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WO2021087579A1 - Compositions pour la détection de lymphocytes t alloréactifs - Google Patents

Compositions pour la détection de lymphocytes t alloréactifs Download PDF

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Publication number
WO2021087579A1
WO2021087579A1 PCT/AU2020/051221 AU2020051221W WO2021087579A1 WO 2021087579 A1 WO2021087579 A1 WO 2021087579A1 AU 2020051221 W AU2020051221 W AU 2020051221W WO 2021087579 A1 WO2021087579 A1 WO 2021087579A1
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Prior art keywords
peptides
allomorph
cells
mhc
alloreactive
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Inventor
Alexandra Sharland
Eric Taeyoung Son
Moumita Paul-Heng
Nicole Mifsud
Anthony Purcell
Pouya Faridi
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Monash University
University of Sydney
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Monash University
University of Sydney
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Priority claimed from AU2019904221A external-priority patent/AU2019904221A0/en
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/0005—Vertebrate antigens
    • A61K39/001—Preparations to induce tolerance to non-self, e.g. prior to transplantation
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
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    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
    • G01N33/5047—Cells of the immune system
    • G01N33/505—Cells of the immune system involving T-cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/536—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/566—Immunoassay; Biospecific binding assay; Materials therefor using specific carrier or receptor proteins as ligand binding reagents where possible specific carrier or receptor proteins are classified with their target compounds
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56966—Animal cells
    • G01N33/56977—HLA or MHC typing
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
    • G01N33/6848—Methods of protein analysis involving mass spectrometry
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
    • C12N2750/00011—Details
    • C12N2750/14011—Parvoviridae
    • C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
    • C12N2750/14141—Use of virus, viral particle or viral elements as a vector
    • C12N2750/14143—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/70503—Immunoglobulin superfamily, e.g. VCAMs, PECAM, LFA-3
    • G01N2333/70539—MHC-molecules, e.g. HLA-molecules
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/10—Screening for compounds of potential therapeutic value involving cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/24—Immunology or allergic disorders
    • G01N2800/245—Transplantation related diseases, e.g. graft versus host disease

Definitions

  • the present invention relates generally to the field of immunology. More specifically, the present invention relates to the fields of organ and/or tissue transplantation and transplant rejection.
  • the invention relates to compositions for the detection of alloreactive T cells in transplant recipients, methods for producing the compositions and methods for the use thereof.
  • Transplantation is a life-saving treatment for end-stage organ failure.
  • recipients To prevent graft rejection, recipients must be treated indefinitely with immunosuppressive medications. These agents attenuate transplant rej ection but also impair the recipient's defences against infections and the development of cancer.
  • immunosuppressive drugs despite the introduction of increasingly powerful immunosuppressive drugs, the long-term outcomes of clinical organ transplantation have not improved significantly over the past 20 years. If specific immunological tolerance to transplanted tissues and organs could be achieved, long-term immunosuppression would not be required, sparing recipients from serious complications, and improving both graft and patient survival.
  • Allorecognition is the process whereby the recipient's T cells recognise donor major histocompatibility complex (MHC) molecules as foreign. Allorecognition may result in either graft rejection or in transplant tolerance induction, depending upon the context in which donor MHC is encountered.
  • the adaptive alloreactive T cell response can be a maj or barrier to tolerance induction but assays that are capable of accurately detecting and monitoring donor antigen-specific T cell responses in tissue and organ transplant recipients are currently lacking.
  • the present invention alleviates at least one of the problems associated with current approaches for detecting and/or monitoring an alloreaction by providing compositions comprising specific peptides that combine with allogeneic MHC molecules to provide the ligands for most alloreactive T cell clones in an alloreaction, and methods for identifying said peptides.
  • MHC I allogeneic MHC class I
  • AAV liver-specific adeno-associated viral
  • the inventors have also shown that direct recognition of the intact donor MHC molecule is required for tolerance induction.
  • the inventors have confirmed that the peptide antigen cargo of these donor MHC molecules (i.e. the endogenous liver peptide repertoire) plays an essential role in tolerance induction following MHC I gene transfer.
  • the present invention provides methods for detecting the specific peptides that combine with allogeneic MHC molecules to bind a high proportion of alloreactive T cells in an alloreaction, and compositions comprising said peptides and MHC molecules.
  • the present inventors have used the methods of the invention to identify a set of endogenous peptides which, when presented by allogeneic donor MHC class I molecules, are recognised by a high frequency of recipient alloreactive T cells.
  • This set of peptides may be incorporated into peptide-MHC multimers (e.g. tetramers, pentamers, dextramers), which may be used for the identification of alloreactive T cells within polyclonal recipient populations.
  • compositions described herein are generally useful for the detection of alloreactive T cells and may find application, for example, in the identification of alloreactive T cells within polyclonal populations in a transplant recipient.
  • Embodiment 1 A composition comprising one or more peptides, wherein each peptide is complexed to a specific MHC allomorph to form a peptide-MHC (pMHC) monomer, and wherein the one or more peptides have been identified by a method comprising:
  • identifying peptides bound to the specific MHC allomorph in: a) hepatocytes expressing allogeneic MHC molecules of the specific MHC allomorph; b) lymphoid tissue expressing the specific MHC allomorph; and c) transplant tissue expressing the specific MHC allomorph;
  • Embodiment 2 The composition according to embodiment 1, wherein at least one of the peptides has the amino acid sequence SNYLFTKL.
  • Embodiment 3 The composition according to embodiment 1 or embodiment 2, wherein at least one of the peptides has an amino acid sequence selected from:
  • Embodiment 4 The composition according to embodiment 1, wherein the peptides comprise or consist of the amino acid sequences:
  • Embodiment 5 The composition according to any one of embodiments 1 to 4, wherein the specific MHC allomorph is a mouse MHC allomorph.
  • Embodiment 6 The composition according to any one of embodiments 1 to 5, wherein the subject who has rejected a transplant expressing the specific MHC allomorph is a mouse.
  • Embodiment 7 The composition according to any one of embodiments 1 to 6, wherein the peptides all have the same amino acid sequence.
  • Embodiment 8 The composition according to any one of embodiments 1 to 6, wherein the peptides comprise a plurality of different amino acid sequences.
  • Embodiment 9 The composition according to any one of embodiments 1 to 8, wherein the pMHC monomers are capable of binding to at least 5% of alloreactive T cells in a recipient of a transplant expressing the specific MHC allomorph when contacted with a biological sample from the recipient.
  • Embodiment 10 The composition according to any one of embodiments 1 to 9, wherein the pMHC monomers are capable of binding to at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55% or at least 60% of alloreactive T cells in a recipient of a transplant expressing the specific MHC allomorph when contacted with a biological sample from the recipient.
  • Embodiment 11 The composition according to any one of embodiments 1 to 10, wherein the pMHC monomers form one or more mul timers.
  • Embodiment 12 The composition according to embodiment 11, wherein the multimers are attached to a reporter molecule.
  • Embodiment 13 The composition according to embodiment 12, wherein the reporter molecule is a fluorophore or a metal isotope.
  • Embodiment 14 The composition according to any one of embodiments 1 to 13, wherein the specific MHC allomorph is MHC class I.
  • Embodiment 15 The composition according to any one of embodiments 1 to 14, wherein the peptides are identified in (i) by immunoaffmity purification and/or mass spectrometry.
  • Embodiment 16 The composition according to any one of embodiments 1 to 15, wherein detecting peptides which bind to at least 2% of alloreactive T cells in (iv) is by:
  • Embodiment 17 The composition according to embodiment 16, wherein said detecting by flow cytometry comprises detection of PD1 expression on alloreactive T cells bound to said peptides.
  • Embodiment 18 The composition according to any one of embodiments 1 to 17, wherein the peptides detected in (iv) bind to at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, or at least 45% of alloreactive T cells in the sample of liver leukocytes.
  • Embodiment 19 The composition according to embodiment 1, wherein the specific MHC allomorph is a human MHC allomorph.
  • Embodiment 20 A method for identifying peptides which, when complexed to a specific MHC allomorph to form a peptide-MHC (pMHC) monomer, are capable of binding to at least 2% of alloreactive T cells in a transplant recipient when contacted with a biological sample from the transplant recipient, the method comprising:
  • identifying peptides bound to the specific MHC allomorph in: a) hepatocytes expressing allogeneic MHC molecules of the specific MHC allomorph; b) lymphoid tissue expressing the specific MHC allomorph; and c) transplant tissue expressing the specific MHC allomorph;
  • Embodiment 21 The method according to embodiment 20, wherein the specific MHC allomorph is a mouse MHC allomorph.
  • Embodiment 22 The method according to embodiment 20, wherein the specific MHC allomorph is a human MHC allomorph.
  • Embodiment 23 The method according to any one of embodiments 20 to 22, wherein the subject who has rejected a transplant expressing the specific MHC allomorph is a mouse.
  • Embodiment 24 The method according to any one of embodiments 20 to 23, wherein the specific MHC allomorph is MHC class I.
  • Embodiment 25 The method according to any one of embodiments 20 to 24 , wherein the peptides are identified in (i) by immunoaffmity purification and/or mass spectrometry.
  • Embodiment 26 The method according to any one of embodiments 20 to 25, wherein detecting peptides which bind to at least 2% of alloreactive T cells in (iv) is by:
  • Embodiment 27 The method according to embodiment 26, wherein said detecting by flow cytometry comprises detection of PD1 expression on alloreactive T cells bound to said peptides.
  • Embodiment 28 The method according to any one of embodiments 20 to 27, wherein the peptides detected in (iv) bind to at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, or at least 45% of alloreactive T cells in the sample of liver leukocytes.
  • Embodiment 29 A peptide which, when complexed to a specific MHC allomorph, is capable of binding to at least 2% of alloreactive T cells in a transplant recipient when contacted with a biological sample from the transplant recipient, wherein the peptide is obtained or obtainable by the method according to any one of embodiments 20 to 28.
  • Embodiment 30 A method for detecting alloreactive T cells in a transplant recipient, the method comprising:
  • Embodiment 31 The method according to embodiment 30, wherein detecting alloreactive T cells is by flow cytometry or mass cytometry.
  • Embodiment 32 A method of inducing tolerance to an MHC allomorph in a subject, the method comprising administering to the subject one or more peptides complexed to one or more components of the MHC allomorph to thereby induce tolerance to the MHC allomorph, wherein the peptide amino acid sequences comprise any one or more of:
  • composition “comprising” means “including”. Variations of the word “comprising”, such as “comprise” and “comprises” have correspondingly varied meanings. Thus, for example, a composition “comprising” peptides complexed to MHC molecules may consist exclusively of peptides complexed to MHC molecules or may include one or more additional components.
  • a plurality means more than one. In certain specific aspects or embodiments, a plurality may mean 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,
  • MHC is an abbreviation of major histocompatibility complex and encompasses the cell surface molecules which present peptide antigens to T cells and the genes encoding said molecules.
  • HLA is an abbreviation of human leukocyte antigen, which is the human version of the major histocompatibility complex.
  • MHC and HLA may be used interchangeably herein and both the cell surface molecules which present peptide antigens to T cells and the genes encoding said molecules may be included within the scope of these terms.
  • allomorph refers to a cell surface molecule that corresponds to a particular MHC allele.
  • FIG. 1 Expression of donor MHC in the recipient liver leads to donor-specific tolerance.
  • naive B10.BR (H-2 k ) recipients were injected with rAAV-expressing H- 2K b (rAAV-K b ) to induce K b expression on hepatocytes 7 days before challenge with a K b+ skin graft.
  • rAAV-K b -treated B10.BR mice accepted K b+ skin grafts.
  • MST median survival time
  • Figure 2 Donor-specific tolerance induction.
