of RT1.A.sup.a/Du51 was performed at room temperature by addition of streptavidin-PE (Jackson ImmunoResearch) or streptavidin-APC (BD Biosciences) at a 4:1-molar ratio, in four equal aliquots added at 15-min intervals. Likewise, the control tetramer RT1.A.sup.a/MTF-E (ILFPSSERLISNR) was conjugated to streptavidin-BV421 (Biolegend) and represented 1.6+/0.7% of non-specific staining among Du51-specific cells. These three reagents were mixed and added to plated cells at 10 g/mL for 1 hour at 4 C. Cells were further stained for CD8 and CD45RC and fluorescence was analyzed on a FACSCanto II cytometer (BD Biosciences, Mountain View, Calif.). Cells were first gated by their morphology and dead cells excluded by selecting DAPI negative cells.
[0228] Peptides Treatment In Vivo:
[0229] 16-mer peptides were dissolved in 0.4% DMSO/PBS before injection. For the first protocol, single doses of peptide (500 g/injection) were injected i.v at different time points before and after transplantation at day 6, 3, 0 +3 and +7 into grafted LEW.1A recipients. In the second protocol, mini osmotic pumps (ALZET, Cupertino, Calif., USA) were implanted intraperitoneally (i.p) in recipients and delivered continuously either 20.83 or 41.66 g/hour of 16-mer peptides for 14 days. A first pump was implanted on day 7 before transplantation and was replaced by a second one at day +7, allowing delivery of 14 or 28 mg of peptide per animal for 28 consecutive days. A depleting anti-CD8 mAb (OX8, IgG1, 3 mg/kg) or an anti-MHC class Ia and Ib mAb (OX18, 3 mg/kg) were injected i.p. twice a week from day 7 until rejection. Allografts were monitored daily by palpation and allograft rejection was defined as complete cessation of palpable heart beat.
[0230] Adoptive Cell Transfer:
[0231] Cell transfers were performed by i.v. injection of purified sorted total or Du51 tetramer.sup. CD8.sup.+CD40Ig Tregs into LEW.1A recipients sublethally irradiated (4.5 Gy whole-body irradiation) on the day before transplantation. Total splenocytes (1.5.Math.10.sup.8 cells) were adoptively transferred i.v. the day before heart transplantation into naive LEW-1A recipients that had received 4.5 Gy of whole-body irradiation the same day. Recipients received splenocytes from Du51-treated rats (defined as 1.sup.st spl-transferred) or from naive rats (naive spl-transferred) or no cells (untreated irradiated).
[0232] Morphometric Analysis of Cardiac Grafts:
[0233] The upper third of the graft was fixed in paraformaldehyde and embedded in paraffin. Five m coronal sections were stained with hematoxylin-eosine-safron. Tissues were analyzed by a pathologist (K.R.) blinded to the groups and chronic rejection was evaluated as previously described (63).
[0234] Donor Specific Alloantibody Detection:
[0235] Alloantibodies were analyzed by cytofluorimetry as described elsewhere (28). Briefly, after digestion by collagenase D and red blood cell lysis, allogeneic spleen cells were incubated with diluted (1/8) heat-inactivated serum, and then with FITC-conjugated goat anti-rat IgG antibodies (H+L chain specific) (Jackson Laboratories), a mouse anti-rat IgG1 MAb (MCA 194, Serotec), IgG2a (MCA 278, Serotec) or IgG2b (MCA 195, Serotec). Antibody binding was revealed using FITC-coupled F(ab)2 goat anti-mouse IgG (Jackson Laboratories). Cells were analyzed using a FACS Canto II cytofluorimeter (BD Biosciences, Mountain View, Calif.) and the results were expressed as mean channel fluorescence for each serum.
