Proteomic Analysis of Human iPSC-Derived Neural Stem Cells and Motor Neurons Identifies Proteasome Structural Alterations.

BACKGROUND: Proteins targeted by the ubiquitin proteasome system (UPS) are identified for degradation by the proteasome, which has been implicated in the development of neurodegenerative diseases. Major histocompatibility complex (MHC) molecules present peptides broken down by the proteasome and are involved in neuronal plasticity, regulating the synapse number and axon regeneration in the central or peripheral nervous system during development and in brain diseases. The mechanisms governing these effects are mostly unknown, but evidence from different compartments of the cerebral cortex indicates the presence of immune-like MHC receptors in the central nervous system. METHODS: We used human induced pluripotent stem cells (iPSCs) differentiated into neural stem cells and then into motor neurons as a developmental model to better understand the structure of the proteasome in developing motor neurons. We performed a proteomic analysis of starting human skin fibroblasts, their matching iPSCs, differentiated neural stem cells and motor neurons that highlighted significant differences in the constitutive proteasome and immunoproteasome subunits during development toward motor neurons from iPSCs. RESULTS: The proteomic analysis showed that the catalytic proteasome subunits expressed in fibroblasts differed from those in the neural stem cells and motor neurons. Western blot analysis confirmed the proteomic data, particularly the decreased expression of the ß5i (PSMB8) subunit immunoproteasome in MNs compared to HFFs and increased ß5 (PSMB5) in MNs compared to HFFs. CONCLUSION: The constitutive proteasome subunits are upregulated in iPSCs and NSCs from HFFs. Immunoproteasome subunit ß5i expression is higher in MNs than NSCs; however, overall, there is more of a constitutive proteasome structure in MNs when comparing HFFs to MNs. The proteasome composition may have implications for motor neuron development and neurodevelopmental diseases that warrant further investigation.

Analysis of the different subpeptidomes presented by the HLA class I molecules of the B7 supertype.

MHC-I molecules of the HLA-B7 supertype preferentially bind peptides with proline at position 2. HLA-B*51:01 and B*51:08 present two predominant subpeptidomes, one with Pro2 and hydrophobic residues at P1, and another with Ala2 and Asp enriched at position 1. Here, we present a meta-analysis of the peptidomes presented by molecules of the B7 supertype to investigate the presence of subpeptidomes across different allotypes. Several allotypes presented subpeptidomes differing in the presence of Pro or another residue at P2. The Ala2 subpeptidomes preferred Asp1 except in HLA-B*54:01, where ligands with Ala2 contained Glu1. Sequence alignment and the analysis of crystal structures allowed us to propose positions 45 and 67 of the MHC heavy chain as relevant for the presence of subpeptidomes. Deciphering the principles behind the presence of subpeptidomes could improve our understanding of antigen presentation in other MHC-I molecules. Running title: HLA-B7 supertype subpeptidomes.

Purification of HLA Immunopeptidomes from Human Thymus.

Mass spectrometry has become an essential technique for the analysis of peptide repertoires presented by MHC molecules to T lymphocytes. Years ago, analyses of MHC peptidomes were performed using a great number of cells, and cell lines were chosen as the main peptide source. Mass spectrometry devices have been improved in terms of sensitivity and resolution, making feasible the analysis of samples with relatively small amounts of cells. Thus, analyses of MHC peptide repertoires from different tissue samples are now available. Here, I describe a protocol to process human thymus samples to purify HLA class I- or HLA-DR-associated peptidomes. For that, cells are lysed using a nonionic detergent together with a mechanical cell rupture. Immunopeptidomes are purified by immunoaffinity chromatography. The peptide pool is fractionated by ionic chromatography. Finally, peptide fragmentation and identification are conducted by LC-MS/MS and the use of MASCOT search engine.

Global Proteomic and Methylome Analysis in Human Induced Pluripotent Stem Cells Reveals Overexpression of a Human TLR3 Affecting Proper Innate Immune Response Signaling.

