Exploring the MHC-peptide matrix of central tolerance in the human thymus

Abstract

Ever since it was discovered that central tolerance to self is imposed on developing T cells in the thymus through their interaction with self-peptide major histocompatibility complexes on thymic antigen-presenting cells, immunologists have speculated about the nature of these peptides, particularly in humans. Here, to shed light on the so-far unknown human thymic peptide repertoire, we analyse peptides eluted from isolated thymic dendritic cells, dendritic cell-depleted antigen-presenting cells and whole thymus. Bioinformatic analysis of the 842 identified natural major histocompatibility complex I and II ligands reveals significant cross-talk between major histocompatibility complex-class I and II pathways and differences in source protein representation between individuals as well as different antigen-presenting cells. Furthermore, several autoimmune- and tumour-related peptides, from enolase and vimentin for example, are presented in the healthy thymus. 302 peptides are directly derived from negatively selecting dendritic cells, thus providing the first global view of the peptide matrix in the human thymus that imposes self-tolerance in vivo.

ARTICLE

Received 20 Dec 2012 | Accepted 21 May 2013 | Published 19 Jun 2013

Eleni Adamopoulou1, Stefan Tenzer2, Nina Hillen3, Paula Klug1, Ioanna A. Rota1, Silvia Tietz1, Madlen Gebhardt1, Stefan Stevanovic3, Hansjrg Schild2, Eva Tolosa4, Arthur Melms1,w & Christina Stoeckle1,w

Ever since it was discovered that central tolerance to self is imposed on developing T cells in the thymus through their interaction with self-peptide major histocompatibility complexes on thymic antigen-presenting cells, immunologists have speculated about the nature of these peptides, particularly in humans. Here, to shed light on the so-far unknown human thymic peptide repertoire, we analyse peptides eluted from isolated thymic dendritic cells, dendritic cell-depleted antigen-presenting cells and whole thymus. Bioinformatic analysis of the 842 identied natural major histocompatibility complex I and II ligands reveals signicant crosstalk between major histocompatibility complex-class I and II pathways and differences in source protein representation between individuals as well as different antigen-presenting cells. Furthermore, several autoimmune- and tumour-related peptides, from enolase and vimentin for example, are presented in the healthy thymus. 302 peptides are directly derived from negatively selecting dendritic cells, thus providing the rst global view of the peptide matrix in the human thymus that imposes self-tolerance in vivo.

DOI: 10.1038/ncomms3039 OPEN

Exploring the MHC-peptide matrix of central tolerance in the human thymus

1 Department of General Neurology, Hertie Institute for Clinical Brain Research, 72076 Tbingen Germany. 2 Institute for Immunology, University Medical Center of the Johannes Gutenberg University of Mainz, 55131 Mainz, Germany. 3 Department of Immunology, Institute of Cell Biology, University of Tbingen, 72076 Tbingen, Germany. 4 Institute for Immunology, University Medical Center Hamburg-Eppendorf, 20246 Hamburg, Germany. w Present addresses:

Department of Neurology, University of Erlangen-Nuremberg, Erlangen, Germany (A.M.); Institute of Pharmacology, University of Bern, Bern, Switzerland (C.S.). Correspondence and requests for materials should be addressed to C.S. (email: mailto:[email protected]

Web End [email protected] ).

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Afunctional immune system must be able to effectively recognize and ght pathogens while staying tolerant to the bodys own components. This is achieved by central

and peripheral tolerance mechanisms. Central tolerance to self is imposed on T cells during their development in the thymus, where they are educated to be self-referential, recognizing peptides bound to self-major histocompatibility complex (MHC) molecules, as well as self-tolerant, meaning that they remain unresponsive to self-peptides displayed on the very same MHC molecules1,2.

In order to full both criteria, the ab T cells must pass through two major checkpoints in their development, which are controlled by antigen-presenting cells (APCs) in distinct thymic micro-environments. These APC comprise cortical (cTECs) and medullary thymic epithelial cells (mTECs), as well as both resident and immigrant dendritic cells (DCs). During development, thymocytes must rearrange their genes in order to express a functional T-cell receptor (TCR) that can successfully interact with self-peptide-MHC ligands on cTEC, from which they will then receive a survival signal (positive selection). Positively selected thymocytes migrate into the thymic medulla, where they interact with self-peptideMHC complexes displayed by mTECs and DCs. T cells expressing a high afnity TCR for these complexes will be negatively selected and undergo apoptosis. Only T cells with a low/intermediate afnity TCR for self-peptides will be allowed to leave the thymus as naive or regulatory T cells1,2. If a T cell that strongly recognizes self-peptides fails to be deleted, for example, because the corresponding self-protein is not expressed in the thymus or the epitope is destroyed during antigen processing, autoreactive T cells can escape into the periphery, where they can be activated and cause autoimmune disease3. Therefore, the interaction between the TCR of developing thymocytes and the MHC peptide ligandome of distinct thymic APCs is crucial for shaping the T-cell repertoire and central tolerance.