[0236] Statistical Analysis:
[0237] 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 TCR V11 expression, phenotype of activated cells, cytokine expression, proliferation assay and tetramer staining was evaluated by a two-tailed Mann Whitney t test. Graft survival was analyzed by Kaplan-Meier log-rank test. The Two-Way ANOVA test and Bonferroni post-tests were used for donor-specific antibody analysis and splenocyte phenotypic characterization. Analyses were made with GraphPad Prism 5.04 software (GraphPad, San Diego, Calif., USA). For the diversity analysis, Kruskal-Wallis and Dunn's multiple comparison post-test were performed using GraphPad Prism 6.0c. A P-value less than 0.05 was considered significant.
[0238] Results
[0239] CD8.sup.+CD40Ig Tregs Activation In Vitro.
[0240] In order to identify TCR recognition of allogeneic MHC/peptide complexes by CD8.sup.+CD40Ig Tregs and subsequent activation of their function, we had to select a specific marker of activation allowing analysis by flow cytometry following exposure to antigenic stimulation. Therefore, we screened molecules expressed at different time points by CD8.sup.+CD40Ig Tregs upon stimulation with polyclonal anti-CD3 and anti-CD28 antibodies. Expression of molecules on freshly isolated CD8.sup.+CD40Ig Tregs has been previously assessed by Q RT-PCR (5) and demonstrated that among these molecules, CD25 and IFN were markers distinguishing CD8.sup.+CD40Ig Tregs from other cell populations. We analyzed by flow cytometry at day 0, 1, 2, 3 and 6 their expression of CD71, CD25 and IFN.
[0241] We confirmed, at day 0, that CD8.sup.+CD40Ig Tregs expressed low levels of CD71 (0.830.1%), CD25 (12.746.1%), and IFN (5.573.3%). After polyclonal stimulation, CD71, CD25 and IFN expression increased significantly from the first day and remained stable over time with respectively 824.5%, 98.11.9% and 91.77% of positive cells at day 6.
[0242] In conclusion, we identified three markers of interest to CD8.sup.+CD40Ig Tregs with low basal expression and significant up-regulation upon stimulation. Since CD25 was the most and the earliest up-regulated marker and since it was a marker previously described by us and others (5, 12), we selected this marker to assess CD8.sup.+CD40Ig Treg activation for the remaining aspects of this study.
[0243] CD8.sup.+CD40Ig Tregs Cells Recognized Two Donor MHC Class 11-Derived Peptides.
[0244] In the rat MHC-mismatched heart allograft model, donors (RT1.sup.u) and recipients (RT1.sup.a) are mismatched for all MHC molecules. We therefore aligned donor and recipient MHC I and II amino acids (aa) sequences and designed 82 overlapping 16-mer peptides matching the polymorphic domains of donor MHC I and II molecules (17-19). Peptides were first grouped into pools of 6 to 8 peptides (30 g/ml of each peptide) and tested in an in vitro assay where immature or mature syngeneic recipient pDCs and sorted-CD8.sup.+CD40Ig Tregs from CD40Ig-treated long-term allograft bearing recipients were cocultured for 3 or 6 days. With immature pDCs, we observed no significant activation of CD8.sup.+CD40Ig Tregs at day 3 or day 6 with any of the allogeneic pools of peptides. After stimulation with mature pDCs and pools of allogeneic peptides we observed at day 3 a slight upregulation of CD25 expression of a small population of CD8.sup.+CD40Ig Tregs, and at day 6, a significant up-regulation of CD25 expression following allogeneic stimulation. These results suggested that some allogeneic peptides were efficiently recognized by CD8.sup.+CD40Ig Tregs and that this recognition led to increased CD25 expression. It also demonstrated that pDCs must be matured in our assay.
[0245] We next tested the stimulatory capacity of the 82 individual allopeptides in the presence of naive matured syngeneic pDCs and CD8.sup.+CD40Ig Tregs purified from long-term survivors in a 6 days culture (
[0246] CD8.sup.+CD40Ig Tregs Recognized an Unusually Long Allogeneic 15-Mer Peptide.