When considering the clinical applications of autologous cell replacement therapy of human induced pluripotent stem cells (iPSC)-derived cells, there is a clear need to better understand what the immune response will be before we embark on extensive clinical trials to treat or model human disease. We performed a detailed assessment comparing human fibroblast cell lines (termed F1) reprogrammed into human iPSC and subsequently differentiated back to fibroblast cells (termed F2) or other human iPSC-derived cells including neural stem cells (NSC) made from either retroviral, episomal, or synthetic mRNA cell reprogramming methods. Global proteomic analysis reveals the main differences in signal transduction and immune cell protein expression between F1 and F2 cells, implicating wild type (WT) toll like receptor protein 3 (TLR3). Furthermore, global methylome analysis identified an isoform of the human TLR3 gene that is not epigenetically reset correctly upon differentiation to F2 cells resulting in a hypomethylated transcription start site in the TLR3 isoform promoter and overexpression in most human iPSC-derived cells not seen in normal human tissue. The human TLR3 isoform in human iPSC-NSC functions to suppress NF-KB p65 signaling pathway in response to virus (Poly IC), suggesting suppressed immunity of iPSC-derived cells to viral infection. The sustained WT TLR3 and TLR3 isoform overexpression is central to understanding the altered immunogenicity of human iPSC-derived cells calling for screening of human iPSC-derived cells for TLR3 expression levels before applications. Stem Cells 2019;37:476-488.

PRBAM: a new tool to analyze the MHC class I and HLA-DR anchor motifs.

Major histocompatibility complex (MHC) genes are highly polymorphic, which makes each MHC molecule different regarding their peptide repertoire, so they can bind and present to T lymphocytes. The increasing importance of immunopeptidomics and its use in personalized medicine in different fields such as oncology or autoimmunity demand the correct analysis of the peptide repertoires bound to human leukocyte antigen type 1 (HLA-I) and HLA-II molecules. Purification of the peptide pool by affinity chromatography and individual peptide sequencing using mass spectrometry techniques is the standard protocol to define the binding motifs of the different MHC-I and MHC-II molecules. The identification of MHC-I binding motifs is relatively simple, but it is more complicated for MHC-II. There are some programs that identify the anchor motifs of MHC-II molecules. However, these programs do not identify the anchor motif correctly for some HLA-II molecules and some anchor motifs have been deduced using subjective interpretation of the data. Here, we present a new software, called PRBAM (Peptide Repertoire-Based Anchor Motif) that uses a new algorithm based on the peptide-MHC interactions and, using peptide lists obtained by mass spectrometry sequencing, identifies the binding motif of MHC-I and HLA-DR molecules. PRBAM has an easy-to-use interface, and the results are presented in graphics, tables and peptide lists. Finally, the fact that PRBAM uses a new algorithm makes it complementary to other existing programs.

Human Leukocyte Antigen (HLA)-DRB1*15:01 and HLA-DRB5*01:01 Present Complementary Peptide Repertoires.

Human leukocyte antigen (HLA)-DR15 is a haplotype associated with multiple sclerosis. It contains the two DRB* genes DRB1*1501 (DR2b) and DRB5*0101 (DR2a). The reported anchor motif of the corresponding HLA-DR molecules was determined in 1994 based on a small number of peptide ligands and binding assays. DR2a could display a set of peptides complementary to that presented by DR2b or, alternatively, a similar peptide repertoire but recognized in a different manner by T cells. It is known that DR2a and DR2b share some peptide ligands, although the degree of similarity of their associated peptidomes remains unclear. In addition, the contribution of each molecule to the global peptide repertoire presented by the HLA-DR15 haplotype has not been evaluated. We used mass spectrometry to analyze the peptide pools bound to DR2a and DR2b, identifying 169 and 555 unique peptide ligands of DR2a and DR2b, respectively. The analysis of these sets of peptides allowed the refinement of the corresponding binding motifs revealing novel anchor residues that had been overlooked in previous analyses. Moreover, the number of shared ligands between both molecules was low, indicating that DR2a and DR2b present complementary peptide repertoires to T cells. Finally, our analysis suggests that, quantitatively, both molecules contribute to the peptide repertoire presented by cells expressing the HLA-DR15 haplotype.

