Abstract
While direct allorecognition underpins both solid organ allograft rejection and tolerance induction, the specific molecular targets of most directly-alloreactive CD8+ T cells have not been defined. In this study, we used a combination of genetically-engineered MHC I constructs, mice with a hepatocyte-specific mutation in the class I antigen-presentation pathway and immunopeptidomic analysis to provide definitive evidence for the contribution of the peptide cargo of allogeneic MHC I molecules to transplant tolerance induction. We established a systematic approach for the discovery of directly-recognised pMHC epitopes, and identified 17 strongly immunogenic H-2Kb-associated peptides recognised by CD8+ T cells from B10.BR (H-2k) mice, 13 of which were also recognised by BALB/c (H-2d) mice. As few as five different tetramers used together were able to identify almost 40% of alloreactive T cells within a polyclonal population. To our knowledge, this represents the first example of such an approach in the context of direct allorecognition.
Introduction
Allorecognition may result in either graft rejection, graft versus host disease or transplantation tolerance, depending upon the context in which the recipient immune system encounters the allogeneic major histocompatibility complex (MHC). When donor MHC class I (MHC I) molecules are expressed in the hepatocytes of recipient mice, subsequent skin or pancreatic islet grafts bearing the same donor allomorph are accepted indefinitely1–3. Insights from this model can inform our understanding of allorecognition and transplant tolerance induction more broadly. MHC I molecules are ubiquitously expressed, and display a range of endogenous peptides (the class I immunopeptidome) reflecting protein turnover and normal cellular processes4. While tolerance induction depends upon recognition of intact donor MHC I molecules by recipient CD8+ T cells3, the contribution of the self-peptide cargo of these molecules to tolerance induction in this setting is unknown.
Many individual alloreactive T cell clones recognise epitopes comprising allogeneic MHC I molecules complexed with self-peptides5–12. Conversely, peptide-independent direct allorecognition is also described7, 13, 14, and the ability of peptide-independent cytotoxic T lymphocyte (CTL) clones to bring about rapid destruction of allogeneic skin grafts has been demonstrated15. At the level of a polyclonal alloresponse in vivo, there is limited information about the role of the donor’s tissue-specific class I immunopeptidome in allorecognition and consequent immune responses that impact on graft survival.
In preceding studies1, 3, we had used liver-specific adeno-associated viral (AAV) vectors encoding the donor MHC I heavy chain (HC). Within transduced hepatocytes, allogeneic HC associated with native β2 microglobulin (β2m) and the resulting heterodimers were loaded with a repertoire of endogenous peptides (Figure 1a). Here, we examined the role of the liver immunopeptidome in transplantation tolerance induction using two different approaches. In the first of these, we engineered liver-specific AAV vectors which would express H-2Kb or H-2Kd as a single chain trimer (SCT) comprising the polymorphic HC, β2m light chain and a defined covalently-bound peptide species16, 17, thus excluding presentation of endogenous peptides by the allogeneic MHC I when these constructs were expressed in B10.BR (H-2k) or C57BL/6 (H-2b) recipient hepatocytes (Figure 1a). In parallel, we introduced a global shift in the repertoire of peptides bound to allogeneic H-2Kd. To accomplish this, we generated recipient mice in which the transporter associated with antigen processing (TAP1) protein was deficient only in hepatocytes (TAP1KOHep, H-2b) to reduce the availability of high affinity Kd-binding peptides within the endoplasmic reticulum, and designed a construct expressing the H-2Kd HC with a modification (Y84C, A139C; subsequently termed YCAC) which stabilises the molecule and permits occupancy by lower affinity peptides18. Induction of tolerance to subsequent donor skin grafts was blocked by these manipulations, establishing that the endogenous peptide repertoire of hepatocytes makes an essential contribution to transplant tolerance induction.
Differential responses by alloreactive T cells to MHC I allomorphs expressed by various target tissues has been cited as evidence in support of peptide-specific allorecognition6, 19, 20. Based on the ability of H-2Kb or Kd HC expressed in recipient hepatocytes to induce tolerance to allogeneic skin grafts and to downmodulate responses against donor splenocyte stimulators1, 3, we hypothesised that the peptides critical for allorecognition and tolerance induction would be found within a subset common to these three tissue types. We identified these peptides using immunoaffinity purification coupled with mass spectrometry and determined their ability to bind activated alloreactive T cells using tetramer staining. Of 100 peptides selected for tetramer screening, 17 bound more than 5% of this T cell population, and 13 of these peptide-MHC (pMHC) epitopes were also recognised by BALB/c mice. Strikingly, we demonstrated a cumulative increase in the proportion of alloreactive T cells bound when multiple pMHC tetramers were used together, whereby as few as five different pMHC tetramers were able to identify around 40% of alloreactive T cells within a polyclonal population. Development of multimer panels to capture a substantial proportion of directly alloreactive T cells will enable immune monitoring and a range of other downstream applications including T cell receptor (TCR) repertoire analysis, cloning and biophysical and structural studies. In short, the findings of this study represent a significant advance in our understanding of the role of endogenous peptides in direct T cell alloreactivity, and a springboard for further knowledge gain and technological development.