  • Figure 2 provides a non-limiting schematic of expression of donor MHC in a recipient liver leading to donor-specific tolerance.
  • Figure 3 Tolerance induction depends upon recognition of intact MHC class I.
  • Figure 3 provides a non-limiting schematic of direct and indirect allorecognition.
  • Allogeneic MHC class I may be recognized by recipient T cells as an intact molecule (direct recognition) or may be processed and presented as an allogeneic peptide in the context of self-MHC (indirect recognition).
  • Direct allorecognition is required for tolerance induction.
  • Figure 4 Direct allorecognition can be peptide-dependent or peptide-independent.
  • Figure 4 provides a non-limiting schematic of peptide-dependent and peptide-independent allorecognition.
  • Figure 5 Single chain trimer MHC vectors exclude presentation of endogenous peptides.
  • Figure 5a shows transduced hepatocytes in which the H-2 K b HC assembles with native ⁇ 2m and is loaded with a large repertoire of self-peptides.
  • the K b -KIITYRNL single- chain trimer (SCT) molecule excludes binding of naturally-processed endogenous peptides.
  • a single type of pMHC epitope is presented on the cell surface.
  • Figure 6 Generation of SCT constructs.
  • Figure 6 provides a non-limiting schematic for the process of generating a single-chain trimer (SCT) of ⁇ 2-microglobulin ( ⁇ 2m ), H-2K b heavy chain and a defined peptide sequence.
  • SCT single-chain trimer
  • FIG. 7 SCT-K b -KHT constructs exclude presentation of endogenous peptides.
  • FIGs 7a, 7b and 7c B10.BR mice were transduced with either AAV-sct-K b -SIIN, AAV-sct-K b -KIIT or AAV-HC-K b with or without AAV-OVA. Hepatocytes were isolated on day 7 post-inoculation, then subjected to antibody staining and flow cytometric analysis.
  • Figure 7a shows that equivalent levels of H-2K b cell surface expression were demonstrated using the mAh (AF6-88.5).
  • Figures 7b and 7c show detection of SIINFEKL (OVA257-264) peptide bound to H- 2K b carried out using anti-K b -SIINFEKL mAh (25D-1.16).
  • Control B10.BR hepatocytes B10.BR hepatocytes transduced with either AAV-HC-K b or AAV- sct-K b -KIIT and control C57BL/6 hepatocytes underwent immunoaffmity purification in order to identify unique H2-K b and H2-K k peptides (1% FDR cut-off).
  • Data from a single experiment are shown in Figures 7e to 7i. A total of 4 mice were pooled per group.
  • Figure 7e shows peptide- binding motifs for H2-K b peptides were generated from a non-redundant list of 8 ⁇ 11 -mer peptides using the GibbsCluster-2.0 (DTU bioinformatics) algorithm.
  • Figure 8 Exclusion of binding of naturally-processed endogenous peptides by the K b - KIITYRNL SCT molecule.
  • Figure 8 shows the numbers of unique K b -restricted peptides in B10.BR, B10.BR + K b -KIITYRNL SCT, B10.BR + K b -HC and C57BL/6 mice.
  • Figure 9 Peptides bound to hepatocytes expressing K b -WT and SCT-K b -KIITYRNL.
  • Figure 9 provides a schematic of peptides bound to hepatocytes expressing K b -WT and SCT-K b - KIITYRNL.
  • Figure 10 SCT can be recognised by peptide-specific transgenic T cells.
  • Figure 10 shows that B10.BR-RAG mice reconstituted with Des-RAG lymphocytes recognise KIITYRNL and allogeneic K b -KIITYRNL, and that OTI-RAG T cells recognise SIINFCKL and K b - SIINFCKL.
  • Figure 11 178.3 skin grafts survived indefinitely on unreconstituted B10.BR-RAG hosts.
  • a dose of 50,000 cells yielded skin graft survival approximating that in immunosufficient B10.BR mice, and was used in subsequent experiments.
  • FIG. 12 The liver immunopeptidome plays an important role in tolerance induction.
  • B 10.BR H-2 k background
  • B 10.BR-RAG mice reconstituted with Des-RAG cells which recognise the K b -KIITYRNL epitope
  • Des-RAG cells which recognise the K b -KIITYRNL epitope
  • K b -HC K b -HC
  • SCT-K b - KIITYRNL SCT-K b -SIINFEKL vectors
  • All vectors produced strong expression of H-2K b on the hepatocyte surface ( Figure 12a).
  • MST median survival time
  • Figure 12d provides a schematic of the experimental timeline.
  • Figure 13 Transplanted skin grafts from reconstituted B10.BR-RAG mice + SCT-K b - KIITYRNL were macroscopically and histologically normal with robust expression of K b through day 100.
  • Figure 13 shows that transplanted skin grafts from reconstituted B10.
  • BR-RAG mice + SCT-K b -KIITYRNL (syngeneic) and 178.3 mice + SCT-K b -KIITYRNL (allogeneic) were macroscopically (Figure 13a) and histologically (Figure 13b) normal with robust expression of K b through day 100, demonstrating continued allograft survival (Figure 13a shows n 6 per group).
  • FIG 14 Recognition of dt-SCT peptide-MHC ligands in vitro and in vivo.
  • RMA-S cells were pulsed with different concentrations of the peptides KIITYRNL (Pcid23i8- 325), SIINFEKL (OVA257-264) or AAAAFAAL (synthetic negative control), or were unpulsed. Stabilisation of H-2K b surface expression was assessed by flow cytometry following staining with a conformation-dependent anti-H-2K b mAh (clone Y3). Samples were acquired using a LSR Fortessa X-20 (BD Biosciences) and analysed using FlowJo vlO (BD).
  • FIG. 14b The flow plots shown in Figure 14b are representative of 3 separate experiments. Peptide concentrations required to achieve equivalent H-2K b surface expression levels were determined.
  • Figure 14c RMA-S cells were transiently transfected with constructs encoding sct-K b -KIIT, sct-K b -SIIN and sct-K b -AAAA using a Lonza-AMAXA Nucleofector 2b. Transgene expression was assessed by flow cytometry 24 hours after transfection.
  • Flow plots shown are representative of 3 separate experiments.
  • Figure 14d the frequency of interferon-g secreting cells upon recognition of their cognate antigens was determined using Interferon-g ELISPOT assays.
  • Splenocytes from Des-RAG or OT-I-RAG mice were cultured with irradiated stimulators; RMA-S pulsed with selected peptides or expressing dt-SCT constructs after transient transfection.
  • dt-SCT recognition by cognate TCRs mirrored recognition of the native H-2K b -peptide complex.
  • Des-RAG lymphocytes were labelled with CFSE, adoptively transferred into recipient mice and recovered from the recipient liver two days later. Some recipient mice were treated with AAV encoding sct-K b -KIIT or sct-K b -SIIN prior to adoptive transfer, as shown.
  • Figure 14f shows flow cytometry analysis of CFSE-labelled Des-RAG lymphocytes which demonstrates peptide-specific proliferation and activation of adoptively transferred CD8+ Des- RAG T-cells upon encounter with cognate antigen in the liver.
  • the sct-K b -KIIT construct was recognised in a peptide-specific manner in vivo.
  • FIG. 14g B10.BR mice were primed against allogeneic H-2Kb (178.3 skin graft) Approximately 30 days post-graft rejection, primed B10.BR or C57BL/6 mice were inoculated with AAV-sct-Kb-KIIT. Liver leukocytes were analysed on day 7 post-inoculation.
  • Figure 14h shows that inoculation of primed B10.BR mice with AAV-sct-K b - KIIT generated a population of activated (CD44 + PD1 hi ) CD8 + T cells which bound K b - KIITYRNL dextramers specifically.
  • Figure 15 Data demonstrating the safety and the effectiveness of dt-SCT constructs.
  • Figure 15a provides a schematic diagram illustrating the time-course experiment performed to assess the safety and the effectiveness of dt-SCT constructs.
  • FIG. 16 1 Flow cytometry analysis of H-2K b stabilisation levels.
  • Figure 16 shows flow cytometry analysis of H-2K b stabilisation levels on RMA-S (H-2K b ) cells which had been either unpulsed or pulsed with SIINFEKL (OVA257-264) orKIITYRNL (Pcid2 318-325 ) peptides.
  • H-2K b expression Equivalent levels of H-2K b expression were achieved with the indicated peptide concentrations (0.02 ⁇ M for SIINFEKL and 10 ⁇ M for KIITYRNL). (middle & right) Detection of SIINFEKL peptide bound to H-2K b was carried out using anti-K b -SIINFEKL mAh (25D-1.16). The mAh clone 25D-1.16 was validated before use.
  • Figure 17 In the absence of TAP, an altered peptide repertoire gains access to the ER through alternative routes.
  • Figure 17 provides a schematic of the altered peptide repertoire in TAPI knockout mice.
  • Figure 18 Disulphide bridging of MHC-I permits binding of lower affinity peptides and stabilises the expression of sub-optimally loaded MHC-I at the cell surface.
  • TCR recognition of a given pMHC is not altered by disulphide bridging of MHC-I.
  • FIG. 19 Characterisation of TaplKOHep mice. Hepatocytes were isolated from Tap1KOHep or Tap1 fl/fl mice transduced with either AAV-HC-K d or rAAV-K d -YCAC vectors. The effect of the YCAC modification is to stabilise surface expression of H-2 K d when loaded with low-affinity peptides or even when empty. While surface expression of H-2K d in Tap1KOHep mice transduced with AAV-HC-K d was clearly increased above background levels, expression levels equivalent to those in Tap1 fl/fl control mice could not be achieved, and raising the dose of vector from 5 x 10 11 vgc to 2 x 10 12 vgc did not further augment expression. In contrast, comparable strong surface expression of H2-K d was achieved in all mice receiving 5 x 10 11 vgc AAV-Kd-YCAC. Flow plots shown are representative of 3 separate experiments.
  • FIG. 20 Increasing perturbation of the K d -bound peptide repertoire in hepatocytes progressively shortened graft survival of B6.K d skin grafts.
  • TAP1 an altered peptide repertoire gains access to the antigen processing pathway.
  • Figure 21 Amino acid sequences for the sct-K b -SIIN, sct-K b -KIIT, sct-K d -SYFP and HC-K d -YCAC constructs.
  • Figure 22 Immunopeptidome analysis. The repertoire of bound peptides for the mouse MHC molecule H-2K b was determined using immunoaffmity purification and tandem mass spectrometry. Figure 22 provides a non-limiting schematic of the elution of K b -bound peptides from transduced hepatocytes, donor skin and donor spleen prior to mass spectrometry to identify peptides bound to allogeneic MHC molecules expressed in these tissues.
  • Figure 23 Peptide screening.
  • Figure 23 shows unique H-2K b peptides identified from B10.BR hepatocytes transduced with AAV-HC-K b , 178.3 spleen and 178.3 skin grafts collected 7 days after transplantation. Data from a single experiment are shown. Samples from 4 - 7 mice were pooled per condition. 332 peptides were found to be shared across all three tissue types and were screened by tetramer binding.
  • Figure 24 Length distribution of filtered H-2K b peptides from hepatocytes, spleen and skin graft tissue samples. Number of peptides of each length identified with a 1% FDR cut-off are shown.
  • FIG. 25 Peptide-binding motifs for H2-K b peptides.
  • peptide-binding motifs for H2-K b peptides were generated from a non-redundant list of 8-11 mer peptides using the GibbsCluster-2.0 (DTU bioinformatics) algorithm.
  • FIG. 26 pMHC epitope screening by tetramer-binding.
  • B10.BR-RAG mice were reconstituted with doses of Des-RAG lymphocytes ranging from 1.0 ⁇ 10 4 to 2.5 ⁇ 10 5 cells, 3 days prior to receiving a skin graft from a K b -bearing 178.3 donor. Some mice were also inoculated with AAV vectors as shown in Figure 26.
