Approach for Identifying Human Leukocyte Antigen (HLA)-DR Bound Peptides from Scarce Clinical Samples.

Immune-mediated diseases strongly associating with human leukocyte antigen (HLA) alleles are likely linked to specific antigens. These antigens are presented to T cells in the form of peptides bound to HLA molecules on antigen presenting cells, e.g. dendritic cells, macrophages or B cells. The identification of HLA-DR-bound peptides presents a valuable tool to investigate the human immunopeptidome. The lung is likely a key player in the activation of potentially auto-aggressive T cells prior to entering target tissues and inducing autoimmune disease. This makes the lung of exceptional interest and presents an ideal paradigm to study the human immunopeptidome and to identify antigenic peptides.Our previous investigation of HLA-DR peptide presentation in the lung required high numbers of cells (800 × 10(6) bronchoalveolar lavage (BAL) cells). Because BAL from healthy nonsmokers typically contains 10-15 × 10(6) cells, there is a need for a highly sensitive approach to study immunopeptides in the lungs of individual patients and controls.In this work, we analyzed the HLA-DR immunopeptidome in the lung by an optimized methodology to identify HLA-DR-bound peptides from low cell numbers. We used an Epstein-Barr Virus (EBV) immortalized B cell line and bronchoalveolar lavage (BAL) cells obtained from patients with sarcoidosis, an inflammatory T cell driven disease mainly occurring in the lung. Specifically, membrane complexes were isolated prior to immunoprecipitation, eluted peptides were identified by nanoLC-MS/MS and processed using the in-house developed ClusterMHCII software. With the optimized procedure we were able to identify peptides from 10 × 10(6) cells, which on average correspond to 10.9 peptides/million cells in EBV-B cells and 9.4 peptides/million cells in BAL cells. This work presents an optimized approach designed to identify HLA-DR-bound peptides from low numbers of cells, enabling the investigation of the BAL immunopeptidome from individual patients and healthy controls in order to identify disease-associated peptides.

Signal sequence directs localized secretion of bacterial surface proteins.

All living cells require specific mechanisms that target proteins to the cell surface. In eukaryotes, the first part of this process involves recognition in the endoplasmic reticulum of amino-terminal signal sequences and translocation through Sec translocons, whereas subsequent targeting to different surface locations is promoted by internal sorting signals. In bacteria, N-terminal signal sequences promote translocation across the cytoplasmic membrane, which surrounds the entire cell, but some proteins are nevertheless secreted in one part of the cell by poorly understood mechanisms. Here we analyse localized secretion in the Gram-positive pathogen Streptococcus pyogenes, and show that the signal sequences of two surface proteins, M protein and protein F (PrtF), direct secretion to different subcellular regions. The signal sequence of M protein promotes secretion at the division septum, whereas that of PrtF preferentially promotes secretion at the old pole. Our work therefore shows that a signal sequence may contain information that directs the secretion of a protein to one subcellular region, in addition to its classical role in promoting secretion. This finding identifies a new level of complexity in protein translocation and emphasizes the potential of bacterial systems for the analysis of fundamental cell-biological problems.

Enhanced Inflammasome Activity in Patients with Psoriasis Promotes Systemic Inflammation.

Psoriasis is linked to systemic inflammation and cardiovascular comorbidities, but studies of the underlying cellular mechanisms are lacking. The NLRP3 inflammasome is genetically associated with psoriasis, and its activation is increasingly linked with cardiovascular disease. In this study, we show that patients with psoriasis exhibited higher plasma levels of inflammasome-generated IL-1ß and IL-18, without any correlation to skin lesion severity. Increased constitutive expression of the inflammasome sensors NLRP3, NLRP1, and AIM2 was found in peripheral blood cells of the patients and also of those with mild disease, and this was accompanied by an increased caspase-1 reactivity in the myeloid blood subsets. TNF-α was found to activate selectively the NLRP3 inflammasome without the requirement for a priming signal. TNF-α was found to signal through the TNFR‒caspase-8‒caspase-1 alternative inflammasome pathway, which proceeds independently of pyroptosis. Patients who received anti-TNF therapy had normalized plasma IL-1ß and IL-18 levels as well as normalized caspase-1 reactivity. This was in contrast to the patients treated with methotrexate who exhibited persistent, increased caspase-1 reactivity. Thus, we show that the TNF-α-mediated activation of NLRP3 inflammasomes in patients with psoriasis may contribute to systemic inflammation. Anti-TNF therapy normalized inflammasome function, suggesting a mechanism for the cardiovascular risk‒reducing effect.