Results
Single chain trimer constructs exclude presentation of endogenous peptides
To express allogeneic MHC I at high levels on recipient hepatocytes, while excluding the presentation of naturally processed peptides, we engineered SCT constructs, each encoding the HC of H-2Kb, β2m and a single, defined H-2Kb-restricted peptide [SIINFEKL (SIIN) or KIITYRNL (KIIT), Figure 1a], and packaged them in hepatocyte-specific AAV2/8 vectors. Sequences are shown in supplementary Figure 1. Transgene expression in hepatocytes was close to maximal by d7 following intravenous (iv) inoculation, and persisted through to at least d100, no significant increases in serum aspartate aminotransferase (AST) or alanine aminotransferase (ALT) levels were observed, and minimal cellular infiltration was detected by histology (Figure 1b-c). SCT molecules were expressed on transduced hepatocytes at equivalent levels to the heterotrimer formed by transgenic H-2Kb HC with native β2m and peptide (Figure 2a). To demonstrate exclusion of naturally processed peptides, we co-transduced B10.BR (H-2k) hepatocytes with AAV vectors encoding full-length chicken ovalbumin (OVA) and either HC-Kb or SCT-Kb-KIIT, and stained them with a monoclonal antibody, 25D-1.16, which is specific for the OVA peptide SIINFEKL complexed with Kb. Kb-SIINFEKL was only detected at the surface of cells co-transduced with HC-Kb and not those expressing SCT-Kb-KIIT (Figure 2b-c). We extended this analysis to the broader endogenous peptide repertoire of Kb-transduced hepatocytes using immunoaffinity purification with the H-2Kb-specific antibody K9-178, followed by reverse phase high performance liquid chromatography (RP-HPLC) to collect peptide-containing fractions that were then analysed using liquid chromatography with tandem mass spectrometry (LC-MS/MS) to identify bound peptides (Figure 2d). The Kb binding motif of peptides eluted from HC-Kb transduced hepatocytes mirrored that obtained from C57BL/6 (H-2b) hepatocytes (Figure 2e). While there was greater diversity among the unique peptides isolated from HC-Kb transduced hepatocytes (Figure 2f), there was substantial overlap between the peptide repertoires, with >90% of the peptides from C57BL/6 hepatocytes also identified in HC-Kb transduced B10.BR (Figure 2g). In contrast, almost no Kb-bound peptides could be identified in association with SCT-Kb-KIIT, confirming exclusion of the endogenous peptide repertoire from presentation by this molecule (Figure 2f). Hepatocytes transduced with SCT-Kb-KIIT did not manifest any generalised defect in antigen processing, with both the numbers and repertoire of peptides isolated from the native allomorph H-2Kk being similar across all groups (Figures 2h-i). The full peptide dataset can be found in supplementary data 1.
SCT-Kb-KIIT induces tolerance in alloreactive T cells expressing the cognate T cell receptor but not in a polyclonal alloreactive population
We first verified that SCT molecules were recognised by cells bearing their cognate TCRs in a manner analogous to the native epitope. Pulsing RMA-S cells with synthetic peptides KIITYRNL, SIINFEKL and AAAAFAAL (minimum binding requirement for H-2Kb) stabilised Kb expression on the cell surface (Supplementary Figure 2a-b). Similar levels of surface expression were achieved by transient transfection of RMA-S cells with the corresponding SCT constructs (Supplementary Figure 2c). Using interferon-gamma (IFN-γ) ELISPOT, we found a strong, specific response to SCT-Kb-KIIT by Des-RAG TCR-transgenic T cells which express the cognate TCR; SCT-Kb-SIIN was similarly recognised by OT-I-RAG T cells (Supplementary Figure 2d). Following adoptive transfer into B10.BR mice transduced with SCT-Kb-KIIT, Des-RAG T cells proliferated vigorously, expanding more than those transferred to the positive control 178.3 (H-2k + Kb) mice after 2 days (p < 0.0001, Supplementary Figure 2e-f). The polyclonal responder population of liver leukocytes from B10.BR mice also contained CD8+ T cells, which were activated in response to transduction with SCT-Kb-KIIT. Naïve or primed B10.BR mice were inoculated with SCT-Kb-KIIT, and liver leukocytes isolated 7 days later (Figure 3a). Activated CD8+ T cells, defined as CD44+PD-1hi, increased upon priming or transduction with SCT-Kb-KIIT, with a further augmentation when primed mice received SCT-Kb-KIIT vector (Figure 3b). Activated CD8+ T cells from transduced mice recognised Kb-KIIT dextramers but not dextramers of the self-pMHC complex Kk-EEEPVKKI, while PD-1- bystander CD8+ T cells did not bind either dextramer (Figure 3c-d).