  • the Figure shows that, after allogeneic skin graft rejection, the liver contained a population of central memory CD8+ T cells, which could be expanded by boosting with a liver-specific AAV vector encoding the allogeneic MHC molecule to yield a highly-enriched population of activated alloreactive cells that could be used with pMHC multimers to determine which pMHC epitopes are recognised.
  • Figure 27 Screening of candidate allostimulatory peptides was carried out by tetramer binding/flow cytometry.
  • Figure 27 shows the gating strategy that was employed to identify and stratify CD8 + T cells into two groups; CD44 + PD1 hi and CD44 + PD1-.
  • CD44 + PD1 hi cells are activated alloreactive CD8+ T cells and CD44 + PD1- cells are non-activated internal control CD8+ T cells.
  • the bottom panel displays examples of pMHC tetramer binding by CD44 + PD1- and CD44 + PD1- CD8+ T-cells. Representative FACS plots for the selected pMHC epitopes are shown.
  • K b -SNYLFTKL (Epas387-394), K b - V GPRYTNL (Mapkl 19-20) and K b -RTYTYEKL (Ctnnbl 329-335) were recognised by a large proportion of activated alloreactive T cells, whereas few T cells bound K b -ASYEFVQRL (Dynclhl 1379-1387).
  • Figure 28 12/30 peptides were recognised in association with H-2K b by > 5% of alloreactive T cells.
  • Figure 28 shows that 30 of the 332 peptides common to all three tissue types were screened to determine whether they are recognised in association with H-2K b by alloreactive T cells. 12/30 peptides were recognised by > 5% of these T cells.
  • Figure 29 Screening results for the first 30 peptides.
  • Figure 29 provides a summary of pMHC tetramer binding by CD44 + PD1 hi (coloured or black dots) and CD44 + PD1- (grey dots) CD8+ T-cells for 30 pMHC epitopes screened.
  • Thresholds were set at 2% and 5% ofPD1 hi CD44 + CD8+ T cells. Of the 30 pMHC epitopes, 12 bound > 5% of these cells, and a further 8 bound between 2 and 5%. Figure 30. Combining pMHC tetramers yielded a cumulative increase in alloreactive
  • Figure 31 pMHC tetramer binding levels were strongly correlated with the product of their spectral intensity values across three different tissue immunopeptidomes.
  • Figure 31 shows that pMHC tetramer binding levels were strongly correlated with the product of their spectral intensity values across three different tissue immunopeptidomes (Pearson correlation coefficient - 0.6207, ***p - 0.00025).
  • data was obtained from one - two independent experiments with a total of 3 - 4 biological replicates. Values represent mean ⁇ SEM. ns, not significant, *p ⁇ 0.05, ** p ⁇ 0,01, *** p ⁇ 0.001.
  • Figure 32 pMHC epitopes recognised by alloreactive CD8+ T cells are conserved across different sexes and strains.
  • Figure 32 shows a summary of pMHC tetramer binding by CD44 + PD1 hi (coloured or black dots) and CD44 + PD1- (grey squares) CD8+ T-cells from male B10.BR mice, female B10.BR mice and male BALB/c mice for the pMHC epitopes screened.
  • Figure 33 provides a heatmap generated in order to compare how pMHC epitopes are recognised by alloreactive CD8+ T cells across different sexes and strains. pMHC epitopes are ordered from the top to the bottom by the average of pMHC tetramer binding across all samples.
  • Figure 34a shows allogeneic graft rejection tempos of male B10.BR, female B10.BR and male BALB/c mice.
  • pMHC epitopes were found to be largely conserved across different sexes and strains. For each pMHC epitope, data was obtained from one-two independent experiments with a total of 3 biological replicates. Values represent mean ⁇ SEM. ns, not significant; *p ⁇ 0.05, ** p ⁇ 0.01, *** p ⁇ 0.001.
  • Figure 35 provides a schematic diagram illustrating the immunoaffmity purification workflow'.
  • Figure 36 provides a diagram showing unique peptides identified from transduced hepatocytes, skin and spleen.
  • H-2K d the peptide repertoires of C57BL/6 hepatocytes transduced with AAV-HC-K d , B6.K d spleen and B6.K d skin grafts collected 7 days after transplantation were determined. Data from two experiments are shown. Within each replicate experiment, samples from 3-4 mice were pooled per condition. 8809-mer peptides w'ere found to be shared across all three tissue types,
  • Figure 37 provides a diagram showing unique peptides identified from transduced hepatocytes, skin and spleen.
  • H-2K b the peptide repertoires of B10.BR hepatocytes transduced with AAV-HC-K b , 178,3 spleen and 178.3 skin grafts removed 7 days post-transplant. Data from two experiments are shown. Within each replicate experiment, samples from 3-4 mice were pooled per condition. 1083 K b peptides (8-11 mer, IC50 ⁇ 500nM) were common to the three tissues.
  • Figure 38 provides a graph of the length distribution of filtered H-2K d peptides from hepatocytes, spleen and skin graft tissue samples. The number of peptides of each length identified with a 5% FDR cut-off are shown.
  • Figure 39 provides a graph of the length distribution of H-2K b peptides from hepatocytes, spleen and skin graft tissue samples. The number of peptides of each length identified with a 5% FDR cut-off are shown. Most eluted peptides are 8-mers, with 9-rners also relatively frequent.
  • Figure 40 provides a diagram of the peptide-binding motifs for H2-K d peptides generated from a non-redundant list of 9-mer peptides using GibbsCluster-2.0 (DTU bioinformatics) algorithm. The canonical binding motifs were observed for all three tissues
  • Figure 41 provides a diagram of the peptide-binding motifs for H2-K b peptides generated from a list of 8-11-mer beptides using GibbsCiuster-2.0 (DTU bioinformatics) algorithm. The canonical binding motifs were observed for all three tissues.
  • Figure 42 provides a diagram showing that the extent of peptide sharing between the H2-K d repertoires of hepatocytes, spleen and skin was substantially reduced when Tap1KOHep, Tap1fl/fl or C57BL6 mice inoculated with AAV-HC-Kd-YCAC were substituted for C57BL/6 transduced with AAV-HC-K d .
  • Figure 43 provides example spectra for three pairs of synthetic and eluted peptides (left). The Pearson correlation coefficients between the log 10 intensities of identified b- and y-ions in the synthetic and sample-derived spectra are shown on the right. The corresponding p-value was ⁇ 0,05 for each peptide pair.
  • Figure 44 provides a schematic of an experiment in which male B10.BR mice were primed with male 178.3 skin grafts and 30 days after rejection, mice were transduced with AAV-HC-K b . Liver leukocytes were isolated and pMHC tetramer binding was analysed using flow cytometry
  • Figure 45 provides plots showing the gating strategy that was employed to identify and stratify CD8+ T cells into two groups; CD44+ PD1hi and CD44+ PD1-.
  • CD44+ PD1hi cells are activated alloreactive CD8+ T cells and CD44+ PD1- cells are non-activated bystander CD8+ T cells which serve as internal controls.
  • Figure 46 provides representative FACS plots for selected pMHC epitopes.
  • K b -SNYLFTKL Espas 387-394
  • K b -VGPRYTNL Mapkl 19-26
  • K b -RTYTYEKL Ctnnb1 329-336
  • Figure 47 provides a heatmap which compares how pMHC epitopes are recognised by alloreactive CD8+ T cells across different sexes and strains. pMHC epitopes are ordered from the top to the bottom by the average of pMHC tetramer binding across ail samples. 13 pMHC were recognised by ⁇ 5% of activated alloreactive T cells across all three groups.
  • data w'as obtained from one - two independent experiments with a total of 3 biological replicates. Values represent mean + SEM. ns, not significant; *p ⁇ 0.05, ** p ⁇ 0.01, *** p ⁇ 0.001.
  • Predicted peptide binding affinity for H-2K b did not differ significantly between strongly, moderately or non-immunogenic peptides (p 0098 by one-way ANOVA, line at mean).
  • Figure 52 provides plots of the results of staining with two different pMHC tetramers to evaluate the proportion of T cells recognising more than one pMHC specificity.
  • Six strongly immunogenic peptides were tested. 86.7% of SNYLFTKL+ T cells recognised SGYIYHKL in addition to SNYLFTKL, while SVYVYKVL tetramers bound 66.8% of SNYLFTKL+ T cells and SGYIYHKL tetramers bound 75.8% of SVYVYKVL+ cells.
  • Figure 53 provides a heatmap of the results of staining with two different pMHC tetramers was used to evaluate the proportion of T cells recognising more than one pMHC specificity. Six strongly immunogenic peptides were tested.
  • the present inventors have developed methods which can identify the specific endogenous peptides, from the large self-peptide repertoire, that combine with allogeneic MHC molecules to form T cell epitopes in the context of an alloreaction.
  • the peptides identified by the methods may be combined with the allogeneic MHC molecules to form peptide-MHC (pMHC) multimers which may be used with flow cytometry or mass cytometry for the detection of alioreactive T cells in transplant recipients.
  • pMHC peptide-MHC
  • the present invention provides methods for identifying peptides which, when complexed to a specific MHC allomorph, are capable of binding to at least 2% of alioreactive T cells in a recipient of a transplant expressing the specific MHC allomorph when contacted with a biological sample from the transplant recipient.
  • the methods of the present invention may involve the identification of a set of endogenous peptides bound to a specific MHC allomorph common to recipient hepatocytes, donor lymphoid tissue and the graft.
  • the identification of specific peptides bound to allogeneic MHC molecules of a specific MHC allomorph in recipient hepatocytes could be achieved by the introduction of the allogeneic MHC molecule to a recipient liver.
  • the allogeneic MHC molecule of a specific MHC allomorph may be introduced to a recipient liver by a vector carrying the gene encoding the specific MHC allomorph.
  • the vector may be a viral vector, for example, an adeno-associated virus (AAV), a lentivirus or a retrovirus.
  • AAV adeno-associated virus
  • the DNA could be introduced via plasmids, mini-circles and/or nanoparticles.
  • the transgene may be cDNA and may be codon-optimised in order to achieve higher expression of the transgene product.
  • Various enhancers and promoters may be used to control the level and/or cell type specificity of expression. Liver-specific expression of the gene may be achieved with the use of a liver-specific promoter.
  • the vector may be introduced via injection, which could be local or intravenous.
  • Intravenous delivery could be via the hepatic artery or portal vein and cannulation may be used. Additionally or alternatively, intravenous delivery may be via a peripheral vein.
  • intravenous delivery may be achieved via the tail vein.
  • the penile vein may be used.
  • Intraperitoneal injection may also be used in the methods of the present invention. Doses of the vector can vary, and high doses can be used where a severe immune response would not be problematic. The person skilled in the art would be familiar with many techniques which could be used to express an allogeneic MHC molecule in recipient tissue.
  • the methods of the present invention may involve the identification of a set of endogenous peptides bound to the specific MHC allomorph in recipient hepatocytes which are also common to donor lymphoid tissue and the graft. Methods for isolating hepatocytes, splenocytes and lymphocytes from draining lymph nodes are standard in the art.
  • Immunoaffinity purification also known as immunoadsorption chromatography, is a commonly used technique in the art.
  • Immunoadsorption chromatography is a form of affinity chromatography which utilises an antibody or antibody fragment as a ligand immobilized onto a solid support matrix in a manner that retains its binding capacity. This method is commonly performed for the identification, quantification, or purification of antigens and may be used in some embodiments of the present invention to isolate the peptides bound to a specific MHC allomorph across all three tissue types of interest.