[0247] To determine the sequence of the natural dominant donor peptide recognized by antigen-specific CD8.sup.+CD40Ig Tregs, we used a library of degenerated peptides, ranging from 9-mer peptides with one aa lagging to 15-mer peptides with two or more aa lagging derived from the dominant 16-mer Du51 (labeled #51-1 to #51-18) (
TABLE-US-00011 TABLE1 Listofpeptidesusedinthepresentstudy: Length SEQ Name Sequence (aa) IDNO: Du51 NREEYARFDSDVGEYR 16 2 51-1 NREEYARFD 9 11 51-2 REEYARFDS 9 12 51-3 EEYARFDSD 9 13 51-4 EYARFDSDV 9 14 51-5 YARFDSDVG 9 15 51-6 ARFDSDVGE 9 16 51-7 RFDSDVGEY 9 17 51-8 FDSDVGEYR 9 18 51-9 YARFDSDVGE 10 19 51-10 EYARFDSDVG 10 20 51-11 EYARFDSDVGE 11 21 51-12 YARFDSDVGEY 11 22 51-13 YARFDSDVGEYR 12 23 51-14 EYARFDSDVGEY 12 24 51-15 EEYARFDSDVGE 12 25 51-16 EEYARFDSDVGEY 13 26 51-17 EEYARFDSDVGEYR 14 27 51-18 REEYARFDSDVGEYR 15 1
[0248] None of the derivative 9-mer peptides #51-1 to #51-8 was able to induce activation of CD8.sup.+CD40Ig Tregs equivalent to the one observed with the 16-mer Du51. However, we were able to induce a much stronger and significant CD25 upregulation with a 15-mer derivative peptide (51-18) (2.040.3 fold vs. no peptide) missing the N-term asparagine (N). Contrary to other derivatives, CD25 upregulation induced by peptide #51-18 was not significantly different from that induced by Du51 (
[0249] Altogether, these results showed that a dominant MHC class II-derived 15-mer natural peptide (REEYARFDSDVGEYR) was presented to the CD8.sup.+CD40Ig Tregs and that such presentation induced activation of the specific cells.
[0250] Du51-Activated CD8.sup.+CD40Ig Treg Cells Displayed a Modified Phenotype and Efficiently Suppressed Antigen-Specific Activated T Cells.
[0251] The phenotype of Du51-activated CD8.sup.+CD40Ig Tregs was analyzed 6 days after stimulation. We previously demonstrated that CD8.sup.+CD40Ig Tregs acted through secretion of high levels of IFN, that in turn induced IDO expression by DCs and graft ECs and this action was necessary for tolerance induction in vivo (5). According to these results, we observed that stimulation of CD8.sup.+ Tregs by the peptide in the presence of pDCs led to significant increased expression of IFN, most likely by activated CD8.sup.+CD40Ig Tregs. In the same culture supernatants, we observed decreased IL-12 production, likely of pDC origin, but no modification of IL-10 and TGF expression that could be produced by both CD8.sup.+CD40Ig Tregs and pDCs. We also observed an upregulation of CD71, CD28 and MHC class II, but no modification of Foxp3 expression after 6 days of peptide stimulation.
[0252] We previously demonstrated that CD8.sup.+CD40Ig Tregs, in the presence of allogeneic pDCs or syngeneic pDC and a lysate of donor cells, could suppress the proliferation of syngeneic effector CD4.sup.+CD25.sup. T cells, showing that CD8.sup.+CD40Ig Tregs acted through the direct and indirect pathway of allorecognition, and that they are more efficient suppressor cells than naive CD8.sup.+CD45RC.sup.low Tregs (6). Here, we investigated whether Du51-stimulated CD8.sup.+CD40Ig Tregs could efficiently suppress effector T cell proliferation after 6 days coculture and thus maintained their suppressor activity, compared to CD8.sup.+CD40Ig Tregs stimulated with a non-activating control peptide. We performed a MLR assay stimulating CD8.sup.+CD40Ig Tregs for 6 days in the presence of syngeneic pDCs and Du51 or non-activating peptide. Peptides-stimulated CD8.sup.+CD40Ig Tregs were then sorted using a FACS Aria and added in a direct MLR assay of sorted allogeneic pDCs and syngeneic CFSE-labeled CD4.sup.+CD25.sup. effector T cells. We hypothesized that expanded-CD8.sup.+CD40Ig Tregs would exert bystander regulation of the proliferation of effector CD4.sup.+CD25.sup. T cells stimulated by the direct allorecognition pathway, that was measured 6 days later (24, 25). In the absence of Tregs, 80.5% of CD4.sup.+CD25.sup.effector T cells proliferated. The addition of Du51-stimulated Tregs resulted in a significant inhibition of the proliferation of effector T cells compared to Tregs stimulated by a non-activating peptide. Thus, Du51 antigen-specific CD8.sup.+CD40Ig Tregs maintained an efficient suppressive activity after in vitro activation and Tregs that remained unstimulated for 6 days lost their suppressive activity and started to die by neglect.