Corrigendum: Functional and Structural Characterization of a Novel HLA-DRB1*04:01-Restricted α-Enolase T Cell Epitope in Rheumatoid Arthritis.

[This corrects the article on p. 494 in vol. 7, PMID: 27895642.].

Functional and Structural Characterization of a Novel HLA-DRB1*04:01-Restricted α-Enolase T Cell Epitope in Rheumatoid Arthritis.

Antibodies to citrullinated proteins, common in rheumatoid arthritis (RA) patients, are strongly associated to a specific set of HLA-DR alleles including HLA-DRB1*04:01, *04:04, and *01:01. Here, we first demonstrate that autoantibody levels toward the dominant citrullinated B cell epitope from α-enolase are significantly elevated in HLA-DRB1*04:01-positive RA patients. Furthermore, we identified α-enolase-derived T cell epitopes and demonstrated that native and citrullinated versions of several peptides bind with different affinities to HLA-DRB1*04:01, *04:04, and *01:01. The citrulline residues in the eight identified peptides are distributed throughout the entire length of the presented epitopes and more specifically, localized at peptide positions p-2, p2, p4, p6, p7, p10, and p11. Importantly, in contrast to its native version peptide 26 (TSKGLFRAAVPSGAS), the HLA-DRB1*04:01-restricted citrullinated peptide Cit26 (TSKGLFCitAAVPSGAS) elicited significant functional T cell responses in primary cells from RA patients. Comparative analysis of the crystal structures of HLA-DRB1*04:01 in complex with peptide 26 or Cit26 demonstrated that the posttranslational modification did not alter the conformation of the peptide. And since citrullination is the only structural difference between the two complexes, this indicates that the neo-antigen Cit26 is recognized by T cells with high specificity to the citrulline residue.

Accelerating translational research by clinically driven development of an informatics platform--a case study.

Translational medicine is becoming increasingly dependent upon data generated from health care, clinical research, and molecular investigations. This increasing rate of production and diversity in data has brought about several challenges, including the need to integrate fragmented databases, enable secondary use of patient clinical data from health care in clinical research, and to create information systems that clinicians and biomedical researchers can readily use. Our case study effectively integrates requirements from the clinical and biomedical researcher perspectives in a translational medicine setting. Our three principal achievements are (a) a design of a user-friendly web-based system for management and integration of clinical and molecular databases, while adhering to proper de-identification and security measures; (b) providing a real-world test of the system functionalities using clinical cohorts; and (c) system integration with a clinical decision support system to demonstrate system interoperability. We engaged two active clinical cohorts, 747 psoriasis patients and 2001 rheumatoid arthritis patients, to demonstrate efficient query possibilities across the data sources, enable cohort stratification, extract variation in antibody patterns, study biomarker predictors of treatment response in RA patients, and to explore metabolic profiles of psoriasis patients. Finally, we demonstrated system interoperability by enabling integration with an established clinical decision support system in health care. To assure the usefulness and usability of the system, we followed two approaches. First, we created a graphical user interface supporting all user interactions. Secondly we carried out a system performance evaluation study where we measured the average response time in seconds for active users, http errors, and kilobits per second received and sent. The maximum response time was found to be 0.12 seconds; no server or client errors of any kind were detected. In conclusion, the system can readily be used by clinicians and biomedical researchers in a translational medicine setting.

Citrulline-specific Th1 cells are increased in rheumatoid arthritis and their frequency is influenced by disease duration and therapy.

