WO2021087579A1

WO2021087579A1 - Compositions for detecting alloreactive t cells

Info

Publication number: WO2021087579A1
Application number: PCT/AU2020/051221
Authority: WO
Inventors: Alexandra Sharland; Eric Taeyoung Son; Moumita Paul-Heng; Nicole Mifsud; Anthony Purcell; Pouya Faridi
Original assignee: Monash University; University of Sydney
Current assignee: Monash University; University of Sydney
Priority date: 2019-11-08
Filing date: 2020-11-09
Publication date: 2021-05-14
Anticipated expiration: 2022-05-08

Abstract

The present invention relates to compositions for the detection of alloreactive T cells in transplant recipients, methods for producing the compositions, and methods utilising the compositions. Said compositions comprising peptides-MHC (pMHC) complexes can be used for detecting alloreactive T cells in a transplant recipient or inducing tolerance to an MHC allomorph in a subject.

Description

Compositions for detecting alloreactive T cells

Field of the Invention

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.

Background to the Invention The following discussion of the background of the invention is merely provided to aid the reader in understanding the invention, and is not admitted to describe or constitute prior art to the invention.

Transplantation is a life-saving treatment for end-stage organ failure. 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. Moreover, 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.

Various surrogate approaches to the problem have been adopted. Methodologies such as ELISpot or intracellular staining to detect interferon gamma provide a measurement of the magnitude of responses by recipient responder T cells against alloantigens, while next generation sequencing of bulk populations of T cells which have proliferated in a mixed lymphocyte reaction with donor stimulator cells yields partial sequence information about T cell receptor chains which are over-represented in an alloreaction. However, none of these methods allow the definitive detection of alloantigen-specific T cells. The inability to accurately detect and monitor donor antigen-specific T cell responses impacts upon basic research in animal models and upon patient care in the clinical setting. The availability of assays which can detect and track alloantigen-specific T cell responses would permit a detailed mechanistic understanding of the various tolerance induction protocols currently in development or already in clinical trial, facilitate optimisation of immunosuppression, and help to identify those in whom a trial of immunosuppression withdrawal may be safely undertaken.

Whether in the context of tolerance induction or conventional immunosuppression, there is an unmet need for assays to accurately detect and monitor donor antigen-specific T cell responses in tissue and organ transplant recipients.

Summary of the Invention

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. The inventors of the present invention have demonstrated that tolerance to allogeneic MHC class I (MHC I) molecules can be achieved when these molecules are expressed in the hepatocytes of a transplant recipient using a liver-specific adeno-associated viral (AAV) vector. 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.

Without limitation, the methods and 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.

The present invention relates at least in part to the following embodiments: 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:

(i) 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;

(ii) complexing the pMHC monomers to form one or more multimers, wherein each multimer is attached to one or more reporter molecules;

(iii) contacting the multimers with a sample of liver leukocytes from a subject who has rejected a transplant expressing the specific MHC allomorph; and

(iv) detecting peptides which bind to at least 2% of alloreactive T cells in the sample of liver leukocytes, wherein alloreactive T cells in the subject who has rejected a transplant expressing the specific MHC allomorph are boosted by introducing the specific MHC allomorph to the subject prior to contacting the multimers with a sample of liver leukocytes from the subject.

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:

(i) ATLVFHNL;

(ii) VGPRYTNL;

(iii) RTYTYEKL;

(iv) INFDFPKL;

(v) SVYVYKVL;

(vi) HIYEFPQL;

(vii) VAFDFTKV;

(viii) VSFTYRYL;

(ix) RNYSYEKL;

(x) SGYIYHKL;

(xi) SSYTFPKM;

(xii) SAFSFRTL;

(xiii) VSPLFQKL;

(xiv) VSQYYPKL; (xv) VSYLFSHV;

(xvi) HGYTFANL;

(xvii) VGPRYTQL;

(xviii) ATQVYPKL;

(xix) ATRSFPQL;

(xx) AVLSFSTRL;

(xxi) LQYEFTKL; and (xxii) VNVDYSKL.

Embodiment 4. The composition according to embodiment 1, wherein the peptides comprise or consist of the amino acid sequences:

(i) SNYLFTKL;

(ii) ATLVFHNL;

(iii) VGPRYTNL;

(iv) RTYTYEKL;

(v) INFDFPKL;

(vi) SVYVYKVL;

(vii) SGYIYHKL;

(viii) RNYSYEKL;

(ix) SAFSFRTL;

(x) VSFTYRYL;

(xi) VSPLFQKL;

(xii) VSQYYPKL;

(xiii) VSYLFSHV;

(xiv) HIYEFPQL;

(xv) HGYTFANL;

(xvi) SSYTFPKM;

(xvii) VGPRYTQL;

(xviii) ATQVYPKL;

(xix) ATRSFPQL;

(xx) VAFDFTKV;

(xxi) AVLSFSTRL;

(xxii) LQYEFTKL; and (xxiii) VNVDYSKL. 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:

- flow cytometry;

- mass cytometry; and/or

- PCR amplification of oligonucleotide tags attached to each multimer. 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:

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:

- flow cytometry;

- mass cytometry; and/or

- PCR amplification of oligonucleotide tags attached to each multimer.

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:

(i) obtaining a biological sample from the transplant recipient;

(ii) contacting the biological sample with the composition of any one of embodiments

1 to 19; and

(iii) detecting alloreactive T cells bound to the pMHC monomers.

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:

(i) SNYLFTKL;

(ii) ATLVFHNL;

(iii) VGPRYTNL;

(iv) RTYTYEKL; (v) INFDFPKL;

(vi) SVYVYKVL;

(vii) SGYIYHKL;

(viii) RNYSYEKL;

(ix) SAFSFRTL;

(x) VSFTYRYL;

(xi) VSPLFQKL;

(xii) VSQYYPKL;

(xiii) VSYLFSHV;

(xiv) HIYEFPQL;

(xv) HGYTFANL;

(xvi) SSYTFPKM; (xvii) VGPRYTQL; (xviii) ATQVYPKL;

(xix) ATRSFPQL;

(xx) VAFDFTKV;

(xxi) AVLSFSTRL; (xxii) LQYEFTKL; and (xxiii) VNVDYSKL.

Definitions

Certain terms are used herein which shall have the meanings set forth as follows.

As used in this application, the singular form “a”, “an” and “the” include plural references unless the context clearly dictates otherwise.

As used herein, the term “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.

As used herein, the term “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,

21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46

47, 48, 49, 50, 51, or more, and any numerical value derivable therein, and any range derivable therein. As used herein, the term “between” when used in reference to a range of numerical values encompasses the numerical values at each endpoint of the range.

As used herein, “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.

As used herein, the term “allomorph” refers to a cell surface molecule that corresponds to a particular MHC allele.

Any description of prior art documents herein, or statements herein derived from or based on those documents, is not an admission that the documents or derived statements are part of the common general knowledge of the relevant art.

For the purposes of description, all documents referred to herein are hereby incorporated by reference in their entirety unless otherwise stated.