[0253] Identification of Du51-Specific CD8.sup.+CD45RC.sup.low Tregs Using MHC Class I Tetramer Revealed their Enrichment in CD40Ig-Treated Recipients.
[0254] An important challenge in transplantation remains the identification of antigen-specific Tregs as they represent a more potent suppressive population and very few natural epitopes have been identified so far. In addition, to date, in the rat and transplantation settings, no tools were available to directly visualize and detect antigen-specific CD8.sup.+ Treg populations. To that end, we generated a MHC class I tetramer RT1.A.sup.a/Du51 that was labeled with phycoerythrin (PE) and allophycocyanin (APC) and stained specific populations in the spleen and graft.
[0255] Cells were first stained with a mixture of PE-conjugated and APC-conjugated RT1.A.sup.a/Du51 tetramers, together with a control tetramer RT1.A.sup.a/MTF-E labeled with BV421. This dual fluorochrome strategy, previously described for antigen-specific CD8.sup.+ effector T cells, allows the discrimination of signal and noise staining, as specific CD8.sup.+ Tregs will bind equally to each version of RT1.A.sup.a/Du51 tetramer whereas random elements will not (26). Among double positive stained events, non peptide-specific cells can be visualized and excluded as they bind to a control tetramer bearing the same heavy chain RT1.A.sup.a but with an irrelevant peptide associated. Cells were secondary stained with CD8-PeCy7 and CD45RC-FITC to identify either CD8.sup.+CD45RC.sup.low Tregs or CD8.sup.+CD45RC.sup.high T cells. With such a strategy, we were able to identify 2.190.6% in the spleen and 1.160.25% in the graft of Du51-specific cells among CD8.sup.+CD40Ig Tregs. In the naive splenic CD8.sup.+CD45RC.sup.low Tregs population, we evaluated the precursor frequency at 0.730.2%, demonstrating that even 120 days following transplantation and CD40Ig treatment, the frequency was still increased around 3 times and that in naive animals, we were able to identify a pool of donor-specific Tregs. This difference was true when looking at the percentage and absolute number of tetramer-positive CD8.sup.+CD40Ig Tregs or naive CD8.sup.+CD45RC.sup.low T cells in the spleen (0.6240.128 vs. 0.1730.071 respectively for percentage and 623800127700 vs. 17260070500 for absolute number in the spleen, p<0.05). There was also significantly more Du51-specific cells among CD8.sup.+CD40Ig Tregs than among CD8.sup.+CD45RC.sup.high T cells from naive or CD40Ig-treated animals, in terms of the percentage of positive cells among each population and percentage or absolute number in the total spleen, and the percentage in the graft. There was no difference between CD8.sup.+CD45RC.sup.low and CD8.sup.+CD45RC.sup.high T cells from naive animals in the spleen. Interestingly, tetramer-positive cells were localized both in the graft and the spleen within the CD8.sup.high subset of CD8.sup.+CD45RC.sup.low Tregs.
[0256] Altogether, these results demonstrated that we were able to generate a functional RT1A.sup.a/Du51 tetramer to detect alloantigen-specific CD8.sup.+ Tregs, a population that was significantly increased upon transplantation and CD40Ig treatment.