  • the crude extract from hepatocytes, lymphoid tissue or the graft may be pumped through an immunoaffinity column and the unbound material washed clear prior to elution of the retained antigen, i.e. the peptide complexed to the specific MHC allomorph, by alterations to the mobile-phase conditions that weaken the antibody-antigen interaction.
  • Immunoaffinity purification of the peptides of the present invention may be achieved with the use of antibodies which bind specifically to the specific MHC allomorph.
  • the present inventors have provided non-limiting examples of antibodies which bind specifically to mouse MHC molecules which may be used in some embodiments of the present invention.
  • Antibodies may be easily purchased which bind to specific MHC molecules in a variety of species including, but not limited to, mouse, human, rat, cynomolgus monkey and macaque.
  • Antibodies which bind specifically to a specific MHC allomorph may be used, or antibodies which will bind an epitope common to several MHC types within a species may be employed.
  • an affinity tag may be engineered into the allomorph to facilitate immunoprecipitation.
  • Peptides may be eluted from the MHC molecules using, for example, acetic acid, or may be washed with a mild acid. Peptides may be reconstituted in, for example, formic acid for further analysis. In some embodiments, 0.1% formic acid may be used to reconstitute the peptides for further analysis.
  • Alternative methods for identifying peptides bound to MHC allomorphs may be found in Purcell et al. 2019 Nature Protocols 14(6): 1687-1707. The entire contents of this publication are incorporated herein by cross reference.
  • Elution of peptides bound to specific MHC allomorphs from hepatocytes transduced with a gene encoding the specific MHC allomorph, graft tissue expressing the specific MHC allomorph and lymphoid tissue expressing the specific MHC allomorph may be followed with mass spectrometry (MS) to identify the peptides.
  • MS mass spectrometry
  • Reconstituted peptides may be analysed by LC- MS/MS using an information-dependent acquisition strategy. Data-independent methods may also be used, for example, SWATH-MS.
  • targeted MS methods may be used such as selected reaction monitoring (SRM), parallel reaction monitoring (PRM) or multiple reaction monitoring (MRM).
  • SRM selected reaction monitoring
  • PRM parallel reaction monitoring
  • MRM multiple reaction monitoring
  • Peptides identified by immunoaffmity purification and mass spectrometry as binding to a specific MHC allomorph across all three tissue types (liver, lymphoidtissue and the graft) may be screened for recognition, in association with the specific MHC allomorph, for binding to alloreactive T cells.
  • the peptides identified as binding to a specific MHC allomorph across all three tissue types may be screened for binding to alloreactive T cells by the creation of peptide-MHC (pMHC) multimers comprising the peptide and the specific MHC allomorph for incubation with a sample potentially containing alloreactive T cells.
  • the multimers may be tetramers. In other embodiments, the multimers may be pentamers or dextramers. Any number of MHC monomers may be incorporated into a multimer for use the methods of the present invention.
  • pMHC multimers are widely used in the art for the detection and monitoring of antigen- specific T cells by, for example, flow cytometry or mass cytometry.
  • Multimerisation of soluble pMHC extends the interaction between pMHC and the TCR by increasing avidity.
  • pMHC multimers may be easily manufactured in the laboratory by the skilled artisan. Monomeric proteins of the same pMHC type are commonly assembled into, for example, tetramers or dextramers.
  • soluble pMHC is complexed with biotin.
  • a reporter molecule conjugated to streptavidin may be added to biotinylated pMHC molecules.
  • Other pairs of molecules with strong binding affinity may be substituted for biotin and streptavidin in the creation of multimers, for example, Ni2+nitrilotriacetic acid and histidine can also be used in this way.
  • the reporter molecule may be a fluorophore.
  • fluorophores which could be incorporated into the pMHC multimers of the present invention include R-phycoerythrin (PE), allophycocyanin (APC), and members of the BD Horizon Brilliant Violet family, for example, BV421 and BV605.
  • the fluorophores exemplified herein and others may be purchased with or without conjugated streptavidin. No limitation exists regarding fluorophores used in the multimers.
  • the reporter molecule may be a metal isotope.
  • the metal isotope may be a lanthanide.
  • the pMHC monomers and reporter molecules may be attached to a dextran backbone. Ratios of reporter molecules to pMHC monomers may be varied according to the application. Many publications exist to aid the skilled worker in creating multimers according to the present invention (see, for example, Dolton et al.2014 Clinical and Experimental Immunology 177(1): 47-63). No limitation exists in relation to the multimer technology used in the invention in general.
  • multimers may be tagged with an oligonucleotide barcode enabling differentiation of T cells bearing TCRs with various pMHC specificities within an alloreactive T cell population.
  • a non-limiting example of such multimers are Immudex DCode dextramers.
  • Synthetic peptides may be used in the pMHC multimers.
  • the peptides may be created by solid-phase peptide synthesis, which is an approach commonly used in the art (Palomo 2014 RSC Advances 4: 32658-32672).
  • peptides may be ordered from one of the many suppliers or providers of custom peptide synthesis.
  • a sample containing T cells for use in screening could be obtained from a subject who has rejected a transplant expressing the specific MHC allomorph.
  • the liver of the subject may contain a population of central memory CD8+ T cells specific for the MHC allomorph. This population of memory T cells could be expanded by introducing specific MHC allomorph to the subject using a liver-specific vector.
  • the vector may be a viral vector, for example, an adeno-associated virus (AAV), a lentivirus or a retrovirus and may contain a gene encoding the specific MHC allomorph. Introducing the allomorph to the subject may boost the immunological memory of the subject in relation to the specific allomorph.
  • AAV adeno-associated virus
  • a sample may then be obtained from the subject using one of the many methods known in the art for use in pMHC multimer staining using the pMHC multimers incorporating the peptides bound to the specific MHC allomorph across all three tissue types (liver, lymphoid tissue and the graft).
  • antibodies which bind to cell surface markers on T cells may be used to identify alloreactive T cells bound to the multimers.
  • the antibodies specific for the cell surface markers may be attached to a reporter molecule, for example, a fluorophore or a metal isotope.
  • the antibodies may be conjugated to biotin.
  • Non-limiting examples of cell surface markers which could be used for identification include CD3, CD4, CD8, CD19, CD44, CD90, Tim-3, CD69 and PD1.
  • Cell surface markers may be positive or negative markers for the T cells of interest.
  • protein kinase inhibitors may be used prior to staining to increase the efficiency of staining.
  • a non-limiting example of a protein kinase inhibitor which may be used is dasatinib.
  • Stabilisation of multimer binding at the cell surface may be achieved in some embodiments of the invention with an antibody against the fluorophore (eg anti-PE or anti-APC).
  • the antibody against the fluorophore would be biotinylated, and could be followed by a secondary reagent (eg streptavidin-PE could follow anti-PE-biotin) to further amplify the multimer staining.
  • pMHC multimer staining of a sample may be followed by analysis of the sample by flow cytometry or mass cytometry.
  • flow cytometry is used to detect binding of the peptides to alloreactive T cells. Detection may be achieved by lymphocyte gating, which is an immunophenotyping technique well known to those skilled in the art (Loken et al. 1990 Cytometry 11 : 453-459). An initial subset of lymphocytes may be identified by light scattering, for example, using the parameters of forward scatter and side scatter. T cells may then be detected using the presence of T cell-specific markers such as CD3 and CD8.
  • activated T cells may be identified by the presence of cell surface markers such as CD44 and/or PD1.
  • cell surface markers such as CD44 and/or PD1.
  • FACS fluorescence activated cell sorting
  • peptides will be selected for inclusion in the compositions of the invention based on their ability, when complexed to a specific MHC allomorph, to bind to at least 2%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, or at least 45% of alloreactive T cells in a recipient of a transplant expressing the specific MHC allomorph who has rejected the transplant, when contacted with a biological sample from the recipient.
  • compositions of the present invention may comprise peptides identified by the methods described above complexed to a specific MHC allomorph.
  • the compositions may comprise peptides with the same amino acid sequence.
  • the peptide may be capable of binding to at least 2%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, or at least 45% of alloreactive T cells in a recipient of a transplant expressing the specific MHC allomorph when contacted with a biological sample from the recipient.
  • the peptide may have the amino acid sequence SNYLFTKL, ATLVFHNL, VGPRYTNL, RTYTYEKL, INFDFPKL, SVYVYKVL, HIYEFPQL, VAFDFTKV, VSFTYRYL, RNYSYEKL, SGYIYHKL, SSYTFPKM, SAFSFRTL, VSPLFQKL, VSQYYPKL, VSYLFSHV, HGYTFANL, VGPRYTQL, ATQYYPKL, ATRSFPQL, AVLSFSTRL, LQYEFTKL, or VNVDYSKL.
  • the peptide may be another peptide identified by the methods of the present invention.
  • the specific MHC allomorph may be MHC class I.
  • the peptide complexed to a specific MHC allomorph may be further complexed to form a multimer as described above.
  • the multimers may be tetramers. In other embodiments, the multimers may be pentamers or dextramers. Any type of multimer is encompassed by the compositions of the invention.
  • pMHC multimers may be easily manufactured in the laboratory by the skilled artisan. Monomeric proteins of the same pMHC type are commonly assembled into, for example, tetramers or dextramers.
  • soluble pMHC is complexed with biotin.
  • a reporter molecule conjugated to streptavidin may be added to biotinylated pMHC molecules. Other pairs of molecules with strong binding affinity may be substituted for biotin and streptavidin in the creation of multimers for the compositions of the invention.
  • the reporter molecule may be a fluorophore.
  • fluorophores which could be incorporated into the pMHC multimers of the present invention include R-phycoerythrin (PE), allophycocyanin (APC), fluorescein isothiocyanate (FITC) and Alexa Fluor 488 (Alexa488).
  • the fluorophores exemplified herein and others may be purchased with or without conjugated streptavidin. No limitation exists regarding fluorophores used in the multimers.
  • the reporter molecule may be a metal isotope. In some embodiments, the metal isotope may be a lanthanide.
  • the pMHC monomers and reporter molecules may be attached to a dextran backbone. Ratios of reporter molecules to pMHC monomers may be varied according to the application. Many publications exist to aid the skilled worker in creating multimers for the compositions of the present invention (see, for example, Dolton et al. 2014 Clinical and Experimental Immunology 177(1): 47-63). No limitation exists in relation to the multimer technology used in the compositions of the invention.
  • the multimers may be combined to create a panel combining a plurality of peptides.
  • the peptides used could be SNYLFTKL, ATLVFHNL, VGPRYTNL, RTYTYEKL, INFDFPKL, SVYVYKVL, HIYEFPQL, VAFDFTKV, VSFTYRYL, RNYSYEKL, SGYIYHKL, SSYTFPKM, SAFSFRTL, VSPLFQKL, VSQYYPKL, VSYLFSHV, HGYTFANL, VGPRYTQL, ATQVYPKL, ATRSFPQL, AVLSFSTRL, LQYEFTKL, or VNVDYSKL or any combination thereof.
  • the composition includes SNYLFTKL.
  • the composition comprises SNYLFTKL, ATLVFHNL, VGPRYTNL, RTYTYEKL, INFDFPKL and SVYVYKVL.
  • the pMHC monomers are capable of binding to at least 2%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55% or at least 60% of alloreactive T cells in a recipient of a transplant expressing the specific MHC allomorph when contacted with a biological sample from the recipient.
  • compositions of the present invention may be provided in any suitable diluent and/or additive.
  • the compositions may be provided in PBS or TBS to maintain a constant pH.
  • Bovine serum albumin (BSA) may be added to the compositions.
  • suitable diluents and/or additives include HBSS, glycerol, EDTA and sodium azide (NaN 3 ).
  • the additive is a preservative.
  • the compositions may also contain protease inhibitors, such as leupeptin or pepstatin.
