Stabilizing role of platelet P2Y(12) receptors in shear-dependent thrombus formation on ruptured plaques

Background: In most models of experimental thrombosis, healthy blood vessels are damaged. This results in the formation of a platelet thrombus that is stabilized by ADP signaling via P2Y(12) receptors. However, such models do not predict involvement of P2Y(12) in the clinically relevant situation of thrombosis upon rupture of atherosclerotic plaques. We investigated the role of P2Y(12) in thrombus formation on (collagen-containing) atherosclerotic plaques in vitro and in vivo, by using a novel mouse model of atherothrombosis. Methodology: Plaques in the carotid arteries from Apoe(-/-) mice were acutely ruptured by ultrasound treatment, and the thrombotic process was monitored via intravital fluorescence microscopy. Thrombus formation in vitro was assessed in mouse and human blood perfused over collagen or plaque material under variable conditions of shear rate and coagulation. Effects of two reversible P2Y(12) blockers, ticagrelor (AZD6140) and cangrelor (AR-C69931MX), were investigated. Principal Findings: Acute plaque rupture by ultrasound treatment provoked rapid formation of non-occlusive thrombi, which were smaller in size and unstable in the presence of P2Y(12) blockers. In vitro, when mouse or human blood was perfused over collagen or atherosclerotic plaque material, blockage or deficiency of P2Y(12) reduced the thrombi and increased embolization events. These P2Y(12) effects were present at shear rates >500 s(-1), and they persisted in the presence of coagulation. P2Y(12)-dependent thrombus stabilization was accompanied by increased fibrin(ogen) binding. Conclusions/Significance: Platelet P2Y(12) receptors play a crucial role in the stabilization of thrombi formed on atherosclerotic plaques. This P2Y(12) function is restricted to high shear flow conditions, and is preserved in the presence of coagulation

Varicellovirus UL 49.5 proteins differentially affect the function of the transporter associated with antigen processing, TAP

Cytotoxic T-lymphocytes play an important role in the protection against viral infections, which they detect through the recognition of virus-derived peptides, presented in the context of MHC class I molecules at the surface of the infected cell. The transporter associated with antigen processing (TAP) plays an essential role in MHC class I–restricted antigen presentation, as TAP imports peptides into the ER, where peptide loading of MHC class I molecules takes place. In this study, the UL49.5 proteins of the varicelloviruses bovine herpesvirus 1 (BHV-1), pseudorabies virus (PRV), and equine herpesvirus 1 and 4 (EHV-1 and EHV-4) are characterized as members of a novel class of viral immune evasion proteins. These UL49.5 proteins interfere with MHC class I antigen presentation by blocking the supply of antigenic peptides through inhibition of TAP. BHV-1, PRV, and EHV-1 recombinant viruses lacking UL49.5 no longer interfere with peptide transport. Combined with the observation that the individually expressed UL49.5 proteins block TAP as well, these data indicate that UL49.5 is the viral factor that is both necessary and sufficient to abolish TAP function during productive infection by these viruses. The mechanisms through which the UL49.5 proteins of BHV-1, PRV, EHV-1, and EHV-4 block TAP exhibit surprising diversity. BHV-1 UL49.5 targets TAP for proteasomal degradation, whereas EHV-1 and EHV-4 UL49.5 interfere with the binding of ATP to TAP. In contrast, TAP stability and ATP recruitment are not affected by PRV UL49.5, although it has the capacity to arrest the peptide transporter in a translocation-incompetent state, a property shared with the BHV-1 and EHV-1 UL49.5. Taken together, these results classify the UL49.5 gene products of BHV-1, PRV, EHV-1, and EHV-4 as members of a novel family of viral immune evasion proteins, inhibiting TAP through a variety of mechanisms

Mass cytometric analysis unveils a disease-specific immune cell network in the bone marrow in acquired aplastic anemia

