Disulfide bond reduction and exchange in C4 domain of von Willebrand factor undermines platelet binding

Background The von Willebrand factor (VWF) is a key player in regulating hemostasis through adhesion of platelets to sites of vascular injury. It is a large, multi-domain, mechano-sensitive protein that is stabilized by a net of disulfide bridges. Binding to platelet integrin is achieved by the VWF-C4 domain, which exhibits a fixed fold, even under conditions of severe mechanical stress, but only if critical internal disulfide bonds are closed. Objective To determine the oxidation state of disulfide bridges in the C4 domain of VWF and implications for VWF’s platelet binding function. Methods We combined classical molecular dynamics and quantum mechanical simulations, mass spectrometry, site-directed mutagenesis, and platelet binding assays. Results We show that 2 disulfide bonds in the VWF-C4 domain, namely the 2 major force-bearing ones, are partially reduced in human blood. Reduction leads to pronounced conformational changes within C4 that considerably affect the accessibility of the integrin-binding motif, and thereby impair integrin-mediated platelet binding. We also reveal that reduced species in the C4 domain undergo specific thiol/disulfide exchanges with the remaining disulfide bridges, in a process in which mechanical force may increase the proximity of specific reactant cysteines, further trapping C4 in a state of low integrin-binding propensity. We identify a multitude of redox states in all 6 VWF-C domains, suggesting disulfide bond reduction and swapping to be a general theme. Conclusions Our data suggests a mechanism in which disulfide bonds dynamically swap cysteine partners and control the interaction of VWF with integrin and potentially other partners, thereby critically influencing its hemostatic function

Compartmentalization of total and virus-specific tissue-resident memory CD8+ T Cells in human lymphoid organs

Disruption of T cell memory during severe immune suppression results in reactivation of chronic viral infections, such as Epstein Barr virus (EBV) and Cytomegalovirus (CMV). How different subsets of memory T cells contribute to the protective immunity against these viruses remains poorly defined. In this study we examined the compartmentalization of virus-specific, tissue resident memory CD8+ T cells in human lymphoid organs. This revealed two distinct populations of memory CD8+ T cells, that were CD69+CD103+ and CD69+CD103-, and were retained within the spleen and tonsils in the absence of recent T cell stimulation. These two types of memory cells were distinct not only in their phenotype and transcriptional profile, but also in their anatomical localization within tonsils and spleen. The EBV-specific, but not CMV-specific, CD8+ memory T cells preferentially accumulated in the tonsils and acquired a phenotype that ensured their retention at the epithelial sites where EBV replicates. In vitro studies revealed that the cytokine IL-15 can potentiate the retention of circulating effector memory CD8+ T cells by down-regulating the expression of sphingosine-1-phosphate receptor, required for T cell exit from tissues, and its transcriptional activator, Kruppel-like factor 2 (KLF2). Within the tonsils the expression of IL-15 was detected in regions where CD8+ T cells localized, further supporting a role for this cytokine in T cell retention. Together this study provides evidence for the compartmentalization of distinct types of resident memory T cells that could contribute to the long-term protection against persisting viral infections

Identification of putative functional motifs in viral proteins essential for human cytomegalovirus (HCMV) DNA replication

Human cytomegalovirus (HCMV) is a ubiquitous virus that causes significant morbidity and mortality in immunocompromised individuals. Although there are prophylactic treatments available, all current antiviral drugs ultimately target the DNA polymerase, resulting in the increasing emergence of antiviral resistant strains in the clinical setting. There is a fundamental need for understanding the role of other essential genes in DNA replication as a foundation for developing new antiviral treatments that are safe and which utilize a mechanism of action different to existing therapies. In this study we looked at six HCMV replication genes encoding for the DNA polymerase accessory protein (UL44), single stranded DNA binding protein (UL57), primase (UL70), helicase (UL105), primase-helicase associated protein (UL102), and the putative initiator protein (UL84) in order to increase our understanding of their role in DNA replication. The aim of this project was to identify variation within these genes as well as to predict putative domains and motifs in order to ultimately express and study the functional properties of the HCMV primase (UL70) through the use of recombinant mutants. Sequencing of these genes revealed a high degree of conservation between the isolates with amino acid sequence identity of >97% for all genes. Using ScanProsite software from the Expert Protein Analysis System (ExPASy) proteomics server, we have mapped putative motifs throughout these HCMV replication genes. In particular, highly conserved putative Nlinked glycosylation sites were identified in UL105 that were also conserved across 33 homologues as well as several unique motifs including casein kinase II phosphorylation sites (CKII) in UL105 and UL84, a microbodies signal motif in UL57 and an integrin binding site in the UL102 helicase-primase associated protein. Our investigations have also elucidated motif-rich regions of the UL44 DNA polymerase accessory protein, mapped functionally important domains of the UL105 helicase and identified cysteine motifs that have implications for folding of the UL70 primase. Taken together, these findings provide insights to regions of these HCMV replication proteins that are important for post-translation modification, activation and overall function, and this information can be utilized to target further research into these proteins and advance the development of novel antiviral agents that target these processes