  • Synthetic peptides may be used in the pMHC multimers.
  • the peptides may be created by solid-phase peptide synthesis, which is an approach commonly used in the art (Palomo 2014 RSC Advances 4: 32658-32672).
  • peptides may be ordered from one of the many suppliers or providers of custom peptide synthesis.
  • the present invention also provides methods using the compositions of the invention for detecting alloreactive T cells in a transplant recipient.
  • the transplant is not limited to a particular type of organ or tissue.
  • the recipient of the transplant could be a mammal.
  • the transplant recipient is a mouse, human, rat, cynomolgus monkey or macaque.
  • the method comprises obtaining a biological sample from the transplant recipient and contacting the sample with the compositions of the present invention. Alloreactive T cells may then be detected using flow cytometry or mass cytometry.
  • the methods may be used to identify, track and/or characterise alloreactive T cells from a transplant recipient. T cell receptors of alloreactive T cells detected by the methods could be cloned for future study of the alloreaction.
  • the peptides of the present invention may find application in the induction of tolerance to a specific MHC allomorph in a subject prior to organ or tissue transplantation.
  • the peptides described herein, or other peptides identified by the methods of the invention may be used to induce tolerance to a specific MHC allomorph expressed by an organ or tissue. Any one or more peptides identified by the methods of the invention may be used with the specific allomorph to induce tolerance.
  • one, two or any number of peptides identified by the methods described herein may be complexed to the entire MHC allomorph and administered to a subject to thereby induce tolerance.
  • one, two or any number of peptides identified by the methods of the invention may be complexed to one or more components of the MHC allomorph.
  • suitable components of the MHC allomorph include ⁇ 2-microglobulin, or the heavy chain of the allomorph.
  • the peptide/s and MHC allomorphs, or components thereof may be expressed in the liver of the subject to induce tolerance to the allomorph.
  • a non-limiting example of a way in which the peptides of the invention may be used to induce tolerance to an MHC allomorph is provided in Example One. The person skilled in the art will recognise that the peptides of the invention and the allomorphs, or components thereof, may be administered to the subject in various ways in order to induce tolerance to the allomorph in the subject.
  • Example One The endogenous peptide repertoire of hepatocytes plays a critical role in tolerance induction
  • AAV-mediated expression of donor MHC I heavy chain (K b -HC) in MHC-mismatched recipient hepatocytes (H-2 k ) has been shown to induce donor-specific tolerance in a mouse skin transplant model ( Figures 1 and 2).
  • Tolerance can be induced in mice primed by prior rejection of a donor-strain skin graft, as well as in naive recipients.
  • Allogeneic MHC class I may be recognised by recipient T cells as an intact molecule (direct recognition) or may be processed and presented as an allogeneic peptide in the context of self-MHC (indirect recognition). Tolerance induction depends upon recognition of the intact donor MHC molecule by alloreactive CD8 + T cells. ( Figure 3). Direct allorecognition can be peptide-dependent or peptide-independent ( Figure 4). Most alloreactive CD8 + T cell clones recognise specific peptides in association with allogeneic
  • the endogenous peptide repertoire of hepatocytes plays a critical role in tolerance induction.
  • AAV vectors were engineered expressing a single-chain trimer (SCT) of ⁇ 2- microglobulin ( ⁇ 2m), H-2K b heavy chain and a defined peptide sequence (KIITYRNL or SIINFEKL) ( Figures 5 and 6).
  • SCT single-chain trimer
  • ⁇ 2m microglobulin
  • H-2K b heavy chain H-2K b heavy chain
  • SIINFEKL SIINFEKL
  • FIG. 9 A schematic showing the immunoaffmity purification and elution of peptides bound to hepatocytes expressing K b -WT and SCT-K b -KIITYRNL is provided in Figure 9.
  • the SCT can be recognised by peptide-specific transgenic T cells.
  • OT-I-RAG T cells can recognise SIINFEKL.
  • Des-RAG T cells can recognise KIITYRNL ( Figure 10).
  • B10.BR H-2 k background
  • B10.BR-RAG mice reconstituted with Des-RAG cells (which recognise the K b -KIITYRNL epitope), were inoculated with either K b -HC, SCT-K b -KIITYRNL or SCT-K b -SIINFEKL vectors, then challenged with K b -bearing skin grafts.
  • a dose of 50,000 cells yielded skin graft survival approximating that in immunosufficient B10.BR mice, and was used in subsequent experiments.
  • a global change in peptide loading was introduced, altering the repertoire of presented peptides.
  • a construct was designed expressing the H-2K d heavy chain with a modification (Y84C, A139C, subsequently termed YCAC) which stabilises the molecule in a peptide-receptive configuration, and permits the binding of lower affinity peptides.
  • Mice with hepatocyte-specific absence of TAP1 (Tap1KOHep, H-2 b ), generated using the Cre-Lox system, and controls, were transduced with AAV-K d -YCAC.
  • TAP transports high affinity peptides for MHC-I loading.
  • an altered peptide repertoire gains access to the ER through alternative routes ( Figure 17).
  • the YCAC modification permits binding of lower affinity peptides and stabilises the expression of suboptimally loaded MHC-I at the cell surface via disulphide bridging of MHC-I, without altering TCR recognition of a given pMHC ( Figure 18 (Hein el al. 2014 Journal of Cell Science 127: 2885-2897)).
  • the effect of the YCAC modification is to stabilise surface expression of H-2 K d when loaded with low-affinity peptides or even when empty.
  • mice were grafted with allogeneic K d -bearing B6.K d skin.
  • an altered peptide repertoire gains access to the antigen processing pathway.
  • Increasing perturbation of the K d -bound peptide repertoire in hepatocytes progressively shortened graft survival of B6.K d skin grafts.
  • Expression of K d -YCAC in the hepatocytes of Tap1KOHep mice perturbed the bound peptide repertoire of H-2K d to a greater extent than expression inTap1 fl/fl control or WT C57BL/6 ( Figure 20).
  • Peptides were synthesised with an average of 98% purity (GL Biochem Shanghai Ltd.).
  • the lyophilised peptides were reconstituted in 10% DMSO and all peptides were stored at -80°C.
  • Dasatinib (Sigma- Aldrich, catalogue# CDS023389) was reconstituted in DMSO and 5 mM stocks were stored at -80°C.
  • T lymphoma cell line RMA-S cells were cultured in RPMI 1640 medium supplemented with L-glutamine (Lonza, catalogue# 12-702F), penicillin-streptomycin (Invitrogen, catalogue# 15140) and 10% FCS (Sigma-Aldrich, catalogue# 13K179) at 37°C with 5% CO 2 .
  • RMA-S cells were grown to confluence and passaged to a concentration of 3 ⁇ 10 5 cells/mL, then transferred to flat-bottomed 24 well culture plates. The cells were incubated at 27°C for 20 hours with 5% CO 2 , then pulsed with 6 different concentrations of peptides ranging from 0.0001 - 10 ⁇ M. The cells were incubated with peptides at 27°C for 1 hour then returned to 37°C for 2 hours. The surface expression of stabilised H-2 K b was quantified using flow cytometry with the conformation-dependent mAb Y-3 antibody which binds to correctly folded K b molecules stabilised with peptides.
  • B10.BR (H-2 k ), 178.3 and Des-RAG mice were used. 178.3 mice express a transgenic MHC class I molecule H-2K b ubiquitously, under the control of its own promoter, on a B10.BR (H-2 k ) background. Des-TCR mice express an H-2K b -specific TCR which recognises the peptides KVITFIDL, KVLHFYNV and KIITYRNL restricted by H-2K b and Des-TCR is identifiable by a clonotypic mAb (Desire). Des-RAG mice were obtained by crossing Des-TCR mice with CD45.1 +/+ RAG1 -/- mice which are both on a B10.BR (H-2 k ) background.
  • C57BL/6JArc (H-2 b ) and BALB/c (H-2 d ) mice were purchased from the Animal Resources Centre.
  • B6.Kd mice express an H-2K d transgene ubiquitously on a C57BL/6 (H-2 b ) background.
  • B6.Kd mice were backcrossed for 3 generations to C57BL/6J prior to use.
  • Tap1KOHep mice are homozygous for the floxed Tap1 allele and have one copy of albumin-Cre which allows specific deletion of the floxed Tap1 allele in the hepatocytes.
  • Tap1KOHep mice were generated by crossing Tap1 fl/fl mice with albumin promoter-driven Cre (Alb-Cre) transgenic mice which are both on a C57BL/6 (H2-K b ) background. Male mice aged between 8 and 12 weeks were used in these experiments. Male mice were used unless stated otherwise. At the termination of each experiment, all tissues were harvested under general anaesthesia. Frozen tissues were stored at -80°C.
  • a single chain trimer construct with a disulphide trap consists of a defined peptide sequence, ⁇ 2-microglobulin and MHC-I heavy chain were joined together by flexible linkers.
  • the construct encodes a signal peptide sequence followed immediately by a defined peptide sequence, then a linker of GCGAS(G4S)2, a ⁇ 2 m sequence, a linker of (G4S)4 and either a heavy chain H2- K b or H2-K d sequence.
  • a tyrosine to cysteine substitution at heavy chain position 84 and a cysteine at the second position of the peptide- ⁇ 2 m linker form the disulphide trap.
  • dt-SCT (H-2K b ) constructs with defined peptide sequences KIITYRNL or SIINFEKL and a dt-SCT (H-2K d ) construct with a defined peptide sequence SYFPEITHI were designed in-silico (termed sct-K b - KIIT, sct-K b -SIIN and sct-K d -SYFP respectively), codon-optimised and were synthesised by GeneArt (Thermo Fisher Scientific).
  • H2-K d sequences incorporating the Y84C and A139C mutations were created in-silico (termed K d -YCAC), codon optimised and were synthesised by GenScript.
  • the amino acid sequences for the sct-K b -SIIN, sct-K b -KIIT, sct-K d -SYFP and HC-K d - YCAC constructs are provided in Figure 21.
  • All synthesised genes were delivered in pcDNA3.1 + plasmids.
  • the full-length native chicken ovalbumin (OVA) gene inserted in a pcDNA3.1 + plasmid (clone ID: OGa28271) was purchased from GenScript.
  • Gene inserts from pcDNA3.1 + plasmids were cloned into the pAM2AA backbone incorporating the human ⁇ -1 antitrypsin, liver-specific, promoter and human ApoE enhancer flanked by AAV2 inverted terminal repeats. Each gene was then packaged into an AAV2/8 vector, purified, and quantitated.
  • AAV2/8 vector aliquots were stored at -80°C.
  • AAV2/8 vectors in 500 ⁇ l sterile PBS were administered to male mice via penile vein intravenous injection and to female mice via tail vein intravenous injection under general anaesthesia.
  • OCT embedded frozen tissues were cut into 6 ⁇ m thick sections. Sections were allowed to air dry for 1 hour at room temperature (RT) prior to fixation in acetone for 8 minutes at RT. Sections were blocked with 20% normal mouse serum (MilliporeSigma, catalogue# M5905) and 5% normal porcine serum (Thermo Fisher Scientific, catalogue# 31890) for 20 minutes at RT and they were stained with FITC-conjugated primary antibodies against H2-K b (AF6-88.5, BioLegend), H2-K d (SFl-1.1, BD Biosciences), CD4 (GK 1.5, BD Biosciences), CD8a (53-6.7, Biolegend), F4/80 (BM8, Biolegend), B220 (RA3-6B2, BD Biosciences), CD11c (N418, Biolegend) or CD19 (6D5, Biolegend) (Table 1) or the corresponding isotype controls for 30 minutes at RT.