Next, we determined that Des-RAG T cells alone were capable of rejecting Kb-bearing 178.3 skin grafts upon adoptive transfer to B10.BR-RAG recipients (Figure 3e-f). Graft survival was inversely related to the T cell dose; 50,000 transferred cells yielded a median graft survival comparable to that in B10.BR mice with a polyclonal Kb-reactive T cell repertoire and this dose was used in subsequent experiments. All grafts to reconstituted B10.BR-RAG mice treated with SCT-Kb-KIIT survived indefinitely, whereas no graft survival prolongation was observed in reconstituted mice receiving the control vector SCT-Kb-SIIN (Figure 3g). Surviving grafts appeared normal macroscopically (Figure 3h) and upon histology (Figure 3i) with continued expression of H-2Kb. In contrast, transduction of wild-type B10.BR mice with either SCT-Kb-KIIT or SCT-Kb-SIIN only briefly delayed graft rejection (Figure 3j).
The YCAC mutation alters the repertoire of H-2Kd-bound peptides in both TAP1KOHep and C57BL/6 mice
Mice with a conditional deletion of Tap1 in hepatocytes (TAP1KOHep, H-2b) express trace amounts of the native MHC I allomorphs H-2Kb and H-2Db at the hepatocyte surface (Figure 4a-d). Importantly, expression of these molecules on most other cell types within the liver, in other tissues (such as spleen, thymus and lymph node), and in TAP1fl/fl control mice was normal (Figure 4a-d and supplementary Figure 3a). Transduction of TAP1KOHep mice with a vector encoding the H-2Kd HC (HC-Kd) resulted in surface expression of H-2Kd which was clearly positive with respect to untransduced controls, yet was reduced in comparison to that in C57BL/6 or TAP1fl/fl mice (Figure 4e-f). Increasing the vector dose did not yield an appreciable increase in surface expression (data not shown), most likely because of instability of suboptimally-loaded H-2Kb molecules and their rapid recycling from the cell surface21–23. To counter this, we designed a construct where the point mutations Y84C and A139C (YCAC) in the H-2Kd HC result in the formation of a disulphide bridge, which stabilises the molecule when empty or loaded with lower affinity peptides18. This modification does not interfere with TCR recognition of the bound peptide18. The construct sequence is shown in supplementary Figure 1. Comparable strong expression of H-2Kd on the surface of hepatocytes was achieved in TAP1KOHep mice treated with HC-Kd-YCAC and in TAP1fl/fl mice receiving either the HC-Kd or HC-Kd-YCAC vectors (Figure 4e-g). Immunoaffinity purification and tandem LC-MS/MS were used to characterise the bound self-peptide repertoire of transduced hepatocytes. 9570 unique peptides were identified from C57BL/6 hepatocytes transduced with HC-Kd (B6/HC), compared with 7690, 7776, and 6417 unique peptides respectively, from C57BL/6, TAP1fl/fl and TAP1KOHep transduced with HC-Kd-YCAC (i.e. B6/YCAC, TAP1fl/fl/YCAC and TAP1KO/YCAC). The full dataset can be found in supplementary data 1, while distribution of peptide lengths and spectral intensities is shown in supplementary Figure 4a-b, along with the gene ontology analysis of the subcellular location and function of the source proteins. Whilst the sequences of eluted 9-mer peptides from B6/HC, B6/YCAC and TAP1fl/fl/YCAC corresponded to the canonical motif for H-2Kd, with tyrosine (Y) residues predominant at position 2, and leucine (L) and isoleucine (I) most frequently found at position 9, this motif was not observed for the peptides eluted from TAP1KO/YCAC (Figure 5a-b). Compared to B6/HC, just under 45% of peptides were common to B6/YCAC or TAP1fl/fl /YCAC, while 22% were shared with TAP1KOHep/YCAC (Figure 5c). Similarity across peptide repertoires was increased when the comparison was weighted for peptide abundance (Figure 5c). The peptide SYFPEITHI (SYFP) was common to all Kd repertoires, and comparable proportions of CD8+ T cells recognising Kd-SYFP could be detected among the liver leukocytes isolated from primed TAP1KO/YCAC as well as from primed B6/YCAC and B6/HC (Figure 5d) consistent with published reports that the YCAC mutation alters the peptide repertoire but does not interfere with TCR recognition of presented peptides18, 24.