[0257] Superior Suppressive Capacity of Du51-Specific CD8.sup.+CD45RC.sup.low Tregs Mediated by Direct and Indirect Pathways of Allorecognition and Requirement for In Vivo Tolerance Induction.
[0258] We previously demonstrated the suppressive capacity of Du51-stimulated CD8.sup.+CD40Ig Tregs. In this experiment, we wanted to study the suppressive capacity of freshly sorted RT1A.sup.a/Du51 tetramer-specific CD8.sup.+CD40Ig Tregs (
[0259] To study the in vivo relevance of the differential ex vivo suppressive effect observed for Du51-specific CD8.sup.+CD40Ig Tregs, we performed adoptive cell transfer experiments. Total CD8.sup.+CD40Ig or RT1A.sup.a/Du51 tetramer negative (tet.sup.) CD8.sup.+CD40Ig Tregs, were sorted and adoptively transferred into naive grafted irradiated recipients (
[0260] Tolerance Induction by In Vivo Peptide Treatment Correlated with Increased Proportion of CD8.sup.+CD45RC.sup.low Tregs and Total Inhibition of Anti-Donor Antibody Responses.
[0261] To further determine the potential of the immunodominant peptide identified in the in vivo generation of CD8.sup.+CD45RC.sup.low Tregs and in allograft survival, animals were separately treated using two different protocols of peptide administration with no other treatment. In the first one, animals received five intravenous (i.v) injections of 500 g of peptide (
[0262] Interestingly, increased dose of Du51 administered by osmotic pump delivering 41.6 g of peptide per hour induced an indefinite allograft survival in 80% of the recipients of Lewis 1W (LEW.1W) donor hearts (
[0263] Grafted hearts and spleens of rejecting or long-term surviving recipients treated with peptide Du51 were analyzed for signs of chronic rejection, presence of anti-donor antibodies, proportion of total and tetramer-positive CD8.sup.+CD45RC.sup.low Tregs, and in vitro suppression towards CD4.sup.+ effector T cells (
[0264] Infectious Tolerance Following Du51 Monotherapy.
[0265] To assess the dominant suppressive potential of induced regulatory cells involved in the long-term allograft survival generated by 1 mg/day of in vivo peptide Du51 treatment. To do so, we performed adoptive cell transfer experiments using splenocytes of long-surviving recipients into naive grafted sublethaly irradiated recipients, as we have done before (5) (
DISCUSSION
[0266] Our current knowledge on how Tregs recognize peptides and the role of this recognition is very limited and mostly based on either transgenic mouse models using CD4.sup.+ Treg's TCR gene transfer (8, 11) or on murine Qa-1-restricted CD8.sup.+ Tregs involved in autoimmune disease and cancer and whose Qa-1-peptide repertoire has been described in the last few years (29). However, these studies suggest that antigen-specific Tregs are crucially influencing the outcome of long-term transplantation and contribute to the establishment of tolerance (30). Recent studies have characterized peptides recognized by CD8.sup.+ Tregs during cancer (an heme oxygenase-1-derived peptide), autoimmunity (V-derived peptide) or even pregnancy (minor antigen-derived peptide) but not during transplantation (2). Transplantation is a particular setting to identify antigen recognized by Tregs as the presence of the graft is a continuous source of alloantigens and that is most certainly essential for the function and maintenance of regulatory populations and thus the survival of the grafted organ (31). The recognition of alloantigens in the context of regulation has been shown by us and others to occur mainly by the indirect pathway of presentation, and in particular supported by pDCs (6, 30, 32).