Myeloid DCs (mDCs) in the thymus were the rst cells discovered to be responsible for deletion of autoreactive T cells in the thymus and are crucial for maintaining tolerance to self4,5. Although, in contrast to mTECs, they are not specialized in expressing tissue-specic antigens such as proinsulin, they can efciently capture these from mTECs and present them to developing T cells, inducing their deletion, a process that involves autoimmune regulator expression (AIRE) in mTECs6,7. A role of plasmacytoid DCs (pDCs) in negative selection has only been described very recently8 and thymic B cells might also be able to contribute to negative selection9. Although so far there is no direct proof that the different subsets of thymic APCs present different sets of peptides, there are two strong lines of indirect evidence supporting this notion. First, the different APCs express different sets of self-proteins. In addition to constitutively expressed house-keeping proteins and cell-type-specic proteins, mTECs express tissue-restricted antigens such as insulin under the control of the AIRE transcription factor10,11. Also, blood-born mDCs and pDCs can import peripheral antigens into the thymus where they delete self-reactive thymocytes8,12. Second, both the MHC I and II antigen-processing machinery differs between different thymic APCs, which should inuence the epitopes that are generated1316. The different APCs thus contribute to the diversity of the immunological self displayed in the thymus shaping the characteristics of the mature T-cell repertoire of each individual.

Our current knowledge regarding the self-peptide repertoire in the thymus derives mostly from studies in TCR transgenic mouse models, where expression of a particular antigen is a prerequisite for positive/negative selection. However, none of these studies provides evidence that the respective peptides actually contribute

to negative selection under physiological conditions. In the early nineties, Marrack et al.17 successfully identied B20 MHC II-bound peptides in pooled thymi of several hundred mice and a few natural ligands for positive selection of mouse OT-I cells are known18,19. More recently, another group has eluted peptides from whole-mouse thymus, using bioinformatics to narrow down on likely MHC I ligands20. In humans, presentation of an epitope from proinsulin on thymic DCs has been reported using monoclonal antibodies21, but beyond this, the human thymic peptide repertoire is unknown.

In this work we set out to explore the human thymus MHC ligandome. We describe an in depth analysis of the peptide repertoire presented by both MHC class I and class II molecules in the healthy human thymus, both for whole thymus as well as isolated thymic mDCs. In summary, we here present the rst data on naturally occurring peptides on which human T cells are selected under physiological conditions, including peptides from DCs which are responsible for deletion of autoreactive T cells.

ResultsIdentication of peptides presented by thymic APCs. To address the question which peptides are naturally presented to developing thymocytes to induce negative selection under physiological conditions in humans, we analysed peptides eluted from both MHC I (pan-class I human leucocyte antigen; HLA) and II (HLADR) complexes from isolated thymic mDCs by mass spectrometry and compared them with the repertoire of the MHC-bound peptides eluted from cells of the APC-enriched, mDC-depleted (containing pDCs, TECs and other thymic APCs, that is, ligands for both positive and negative selection) as well as whole-thymus fractions (Fig. 1).

One major challenge for MS-based analysis of peptides presented by thymic APCs is their low frequency and their purication in sufcient numbers to allow ligandome analysis. Typically, the number of cells needed for such experiments is in the order of 108, limiting the possibilities for analysis of primary puried cell types. In our experiments, we identied a total of 842 peptides from the different cell preparations of the eight individual thymi investigated, of which 621 were eluted from MHC I and 221 from MHC II molecules. In the individual mDC preparations, we successfully identied up to 90 peptide sequences eluted from MHC I molecules and up to 48 from MHC II from as little as 30 106 cells from four different donors

(Table 1). Complete lists of the peptide sequences obtained from the different samples and their source proteins are given in Supplementary Data 1.

Cross-talk between MHC I and MHC II pathways. Peptides presented by MHC I and II derive from different cellular compartments. Typically, nuclear and cytosolic proteins are presented by MHC I, while proteins that are present in the vesicular compartments or the cell membrane or are taken up from the extracellular environment are presented by MHC II molecules. However, especially in professional APCs, there is considerable cross-talk between those two pathways22. Mechanisms by which cytosolic/nuclear proteins gain access to the MHC II pathway include autophagy and uptake of apoptotic cells23,24. Vice versa, proteins or peptides can shuttle out of the lysosome into the cytosol and thus gain access to the MHC I pathway22. In addition to this intracellular cross-talk, DCs may take up antigens from other cells such as mTECs, further broadening their peptide repertoire25,26.

To gain insight into the origin of the presented peptides as well as the dynamics of antigenic cross-talk between MHC presentation pathways in thymic mDCs and non-mDC APCs, we

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Percoll

Thymus tissue

Mechanical disruption

Enzymatic digestion APC enrichment

MACS

CD11c+ DC

DC-depleted APC

LC-based peptide separation and

MS/MS sequencing

Disruption of MHC/peptide complexes

Isolation of MHC/peptide complexes on antibody column

Intensity

Peptides

m/z

MHC I

MHC II MHC I/W6/32 MHC II/L243

MHC I

mDC

Peptides

MHC II MHC I/peptide MHC II/peptide

CD11c-PE Ab + anti-PE beads

L243 antibody

W6/32 antibody

Figure 1 | Workow diagram for the isolation of MHC I and MHC II ligands from CD11c mDC and the DC depleted, APC-enriched fractions.