Brief Description of the Figures

Preferred embodiments of the present invention will now be described, by way of example only, with reference to the accompanying Figures as set out below:

Figure 1: Expression of donor MHC in the recipient liver leads to donor-specific tolerance. In Figure 1a, 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. Figure 1b shows the increased median survival time (MST) of the rAAV-K^b-treated B10.BR mice, which was more than 100 days, compared to uninoculated controls (MST=18.5 days).

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 β2m and is loaded with a large repertoire of self-peptides. In Figure 5b, 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 β2-microglobulin ( β2m ), H-2K^b heavy chain and a defined peptide sequence.

Figure 7: SCT-K^b-KHT constructs exclude presentation of endogenous peptides.

In Figures 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). Co-transduction with a vector encoding full length chicken ovalbumin (AAV-OVA) permitted presentation of SIINFEKL by hepatocytes transduced with AAV-HC-K^b. Conversely, the sct-Kb-KIIT construct excluded presentation of endogenously processed SIINFEKL peptide. For Figures 7a and 7c, data were obtained from three independent experiments with a total of n = 3 biological replicates per group, and are displayed as the mean ± SEM of the geometric mean fluorescent intensity (MFI). Figure 7c depicts one representative experiment (from n = 3). Figure 7d provides an exemplary workflow for the immunoaffmity purification assay.

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. The binding motif obtained from B10.BR hepatocytes transduced with AAV-HC-K^b closely reflected that from C57BL/6 (H- 2^b) hepatocytes. In Figures 7f and 7g, whereas similar numbers of K^b -binding peptides were isolated from AAV-HC-K^b -transduced B10.BR hepatocytes and C57BL/6 hepatocytes, with significant overlap between the two repertoires, very few K^b -binding peptides could be eluted from B10.BR hepatocytes transduced with AAV-sct-K^b-KIIT compares to the untransduced control B10.BR group. In Figures 7h and 7i, comparable numbers of K^k-binding peptides were eluted from all B10.BR hepatocyte samples, but not from C57BL/6 mice which do not express this allomorph. Values represent mean ± SEM. Statistical analysis was performed, ns, not significant; *p < 0.05, **p < 0.01, ***p < 0.001, ***p < 0.0001. In Figures 7a and 7c, a one-way analysis of variance (ANOVA) was used in conjunction with Sidak's multiple comparison test.

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. Figure 11 shows that a progressive shortening in survival accompanied the adoptive transfer of increasing cell numbers (****p < 0.0001, Mantel-Cox log-rank test for trend, n = 6 per group).

A dose of 50,000 cells yielded skin graft survival approximating that in immunosufficient B10.BR mice, and was used in subsequent experiments.

Figure 12: The liver immunopeptidome plays an important role in tolerance induction. In Figure 12, B 10.BR (H-2^k background) or B 10.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. All vectors produced strong expression of H-2K^b on the hepatocyte surface (Figure 12a). B10.BR- RAG mice reconstituted with Des-RAG cells accepted K^b -bearing skin grafts indefinitely when transduced with SCT-K^b -KIITYRNL but rejected grafts with no survival prolongation compared with that of untransduced recipients (median survival time (MST) of 18.5 days versus 20 days after inoculation with SCT-K^b-SIINFEKL) days (ns, p = 0.2, Mantel-Cox log-rank test, n = 6 per group). ***p = 0.0005, Mantel-Cox log-rank test between B10.BR-RAG mice receiving AAV- sct-K^b-KIIT versus AAV-sct-K^b-SIIN (n = 6 per group) (Figure 12b). Conversely, Figure 12c shows that while inoculation of B10.BR mice with AAV-K^b HC induced tolerance, treatment with either of the K^b-SCT vectors only prolonged graft survival by a few days (MST 25d vs 17d in no vector controls) (n=6, p=0.0007, Mantel-Cox log-rank test between B10.BR inoculated with AAV-HC-K^b and B10.BR inoculated with either AAV-sct-K^b- KIIT or AAV-sct-K^b-SIIN). Figure 12d provides a schematic of the experimental timeline. B10.BR mice were inoculated with either AAV-HC-K^b, AAV-sct-K^b-KIIT or AAV-sct-K^b-SIIN or were not transduced (n = 6 per group). 7 days post-inoculation, the mice received 178.3 skin grafts.

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). Figure 13b shows representative IHC and H&E images showing sections of syngeneic (B10. BR-RAG) and allogeneic (178.3) skin grafts on transduced B10. BR-RAG mice reconstituted with Des-RAG lymphocytes 100 days post-transplant (n = 3 per group). Skin grafts are morphologically normal, with persistent expression of H2-K^b within allogeneic 178.3 transplants. Other groups did not produce these normal results.

Figure 14: Recognition of dt-SCT peptide-MHC ligands in vitro and in vivo. In Figure 14, 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). 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. In 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. In 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. dt-SCT constructs were recognised in a peptide- specific manner in vitro. Data from two independent experiments with a total of n = 3 biological replicates per group are shown (one-way analysis of variance (ANOVA) in conjunction with Sidak's multiple comparison test).

In Figure 14e, 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. Data from a single experiment with a total of n = 1 biological replicate per group are shown. In Figure 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. Dextramers of the syngeneic pMHC K^k-EEEPVKKI were used as negative controls. Data from one - two independent experiments with a total of n = 1 - 4 biological replicates per group are shown. Inoculation with the sct-Kb-KIIT vector not only activated a clone of transgenic Des-RAG T cells bearing the cognate receptor, but also activated a proportion of the polyclonal T cell repertoire of normal B10.BR mice (unpaired Student's t-test). All values in Figure 14 represent mean ± SEM. Statistical analysis was performed, ns, not significant; *p < 0.05, ** p < 0.01, *** p < 0.001, *** p < 0.0001.

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. B10.BR mice were inoculated with AAV-sct-K^b-KIIT and at day 2, 4, 7, 14, 28 and 100 post-inoculation, tissues were collected for analysis (n = 3 for each time point). Figure 15b shows quantification of aspartate transaminase (AST) and alanine transaminase (ALT) levels at different timepoints (n = 3 for each time point). Figure 15c provides representative IHC and H&E images showing transduced liver sections (n = 3 for each time point). Robust expression of H2-K^b was present through day 100 post-inoculation. Histologic examination of the liver sections was normal. F480, CD4, CD8, CD11c and CD19 markers demonstrate no infiltration or abnormality with cells expressing these markers. Figure 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. Equivalent levels of H-2K^b expression were achieved with the indicated peptide concentrations (0.02 μM for SIINFEKL and 10 μM 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. In Figure

18, TCR recognition of a given pMHC is not altered by disulphide bridging of MHC-I.

Figure 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 10¹¹ vgc to 2 x 10¹² vgc did not further augment expression. In contrast, comparable strong surface expression of H2-K^d was achieved in all mice receiving 5 x 10¹¹ vgc AAV-Kd-YCAC. Flow plots shown are representative of 3 separate experiments.