CD69⁺CD103⁺CD8⁺ T cells localize near the epithelial barrier in tonsils.

<p>The locations of CD69⁺CD8⁺ T cell subsets were determined by immunohistochemistry. (A) Immunofluorescence microscopy images of human tonsils show the localization of CD8 (green), CD69 (blue) and CD103 (red). Scale bar represents 100 μm. (B) Higher magnification of areas 1 & 2 show the localization of CD103⁺CD69⁺CD8⁺ and CD103CD69⁺CD8⁺ T cells within the tonsils. (C) White arrowheads point to CD103⁺CD69⁺CD8⁺ T cells in areas 1 and CD103^—CD69⁺CD8⁺ T cells in area 2. (C) Quantitative analysis of the distance of CD103⁺CD3⁺CD8⁺ T cells from the epithelium shows majority localizing near the epithelial surface (P = 0.0022 by two-tailed Mann Whitney U-test). (D) Immunofluorescence microscopy of human spleen sections shows the localization of CD8 (green), CD69 (blue) and CD103 (red). Scale bar represents 100 μm. Higher magnification of areas 1 and 2 show the distribution of CD103⁺CD69⁺CD8⁺ and CD103^—CD69⁺CD8⁺ T cells. White arrowheads in area 1 show the CD103⁺CD69⁺CD8⁺ T cells and in area 2 show the CD103^—CD69⁺CD8⁺ T cells.</p

Two subsets of CD8⁺ T cells are retained within human lymphoid tissues.

<p>The co-expression of CD69 and CD103 by CD8⁺ T cells isolated from spleen and tonsils were determined by flow cytometry. (A and B) Representative flow cytometry plots (left) and graphs (right; mean ± SEM; n = 10) show the proportion of CD69⁺CD8⁺ T cells expressing CD103 in human spleen (A) and tonsils (B). (C—E) Representative flow cytometry plots showing the expression levels of CCR7, CD45RA and CD11a between CD69^—CD103^—(blue), CD69⁺CD103^—(red) and CD69⁺CD103⁺ (green) CD8⁺ T cell subsets from the spleen (C) and tonsils (D) and the expression of PD-1, TIM3 and BTLA in tonsils (E). Data is representative of 3–5 independent experiments. (F) Relative expression of <i>KLF2</i> and <i>S1PR1</i> in purified CD8⁺ T subsets from the spleen (left panels) and tonsils (right panels). Individual dots represent different donor samples (n = 8 for spleen and n = 3 for tonsils). Statistical analysis was performed using one-way ANOVA and Tukey test. P<0.05 is noted with * and P<0.0005 is noted with ***.</p

IL-15 and TGF-β co-operate to extinguish expression of <i>KLF2</i> and <i>S1PR1</i>.