Table 1 | MHC I and MHC II ligands identied on thymic CD11c dendritic cells of individual donors.

Tissue number

DC cell number

No. of MHC II ligands

TT222 36 106 43 47

TT251 66 106 90 35

TT259 33 106 15 19

TT260 30 106 29 24

DC, dendritic cell; MHC I, major histocompatibility complex I; MHC II, major histocompatibility complex II.

The table contains the number of identied MHC I- and MHC II-derived peptides, the source tissue and the number of isolated DCs from which they were eluted.

No. of MHC I ligands

performed automated database analysis supplemented by manual annotation of the source proteins and their subcellular localization. Depending on their topological origin, proteins were assigned either to the classical MHC class I (cytosolic, nuclear) or the MHC class II (extracellular, secretory, endocytic, and cell

membrane) pathway, the mitochondrial and the ER compartment (Fig. 2a). As expected, the majority of MHC I ligands presented by both DCs and non-DC APCs derived from cytosolic/nuclear proteins, that normally do not intersect the endocytic compartment, such as histones, cytoskeletal proteins (actin, tubulin and fascin), metabolic enzymes (protein phosphatase 1, hydroxypyruvate reductase), and cytosolic chaperones (heat shock proteins). MHC class II-bound peptides originated from proteins present in different cellular compartments, including the extracellular milieu, the plasma membrane and the vesicular compartment with membrane proteins being the most abundant peptide generators (mostly from MHC class II proteins) in agreement with previous studies with other cell types24. A substantial number of MHC II presented peptides originated from blood or serum proteins, such as serum albumin, haemoglobin and apolipoprotein A2, providing direct evidence that thymic DCs sample the blood stream.

Interestingly, a considerable fraction of the MHC II ligands derived from source proteins localized to the cytosol or nucleus(35.4% in DCs and 21.8% in non-DCs), which are topologically separated from the MHC class II pathway. Similarly, 22.4% of MHC I ligands on DCs derived from the vesicular/extracellular

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a

100%

90% 29

209 105 118 40 17

33 23 69 57

80% 70% 60% 50% 40% 30% 20% 10%

0%

100%

90% 80% 70% 60% 50% 40% 30% 20% 10%

0%

Whole-

thymus

MHC I

Unknown Mitochondrial MHC IIMHC IER

Whole-

thymus

MHC I

CD11c+

DC

MHC I

DC-

depleted

MHC I

CD11c+

DC

MHC II

DC-

depleted

MHC II

b

Cell death

Cellular component organization

Other/unknown

Translation

Response to stress

Cell differentiation and development

Cell cycle, growth and proliferation

Metabolic process

Transport

Viral reproduction

Signalling

Antigen processing and presentation

Immune response

CD11c+

DC

MHC I

DC-

depleted

MHC I

CD11c+

DC

MHC II

DC-

depleted

MHC II

Figure 2 | MHC pathway assignment and functional categorization of the identied source proteins isolated from the different thymic sample preparations. The cellular localization (a) and biological processes (b) of the protein categories represented graphically as percentages are based on n the number of the individual proteins identied in each thymic APC preparation.

compartment (compared with 15.5% in the DC-depleted fraction and 11.4% in the complete thymus samples). Peripheral DCs are highly efcient at cross-presenting endocytosed antigens on MHC I and cytosolic/nuclear antigens on MHC II. It is therefore crucial to induce central tolerance also to cross-presented self-proteins. The high degree of cross-talk between the two pathways that we observe in thymic DCs, as well as to a lesser extent in the DC-depleted fraction, reects this need to avoid peripheral autoimmune responses by deleting T cells that recognize cross-presented self-proteins.

A recent study has underscored the importance of autophagy for MHC II presentation during central tolerance induction. In mice, genetic interference with autophagy leads to an altered selection of certain CD4 T-cell specicities that give rise to an autoreactive T-cell repertoire27. Most of the studies so far have addressed the in vivo relevance of autophagy mainly in TECs, and not thymic DCs, but in peripheral DCs autophagosomes have been shown to fuse with the MHC II loading compartment23. Our peptide data show considerable presentation of peptides derived

from nuclear/cytosolic as well as some mitochondrial proteins on MHC class II by thymic mDCs, suggesting a role for autophagy in MHC II ligands sampling in these cells. Alternatively, these proteins could enter the MHC II pathway by uptake from other cells such as apoptotic thymocytes or live/dying mTECs7,28,29 or transfer of peptideMHC complexes from mTECs30.