Figure 20: Increasing perturbation of the K^d-bound peptide repertoire in hepatocytes progressively shortened graft survival of B6.K^d skin grafts. Tap1KOHep, Tap1^fl/fl or C57BL6 mice were inoculated with AAV-HC-K^d-YCAC or were not transduced. Additional groups included C57BL6 mice and Tap 1^11/11 controls transduced with AAV-HC-K^d and C57BL/6 transduced with AAV-sct-K^d-SYFP (n = 6 per group). 7 days post inoculation, the mice were challenged with B6.Kd skin grafts. In normal C57BL/6, only expression of HC-K^d loaded with the endogenous peptide repertoire was able to induce tolerance to B6.Kd skin grafts.

Increasing perturbation of the H-2K^d bound peptide repertoire progressively shortened graft survival of H-2K^d bearing skin grafts. Statistical analysis of graft survival was carried out using the log-rank (Mantel-Cox) test. ***p = 0.0007, between C57BL/6 inoculated with AAV-HC-K^d (MST: indefinite) and C57BL/5 inoculated with either AAV-HC -K^d-YCAC (MST: 67 days), n = 6 per group. ***p = 0.0007, between C57BL/6 inoculated with AAV-HC -K^d (MST: indefinite) and Tap1^fl/fl inoculated with either AAV-HC-K^d-YCAC (MST: 53 days), n = 6 per group. ***p = 0.0005, between C57BL/6 inoculated with AAV-HC-K^d (MST: indefinite) and Tap1KOHep inoculated with AAV-HC -K^d-YCAC (MST: 20 days), n = 6 per group. In the absence of 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.

Figure 25. Peptide-binding motifs for H2-K^b peptides. In Figure 25, 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 26. pMHC epitope screening by tetramer-binding. B10.BR-RAG mice were reconstituted with doses of Des-RAG lymphocytes ranging from 1.0 × 10⁴ to 2.5 × 10⁵ 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

T cell detection compared to single SNYLFTKL pMHC tetramer. In Figure 30a, 10 pMHC tetramers each binding >5% of CD44⁺ PD1^hi CD8+ T cells were combined into a test panel. Each tetramer in the 10 pMHC test panel was four-fold less concentrated than when the tetramers were used individually due to the obligatory increase in staining volume (0.5 μg/200 μl versus 0.5 μg/50 μl, respectively). The proportion of cells stained by SNYLFTKL pMHC tetramer at this reduced concentration (0.5 μg/200 μl) has fallen compared to the SNYLFTKL pMHC tetramer used at its original concentration (0.5 μg/50 μl), (19.3 ± 0.9 versus 14.1 ± 1.7, *p = 0.034). Combining pMHC tetramers yielded a cumulative increase in alloreactive T cell detection compared to single SNYLFTKL pMHC tetramer (45.4 ± 1.2 versus 14.1 ± 1.7, ***p = 0.0001). Figure 30b provides a representative flow plot for the test panel.

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). For each pMHC epitope, 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. (c) unpaired Student's t-test.

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. Figure 34b show's that, for male B10.BR mice, 8-mer pMHC tetramer binding levels were strongly correlated with the product of the spectral intensity values for each peptide across three different tissue immunopepti domes (Pearson correlation coefficient = 0.64, ***p < 0.0001). Conversely, 9-mer pMHC tetramer binding levels did not correlate with peptide spectral intensity. For male BALB/c mice, 8-mer pMHC tetramer binding levels for each peptide also correlated with the product of the spectral intensity values, albeit less strongly than for B10.BR males (Pearson correlation coefficient = 0.26, **p = 0.004) and similarly, 9-mer pMHC tetramer binding levels did not correlate with spectral intensity values. Figure 34c shows pMHC epitopes recognised by alloreactive CD8+ T cells from male B10.BR were also recognised by female B10.BR and male BALB/c mice (Pearson correlation coefficient = 0.80, ***p < 0.0001, Pearson correlation coefficient = 0.64, ***p < 0.0001, respectively). 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. For 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. ForH-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 log₁₀ 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 (Epas_387-394), K^b-VGPRYTNL (Mapkl 19-26) and K^b-RTYTYEKL (Ctnnb1_329-336) were recognised by a large proportion of activated alloreactive T cells.

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. Figure 48 provides graphs showing strong correlation of T cell binding to each pMHC between male and female B10.BR (Pearson correlation coefficient (r) = 0.76, ***p < 0.0001) and between male B10.BR and male BALB/c (r = 0.62, ***p < 0.0001).

Figure 49 provides graphs which show that, for male B10.BR mice, 8-mer pMHC tetramer binding levels were proportional to the product of the spectral intensity values for each peptide across three different tissues (r = 0.52, ***p < 0.0001). Conversely, 9-mer pMHC tetramer binding levels did not correlate with peptide spectral intensity (r = 0.059, p = 0.65). For each pMHC epitope, 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 (measured by IC50) did not differ significantly between strongly, moderately or non-immunogenic peptides (p 0098 by one-way ANOVA, line at mean).

Figure 50 provides a graph which shows that predicted peptide binding affinity for H-2K^b (measured by IC50) did not differ significantly between strongly, moderately or non-immunogenic peptides (p^:=0.098 by one-way ANOVA, line at mean).

Figure 51 provides a graph which shows that a correlation was not observed between IC50 and spectral abundance (r = 0.11, p = 0.34).

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. Conversely, cross-reactivity between VGPRYTNL, INFDFPKL and RTYTYEKL was considerably lower (4.0 and 8.4% of T cells recognised VGPRYTNL in addition to INFDFPKL and RTYTYEKL, respectively), 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. 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. Conversely, cross-reactivity between VGPRYTNL, INFDFPKL and RTYTYEKL was considerably lower (4.0 and 8.4% of T cells recognised VGPRYTNL in addition to INFDFPKL and RTYTYEKL, respectively).

Figure 54 provides plots showing that when 5 of these 6 peptides (excluding SGYIYHKL), each binding between 7.2 and 15.2% of T cells, were used together as a panel, the proportion of alioreactive CD8+ T cells bound increased to 39.1% (p = 0.002 compared with SNYLFTKL). Each tetramer was two-fold less concentrated compared to other multimer staining experiments due to the obligatory increase in staining volume (0.5 μg/100 μL versus 0.5 μg/50 μL, respectively).

Figure 55 provides a graph showing that, when 5 of these 6 peptides (excluding SGYIYHKL), each binding between 7.2 and 15.2% of T cells, were used together as a panel, the proportion of alioreactive CD8⁺ T cells bound increased to 39.1% (p= 0.002 compared with SNYLFTKL). Each tetramer was two-fold less concentrated compared to other multimer staining experiments due to the obligatory increase in staining volume (0.5 μg/100 μL versus 0.5 μg/50 μL, respectively).

Detailed Description of the Invention

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.

There is a need in the art for assays to accurately detect and monitor donor antigen-specific T cell responses in tissue and organ transplant recipients. However, prior to the present invention, the specific peptides which combine with allogeneic MHC molecules to provide the ligands for most alioreactive T cell clones had not been identified, and methods for identifying said peptides were not known. 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.

Identification of self-peptides bound to allogeneic MHC molecules in the liver, lymphoid tissue and the graft

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. In some embodiments of the invention, 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. 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.