[0267] In this report, we demonstrated for the first time that CD8.sup.+CD45RC.sup.low Tregs, through the indirect pathway of presentation, can recognize one dominant allopeptide, named Du51, (and one sub-dominant) derived from the 1 domain of natural donor MHC class II molecules. These peptides share 80-90% homology with human HLA class II molecules and thus could be used to detect specific CD8.sup.+ Tregs in humans. By the use of a MHC-I specific tetramer, we showed that Du51-specific CD8.sup.+CD45RC.sup.low Tregs were enriched in CD40Ig-treated long-term surviving recipients, expressed a biased restricted V11 chain, displayed a strong suppressive activity ex-vivo and played a crucial role in tolerance induction upon adoptive transfer. Finally, peptide Du51 was shown to induce prolongation of allograft survival in vivo, inducing donor-specific CD8.sup.+ Tregs.
[0268] Here, we described that the peptide Du51 displayed an unusual length of 15 aa and that shorter peptides tested failed to induce significant recognition by CD8.sup.+CD40Ig Tregs. Most of the literature has focused on short peptides (8-10 aa) bound to MHC class I, although it is known that 5-10% of peptides are longer peptides (more than 10 aa) that can be presented by MHC class I molecules (33, 34). So far, such peptides have been identified for CTL models and are mostly derived from viral antigens. To our knowledge, we provide the first description of a 15 aa peptide that can be structurally recognized by CD8.sup.+ Tregs, as shown with our RT1A.sup.a/Du51 tetramers. Recent studies suggested that these long peptides drive a TCR recognition more focused on the peptide (35) and that TCRs recognize MHC class I peptides of a preferential length (36). The rat MHC class I molecule RT1.A.sup.a has been known to accommodate particularly long peptides, with key-position residues as Gln, Met or Leu at P2, Phe at P3, Pro at P4 and Arg at the C terminus (21, 22). Speir et al has demonstrated previously in a model of maternally transmitted minor histocompatibility antigen (MTF-E) of 13 residues that important anchor residues (in particular arginine at position 13 (P13)) allowed binding with considerable bulged conformation (37). We also observed that Du51 displayed an Arg at the C-term end and thus could help for RT1.A.sup.a accommodation of the peptide. Interestingly, some of the peptides that were tested by us were also tested by Ballet et al. on CD4.sup.+ and CD8.sup.+ T cells isolated from rejected untreated animals (including dominant peptide Du51) in the same mismatched cardiac allograft model (LEW.1W into LEW.1A) as us (18). They found two immunodominant peptides, referred by us as peptide #47 and peptide #55, all derived from LEW.1W RT1.D.sup.u molecules, involved in acute rejection of grafts from unmodified LEW.1A recipients. Importantly, peptide Du51 was not involved in acute rejection in their model and the two immunodominant peptides identified by Ballet et al. were not recognized by our CD8.sup.+CD40Ig Tregs, suggesting that Tregs and non-Tregs did not recognize the same antigens.
[0269] We were able to produce a RT1.A.sup.a/Du51-tetramer, which in addition to being a valuable tool to track antigen-specific cells, can also be used to determine TCR fine specificity and affinity. With this tetramer, we identified in naive animals a pool of antigen-specific Treg precursors of 0.73%, which was expanded around three times by 120 days following transplantation and CD40Ig treatment. This precursor frequency correlated with the observations of Leavenworth et al. made in a model of arthritis in mice where they analyzed the occurrence of Qa-1-Hsp60.sub.p216 and Qa-1.R72A-Qdm tetramer-specific CD8.sup.+ Tregs and described a naive frequency of respectively 1.65% and 0.46% of positive CD8.sup.+ T cells for each (38). These observations suggest that precursor frequency of antigen specific cells in a Treg population might be higher than the frequency of a given antigen-reactive non-Treg T cells.