Nature of DC-presented peptides. Analysis of the biological processes associated with the peptides source proteins revealed a prominent fraction of proteins participating in immune response and antigen processing/presentation, biological functions reecting the nature of the thymus as an immune organ (Fig. 2b). The most highly represented class of proteins in these groups were HLA molecules and, not surprisingly, their presence was more pronounced in the DC and DC-depleted samples (enriched for other thymic APCs), compared with whole-thymus samples. Most of the remaining class I and II natural ligands derive from house-keeping proteins (signalling,

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translation, metabolism, cell cycle and proliferation). Interestingly, peptides derived from proteins associated with metabolic processes are preferentially presented on MHC class II molecules, and peptides derived from proteins involved in cell cycle, growth, proliferation and differentiation are more abundantly presented on MHC class I molecules. The explanation for the preferential presentation of peptides derived from proteins involved in these processes by MHC I is that in these groups the majority of peptides derive from nuclear and cytosolic proteins involved in maintaining cellular structure (for example, the cytoskeleton) and transcription. These are typically degraded by the proteasome and presented via the classical MHC I pathway22. A closer look at the metabolic processes-associated proteins revealed that many of the MHC II-bound identied peptides in this group derived from cytosolic proteins. The sources of these peptides included a considerable number of long-lived constitutive metabolic enzymes like glyceraldehyde 3-phosphate dehydrogenase and phosphoglycerate kinase 1. Interestingly, long-lived proteins have been suggested to be disposed by autophagic rather than proteasomal protein degradation31. Autophagic degradation of cellular material has been shown to signicantly contribute to the peptide pool presented by MHC II (refs 23,24). In agreement with our results and in support of this idea, glyceraldehyde 3-phosphate dehydrogenase was previously identied as a source of natural MHC class II, but not class I ligands24,32. Hence, autophagic degradation of long-lived metabolic proteins might explain why these cytosolic proteins are presented by MHC class II rather than class I. The observed wide cellular functional distribution of proteins reects the wide range of proteins that serve as a matrix for self-protein presentation in the thymus.

Peptides differ between individuals and APC. It has been suggested that different types of thymic APC present different non-overlapping set of self-proteins, despite the existence of pathways allowing protein/peptide transfer between cells and despite the fact that a lot of basic cellular components like metabolic enzymes, and structural proteins should be expressed at similar levels in most cells. One reason for this might be the differences in the antigen-processing machinery between the different cell types1316. To address this question, we compared the source proteins of peptides presented on DCs to that of the DC-depleted fraction (enriched for other thymic APCs, for composition see Supplementary Table S1 and Supplementary Fig. S1). In total, we identied 302 peptides originating from 187 different proteins on DCs and 265 peptides from 184 different proteins in the DC-depleted fraction, 71 of which were present in both fractions (Fig. 3a). These include proteins such as HLA molecules, histones, proliferating cell nuclear antigen, actin, pyruvate kinase, serum

albumin and b2 microglobulin. Although, B1/3 of the protein pool was shared between DCs and non-DC cells, the majority of the identied proteins were unique for each group, supporting the idea that different thymic APCs present different peptides.

Data from whole thymus indicated a large degree of variability of the source proteins from which peptides were presented in different donors (Supplementary Fig. S2), probably partially due to differences in HLA-binding preferences and protein expression. Quantitative differences in expression levels of individual MHCpeptide complexes might also play a role as our experimental approach preferentially detects high abundance peptides, especially when source material is limiting. To exclude the possibility that the limited overlap observed was due to issues of technical reproducibility rather than bona de biological variability, we performed extensive validation experiments which showed that technical reproducibility was in the order of 85%, while the overlap between samples derived from different donors was much lower (Supplementary Fig. S3), demonstrating that the differences observed were indeed due to biological, and not technical, variability.

To clarify whether this variability was also observed for the self-proteins directly involved in negative selection of T cells, we compared the source proteins of the peptides eluted from thymic DCs from different donors (Fig. 3b). Of the 187 proteins whose peptides were presented by mDCs, only 27 were detected in more than one donor, and only one (HLA-DRB1) was shared by the four individual thymic DC samples. Peptides from seven proteins (HLA-DRA, serum albumin, tubulin b 2 A chain, serine/

threonine-protein phosphatase PP1, astrocytic phosphoprotein PEA-15, bax inhibitor 1, signal transducer and activator of transcription 1-a/b) could be detected in three out of four DC samples, but peptides from the vast majority of proteins (160) were only detected in one sample, reecting a high inter-individual variability in the peptide matrix for negative selection. A similar picture is observed in the DC-depleted-derived peptide pool (Fig. 3c).