In some embodiments of the present invention, 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. In mice, intravenous delivery may be achieved via the tail vein. In male animals, for example, mice, 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. If suitable antibodies are not available for a specific allomorph, 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. 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. In some embodiments of the invention, targeted MS methods may be used such as selected reaction monitoring (SRM), parallel reaction monitoring (PRM) or multiple reaction monitoring (MRM). Peptide identities may be determined using specific bioinformatic criteria. The types of analysis that can be performed using mass spectrometry are well known in the art (Zhang et al. 2014 Current protocols in molecular biology 108:10.21.1- 10.21.30). Other non-limiting examples of methods which may be used to identify peptides include Edman sequencing, and T-Scan (see, for example, Kula et al.2019 Cell 178(4): 1016-1028).

Screening of peptides bound to allogeneic MHC molecules for binding to alloreactive T cells 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.

In some embodiments of the invention, 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. In some embodiments of the present invention, 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. Nitrilotriacetic acid in neutral aqueous solutions chelates bivalent cations such as Ni2+, which in turn bind the side chains of histidine. In some embodiments, the reporter molecule may be a fluorophore. Non-limiting examples of 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. In some embodiments, the reporter molecule may be a metal isotope. In some embodiments, the metal isotope may be a lanthanide. In some embodiments of the present invention, 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.

Additionally or alternative, 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). Alternatively, 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. 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).

Techniques for pMHC multimer staining of T cells are also commonly known in the art (Dolton et al. 2015 Immunology 146(1): 11-22). In some embodiments of the present invention, 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. In some embodiments, 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. In some embodiments, 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). In some embodiments 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.

In some embodiments of the present invention, pMHC multimer staining of a sample may be followed by analysis of the sample by flow cytometry or mass cytometry. In some embodiments, 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. In some embodiments, activated T cells may be identified by the presence of cell surface markers such as CD44 and/or PD1. The person skilled in the art will recognise that a variety of cell surface markers may be used to immunophenotype T cells which bind the multimers in terms of activation status, memory status, etc. In some embodiments of the invention, fluorescence activated cell sorting (FACS) may be used to determine the percentage of alloreactive T cells bound by multimers comprising each peptide.

In some embodiments of the present invention, 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.

In some embodiments of the present invention, there may be a correlation between the percentage of alloreactive T cells bound by multimers comprising each peptide and the product of the spectral intensity produced by that peptide during mass spectrometry across all three tissue types (liver, lymphoid tissue and the graft).

Compositions The 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. As described above, 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. In some embodiments of the present invention, 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.

In some embodiments, the reporter molecule may be a fluorophore. Non-limiting examples of 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. In some embodiments, the reporter molecule may be a metal isotope. In some embodiments, the metal isotope may be a lanthanide. In some embodiments of the present invention, 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.

In some embodiments of the present 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. In some embodiments, the composition includes SNYLFTKL. In some embodiments, the composition comprises SNYLFTKL, ATLVFHNL, VGPRYTNL, RTYTYEKL, INFDFPKL and SVYVYKVL. In some embodiments, 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. In some embodiments of the present invention, a cumulative increase in binding to alloreactive T cells is achieved by combining multimers into a panel. The 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. Other non-limiting examples of suitable diluents and/or additives include HBSS, glycerol, EDTA and sodium azide (NaN₃). In some embodiments, the additive is a preservative. The compositions may also contain protease inhibitors, such as leupeptin or pepstatin.

Applications of the compositions

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. In some embodiments, the transplant recipient is a mouse, human, rat, cynomolgus monkey or macaque. In some embodiments, 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. In some embodiments, 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. In some embodiments of the invention 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. Additionally or alternatively, 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. Non-limiting examples of suitable components of the MHC allomorph include β2-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.

Examples

The present invention will now be described with reference to the following specific examples, which should not be construed as in any way limiting. 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

MHC. However, the role of endogenous peptides in direct allorecognition has been difficult to study in the past at the level of a polyclonal population.

The endogenous peptide repertoire of hepatocytes plays a critical role in tolerance induction. To express high levels of donor MHC I while excluding binding of naturally-processed endogenous peptides, AAV vectors were engineered expressing a single-chain trimer (SCT) of β2- microglobulin (β2m), H-2K^b heavy chain and a defined peptide sequence (KIITYRNL or SIINFEKL) (Figures 5 and 6). Exclusion of binding of naturally-processed endogenous peptides by the K^b -KIITYRNL SCT molecule can be seen in Figures 7 and 8. 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) or 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. Figure 11 shows that a progressive shortening in survival accompanied the adoptive transfer of increasing cell numbers (****p < 0.0001 , Mantel-Cox log-rank test for trend, n = 6 per group).

All vectors produced strong expression of H-2K^b on the hepatocyte surface (Figure 12a). B 10.BR-RAG mice reconstituted with Des-RAG cells accepted K^b-bearing skin grafts indefinitely when transduced with SCT-K^b-KIITYRNL but rejected grafts with a median survival time (MST) of 20 days after inoculation with SCT-K^b-SIINFEKL (Figure 12b). Conversely, while inoculation of B 10. BR mice with AAV-K^b HC induced tolerance, treatment with either of the K^b-SCT vectors only prolonged graft survival by a few days (Figure 12c). Figure 12d provides a schematic of the experimental timeline. Transplanted skin grafts from reconstituted B10. BR-RAG mice + SCT-K^b- KIITYRNL were macroscopically (Figure 13a) and histologically (Figure 13b) normal with robust expression of K^b through day 100. Data demonstrating recognition of dt-SCT peptide-MHC ligands in vitro and in vivo are provided in Figure 14. Data demonstrating the safety and the effectiveness of dt-SCT constructs are provided in Figure 15. Figure 16 provides data from an assessment of H-2K^b stabilisation levels.

In a setting where all the alloreactive T cells recognise the K^b-KIITYRNL epitope, rejection of K^b -bearing skin grafts is abrogated by inoculation with SCT K^b-KIITYRNL and not SCT K^b- SIINFEKL. However, in mice with a polyclonal repertoire of alloreactive T cells, only treatment with the K^b-HC vector which permits presentation of a wide repertoire of endogenous liver peptides by the allogeneic H-2K^b induces tolerance.

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. In the absence of TAP, 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. 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 × 10¹¹ vgc to 2 × 10¹² vgc did not further augment expression. In contrast, comparable strong surface expression of H2-K^d was achieved in all mice receiving 5 × 10¹¹ vgc AAV-Kd-YCAC (Figure 19).

Following inoculation, mice were grafted with allogeneic K^d-bearing B6.K^d skin. In the absence of TAP1, 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 and reagents

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.

Cells

Tap2 deficient 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% CO₂.