[0270] By the use of this MHC-I tetramer, we compared the suppressive capacity of Du51-specific and non-specific CD8.sup.+CD40Ig Tregs ex vivo in a coculture assay and in vivo through adoptive transfer of tetramer.sup. CD8.sup.+CD40Ig Tregs (depleted in tetramer.sup.+ CD8.sup.+CD40Ig Tregs). We demonstrated ex vivo the superior suppressive potential of tetramer over tetramer.sup. CD8.sup.+CD40Ig Tregs, which significantly inhibit CD4.sup.+ effector T cells proliferation stimulated by both the direct and indirect pathways of presentation, and most efficiently in the indirect allorecognition setting. These results are in agreement with several studies demonstrating the superior suppressor potential of Tregs of indirect specificity in vitro (39, 40). In vivo, we observed a rapid allograft rejection in naive grafted irradiated recipients transferred with tetramer.sup. CD8.sup.+CD40Ig Tregs compared to recipients adoptively transferred with total CD8.sup.+CD40Ig Tregs. In a similar manner, adoptive transfer of tet.sup.CD8.sup.+ Tregs (depleted in tetramer Qa-1/Hsp60.sub.p216-specific Tregs) could not prevent the development of autoimmune arthritis in a mouse model (38). Also, Tsang et al. showed a crucial role of CD4.sup.+ Tregs of indirect allospecificity in transplantation tolerance, demonstrating that only adoptive transfer of TCR-transduced CD4.sup.+ Tregs of both direct and indirect specificity, and not CD4.sup.+ Tregs of direct specificity only, can inhibit cardiac allograft rejection (11).
[0271] We have also analyzed different markers potentially expressed by antigen-specific regulatory T cells. Foxp3 does not seem to be an interesting marker of CD8.sup.+CD40Ig Tregs unstimulated or stimulated with peptide, although upon several rounds of strong anti-CD3/anti-CD28/IL-2 stimulation, we were able to detect substantial levels of Foxp3 (data not shown). We also found increased IFN production and decreased IL-12 expression after peptide-specific stimulation. IFN was already shown by us as a crucial cytokine in our model (6).
[0272] Regarding the repertoire of induced CD8.sup.+CD40Ig Tregs, we previously showed that these cells preferentially used a TCR that recombined the V11 chain and displayed a CDR3 of 9 aa in the spleen, suggesting the expansion of an oligoclonal population of Tregs (5). However, sequencing of around 700 CDR3 across six long-surviving animals demonstrated that total V11.sup.+CD8.sup.+CD40Ig Tregs displayed a relatively diverse repertoire in the spleen. Nevertheless, a more frequent repeated sequence was found in some animals. Analysis of the TCR repertoire in the graft revealed the predominance of two TCR-V chains: V11 (as in the spleen) and V18. Unlike the spleen, sequencing revealed biased and restricted repertoires for both chains, with some shared clonotypes for the V18 TCR, as in the donor-specific blood transfusion model of induction of long-term allograft survival (41). However, the public sequence described by Douillard et al. was only found once in one animal of our study and did not represent the V18-repertoire in our model. One interesting hypothesis would be that CD8.sup.+CD40Ig Tregs bearing such biased and restricted clonotypes are more potent suppressors and migrate early in the graft to exert their inhibitory activity, and then stay localized in the tolerated transplant where immune regulation is required (42). In contrast, total CD8.sup.+CD40Ig Tregs that reside in the spleen but displayed a non (V18) or less (V11) restricted repertoire (and consequently closer to the naive Tregs' repertoire) potentially identify distinct regulatory populations (that possibly do not express the same chemokine receptors) being recruited upon subsequent inflammation. Despite some shared clonotypes for both V11 and V18 (Suppl. Table 1), we could not find public CDR3 sequences. Analyzing more precisely the repertoire of antigen-specific CD8.sup.+CD40Ig Tregs in the spleen by focusing on tetramer Du51.sup.+ Tregs, we demonstrated reduced clonotypic diversity of Du51-specific V11.sup.+CD8.sup.+CD40Ig Tregs (compared to the total CD8.sup.+CD40Ig Tregs from naive and CD40Ig-treated spleen and similar to the graft CD8.sup.+CD40Ig Tregs), but not of Du51-specific V18.sup.+CD8.sup.+CD40Ig Tregs. Thus, the V18 chain usage might not be optimal for recognition of this particular antigen. However, even this analysis of the antigen-specific population did not reveal public clonotypes for this Treg population. TCR sequences that are shared between individuals tend to be more efficiently produced by a process of convergent recombination (43) and thus present at a higher frequency in the naive T cell repertoire (44-46). However, inter-individually shared TCR sequences are not necessarily dominant in the immune response to antigen (47). This appears to be the case in our model, suggesting that this process may be modified by either TCR expansion or TCR-chain pairing. More recent publications have revealed the important contribution of the alpha-chain in the specific pMHC recognition and how TCR diversity should be taken into consideration, as some specific TCR V pairings dictate and alter MHC restriction (48, 49). In the literature, a consensus on TCR diversity of naive CD4.sup.+ Tregs was obtained stating that more is better. Authors demonstrated that a high TCR diversity ensures optimal Treg expansion and function by increasing the probability of having antigen-specific clones responding (13, 16, 50, 51). In our model, naive CD8.sup.+ Tregs displayed a highly diverse repertoire that was remodeled and biased by expansion of Du51-specific CD8.sup.+ Tregs after transplantation and CD40Ig treatment.