To further explore the differences between the HLA ligandome of DCs and non-DCs, we directly compared the source proteins (Fig. 4a) and the protein-derived peptides (Fig. 4b) that are present on the respective fractions from the same donors, eliminating the contribution of HLA-binding preference to peptide variability between fractions. Interestingly, only about a fth of the detected proteins were presented by both DC and non-DC APCs, and for donor TT259 the overlap was even smaller. In summary, we demonstrate a high inter-individual variability of the MHC ligandome and furthermore, our results agree with the notion that different, but overlapping spectra of self-proteins on different thymic APCs results in partially non-redundant contributions of different APCs to negative selection.

a b c

TT251

CD11c+ DC DC depleted

TT222

TT251

CD11c+ DC DC depleted

TT259

TT259

TT260

88

2 0 3

4

102 6 23

33

0

10

3

18

0

116 71

113 44

6

16 4

1

1

6

1

TT260

Figure 3 | Inter-individual variation in presentation of peptides from different source proteins presented by MHC I and II. The diagrams depict the number of different source proteins identied in the different cell preparations as well as overlap between samples. (a) Overlap of identied proteins between mDC and mDC-depleted (enriched for other APC) fractions (individual donors taken together). Inter-individual variations of proteins presented on (b) DC of four different donors and (c) DC-depleted fractions of three individuals, respectively.

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T-cell epitopes linked to autoimmunity. Incomplete representation of the full spectrum of self-proteins (the immunological self) on MHC molecules of mTECs and DCs will result in a lack

of central tolerance to those epitopes that are not displayed, increasing the propensity for autoreactivity among mature T cells in the periphery1,33.

We were therefore particularly interested whether we could identify peptides corresponding to known T-cell epitopes that have been described as targets in human autoimmune diseases. Literature search yielded a number of T-cell autoantigens that were also detectable in our samples (Table 2). The table only includes T-cell autoantigens for which reactivity has been experimentally veried in humans, such as vimentin (rheumatoid arthritis, sarcoidosis)34,35, ATP synthase (sarcoidosis)35, enolase 2 (multiple sclerosis)36 and heat shock protein 70 (type 1 diabetes mellitus)37. For those autoantigens where reactivity against specic epitopes (rather than whole protein) was reported, the epitopes were identical to, or contained within the peptide sequences that we identied in our samples, mostly on DCs. This clearly demonstrates presentation of these epitopes in normal human thymus, which should lead to deletion of autoreactive T cells. We therefore hypothesize that in patients with autoimmune disease, presentation of these epitopes could be impaired, resulting in failure of central tolerance towards these epitopes.

Tumour-associated antigens in the thymus. Central tolerance is a double-edged sword. Although it protects us from autoimmune disease, it can also impede anti-tumour immunity. It is likely that presentation of self-proteins which tolerize the T-cell repertoire to self-antigens in order to avoid autoimmunity may also negatively impact the ability of T cells to mediate tumour immunity. This prediction has been conrmed by studies demonstrating the expression of tumour-associated antigens, including differentiation antigens and cancer-germline antigens, in human mTECs38, and moreover, expression of the human carcinoembryonic antigen (CEA), in mTECs of transgenic mice induces central tolerance39. A thorough literature search on natural MHC ligand repertoires in tumours revealed that a signicant number of the MHC-bound peptides detected in our experiments are also found to be (over)presented in renal cell carcinomas40,41 or other disease tissues (Supplementary Data 2), including the SET nuclear oncogene and APOL1. Our ndings provide an explanation why only low-avidity T cells against many cancer-associated antigens are present in the periphery42, as presentation of tumour-associated antigens by negatively selecting cells of the thymus should result in the deletion of high-avidity T cells. Therefore, even if they are found to be overexpressed on tumour tissues, they might not be the rst choice as a target for immunotherapy. An

Overlap of source proteins

All MHC I MHC II

CD11c+

DC

DC depleted

CD11c+

DC

DC depleted

CD11c+

DC

DC depleted

TT251

TT259

TT260 22

57 48 80 48 36 61 13 11 23

CD11c+

DC

DC depleted

CD11c+

DC

DC depleted

CD11c+

DC

DC depleted

18 3 30 15 0 18 4 3 12

CD11c+

DC

DC depleted

CD11c+

DC

DC depleted

CD11c+

DC

DC depleted

15 38 16 13 31 6 3 7

Overlap of presented peptides

All

MHC I MHC II

CD11c+

DC

DC depleted

CD11c+

DC

DC depleted

CD11c+

DC

DC depleted

TT251

TT259

TT260

80

33

35 18 44 17 12 35 18 6 9

45

114 57 33 71 24 11 44

CD11c+

DC

DC depleted

CD11c+

DC

DC depleted

CD11c+

DC

DC depleted

1

42 15 0 1

18 18 24

CD11c+

DC

DC depleted

CD11c+

DC

DC depleted

CD11c+

DC

DC depleted

Figure 4 | Comparison of mDC and non-mDC fractions in each donor. Overlap of (a) source proteins and (b) presented peptides between mDC and mDC-depleted cells in three individual thymi.

Table 2 | T-cell autoantigens/epitopes in humans identied in different thymus samples.