A Comparative Analysis of the Peptide Repertoires of HLA-DR Molecules Differentially Associated With Rheumatoid Arthritis.

OBJECTIVE: To evaluate similarity of the peptide repertoires bound to HLA-DR molecules that are differentially associated with rheumatoid arthritis (RA), and to define structural features of the shared peptides. METHODS: Peptide pools bound to HLA-DRB1*01:01, HLA-DRB1*04:01, and HLA-DRB1*10:01 (RA associated) and those bound to HLA-DRB1*15:01 (non-RA-associated) were purified and analyzed by liquid chromatography (LC) matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MS) and LC-ion-trap MS. Peptide pools from each allotype were compared in terms of size, protein origin, composition, and affinity (both theoretical and experimental with some peptides). Finally, 1 peptide sequenced from DR1, DR4, and DR10, but not from DR15, was modeled in complex with all 4 HLA-DRB1 molecules and HLA-DRB5*01:01. RESULTS: A total of 6,309 masses and 962 unique peptide sequences were compared. DR10 shared 29 peptides with DR1, 9 with DR4, and 1 with DR15; DR1 shared 6 peptides with DR4 and 9 with DR15; and DR4 and DR15 shared 4 peptides. The direct identification of peptide ligands indicated that DR1 and DR10 were the most similar molecules regarding the peptides that they could share. The peptides common to these molecules contained a high proportion of Leu at P4 and basic residues at P8 binding core positions. CONCLUSION: The degree of overlap between peptide repertoires associated with different HLA-DR molecules is low. The repertoires associated with DR1 and DR10 have the highest similarity among the molecules analyzed (∼10% overlap). Among the peptides shared between DR1 and DR10, a high proportion contained Leu(4) and basic residues at the P8 position of the binding core.

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.

Factors Associated with the Selection of First-line Bevacizumab plus Chemotherapy and Clinical Response in HER2-negative Metastatic Breast Cancer: ONCOSUR AVALOX Study.

AIM: To evaluate factors associated with the selection of first-line bevacizumab plus chemotherapy and clinical response in HER2-negative metastatic breast cancer (MBC) in clinical practice in Spain. PATIENTS AND METHODS: All consecutive adult female patients with HER2-negative MBC who had received first-line bevacizumab plus chemotherapy for at least 3 months were enrolled in the present study. RESULTS: A total of 292 evaluable patients were included; 25% had triple-negative breast cancer (TNBC) and 75% had hormone receptor-positive breast cancer (HRPBC). Nearly 40% of patients had ≥3 metastatic sites, mainly located in the bone (48%) and liver (40%). Bevacizumab was mostly combined with paclitaxel (67.1%). ER-positive tumors were only identified as an independent factor associated with the choice of treatment (odds ratio (OR): 0.538; p=0.02). The overall response rate (ORR) was 63.7% (TNBC: 57.5%; HRPBC: 65.9%). Patients aged 36-50 years (OR: 3.03; p=0.028) and those with metastases at sites other than the bone (OR: 0.38; p=0.001) and ≥3 metastatic sites (OR: 1.41; p=0.018) were more likely to achieve objective responses. CONCLUSION: First-line bevacizumab plus chemotherapy, mainly paclitaxel, is an effective and well-tolerated treatment option for HER2-negative MBC, particularly in more aggressive disease.

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.

The Power and the Promise of Cell Reprogramming: Personalized Autologous Body Organ and Cell Transplantation.