[0273] At last, from a therapeutic point of view, the identification of natural peptide recognized by regulatory T cells in transplantation is an important goal as new strategies using amplified CD4.sup.+ Tregs are being tested currently in human transplantation (30, 52). The difficulty is that these human CD4.sup.+ Tregs amplified in a polyclonal way by non-specific stimulus such as anti-CD3/CD28 antibodies are less efficient than antigen-specific Tregs (2, 53), display limited expansion capacity and are usually outgrown by conventional effector T cells (54). The use of specific antigen to expand Tregs in short-term culture would surely improve clinical settings. As a proof of principle, we administered the dominant peptide Du51 in naive grafted recipients, without immunosuppressive treatment, and observed significant prolongation of allograft survival. This is the first time this extended survival can be obtained with allopeptide alone (i.e. without immunosuppressive drugs) in rodents and clearly demonstrates both the efficiency of this peptide and the implication of the indirect pathway of presentation in tolerance induction. Some studies had described earlier immunomodulatory effects of HLA-derived peptides on alloimmune responses (55). Especially, an HLA-B7 derived-peptide, called Allotrap, was shown to prolong skin and heart allograft survival when associated with ciclosporin administration in mice and rat respectively (56, 57). Its effect was associated with modulation of heme-oxygenase 1 activity (58). In our model, administration of either anti-CD8 or anti-MHC-I antibodies in combination with peptide Du51 completely abolished allograft survival. Thus, the in vivo therapeutic effect obtained with peptide Du51 infusion was directly linked to MHC class I presentation and CD8.sup.+ T cell induction. Moreover, the immunodominant peptide Du51 induced a donor-specific inhibition of alloimmune responses as third party grafts were promptly rejected. In addition, the peptide Du51-induced allograft survival was accompanied by a total inhibition of anti-donor antibodies that is probably related to the occurrence of antigen-specific CD8.sup.+ Tregs after infusion of the allopeptide. This result is important as it shows the potency of peptide-induced Tregs to inhibit acute and chronic allograft rejection occurrence, and opens new possibilities in human transplantation. In addition, such expanded highly suppressive Tregs could provide us with more fundamental information on new and poorly described genes overexpressed with such conditions and that could be used as biomarkers (6). Finally, we could assume that the presence of this CD8.sup.+CD45RC.sup.low Treg population in some groups of patients could be associated with a better prognosis during the course of diseases (59, 60).
[0274] In conclusion, our study indicates that MHC class II donor antigen can be used to boost antigen-specific CD8.sup.+ Treg generation and/or function, and that in turn, these Tregs inhibit anti-donor immune responses allowing the establishment of a true tolerance. We also demonstrated that antigen-specific CD8.sup.+ Tregs' TCRs display a private and restricted repertoire that ensures efficient expansion and suppression of alloreactive immune responses. Altogether, these results highlight the importance of the TCR, of its interaction with MHC/peptide and open new possibilities in the generation of this population that could be transferrable to human settings.
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