Iñaki Alvarez

Dissection of the HLA-DR4 Peptide Repertoire in Endocrine Epithelial Cells: Strong Influence of Invariant Chain and HLA-DM Expression on the Nature of Ligands

Class II MHC (MHC II) expression is restricted to professional APCs and thymic epithelium but it also occurs in the epithelial cells of autoimmune organs which are the unique targets of the CD4 autoreactive T cells in endocrine autoimmune diseases. This specificity is presumably conditioned by an epithelium-specific peptide repertoire associated to MHC II at the cell surface. MHC II expression and function is dependent on the action of two main chaperones, invariant chain (Ii) and DM, whose expression is coregulated with MHC II. However, there is limited information about the in vivo expression levels of these molecules and uncoordinated expression has been demonstrated in class II-positive epithelial cells that may influence the MHC-associated peptide repertoires and the outcome of the autoimmune response. We have examined the pool of peptides associated to DR4 molecules expressed by a neuroendocrine epithelial cell and the consequences of Ii and DM coexpression. The RINm5F rat insulinoma cell line was transfected with HLA-DRB1*0401, Ii, and DM molecules in four different combinations: RIN-DR4, -DR4Ii, -DR4DM, and -DR4IiDM. The analysis of the peptide repertoire and the identification of the DR4 naturally processed ligands in each transfected cell were achieved by mass spectrometry. The results demonstrate that 1) the expression of Ii and DM affected the DR4 peptide repertoires by producing important variations in their content and in the origin of peptides; 2) these restrictions affected the stability and sequence of the peptides of each repertoire; and 3) Ii and DM had both independent and coordinate effects on these repertoires.

Thyroglobulin Peptides Associate In Vivo to HLA-DR in Autoimmune Thyroid Glands

Endocrine epithelial cells, targets of the autoimmune response in thyroid and other organ-specific autoimmune diseases, express HLA class II (HLA-II) molecules that are presumably involved in the maintenance and regulation of the in situ autoimmune response. HLA-II molecules thus expressed by thyroid cells have the “compact” conformation and are therefore expected to stably bind autologous peptides. Using a new approach to study in situ T cell responses without the characterization of self-reactive T cells and their specificity, we have identified natural HLA-DR-associated peptides in autoimmune organs that will allow finding peptide-specific T cells in situ. This study reports a first analysis of HLA-DR natural ligands from ex vivo Graves’ disease-affected thyroid tissue. Using mass spectrometry, we identified 162 autologous peptides from HLA-DR-expressing cells, including thyroid follicular cells, with some corresponding to predominant molecules of the thyroid colloid. Most interestingly, eight of the peptides were derived from a major autoantigen, thyroglobulin. In vitro binding identified HLA-DR3 as the allele to which one of these peptides likely associates in vivo. Computer modeling and bioinformatics analysis suggested other HLA-DR alleles for binding of other thyroglobulin peptides. Our data demonstrate that although the HLA-DR-associated peptide pool in autoimmune tissue mostly belongs to abundant ubiquitous proteins, peptides from autoantigens are also associated to HLA-DR in vivo and therefore may well be involved in the maintenance and the regulation of the autoimmune response.

The rheumatoid arthritis–associated allele HLA–DR10 (DRB1*1001) shares part of its repertoire with HLA–DR1 (DRB1*0101) and HLA–DR4 (DRB*0401)

OBJECTIVE To identify the peptide anchor motif for the rheumatoid arthritis (RA)-related HLA allele, DR10, and find shared natural ligands or sequence similarities with the other disease-associated alleles, DR1 and DR4. METHODS The HLA-DR10-associated peptides were purified, and a proportion of these natural ligands were de novo sequenced by mass spectrometry. Based on crystallographic structures, the complexes formed by peptide influenza virus hemagglutinin HA306-318 with DR1, DR4, and DR10 were modeled, and binding scores were obtained. RESULTS A total of 238 peptides were sequenced, and the anchor motif of the HLA-DR10 peptide repertoire was defined. A large proportion of the DR10-associated peptides had the structural features to bind DR1 and DR4 but were theoretical nonbinders to the negatively associated alleles DR15 and DR7. Among the sequenced ligands, 10 had been reported as ligands to other RA-associated alleles. Modeling data showed that peptide HA306-318 can bind DR1, DR4, and DR10 with similar affinities. CONCLUSION The data show the presence of common peptides in the repertoires of RA-associated HLA alleles. The combination of the shared epitope present in DR1, DR4, and DR10 together with common putative arthritogenic peptide(s) could influence disease onset or outcome.

Composition of the HLA-DR-associated human thymus peptidome.