Idiopathic acquired aplastic anemia (AA) is considered an immune-mediated syndrome of bone marrow failure since approximately 70% of patients respond to immunosuppressive therapy (IST) consisting of a course of anti-thymocyte globulin (ATG) followed by long-term use of ciclosporin. However, the immune response that underlies the pathogenesis of AA remains poorly understood. In this study, we applied high-dimensional mass cytometry on bone marrow aspirates of AA patients pre-ATG, AA patients post-ATG and healthy donors to decipher which immune cells may be implicated in the pathogenesis of AA. We show that the bone marrow of AA patients features an immune cell composition distinct from healthy donors, with significant differences in the myeloid, B-cell, CD4+ and CD8+ T-cells lineages. Specifically, we discovered that AA pre-ATG is characterized by a disease-specific immune cell network with high frequencies of CD16+ myeloid cells, CCR6++ B-cells, Th17-like CCR6+ memory CD4+ T-cells, CD45RA+CCR7+CD38+ CD8+ T-cells and KLRG1+ terminally differentiated effector memory (EMRA) CD8+ T-cells, compatible with a state of chronic inflammation. Successful treatment with IST strongly reduced the levels of CD16+ myeloid cells and showed a trend toward normalization of the frequencies of CCR6++ B-cells, CCR6+ memory CD4+ T-cells and KLRG1+EMRA CD8+ T-cells. Altogether, our study provides a unique overview of the immune landscape in bone marrow in AA at a single-cell level and proposes CCR6 as a potential new therapeutic target in AA

High-Throughput Identification of Potential Minor Histocompatibility Antigens by MHC Tetramer-Based Screening: Feasibility and Limitations

T-cell recognition of minor histocompatibility antigens (MiHA) plays an important role in the graft-versus-tumor (GVT) effect of allogeneic stem cell transplantation (allo-SCT). However, the number of MiHA identified to date remains limited, making clinical application of MiHA reactive T-cell infusion difficult. This study represents the first attempt of genome-wide prediction of MiHA, coupled to the isolation of T-cell populations that react with these antigens. In this unbiased high-throughput MiHA screen, both the possibilities and pitfalls of this approach were investigated. First, 973 polymorphic peptides expressed by hematopoietic stem cells were predicted and screened for HLA-A2 binding. Subsequently a set of 333 high affinity HLA-A2 ligands was identified and post transplantation samples from allo-SCT patients were screened for T-cell reactivity by a combination of pMHC-tetramer-based enrichment and multi-color flow cytometry. Using this approach, 71 peptide-reactive T-cell populations were generated. The isolation of a T-cell line specifically recognizing target cells expressing the MAP4K1IMA antigen demonstrates that identification of MiHA through this approach is in principle feasible. However, with the exception of the known MiHA HMHA1, none of the other T-cell populations that were generated demonstrated recognition of endogenously MiHA expressing target cells, even though recognition of peptide-loaded targets was often apparent

Transduction of Human T Cells with a Novel T-Cell Receptor Confers Anti-HCV Reactivity

Hepatitis C Virus (HCV) is a major public health concern, with no effective vaccines currently available and 3% of the world's population being infected. Despite the existence of both B- and T-cell immunity in HCV-infected patients, chronic viral infection and HCV-related malignancies progress. Here we report the identification of a novel HCV TCR from an HLA-A2-restricted, HCV NS3:1073–1081-reactive CTL clone isolated from a patient with chronic HCV infection. We characterized this HCV TCR by expressing it in human T cells and analyzed the function of the resulting HCV TCR-transduced cells. Our results indicate that both the HCV TCR-transduced CD4+ and CD8+ T cells recognized the HCV NS3:1073–1081 peptide-loaded targets and HCV+ hepatocellular carcinoma cells (HCC) in a polyfunctional manner with cytokine (IFN-γ, IL-2, and TNF-α) production as well as cytotoxicity. Tumor cell recognition by HCV TCR transduced CD8− Jurkat cells and CD4+ PBL-derived T cells indicated this TCR was CD8-independent, a property consistent with other high affinity TCRs. HCV TCR-transduced T cells may be promising for the treatment of patients with chronic HCV infections

Varicellovirus UL49.5 Proteins Differentially Affect the Function of the Transporter Associated with Antigen Processing, TAP