Autoantigen Epitope (thymus) Thymus fraction Disease Epitope used in T-cell assay Assay type Ref. Vimentin RETNLDSLPL (424433) DC Rheumatoid arthritis LPNFSSLNLRETNLDSLPL (415-433) ICS

IFN-g

34

H /K ATPase (ATP synthase)

VPPVQVSPLIKLG RYSAL (219)

DC depleted Sarcoidosis VPPVQVSPLIKLGRYSAL (219) ELISPOT

IFN-g

51

b-actin RVAPEEHPVL (95104) DC Sarcoidosis NELRVAPEEHPVLLT (92106) ELISPOT

IFN-g

51

Heat shock protein 70 NVLRIINEP

TAAAIA(168182)

DCDC depleted

Type 1 diabetes mellitus GLNVLRIINEPTAAAIAYGL (166185) 3H-thymidine 37

Enolase 2 IVGDDLTVTNPK DC Multiple sclerosis Whole protein 3H-thymidine 36 Cytokeratin 17 RLASYLDKV (125133) Whole thymus Psoriasis RLASYLDKV (125133) ICS

IFN-g

52

DC, dendritic cell; ICS, intracellular cytokine staining; IFN-g, interferon-g.

The table shows the autoantigens and the derived peptides identied in our samples as well as the preparation in which we could identify them, the autoimmune disease in which T-cell responses against the autoantigen/epitope have been experimentally veried, the epitope (where available) as well as the experimental method by which T-cell responses were measured in the respective publication.

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example is p53, a tumour suppressor with a central role in tumour development. Here, we detected a p53-derived peptide presented on MHC I on DCs (Supplementary Data 1 and 2), which should lead to thymic deletion of p53-specic CD8 T cells. This is in line with a recent ovarian cancer study describing the lack of spontaneous p53-specic CD8 , but not CD4specic T-cell responses in healthy donors and patients with high antibody titres against p53 (ref. 43).

DiscussionIn summary, we provide here the rst characterization of the peptide repertoire presented by MHC molecules in the healthy human thymus in vivo with a focus on those epitopes presented by mDCs that are important for negative selection. Interestingly, both known autoantigens as well as tumour-associated antigens were detected. We observed a high degree of inter-individual variability between donors that can in part be explained by differences in HLA type but might also be partially due to differences in protein expression. Furthermore, mDCs and nonmDC APCs present different, only partially overlapping sets of peptides, probably due to differences both in self-protein expression as well as differences in their antigen-processing machinery13,15.

Although our data contribute to answer the long-standing question of which peptides are physiologically involved in T-cell selection in the human thymus, many issues still remain to be addressed before we fully understand the process of central tolerance. For example, analysis of ligands derived from isolated mTECs or cTECs as well as low abundance ligands presented by DCs is currently limited by available technology. For this reason, the data presented here are from mDCs only, and do not include ligands from isolated mTECs, which also contribute to central tolerance. Another question that remains to be addressed is whether the thymic ligandome changes considerably during the lifetime of an individual. For obvious reasons it will be difcult to answer this question experimentally in humans, but some previous studies suggest that variation is likely to occur. Thymic DCs and mTECs have a half-life of about 23 weeks and at least for mTECs, considerable differences in tissue-restricted antigen expression between different cells have been described44. Our and other data support the idea that thymic DCs sample the blood stream for proteins, and peripheral DCs can take up antigens, migrate to the thymus and delete the corresponding T cells8,12. Changes in the periphery would therefore be expected to impact on the peptides presented in the thymus.

Our data should pave the way for a comprehensive understanding of central tolerance and might one day help to improve the design of therapeutic interventions such as cancer vaccines. The expression of ligands derived from tumour-associated antigens on thymic APCs highlights the need for a more systematic analysis of potential cancer vaccine candidates, employing our data to preselect or exclude antigens from the growing list of candidates in future vaccine trials.

Methods

Thymic samples. Normal human thymus samples were obtained from children undergoing corrective cardiac surgery (Department of Cardiac Surgery, University of Tbingen, Germany) after parents informed consent and approval by the Institutional Review Board of the Medical Faculty of the University of Tbingen. For details of donors see Table 3.

Genotyping of thymic tissues. DNA for HLA genotyping was extracted from thymus tissue using the QIAamp DNA Blood Mini kit (Qiagen). Low-resolution HLA typing was performed by the Institute for Clinical and Experimental Transfusion Medicine (IKET), University of Tbingen, Germany.

Isolation of thymic DCs. mDCs were isolated from thymus tissue after mechanical and enzymatic disruption (collagenase (1 mg ml 1) and DNase (50 mg ml 1, both from Roche), at 37 C, two to three times for 3045 min)14. APCs were enriched from the resulting single-cell suspension by Percoll gradient centrifugation (density: 1.07 g ml 1, 30 min, 3,500 g, 4 C) and DCs were isolated by magnetic beads (CD11c-PE and anti-PE beads, Miltenyi Biotec) from the low-density fraction following the manufacturers instructions. Both the positive (DC) and negative (DC-depleted) fractions (for composition see Supplementary Table S1 and Supplementary Fig. S1) were collected. A summary of the workow isgiven in Fig. 1.