  • H2-K b AF6-88.5, BioLegend
  • Sections were then stained with horseradish peroxidase- conjugated rabbit-anti-FITC secondary antibody (Bio-Rad, catalogue# 4510-7864) before development with diaminobenzidine (DAB) substrate chromogen system (Dako, catalogue# K3468). Sections were counterstained in Mayer's hematoxylin solution (MilliporeSigma, catalogue# MHS16) for 2 minutes and mounted with Fronine safety mount No.4 (Thermo Fisher Scientific, catalogue# FNNII068). For H&E staining, 5 ⁇ m thick sections from formalin fixed paraffin embedded tissues were used.
  • IFN-g ELISpot assays were performed according to the manufacturer's protocol (U-Cytech, catalogue# CT317-PR5).
  • RMA-S cells either pulsed with peptides or transiently transfected with dt-SCT plasmids, were irradiated with a dose of 3000 rad.
  • Responders were either OT-I-RAG or Des-RAG splenocytes.
  • 1 ⁇ 10 6 irradiated stimulator cells and 1 ⁇ 10 6 responder splenocytes were suspended in 250 ⁇ l of RPMI/FCSIO medium with penicillin- streptomycin in each well of a 96-well El-bottom plate (Coming, catalogue# 3788).
  • Example Two An innovative strategy combining mass spectrometry with pMHC multimer staining permits discovery of the individual pMHC epitopes recognised by alloreactive T cells.
  • This example describes a screening strategy to identify allostimulatory pMHC epitopes.
  • the H-2K b immunopeptidomes of various tissues were determined using immunoaffmity purification and tandem mass spectrometry. Given that expression of donor MHC I in recipient hepatocytes induces tolerance to skin grafts and suppresses production of IFN- ⁇ by alloreactive T cells upon stimulation with donor splenocytes, it was hypothesised that the peptides which are critical for allorecognition and tolerance induction would be shared between all three tissue types (i.e.
  • liver contained a population of central memory CD8+ T cells, which could be expanded by boosting with a liver-specific AAV vector encoding the allogeneic MHC molecule to yield a highly- enriched population of activated alloreactive cells that could be used with pMHC multimers to determine which pMHC epitopes are recognised.
  • B10.BR mice were primed with 178.3 skin grafts and 30 days after rejection, mice were transduced with AAV-HC-K b . Liver leukocytes were isolated and pMHC tetramer binding was analysed using flow cytometry (Figure 25).
  • CD44 + PD1 hi and CD44 + PD1- are activated alloreactive CD8+ T cells and CD44 + PD1- cells are non-activated internal control CD8+ T cells.
  • K b -SNYLFTKL Espas387-394
  • K b - VGPRYTNL Mapk1 19-26
  • K b -RTYTYEKL Ctnnbl 329-336
  • Retrograde perfusion of the liver was achieved by cannulating the IVC and allowing the perfusate to flow out of the liver via the transected hepatic portal vein.
  • the liver was sequentially perfused with the following solutions at a flow rate of 5 ml/min (administered using a Masterflex L/S 7528-30, Thermo Fisher Scientific); firstly with 25 ml of HBSS (Lonza, catalogue# 10-543F), then with 25 ml of HBSS with 0.5 mM EDTA (MilliporeSigma, catalogue# E6758), followed by 25 ml of HBSS, and finally with 25 ml of HBSS plus 5 mM CaCl 2 (calcium chloride, MilliporeSigma, catalogue# C5670) and 0.05% of collagenase IV (collagenase Type IV, Thermo Fisher Scientific, catalogue# 17104019).
  • liver leukocyte isolation the IVC was cannulated and the hepatic portal vein was transected.
  • the liver was flushed with 20 ml of PBS at RT and after gall bladder removal, the liver was meshed through a 100 ⁇ m cell strainer and washed through with cold RMPI/FCS2 medium.
  • the liver slurry was centrifuged at 400 g for 10 minutes and washed twice.
  • the liver slurry was purified using isotonic Percoll PLUS gradient separation. The supernatant was discarded, and the liver leukocyte pellet was then washed before being resuspended in red cell lysis buffer for 2 minutes at RT. Following this, the liver leukocytes were washed twice and analysed using flow cytometry.
  • the spleen was pressed through a 70 ⁇ m nylon mesh strainer, washed and resuspended in red cell lysis buffer for 2 minutes at RT. The splenocytes were washed twice and analysed using flow cytometry. For isolating lymphocytes from draining lymph nodes, the nodes were ruptured through a 40 ⁇ m nylon mesh strainer and then prepared as for splenocytes, with the omission of the red cell lysis step.
  • Cells were incubated with a protein kinase inhibitor, 50 nM dasatinib, for 30 minutes at 37°C.
  • PE- or APC-conjugated tetramers or dextramers were centrifuged at 16,000 g for 1 minute to remove aggregates.
  • the cells were stained with indicated concentrations of pMHC multimers for 30 minutes on ice. Following pMHC multimer staining, the cells were washed with cold FACS staining buffer twice.
  • mouse Fc Block Blocking with mouse Fc Block (BD Biosciences, catalogue# 553141) was performed for 10 minutes at 4°C and either or both of mouse anti -PE (clone PE001, BioLegend) and anti-APC (clone APC003, BioLegend) biotin-conjugated antibodies were added at 0.5 ⁇ g/100 ul to the cells depending on the pMHC multimer conjugates used.
  • mouse anti -PE clone PE001, BioLegend
  • anti-APC clone APC003, BioLegend
  • the cells were washed and the following antibodies were then added for 30 minutes at 4°C: anti-PD1-BV421 (29F.1A12, Biolegend), anti-CD8-FITC (KT-15, Invitrogen), anti-CD14-BV605 (rmC5-3, BDBioscience), anti-CD 19-BV605 (6D5, Biolegend), anti-CD44 (IM7, Biolegend) and anti-CD90.2-PerCPCy5.5 (53-2.1, Biolegend) (Table 3).
  • Cells were washed twice with PBS before staining with Zombie NIR viability dye (BioLegend, catalogue# 423105) for 15 minutes at RT. Cells were washed with staining buffer before analysis. The samples were analysed using LSR Fortessa X-20 (BD Biosciences) and analysis of data was performed using FlowJo v10.
  • Dextramers were purchased from Immudex. QuickSwitch Custom Tetramer Kits (MBL International) were utilised to generate multiple tetramers with selected peptides in order to screen an array of pMHC epitopes. Quantitation of peptide exchange with selected peptides was performed according to the manufacturer's protocol.
  • hepatocytes from 4 - 5 mice were pooled per group. Hepatocytes were lysed in 0.5% IGEPAL, 50 mM Tris (pH 8), 150 mMNaCl and protease inhibitors (Complete Protease Inhibitor Cocktail Tablet; Roche Molecular Biochemicals). Spleens and tail skins from 5 - 9 donors were pooled per group. Spleens and tail skin samples were ground in a Retsch Mixer Mill MM 400 under cryogenic conditions and lysed in 0.5% IGEPAL, 50 mM Tris (pH 8), 150 mM NaCl, and protease inhibitors. The lysates were incubated for 1 hour at 4°C.
  • the lysates were cleared by ultracentrifugation (40,000 rpm, 30 min) and supernatant containing MHC complexes were isolated by immunoaffmity purification using solid-phase-bound monoclonal antibodies SFl.1.10 (anti H-2K d ), K9-178 (anti H-2K b ), Y3 (anti H-2K b /K k ) and 28.14.8s (anti H- 2D b ).
  • Peptides were eluted from the MHC with 10% acetic acid.
  • HPLC high-performance liquid chromatography
  • the mixture of peptides, class I heavy-chain and ⁇ -2 microglobulin was fractioned on a 4.6 mm internal diameter x 100 mm monolithic reverse-phase C18 high-performance liquid chromatography (HPLC) column (Chromolith SpeedROD; Merck Millipore) using an AKTAmicro HPLC (GE Healthcare) system, running a mobile phase consisting of buffer A (0.1% trifluoroacetic acid; Thermo Fisher Scientific) and buffer B (80% acetonitrile, 0.1% trifluoroacetic acid; Thermo Fisher Scientific), running at 1 mL min -1 with a gradient of B of 2-40% over 4 min, 40-45% over 4 min and 45-99% over 2 min, collecting 500 ⁇ L fractions.
  • Peptide-containing fractions were either unpooled or combined into pools, vacuum-concentrated and reconstituted in 0.1% formic acid (Thermo Fisher Scientific) for mass spectrometry analysis.
  • the mixture of peptides, class I heavy-chain and ⁇ -2 microglobulin was purified using 5 kDa Amicon centrifugal units (Millipore) in 0.1% trifluoroacetic acid.
  • Peptides were extracted and desalted from the filtrate using Millipore ZipTip C18 pipette tips (Millipore) in a final buffer of 30% acetonitrile, 0.1% trifluoroacetic acid.
  • Peptide samples were vacuum-concentrated and reconstituted in 0.1% formic acid for mass spectrometry analysis.
  • Reconstituted peptides were analyzed by LC-MS/MS using an information-dependent acquisition strategy on a Q-Exactive Plus Hybrid Quadrupole Orbitrap (Thermo Fisher Scientific) coupled to a Dionex UltiMate 3000 RSLCnano system (Thermo Fisher Scientific). Data were analysed using PEAK software and peptide identities determined by strict bioinformatic criteria. A false discovery rate cut-off of 1% was applied. 8-mer to 11-mer peptides were analysed and visualised using GibbsCluster2.0 algorithm (NetMHC4.0). Probable MHC binders were determined based on predicted half-maximum inhibitory concentration (IC 50 ) of binding to MHC having a value less than 500 nM using NetMHC4.0 database. Data analysis
  • mice Alloreactive T cells bound using the 10 pMHC test panel pMHC epitopes recognised by alloreactive CD8+ T cells were conserved across different sexes and strains.
  • Female B10.BR mice were primed with female 178.3 skin grafts and male BALB/c mice were primed with male C57BL/6 skin grafts. 30 days after rejection, mice were transduced with AAV-HC-Kb. Liver leukocytes were isolated and pMHC tetramer binding was analysed using flow cytometry. Their pMHC tetramer binding responses were compared to that of male B10.BR mice primed with male 178.3 skin grafts as described above.
  • FIG. 32 A summary of pMHC tetramer binding by CD44 + PD1 hi and CD44 + PD1- CD8+ T-cells from male B10.BR mice, female B10.BR mice and male BALB/c mice for the pMHC epitopes screened is shown in Figure 32.
  • a heatmap was generated in order to compare how pMHC epitopes are recognised by alloreactive CD8+ T cells across different sexes and strains (Figure 33). Allogeneic graft rejection tempos of male B10.BR, female B10.BR and male BALB/c mice are shown in Figure 34a.
  • pMHC epitopes recognised by alloreactive CD8+ T cells from maleB10.BR were also recognised by female B10.BR and male BALB/c mice. pMHC epitopes were found to be largely conserved across different sexes and strains ( Figure 34c).
  • Example Three Further profiling of the tissue-specific immunopeptidomes of hepatocytes, skin and spleen
  • Peptides were synthesised with an average of 98% purity (GL Biochem Shanghai Ltd.).
  • the lyophilised peptides were reconstituted in 10% DMSO and all peptides were stored at -80°C.
  • Dasatinib (Sigma-Aldrich, catalogue# CDS023389) was reconstituted in DMSO and 5 mM stock was stored at -80°C.
  • the Tap2 deficient T lymphoma cell line RMA-S was cultured in RPMI 1640 medium supplemented with L-glutamine (Lonza, catalogue# 12-702F), penicillin-streptomycin (Invitrogen, catalogue# 15140) and 10% FCS (Sigma-Aldrich, catalogue# 13K179) at 37°C with 5% CO 2 .