<p>(A) Representative flow cytometry plots show the expression of CD69 and CD103 by CD8⁺ T cells following 7 day culture under different conditions: unstimulated (US), IL-15 or IL-15 + TGF-β and polyclonal stimulation (TAE). (B) Plot shows the proportion of CD69⁺CD103^- (left panel) and CD69⁺CD103⁺ (right panel) CD8⁺ T cells following 7 day culture with different cytokines. Data are represented as the mean and SEM of 5–11 donors. (C) Representative flow cytometry plots show the expression of CD69, CD137 and dilution of cell trace violet (CTV) dye following stimulation of circulating CD8⁺ T cells for 7 days with TAE beads (upper panels) or IL-15 (lower panels). (D) Representative flow cytometry plot and graph show the expression of CD69 and the dilution of cell trace violet dye following stimulation of purified circulating naïve (n = 4), TCM (n = 2), TEM (n = 8) and TEMRA (n = 5) CD8⁺ T cells for 7 days with IL-15. (E-F) Plots show the relative expression of <i>S1PR1</i> (E) and <i>KLF2</i> (F) in CD69⁺ or CD69^—CD8⁺ T cells following culture for 7 days with no stimulation or stimulation with IL-15 with and without TGF-β. Purified circulating CD8⁺ T cells were cultured for 7 days and the resulting CD69⁺ and CD69^—populations were purified by cell sorting. The expression levels of KLF2 and S1pr1 were quantified by RT-PCR. Individual dots represent different samples and the data is represented as the mean ± SEM. Statistical analysis was performed using one-way ANOVA and Tukey test. P<0.05 is noted with * and P<0.005 is noted with **. (G-H) The ability of IL-15 induced CD69⁺CD8⁺ T cells to migrate to S1P and CCL5 (20 nM) was tested in trans-well migration assays. Cultured cells were sorted as stated above (for F) and their ability to migrate towards different concentrations of S1P (G) or CCL5 (20 nM) (H) was determined. Data represent the mean and SEM of three independent experiments using three different donors. Statistical analysis was performed using two-way ANOVA and the p value was < 0.05.</p

Constitutive expression of IL-15 within tonsils.

<p>Immunofluorescence microscopy of frozen section of the tonsils show the presence of IL-15 expressing cells in the T cell areas and the epithelial lining of the tissue. Sections were stained with anti-IL-15 (red), anti-CD8 (green) and anti-IgM (blue). Yellow dashed line marks the epithelial barrier surface and the white dashed-lines show B cell follicles (B). ‘T” indicates the extra-follicular regions where T cells localize.</p

CD69 is expressed on CD8⁺ T cells in the absence of recent T cell activation.

<p>The phenotype of CD8⁺ T cells isolated from peripheral blood, spleen and tonsils were examined by flow cytometry. (A) The mean (± SD; n = 13 for blood and spleen and n = 7 for tonsils) percent of CD69⁺CD8⁺ T cells in these compartments. (B) CD69 expression on naïve (CCR7⁺CD45RA⁺), central memory (TCM, CCR7⁺CD45RA^—), effector memory (TEM, CCR7^—CD45RA^—) and TEMRA (CCR7^—CD45RA⁺) CD8⁺ T cells from blood (blue), spleen (red) and tonsils (green). Individual dots represent different donor samples. (C and D) Representative flow cytometry histogram plots show the expression levels of activation markers CD25, CD137, HLA-DR and KLRG1 between the CD69⁺CD8⁺ T cells (red) and CD69^—CD8⁺ T cells (blue) in the spleen (C) and tonsils (D). Data is representative of 5 independent experiments. (E) Graph shows the relative expression levels of <i>BCL2</i> between CD69⁺ TEM CD8⁺ T cells and CD69^—TEM CD8⁺ T cells from human spleen (n = 5). P = 0.0625 by one-way ANOVA.</p

Selective retention of EBV-specific CD8⁺ T cells in tissues.

<p>Virus-specific cells were determined by staining single cell suspensions with soluble peptide-MHC complexes (dextramers). (A) The graphs show the normal range and the line at mean value for each virus-specific population in blood (blue), spleen (red) and tonsils (orange). (B) Representative flow cytometry plots show the dual expression of CD69 and CD103 on EBV and CMV-specific CD8⁺ T cells in blood, spleen and tonsils. (C) Graphs show the proportions of CD69^—CD103^—, CD69⁺CD103^—and CD69⁺CD103⁺ EBV-specific and CMV-specific CD8⁺ T cells from spleen and tonsils. Data is presented as the mean ± SEM of n = 5 (for EBV specific cells in tonsils and CMV-specific cells in spleen), n = 6 (EBV-specific cells in spleen) and n = 3 (CMV-specific cells in tonsils) different samples. (D) Representative flow cytometry overlayed histogram plots show no difference in the expression of HLA-DR between CD69^—(blue) and CD69⁺ (red) EBV-specific CD8⁺ T cells in the tonsils (left) and spleen (right).</p