RMA-S peptide stabilisation and transfection

RMA-S cells were grown to confluence and passaged to a concentration of 3×10⁵ cells/mL, then transferred to flat-bottomed 24 well culture plates. The cells were incubated at 27°C for 20 hours with 5% CO₂, then pulsed with 6 different concentrations of peptides ranging from 0.0001 - 10 μM. 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. For transient transfection of RMA-S cells, 2 × 10⁶ cells were nucleofected with 2 μg of pcDNA3.1⁺ plasmid carrying a gene insert of interest per reaction (Lonza- AMAXA program X-001, Nucleofector 2b). 24 hours after transfection, expression of the transgene was confirmed using flow cytometry. Mice

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 (termed C57BL/6 and BALB/c) 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.

AAV vectors A single chain trimer construct with a disulphide trap (dtSCT) consists of a defined peptide sequence, β2-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 β₂m 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-β₂m 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 α-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 μl sterile PBS were administered to male mice via penile vein intravenous injection and to female mice via tail vein intravenous injection under general anaesthesia.

Histology and immunostaining

For immunohistochemical staining, OCT embedded frozen tissues were cut into 6 μm 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. 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 μm thick sections from formalin fixed paraffin embedded tissues were used.

Table 1. Antibodies used for IHC.

Skin transplantation Full-thickness grafts of 1 × 1cm² tail skin from donor mice were grafted onto the dorsum of anaesthetised recipient mice whose graft bed has been shaved and a small 1 × 1cm² area excised to accommodate the donor skin graft. The graft was glued 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 0.5mL of warmed saline. The bandage was removed 7-10 days later and the grafts were regularly monitored for 100 days post-transplant. Grafts were deemed rejected when less than 20% of the viable skin graft remained. B10.BR and B10.BR-RAG mice on an H-2k background received H-2Kb singly-mismatched allogeneic skin grafts from 178.3 strain donor mice. C57BL/6, Tap1^fl/fl and Tap1KOHep mice on an H-2b background received H- 2Kd singly-mismatched allogeneic skin grafts from B6.Kd donor mice. BALB/c mice on an H-2d background received fully allogeneic skin grafts from C57BL/6 donor mice. Skin transplantation donors and recipients were sex-matched. ELI Spot

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. For pre-stimulation, 1 × 10⁶ irradiated stimulator cells and 1 × 10⁶ responder splenocytes were suspended in 250 μl of RPMI/FCSIO medium with penicillin- streptomycin in each well of a 96-well El-bottom plate (Coming, catalogue# 3788). They were cultured at 37°C with 5% CO₂ for 24 hours and then transferred into an antibody-coated polyvinylidene difluoride (PVDF) plate, serially diluted, and incubated for a further 16 hours. The plates were then developed, and the spots were counted using an AID iSpot plate reader. Taken together, the results in this example demonstrate that the liver immunopeptidome plays an important role in allorecognition and tolerance induction.

Example Two: An innovative strategy combining mass spectrometry with pMHC multimer staining permits discovery of the individual pMHC epitopes recognised by alloreactive T cells.

Distinguishing the peptides which are specifically recognised by recipient alloreactive T cells from the large background of bound peptides was a challenging task. 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-γ 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, spleen and skin), and that the search for the relevant peptides should be targeted to the subset that is common to hepatocytes, lymphoid tissue and the graft (Figure 22). Peptides common to all three subsets were selected for screening. 332 peptides were common to all tissue types and were screened as candidate allostimulatory peptides by tetramer binding (Figure 23). The length distribution of these peptides is shown in Figure 20. Peptide-binding motifs were generated from a non-redundant list of 8-11 mer peptides using the GibbsCluster-2.0 (DTU bioinformatics) algorithm (Figure 24).

A further relevant observation was 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. 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).

A gating strategy 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. K^b-SNYLFTKL (Epas387-394), K^b- VGPRYTNL (Mapk1_19-26) and K^b-RTYTYEKL (Ctnnbl 329-336) were recognised by a large proportion of activated alloreactive T cells, whereas few T cells bound K^b-ASYEFVQRL (Dync1h1_1379-1387) (Figure 26).

Hepatocyte isolation

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 CaCl₂ (calcium chloride, MilliporeSigma, catalogue# C5670) and 0.05% of collagenase IV (collagenase Type IV, Thermo Fisher Scientific, catalogue# 17104019). All solutions were warmed to 37°C. The gall bladder was removed and the liver was gently agitated in cold RPMI 1640 medium containing 2% FCS (RPMI/FCS2) to collect the hepatocytes. The hepatocyte slurry was centrifuged at 50 g for 3 minutes and washed twice. The hepatocyte slurry was resuspended in isotonic Percoll PLUS (GE Healthcare Life Sciences)/PBS and centrifuged at 500 g for 15 minutes at RT. The hepatocyte pellet was collected, washed twice, resuspended in RPMI/FCS2 medium and analysed using flow cytometry. Hepatocytes for immunoaffmity purification experiments underwent further washing with cold PBS before being stored at -80°C. Leukocyte isolation from liver, spleen, and draining lymph nodes

For 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 μm 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. For splenocyte isolation, the spleen was pressed through a 70 μm 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 μm nylon mesh strainer and then prepared as for splenocytes, with the omission of the red cell lysis step.

Flow cytometry Cells resuspended in cold staining buffer (2% FCS in PBS) were blocked with mouse Fc

Block (BD Biosciences, catalogue# 553141) for 10 minutes at 4°C and stained with a panel of antibodies (Table 2). 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 the staining buffer before analysis. The samples were analysed using LSR Fortessa X-20 (BD Biosciences) and analysis of data was performed using FlowJo v10.

Table 2. Antibodies used for hepatocyte/RMA-S staining (flow)

pMHC multimer staining

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. 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 μg/100 ul 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, 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.

Table 3. Antibodies used for multimer staining.

Immunopeptidome analysis

Around 1 × 10⁸ purified 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. For purified hepatocyte and spleen samples, the mixture of peptides, class I heavy-chain and β-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 μL 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. For tail skin samples, the mixture of peptides, class I heavy-chain and β-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 (IC₅₀) of binding to MHC having a value less than 500 nM using NetMHC4.0 database. Data analysis

Statistical tests were performed using GraphPad Prism version 7 (GraphPad Software, La Jolla California USA). A heatmap was generated using Morpheus, https://software.broadinstitute.org/morpheus.

Results

30 of the 332 peptides common to all three tissue types were screened to determine whether they are recognized in association with H-2K^b by alloreactive T cells. In the initial stage of screening, 12/30 peptides were recognized by > 5% of these T cells (Figure 27). Table 4 shows the shows the sequences of the 30 peptides screened, along with the proportion of alloreactive CD8+ T cells that were labelled by each individual pMHC tetramer. Sequences coloured in red in the table correspond to epitopes which were recognised by >5% of activated cells, while those sequences coloured in blue correspond to the epitopes recognised by 2-5% of activated cells. A summary of the screening results is provided in Figure 28.