Increasing perturbation of the hepatocyte H-2Kd peptide repertoire correlates with progressive reduction of Kd-bearing skin graft survival in transduced mice
Mice were inoculated with AAV-HC-Kd, AAV-Kd-YCAC, or an SCT vector encoding Kd with the single peptide SYFPEITHI (AAV-SCT-Kd-SYFP), seven days prior to transplantation with a B6.Kd skin graft. The construct sequence and expression data for AAV-SCT-Kd-SYFP are shown in supplementary Figures 1 and 5, respectively. The majority of C57BL/6 or TAP1fl/fl mice transduced with AAV-HC-Kd accepted Kd-bearing B6.Kd skin grafts indefinitely, whereas median survival of B6.Kd skin grafts to C57BL/6 or TAP1fl/fl mice treated with AAV-Kd-YCAC was reduced to 62.5 ± 5.3 days and 52.5 ± 9.2 days respectively (p = 0.009 and p = 0.007 compared to HC-Kd) (Figure 5e). Graft survival in TAP1KOHep mice inoculated with AAV-Kd-YCAC was further reduced to 20 ± 1.9 days, similar to that in C57BL/6 mice which received a vector encoding SCT-Kd-SYFP (17 ± 1.2 days). Median graft survival in no vector control mice was 12.5 ± 0.6 days for C57BL/6 and 10 ± 0.6 days for TAP1KOHep (Figure 5e). Failure to induce tolerance was not associated with loss of transgene expression in the transduced livers (supplementary Figure 5). Instead, progressive loss of ability of H-2Kd gene transfer to prolong B6.Kd skin graft survival correlated with increasing disturbance of the hepatocyte Kd peptide repertoire in the various experimental groups. These findings, along with the inability of SCT-Kb vectors to induce tolerance in polyclonal alloreactive T cell populations demonstrate the importance of the hepatocyte immunopeptidome in transplantation tolerance induction following donor MHC I gene transfer to the liver.
Profiling the tissue-specific immunopeptidomes of hepatocytes, skin and spleen
Given the ability of H-2Kb or Kd HC expressed in recipient hepatocytes to induce tolerance to allogeneic skin grafts and to downmodulate responses against donor splenocyte stimulators1, 3, we postulated that the peptides critical for allorecognition and tolerance induction would be found within a subset common to these three tissue types. The self-peptide repertoires of transduced hepatocytes, grafted donor skin and donor spleen were determined using a combination of immunoaffinity purification and RP-HPLC to liberate and collect peptide containing fractions from associated MHC I with LC-MS/MS for peptide identification, for both the 178.3 to B10.BR (Kb mismatch, H-2k background) and B6.Kd to C57BL/6 (Kd mismatch, H-2b background) strain combinations, as outlined in Figure 6a. The lists of common peptides are found in supplementary Table 2 (H-2Kd) and supplementary Table 3 (H-2Kb). For H-2Kd, 880 common peptides were identified across the three tissue types (Figure 6b), whereas there were 1083 common Kb-binding peptides (Figure 6c). The peptide length distributions (Figure 6d and f) and binding motifs (Figure 6e and g) 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-Kd-YCAC (324, 347 and 36 unique peptides respectively), compared to TAP-sufficient hepatocytes transduced with HC-Kd (880 unique peptides) (Figure 6h). 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-2Kd (Figure 6b) and H-2Kb (Figure 6c), respectively. Conversely, only 1.6% of skin peptides were also found in TAP1KOHep hepatocytes transduced with AAV-HC-Kd-YCAC, while in the 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%. Gene ontology analysis of source proteins is shown in supplementary Figure 4c.