OBJECTIVE: Rheumatoid arthritis (RA) is thought to be a T cell-mediated disease, based on its strong association with HLA class II alleles, clinical responsiveness to T cell-directed therapies, and the presence of CD4+ T cells in rheumatoid joints. The presence of anti-citrullinated protein antibodies (ACPAs) in RA serum and the association of these antibodies with HLA-DR4 alleles implicate citrulline-specific autoreactive T cells in the development and progression of RA. The goal of this study was to determine the characteristics and specificity of autoreactive T cell responses in RA. METHODS: We developed a panel of HLA-DRB1*04:01 tetramers, selecting citrullinated peptides from synovial antigens and verifying their immunogenicity in DRB1*04:01-transgenic mice. Seven tetramers were used to examine the ex vivo frequency and surface phenotype of citrulline-specific (Cit-specific) T cells in patients with RA and healthy subjects with DRB1*04:01 haplotypes, using a magnetic enrichment procedure. RESULTS: Cit-specific T cells were detectable in peripheral blood samples from both healthy subjects and RA patients. In comparison to healthy subjects, RA patients had significantly higher frequencies of Cit-specific T cells, and a greater proportion of these cells displayed a Th1 memory phenotype. Among RA patients, the frequency of Cit-specific T cells was highest within the first 5 years after diagnosis of RA and was decreased in patients taking biologic agents, irrespective of disease duration. CONCLUSION: These findings link the presence of ACPAs in RA with Th1 cells specific for citrullinated epitopes and provide tools for disease-specific immunomonitoring of autoreactive T cells.

Functional dissection of Streptococcus pyogenes M5 protein: the hypervariable region is essential for virulence.

The surface-localized M protein of Streptococcus pyogenes is a major virulence factor that inhibits phagocytosis, as determined ex vivo. Because little is known about the role of M protein in vivo we analyzed the contribution of different M protein regions to virulence, using the fibrinogen (Fg)-binding M5 protein and a mouse model of acute invasive infection. This model was suitable, because M5 is required for mouse virulence and binds mouse and human Fg equally well, as shown here. Mixed infection experiments with wild type bacteria demonstrated that mutants lacking the N-terminal hypervariable region (HVR) or the Fg-binding B-repeat region were strongly attenuated, while a mutant lacking the conserved C-repeats was only slightly attenuated. Because the HVR of M5 is not required for phagocytosis resistance, our data imply that this HVR plays a major but unknown role during acute infection. The B-repeat region is required for phagocytosis resistance and specifically binds Fg, suggesting that it promotes virulence by binding Fg. However, B-repeat mutants were attenuated even in Fg-deficient mice, implying that the B-repeats may have a second function, in addition to Fg-binding. These data demonstrate that two distinct M5 regions, including the HVR, are essential to virulence during the early stages of an infection. In particular, our data provide the first in vivo evidence that the HVR of an M protein plays a major role in virulence, focusing interest on the molecular role of this region.

Binding of human plasma proteins to Streptococcus pyogenes M protein determines the location of opsonic and non-opsonic epitopes.

Antibodies directed against a pathogenic microorganism may recognize either protective or non-protective epitopes. Because antibodies elicited by a vaccine must be directed against protective epitopes, it is essential to understand the molecular properties that distinguish the two types of epitope. Here we analyse this problem for the antiphagocytic M protein of Streptococcus pyogenes, using the opsonizing capacity of antibodies to estimate their ability to confer protection in vivo. Our studies were focused on the M5 protein, which has three surface-exposed regions: the amino-terminal hypervariable region (HVR) and the B- and C-repeat regions. We first analysed the role of different M5 regions in phagocytosis resistance under non-immune conditions, employing chromosomal mutants expressing M5 proteins with internal deletions, and demonstrate that only the B-repeat region is essential for phagocytosis resistance. However, only antibodies to the HVR were opsonic. This apparent paradox could be explained by the ability of fibrinogen and albumin to specifically bind to the B- and C-repeats, respectively, causing inhibition of antibody binding under physiological conditions, while antibodies to the HVR could bind and promote deposition of complement. These data indicate that binding of human plasma proteins plays an important role in determining the location of opsonic and non-opsonic epitopes in streptococcal M protein.