Major histocompatibility complex class II (MHC‐II) molecules bind to and display antigenic peptides on the surface of antigen‐presenting cells (APCs). In the absence of infection, MHC‐II molecules on APCs present self‐peptides and interact with CD4+ T cells to maintain tolerance and homeostasis. In the thymus, self‐peptides bind to MHC‐II molecules expressed by defined populations of APCs specialised for the different steps of T‐cell selection. Cortical epithelial cells present peptides for positive selection, whereas medullary epithelial cells and dendritic cells are responsible for peptide presentation for negative selection. However, few data are available on the peptides presented by MHC molecules in the thymus. Here, we apply mass spectrometry to analyse and identify MHC‐II‐associated peptides from five fresh human thymus samples. The data show a diverse self‐peptide repertoire, mostly consisting of predicted MHC‐II high binders. Despite technical limitations preventing single cell population analyses of peptides, these data constitute the first direct assessment of the HLA‐II‐bound peptidome and provide insight into how this peptidome is generated and how it drives T‐cell repertoire formation.

The Repertoires of Peptides Presented by MHC-II in the Thymus and in Peripheral Tissue: A Clue for Autoimmunity?

T-cell tolerance to self-antigens is established in the thymus through the recognition by developing thymocytes of self-peptide-MHC complexes and induced and maintained in the periphery. Efficient negative selection of auto-reactive T cells in the thymus is dependent on the in situ expression of both ubiquitous and tissue-restricted self-antigens and on the presentation of derived peptides. Weak or inadequate intrathymic expression of self-antigens increases the risk to generate an autoimmune-prone T-cell repertoire. Indeed, even small changes of self-antigen expression in the thymus affect negative selection and increase the predisposition to autoimmunity. Together with other mechanisms, tolerance is maintained in the peripheral lymphoid organs via the recognition by mature T cells of a similar set of self-peptides in homeostatic conditions. However, non-lymphoid peripheral tissue, where organ-specific autoimmunity takes place, often have differential functional processes that may lead to the generation of epitopes that are absent or non-presented in the thymus. These putative differences between peptides presented by MHC molecules in the thymus and in peripheral tissues might be a major key to the initiation and maintenance of autoimmune conditions.

The peptide-binding motif of HLA-DR8 shares important structural features with other type 1 diabetes-associated alleles

The objective of this study was to characterize the peptide-binding motif of the major histocompatibility complex (MHC) class II HLA-DR8 molecule included in the type 1 diabetes-associated haplotype DRB1*0801-DQA1*0401/DQB1*0402 (DR8-DQ4), and compare it with that of other diabetes-associated MHC class II alleles; DR8-bound peptides were eluted from an HLA-DR homozygous lymphoblastoid cell line. The repertoire was characterized by peptide sequencing using a LTQ ion trap mass spectrometer coupled to a multidimensional liquid chromatography system. After validation of the spectra identification, the definition of the HLA-DR8 peptide-binding motif was achieved from the analysis of 486 natural ligands, based on serial alignments of all possible HLA-DR-binding cores. The DR8 motif showed a strong similarity with the peptide-binding motifs of other MHC class II diabetes-associated alleles, HLA-DQ8 and H-2 I-Ag7. Similar to HLA-DQ8 and H-2 I-Ag7, HLA-DR8 preferentially binds peptides with an acidic residue at position P9 of the binding core, indicating that DR8 is the susceptibility component of the DR8-DQ4 haplotype. Indeed, some DR8 peptides were identical to peptides previously identified as DQ8- or I-Ag7 ligands, and several diabetes-specific peptides associated with DQ8 or I-Ag7 could theoretically bind to HLA-DR8. These data further strengthen the association of HLA-DR8 with type I diabetes.

Central T cell tolerance: Identification of tissue-restricted autoantigens in the thymus HLA-DR peptidome

Promiscuous gene expression (pGE) of tissue-restricted self-antigens (TRA) in medullary thymic epithelial cells (mTECs) is in part driven by the Autoimmune Regulator gene (AIRE) and essential for self-tolerance. The link between AIRE functional mutations and multi-organ autoimmunity in human and mouse supports the role of pGE. Deep sequencing of the transcriptome revealed that mouse mTECs potentially transcribe an unprecedented range of >90% of all genes. Yet, it remains unclear to which extent these low-level transcripts are actually translated into proteins, processed and presented by thymic APCs to induce tolerance. To address this, we analyzed the HLA-DR-associated thymus peptidome. Within a large panel of peptides from abundant proteins, two TRA peptides were identified: prostate-specific semenogelin-1 (an autoantigen in autoimmune chronic prostatitis/chronic pelvic pain syndrome) and central nervous system-specific contactin-2 (an autoantigen in multiple sclerosis). Thymus expression of both genes was restricted to mTECs. SEMG1 expression was confined to mature HLA-DR(hi) mTECs of male and female donors and was AIRE-dependent, whereas CNTN2 was apparently AIRE-independent and was expressed by both populations of mTECs. Our findings establish a link between pGE, MHC-II peptide presentation and autoimmunity for bona fide human TRAs.