H-2Kb peptides from the common peptide pool are recognised by activated alloreactive CD8+ T cells
A total of 100 peptides were selected for screening (listed in supplementary Table 4). 96 peptides were drawn from the common peptide pool, and their identity was confirmed by a direct comparison between the mass spectra obtained from synthetic and eluted natural peptides. Representative spectra from three peptide pairs, along with the Pearson correlation coefficient and the corresponding p-value between the log10 intensities of identified b- and y-ions in synthetic and sample-derived spectra is shown in Figure 7a. For all peptides, p < 0.05. A further four peptides had been previously identified as alloreactive CD8+ T cell epitopes in B10.BR mice11, 25. Three of these four epitopes were detected within the common pool. Binding of pMHC tetramers was used to determine which peptides combined with H-2Kb to form immunogenic epitopes recognised by alloreactive B10.BR CD8+ T cells. B10.BR mice were first primed by placement of a Kb-bearing 178.3 skin graft. Approximately 30 days after graft rejection, mice were inoculated with AAV-HC-Kb, and after a further 7 days, liver leukocytes were isolated and stained by flow cytometry (Figure 7b). The gating strategy is shown in Figure 7c. Activated CD8+ T cells were defined as CD44+PD-1hi, whereas PD-1- cells were considered to be an internal control population, which had been exposed in vivo to H-2Kb expressed on hepatocytes but were not activated. Peptides were deemed immunogenic when ≥2% of CD44+PD-1hi 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 7d, and data summarising the results are shown in Figure 8a and supplementary Table 4. Allorecognition of Kb-bound peptides was then examined in recipient mice of a second background haplotype (BALB/c, H-2d) (Figures 8a-b and Supplementary Table 4).
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 8a-b). A number of pMHC epitopes were recognised by BALB/c mice as well as B10.BR (Figures 8a-b). 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 8c). No such relationship was observed for 9-mers. Predicted peptide binding affinity for H-2Kb (measured by IC50) did not differ significantly between strongly, moderately or non-immunogenic peptides (Figure 8d, p = 0.098 by one-way ANOVA), nor was a correlation observed between IC50 and spectral abundance (Figure 8e). 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), whereas cross-reactivity between VGPRYTNL, INFDFPKL and RTYTYEKL was considerably lower (Figure 8f-g). 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 8i, 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.
Discussion
Direct allorecognition refers to the engagement of recipient TCRs by intact allogeneic MHC molecules on the surface of donor cells. In some settings, intact, unprocessed donor MHC is transferred to the surface of recipient APC, a variant known as the semi-direct pathway of allorecognition26. While direct/semi-direct allorecognition is a critical driver of both solid organ allograft rejection26 and tolerance induction3, the specific molecular targets of most directly-alloreactive CD8+ T cells have not been defined, limiting our understanding of these phenomena. In this study, we provide definitive evidence for the contribution of the peptide cargo of allogeneic MHC I molecules to transplant tolerance induction. Moreover, we demonstrate the feasibility of a systematic approach incorporating mass spectrometry-based identification of alloantigen-presented peptides, alloreactive T cell enrichment and pMHC multimer screening for the discovery of pMHC epitopes that are directly recognised by alloreactive CD8+ T cells. To our knowledge, this represents the first example of such an approach in the context of direct allorecognition.
Studies over many years using T cell clones suggested that most directly alloreactive CD8+ clones recognise allogeneic MHC I molecules complexed with an endogenous peptide, while peptide-independent direct allorecognition was also reported. Our recent findings that direct recognition of hepatocyte-expressed donor MHC I by recipient CD8+ T cells was required for donor skin graft tolerance induction provided us with a unique opportunity to examine the role of the hepatocyte immunopeptidome in transplant tolerance induction, and by extension, in alloresponses more broadly. We used two complementary approaches to separate expression of donor MHC I molecules from presentation of their usual peptide cargo. In the first approach, we utilised an AAV vector encoding H-2Kb HC, β2m and the single peptide KIITYRNL to express high levels of Kb on the surface of recipient hepatocytes, while excluding presentation of naturally-processed endogenous peptides. Tolerance to Kb-bearing skin grafts was able to be induced in reconstituted mice with a monoclonal population of T cells all recognising the Kb-KIIT epitope, but not in wild-type mice possessing a full TCR repertoire. Secondly, we expressed a mutated version of the H-2Kd HC, which is permissive to loading with suboptimal peptides in a purpose-bred mouse strain (TAP1KOHep) with a hepatocyte-specific deletion of Tap1. This combination resulted in a significant alteration of the Kd-bound peptide repertoire and prevented tolerance induction. Graft survival was shortened to a lesser extent in TAP1-sufficient mice transduced with AAV-HC-Kd-YCAC, in keeping with the lesser perturbation of the Kd peptide repertoire observed in these mice. The point mutations Y84C and A139C in the Kd HC permit the creation of a disulphide bridge which stabilises the peptide binding groove when empty or populated by a low-affinity peptide18. The presence of these mutations alters the composition of the Kd-associated peptide repertoire but does not impede T cell responses to a given pMHC ligand24. Accordingly, the ubiquitous peptide SYFPEITHI was present within the repertoires of all hepatocytes transduced with either AAV-HC-Kd or AAV-HC-Kd-YCAC and comparable proportions of T cells isolated from the livers of these mice bound Kd-SYFP dextramers.