FACS Analysis. Single-cell suspensions from whole thymus, the APC-enriched fraction after Percoll, DC and DC-depleted fractions were stained with antibodies against HLADR (PerCP, 307628, 1:50), CD19 (FITC, 302205, 1:20), CD14 (APC, 301807, 1:50), CD45RA (FITC, 304105, 1:10), CD123 (PE/Cy7, 306009, 1:10), (all from Biolegend), CD45 (PB, PB986, 1:10, DAKO), BDCA-4 (PE, 130-090-533, 1:10), CD11c (PE, 130-092-411, 1:10), EpCAM (APC, 130-091-254, 1:10, from Miltenyi Biotec), CDR2 (1:50, Bruno Kyewski, DKFZ, Heidelberg; labelled with Alexa488 using the Alexa Fluor 488 Protein Labelling kit from Molecular Probes according to manufacturers instructions). mDCs were gated as CD11c , B cells were gated as CD19 , pDCs as CD45RA BDCA-4 , monocytes as CD14 , mTEC and cTEC as CD45lo-negEpCAMhi or CD45lo-negEpCAMloCDR2 , respectively.

Elution of MHC ligands. MHC-bound peptides were isolated using antibodies L234 (HLADR) and W6/32 (pan-HLA class I)45,46. We have also published a detailed step-by-step protocol of this procedure45. Frozen-cell pellets or tissue pieces were lysed with 0.6% CHAPS with protease inhibitors in PBS, stirring for 2 h at 4 C. The resulting lysate was ultrasonicated, and debris pelleted by centrifugation (4,000 g for 20 min, followed by ultracentrifugation at 40,000 r.p.m. for 1 h). The supernatant was claried by ultraltration (0.2 mm pore size, Sartolab

P plus, Sartorius) and peptideMHC complexes were isolated from the supernatant by afnity chromatography (antibodies L234 (MHC II) or W6/32 (MHC I) coupled to CNBr-Sepharose, overnight, 4 C). Bound MHCpeptide complexes were eluted with 0.1% TFA. Eluted peptides were separated from MHC proteins by centrifugation though a lter with a MW cutoff of 10 kDa and analysed by LC-MS/MS.

Identication of MHC ligands. Nanoscale liquid chromatography of MHC ligands was performed with a Waters NanoAcquity UPLC system equipped with a 75 mm 150 mm BEH C18 reversed phase column and a 2.6-ml PEEKSIL-sample

loop (SGE, Darmstadt, Germany). The aqueous mobile phase (mobile phase A) was H2O with 0.1% formic acid. The organic mobile phase (mobile phase B) was 0.1%

Table 3 | Age, gender and HLA typing of thymus tissue donors included in this study.

Tissue no. Age Gender HLA typingTT208 15 years F A*01, A*33, B*49:01, B*51,C*07, C*16

DRB1*11, DRB1*13, DRB3

TT222 6 months F A*01, A*24, B*40, B*44DRB1*04, DRB1*12, DRB3, DRB4

TT233 6 years F A*02, B*78, C*16 DRB1*13 DRB3

TT246 2 years M A*02, A*25, B*13, B*18, C*06, C*12

DRB1*07:01, DRB1*15:01 TT247 2 years M A*01, A*30, B*08, B*27

DRB1*03:01, DRB1*15:01TT251 8 months M A*26, A*66, B*35, B*38, C*04, C*12

DRB1*03, DRB1*11, DRB3

TT259 5 years M A*02, A*24, B*13, B*27, C*01, C*06DRB1*0103, DRB1*15, DRB5

TT260 11 months F A*24, A*26, B*18, B*55, C*02, C*03DRB1*11, DRB3

F, female; HLA, human leucocyte antigen; M, male.

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formic acid in acetonitrile. Samples (2.6 ml injection) were loaded onto the column in direct injection mode with 3% mobile phase B for 15 min at 400 nl min 1, followed by an additional 10 min wash (3% B) for 10 min at 300 nl min 1. Peptides were eluted from the column with a gradient from 335% mobile phase B over 120 min at 300 nl min 1 followed by a 20-min rinse of 80% mobile phase B. The column was immediately re-equilibrated at initial conditions (3% mobile phase B)

for 20 min. [Glu1] brinopeptide was used as lockmass at 500 fmol ml 1. Lockmass solution was delivered from the auxiliary pump of the NanoAcquity system at 400 nl min 1 to the reference sprayer of the NanoLockSpray source. MS/MS analysis of peptides was performed by using a Waters Q-Tof Premier mass spectrometer operated in V-mode with typical resolving power of R 10.000 in data-

dependent acquisition mode, selecting the three most intense precursor ions for fractionation in the collision cell. Fragmentation of the parent ions was achieved by collision with argon atoms. Collision energy was varied from 1535 eV dependent on precursor ion mass and charge. The integration time for the TOF analyser was 1 s with an interscan delay of 0.1 s.

