Javier Collado

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.

Increased Apoptosis after Autoimmune Regulator Expression in Epithelial Cells Revealed by a Combined Quantitative Proteomics Approach

Autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED) is a rare autosomal recessive autoimmune disease, affecting many endocrine tissues. APECED is associated to the lack of function of a single gene called AutoImmune REgulator (AIRE). Aire knockout mice develop various autoimmune disorders affecting different organs, indicating that Aire is a key gene in the control of organ-specific autoimmune diseases. AIRE is mainly expressed by medullary thymic epithelial cells (mTECs), and its absence results in the loss of tolerance against tissue restricted antigens (TRAs). Aire induces the transcription of genes encoding for TRAs in mTECs. In this report, the analysis of AIREs effect on the cellular proteome was approached by the combination of two quantitative proteomics techniques, 2D-DIGE and ICPL, using an AIRE-transfected and nontransfected epithelial cell line. The results showed increased levels of several chaperones, (HSC70, HSP27 and tubulin-specific chaperone A) in AIRE-expressing cells, while various cytoskeleton interacting proteins, that is, transgelin, caldesmon, tropomyosin alpha-1 chain, myosin regulatory light polypeptide 9, and myosin-9, were decreased. Furthermore, some apoptosis-related proteins were differentially expressed. Data were confirmed by Western blot and flow cytometry analysis. Apoptosis assays with annexin V and etoposide demonstrated that AIRE-positive cells suffer more spontaneous apoptosis and are less resistant to apoptosis induction.

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.

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.

BA71ΔCD2: A new recombinant live attenuated African swine fever virus with cross-protective capabilities

ABSTRACT African swine fever is a highly contagious viral disease of mandatory declaration to the World Organization for Animal Health (OIE). The lack of available vaccines makes its control difficult; thus, African swine fever virus (ASFV) represents a major threat to the swine industry. Inactivated vaccines do not confer solid protection against ASFV. Conversely, live attenuated viruses (LAV), either naturally isolated or obtained by genetic manipulation, have demonstrated reliable protection against homologous ASFV strains, although little or no protection has been demonstrated against heterologous viruses. Safety concerns are a major issue for the use of ASFV attenuated vaccine candidates and have hampered their implementation in the field so far. While trying to develop safer and efficient ASFV vaccines, we found that the deletion of the viral CD2v (EP402R) gene highly attenuated the virulent BA71 strain in vivo. Inoculation of pigs with the deletion mutant virus BA71ΔCD2 conferred protection not only against lethal challenge with the parental BA71 but also against the heterologous E75 (both genotype I strains). The protection induced was dose dependent, and the cross-protection observed in vivo correlated with the ability of BA71ΔCD2 to induce specific CD8+ T cells capable of recognizing both BA71 and E75 viruses in vitro. Interestingly, 100% of the pigs immunized with BA71ΔCD2 also survived lethal challenge with Georgia 2007/1, the genotype II strain of ASFV currently circulating in continental Europe. These results open new avenues to design ASFV cross-protective vaccines, essential to fight ASFV in areas where the virus is endemic and where multiple viruses are circulating. IMPORTANCE African swine fever virus (ASFV) remains enzootic in most countries of Sub-Saharan Africa, today representing a major threat for the development of their swine industry. The uncontrolled presence of ASFV has favored its periodic exportation to other countries, the last event being in Georgia in 2007. Since then, ASFV has spread toward neighboring countries, reaching the European Unions east border in 2014. The lack of available vaccines against ASFV makes its control difficult; so far, only live attenuated viruses have demonstrated solid protection against homologous experimental challenges, but they have failed at inducing solid cross-protective immunity against heterologous viruses. Here we describe a new LAV candidate with unique cross-protective abilities: BA71ΔCD2. Inoculation of BA71ΔCD2 protected pigs not only against experimental challenge with BA71, the virulent parental strain, but also against heterologous viruses, including Georgia 2007/1, the genotype II strain of ASFV currently circulating in Eastern Europe.

Unraveling features of the natural MHC class II peptidome of skin-migrated dendritic cells

Dendritic cells (DCs) migrating from peripheral tissues at steady state are considered the most efficient antigen-presenting cells (APCs) involved in the induction of peripheral T-cell tolerance via self-antigen presentation on MHC class II molecules. However, difficulties in obtaining sufficient numbers of such DCs have precluded previous analyses of their natural MHC class II peptidome in laboratory animals or humans. Here, we overcome this difficulty by collecting the large quantities of sheep DCs that migrate from the skin via the afferent lymphatics at steady state to the draining lymph node. We compared the repertoire of MHC class II-bound peptides from afferent lymph DCs with autologous APCs derived from peripheral blood. A large fraction of the MHC class II peptidome from skin DCs was derived from membrane-recycling proteins (59%) and from proteins of the antigen presentation machinery (50%), whereas these types of peptides constituted a more limited fraction in blood APCs (21 and 11%, respectively). One sheep cytokeratin peptide was identified in the skin DC peptidome indicating active processing of epithelium-derived antigens. Conversely, peptides derived from cytosolic and soluble antigens of the extracellular milieu were more represented in blood APCs than skin DCs. The biased peptidome of skin-migrated DCs indicates that these cells express a peptide repertoire for the generation of self-reactive and/or regulatory T cells mainly directed toward DC molecules from internal and external membranes and to a lesser extent toward antigens of the extracellular milieu, including some tissue-specific peptides.