Having demonstrated the importance of the peptide cargo of allogeneic MHC I molecules for transplant tolerance induction, we set about identifying the subset of peptides responsible for modulating the anti-graft alloresponse, using an immunoproteomic approach to identify and characterise the immunopeptidomes of relevant tissues. We hypothesised that hepatocyte expression of peptides that were also presented by cells in the grafted skin underpinned tolerance induction. Consistent with this hypothesis, in conditions where tolerance was achieved, 43% of Kd-associated peptides and 45% of Kb-bound peptides from transduced hepatocytes were also expressed in donor skin. When survival prolongation was minimal, only 1.6% of hepatocyte Kd-bound peptides were common to donor skin, and this shared proportion increased to 15-17% in the groups with intermediate graft survival. This data in combination with our previous observations that expression of allogeneic MHC I in recipient liver reduces IFN-γ ELISPOT responses against donor splenocyte stimulators, implied that the critical set of self-peptides would be concentrated amongst those common to all three tissue types.
We next set out to determine which peptides from this shared subset would be recognised by alloreactive T cells using tetramer binding assays. We generated an enriched population of alloreactive CD8+ T cells in the liver by priming mice with a skin graft expressing H-2Kb, followed by boosting with liver-specific AAV-HC-Kb. Co-expression of CD44 with high levels of PD-1 allowed us to distinguish between activated cells and bystander cells which had been exposed to the alloantigens, but had not responded. Of the 100 Kb-binding peptides selected for tetramer screening, 17 were recognised by >5% of the enriched alloreactive T cell population. INFDFNTI, previously reported to be recognised by a large proportion of the polyclonal alloreactive T cell population of B10.BR mice25 was bound by an average of 3.7% of male B10.BR cells.
To examine factors which might contribute to the immunogenicity of different pMHC, we used spectral intensity as an approximation for relative peptide abundance and IC50 to predict binding affinity of the peptide for H-2Kb. An overall measure of abundance, was derived by multiplying the spectral intensity recorded in each of the three tissues. For 8-mer peptides, a strong correlation was found between abundance and the proportion of CD8+ T cells recognising particular peptides. This is at variance with observations of CD8+ T cell recognition of Kb-associated viral epitopes, where the frequency of T cells responding to a given epitope corresponds weakly at best to epitope abundance27, 28. In contrast, while there was a tendency for the strongly immunogenic peptides to have high predicted affinity for Kb, there was no significant difference in IC50 between the strongly-, moderately- and non-immunogenic groups, nor was there an association between IC50 and peptide abundance. While a prediction of immunogenicity based on peptide abundance is no substitute for direct measurement, these findings suggest that the likelihood of finding strongly immunogenic peptides is greater if the search is targeted towards the most highly-represented peptide species. The relationship between abundance and immunogenicity did not apply to 9-mer peptides. A staining panel comprising only five strongly immunogenic pMHC epitopes identified nearly 40% of T cells in an enriched alloreactive population; we anticipate that an expanded panel will capture an even greater proportion of alloreactive cells, and that such panels could be used to enumerate, track and phenotype polyclonal alloreactive T cell populations.
One intriguing finding of this study was the broad overlap between strongly immunogenic Kb-peptide epitopes recognised by BALB/c and those first identified in B10.BR mice (13/17 peptides). Further studies in mouse and human subjects will be needed to establish the prevalence of such ‘super-epitopes” recognised by allogeneic T cells across several genetic backgrounds. While extensive cross-reactivity is a feature of self MHC-restricted T cells, the range of possible cross-reactive epitopes is constrained by peptide length, with T cell clonotypes showing a strong preference for a single length peptide29. Alloreactive T cell clones have undergone positive selection during thymic development based on self-MHC. The majority of peptides eluted from H-2Kk are 8-mers (30 and supplementary data 1) which aligns with the result that in male B10.BR mice (H-2k) 17/79 8-mer peptides but 0/21 9-mers were strongly immunogenic. Most peptides associated with the H-2d allomorphs Dd, Ld and Kd are nonamers30, yet 42/79 8-mers and only 1/21 9-mers were strongly immunogenic in BALB/c (H-2d) mice. Possible explanations for this asymmetry are that cross-reactivity involving an allogeneic MHC molecule is not subject to the same peptide length limitations as cross-reactivity between self MHC-restricted peptide epitopes, or that a small number of octamer-preferring CD8+ T cells from BALB/c mice expand preferentially in the presence of H-2Kb presenting mainly 8-mers. While the mean H-2Kb IC50 among the 9-mer peptides was greater than that of the 8-mers, a number of 9-mer peptides were in the same IC50 range as the strongly immunogenic 8-mers, so this is unlikely to fully explain the reduced frequency of recognition of 9-mer peptides.