Human fibrinogen bound to Streptococcus pyogenes M protein inhibits complement deposition via the classical pathway.

Human fibrinogen (Fg) binds to surface proteins expressed by many pathogenic bacteria and has been implicated in different host-pathogen interactions, but the role of bound Fg remains unclear. Here, we analyse the role of Fg bound to Streptococcus pyogenes M protein, a major virulence factor that confers resistance to phagocytosis. Studies of the M5 system showed that a chromosomal mutant lacking the Fg-binding region was completely unable to resist phagocytosis, indicating that bound Fg plays a key role in virulence. Deposition of complement on S. pyogenes occurred via the classical pathway even under non-immune conditions, but was blocked by M5-bound Fg, which reduced the amount of classical pathway C3 convertase on the bacterial surface. This property of M protein-bound Fg may explain its role in phagocytosis resistance. Previous studies have shown that many M proteins do not bind Fg, but interfere with complement deposition and phagocytosis by recruiting human C4b-binding protein (C4BP), an inhibitor of the classical pathway. Thus, all M proteins may share ability to recruit a human plasma protein, Fg or C4BP, which inhibits complement deposition via the classical pathway. Our data identify a novel function for surface-bound Fg and allow us to propose a unifying mechanism by which M proteins interfere with innate immunity.

Induction of a regulatory phenotype in human CD4+ T cells by streptococcal M protein.

Regulatory T cells (Tregs) participate in the control of the immune response. In the human system, an IL-10-secreting, T regulatory type 1 cell (Tr1)-like subset of Tregs can be induced by concurrent cross-linking of the TCR and CD46 on naive CD4(+) T cells. Because many viral and bacterial pathogens, including the major human pathogen Streptococcus pyogenes, bind to CD46, we asked whether this bacterium can directly induce Tr1-like cells through the streptococcal ligand for CD46, the M protein. The M5 and M22 proteins were found to induce T cells to develop into the IL-10-producing Tr1-like phenotype. Moreover, whole M5-expressing bacteria, but not isogenic M-negative bacteria, led to proliferation and IL-10 secretion by T cells. The interaction between the M5 protein and T cells was dependent on CD46 and the conserved C repeat region of M5. Supernatants derived from T cells stimulated with M proteins or M protein-expressing bacteria suppressed bystander T cell proliferation through IL-10 secretion. In addition, activation of CD46 through streptococcal M protein induced the expression of granzyme B, providing a second means for these cells to regulate an immune response. These findings suggest that binding to CD46 and exploiting its signaling pathway may represent a strategy employed by a number of important human pathogens to induce directly an immunosuppressive/regulatory phenotype in T cells.

Isolation and detection of human IgA using a streptococcal IgA-binding peptide.

Bacterial proteins that bind to the Fc part of IgG have found widespread use in immunology. A similar protein suitable for the isolation and detection of human IgA has not been described. Here, we show that a 50-residue synthetic peptide, designated streptococcal IgA-binding peptide (Sap) and derived from a streptococcal M protein, can be used for single-step affinity purification of human IgA. High affinity binding of IgA required the presence in Sap of a C-terminal cysteine residue, not present in the intact M protein. Passage of human serum through a Sap column caused depletion of >99% of the IgA, and elution of the column allowed quantitative recovery of highly purified IgA, for which the proportions of the IgA1 and IgA2 subclasses were the same as in whole serum. Moreover, immobilized Sap could be used for single-step purification of secretory IgA of both subclasses from human saliva, with a recovery of approximately 45%. The Sap peptide could also be used to specifically detect IgA bound to Ag. Together, these data indicate that Sap is a versatile Fc-binding reagent that may open new possibilities for the characterization of human IgA.