Interpretation of MS/MS fragmentation spectra was carried out manually using MassLynx 4.0 software47. For manual peptide identication, sequence tag searches were done using Mascot 2.0 software (peptide and MS/MS tolerance, 0.2 Da; National Center for Biotechnology Information non-redundant (NCBInr) database updated monthly and restricted for search to human entries), and relevant hits were assessed manually and not by evaluation of the Mascot score. Criteria for manual identication were: a reasonable interpretation of at least 95% of all fragment peaks, complete sequence coverage with MS/MS fragments, and signal intensities of fragment ion peaks that match breakage probabilities of the respective sequence.

The obtained peptide sequences were assigned to source proteins using the BLAST algorithm and further bioinformatics analysis48,49 was performed. The MHC class I and MHC class II ligands in different thymic cell preparations were assigned to subcellular compartments or functional categories according to Gene Ontology annotations (http://www.geneontology.org

Web End =www.geneontology.org). In cases where this search yielded no or incomplete information, annotation was performed manually basedon information from WoLF PSORT48 (wolfpsort.org), SubLoc49 (http://www.bioinfo.tsinghua.edu.cn/SubLoc/

Web End =www.bioinfo.tsinghua.edu.cn/SubLoc/), SwissProt and published literature. Source proteins were then grouped to the classical MHC I (cytosolic, nuclear) or MHC II pathway (extracellular, vesicular and cell membrane), the mitochondrial or ER compartment. To facilitate interpretation of data for biological function, related subcategories were grouped together, for example, signalling, cellcell signalling and signal transduction were grouped together as signalling. All peptides were assigned to a specic HLA allotype by taking the HLA type of the patient into consideration as well as known binding motifs of different allotypes50. Peptides were assigned to the presenting HLA molecule by comparison of their anchor amino acids with known HLA allele-specic peptide motifs. In order to carry out an automated, reproducible assignment to the correct HLA allotype, all peptide sequences were subjected to epitope prediction using the database SYFPEITHI (ref. 50) (http://www.syfpeithi.de

Web End =www.syfpeithi.de), evaluating the preferential binding to one of the HLA allotypes dened by HLA typing. SYFPEITHI calculates scores according to the preferred amino acids in each sequence position. Peptides receiving more than the half-maximal score for a given HLA molecule were assigned to the respective molecule. It has to be noted that ligands of different HLA molecules may achieve different maximal scores. The maximal scores are 44 for HLA-A*03, 44 for HLA-A*26, 29 for HLA-A*31, 37 for HLA-A*32, 33 for HLA-B*07, 30 for HLA-B*38 and 38 for HLA-B*51. If epitope prediction was not possible by the web version of SYFPEITHI, an in-house version with extended options was used. This was the case for predictions of HLA-A*31 and HLA-A*32 ligands and for rarely occurring peptide lengths (8 mer, 11 mer) of other HLA allotypes.

Reproducibility analysis. For method validation and reproducibility analysis, MHC I peptides from PBMC of healthy volunteers were isolated and technical replicates performed. For statistical analysis of the intra-sample (that is, technical) reproducibility, the overlaps of every conceivable pair of technical replicates were calculated for one randomly selected sample. For the assessment of inter-individual variability we analysed the overlaps of HLA ligand source proteomes of ve healthy volunteer PBMC samples. We performed comprehensive overlap analysis of all ve samples. For the statistical analysis of inter-individual variability, paired overlap analyses of all 10 possible combinations of source proteomes were performed. The two populations (technical replicate overlaps versus inter-individual overlaps) were compared using a two-sided, unpaired Students t-test not assuming equal standard deviations (Supplementary Fig. S3).

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Acknowledgements

We thank the young patients and their parents for donating thymus samples, Professor Gerhard Ziemer, Professor Hermann Aebert, Dr Volker Steger and Dr Wilke Schneider (Tbingen, Germany) for providing us with the specimens, Professor Dorothee Wernet (Tbingen, Germany) for HLA typing, Simone Pschel for technical assistance, Daniel Kowalewski (Tbingen, Germany) for methodological support and Professor Bruno Kyewski (Heidelberg, Germany) for the CDR2 antibody. This work was supported by the Deutsche Forschungsgemeinschaft (SFB 685, Graduate School 794, INST 371/23-1 FUGG), the Hertie Foundation, the BMBF (e:Bio Express2Present, 0316179C) and the Forschungszentrum Immunologie of the Johannes Gutenberg University Mainz.

Author contributions

C.S. designed the project with support from E.T. and coordinated research. C.S., E.A. and N.H. designed and performed experiments and de novo sequencing data analysis with P.K., S.T., I.R. and M.G. providing assistance. LC-MS/MS and bioinformatic analysis was carried out by S.T., S.S. provided valuable help with peptide HLA assignment and methodology, E.T., S.T. and H.S. contributed scientic discussion. A.M. coordinated thymi sample collection and contributed to the direction of the study. E.A. and C.S. evaluated the data and wrote the manuscript, which was discussed, critically edited and approved by all co-authors. C.S. and A.M. share senior authorship.

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How to cite this article: Adamopoulou, E. et al. Exploring the MHC-peptide matrix of central tolerance in the human thymus. Nat. Commun. 4:2039 doi: 10.1038/ncomms3039 (2013).

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