Peptides presented by HLA class I molecules in the human thymus

UNLABELLED The thymus is the organ in which T lymphocytes mature. Thymocytes undergo exhaustive selection processes that require interactions between the TCRs and peptide-HLA complexes on thymus antigen-presenting cells. The thymic peptide repertoire associated with HLA molecules must mirror the peptidome that mature T cells will encounter at the periphery, including peptides that arise from tissue-restricted antigens. The transcriptome of specific thymus cell populations has been widely studied, but there are no data on the HLA-I peptidome of the human thymus. Here, we describe the HLA-I-bound peptide repertoire from thymus samples, showing that it is mostly composed of high-affinity ligands from cytosolic and nuclear proteins. Several proteins generated more than one peptide, and some redundant peptides were found in different samples, suggesting the existence of antigen immunodominance during the processes that lead to central tolerance. Three HLA-I ligands were found to be derived from proteins expressed by stromal cells, including one from the protein TBATA (or SPATIAL), which is present in the thymus, brain and testis. The expression of TBATA in medullary thymic epithelial cells has been reported to be AIRE dependent. Thus, this report describes the first identification of a thymus HLA-I natural ligand derived from an AIRE-dependent protein with restricted tissue expression. BIOLOGICAL SIGNIFICANCE We present the first description of the HLA-I-bound peptide repertoire from ex vivo thymus samples. This repertoire is composed of standard ligands from cytosolic and nuclear proteins. Some peptides seem to be dominantly presented to thymocytes in the thymus. Most importantly, some HLA-I associated ligands derived from proteins expressed by stromal cells, including one peptide, restricted by HLA-A*31:01, arising from an AIRE-dependent protein with restricted tissue expression.

Analysis of the HLA class I associated peptide repertoire in a hepatocellular carcinoma cell line reveals tumor-specific peptides as putative targets for immunotherapy.

HLA class I molecules present peptides on the cell surface to CD8+ T cells. The repertoire of peptides that associate to class I molecules represents the cellular proteome. Therefore, cells expressing different proteomes could generate different class I‐associated peptide repertoires. A large number of peptides have been sequenced from HLA class I alleles, mostly from lymphoid cells. On the other hand, T cell immunotherapy is a goal in the fight against cancer, but the identification of T cell epitopes is a laborious task. Proteomic techniques allow the definition of putative T cell epitopes by the identification of HLA natural ligands in tumor cells. In this study, we have compared the HLA class I‐associated peptide repertoire from the hepatocellular carcinoma (HCC) cell line SK‐Hep‐1 with that previously described from lymphoid cells. The analysis of the peptide pool confirmed that, as expected, the peptides from SK‐Hep‐1 derive from proteins localized in the same compartments as in lymphoid cells. Within this pool, we have identified 12 HLA class I peptides derived from HCC‐related proteins. This confirms that tumor cell lines could be a good source of tumor associated antigens to be used, together with MS, to define putative epitopes for cytotoxic T cells from cancer patients.

Comparative Analysis of the Endogenous Peptidomes Displayed by HLA-B*27 and Mamu-B*08: Two MHC Class I Alleles Associated with Elite Control of HIV/SIV Infection

Indian rhesus macaques are arguably the most reliable animal models in AIDS research. In this species the MHC class I allele Mamu-B*08, among others, is associated with elite control of SIV replication. A similar scenario is observed in humans where the expression of HLA-B*27 or HLA-B*57 has been linked to slow or no progression to AIDS after HIV infection. Despite having large differences in their primary structure, it has been reported that HLA-B*27 and Mamu-B*08 display peptides with sequence similarity. To fine-map the Mamu-B*08 binding motif and assess its similarities with that of HLA-B*27, we affinity purified the peptidomes bound to these MHC class I molecules and analyzed them by LC-MS, identifying several thousands of endogenous ligands. Sequence analysis of both sets of peptides revealed a degree of similarity in their binding motifs, especially at peptide position 2 (P2), where arginine was present in the vast majority of ligands of both allotypes. In addition, several differences emerged from this analysis: (i) ligands displayed by Mamu-B*08 tended to be shorter and to have lower molecular weight, (ii) Mamu-B*08 showed a higher preference for glutamine at P2 as a suboptimal binding motif, and (iii) the second major anchor position, found at PΩ, was much more restrictive in Mamu-B*08. In this regard, HLA-B*27 bound efficiently peptides with aliphatic, aromatic (including tyrosine), and basic C-terminal residues while Mamu-B*08 preferred peptides with leucine and phenylalanine in this position. Finally, in silico estimations of binding efficiency and competitive binding assays to Mamu-B*08 of several selected peptides revealed a good correlation between the characterized anchor motif and binding affinity. These results deepen our understanding of the molecular basis of the presentation of peptides by Mamu-B*08 and can contribute to the detection of novel SIV epitopes restricted by this allotype.