Table 4: pMHC epitope screening

10 pMHC tetramers each binding >5% of CD44⁺ PD1^hi CD8+ T cells were combined into a test panel (Figure 29). Each tetramer in the 10 pMHC test panel was four-fold less concentrated than when the tetramers were used individually due to the obligatory increase in staining volume (0.5 μg/200 μl versus 0.5 μg/50 μl, respectively). The proportion of cells stained by the

SNYLFTKL pMHC tetramer at this reduced concentration (0.5 μg/200 μl) fell compared to

SNYLFTKL pMHC tetramer used at its original concentration (0.5 μg/50 μl) (19.3 ± 0.9 versus

14.1 ± 1.7, *p = 0.034). Combining pMHC tetramers yielded a cumulative increase in alloreactive

T cell detection compared to the single SNYLFTKL pMHC tetramer (45.4 ± 1.2 versus 14.1 ± 1.7, ***p = 0.0001). (Figure 30). For the common 332 peptides, the product of their spectral intensity values (peak area) across three different tissue immunopeptidomes were calculated. Peptides were ordered from the highest to the lowest product of spectral intensity values and peptide-MHC (H-

2Kb) tetramers were generated to screen pMHC epitopes in that order. pMHC tetramer binding levels were strongly correlated with the product of their spectral intensity values across three different tissue immunopeptidomes (Figure 31).

Results obtained using the 10 pMHC test panel are shown in Table 5.

Table 5: 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.

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. For male B10.BR mice, 8-mer pMHC tetramer binding levels were strongly correlated with the product of the spectral intensity values for each peptide across three different tissue immunopeptidomes. Conversely, 9-mer pMHC tetramer binding levels did not correlate with peptide spectral intensity. For male BALB/c mice, 8-mer pMHC tetramer binding levels for each peptide also correlated with the product of the spectral intensity values, albeit less strongly than for B10.BR males and similarly, 9-mer pMHC tetramer binding levels did not correlate with spectral intensity values (Figure 34b). 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

Methods

Peptides, antibodies and reagents

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.

Cell Lines

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% CO₂.

RMA-S peptide stabilisation and transfection

RMA-S cells were grown to confluence and passaged to a concentration of 3 × 10⁵ cells/mL then transferred to flat-bottomed 24 well culture plates. The cells were incubated at 27°C for 20 hours with 5% CO₂, then pulsed with 6 different concentrations of peptides ranging from 0.0001 - 10 μM. 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. For transient transfection of RMA-S cells, 2 × 106 cells were nucleofected with 2 μg of pcDNA3.1+ plasmid carrying a gene insert of interest per reaction (Lonza-AMAXA program X-001, Nucleofector 2b). 24 hours after transfection, the expression of the transgene was checked using flow cytometry.

Mice

Unless otherwise stated, 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 Centenary 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 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.

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.

AAV vectors

H-2K^b and H-2K^d were cloned. An SCT construct with a disulphide trap consists of a defined peptide sequence, β2m 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 β2m 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-β2m 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). Also, 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 α-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 μL sterile PBS were administered to male mice via penile vein intravenous injection and to female mice via tail vein intravenous injection under general anaesthesia.

Skin transplantation

Full-thickness grafts of 1×1cm² tail skin from donor mice were grafted onto the dorsum of anaesthetised recipient mice whose graft bed has been shaved and a small 1 × 1cm² 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. The bandage was removed 7-10 days later and the grafts were regularly monitored for 100 days post-transplant. Grafts were deemed rejected when less than 20% of the viable skin graft remained. B10.BR and B10.BR-RAG mice (H-2^k) received H-2K^b singly-mismatched allogeneic skin grafts from 178.3 strain donor mice. C57BL/6, Tap1fl/fl and Tap1KOHep mice (H-2^b) received H-2K^d singly-mismatched allogeneic skin grafts from B6.K^d donor mice. BALB/c mice (H-2^d) received fully allogeneic skin grafts from C57BL/6 donor mice. Skin transplant donors and recipients were sex-matched.

Hepatocyte isolation

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). All solutions were warmed to 37°C. The gallbladder was removed and the liver was gently agitated in cold RPMI 1640 medium containing 2% FCS (RPMI/FCS2) to collect the hepatocytes. The hepatocyte slurry was centrifuged at 50 g for 3 minutes and washed twice. The hepatocyte slurry was resuspended in isotonic Percoll PLUS (GE Healthcare Life Sciences)/PBS and centrifuged at 500 g for 15 minutes at RT. The hepatocyte pellet was collected, washed twice, resuspended in RPMI/FCS2 medium and analysed using flow cytometry. Hepatocytes for immunoaffmity purification experiments underwent further washing with cold PBS before being stored at -80°C.

Leukocyte isolation from liver, spleen, and draining lymph nodes

For 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 μm 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. For splenocyte isolation, the spleen was pressed through a 70 μm 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 μm nylon mesh strainer and then prepared as for splenocytes, with the omission of the red cell lysis step.

Adoptive transfers

Lymphocytes from portal and mesenteric lymph nodes were collected and processed. Cells resuspended in RPMI 1640 medium containing 10% FCS were labelled with 10 μM 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 μL 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.

Histology and immunostaining

For immunohistochemical staining, OCT-embedded frozen tissues were cut into 6 μm 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. 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,

5 μm thick sections from formalin fixed paraffin embedded tissues were used.

Confocal imaging The livers of freshly-sacrificed mice were perfused retrogradely via the IVC (as above) with 3 ml ofPBS and then 10ml of 2% paraformaldehyde (Sigma, catalogue# 30525-89-4) inPBS. The gallbladder was removed and the liver was fixed in 2% paraformaldehyde in PBS for 8 hours.

A section of the liver was embedded in 3% agarose (Fisher Biotec, catalogue# AGR-LM-50) and 150 μm 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. 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).

Block (BD Biosciences, catalogue# 553141) for 10 minutes at 4°C and stained with a panel of antibodies. 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 the staining buffer before analysis. The samples were analysed using LSR Fortessa X-20 (BD Biosciences) and analysis of data was performed using FlowJo vlO (BD).

ELISpot

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. For pre-stimulation, 1 × 10⁶ irradiated stimulator cells and 1 × 10⁶ responder splenocytes were suspended in 250 μL of RPMI/FCSIO medium with penicillin- streptomycin in each well of a 96-well U-bottom plate (Coming, catalogue# 3788). They were cultured at 37°C with 5% CO₂ for 24 hours and then transferred into an antibody-coated polyvinylidene difluoride (PVDF) plate, serially diluted, and incubated for a further 16 hours. The plates were then developed, and the spots were counted using an AID iSpot plate reader. pMHC multimer staining

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 μg of pMHC multimers in 50 μL unless stated otherwise for 30 minutes on ice. Following pMHC multimer staining, the cells were washed with cold FACS staining buffer twice. 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 μg/100 μL 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. 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.

Immunoaffinity purification

Two replicate samples were prepared for each tissue or experimental group. Around 1 × 10⁸ purified hepatocytes from 4 - 5 mice were pooled per sample. 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. Spleen and skin samples were ground in a Retsch Mixer Mill MM 400 under cryogenic conditions and then lysed in 0.5% IGEPAL, 50 mM Tris (pH 8), 150 mMNaCl, and protease inhibitors. Lysates were incubated for 1 hour at 4°C, then cleared by ultracentrifugation (40,000 rpm, 30 min) and MHC complexes were isolated from supernatant by immunoaffmity purification using solid-phase-bound monoclonal antibodies SF1- 1.1.10 (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 dissociated from the MHC with 10% acetic acid. For purified hepatocyte and spleen samples, the mixture of peptides, class I HC and β2m was fractionated on a 4.6 mm internal diameter × 100 mm monolithic C18 column (Chromolith SpeedROD; Merck Millipore, catalogue# 1021290001) using an ÄKTAmicro 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 μL 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. For tail skin samples, the mixture of peptides, class I HC and β2m 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.