  • RMA-S cells were grown to confluence and passaged to a concentration of 3 ⁇ 10 5 cells/mL then transferred to flat-bottomed 24 well culture plates. The cells were incubated at 27°C for 20 hours with 5% CO 2 , then pulsed with 6 different concentrations of peptides ranging from 0.0001 - 10 ⁇ M. The cells were incubated with peptides at 27°C for 1 hour then returned to 37°C for 2 hours. The surface expression of stabilised H-2K b was quantified using flow cytometry with the conformation-dependent mAb Y-3 antibody, which binds to correctly folded K b molecules stabilised with peptides.
  • mice were bred at the University of Sydney (Camperdown, Australia). 178.3 mice (originally provided by Drs. W. Health and M. Hoffman, Walter and Eliza Hall Institute, Melbourne, Australia) express a transgenic MHC class I molecule H-2K b ubiquitously, under the control of its own promoter, on a B10.BR (H-2k) background. Des-TCR mice express an H-2K b -specific TCR, which recognises the peptides KVITFIDL, KVLHFYNV and KIITYRNL restricted by H-2K b and Des-TCR is identifiable by a clonotypic mAb (Desire).
  • Des-RAG mice were obtained by crossing Des-TCR mice with CD45.1+/+ RAG1-/- mice, which are both on a B10.BR (H-2 k ) background.
  • OT-I mice carry a TCR which recognises the peptide SIINFEKL presented by H-2K b .
  • OT-I were crossed with RAG1-/- mice to create the OT-I RAG line. These mice were bred at the Conscery Institute.
  • C57BL/6JArc (H-2 b ) and BALB/c (H-2 d ) mice (termed C57BL/6 and BALB/c) were purchased from the Animal Resources Centre, Perth, Australia.
  • B6.K d mice express an H-2K d transgene ubiquitously on a C57BL/6 (H-2 b ) background.
  • B6.K d mice were originally developed by R. Pat Bucy at the University of Alabama (Tuscaloosa, Alabama, USA) and were provided by Robert Fairchild, Cleveland Clinic (Cleveland, Ohio, USA).
  • B6.Kd mice were backcrossed for 4 generations to C57BL/6J, prior to use.
  • Tap1KOHep mice were generated based on the conditional-ready strain 09400, C57BL/6N-
  • Tap1 ⁇ tm2a(EUCOMM)Hmgu>/Ieg developed as part of the European Conditional Mouse Mutagenesis programme (EUCOMM)35.
  • Mice heterozygous for the Tap1tm2a allele on the C57BL/6N genetic background were obtained from the European Mutant Mouse Archive, based at Helmholtz Zentrum. These mice were backcrossed to C57BL/6JArc, then intercrossed with FLPo deleter (B6.129S4-Gt(ROSA)26SORtm2(FLPo)Sor/J) mice36 (imported from the Jackson Laboratory, Bar Harbor, ME) to generate mice carrying the Tap1tm2c (floxed) allele.
  • FLPo deleter B6.129S4-Gt(ROSA)26SORtm2(FLPo)Sor/J mice36 (imported from the Jackson Laboratory, Bar Harbor, ME) to generate mice carrying the Tap1tm2c (floxed) allele.
  • FLPo was bred out by backcrossing to C57BL/6JArc, following which the mice were crossed to Albumin- Cre mice (B6.FVB(129)-Tg(Alb1-cre)1D1r/J)37.
  • Tap1KOHep mice are homozygous for the floxed Tap1 allele (Tap1tm2c) and have one copy of Cre, which is expressed exclusively in hepatocytes resulting in hepatocyte-specific deletion of the floxed Tap1 allele.
  • Genotyping and genetic background testing was performed on earpunch tissue, isolated hepatocytes or spleen by Transnetyx (Cordova, TN, USA).
  • the genetic background of Tap1KOHep and Tap1fl/fl control mice was at least 91.3% C57BL/6J (91.3-97.9%) and these mice did not reject syngeneic skin grafts from C57BL/6J donors.
  • mice Male and female mice aged between 8 and 12 weeks were used in this study. Male mice were used unless stated otherwise. At the termination of each experiment, tissues were collected under general anaesthesia. Frozen tissues were stored at -80°C. All animal procedures were approved by the University of Sydney Animal Ethics Committee (protocol 2017/1253) and carried out in accordance with the Australian code for the care and use of animals for scientific purposes.
  • H-2K b and H-2K d were cloned.
  • An SCT construct with a disulphide trap consists of a defined peptide sequence, ⁇ 2m and MHC I HC joined together by flexible linkers. The construct encodes a signal peptide sequence followed immediately by a defined peptide sequence, then a linker of GCGAS(G4S)2, a ⁇ 2m sequence, a linker of (G4S)4 and either a HC H2-K b or H2-K d sequence.
  • a tyrosine to cysteine substitution at HC position 84 and a cysteine at the second position of the peptide- ⁇ 2m linker form the disulphide trap.
  • dt-SCT (H-2K b ) constructs with defined peptide sequences KIITYRNL or SIINFEKL and a dt-SCT (H-2K d ) construct with a defined peptide sequence SYFPEITHI were designed in silico (termed SCT-Kb-KIIT, SCT-Kb- SIIN and SCT-Kd-SYFP, respectively), codon-optimised and were synthesised by GeneArt (Thermo Fisher Scientific).
  • H2-K d sequence incorporating the Y84C and A139C mutations were created in silico (termed Kd-YCAC), codon optimised and was synthesised by GenScript.
  • All synthesised genes were delivered in pcDNA3.1+ plasmids.
  • the full-length native chicken ovalbumin (OVA) gene inserted in a pcDNA3.1+ plasmid (clone ID: OGa28271) was purchased from GenScript.
  • Gene inserts from pcDNA3.1+ plasmids were cloned into the pAM2AA backbone incorporating the human ⁇ -1 antitrypsin, liver-specific, promoter and human ApoE enhancer flanked by AAV2 inverted terminal repeats. Each gene was then packaged into an AAV2/8 vector, purified, and quantitated.
  • AAV Vectors were either produced in-house or by the Vector and Genome Engineering Facility, Children's Medical Research Institute, Westmead, Australia.
  • AAV2/8 vector aliquots were stored at -80°C.
  • AAV2/8 vectors in 500 ⁇ L sterile PBS were administered to male mice via penile vein intravenous injection and to female mice via tail vein intravenous injection under general anaesthesia.
  • Full-thickness grafts of 1 ⁇ 1cm 2 tail skin from donor mice were grafted onto the dorsum of anaesthetised recipient mice whose graft bed has been shaved and a small 1 ⁇ 1cm 2 area excised to accommodate the donor skin graft.
  • the graft was fixed using cyanoacrylate tissue adhesive (Dermabond, Ethicon, catalogue# ANX12) and bandaged.
  • Mice received analgesia with buprenorphine (Temgesic, Schering-Plough, 0.05 mg kg -1 s.c.), prophylactic ampicillin (Alphapharm, 100 mg kg -1 s.c.) and 0.5 ml of warmed saline.
  • H-2 k received H-2K b singly-mismatched allogeneic skin grafts from 178.3 strain donor mice.
  • Retrograde perfusion of the liver was achieved by cannulating the inferior vena cava (IVC) and allowing the perfusate to flow out of the liver via the transected hepatic portal vein.
  • the liver was sequentially perfused with the following solutions at a flow rate of 5 ml/min (administered using a Masterflex L/S 7528-30, Thermo Fisher Scientific); firstly with 25 ml of HBSS (Lonza, catalogue# 10-543F), then with 25 ml of HBSS with 0.5 mM EDTA (Millipore Sigma, catalogue# E6758), followed by 25 ml of HBSS, and finally with 25 ml of HBSS plus 5 mM CaC12 (calcium chloride, MilliporeSigma, catalogue# C5670) and 0.05% of collagenase Type IV (Thermo Fisher Scientific, catalogue# 17104019).
  • the IVC was cannulated and the hepatic portal vein was transected.
  • the liver was flushed with 20 ml of PBS at RT and after gall bladder removal, the liver was meshed through a 100 ⁇ m cell strainer and washed through with cold RMPI/FCS2 medium.
  • the liver slurry centrifuged at 400 g for 10 minutes and washed twice.
  • the liver slurry was purified using isotonic Percoll PLUS gradient separation. The supernatant was discarded, and the liver leukocyte pellet was then washed before being resuspended in red cell lysis buffer for 2 minutes at RT. Following this, the liver leukocytes were washed twice and analysed using flow cytometry.
  • the spleen was pressed through a 70 ⁇ m nylon mesh strainer, washed and resuspended in red cell lysis buffer for 2 minutes at RT. The splenocytes were washed twice and analysed using flow cytometry. For isolating lymphocytes from draining lymph nodes, the nodes were ruptured through a 40 ⁇ m nylon mesh strainer and then prepared as for splenocytes, with the omission of the red cell lysis step.
  • Lymphocytes from portal and mesenteric lymph nodes were collected and processed. Cells resuspended in RPMI 1640 medium containing 10% FCS were labelled with 10 ⁇ M CFSE dye (Thermo Fisher Scientific, catalogue# C34570) for 4 minutes at RT. The reaction was quenched by adding more RPMI 1640 medium containing 10% FCS. CFSE-labelled lymphocytes were washed with cold RMPI/FCS10 medium, filtered through 40 mih nylon mesh and resuspended in 500 ⁇ L cold sterile PBS. CFSE-labelled lymphocytes were administered via penile vein intravenous injection under general anaesthesia. CFSE-labelling and cell viability were assessed using flow cytometry.
  • OCT-embedded frozen tissues were cut into 6 ⁇ m thick sections. Sections were allowed to air dry for 1 hour at room temperature (RT) prior to fixation in acetone for 8 minutes at RT. Sections were blocked with 20% normal mouse serum (MilliporeSigma, catalogue# M5905) and 5% normal porcine serum (Thermo Fisher Scientific, catalogue# 31890) for 20 minutes at RT and they were stained with FITC-conjugated primary antibodies against H2-K b (AF6-88.5, BioLegend), H2-K d (SFl-1.1, BD Biosciences), CD4 (GK 1.5, BD Biosciences), CD8a (53-6.7, BioLegend), F4/80 (BM8, BioLegend), B220 (RA3-6B2, BD Biosciences), CDllc (N418, BioLegend) or CD19 (6D5, BioLegend) or the corresponding isotype controls for 30 minutes at RT.
  • H2-K b AF6-88.5, BioLe
  • Sections were then stained with horseradish peroxidase- conjugated rabbit-anti-FITC secondary antibody (Bio-Rad, catalogue# 4510-7864) before development with diaminobenzidine (DAB) substrate chromogen system (Dako, catalogue# K3468). Sections were counterstained in Mayer's hematoxylin solution (MilliporeSigma, catalogue# MHS16) for 2 minutes and mounted with Fronine safety mount No.4 (Thermo Fisher Scientific, catalogue# FNNII068). Tissue processing and H&E staining were performed by the Histopathology Laboratory, Discipline of Pathology, Sydney Medical School. For H&E staining,
  • a section of the liver was embedded in 3% agarose (Fisher Biotec, catalogue# AGR-LM-50) and 150 ⁇ m thick sections were cut using a Vibratome 1000 Plus Sectioning System (Harvard Apparatus, Holliston MA). Sections were blocked with 4% bovine serum albumin (Tocris bioscience, catalogue# 9048-46-8), 5% normal goat serum (Invitrogen, catalogue# 31873) and 0.3% Triton-X 100 (Sigma, catalogue# 9002-93-1) in PBS for 20 hours at 4°C.
  • Sections were stained with primary antibodies; anti-mouse CD31-AF488 (PECAM-1, BioLegend), anti-mouse CD45- AF647 (30-F11, BioLegend), anti-mouse CK19 purified (EPNCIR127B, Abeam) and anti-mouse H2-K b purified (Y-3, WEHI), for 20 hours at 4°C.