Reprogramming somatic cells to induced pluripotent stem cells (iPSCs) or direct reprogramming to desired cell types are powerful and new in vitro methods for the study of human disease, cell replacement therapy, and drug development. Both methods to reprogram cells are unconstrained by the ethical and social questions raised by embryonic stem cells. iPSC technology promises to enable personalized autologous cell therapy and has the potential to revolutionize cell replacement therapy and regenerative medicine. Potential applications of iPSC technology are rapidly increasing in ambition from discrete cell replacement applications to the iPSC assisted bioengineering of body organs for personalized autologous body organ transplant. Recent work has demonstrated that the generation of organs from iPSCs is a future possibility. The development of embryonic-like organ structures bioengineered from iPSCs has been achieved, such as an early brain structure (cerebral organoids), bone, optic vesicle-like structures (eye), cardiac muscle tissue (heart), primitive pancreas islet cells, a tooth-like structure (teeth), and functional liver buds (liver). Thus, iPSC technology offers, in the future, the powerful and unique possibility to make body organs for transplantation removing the need for organ donation and immune suppressing drugs. Whilst it is clear that iPSCs are rapidly becoming the lead cell type for research into cell replacement therapy and body organ transplantation strategies in humans, it is not known whether (1) such transplants will stimulate host immune responses; and (2) whether this technology will be capable of the bioengineering of a complete and fully functional human organ. This review will not focus on reprogramming to iPSCs, of which a plethora of reviews can be found, but instead focus on the latest developments in direct reprogramming of cells, the bioengineering of body organs from iPSCs, and an analysis of the immune response induced by iPSC-derived cells and tissues.

Peptides presented by HLA class I molecules in the human thymus.

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.

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.

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 AIRE's 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.

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.

Peptides presented in vivo by HLA-DR in thyroid autoimmunity.

The association of the major histocompatibility complex (MHC) genes with autoimmune diseases together with the ectopic expression of class II molecules by epithelial cells of the target tissue gives to these molecules a central role in the pathogenesis of the disease, in its regulation and in the persistence of the immune response in situ. HLA-DR molecules expressed by thyroid follicular cells in thyroid autoimmune diseases are compact molecules stably associated with peptides. The nature of these peptides is of vital importance in the understanding of the disease, since these MHC-II-peptide complexes are going to be recognized by both effector and regulatory T cells in situ. In this chapter, we review the current state of the analysis of naturally processed peptides presented by MHC class II molecules in the context of autoimmunity and we discuss our data of natural HLA-DR ligands eluted from Graves' disease affected thyroid glands, from where autoantigen-derived peptides have been identified.

Infection with Salmonella typhimurium has no effect on the composition and cleavage specificity of the 20S proteasome in human lymphoid cells.

Human leucocyte antigen (HLA)-B27 is strongly associated with spondyloarthropathies, including reactive arthritis. Several Gram-negative bacteria, such as Salmonella typhimurium, can trigger this disease. It has been suggested that peptides derived from bacterial proteins and presented by HLA-B27 to cytotoxic T lymphocytes might show molecular mimicry with autologous peptides, leading to T-cell cross-reaction and autoimmunity. Antigen presentation in Salmonella-infected cells could be modulated by changes in the composition of the proteasome, which is the major proteolytic system that generates major histocompatibility complex class I ligands. In this study we analysed whether the composition or activity of the 20S proteasome was altered upon infection of lymphoid cells by S. typhimurium. Two-dimensional gel electrophoresis failed to show any differences between the composition of 20S proteasomes from cells infected with S. typhimurium for 24 hr, relative to non-infected cells. In addition, digestions of oxidized insulin B-chain with purified 20S proteasomes from non-infected and infected cells generated the same products, indicating that the proteasomal cleavage specificity was not altered upon infection. These data indicate that infection of lymphoid cells by S. typhimurium fails to induce formation of immunoproteasomes or otherwise alter the proteolytic specificity of the 20S proteasome.