Mass Spectrometry

Reconstituted 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 μm × 50 cm, 3 μm 100 Å 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. Survey full scan MS spectra (m/z 375-1800) were acquired in the Orbitrap with 70,000 resolution (m/z 200) after the accumulation of ions to a 5 × 10⁵ target value with a maximum injection time of 120 ms. For Data Dependant Acquisition (DDA) runs, the 12 most intense multiply charged ions (z ≥ 2) were sequentially isolated and fragmented by higher-energy collisional dissociation (HCD) at 27% with an injection time of 120 ms, 35,000 resolution and target of 2 × 10⁵ counts. An isolation width of 1.8 m/z was applied and underfill ratio was set to 1% and dynamic exclusion to 15 sec. For Data Independent Acquisition (DIA) runs, the 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. The mass spectrometry data will be deposited to the ProteomeXchange Consortium via the PRIDE41 partner repository.

DDA data analysis

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.

DIA data analysis

PEAKS Studio X+ was used to generate a spectral library from all DDA data. DIA raw files were imported to Spectronaut 11 Pulsar (Biognosys). The parent mass error tolerance was set to 10 ppm and the fragment mass error tolerance to 0.02 Da. Oxidation of M was set as variable modifications and a peptide list was exported at Q-value=1%.

Immunopeptidome analysis

Unique peptides from the DDA (replicate 1) and DIA (replicate 2) datasets were combined to increase the coverage of the tissue immunopeptidomes. For analysis requiring spectral intensity values, only DDA datasets were used. Binding motifs of 8-mer peptides from samples that were immunoaffmity purified with K9-178 antibody (H-2K^b group) and 9-mer peptides from samples that had been immunoaffmity purified with SF 1-1.1.10 antibody (H-2K^d group) were visualised using the GibbsCluster2.0 algorithm (NetMHC4.0)43. 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. Validation of peptide identification using retrospectively synthesised peptides The identity of a panel of peptides was validated by comparing chromatographic retention and MS/MS spectra of synthesised peptides (GL Biochem, Shanghai) with those of the corresponding eluted peptides. The PKL files of the synthetic and eluted peptides were exported from PEAKS X plus studio software. To evaluate the similarity between two spectra, all b- and y- ions for each sequence were predicted and the intensity for each ion was then extracted (with a fragment mass error tolerance of 0.02 Da). The Pearson correlation coefficient and the corresponding p-value between the logio intensities of identified b- and y-ions in the synthetic and sample-derived spectra were calculated. The closer the correlation coefficient to 1, the greater identity between paired spectra. All tested peptides were found to have a p-value of less than 0.05.

Statistical Analysis and Data Visualisation

Data are represented as mean ± SEM unless otherwise stated. Unpaired Student's t-tests were performed to calculate statistical differences in a single variable between the means of two groups and one-way analysis of variance (ANOVA) in conjunction with Sidak's multiple comparison tests were used to calculate statistical differences between the means of three or more groups. For analysis of 2 variables, two-way ANOVA with Sidak's multiple comparison tests were used. Graft survival curves were compared using Mantel Cox log-rank tests. Synthetic and corresponding eluted peptide spectra were compared using Pearson correlation tests. The relationship between overall peptide abundance and alloreactive T cell binding, and the impact of differences in sexes and strains on alloreactive T cell binding, were analysed using linear regression and Pearson correlation tests. Statistical tests were performed using GraphPad Prism version 8.01 (GraphPad Software, La Jolla CA). Heatmaps were generated using Morpheus software (https://software.broadinstitute.org/morpheus).

Results

The self-peptide repertoires of transduced hepatocytes, grafted donor skin and donor spleen were determined using a combination of immunoaffmity purification and RP-HPLC to liberate and collect peptide-containing fractions from associated MHC I with LC-MS/MS for peptide identification for both 178.3 to B10.BR (K^b mismatch, H-2^k background) and B6.Kd to C57BL/6 (K^d mismatch, H-2^b background) strain combinations, as outlined in Figure 35. For H-2K^d, 880 common peptides were identified across the three tissue types (Figure 36), whereas there were 1083 common K^b -binding peptides (Figure 37). The peptide length distributions (Figures 38 and 39) and binding motifs (Figures 40 and 41) were as anticipated for the respective allomorphs and were similar across tissue types. Of note, the common peptide pool was more limited when TAP- sufficient, or particularly TAP-deficient hepatocytes had been transduced with HC-K^d-YCAC (324, 347 and 36 unique peptides respectively), compared to TAP-sufficient hepatocytes transduced with HC-Kd (880 unique peptides) (Figure 42). Comparison of the different tissue immunopeptidomes showed that in the two settings where skin graft tolerance was achieved in wild-type recipient mice following expression of allogeneic donor MHC I in hepatocytes, the proportion of skin peptides common to hepatocytes was 43% and 45% for H-2K^d (Figure 36) and H-2K^b (Figure 37), respectively. Conversely, only 1.6% of skin peptides were also found in TAP1KOHep hepatocytes transduced with AAV-HC-K^d-YCAC, while in two groups with intermediate graft survival (TAP1fl/fl or C57BL/6 inoculated with AAV-HC-Kd-YCAC), the proportion of skin peptides present in hepatocytes was in the order of 15-17%.

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. A further four peptides had been previously identified as alloreactive CD8+ T cell epitopes in B10.BR mice. Three of these four epitopes were detected within the common pool. Binding of pMHC tetramers was used to determine which peptides combined with H-2K^b to form immunogenic epitopes recognised by alloreactive B10.BR CD8+ T cells. 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. Activated 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).

Of 100 peptides screened, 17 peptides were recognised by >5% of activated recipient CD8+ T cells from male B10.BR mice (termed strongly immunogenic), and a further 39 were bound by 2-5% of cells (moderately immunogenic). These responses were mirrored in female B10.BR recipients (Figures 47-48). A number of pMHC epitopes were recognised by BALB/c mice as well as B10.BR (Figures 47-48). All peptides recognised by >5% of B10.BR responder cells and 42/43 of those binding >5% of BALB/c cells were 8-mers. For 8-mer peptides, there was a strong correlation between overall peptide abundance (as estimated by the product of the spectral intensity across the three tissue types) and the percentage of T cells with specificity for a given pMHC (r=0.52, p<0.0001, Figure 49). No such relationship was observed for 9-mers. Predicted peptide binding affinity for H-2K^b (measured by IC50) did not differ significantly between strongly, moderately or non-immunogenic peptides (Figure 50, p=0.098 by one-way ANOVA), nor was a correlation observed between IC50 and spectral abundance (Figure 51). Simultaneous staining with two different pMHC tetramers was used to evaluate the proportion of T cells recognising more than one pMHC specificity, with a total of six peptides being evaluated. A substantial proportion of T cells recognised two peptides (SGYIYHKL and/or SVYVYKVL) in addition to SNYLFTKL (86.7% of T cells recognising SGYIYHKL-PE could recognise SNYLFTKL-APC and 66.8% of T cells recognising SVYVYKVL-PE could also recognise SNYLFKTKL-APC) (Figures 52-53). When 5 of these 6 peptides (excluding SGYIYHKL), each binding between 7.2 and 15.2% of T cells, were used together as a panel the proportion of alloreactive CD8+ T cells bound increased to 39.1% (Figure 55, p= 0.002 compared with SNYLFTKL). This cumulative increase in binding is consistent with alloreactive T cell recognition of epitopes comprising both a self-peptide and allogeneic MHC I molecule, and suggests that the development of pMHC multimer panels for the identification and tracking of alloreactive T cell populations is feasible.