Cytotoxic T-lymphocytes play an important role in the protection against viral infections, which they detect through the recognition of virus-derived peptides, presented in the context of MHC class I molecules at the surface of the infected cell. The transporter associated with antigen processing (TAP) plays an essential role in MHC class I–restricted antigen presentation, as TAP imports peptides into the ER, where peptide loading of MHC class I molecules takes place. In this study, the UL49.5 proteins of the varicelloviruses bovine herpesvirus 1 (BHV-1), pseudorabies virus (PRV), and equine herpesvirus 1 and 4 (EHV-1 and EHV-4) are characterized as members of a novel class of viral immune evasion proteins. These UL49.5 proteins interfere with MHC class I antigen presentation by blocking the supply of antigenic peptides through inhibition of TAP. BHV-1, PRV, and EHV-1 recombinant viruses lacking UL49.5 no longer interfere with peptide transport. Combined with the observation that the individually expressed UL49.5 proteins block TAP as well, these data indicate that UL49.5 is the viral factor that is both necessary and sufficient to abolish TAP function during productive infection by these viruses. The mechanisms through which the UL49.5 proteins of BHV-1, PRV, EHV-1, and EHV-4 block TAP exhibit surprising diversity. BHV-1 UL49.5 targets TAP for proteasomal degradation, whereas EHV-1 and EHV-4 UL49.5 interfere with the binding of ATP to TAP. In contrast, TAP stability and ATP recruitment are not affected by PRV UL49.5, although it has the capacity to arrest the peptide transporter in a translocation-incompetent state, a property shared with the BHV-1 and EHV-1 UL49.5. Taken together, these results classify the UL49.5 gene products of BHV-1, PRV, EHV-1, and EHV-4 as members of a novel family of viral immune evasion proteins, inhibiting TAP through a variety of mechanisms

Extracellular domains of CD8α and CD8ß subunits are sufficient for HLA class I restricted helper functions of TCR-engineered CD4(+) T cells.

By gene transfer of HLA-class I restricted T-cell receptors (TCRs) (HLA-I-TCR) into CD8(+) as well as CD4(+) T-cells, both effector T-cells as well as helper T-cells can be generated. Since most HLA-I-TCRs function best in the presence of the CD8 co-receptor, the CD8αß molecule has to be co-transferred into the CD4(+) T-cells to engineer optimal helper T-cells. In this study, we set out to determine the minimal part of CD8αβ needed for optimal co-receptor function in HLA-I-TCR transduced CD4(+) T-cells. For this purpose, we transduced human peripheral blood derived CD4(+) T-cells with several HLA-class I restricted TCRs either with or without co-transfer of different CD8 subunits. We demonstrate that the co-transduced CD8αβ co-receptor in HLA-I-TCR transduced CD4(+) T-cells behaves as an adhesion molecule, since for optimal antigen-specific HLA class I restricted CD4(+) T-cell reactivity the extracellular domains of the CD8α and ß subunits are sufficient

Strong selection of virus-specific cytotoxic CD4+ T-cell clones during primary human cytomegalovirus infection

To obtain insight into human CD4+ T cell differentiation and selection in vivo, we longitudinally studied cytomegalovirus (CMV)-specific CD4+ T cells after primary infection. Early in infection, CMV-specific CD4+ T cells have the appearance of interferon gamma (IFNgamma)-producing T-helper 1 (TH1) type cells, whereas during latency a large population of CMV-specific CD4+ CD28- T cells emerges with immediate cytotoxic capacity. We demonstrate that CD4+ CD28- T cells could lyse CMV antigen-expressing target cells in a class II-dependent manner. To clarify the clonal relationship between early and late CMV-specific CD4+ T cells, we determined their Vbeta usage and CDR3 sequences. The T-cell receptor beta (TCRbeta) diversity in the early CMV-specific CD4+ T-cell population was high in contrast to the use of a very restricted set of TCRbeta sequences in latent infection. T-cell clones found in the late CMV-specific CD4+ T-cell population could not be retrieved from the early CD4+ T-cell population, or were present only at a low frequency. The observation that dominant CMV-specific CD4+ clones during latency were only poorly represented in the acute phase suggests that after the initial control of the virus strong selection and/or priming of novel clones takes place in persistent infections in human