  • anti-mouse CD31-AF488 PECAM-1, BioLegend
  • anti-mouse CD45- AF647 (30-F11, BioLegend
  • anti-mouse CK19 purified
  • EPNCIR127B Abeam
  • anti-mouse H2-K b purified Y-3, WEHI
  • Sections were washed, then incubated with secondary antibodies [anti-rabbit IgG-AF750 (polyclonal, catalogue# A21039, Invitrogen) and anti-mouse IgG2b-PE (RMG2b-1, BioLegend)], for 20 hours at 4°C, followed by staining with DAPI (Sigma, catalogue# 28718-90-3) for 1 hour at 4°C.
  • Primary and secondary antibodies were made in blocking buffer. Washing buffer comprised 0.1% Triton-X 100 in PBS. Images were acquired using a Leica SP8 confocal microscope at 93x objective magnification with a numerical aperture of 1.35. The images were analysed using Imaris v9.5 (Oxford instruments).
  • IFN-c ELISpot assays were performed according to the manufacturer' s protocol (U-Cytech, catalogue# CT317-PR5).
  • RMA-S cells either pulsed with peptides or transiently transfected with dt-SCT plasmids, were irradiated with a dose of 3000 rad.
  • Responders were either OT-I-RAG or Des-RAG splenocytes.
  • 1 ⁇ 10 6 irradiated stimulator cells and 1 ⁇ 10 6 responder splenocytes were suspended in 250 ⁇ L of RPMI/FCSIO medium with penicillin- streptomycin in each well of a 96-well U-bottom plate (Coming, catalogue# 3788).
  • PVDF polyvinylidene difluoride
  • Cells were incubated with a protein kinase inhibitor, 50 nM dasatinib, for 30 minutes at 37°C.
  • PE- or APC-conjugated tetramers or dextramers were centrifuged at 16,000 g for 1 minute to remove aggregates.
  • the cells were stained with 0.5 ⁇ g of pMHC multimers in 50 ⁇ L unless stated otherwise for 30 minutes on ice. Following pMHC multimer staining, the cells were washed with cold FACS staining buffer twice.
  • mouse Fc Blocking with mouse Fc Block was performed for 10 minutes at 4°C and either or both of mouse anti-PE (clone PE001, BioLegend) and anti-APC (clone APC003, BioLegend) biotin-conjugated antibodies were added at 0.5 ⁇ g/100 ⁇ L to the cells depending on the pMHC multimer conjugates used.
  • the cells were washed and the following antibodies were then added for 30 minutes at 4°C: anti-PD1-BV421 (29F.1A12, BioLegend), anti-CD8-FITC (KT-15, Invitrogen), anti-CD14- BV605 (rmC5-3, BD Bioscience), anti-CD 19-BV605 (6D5, BioLegend), anti-CD44 (IM7, BioLegend) and anti-CD90.2-PerCPCy5.5 (53-2.1, BioLegend). Cells were washed twice with PBS before staining with Zombie NIR viability dye (BioLegend, catalogue# 423105) for 15 minutes at RT. Cells were washed with staining buffer before analysis.
  • hepatocytes were lysed in 0.5% IGEPAL, 50 mM Tris (pH 8), 150 mM NaCl and protease inhibitors (Roche cOmplete Protease Inhibitor Cocktail; Merck, catalogue# 11836145001). Spleens, skin grafts (on d7 post-transplant) or tail skins from 5 - 9 donors were pooled per sample.
  • Peptides were dissociated from the MHC with 10% acetic acid.
  • the mixture of peptides, class I HC and ⁇ 2m was fractionated on a 4.6 mm internal diameter ⁇ 100 mm monolithic C18 column (Chromolith SpeedROD; Merck Millipore, catalogue# 1021290001) using an ⁇ KTAmicro RP-HPLC (GE Healthcare) system, running a mobile phase consisting of buffer A (0.1% trifluoroacetic acid; Thermo Fisher Scientific) and buffer B (80% acetonitrile, 0.1% trifluoroacetic acid; Thermo Fisher Scientific), running at 1 mL min -1 with a gradient of B of 2- 40% over 4 min, 40-45% over 4 min and 45-99% over 2 min, collecting 500 ⁇ L fractions.
  • Peptide- containing fractions were either unpooled or combined into pools, vacuum-concentrated and reconstituted in 0.1% formic acid (Thermo Fisher Scientific) for mass spectrometry analysis.
  • the mixture of peptides, class I HC and ⁇ 2m was purified using Millipore 5 kDa Amicon centrifugal units (Human Metabolome Technologies; catalogue# UFC3LCCNB_HMT) in 0.1% trifluoroacetic acid.
  • Peptides were extracted and desalted from the filtrate using ZipTip C18 pipette tips (Agilent Technologies, catalogue# A57003100K) in a final buffer of 30% acetonitrile, 0.1% trifluoroacetic acid.
  • Peptide samples were vacuum-concentrated and reconstituted in 0.1% formic acid for mass spectrometry analysis.
  • peptides were analysed by LC-MS/MS using an information-dependent acquisition strategy on a Q-Exactive Plus Hybrid Quadrupole Orbitrap (Thermo Fisher Scientific, Bremen, Germany) coupled to a Dionex UltiMate 3000 RSLCnano system (Thermo Fisher Scientific). Briefly, peptides were trapped on a 2 cm Nanoviper PepMap100 trap column at a flow rate of 15 min using a RSLC nano-HPLC.
  • the trap column was then switched inline to an analytical PepMap100 C18 nanocolumn (75 ⁇ m ⁇ 50 cm, 3 ⁇ m 100 ⁇ pore size) at a flow rate of 300 nL/min using an initial gradient of 2.5% to 7.5% buffer B (0.1% formic acid 80% ACN) in buffer A (0.1% formic acid in water) over 1 min followed with a linear gradient from 7.5% to 32.5% buffer B for 58 min followed by a linear increase to 40% buffer B over 5 min and an additional increase up to 99% buffer B over 5 min.
  • buffer B 0.1% formic acid 80% ACN
  • buffer A 0.1% formic acid in water
  • DIA Data Independent Acquisition
  • MSI survey scan and fragment ions were acquired using variable windows at 35,000 resolution with an automatic gain control (AGC) target of 3e6 ions.
  • AGC automatic gain control
  • the mass spectrometry data will be deposited to the ProteomeXchange Consortium via the PRIDE41 partner repository.
  • peptide identification For peptide identification, the acquired raw files were searched with PEAKS Studio X+ (Bioinformatics Solutions) against the Mus musculus (SwissProt) database. The parent mass error tolerance was set to 10 ppm and the fragment mass error tolerance to 0.02 Da. Oxidation of methionine (M) was set as variable modifications and a false-discovery rate (FDR) cut-off of 5% was applied.
  • PEAKS Studio X+ Bioinformatics Solutions
  • FDR false-discovery rate
  • H-2K b peptides For comparison of unique H-2K b peptides between different tissues, 8-mer to 11-mer peptides with a predicted half-maximum inhibitory concentration (IC50) of binding to H-2K b less than 500 nM (NetMHC4.0 database) were selected. For comparison of unique H-2K d peptides between tissues, all 9-mer peptides were used. The source proteins associated with the eluted peptides were analysed using the PANTHER Gene Ontology classification system. Function classification analysis and statistical over-representation tests were performed.
  • IC50 half-maximum inhibitory concentration
  • H-2K b peptides from the common peptide pool are recognised by activated alloreactive CD8+ T cells.
  • a total of 100 peptides were selected for screening. 96 peptides were drawn from the common peptide pool, and their identity was confirmed by a direct comparison between the chromatographic retention and mass spectra obtained from synthetic and eluted natural peptides. To evaluate the similarity between two spectra, all b- and y-ions for each sequence were predicted and then the corresponding intensity for each ion was extracted. Representative spectra from three peptide pairs (left), along with the Pearson correlation coefficient and the corresponding p-value between the logio intensities of identified b- and y-ions in synthetic and sample-derived spectra (right) is shown in Figure 43. For all peptides, p ⁇ 0.05.
  • B10.BR mice were first primed by placement of a K b -bearing 178.3 skin graft. Approximately 30 days after graft rejection, mice were inoculated with AAV-HC-K b , and after a further 7 days, liver leukocytes were isolated and stained by flow cytometry (Figure 44). The gating strategy is shown in Figure 45.
  • CD8+ T cells were defined as CD44+PD1hi, whereas PD1- cells were considered to be an internal control population, which had been exposed in vivo to H-2K b expressed on hepatocytes but were not activated. Peptides were deemed immunogenic when >2% of CD44+PD1hi CD8+ T cells were bound by pMHC tetramer. Representative flow plots demonstrating T cell recognition of immunogenic and non- immunogenic peptides are shown in Figure 46, and data summarising the results are shown in Figure 47. Allorecognition of K b -bound peptides was then examined in recipient mice of a second background haplotype (B ALB/c, H-2 d ) ( Figures 47-50).

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Abstract

La présente invention concerne des compositions pour la détection de lymphocytes T alloréactifs chez des receveurs de greffe, des procédés de production des compositions, et des procédés utilisant les compositions. Lesdites compositions comprenant des complexes peptides-CMH (pCMH) peuvent être utilisées pour détecter des lymphocytes T alloréactifs chez un receveur de greffe ou induire une tolérance à un allomorphe du CMH chez un sujet.
PCT/AU2020/051221 2019-11-08 2020-11-09 Compositions pour la détection de lymphocytes t alloréactifs Ceased WO2021087579A1 (fr)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2025155102A1 (fr) * 2024-01-18 2025-07-24 (주)케어젠 Nouveau peptide ayant une activité favorisant la formation musculaire, activité anti-obésité et activité antidiabétique et ses utilisations

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003016905A2 (fr) * 2001-08-16 2003-02-27 Avidex Limited Methodes

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003016905A2 (fr) * 2001-08-16 2003-02-27 Avidex Limited Methodes

Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
Title
CUNNINGHAM EC ET AL.: "Liver transplant tolerance and its application to the clinic: can we exploit the high dose effect?", CLIN DEV IMMUNOL . 2013, 2013, pages 419692, XP055821957 *
OBST R ET AL.: "The role of peptides in T cell alloreactivity is determined by self-major histocompatibility complex molecules", J EXP MED., vol. 191, no. 5, 6 March 2000 (2000-03-06), pages 805 - 12, XP055821956 *
PURCELL AW ET AL.: "Mass spectrometry-based identification of MHC-bound peptides for immunopeptidomics", NAT PROTOC., vol. 14, no. 6, June 2019 (2019-06-01), pages 1687 - 1707, XP036793528, DOI: 10.1038/s41596-019-0133-y *
ROSETTE C ET AL.: "The impact of duration versus extent of TCR occupancy on T cell activation: a revision of the kinetic proofreading model", IMMUNITY, vol. 15, no. 1, July 2001 (2001-07-01), pages 59 - 70, XP055821955 *
WANG B. ET AL.: "A single peptide-MHC complex positively selects a diverse and specific CD 8 T cell repertoire", SCIENCE, vol. 326, no. 5954, 6 November 2009 (2009-11-06), pages 871 - 4, XP055821938 *
YACHI PP ET AL.: "T cell activation enhancement by endogenous pMHC acts for both weak and strong agonists but varies with differentiation state", J EXP MED., vol. 204, no. 11, 29 October 2007 (2007-10-29), pages 2747 - 57, XP055821952 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2025155102A1 (fr) * 2024-01-18 2025-07-24 (주)케어젠 Nouveau peptide ayant une activité favorisant la formation musculaire, activité anti-obésité et activité antidiabétique et ses utilisations

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