INCORPORATION BY CROSS-REFERENCE

The present invention claims priority from Australian provisional patent application number 2019904221, the entire content of which is incorporated herein by cross-reference.

Claims

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:

2. The composition according to claim 1, wherein at least one of the peptides has the amino acid sequence SNYLFTKL.

3. The composition according to claim 1 or claim 2, wherein at least one of the peptides has an amino acid sequence selected from:

(i) ATLVFHNL;

(ii) VGPRYTNL;

(iii) RTYTYEKL;

(iv) INFDFPKL;

(v) SVYVYKVL;

(vi) HIYEFPQL;

(vii) VAFDFTKV;

(viii) VSFTYRYL;

(ix) RNYSYEKL;

(x) SGYIYHKL; (xi) SSYTFPKM;

(xii) SAFSFRTL;

(xiii) VSPLFQKL;

(xiv) VSQYYPKL;

(xv) VSYLFSHV;

(xvi) HGYTFANL;

(xvii) VGPRYTQL;

(xviii) ATQVYPKL;

(xix) ATRSFPQL;

(xx) AVLSFSTRL;

(xxi) LQYEFTKL; and (xxii) VNVDYSKL.

4. The composition according to claim 1, wherein the peptides comprise or consist of the amino acid sequences:

(i) SNYLFTKL;

(ii) ATLVFHNL;

(iii) VGPRYTNL;

(iv) RTYTYEKL;

(v) INFDFPKL;

(vi) SVYVYKVL;

(vii) SGYIYHKL;

(viii) RNYSYEKL;

(ix) SAFSFRTL;

(x) VSFTYRYL;

(xi) VSPLFQKL;

(xii) VSQYYPKL;

(xiii) VSYLFSHV;

(xiv) HIYEFPQL;

(xv) HGYTFANL;

(xvi) SSYTFPKM;

(xvii) VGPRYTQL;

(xviii) ATQVYPKL;

(xix) ATRSFPQL; (xx) VAFDFTKV;

(xxi) AVLSFSTRL;

(xxii) LQYEFTKL; and (xxiii) VNVDYSKL.

5. The composition according to any one of claims 1 to 4, wherein the specific MHC allomorph is a mouse MHC allomorph.

6. The composition according to any one of claims 1 to 5, wherein the subject who has rejected a transplant expressing the specific MHC allomorph is a mouse.

7. The composition according to any one of claims 1 to 6, wherein the peptides all have the same amino acid sequence.

8. The composition according to any one of claims 1 to 6, wherein the peptides comprise a plurality of different amino acid sequences.

9. The composition according to any one of claims 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.

10. The composition according to any one of claims 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.

11. The composition according to any one of claims 1 to 10, wherein the pMHC monomers form one or more mul timers.

12. The composition according to claim 11, wherein the multimers are attached to a reporter molecule.

13. The composition according to claim 12, wherein the reporter molecule is a fluorophore or a metal isotope.

14. The composition according to any one of claims 1 to 13, wherein the specific MHC allomorph is MHC class I.

15. The composition according to any one of claims 1 to 14, wherein the peptides are identified in (i) by immunoaffmity purification and/or mass spectrometry.

16. The composition according to any one of claims 1 to 15, wherein detecting peptides which bind to at least 2% of alloreactive T cells in (iv) is by: - flow cytometry;

- mass cytometry; and/or

- PCR amplification of oligonucleotide tags attached to each multimer.

17. The composition according to claim 16, wherein said detecting by flow cytometry comprises detection of PD1 expression on alloreactive T cells bound to said peptides.

18. The composition according to any one of claims 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.

19. The composition according to claim 1, wherein the specific MHC allomorph is a human MHC allomorph.

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:

(ii) complexing the pMHC monomers to form one or more multimers, wherein each multimer is attached to one or more reporter molecules; (iii) contacting the multimers with a sample of liver leukocytes from a subject who has rejected a transplant expressing the specific MHC allomorph; and

21. The method according to claim 20, wherein the specific MHC allomorph is a mouse MHC allomorph.

22. The method according to claim 20, wherein the specific MHC allomorph is a human MHC allomorph.

23. The method according to any one of claims 20 to 22, wherein the subject who has rejected a transplant expressing the specific MHC allomorph is a mouse.

24. The method according to any one of claims 20 to 23, wherein the specific MHC allomorph is MHC class I.

25. The method according to any one of claims 20 to 24, wherein the peptides are identified in

(i) by immunoaffmity purification and/or mass spectrometry.

26. The method according to any one of claims 20 to 25, wherein detecting peptides which bind to at least 2% of alloreactive T cells in (iv) is by: - flow cytometry;

- mass cytometry; and/or

- PCR amplification of oligonucleotide tags attached to each multimer.

27. The method according to claim 26, wherein said detecting by flow cytometry comprises detection of PD1 expression on alloreactive T cells bound to said peptides.

28. The method according to any one of claims 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.

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 claims 20 to 28.

30. A method for detecting alloreactive T cells in a transplant recipient, the method comprising:

(i) obtaining a biological sample from the transplant recipient;

(ii) contacting the biological sample with the composition of any one of claims 1 to 19; and

(iii) detecting alloreactive T cells bound to the pMHC monomers.

31. The method according to claim 30, wherein detecting alloreactive T cells is by flow cytometry or mass cytometry.

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:

(i) SNYLFTKL;

(ii) ATLVFHNL;

(iii) VGPRYTNL;

(iv) RTYTYEKL;

(v) INFDFPKL;

(vi) SVYVYKVL;

(vii) SGYIYHKL;

(viii) RNYSYEKL;

(ix) SAFSFRTL;

(x) VSFTYRYL;

(xi) VSPLFQKL;

(xii) VSQYYPKL;

(xiii) VSYLFSHV;

(xiv) HIYEFPQL;

(xv) HGYTFANL;

(xvi) SSYTFPKM; (xvii) VGPRYTQL; (xviii) ATQVYPKL;

(xix) ATRSFPQL;

(xx) VAFDFTKV; (xxi) AVLSFSTRL; (xxii) LQYEFTKL; and

(xxiii) VNVDYSKL.