The findings of this study open a number of avenues for future research. Firstly, they suggest an approach for the systematic discovery of pMHC epitopes for directly alloreactive CD8+ T cells which may be adapted to additional MHC I allomorphs in mice, humans and other species. Identification of large numbers of allogeneic pMHC epitopes will enable the characterisation of alloreactive TCR repertoires, including biophysical and structural studies with many more receptor-ligand pairs than the handful currently known, which in turn will provide further insights into the fundamental basis of alloreactivity. Systematic allogeneic pMHC epitope discovery is complemented by the recent advent of methodologies such as oligonucleotide barcoding permitting the use of pMHC multimers for parallel tracking and enumeration of >1000 individual pMHC specificities, with options for coupling to single cell TCR sequence and gene expression analyses31, 32. pMHC multimers conjugated with radionuclides are sensitive tools which could be applied to in vivo imaging of specific alloresponses33. Beyond their potential as research reagents, allospecific pMHC multimers could enhance post-transplant immune monitoring and immunophenotyping of directly alloreactive T cells in a clinical setting. Finally, pMHC multimers are not only reagents for antigen-specific T cell detection, but potential vehicles for antigen-specific therapy, which may be employed for the depletion or immunomodulation of alloreactive T cells34. In conclusion, the findings of this study represent a significant advance in our understanding of the role of endogenous peptides in direct T cell alloreactivity. They will enable exploration of the alloreactive T cell repertoire and potential translation to clinically-applicable tools.
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. Antibodies used in experiments are summarised in Supplementary Table 4.
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-2Kb was quantified using flow cytometry with the conformation-dependent mAb Y-3 antibody, which binds to correctly folded Kb 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-2Kb ubiquitously, under the control of its own promoter, on a B10.BR (H-2k) background. Des-TCR mice express an H-2Kb-specific TCR, which recognises the peptides KVITFIDL, KVLHFYNV and KIITYRNL restricted by H-2Kb and Des-TCR is identifiable by a clonotypic mAb (Désiré). Des-RAG mice were obtained by crossing Des-TCR mice with CD45.1+/+ RAG1-/- mice, which are both on a B10.BR (H-2k) background. OT-I mice carry a TCR which recognises the peptide SIINFEKL presented by H-2Kb. 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-2b) and BALB/c (H-2d) mice (termed C57BL/6 and BALB/c) were purchased from the Animal Resources Centre, Perth, Australia. B6.Kd mice express an H-2Kd transgene ubiquitously on a C57BL/6 (H-2b) background. B6.Kd 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)1Dlr/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 (not shown). Further characterisation of this strain is shown in Figure 4 and Supplementary Figure 3.
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-2Kb and H-2Kd were cloned as previously described3. An SCT construct with a disulphide trap consists of a defined peptide sequence, β2m and MHC I HC joined together by flexible linkers16. 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-Kb or H2-Kd 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 trap17. dt-SCT (H-2Kb) constructs with defined peptide sequences KIITYRNL or SIINFEKL and a dt-SCT (H-2Kd) 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-Kd 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 as previously described38. 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. All vectors were used at a dose of 5×1011 vgc except AAV-SCT-Kb-KIIT which was used at 2×1012 vgc. 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
Skin transplantation was performed as described previously3. In short, 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 × 1 cm2 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-2k) received H-2Kb singly-mismatched allogeneic skin grafts from 178.3 strain donor mice. C57BL/6, TAP1fl/fl and TAP1KOHep mice (H-2b) received H-2Kd singly-mismatched allogeneic skin grafts from B6.Kd donor mice. BALB/c mice (H-2d) 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 (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 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 immunoaffinity 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 as described above. 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 as detailed above. Cells resuspended in RPMI 1640 medium containing 10% FCS was 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 μm 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 H-2Kb (AF6-88.5, BioLegend), H-2Kd (SF1-1.1, BD Biosciences), CD4 (GK 1.5, BD Biosciences), CD8α (53-6.7, BioLegend), F4/80 (BM8, BioLegend), B220 (RA3-6B2, BD Biosciences), CD11c (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 of PBS and then 10 ml of 2% paraformaldehyde (Sigma, catalogue# 30525-89-4) in PBS. 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, Abcam) and anti-mouse H-2Kb 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).
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 (Supplementary Table 1). 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 (BD).
ELISpot
IFN-γ 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/FCS10 medium with penicillin-streptomycin in each well of a 96-well U-bottom plate (Corning, 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 as previously described1.
pMHC multimer staining
The pMHC multimer staining method has been adapted from that of Dolton et al.39. 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 tetramer or pMHC dextramer at 6.4 nM 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-PD-1-BV421 (29F.1A12, BioLegend), anti-CD8-FITC (KT-15, Invitrogen), anti-CD14-BV605 (rmC5-3, BD Bioscience), anti-CD19-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 mM NaCl, 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 immunoaffinity purification using solid-phase-bound monoclonal antibodies SF1-1.1.10 (anti H-2Kd), K9-178 (anti H-2Kb), Y3 (anti H-2Kb/Kk) and 28.14.8s (anti H-2Db) as described previously40. 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) as described previously31. 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 x 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 MS1 survey scan and fragment ions were acquired using variable windows (Supplementary Table 6) 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.42
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
Known contaminants were removed from the 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 immunoaffinity purified with K9-178 antibody (H-2Kb group) and 9-mer peptides from samples that had been immunoaffinity purified with SF1-1.1.10 antibody (H-2Kd group) were visualised using the GibbsCluster2.0 algorithm (NetMHC4.0)43. For comparison of unique H-2Kb peptides between different tissues, 8-mer to 11-mer peptides with a predicted half-maximum inhibitory concentration (IC50) of binding to H-2Kb less than 500 nM (NetMHC4.0 database) were selected. For comparison of unique H-2Kd 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 system44. Function classification analysis and statistical over-representation tests were performed45.
Validation of peptide identification using retrospectively synthesised peptides
We validated the identity of a panel of peptides 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, we predicted all b- and y-ions for each sequence and then extracted the intensity for each ion (with a fragment mass error tolerance of 0.02 Da). The Pearson correlation coefficient and the corresponding p-value between the log10 intensities of identified b- and y-ions in the synthetic and sample-derived spectra were calculated46. 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).
Author contributions
A.W.P., N.A.M., E.T.S., P.B., D.B. and A.F.S. designed experiments. E.T.S., M.P-H., P.F., S.H.R., M.L., K.E., A.B., N.L.D. and N.A.M. performed experiments. A.W.P., L.L., I.E.A. and P.B. provided reagents and/or samples. E.T.S., P.F., S.H.R., A.W.P., N.A.M. and A.F.S. analysed data. P.F., S.H.R., N.A.M., E.T.S. and A.F.S. wrote the manuscript. All authors read and approved the manuscript.
Competing Interests
E.T.S., M.P-H., A.F.S. (University of Sydney) and N.A.M., A.W.P., P.F., N.L.D. (Monash University) are named as co-inventors in a patent application filed by the University of Sydney and Monash University (PCT/AU2020/051221) covering the identification and use of certain peptides described in the publication.
Figure Legends
Supplementary Data 1. Complete list of identified peptides
Acknowledgements
Dr Nathan P. Croft (Monash University) for discussions of RMA-S peptide-pulsing experiments. Technical assistance from R. Ayala and I. Hanchapola (Monash University). The authors acknowledge the provision of instrumentation and technical support by the Monash Biomedical Proteomics Facility, and by Sydney Cytometry. Computational resources were supported by the R@CMon/Monash Node of the NeCTAR Research Cloud, an initiative of the Australian Government’s Super Science Scheme and the Education Investment Fund. We thank Laboratory Animal Services, University of Sydney, for the provision of superb mouse care, and the Vector and Genome Engineering Facility, CMRI for AAV vector production and advice. A.W.P. is supported by an NHMRC principal research fellowship (1137739). M.L. received a Research Training Program Postgraduate Award (SC1999) from the Department of Education and Training, Australian Federal Government, while M.P-H. was supported by an Earl Owen Fellowship from the Sydney Medical School Foundation, and E.T.S. received funding from the Royal Prince Alfred Hospital Transplant Institute. L.L. was supported by project grants from the Australian National Health and Medical Research Council (NHMRC) (APP1108311, APP1156431 and APP1161583) and research grants from the Department of Science and Higher Education of Ministry of National Defense, Republic of Poland, (“Kościuszko” k/10/8047/DNiSW/T – WIHE/3) and from the National Science Centre, Republic of Poland (OPUS13) (UMO-2017/25/B/NZ1/02790). This project is supported by Grants-in-aid from Sydney Medical School Foundation and the Myee Codrington Medical Research Foundation (to A.F.S.) and by NHMRC Ideas Grant #1183806 to A.F.S. and N.A.M.